©octor of ^liilogopl)?Experiment 2 (2002-2003) was conducted according to simple randomized block...
210
•3' EFFECT OF PHOSPHORUS AND CALCIUM ON SELECTED MEDICINALLY IMPORTANT LEGUMINOUS PLANTS / '/.- SUBMITTED FOR THE AWARD OF THE DEGREE OF ©octor of ^liilogopl)? ^\o^\ IN BOTANY BY Mu. NAEEM \X^ ,v^ <\^^' DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2007
Transcript of ©octor of ^liilogopl)?Experiment 2 (2002-2003) was conducted according to simple randomized block...
• 3 '
EFFECT OF PHOSPHORUS AND CALCIUM ON SELECTED MEDICINALLY
IMPORTANT LEGUMINOUS PLANTS
/ '/.-
SUBMITTED FOR THE AWARD OF THE DEGREE OF
©octor of ^liilogopl)? ^\o^\ IN
BOTANY
BY
Mu. NAEEM
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DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2007
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EFFECT OF PHOSPHORUS AND CALCIUM ON SELECTED MEDICINALLY IMPORTANT LEGUMINOUS PLANTS
Mu. NAEEM
Abstract of the thesis submitted to the Aligarh Muslim University,
AHgarh, U.P., India for the award of the degree of Doctor of Philosophy in
Botany, 2007.
Six-pot experiments were carried out during the 'Rabi' (winter) seasons
of 2002-2004 on hyacinth bean, senna sophera and coffee senna. The salient
points are summarized below.
Experiment 1 (2002-2003) was conducted according to simple
randomized block design, to study the effect of five basal levels of phosphorus,
viz. 0, 25, 50, 75 and 100 mg P per kg soil (PQ, PI , P2, P3 and P4, respectively)
on hyacinth bean {Lablab purpureus L.). The aim of this experiment was to
find out the optimum dose of phosphorus so as to get the best response of
hyacinth bean in terms of growth attributes (fresh weight per plant, dry weight
per plant, number of nodules per plant and dry weight of nodules per plant),
biochemical attributes (total chlorophyll content and total carotenoids content,
nitrate reductase activity, leaf-NPK and Ca contents and nodule-nitrogen and
leghemoglobin contents) and yield and quality attributes (number of pods per
plant, number of seeds per pod, 100-seed weight, seed-yield per plant and seed-
protein content). The growth and biochemical attributes were studied at 60, 90
and 120 DAS. Yield and quality attributes were analyzed at the time of harvest
(150 DAS). Out of the five phosphorus levels, P3 proved optimum and
significantly enhanced all growth, biochemical, yield and quality attributes at
all growth stages except leghemoglobin content at 120 DAS, number of seeds
per pod and 100-seed weight at harvest. This treatment increased seed-yield
and seed-protein content by 38.3 and 14.9 %, respectively, over the Control
(Po).
tB r%.>l^»'"
Experiment 2 (2002-2003) was conducted according to simple
randomized block design. The aim of this experiment was to find out the best
dose of calcium out of five levels of calcium, viz. 0, 40, 80, 120 and 160 mg Ca
per kg soil (Cao, Cai, Ca2, Ca^ and Ca4, respectively) so as to get the best
response of hyacinth bean {Lablab purpureus L.). The performance of the crop
was adjudged on the basis of same attributes employing the same stages as in
the case of Experiment 1. The values of growth, biochemical, yield and quality
attributes studied obtained in this experiment (except number of seeds per pod
and 100-seed weight) were significantly enhanced by the basal application of
calcium. Regarding the calcium levels, Cas proved best for all attributes and
gave maximum seed-yield and seed protein content by 30.3 and 11.6 %,
respectively.
Experiment 3 (2002-2003) was performed according to simple
randomized block design. The aim of this experiment was to establish the best
dose out of five basal levels of phosphorus, viz. 0, 25, 50, 75 and 100 mg P per
kg soil (Po, Pi, P2, P3 and P4, respectively) to exploit the full genetic potential of
senna sophera {Cassia sophera L.) in terms of growth (fresh weight per plant
and dry weight per plant), biochemical (total chlorophyll content and total
carotenoids content, nitrate reductase activity and leaf-NPK and Ca contents)
and yield and quality attributes (number of pods, number of seeds per pod, 100-
seed weight, seed-yield per plant, seed-protein-content and total anthraquinone
glycosides content). All the growth and biochemical attributes were studied at
120, 150 and 180 DAS. Yield and quality attributes were recorded at the time
of harvest (210 DAS). Out of the five levels of phosphorus, P3 proved the best
and significantly increased all the growth, biochemical, yield and quality
attributes at all the grov/th stages (except leaf-calcium content at 180 DAS,
number of seeds per pod, 100-seed weight and total anthraquinone glycosides
content which were found non-significant). This treatment exceeded the
Control (Po) in seed-yield and seed-protein content by 30.1 and 13.6 %,
respectively.
Experiment 4 (2002-2003) was conducted to establish the optimum dose
of calcium so as to get the best performance of senna sophera {Cassia sophera
L.) in terms of growth, biochemical, yield and quality attributes. The
experiment was conducted according to simple randomized block design using
five levels of calcium, viz. 0, 40, 80, 120 and 160 mg Ca per kg soil (Cao, Cai,
Ca2, Ca3 and Ca4, respectively). In this experiment, attributes recorded and
sampling stages employed were the same as in Experiment 3. Considering the
five levels of calcium, Cas showed the best response. It significantly enhanced
the values of all the growth, biochemical, yield and quality attributes compared
to the Control, except number of seeds per pod, 100-seed weight and
anthraquinone glycosides content. This treatment increased seed-yield and
seed-protein content by 23.7 and 12.8 %, respectively, over the Control.
Experiment 5 (2003-2004) was conducted according to simple
randomized block design, to find out the optimum dose of phosphorus for
getting the best response of coffee senna {Senna occidentalis L.) in terms of
growth attributes (fresh weight per plant and dry weight per plant), biochemical
attributes (total chlorophyll content and total carotenoids content, nitrate
reductase activity and leaf-NPK and Ca contents) and yield and quality
attributes (number of pods per plant, number of seeds per pod, 100-seed
weight, seed-yield per plant, seed-protein content and total anthraquinone
glycosides content). Five levels of phosphorus, viz. 0, 25, 50, 75 and 100 mg P
per kg soil (PQ, PI, P2, P3 and P4, respectively) were basally applied to the crop.
The growth and biochemical attributes were studied at 120, 270 and 300 DAS.
Yield and quality attributes were determined at the time of harvest (330 DAS).
Most of the attributes studied were considerably improved due to phosphorus
application. P3 was confirmed as the optimum dose for all the growth,
biochemical, yield and quality attributes at all the stages except number of
seeds per pod, 100-secd weight and total anthraquinone glycosides content. The
treatment gave 35.5 and 12.8 % higher values of seed-yield and seed-protein
content, respectively o\'cr the Conlrol (P(,).
Experiment 6 (2003-2004) was conducted according to simple
randomized block design, to investigate the effect of five basal levels of
calcium viz., 0, 40, 80, 120 and 160 mg Ca per kg soil (Cao, Cai, Ca2, Ca.-s and
Ca4, respectively) in terms of growth, biochemical, yield and quality attributes
of coffee senna {Senna occidentalis L.). The aim of this experiment was to
work out the optimum dose of calcium for getting the best performance of
coffee senna. All the growth, biochemical, yield and quality attribute recorded
and sampling stages employed were the same as in the case of Experiment 5.
Out of the five calcium levels, Ca3 proved the best and exhibited maximum
response for all the growth, biochemical, yield and quality attributes at all the
growth stages except leaf-phosphorus content at 300 DAS, number of seeds per
pod, 100-seed weight and total anthraquinone glycosides content. The
treatment increased seed-yield and seed-protein content by 27.6 and 10.6 %,
respectively, over the Control.
< ^ 5^
EFFECT OF PHOSPHORUS AND CALCIUM ON SELECTED MEDICINALLY
IMPORTANT LEGUMINOUS PLANTS
THESIS .,
SUBMITTED FOR THE AWARD OF THE DEGREE OF
II
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iBottor of ^I)tloi$opI)p IN
BOTANY
BY y^:^;^
Mu. NAEEM
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2007
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M. Masroorji, Kfian M.Sc., M. Phil.. Ph. D. (Alig)
Post Doctorate (Ohio State Univ.USA) Ex-Pool Officer (CSIR, Govt.of India) Research Associate (CSIR & UGC)
Reader
Department of Botany Aligarh Muslim University Aligarh-202 002 India Phone: 0571-2702016(0)
0571-2702923 (R) Mobile: 09412878955 Email: masroorl [email protected]
<f>! #
..iS Date: May 28» 2007
Certificate
This is to certify that the tiiesis entitled 'Effect of Phosphorus and
"" ' ous Plants" submitted in
tor of Philosophy in
Calcium on Selected M<
partial flilfihnent of th^ reqiui£mentsu^p4he%eiL
Botany, is a faithful record of the bowafide research work carried out at the
livei^^,' Aligarh Muslim Universil
supervision and that no part 61
diploma.
igadi'byV under my guidance and
mitted for any other degree or
^
(M. Mftsroor A. Khan) Research Supervisor
-C^
^
ACKNOWLEDGEMENT
Most humbly, I express my heartfelt inquisitiveness to my esteemed
supervisor of research Dr. M. Masroor A. Khan, Reader, Department of
Botany, Allgarh Muslim University, Aligarh, who infused in me the spirit of
research, guided and helped me through my difficulties and troubles at every
step. I am falling short of words to express my deepest sense of indebtedness to
him for his invaluable and skillful guidance, persistent interest and consistent
supervision throughout the course of the present work.
I am also grateful to Prof Ainul Haq Khan, Chairman, Department of
Botany, Aligarh Muslim University, Aligarh, for providing me necessary
facilities for my research work.
I must highly appreciate the unstinted help and encouragement which I
received from Prof. M.M.R.K. Afridi, Prof. S.H. Afaq and Dr. Moinuddin. I am
really grateful to them for giving me extremely detailed and insightful
comments, suggestions and inspiration to the final culmination of my work.
I also express my indebtedness to Prof. Aqil Ahmad, Prof. Arif Inam,
Prof. Firoz Mohammad, Prof. Nafees A. Khan, Dr. Shamsul Hayat and Dr.
Qazi Fariduddinfor their moral support.
I shall be failing in my duty if I do not acknowledge my seniors and
friends Drs. Irfan Ahmad, Manzer Hussain Siddiqui, Aqil Ahmad Khan, Mohd
Faisal, Sarvajeet Singh, Abid AH Ansari and Mohd Nasir Khan for their good
wishes, co-operation, valuable suggestions and endless support during this
period.
My colleagues Minu Singh, Shafia Nasir, Mohd Idrees and Masidur
Alam always helped keep my feet planted firmly on the ground. A very big
thank you to each and every one of them. I am also thankful to my other lab
colleagues for their co-operation, moral support and good wishes.
Finally and most importantly I am much indebted to my revered parents,
for their patience, moral support, blessings and inspiration. The credit of what
I do in my life will always go to them. A special mention is required for my
brothers and sister for their selfless affection and motivation. My other family
members also deserve the acknowledgment from the depth of my heart.
I shall be remain ever grateful to Dr. Brad Morris, USD A, USA for
generous supply of seeds for my research work.
(Mu. Naeem)
CONTENTS
Page No.
1. Introduction 1-3
2. Review of Literature 4-45
3. Materials and Methods 46-63
4. Experimental Results 64-86
5. Discussion 87-103
6. Summary 104-107
7. References 108-135
8. Appendix i-iii
% P ^ ' : > V ^ V>
Introduction
CHAPTER 1
INTRODUCTION
Medicines of plant origin have formed the backbone of the traditional
systems of the world for centuries. Even practitioners of modern medicine have
lately started realizing their efficacy, coupled with minimal side-effects, if any
(Bent and Ko, 2004; Dubey et al., 2004). Majority of these medicinal plants
belong to angiospermic families of which legume family (Fabaceae) is the third
largest, with approximately 650 genera and 20,000 species (Doyle, 1994). A
handsome number of medicinal legumes are potential sources of glycosides
(aloe-emodin, chrysophenol, emodin, and rhein etc.), antibiotics, flavonoids,
alkaloids and phytochemicals, which are used in drug manufacturing by the
pharmaceutical industries (Tyler et al., 1976; Morris, 2003). Among these,
natural products of leguminous plant have been and will continue to be
important sources and models of forage, gums, insecticides, phytochemicals
and other industrial, medicinal and agricultural raw materials.
A large number of medicinal plants have had to be cultivated for
decades to ensure continuous supply of quality drugs in traditional systems of
medicine. Among them, hyacinth bean {Lablab purpureiis L.), serma sophera
{Cassia sophera L.) and coffee senna {Senna occidentalis L.) constitute the
most important source of therapeutic agents used in the modem as well as
traditional systems of medicine (Morris, 1996, 1999, 2003). Hyacinth bean is
used in the treatment of cholera, vomiting, diarrhoea, leucorrhoea, edema and
alcohol intoxication. It contains tyrosinase, an enzyme, which has potential use
for the treatment of hypertension (Beckstrom-Stemberg and Duke, 1994). The
plant parts of senna sophera are used in the treatment of skin diseases, diabetes,
jaundice, ringworm and ulcers (Dastur, 1977; Kirtikar and Basu, 1987; The
Wealth of India, 1992). Coffee senna is used as an analgesic, antibacterial, anti-
hepatotoxic, antifungal, anti-intlammatory, antiseptic, antiparasitic, antiviral,
carminative, laxative, purgative, and vermifuge. The leaves and seeds contain
anthraquiiiones, chrysophenol, emodin, physcion and rhein (Kirtikar and Basu,
1987; The Wealth of India, 1992; Morris, 1996, 1997, 1999).
At Aligarh, Afridi, and his associates (Khan and Mohammad, 2006)
have carried out in-depth studies on the mineral nutritional requirements of a
number of medicinal plants {Anethum, Carum, Cassia, Cichorium, Curcuma,
Cyinbopogon, Datura, Foeniculum, Lallemantia, Linum, Mentha, Nigella,
Plantago, Solanum, Trigonella, Withania and Zingiber). However, no work has
been done so far regarding the fertilizer requirements on any of the three useful
medicinal legumes mentioned above. The survey of literature has also revealed
that meagre information is available on the effect of phosphorus and calcium
application on hyacinth bean, senna sophera and coffee seima.
The scientific cultivation of these medicinally important leguminous
plants could improve their yield and quality, particularly through optimal
quantity of fertilizer application and meet the increasing demands. Application
of phosphatic fertilizers is common practice to alleviate soil-phosphorus
deficiency, as 98 % of agricultural soils in India are phosphorus deficient
(Ghosh and Hasan, 1977). This is true for the soils of Aligarh also (Table 1). It
appears that deficiency of phosphorus and calcium poses a serious limitation
for gainful productivity of crops in this region (Khan and Mohammad, 2006).
Thus, there arises the question "Could we improve the productivity and quality
of three selected plants by application of phosphorus and calcium through
soil?". To get the right answer, the present author, decided to undertake
thorough studies with regard to the use of phosphorus and calcium in
enhancing growth, yield and quality attributes by performing six pot
experiments.
The aims of these pot experiments were as follows:
1. To investigate whether phosphorus application could enhance growth,
yield and quality attributes of hyacinth bean i.e. Lablab purpureas L.
(Experiment 1).
2. To find out whether calcium application could enhance growth, yield and
quality attributes of hyacinth bean i.e. Lablab purpureus L. (Experiment
2).
3. To find out whether phosphorus application could enhance growth, yield
and quality attributes of senna sophera i.e. Cassia sophera L. (Experiment
3).
4. To investigate whether calcium application could enhance growth, yield
and quality attributes of senna sophera i.e. Cassia sophera L. (Experiment
4).
5. To find out whether phosphorus application could enhance growth, yield
and quality attributes of coffee senna i.e. Senna occidentalis L.
(Experiment 5).
6. To investigate whether calcium application could enhance growth, yield
and quality attributes of coffee senna i.e. Senna occidentalis L.
(Experiment 6).
^view of Literature
CONTENTS
2.1
2.1.1
2.1.1.1
2.1.2
2.1.2.1
2.1.3
2.1.3.1
2.2
2.2.1
2.3
2.3.1
2.3.2
2.3.3
2.3.4
General description
Hyacinth bean (Lablab purpureus L.)
Medicinal uses
Senna sophera (Cassia sophera L.)
Medicinal uses
Coffee senna {Senna occidentalis L.)
Medicinal uses
Inorganic plant nutrition
Brief history of mineral nutrition
Physiological roles of N P K and Ca in plants
Role of nitrogen
Role of phosphorus
Role of potassium
Role of calcium
Page No
6
6
8
8
9
10
11
11
12
13
13
14
15
16
2.4 Effect of phosphorus application on leguminous
plants (including medicinal legumes) 17
2.5 Effect of calcium application on leguminous plants
(including medicinal legumes) 36
2.5 Concluding Remarks 45
CHAPTER 2
REVIEW OF LITERATURE
The present day knowledge of medicine is considered to be a gift of
ancient men to us as the use of medicinal plants to cure specific ailments has
been in vogue in various indigenous systems of medicine from ancient times.
The modern as well as traditional systems of medicine have provided a large
variety of potent drugs to alleviate suffering from diseases. Our world is
endowed with a rich wealth of medicinal plants that has been exploited locally
for centuries. With easier and safer transportation, medicinal plants continued
to play an extraordinary significant role in the health care of humankind far and
wide and it is no wonder that presently six hundred million people depend
directly or indirectly on plant-derived drugs for their health-care needs. In
recent years, the growing demand for herbal products has led to a quantum
jump in the volume of plant materials traded within and across the countries.
World Health Organization (WHO) has estimated that about 80 % of the
populations of developing countries depend on plant-based traditional systems
of medicine to meet their health-care needs. The demand for medicinal plants is
increasing in both developing and developed countries due to growing
recognition of natural products, being generally non-narcotic, having no or
little side-effects and easy availability at affordable costs (Bent and Ko, 2004;
Dubey et ai, 2004).
There is no reliable figure for the total number of medicinal plants on
earth, in terms of number and percentage; it may vary from country to country
and from region to region. According to Schippmarm et al. (2002), the number
of species used medicinally worldwide and in India is 52,885 and 3,000
respectively. However, WHO's report contains a list of only 20,000 medicinal
plants used in different parts of the globe (Purohit and Vyas, 2004). Whereas
Shiva (1996) has mentioned 7,500 medicinal plant species for India. One the
other hand, total number of medicinal plants in international trade may be only
about 2,500 species (Schipmann et al., 2002).
India is known to possess a rich repository of medicinal plants. Around
70 % of India's medicinal plants are found in tropical areas, mostly in forests
spread across the Western and Eastern Ghats, the Vindhyas, the Chota Nagpur
plateau, the Aravalis and the Himalyas. This leaves only 30 % of the medicinal
plants in temperate and alpine areas and on higher altitudes (Purohit and Vyas.
2004; Seth and Sharma, 2004). Arguably, being the largest producer of
medicinal herbs, India is appropriately called the botanical garden of the world
and the treasure house of biodiversity (Ahmedullah and Nayar, 1999). Two of
the largest users of medicinal plants are China and India. Scientists, physicians
and pharmaceutical companies will be looking in the future also mainly
towards these countries for their requirements, as they have the largest number
of medicinal plant species and are among the top exporters of the commodity.
According to WHO, the international market of herbal products is US $
62 billion which is estimated to reach US $ 5 trillion by the year 2050.
Surprisingly, India although being one of the largest producers of medicinal
plants, has presently less than 0.5 % share in the global export market (Purohit
and Vyas, 2004; Bhattacharjee, 2004).
It is true that due to awareness concerning herbal remedies and some
meticulous results in laboratory and clinical trials, the market and public
demand of herbal drugs has shown a quantum jump, resulting in increased their
collection. Moreover, increasing urbanization threatens many medicinal plants
to the extent of extinction as well as loss of genetic diversity, resulting in acute
shortage. Also, the current trend towards increased commercialization has
resulted in over-harvesting of some important medicinal plants, many of which
have become threatened. Additionally, 22 % of threatened medicinal plant
species of Indian Himalayas are reported to be critically endangered, 16 %
endangered, and 27 % vulnerable. Of these, 32 threatened medicinal plant
species are endemic (Kala, 2005).
There is thus an urgency to give special emphasis and to clearly define
the policies to regulate medicinal plant conservation, cultivation, quality
control standards, processing and preservation, marketing and export. Out of
these useful steps and policies regarding medicinal plants sustenance, the
cultivation of medicinal plants on scientific lines appears to be extraordinarily
effective to obtain authentic, standard and fresh herbal materials. This would be
a safeguard against unauthentic, spurious, denatured, fake and soiled drugs. For
the cultivation of plants, the role of mineral nutrition is of paramount
importance. In fact, yields of most crop plants increase linearly within limits
with the amount of fertilizer that they absorb (Frainz, 1983; Loomis and
Conner, 1992). Using balanced mineral nutrients the pjant's maximum genetic
potential can be realized successfully (Wallace and Wallace, 2003).
In view of the above, this chapter comprises a general description of the
three medicinally important leguminous plants selected for the present study. In
addition, the effect of the application of phosphorus and calcium on
leguminous plants in general and on these three medicinal legumes in particular
has been reviewed.
2.1 General description
A general account of these medicinal leguminous plants (hyacinth bean,
senna sophera and coffee senna) is given below.
2.1.1 Hyacinth bean {Lablab purpureas L.)
According to system of classification of Bentham and Hooker (1862-
1883), hyacinth bean occupies the following systematic position.
Kingdom
Division
Class
Sub-class
Series
Order
Family
Subfamily
Genus
Species
Plantae
Angiospermae
Dicotyledonae
Polypetalae
Calyci florae
Rosales
Leguminosae
Papilionaceae
Lablab
purpureus
Hyacinth bean is also known by different names, 'Bonavist bean".
'Egyptian kidney bean', 'Lablab bean' and 'Indian butter bean' and locally
known as 'Sem' and is the member of family Leguminosae (Fabaceae).
Hyacinth bean is a herbaceous perennial or erect annual herb (Fig. 1) often
grown as an annual crop (Purseglove, 1974). It grows up to 1 metre (3.2 ft)
high with longer stems of climbing types (up to 6 metres or 20 ft). The leaves
are trifoliate, 10-30 cm long, stipules 5 mm, basifixed, lanceolate, petiole 5-20
cm, glabrous, leaflets 5-15 cm long, ovate, acute, entire pale green and
glabrous. Flowers are purple or white, racemes 15-23 cm, calyx 13 mm,
corolla, white or pink, 15 mm long. Pods vary in shape and in colour, flat or
inflated, 5.0 cm long and 1.5 cm wide containing 2-4 seeds. Seeds are variable
in colour, including white, cream, reddish and brown or black (Murphy and
Colucci, 1999). The taxonomic description for hyacinth bean is well described
by Cameron (1988).
Hyacinth bean originated in India (Deka and Sarkar, 1990) and was
introduced to Africa from Southeast Asia during the 8* century (Kay, 1979).
Hyacinth bean has widely distributed in many tropical and subtropical
countries i.e. South and Central America, Asia, China and India. Hyacinth bean
is well suited to most tropical environment and adaptable to a wide range of
rainfall, temperature and altitude (Murphy and Colucci, 1999). It grows well
under warm, humid conditions at temperatures ranging from 18-30°C and
minimum temperature for growth is S'C. It is normally grown from sea level up
to elevations of between 1800-2100 metres. It also grows well in a wide range
of soil types from deep sands to heavy black clays and can tolerate pH ranges
of 5.0-7.5. Soil fertility is important, thus phosphate fertilizers may need to be
applied at planting and therefore, it responds well to superphosphate (Murphy
and Colucci, 1999).
Hyacinth bean provides biological nitrogen-fixation and the natural
action of converting atmospheric nitrogen into forms available for the plant-
animals-soil system and improves productivity in an inexpensive.
V
Fig. 1: Hyacinth bean {Lablab purpureus L.)
environmentally friendly manner (Murphy and Colucci, 1999). The young pods
and tender beans are used as vegetables mainly in India and also in tropical and
warm temperate Asia (Purseglove, 1974). Its leaves are used as a green just like
spinach. Flowers are used in soups and stews. Hyacinth bean has also been
known for its use as a green manure, adding organic matter as well as nitrogen
and mineral to the soil and also used in several forms including edible young
pods, dried seeds, leaves and flowers (Morris, 1997, 2003)
Hyacinth bean contains water, protein, carbohydrate, fat, fibre, ash, P,
K, Ca and Mg. The dried seeds contain: moisture, 8.0; ash, 3.9; fibre, 10.5; fat.
1.3; carbohydrate, 51.3, protein, 20-28 %; phosphorus, 307; potassium, 198.6;
calcium, 191.1; sodium, 8.0 and magnesium, 43.8 mg/lOOg (Deka and Sarkar,
1990; El Siddig, 2002).
2.1.1.1 Medicinal Uses
Hyacinth bean is used in the treatment of cholera, vomiting, diarrhoea,
leucorrhoea, gonorrhoea, edema, alcohol intoxication and globefish poisoning.
The seeds are used as laxative, diuretic, anthelmintic, antispasmodic,
aphrodisiac, anaphrodisiac, digestive, carminative, febrifuge and stomachic
(Chopra et ai, 1986; Kirtikar and Basu, 1995). Hyacinth bean contains
tyrosinase, an enzyme, which has potential use for the treatment of
hypertension. Hyacinth bean fibre is known to prevent cancer, diabetes, heart
disease, obesity, and used as a laxative (Beckstrom-Stemberg and Duke, 1994).
The potential breast cancer fighting chemical known as Kievitone, a flavonoid
is found in hyacinth bean (Hoffman, 1995). The flavonoid, genistein may play
a role in prevention of carcinogenesis (Kobayashi et ai, 2002) and as a
chemotherapeutic and /or chemopreventive agent for head and neck cancer
(Alhasanetal.,2001)
2.1.2 Senna sophera (Cassia sophera L.)
According to Bentham and Hooker (1862-1883), senna sophera occupies
the following systematic position.
Kingdom
Division
Class
Sub-class
Series
Order
Family
Subfamily
Genera
Species
Plantae
Angiospermae
Dicotyledonae
Polypetalae
Calyciflorae
Resales
Leguminosae
Caesalpiniaceae
Cassia
sophera
Senna sophera is locally known as 'Kasunda', 'Banar' and belongs to
family Leguminosae (Fabaceae). It is diffuse, sub-glabrous, shrubby herb or
under shrub, 0.7-3.0 m in height (Fig. 2). Leaves foetid, unipinnate, 20-30 cm
long. Leaflets 8-12 pairs, oblong or lanceolate, flowers yellow in short, axillary
or temiinal, corymbose racemes. Pods slightly falcate, sub-terete or terete, 6.5
cm X 0.6 cm. Seeds 30-40, ovoid, compressed at one end and rounded at the
other, 6 mm long, 4 mm broad, hard, smooth and dark brown in colour
(Kirtikar and Basu, 1987). Plant parts contain anthraquinones, chrysophenol,
physcion and emodin (The Wealth of India, 1992). Senna sophera found
throughout India and Pakistan. The dried seeds contain: moisture, 6.7; protein,
15.8; pentosans, 14.7; and water mucilage, 20.0 % (The Wealth of India, 1992).
2.1.2.1 Medicinal uses
The plant is expectorant, depurative and alterative; its decoction is used
as an expectorant in acute bronchitis. The leaves possess purgative properties.
A paste of leaves and root bark is a useful application in skin diseases like
ringworm, ulcers etc. (The Wealth of India, 1992). The seeds are cathartic,
used as a febrifuge and administered in diabetes (Dastur, 1977; Kirtikar and
Basu, 1987). The roots are considered diuretic. A paste of the roots is
sometimes substituted for the leaf-juice in ringworm. The bark, like the leaves
and seeds is cathartic in action. Its infusion is considered useful in diabetes and
^
^
^ ^ ^ ^
^^jf-'^aE/^ »8
^ ».>»i ^ ^v^
p..fi
^fr
• ' t t ,^^ '
^^X
li
Fig. 2: Senna sophera (Cassia sophera L.)
its juice in asthma and with honey in diabetes. A paste made of the seeds with
sulphur is applied in skin diseases (The Wealth of India, 1992).
2.1.3 Coffee senna {Senna occidentalis L.)
According to Bentham and Hooker (1862-1883), coffee senna occupies
the following systematic position.
Kingdom
Division
Class
Sub-class
Series
Order
Family
Subfamily
Genera
Species
Plantae
Angiospermae
Dicotyledonae
Polypetalae
Calciflorae
Rosales
Leguminosae
Caesalpiniaceae
Senna
occidentalis
Coffee senna is also known as 'Stinking weed', 'Foetid cassia',
'Rubbish cassia' and 'Negro-coffee' and locally known as 'Badi Kasondi' and
belongs to family Leguminosae (Fabaceae). It is an erect, foetid annual herb or
under shrub 60-150 cm in height (Fig. 3). Leaves, 15-20 cm long, leaflets are
ovate to ovate-lanceolate, and inflorescences are few-flowered axillary racemes
with yellow flowers about 2 cm across. The pods are brown, flat, slightly
curved and 5-12 cm long. It contains 40 or more brown to dark-olive green,
hard, shining, ovoid seeds about 4 mm long. It grows throughout the tropics
and subtropics. It also found throughout India up to an altitude of 1,500 m
(Kirtikar and Basu, 1987; The Wealth of India, 1992). The leaves and seeds
contain anthraquinones (Fig. 4), chrysophenol, emodin, their glycosides,
physcion and rhein. The dried seeds contain: crude protein, 35.2; ether extract,
5.0; N-free extract, 51.3; crude fibre, 3.8; ash, 4.7 %; calcium, 160;
phosphorus, 226; iron, 10.98; niacin, 7.59; and ascorbic acid, 4.29 mg/lOOg
(The Wealth of India, 1992).
Fig. 3: Coffee senna (Senna occidentalis L.)
Fig. 4: Structural Formula of Anthraquinone
2.1.3.1 Medicinal Uses
Plants are used as coffee substitute. Leaf powder is used as an analgesic,
antibacterial, anti-hepatotoxic, antifungal, anti-inflammatory, antiseptic,
antiparasitic, antiviral, carminative, laxatilve, purgative and vermifuge (The
wealth of India, 1992). The volatile oil obtained from leaves, roots and seeds
showed antibacterial and antifungal activity (Chopra et al., 1958; Kirtikar and
Basu, 1987). Seeds are useful in cough and whooping cough, convulsions and
in heart diseases. The roots are bitter in taste and considered as tonic, purgative,
anthelmintic and diuretic (The Wealth of India, 1992). The plant parts are used
to cure sore eyes, haematuria, rheumatism, typhoid, asthma, leprosy, ringwonn
and disorders of hemoglobin. A decoction of the plant is used in hysteria,
dysentery, itch, inflammation of the rectum and scorpion sting. The herb is
reported to be used as condiment and in perfumery (The Wealth of India.
1992).
2.2 Inorganic plant nutrition
Mineral nutrition includes the supply, absorption and utilization of
essential nutrients for growth and yield of crop plants. Mineral nutrients are the
major contributor to enhancing crop production and maintaining soil
productivity and necessary for maintenance of the physical organization and
function of livings cells by virtue of their function in the generation of energy,
building molecules, participating in the repair of protoplasm and regulation of
metabolic process (Nason and McElroy, 1963; Marschner, 2002). Plants
contain small amounts of more than forty elements but only 19 elements are
laiown to be essential. These essential plant elements are mentioned below
according to their concentration in the tissue of higher plants in decreasing
order: hydrogen, carbon, oxygen, nitrogen, potassium, calcium, magnesium,
phosphorus, sulphur, silicon, chlorine, iron, boron, manganese, sodium, zinc,
copper, nickel and molybdenum (Salisbury and Ross, 1992; Marschner, 2002).
Recently, two elements have been added in the list viz. sodium and silicon.
The former is required for C-4 and CAM plants (Subbarao, 2003). However,
silicon is essential for diatoms, Equisetaceae (horsetails or scouring rushes) and
"quasi-essential" for such important crops as rice and sugarcane (Epstein, 1999.
2001; Epstein and Bloom, 2005; Rains et al, 2006). In the following pages, a
brief history of mineral nutrition and their important physiological roles in
plants, particularly those demanded in relatively large quantities (nitrogen,
phosphorus, potassium and calcium) are given.
2.2.1 Brief history of mineral nutrition
The beneficial effect of adding mineral elements in the forai of plant ash
and lime to soils to improve plant growth has been known in agriculture for
more than 2,000 years (Marschner, 2002). No one knows with certainly when
human first incorporated organic substances, manure or wood ashes as
fertilizers in soil to stimulate plant growth. However, it is documented in
writings as early as 2500 B.C. that humans recognized the richness and fertility
of alluvial soils in valleys of the Tigris and Euphrates rivers (Hewitt, 1963).
Romans made significant contributions in the years 800 to 200 B.C.
(Marschner, 2002). The seventeenth century is thought to be the beginning of
researches on plant nutrition with Van Helmont (1577-1644) drew the attention
to the importance of nutrients for plants (Bould, 1963). Glauber (1604-1655)
was first person in the history of mineral nutrition, found that saltpetre
(potassium nitrate) obtained from cattle manure was effective for plant growth.
After a century. Home (1755) found that not only saltpetre stimulated plant
growth but epsom salt (Magnesium sulphate) and potassium sulphate also had
similar effects. Home also showed that there are two ways of approach to the
study of plant nutrition, i.e. pot-culture and plant analysis (Bould, 1963). de
Saussure for the first time established a close relation between the minerals
found in the soil and plants growing in it and claimed the importance of
nitrogen for plant growth.
However, in the middle of 19''' century, Justus Von Liebig (1840)
assumed that nitrogen is absorbed from the air (not from humus) and gave the
theory of the mineral nutrition of the plants (Browne, 1943; Bould, 1963). It
was mainly to the credits of Justus Von Liebig (1803-1873) that the scattered
information concerning the importance of mineral nutrients for plants growth
12
was collected and summarized and the mineral nutrition of plants was
established as a scientific discipline. Sachs and Knop in 1860s were able to
visualize the requirement of ten elements for plants with the help of water
culture experiments. These include calcium, carbon, hydrogen, iron,
magnesium, nitrogen, oxygen, phosphorus, potassium and sulphur. The
concentration of these elements (except iron) is 0.01 % or more in the dry
matter of plants are termed macro elements or macronutrients (Reed, 1942;
Bould, 1963). Subsequent progress in analytical chemistry, particularly in the
purification of chemicals and methods of detection and estimation, enabled
plant scientists to discover the essentiality of seven microelements other than
iron. When the science of plant nutrition bloomed in the 20 century, different
researchers, using sophisticated analytical techniques, were able to demonstrate
the essentiality of these elements for plant growth and development.
2.3 Physiological roles of N P K and Ca in plants
Among various nutrients, nitrogen, phosphorus, potassium and calcium
are considered to be of prime importance. These nutrients play several
important roles in metabolic and regulatory process in plants (Marschner,
2002) and the roles of these nutrients (nitrogen, phosphorus, potassium and
calcium) are briefly described individually in the following pages.
2.3.1 Role of Nitrogen
Nitrogen is the fourth most abundant element in plants after carbon,
hydrogen and oxygen. It is absorbed by plants as nitrate ions (NO3'),
ammonium ions (NH/) and urea (Ford and Clarkson, 1999; von Wiren et al.,
2000) and is reduced and incorporated into organic compounds (Bandurski,
1965). Nitrogen is an integral part of a large number of essential, organic
compounds, including amino acids, proteins, co-enzymes, porphyrins, purins,
pyrimidines, nucleosides and nucleotides, chlorophyll, some vitamins,
alkaloids and growth hormones (Bandurski, 1965; Devlin and Witham, 1986;
Marschner, 2002). Nitrogen evidently plays a central role in cellular
13
metabolism. Hence, physiological maturity and yield of many crops have been
governed by the nitrogen supply to the crops (Black, 1973).
When the nitrogen supply is a limiting factor, both the rate and the
extent of protein synthesis are depressed and flowering and fruit setting are
adversely affected (Agarwala and Sharnia, 1976). The deficiency of nitrogen
results in various crop disorders, plant growth and root development are
retarded. The plants become liable to attack of pests and diseases (Black, 1973;
Devlin and Witham, 1986; Salisbury and Ross, 1992; Marschner, 2002).
2.3.2 Role of phosphorus /^
Phosphorus is present in the soil in two forms, inorganic and organic.
Phosphorus absorbed by plants as monovalent (H2PO4)' or divalent (HP04)"''
anions from the soil, which are later converted into organic form upon entr>'
into roots or after transport through the xylem in shoot (Salisbury and Ross,
1992). Phosphorus is found in plants as constituent of nucleic acids,
nucleoproteins, phospholipids, phytin, flavin adenine dinucleotide (FAD),
nicotinamide adenine dinucleotide phosphate (NADP), adenosine triphosphate
(ATP), pyridoxal phosphate (PP), thymine pyrophosphate (TPP),
phosphorylated sugars and their intermediary metabolic products found in the
glycolytic and alternative oxidative pathways (Nason and McElroy, 1963;
Devlin and Witham, 1986; Salisbury and Ross, 1992; Marschner, 2002). It is
associated with cell division and hereditary because it is a constituent of
nucleoproteins. It is one of the key substrates in energy metabolism and
biosynthesis of nucleic acids and membranes, and also plays an important role
in the photosynthesis, respiration and regulation of a number of enzymes
(Raghothama, 1999). Phosphate participates directly in the photochemical
events of photosynthesis. Orthophosphate and NADP being required for the
production of "assimilatory power" i.e. NADPH+ATP (Amon, 1959). Among
the many inorganic nutrients requires by plants, P is one of the most important
elements that significantly affects plant growth and metabolism. Low
availability of phosphate is a major constraint for crop production in many low-
14
input systems of agricultural worldwide. Many soils around the word are
deficient in phosphate, and even in fertile soils, available phosphate seldom
exceeds 10 i M (Bieleski, 1973).
Phosphate deficiency in plants causes' failure to make a quick start and
growth remain stunted. It also causes many visible effects, acute leaf-angle,
lack of tillering, prolonged dormancy of lateral buds, premature leaf-fall,
decrease in size and number of flower primordia and small fruits or seeds.
Phosphorus deficiency also results in an increase in the accumulation of free
reducing sugar, suggesting an involvement of phosphorus in carbohydrate
metabolism. Anthocyanins sometimes accumulate with the oldest leaves
becoming dark brown and such plants suffer from premature leaf-fall, and
delay in flowering and fruiting (Devlin and Witham, 1986; Raghothama, 1999).
2.3.3 Role of potassium ^
Potassium is abundantly present in soluble form in the cytoplasm and in
the vacuolar cell sap. It exists in the soil in non-exchangeable (fixed form),
exchangeable form and soluble form. Potassium plays an important role in
activating numerous enzyme systems, such as fructokinase, pyruvic kinase and
transacetylase (Nason and McElroy, 1963). Potassium affects several other
important functions, like osmoregulation, water transport and the translocation
of carbohydrates in plants (Clarkson and Hanson, 1980; Maathuis and Sanders,
1996) and it also enhances the ability of the plants to resist diseases, insect
attacks, cold and adverse conditions. Potassium plays an important role in
tissue hydration and thus helps in opening and closing of stomata (Fischer and
Hsiao, 1968; Humble and Hsiao, 1969; Webb and Mansfield, 1992). Potassium
plays a crucial role in legumes by enhancing the production of starch and sugar
that benefit the symbiotic bacteria and thus enhanced the fixation of nitrogen.
Deficiency of potassium results in a decrease in the reducing sugars and
proteins. Potassium deficiency limits CO2 diffusion through stomata thus
resulting in the increase in stomatal resistance. Potassium deficiency creates
rosette habit of vegetables, shortening of internodes in oilseed and cereals.
15
increased tillering in barley and death of internal bud and marginal scorching of
leaves in many crops (Hewitt, 1963).
2.3.4 Role of calcium
Calcium is absorbed by plants as a divalent cation (Ca^^) and as a
constituent of cell walls in the form of calcium pectate. The concentration of
calcium in plants varies between 0.1 and > 5.0 % (dry wt basis) depending on
the growing conditions, plant species and plant organ. Calcium is involved in
cell elongation and cell division (Burstrom, 1968; Hirshi, 2004), influences the
pH of cells, and also acts as a regulatory ion in the source-sink translocation of
carbohydrates through its effects on cells and cell walls (Hirshi, 2004).
Calcium has many important structural and physiological roles in plants. It is
important in maintaining the stability of the cell walls and cell membranes and
also required in the maintenance of cell integrity through membranes bound
proteins (Epstein, 1972; Kirkby and Pilbeam, 1984; Hepler and Wayne, 1985;
Marschner, 2002; White and Broadly, 2003). Calcium is a second messenger
and controls the growth and differentiation of the cells (Berridge et al., 1998).
Calcium in the cell binds with a protein known as calmodulin (CaM).
Calmodulin activates a number of key enzymes and might also regulate
calcium transport within the cell and mediate transfer to the vacuoles (Manne,
1983) and plays crucial role in the growth and development of the plant by
regulating mitosis and cytokinesis of plant cell (Ramsussen and Means, 1989).
There are increasing evidences for a close link between calcium and growth
hormone action in plants. Calcium plays an importeint role in auxin transport
and secretion (Dela Fuente, 1984), ethylene synthesis (Mattoo and Lieberman,
1977), secretion of enzymes (Caspar et al., 1983; Jones and Jacobsen, 1983)
and senescence (Lesham el al., 1984). Senescence in maize leaves can be
deferred by the addition of either calcium or cytokinin (Poovaiah and Leopold,
1973). In general, calcium stimulates membrane bound enzyme ATPase at the
plasma membrane of roots of certain plant species (Kuiper et al., 1974).
16
A quite small deficiency might be sufficient to prevent the normal
functioning of cells and lead to the expression of the symptoms by which the
deficiency is recognized. Calcium deficiency is rare in nature, but may occur
on soils with low base saturation and/ or high levels of acidic deposition.
Several costly Ca-deficiency disorders occur in horticulture crops (Shear,
1975). There are several symptoms of calcium deficiency, viz. cracking of
fruits i.e. fruits of apple (Shear, 1975), tomato (Dickinson and McCoUum,
1964), 'bitter pit' of apples, 'black heart' of celery, 'blossom end rot' of
tomatoes, 'internal browning of brussels sprouts', 'tip bum' of lettuce and
'gold spot' in tomato fruit etc. (Simon, 1978; Bekreij et al, 1992; White and
Broadly, 2003). Cell wall may become rigid or brittle in calcium deficient
plants (Kalra, 1956). Calcium deficiency symptoms appear first in the younger
leaves and the growing apices, probably as a consequence of the immobility of
calcium in the plant.
2.4 Effect of phosphorus application on leguminous plants (including medicinal legumes)
Among various plant nutrients, nitrogen, phosphorus, potassium and
calcium are considered of primary importance as they are required by plants in
large quantities. In the following pages, an effort has therefore, been made to
review the relevant available Indian and abroad literature on phosphorus
application on the leguminous plants.
Verma (1975) carried out an experiment at Jodhpur (Rajasthan), to study
the effect of four levels of phosphorus, viz. 0, 20, 40 and 80 kg P2O5 (0, 8.7,
17.5 and 43.9 kg P respectively) per ha on Dolichos lablab (Lablab purpureas
var. Lignosus). Application of phosphorus (40 and 80 kg P2O5 per ha)
increased seed yield (23.3 and 39.8 % respectively) over the Control.
Kalyanasundram (1981) studied the effect of four levels of nitrogen (0,
25, 50 and 75 kg N) per ha and four levels of phosphorus, viz. 0, 50, 100 and
150 kg P2O5 per ha (equivalent to 0, 21.8, 43.6 and 65.5 Kg P respectively) per
ha in goradu soil of Gujarat on Tinnevelly senna (Cassia angustifolia Vahl.).
V - 17
Half of the N and entire P were given as the basal doses and the remaining half
dose of N was top dressed prior to flower initiation, i.e. 30 days after sowing.
The parameters, studied were green yield of leaflets, dry yield of leaflets,
sennoside percentage and total increased yield. It was reported that 50 Kg N
per ha increased the dry yield of leaflets by 50 % and sennoside yield by 44 %
compared with Control. Application of phosphorus, however, had a slight
beneficial effect on sennoside yield only. The interaction effect between N and
P was non-significant for all parameters studied.
Wasiuddin et al. (1982) conducted a field experiment at Aligarh (Uttar
Pradesh), to study the effect of four levels of basal nitrogen (0, 10, 20 and 30
kg N per ha) and phosphorus, viz. 0, 20, 30 and 40 kg P2O5 (equivalent to 0,
8.7. 13.1 and 17.5 kg P respectively) per ha on the growth of root and shoot and
on the seed yield per plant of Kasondi {Cassia occidentalis L.). They observed
that 30 kg each of N and P2O5 were optimum for better growth and yield of this
important medicinal plant.
Dadson and Acquaah (1984) conducted an experiment at Legon
(Ghana), to study the effect of 3 levels of nitrogen (40, 80 and 160 kg N per ha)
and phosphorus (30, 60 and 90 kg P per ha) and inoculation on nodulation,
symbiotic nitrogen fixation and yield of soybean {Glycine max (L.) Merrill) in
the Southern Savanna of Ghana. Nitrogen and phosphorus were applied in the
fonn of urea and triple superphosphate. These treatments significantly
increased plant height, number of nodules and number of pods per plant, leaf
area index, total dry matter per plant, grain yield and seed weight. Low rates of
nitrogen and medium to high rates of phosphorus promoted nodule number, dry
weight and leghemoglobin content.
Rao and Singh (1985) conducted an experiment at Varansi (Uttar
Pradesh), to find out the effect of four levels of phosphorus, viz. 0, 20, 40 and
60 P2O5 per ha (equivalent to 0, 8.7, 17.5 and 26.2 kg P per ha respectively)
and three basal inoculations viz. no culture, Rhizobium culture and
Phosphobacterin culture on nodulation in chickpea {Cicer arietinum L.). The
18
twelve treatment combinations were laid out in a randomized block design with
three replications. Application of phosphorus at the rate of 40 Kg per ha and
Rhizobium culture significantly increased the dry weight and nitrogen content
of nodules in chickpea.
Kulkarni et al. (1986) laid out a field experiment at Junagadh (Gujarat),
to study the effect of five levels of phosphorus, viz. 0, 12.5, 25.0, 50.0 and 80.0
kg P2O5 (equivalent to 0, 5.4, 10.9, 21.8 and 34.9 kg P respectively) per ha and
potassium, viz. 0, 25 and 50 kg K2O (equivalent to 0, 20.8 and 41.5 kg K per ha
respectively) on nodulation, nitrogen accumulation, dry matter weight and pod
yield of 'GAUG 10' a Virginia runner groundnut. Phosphorus applicafion (50
kg P per ha) increased the number and weight of nodules. Application of 50 kg
P2O5 per ha resulted in an increase in the nodule count and higher nitrogen and
dry matter accumulation in the plant. Pod yield was also higher in this
treatment.
Sawant et al. (1987) carried out field trials, to study the effect of
spacing, N and P on Dolichos lablab {Lablab purpureas) at Konkan
(Maharastra). Application of 0, 30 and 60 kg P2O5 (equivalent to 0, 13.1 and
26.2 kg P respectively) per ha gave average yields of 0.47, 0.52 and 0.58 tons
per ha respectively. The highest yield of 0.75 ton per ha was given by crops
grown at a spacing of 30^20 cm and fertilized with 30 kg N and 60 kg P2O5 per
ha.
Yadav et al. (1987) conducted a pot experiment to find out the effect of
phosphorus and zinc on chickpea variety 'G 130'. They applied four levels of
phosphorus, i.e. 25, 50, 100 and 250 ppm and five levels of zinc i.e. 5, 10, 20,
40 and 100 ppm. They studied flowering behavior, number of pods per plant,
number of seeds per pod and seed yield. Time of flowering was delayed by the
deficiency as well as excess of phosphorus. Production of flower per plant,
pods per plant and seeds per pod were maximum at 25 and 50 ppm P, but
decreased with higher phosphorus levels. However, the values for seed weight
per plant given by different levels of phosphorus did not significantly differ
19
from each other. Apphcation of zinc between 5 and 20 ppm seemed to be
beneficial for flower production and seed yield.
Pandey and Babu (1988) conducted a pot experiment to study the effect
of phosphorus application on different enzymes at various growth stages of
chickpea variety 'JG 62'. They applied three levels of phosphorus, viz. 30, 60
and 80 kg P2O5 per ha (equivalent to 13.1, 26.2 and 34.9 P per ha respectively)
along with a unifomi basal dose of nitrogen (25 kg N per ha) and potassium 40
kg K2O per ha that was equivalent to 33.2 K per ha. The sources of phosphorus,
nitrogen and potassium were single superphosphate, urea and muriate of potash
respectively. The enzymes studied were nitrate reductase, glutamate
dehydrogenase and glutamine synthetase. Phosphorus application significantly
increased nitrate reductase and glutamate dehydrogenase activity at the rate of
60 kg P2O5 per ha. It was also observed that nitrate reductase activity increased
consistently till flowering (13* week), thereafter, the activity declined
drastically. Glutamate dehydrogenase activity in leaves was maximum during
pod development stage, but in nodules maximum activity was noticed during
vegetative stage. Lower dose (30 kg P2O5 per ha) was found most favourable
for glutamine synthetase. Higher dose of phosphorus (80 kg P2O5 per ha) was
proved detrimental for the activity of enzymes.
Idris et al. (1989) conducted a field experiment at Faisalabad (Pakistan),
to study the effect of phosphorus application on chickpea variety 'CM 88'.
They applied five levels of phosphorus, i.e. 0, 40, 60, 80 and 100 kg P2O5 per
ha (equivalent to 0, 17.5, 26.2, 34.9 and 43.7 P respectively) per ha along with
a uniform basal application of 20 kg N and 24 kg K2O per ha (equivalent to
19.9 K per ha) at the time of sowing. The sources of phosphorus, nitrogen and
potassium were single superphosphate, urea and potassium sulphate
respectively. Application of phosphorus increased significantly the number and
weight of nodules per plant over Control (0 kg P2O5 per ha). It was also
observed that phosphorus at 40, 60, 80 and 100 kg P2O5 per ha increased
significantly the grain yield (kg per ha) by 18, 59, 40 and 14 % and biomass
20
yield (kg per ha) by 32, 32, 53 and 14 % respectively as compared to Control.
Among different doses of phosphorus, 60 kg P2O5 per ha appeared to be the
most economical for grain yield and 80 kg P2O5 per ha for straw yield. Highest
dose of phosphorus (100 kg P2O5 per ha) depressed both the grain and biomass
yield of chickpea. Application of phosphorus did not cause any increase in
percentage of nitrogen and phosphorus in shoot as well as in grain. However,
the content of nitrogen in shoot (kg per ha) increased significantly by 46, 39.
58, and 23 % as a result of 40, 60, 80 and 100 kg P2O5 per ha respectively in
comparison to Control. Similarly the phosphorus content in shoot increased b\'
43, 29, 55 and 16 % as a result of 40, 60, 80 and 100 kg P2O5 per has
respectively over the Control.
Maliwal and Gupta (1989) studied the effect of phosphorus on yield and
yield attributes of Trigonella foenum-graecum L. cultivar 'Lagour Local' at
Jobner (Rajasthan). Phosphorus treatments consisted of 0 and 40 kg P2O5
(equivalent to 0 and 17.5 kg P respectively) per ha. Application of phosphorus
at 17.5 kg P per ha increased number of pods per plant, straw yield and seed
yield over the Control. However, 1,000-seed weight and number of seeds per
pod were found to be not affected by phosphorus application.
Rathore and Manohar (1989) reported the effect of two levels of
nitrogen and four levels of phosphorus on growth and yield of Trigonella
foneum-graecum L. cultivar 'Nagour Local' at Jobner (Rajasthan). Nitrogen
was applied at 0 and 20 kg N per ha and phosphorus at 0, 25, 50 and 75 kg
P2O5 (equivalent to 0, 10.9, 21.8 and 32.8 kg P respectively) per ha.
Application of nitrogen at 20 kg N per ha and phosphorus at 21.8 kg P per ha
proved best for most of growth and yield parameters, including number of
branches, dry matter, number of pods and seed yield. They further reported in
other communication that nitrogen and phosphorus had beneficial effect on dry
weight of nodules at 75 days after sowing and nitrogen and phosphorus
percentage in straw and seeds at harvest (Rathore and Manohar, 1990).
21
Patel et al. (1991) conducted a field experiment at Sardar Krushinagar
(Gujarat), to study the effect of different levels of nitrogen, phosphorus and
potash on yield and yield attributes of 'IC 9955' cultivar of Trigonella foenum-
graecum L. They applied three levels each of nitrogen (0, 10 and 20 kg N per
ha) and phosphorus, viz. 0, 20 and 40 kg P2O5 (equivalent to 0, 8.7 and 17.5 kg
P respectively) per ha and two levels of potassium, viz. 0 and 20 kg KiO
(equivalent to 0 and 16.6 kg K respectively) per ha. It was found that
application of nitrogen, phosphorus and potassium did not affect the yield and
yield attributes of fenugreek.
Raju et al. (1991) conducted a field experiment to study the effect of
phosphorus on three chickpea varieties, viz. 'Type 3', 'Pant G 110' and 'K
468'. The treatments consisted of three levels of phosphorus, viz. 20, 40, and
60 kg P2O5 (equivalent to 8.7, 17.5 and 26.2 kg P) per ha, farmyard manure
(F.Y.M.) at the rate of 10 kg N per ha and Rhizobium inoculation. The source
of phosphorus was single superphosphate. It was observed that application of
phosphorus increased seed yield upto 60 kg P2O5 per ha, which was 610 and
890 kg per ha more than obtained with 20 kg P2O5 per ha. Like seed yield,
straw yield, number of nodules per plant and dry weight of nodules per plant
increased significantly upto 60 kg P2O5 per ha. They also found increasing
levels of phosphorus from 20 to 60 kg P2O5 per ha significantly increased the
uptake of nitrogen, phosphorus and potassium.
Singh and Sudhakar (1991) conducted a field experiment at Hyderabad
(Andhra Pradesh) on black gram cultivars 'T 9 and PDU 3' in sandy loam soil
with three levels of phosphorus 0, 20 and 40 kg P per ha with and without
inoculation. Application of 40 kg per ha with inoculation recorded significantly
higher values for dry matter accumulation compare to other combination of
phosphorus and inoculation separately. It was found significant increase in seed
yield and percentage of N and P (stems, leaves and seeds) as a result of
inoculation and phosphorus over the Control.
22
Verma et al. (1991) studied the effect of nitrogen and phosphorus on
Trigonella foenum-graecum L. (cultivar not mentioned) at Agra (Uttar
Pradesh). They applied four levels each of nitrogen (0, 20, 40 and 60 kg N per
ha) and phosphorus, viz. 0, 20, 40 and 60 kg P2O5 (equivalent to 0, 8.7, 17.5
and 26.2 kg P respectively) per ha. Seed yield increased with increasing level
of nitrogen upto 20 kg per ha. However, increasing levels of phosphorus
enhanced the seed yield linearly.
Noor et al. (1992) conducted a field experiment on Dolichos lablab
{Lablab purpureas) cultivar 'line HCOOLO' at Joydebpur (Bangladesh), to
study the effect of different combinations of 0, 30 or 60 kg P2O5 (equivalent to
0, 13.1 or 26.2 kg P respectively) per ha. 0, 50, 100 or 150 kg K2O (equivalent
to 0, 41.5, 83.0 or 124.5 kg K respectively) per ha.; 0 or 20 kg S; 0 or 5 kg Zn
and 5 or 10 ton FYM per ha., except for no fertilizers controls. All plots
received 15 kg N per ha compared with controls. All fertilizer treatments gave
maximum pod yield with 60 kg P2O5 + 100 kg K2O +20 kg S + 5 kg Zn + 5
tons FYM per ha.
Bhati (1993) studied the effect of phosphorus application on Trigonella
foenum-graecum L. cultivar 'Prabha' in an experiment conducted at Jobner
(Rajasthan). Phosphorus was applied at 0, 20, 40 and 60 kg P2O5 (equivalent to
0, 8.7, 17.5 and 26.2 kg P) per ha along with a uniform dose of 30 kg N per ha.
The data revealed that application of phosphorus at 8.7 kg P per ha increased
pods per plant, 1000-seed weight, straw yield and harvest index and water use
efficiency over the Control. However, seed and biological yields increased
significantly upto 17.5 kg P per ha. Phosphorus application did not affect the
plant height, seeds per pod and pod length
Chaudhary and Singh (1994) studied the effect of nitrogen, phosphorus
and zinc on yield of horse gram (Macrotyloma uniflorum). The treatments
comprised three levels each of N (0, 10 and 20 kg per ha), P (0, 8.8 and 17.6 kg
per ha) and 2 levels of Zn (0 and 4.1 kg per ha). Each treatment was replicated
3 times in factorial randomized block design. Application of P upto 8.8 kg per
23
ha increased the seed yield significantly. Pods per plant, pod length and seeds
per pod showed a similar trend. The maturity of crop decreased significantly by
5-6 days at 17.6 kg P per ha compared with the Control.
Patel et al. (1994a) conducted a field experiment at Navasari (Gujarat),
to study the effect of different row spacings (30, 45 or 60 cm) and three levels
of N (15, 25 or 35 kg N) and P, viz. 30, 50 or 70 kg P2O5 (equivalent to 13.1,
21.8 or 30.6 P respectively) per ha on growth of Indian butter bean {Dolichos
lablab var. Typicus). Dry matter production per plant, seeds per pod, pods per
plant and 1000-seed weight were found highest with 25 kg N and 50 kg P2O5.
Patel et al. (1994b) also worked out the yield and quality of Indian
butter bean {Dolichos lablab var. Typicus) with different row spacings (30, 40
or 60 cm), and three levels of N (15, 25 and 35 kg N) and P, viz. 30, 50 and 70
kg P2O5 (equivalent to 13.1, 21.8 and 30.6 kg P respectively) per ha. 25 kg N
and 50 kg P2O5 gave maximum seed yield and seed protein content compared
with 15 or 35 kg N and 30 or 70 kg P2O5.
Banafar et al. (1995) conducted a field trial to study the effect of
nitrogen and phosphorus on Trigonella foenum-graecum L. cutivar 'Plume 55'
at Sehore (Madhya Pradesh). Nitrogen was applied at the rate of 0, 20, 40 and
80 kg N per ha and P, viz. 0, 15, 30 and 45 kg P2O5 (equivalent to 0, 6.6, 13.1
and 19.7 kg P respectively) per ha. Application of 80 kg N and 19.7 kg P per ha
proved best for seed yield and vegetable leaf yield.
Detroja et al. (1995) studied the effect of three levels of nitrogen,
phosphorus and two levels of potassium on seed yield of cultivar IC 9955 of
fenugreek at Junagadh (Gujarat). They applied 0, 30 and 60 kg N per ha, 0, 60
and 120 kg P2O5 (equivalent to 0, 26.2 and 52.4 kg P respectively) per ha and 0
and 30 kg K2O (0 and 24.9 kg K respectively) per ha. Applicafion of 30 kg N,
26.2 kg P and 24.9 kg K per ha proved best for seed yield.
Patel et al. (1995) conducted a field trial at Navsari (Gujarat), to study
the effect of different rows (30, 45 or 60 cm) and three levels of N (15, 25 or 35
kg N per ha) and three levels of P, viz. 30, 50 or 70 kg P2O5 (equivalent to 0,
24
13.1, 21.8 or 30.6 kg P respectively) per ha on Dolichos lablab {Lablab
purpureus). Seed yield and uptake of N and P in seeds were highest with 45 cm
row spacing and application of 25 kg N and 50 kg P2O5 per ha.
Yahiya and Samiullah (1995) conducted a field experiment at Aligarh
(Uttar Pradesh), to examine how P application at the rate of 0, 20, 40 and 60 kg
P2O5 (equivalent to 0, 8.7, 17.5 and 26.2 kg P respectively) per ha affects
nodulation and N2-fixation in chickpea. Number of nodules and dry weight of
nodules per plant were increased significantly due to P application. Among P
levels, 40 kg P2O5 per ha proved to be the most effective dose for these
parameters. The reduced nodulation and N2-fixation in P-deficient plants of
chickpea seem to be impaired shoot metabolism and not by a direct effect of P
deficiency of the nodules.
Singh and Bishnoi (1996) conducted a pot experiment at Ludhiana
(Punjab), to study the response of Trigonella foenum-graecum L. cultivar 'ML
150' to phosphorus application on soils differing in available phosphorus
status-low, medium and high. They applied five levels of phosphorus, viz. 0,
10, 20, 30 and 40 mg P2O5 (equivalent to 0, 4.4, 8.7, 13.1 and 17.5 mg P
respectively) per kg soil along with a uniform dose of 10 mg N and 10 mg K
per kg soil. The results showed that dry matter and yield increased significantly
upto 17.5 mg P per kg soil in low phosphorus soil. In medium and high
phosphorus soil, a significantly increase in dry matter yield was observed upto
4.4 mg P per kg soil application.
Arya et al. (1997) conducted an experiment during 3 seasons of 1992,
1993 and 1994 at Jhansi (Uttar Pradesh), to study the effect of three nitrogen
levels (20, 30 and 60 kg N per ha) and two phosphorus levels, viz. 20 and 40 kg
P2O5 (equivalent to 8.7 and 17.5 kg P respectively) per ha on productivity of
grain sorghum {Sorghum bicolor) and dolichos {Dolichos) intercropping
systems. They observed that higher grain and stover yields of sorghum were
obtained with pure crop of sorghum with 60 kg N + 40 kg P2O5 per ha than the
other intercropping systems with different fertilizer schedules. To an equivalent
25
basis the productivity of grain Sorghum + Dolichos with 60 kg N +20 kg P2O5
per ha (4.21 ton per ha) was more than that of grain Sorghum + Cowpea [Vigna
unguculata (L.) Moench.] with 60 kg N +40 kg P2O5 per ha (4.16 ton per ha).
Ihe minimum sorghum gram-equivalent yield was obtained in pure crops of
sorghum and dolichos.
Kamal and Mehra (1997) studied the effect of fertilizers and foliar spray
of naphthalene acetic acid (NAA) on yield of Trigonella foenum-graecum L
cultivar 'NLM' (Prabha) at Jaipur (Rajarthan). They applied three levels of
nitrogen, viz. 0, 20 and 40 kg N and four levels of phosphorus, viz. 0, 30, 60
and 90 kg per ha and two sprays of 10 ppm naphthalene acetic acid. Higher
seed yield was given by the combination 40 kg N per ha + 90 kg P per ha +
NAA spray.
Dayanand et al. (1998) studied the effect of four levels of phosphorus on
the growth and yield attributes of Trigonella foenum-graecum L. cultivar
'RMT r at Jobner (Rajasthan). Phosphorus was applied at 0, 20, 40 and 60 kg
P2O5 (equivalent 0, 8.7, 17.5 and 26.2 kg P respectively) per ha along with a
uniform basal dose of 25 kg N per ha. Application of 40 kg P2O5 per ha
increased plant height, dry mater accumulation, number of branches per plant,
number of pods per plant, number of seeds per pod over its preceding doses.
However, test weight was not affected by phosphorus application.
Gogoi et al. (1998) conducted an experiment at Biswanath Chariali
(Assam) to study the effect of phosphorus fertilization on Trigonella foenum-
graecum L. cultivar 'Pusa Early Bunching'. Six levels of phosphorus, viz. 0,
10, 20, 30, 40 and 50 kg P2O5 (equivalent 0, 4.4, 8.7, 13.1, 17.5 and 21.8 kg P
respectively) per ha were applied. The plant height and number of branches per
plant did not show any significant change with increasing levels of phosphorus.
Plants applied with 50 kg P2O5 per ha took maximum time to flower. To obtain
the higher seed yield, 30 kg P2O5 per ha should be applied to the soils of north
blank planes of Assam for optimum fenugreek production.
26
Halesh et al. (1998) performed a field trail to study the response of
Trigonella foenum-graecum L. cultivar "COT to the application of nitrogen
and phosphorus at Bangalore (Karnataka). The treatment comprised four levels
of nitrogen, i.e. 0, 30, 60 and 90 kg N per ha and four levels of phosphorus, viz.
0. 30, 60 and 90 kg P2O5 (equivalent to 0, 13.1, 26.2 and 39.3 kg P
respectively) per ha, with a constant dose of 50 kg K2O (41.45 kg K) per ha.
Higher plant growth and seed production were obtained by the application of
60 kg N and 39.3 kg P per ha. Higher dose of nitrogen delayed flowering and
prolonged the crop duration.
Jat et al. (1998) studied the effect of two levels of phosphorus, viz. 20
and 40 kg P2O5 (equivalent to 8.7 and 17.5 kg P respectively) per ha on yield
attributes, yield and nutrient content of Trigonella foenum-graecum L. cultivars
•RMT r at Jobner (Rajaslhan). Application of phosphorus at 40 kg P2O5 per ha
increased the number of pods per plant, seeds per pod, seed and straw yield.
Phosphorus at 40 kg P2O5 per ha also increased nitrogen and phosphorus
content in seeds and straw.
Kumawat et al. (1998) studied the effect of phosphorus application on
Trigonella foenum-graecum L. cultivars 'RMT 1' at Jobner (Rajasthan). Three
levels of phosphorus, viz. 20, 40 and 60 kg P2O5 (equivalent to 8.7, 17.5 and
26.2 kg P respectively) per ha were applied along with a uniform basal dose of
25 kg N per ha. Application of 40 kg P2O5 per ha proved best for plant height,
branches per plant, pods per plant, seeds per pod, test weight, seed yield and
straw yield. Nitrogen content in seed and phosphorus content in both seed and
straw were also maximum with 40 kg P2O5 per ha.
Rana et al. (1998) conducted an experiment at Lakhaoti (Uttar Pradesh),
to study the effect of six nitrogen levels with and without Rhizobium
inoculation (0, 15 and 30 kg N per ha) and three phosphorus levels, viz. 0, 40
and 80 kg P2O5 (equivalent to 0, 17.5 and 35.0 kg P respectively) per ha on root
nodulation, dry matter production and N and P uptake in pigeon pea {Cajanus
cajan L. Mill sp.) on sandy loam soil. Phosphorus at the rate of 40 kg P2O5 per
27
ha enhanced root length, number of nodules, dry matter yield and N and P
content over their respective controls.
Srivastava et al. (1998) conducted a field trial at New Delhi, to study the
effect of three levels of P (0, 13.1 and 26.2 kg P per ha) and two levels of Mo
(0 and 0.5 kg per ha, biofertilizers Rhizobium and Phosphate solubilizing
bacteria (PSB) on growth attributes, yield and harvest index of pea in a sandy
loam soil under semi-arid and sub-tropical climate condition. Application of P
at 26.2 kg per ha. Mo at 0.5 kg per ha and seed inoculation, with Rhizobium
and or PSB resulted in marked increase in number of nodulation, nitrogenase
activity, growth and grain yield over no inoculation.
Santhaguru and Hariram (1998) conducted an experiment at Madurai, to
study the effect of Glomus mosseae and Rhizobium on growth, nodulation and
nitrogen-fixation in Lablab purpureus L. 'Sweet' under different phosphorus
regimes (0 and 150 mg per kg soil). They observed that the nodule activity viz.
the number and dry weight of nodules, leghemoglobin content and nitrogen
content was higher in Glomus mosseae and Rhizobium inoculated Lablab
purpureus as compared to the single inoculation with Rhizobium alone. The
nodule activity was a function of supplemental P in both the dual inoculated
and Rhizobium inoculated plants. Accumulation of plant biomass, total N and P
in Lablab purpureus was higher with dual inoculation than with either
Rhizobium or Glomus mosseae inoculation.
Chaudhary (1999a) studied the response of Trigonella foenum-graecum
L. cultivar 'RMT 1' to nitrogen and phosphorus at Jobner (Rajasthan). Three
levels of nitrogen (0, 20 and 40 kg N) per ha and two levels of phosphorus, viz.
20 and 40 kg P2O5 (equivalent to 8.7 and 17.5 kg P respectively) per ha were
applied. Application of 40 kg N per ha and 40 kg P2O5 per ha proved best for
branches per plant, pods per plant, test weight, seed yield and straw yield.
Chaudhary (1999b) studied the effect of six combinations of nitrogen
and phosphorus on Trigonella foenum-graecum L. cultivar 'RMT 1' at Jobner
(Rajasthan). The combinations of nitrogen (kg N per ha) + phosphorus, i.e. kg
28
P.Os per ha included 0+0, 20+20, 20+40, 20+60, 40+40 and 40+60.
Application of 40 kg N per ha + 40 kg P2O5 per ha proved best for pods per
plant, test weight and seed yield. However, the combinations did not affect for
plant height, branches per plant, pod length and seeds per pod.
Parmar et al. (1999) conducted a field experiment in cold desert area of
Himachal Pradesh, to study the effect of three levels of nitrogen (0, 15 and 30
kg N per ha) and four levels of phosphorus, viz. 0, 30, 60 and 90 kg P2O5
(equivalent to 0, 13.1, 26.2 and 39.3 kg P respectively) per ha on growth, yield
and nutrients uptake in french bean or 'Rajma' {Phaseolus vulgaris L.). Plant
height, nodules per plant, pods per plant, seeds per pod and seed yield
increased significantly upto 15 kg N and 60 kg P2O5 per ha. The uptake of N
and P significantly increased upto 60 kg P2O5 per ha. Nutrient use efficiently
however was significantly highest at their lower levels i.e. 15 kg N and 30 kg
P2O5 gave significantly highest N and P-use efficiency respectively.
Singh et al. (1999) conducted a field experiment on greengram
{Phaseolus radiatus L.) with four levels of P, viz. 0, 8, 17.6 and 26.4 kg per ha
and four levels of S (0, 20, 40 and 60) kg per ha at Faizabad (Uttar Pradesh).
Increasing levels of phosphorus (26.4 kg per ha) significantly increased number
of nodules per plant, dry weight of nodules, number of grains per pod and test
weight. Grain yield also increased significantly upto 26.40 kg P per ha.
Phosphorus at the level of 26.4 kg P per ha significantly increased the
concentration and uptake of N, P and S and protein content.
Sheoran et al. (1999) studied the effect of four levels phosphorus, viz. 0,
30, 60 and 90 kg P2O5 per ha (equivalent to 0, 13.1 26.2 and 39.3 kg P
respectively) per ha with three dates (16, November, 1 and 16 December) on
two fenugreek genotypes 'HM 65' and 'T 8' at Hisar (Haryana). Phosphorus
was applied in the form of single superphosphate and a basal dose of 20 kg N
per ha as urea. Application of phosphorus (60 kg P per ha) significantly
increased seed-yield and productivity of the crop. Based on the pooled data, an
29
increase in seed yield up to 19.4, 27.6 and 28.8 % was recorded by the
application of 30, 60 and 90 kg P2O5 per ha respectively.
Kumar et al. (2000) conducted a field trial on sandy loam soil at
Aliahbad (Uttar Pradesh) to observe the effect of Rhizobium culture and
different levels of phosphorus, viz. 0, 40, 60 and 80 kg P2O5 (equivalent to 0,
17.5, 26.2 and 34.9 kg P) per ha on growth, nodulation and yield of groundnut
cultivar 'Amber'. The application of 60 kg phosphorus per ha coupled with
effective strain of Rhizobium gave significantly higher number of nodule
counts (142-177) and higher growth over rest of the treatment combinations,
where as maximum pod yield (3514 kg per ha) was found with 80 kg P per ha
in Rhizobium inoculated one. Thus the application of phosphorus at the rate of
60-80 kg per ha coupled with effective strain of Rhizobium is essential for
optimum growth, higher yield and for the maintenance of soil fertility.
Reddy and Swamy (2000) conducted a field experiment on blackgram
(Phaseolus mungo) at Hyderabad. They applied three levels of phosphorus (0,
13.1 and 26.2 kg P per ha), two levels of farmyard manure (0 and 10 tons per
ha) and two levels of PSB (inoculated and uninoculated). Application of 26.2
kg P per ha increased growth and yield attributes. With increase in phosphorus
levels from 0 to 26.2 kg P per ha yield attributes like pods per plant, seeds per
pod and seed yield increased by 53.5, 15.3 and 50.6 % respectively over the
Control.
Sinha et al. (2000) conducted a field experiment with three dates of
sowing viz. D, (1 November 1997), D2 (15 November 1997), D3 (30 November
1997) and three levels of phosphorus, viz. 45, 60 and 75 kg P2O5 (equivalent to
19.6, 26.2 and 32.7 kg P respectively) per ha on garden pea (Pisum sativum) at
Nauni-Solan (Himachal Pradesh). Each incremental dose of phosphorus (from
45 to 75 kg P per ha) significantly increased plant height, reduced the number
and days to 50 % flowering and days to harvest of seeds. Yield attributing
traits, viz. pod length, pods per plant, seeds per pod, and seed yield per plant
30
were also remarkably increased with the application of moderate level of
phosphorus (60 kg per ha).
Menna et al. (2001) conducted a field experiment at Jobner (Rajasthan).
to find out response of chickpea to four levels of phosphorus, viz. 0. 20, 40 and
60 P2O5 kg (equivalent to 0, 8.7, 17.5 and 26.2 kg P respectively) per ha and
four biofertilizer levels. Application of 40 kg P2O5 per ha significantly
enhanced pods per plant, seeds per pod, test weight, seed yield and straw yield.
Seed yield increased by 86.3 and 8.57 % by the applicafion of 20-40 and 40-60
kg P2O5 per ha respectively. Protein content was significantly higher with 60 kg
P2O5 per ha over the Control.
Rao and Shaktawat (2001) conducted a field experiment at Udaipur
(Rajasthan), to study the effect of three combinations of organic manure
(Control, 10 ton FYM and 5 ton poultry manure) per ha, three levels of
phosphorus, viz. 20, 40 and 60 kg P2O5 per ha (equivalent to 8.7, 17.5 and 26.2
kg P respectively) per ha and three levels of gypsum (250 kg at sowing (single)
and 125 kg at sowing + at 35 DAS (split) per ha along with Control on growth,
yield and quality of groundnut under rainfed conditions. A mean increase of
14.0 and 11.3 % in pod yield was recorded under FYM and poultry manure
application over the Control (1622 kg per ha). Application of 60 kg P2O5 per ha
significantly increased growth, yield and quality parameters compared to 20 kg
P2O5 per ha. The effect of gypsum either in single or in split doses on dry
matter accumulation at 45 DAS was not evident but as crop growth advanced,
the effect on all growth parameters was evident. The two modes of gypsum
application proved equally effective in increasing pod yield of groundnut.
Gypsum treatment significantly increased harvest index of groundnut on
pooled basis. Oil and protein content groundnut kernel increased significantly
under gypsum treatment.
Ram and Verma (2001) conducted a field experiment at Bichpuri
(Agra), to study the effect of four levels of phosphorus, viz. 0, 26.4, 52.8 and
79.2 kg P per ha on the performance of seed yield of fenugreek (Trigonella
31
foenum-graecum L.) cultivar 'Pusa Early Bunching'. The phosphorus applied
at the rate of 52.8 kg per ha gave significantly more seed yield (2.1 ton per ha),
seeds per plant (1158), weight of seeds per plant (10.2 g), test weight of 100
seeds (1.18 g), seeds per pod (14.1), weight of seeds per pod (0.123 g), weight
of pods per plant (16.4 g), fresh weight of whole plant (35.1 g). It is quite
evident that 52.8 kg P per ha was optimum dose for growing seed crop of
fenugreek.
Singha and Sarma (2001) studied the response of blackgram {Phaseolus
mungo) to phosphorus fertilization and Rhizobium inoculation in the hill soils
of Assam. Four levels of phosphorus, viz. 0, 25, 35 and 45 kg P2O5 (equivalent
to 0, 10.9 15.3 and 19.6 kg P respectively) per ha were applied through single
superphosphate with and without Rhizobium inoculation of seeds. Application
of 45 kg P2O5 per ha produced the highest grain and straw yield and was at par
with that of the application of 25 and 35 kg P2O5 per ha. The N uptake by the
crop increased the grain yield from 20 to 30 kg per ha with that of the
application of 25 to 45 kg P2O5 per ha, respectively over the Control. The P and
K uptake by the crop also increased with increasing levels of P application.
Number of nodules and mass of nodules per plant increased with increasing
levels of P application upto 35 kg P2O5 per ha.
Kumar and Puri (2002) studied the response of two varieties of french
bean or 'Rajma' {Phaseolus radiatus L.) to phosphorus levels, viz. 0, 25 and 50
kg P2O5 (equivalent to 0, 10.9 and 21.8 kg P respectively) per ha and FYM (0
and 10 tons per ha) application under rainfed condition at Salaoni (Himachal
Pradesh). Application of 50 kg P2O5 per ha with 10 ton of FYM per ha resulted
in the maximum plant height, pods per plant, seeds per pod and 1000-seed
weight and thereby higher yields.
Sharma et al. (2002) carried out a field experiment at Udaipur
(Rajasthan), to study the effect of three phosphorus levels viz. 30, 60 and 90 kg
P2O5 (equivalent to 13.1, 17.5 and 21.8 kg P respectively) per ha and sources
with and without FYM in soybean {Glycine max (L.) Merrill.). Phosphorus
32
application significantly enhanced growth determinants and seed yield of
soybean. Application of 60 kg P2O5 per ha significantly improved number of
nodules per plant, weight of nodules per plant, dry matter accumulation (DMA)
per plant and seed yield over the Control.
Singh and Verma (2002) conducted two field experiments at Bari Bagh
(Gazipur) nitrogen (0, 30, 60, 90 and 120 kg per ha) and three phosphorus
levels (0, 30 and 60 kg P per ha) to study their impact on growth, yield
attributes, yield and economic of french bean (Phaseolus vulgaris L.) grown
under late-sown condition in plains of Eastern Uttar Pradesh. Higher dose of
nitrogen (120 kg per ha) and phosphorus (60 kg per ha) resulted in higher yield
and was found more profitable.
Gautum et al. (2003) conducted a field experiment at Pantnagar, to study
the effect of phosphorus rates, (13.2, 19.8 and 26.4 kg P per ha) and
Pseudomonas sp in combination with Bradyrhizobium and farmyard manure at
the rate of 1 ton per ha on seed yield and yield attributes of soybean {Glycine
max (L) Merrill.). The yield attributes i.e. seeds per plant, pod length, number
of pods and pod dry weight, differed significantly due to different treatments.
Seed yield also increased (22.2-34.2 %) significantly owing to variation in
treatments.
Hussein (2003) conducted an experiment at Cairo (Egypt) to investigate
the effect of NPK fertilizer ratios of 1:1:1 (6% N- 6% P2O5 - 6% K2O), 2:1:1
(12% N- 6% P2O5 -6% K2O) or 3:1:1 (18% N- 6% P2O5 - 6% K2O) and soil
amendments (taffla, clay, composted sewage sludge, cattle manure or Agrosil)
on the growth and chemical composition of Senna occidentalis L. grown in
sandy soil. He applied fertilizers at the rate of 5 g per plant per month. He
reported that all fertilizer treatments significantly increased the vegetative
growth (plant height, stem diameter, number of branches and leaves per plants,
and fresh and dry weight of leaves, stems and roots per plant) and NPK at 3:1:1
fertilizer gave the best results. In general, the addition of amendments to the
sandy soil significantly increased the different growth parameters of the plant.
33
He further reported that the different NPK fertilizer treatments and soil
amendments increased the contents of total chlorophyll, total carbohydrates, N.
P and K in the leaves. Raising the N level in the fertilizer increased the contents
of total chlorophyll, total carbohydrates and N, and decreased the P and K
percentages. The phosphorus percentage showed different response to soil
amendments in the 2 seasons. Thus, for the best vegetative growth of Senna
occidentalis L., plants grown in a sandy soil, the soil should be amended with
clay and the plant should be supplied with 5 g per plant per month of 3:1:1
NPK fertilizer.
Khiriya and Singh (2003) conducted a field experiment at Hisar
(Haryana), to study the effects of four levels of farmyard manure (0, 5, 10 and
15 tons per ha) and phosphorus, viz. 0, 20, 40 and 60 kg P2O5 (equivalent to 0,
8.6, 17.2 and 25.8 kg P respectively) per ha on two fenugreek (Trigonella
foenum-graecum L.) cuUivar 'HM 65 and NLM'. Cultivar 'NLM' was
significantly superior to 'HM 65' in tenns of more number of branches per
plant, pods per plant and seeds per pod. Increasing levels of phosphorus up to
40 kg P2O5 per ha significantly increased the yield attributing characters, seed
yield and quality parameter of fenugreek.
Samiullah and Khan (2003) conducted a field experiment at Aligarh
(Uttar Pradesh), to study the individual and combined effect of phosphorus and
potassium, viz. 0, 20, 40 and 60 kg per ha each of P (P2O5) and K (K2O)
(equivalent to 0, 8.6 and 17.2 kg P and 0, 16.6, 33.2 and 49.8 kg K
respectively) per ha and on morphophysiological traits and yield of chickpea
(Cicer arietinum L.) cv. 'Pusa 417'. The observations were recorded on
number of branches, shoot dry weight, root dry weight and number of nodules
per plant, nitrogenase activity of nodules, leaf area index, crop growth rate and
net assimilation rate. At maturity, pod number, 100-seed weight, seed yield and
seed-yield-merit were recorded. A combination of 40 kg P2O5 and 20 kg K2O
per ha produced maximum yield. The low input of P2O5 was compensated by
high input of K2O and vice versa. At lower phosphorus levels (0 or 20 P2O5 per
34
lia) the requirement of potassium was high (40 kg K2O per ha). However, at
higher levels of phosphorus (40 or 60 kg P2O5 per ha) the application of only
20 kg K2O per ha proved effective and seed yield and seed yield merit were
enhanced by 82 and 85 % by 40 kg P2O5 x 20 kg K2O over Control.
Turk et al. (2003) conducted a field experiment in the semi-arid region
in the north of Jordan, to study the effect of sowing dates (14 Jan, 28 Jan and
12 Feb.), seeding rates (40, 60 and 80 plants m"" ), phosphorus levels (17.5, 35.0
and 52.5 kg P per ha) and two methods of P placement on yield response of
narbon vetch {Vicia narbonensis). Phosphorus application had only a
significant effect on seed yield, number of pods per plant, number of seeds per
pod, number of primary branches per plant, lOO-seed weight, pod length and
seed weight per plant. In general, the results revealed that a combination of
early sowing (14 Jan), a high seeding rate (80- plant m ' ) and P application
(52.5 kg P per ha) gave maximum yield of narbon vetch.
Menna et al. (2004) studied the response of chickpea {Cicer arietinum
L.) to soil moisture conservation, three levels of phosphorus, viz. 0, 30 and 60
kg P2O5 (equivalent to 0, 13.1 and 26.2 kg P respectively) per ha and bacterial
{Pseudomonas straiata) inoculation in seed at New Delhi. Application of
phosphorus at the rate of 60 kg P2O5 per ha resulted higher in growth and yield
attributes, grain and straw yields. The application of phosphorus at 60 kg P2O5
per ha had positive effect on protein content in grain and gave 21.7 % higher
value than that of Control. Total uptakes of N, P and K were higher in 60 kg
P2O5 per ha than that of former levels.
Singh and Chauhan (2004) conducted a field experiment on sulphur and
phosphorus deficient sandy loam oil on lentil crop to evaluate the effect of S
and P in the presence and absence of Rhizobium inoculation at Tonk
(Rajasthan). They applied four levels each of sulphur (0, 30, 60 and 90 kg per
ha) and phosphorus (0, 13.1, 26.2 and 39.3 kg P per ha respecfively). Sulphur
and phosphorus application increased the growth and yield of lentil
significantly upto 60 kg S and 26.2 kg P per ha. The maximum and
35
significantly higher N and K content uptake by grain and straw of lentil and
biological N fixation were found with 60 kg S and 26.2 kg P per ha and at par
with that of 90 kg S and 39.3 kg P per ha.
Shubhra et al. (2004) studied the influence of three levels of phosphorus
(75, 150 and 300 mg per pot) on relative water content, leaf water-potential and
osmotic potential, contents of chlorophyll, chl a, chl b, soluble sugars and seed
quality (gum content) in alleviating the deleterious effect of water deficit in
cluster bean at Hisar (Haryana). Under water stress, leaf water-potential,
osmotic potential, chlorophyll and gum contents decreased and sugar contents
increased. Phosphorus application increased chlorophyll content and sugar
content in Control plants and ameliorated the negative effects of water stress.
Naeem and Khan (2005) conducted a pot experiment at Aligarh (Uttar
Pradesh), to find out whether phosphorus application could augment the
growth, physiology and seed yield of Cassia tora (syn.. Cassia obtiisifolia).
Out of five levels of phosphorus, viz. 0, 0.1, 0.2, 0.3 and 0.4 g P per kg soil (PQ,
PI, P2, P3 and P4, respectively). P3 significantly enhanced fresh and dry weights,
number of leaves, leaf-area per plant, total chlorophyll content, leaf-NPK
content, nitrate reductase activity in leaf, pod length, number of pods, pod
weight per plant, seed weight per pod, seed-yield, seed-yield-merit and seed-
protein content respectively. However, seed number per pod, 100-seed weight
and harvest index were not affected by different levels of phosphorus. Seed
yield and seed yield merit were enhanced by 30.3 % and 34.2 % respectively
by P3 level of phosphorus. It is revealed that P3 proved best for most of the
parameters.
2.5 Effect of calcium application on leguminous plants (including medicinal legumes)
The literature on the effect of calcium on medicinal leguminous plants is
meager, therefore, the reference on the effect of calcium on legumes have also
been mentioned in the following pages.
36
Andrew (1976) studied the effect of a factorial combination of calcium
(0.125 and 2.0 mM as calcium nitrate), pH (4.0, 4.5, 5.0 and 6.0) and nitrogen
levels (nil and 2.0 mM as ammonium nitrate) on the nodulation and growth of
some tropical and temperate pasture legumes. In his study, he observed that
Stylosantl eshumilis, Trifolium semipilasum and T. rueppellianum reduced
nodulation only at pH 4.0, while Desmodium uncinatunl and Trifolium repens
were sensitive to low pH, and nodulation were markedly reduced at pH 4.0 and
below 5.0 respectively. Further, he observed that the dry matter yield of
nodulated plants under inferior treatment vv ere less than those of plants
nodulated under optimum treatments. Nodulation and growth were strongly
controlled by pH, and although interactions occurred with calcium treatment.
He found that dry matter yield of nodulated plants of S. eshumilis and L.
hainesii were greater at the low calcium sulphate treatment (0.125 mM) than at
the high treatment (2.0 mM). The remaining legumes gave varying responses to
the high calcium treatment. He further reported that root weight ratios of
nodulated plants were decreased by the higher treatments, the minimum
occurring at those treatments producing the highest dry matter. In the presence
of applied nitrogen (no nodulation), the effects of pH and calcium on dry
matter were small. He considered that both nodulation and subsequent plant
growth were dominated by the hydrogen ion effect, and that were beneficial
effect of calcium operates within certain pH limits. pH had little effect on
plants that were well supplied with nitrogen.
Dahiya and Singh (1980) conducted a pot experiment on loamy sand soil
to find out the effect of different levels of CaCOa and farmyard manure (FYM)
on the dry matter yield and nutrient uptake by oats {Avena saliva cv. HFO-114)
at Hisar (Haryana). They applied FYM (0, 0.5, 1, 2 and 4 %) and four levels of
CaCOs viz. 0, 2, 4 and 8 % (equivalent to 0, 0.8, 1.6 and 3.2 % Ca) to each pot,
which contained 4 kg of soil. A uniform basal dose of N, P, K, Fe and Mn at
the rate of 120, 60, 60, 5 and 10 ppm, respectively was also applied to each pot.
In general, application of CaC03 and FYM significantly increased dry matter
yield of oats. The concentration and uptake of P, Fe and Mn decreased
37
significantly with increasing levels of CaC03; but application of FYM resulted
in a significant increase in the concentration and uptake of the nutrients.
However, application of CaCOs significantly increased the concentration and
uptake of calcium, but the effect of FYM application in the presence (as well as
in absence) of CaCOi was found negative. Application of increasing levels of
CaC03 and FYM significantly decreased the concentration, as well as uptake of
Mn by the plants.
Ssali (1981) studied the effect of various levels of CaCOs^ inoculation
and lime pelleting on nodulation, dry matter yield and nitrogen content of bean
plant {Phaseolns vulgaris cv. Candian wander) in five acid soils in green house
condition in Nairobi (Kenya). The soil represented a range of 3.89 to 5.1 with
exchangeable aluminium from 0.0 to 4 mg per 100 g, exchangeable manganese
from 0.35 to 2.32 Mg per 100 g and percent carbon from 6.69 to 5.60. It was
observed that application of CaCOs increased soil pH and exchangeable
calcium in all soils but exchangeable aluminium and manganese decreased with
increasing amount of CaCOs. Inoculation increased nodule weight, dry matter
yield and percent nitrogen particularly at the low pH level. However, nodule
weight and dry matter yield increased with soil pH where the seeds were not
inoculated. At low organic matter content and with substantial amounts of
aluminium and/or manganese, liming increased nodule weight and dry matter
yield and decreased exchangeable aluminium and/or manganese. However, low
lime rates had little effects on exchangeable aluminium and calcium and dry
matter yield, but higher lime rates decreased exchangeable aluminium and dry
matter yield but increased exchangeable calcium.
Bell et al. (1989) studied the effect of six constant solutions of calcium
concentrations on six tropical food legumes, peanut {Arachis hypogea (L) cv.
Red Spanish), pigeonpea {Cajanus cajan (L). Millsp. cv. Royes), guar
{Cyamopsis tetragonoloba (L.) Taub. cv. Brooks), soybean {Glycine max (L.)
Merr. cv. Fitzroy) and cowpea (Vigna unguiculata (L.) Walp. cv. Vita 4 and
CPI 28215) at Murdoch (Western Australia). They applied six levels of calcium
38
(2, 12, 50, 100, 500 and 2500 [iM) in the flowing solution culture at pH 5.5 ±
0.1 with adequate inorganic nitrogen and controlled based nutrient
concentrations. They observed that increase in solution of calcium
concentration from 2 to !2 j M generally increased rates of absorption of
nitrogen, phosphorus and potassium. Further increases in solution calcium
concentration from 12 to 2500 ji M generally had no effect on potassium
absorption rate, but increased absorption rates of nitrogen (by 20-130 %) and
phosphorus (by 90-500 %), and with guar, rates of phosphorus absorption at <
2500 fiM. Calcium were too low to maintain adequate concentrations of
phosphorus in tops for maximum growth.
Pijnenborg et al. (1990) conducted an experiment on lucerne (Medicago
sativa cv. Resis), to study the effect of calcium on the nodulation using EGTA,
a specific calcium chelator at Wageningen (The Netherlands). They observed
that the number of nodules was optimum at 0.2 mM calcium was given in the
form of CaCb, but when calcium was not given, the number of nodules
decreased by 70 % and similar decrease was observed with 0.2 mM calcium
solution in the presence of 0.2 mM EGTA. However, nodulation was restored
specially by the addition of a higher dose of calcium, viz. 0.8 M CaCl2, to the
0.2 mM calcium solution containing 0.4 EGTA but not with MgCli- They
observed that depletion of soil calcium could depress nodulation only during
the first day after inoculation and concluded that calcium had different modes
of action in the symbiotic process during the initiation and formation of the
nodules.
Pijnenborg and Lie (1990) studied the effect of lime pelleting on the
nodulation of lucerne (Medicago sativa) in an acid soil. Their comparative
study was carried out in the field, in pots and in rhizotrons at Wageningen (The
Netherlands). The seeds were either inoculated with Rhizobium meliloti (R) or
inoculated and pelleted with lime (RP). They observed that in the field
conditions, lime-pelleting (PR) was found superior to inoculation (R) with
regard to seedling establishment and nitrogen yield. The number of seedlings
39
carrying crown nodules increased from 18 % (R) to 56 % (PR) at 26 days after
sowing. They ftirther reported that like field conditions, in pots and rhizotrons
also, lime-pelleting increased crown nodulation. In pots, the increase was from
32 % (R) to 60 % (PR) and in rhizotrons from 5 % (R) to 90 % (PR). They
concluded that crown nodulation may be used to quantify the benefit of lime-
pelleting.
Adams and Hartzog (1991) studied to detennine the effect of applied Ca
on seed Ca, seed germination, and seedling vigor on sites varying in extractable
soil Ca and to determine the minimum seed Ca concentration required for
maximum germination and seedling vigor of runner peanuts (Arachis hypogea
L.) at Auburn. Gypsum were established on soils with 130 to 625 mg per kg
Ca. they reported that yield and SMK (Sound mature kernel) were generally
unaffected by the gypsum topdressing, but there were effects on seed
concentrations of Ca, Mg and K. The minimum seed-Ca needed for maximum
germination was 282 mg per g, while the minimum needed for maximum
seedling survival was 260 mg per g. They observed that the higher level of
available soil Ca is needed for maximum seed quality than is needed to obtain
maximum yield or SMK.
Keiser and Mullen (1993) studied the response of soybean to calcium
and relative humidity. Plants were grown in solution culture in a controlled
environment growth chamber at Ames (lA.), with Ca concentration of 0, 0.6,
1.2 and 2.5 mM in the root medium from beginning seed (R5) to beginning
maturity (R7). Seed-Ca concentration increased with increased Ca supply to the
plant. Relative humidity had not effect on either seed Ca concentration or
germination. A decrease in the percentage of normal seedlings from 96.7 to
41.8 % coincided with a decrease in seed Ca from 2.37 to 0.87 mg per g. Their
results indicated that reduced Ca-supply to the plant may reduce seed-Ca
concentration in addition to altering other seed nutrients. Reduced Ca was
associated with poorer seed germination, but additions studies are needed to
40
clarify the role of Ca and other nutrients in the gemiination of seed and
seedling establishment,
Awada et al. (1995) studied the response of Phaseohis vulgaris cv.
'Contender' seedlings exposed to NaCl or Na2S04 at concentration of 0, 15. 30,
45 or 60 mM per litre combined with either 15 or 30 mM per litre of CaSo4 or
CaCl2 at Logan (USA). Increasing Na concentration decreased shoot dry
weight, number and weight of pods and number of nodules. The photosynthesis
rate decreased with increasing Na-salt concentrations, the decrease was greater
with NaCl than with Na2S04. They observed that calcium sulphate treatments
ameliorated Na-induced salinity in P. vulgaris more than did comparable CaCl2
treatment.
Grusak et al. (1996) investigated the factors, which might enhance the
concentration of Ca in snap bean {Phaseolus vulgaris L.) by measuring whole-
plant net Ca influx, whole-plant Ca partitioning, and various growth parameters
of two bean cultivars (Hystyle and Labrador) at Haustan, Taxas (USA). Plants
were grown hydroponically under environmental conditions while being
provided adequate quantities of Ca. Calcium-flux analysis also revealed that
daily rates of whole-plant net-Ca influx gradually declined throughout the
period of pod growth in both cultivars, this decline was not related to whole-
plant water influx. These results suggest that enhancements in whole-plant net
Ca-influx during pod growth and/or enhancement in the xylem transport of
absorbed Ca to developing pods could increase the Ca concentration of snap
bean pods.
Garg et al (1997) conducted a pot experiment on cluster bean
(Cyamopsis tetragonoloba Taub cv. HF G-I82) at Jodhpur (Rajasthan), to
study the alleviation of sodium chloride induced inhibition of growth and
nitrogen metabolism of cluster bean by calcium. They observed that the
increasing NaCl concentration (0, 50, 100 and 150 mM) progressively
decreased growth and seed yield of cluster bean, which was associated with
decreased concentration of potassium and calcium and increased concentration
41
of sodium in the shoots. Supplemental calcium (0, 2.5 and 5.0 mM)
significantly ameliorated the adverse effects of NaCl due to enhanced Ca and K
uptake and reduced sodium (Na) uptake. Calcium also alleviated the negative
effects of NaCl on activities of nitrogen metabolism enzymes as well as
contents of soluble protein and free amino acids.
Taufiq and Sudaryono (1997) conducted a field experiment at East Java
(Indonesia), to study the role of potassium, calcium and magnesium application
on productivity of groundnut. They reported that an increase in the application
rate of K, Ca and Mg to 15 % more than soil exchangeable K, Ca and Mg
contents that significantly increased the seed yield and number of filled pods
per plant of groundnuts cv. 'Tuban', but did not reduce the number of empty
pods per plant. Yield of groundnuts was significantly correlated with the
concentration of K in the shoot at the pod filling stage.
Carpena et al. (2000) conducted a glass experiment on Pisum sativum L.
cv. 'Argona' with different B and Ca levels in order to elucidate a specific role
for B and Ca on the N2-fixation at Madrid (Spain). The treatments were applied
as follows: Control (9.3 |iM B and 2 mM Ca), - B (without B and 2 mM Ca), -
B + Ca (without B and 3.6 mM Ca), + Ca (9.3 iM B and 3.6 mM Ca), - Ca (9.3
HM B and 0.4 mM Ca) and - Ca + B (46.5 nM B and 0.4 mM Ca). The supply
of - Ca and + Ca did not affect nitrogenase activity, but weight of old shoots
and total N content increased with the Ca treatment. The cell wall and total B
concentrations in the nodules were found to be 4-fold compared with those of
the roots. By contrast the Ca root wall was 2.5 times higher than the nodules
levels although total pectin was higher in nodule than in the root. Their results
showed that a high supply of Ca induce B mobilization from root to shoot.
Adhikari et al. (2003) conducted a field experiment at Gayesphur (West
Benga), to study the effect of gypsum on growth, yield and quality of
groundnut {Arachis hypogaea L.) varieties. The varieties tested were 'TGS 1',
'TKG 19A', 'TG 22' and TCGS 49' and levels of gypsum were 0, 200 and 400
kg per ha (equivalent 0, 46.6 and 93.2 kg Ca respectively) per ha. They
42
reported that all the varieties performed well at 400 kg gypsum level compared
with the lower levels. The highest pod yield (2.38 ton per ha) was found with
400 kg gypsum per ha.
El-Hamdaoui et al. (2003) studied the effects of different levels of B
(from 9.3, 55.8 and 93 nM B) and Ca (0.68, 1.36, 2.72 and 5.44 mM Ca) on
plant development, nitrogen fixation, and mineral composition of pea {Pisum
sativum L. cv. Argona) grown in symbiosis with Rhizobium leguminosarum bv.
'viciae 3841' under salt stress. The experiment was carried out at Madrid
(Spain). They reported that the extra addition of B and Ca to the nutrient
solution prevented the reduction caused by 75 mM NaCl of plant growth and
the inhibition of nodulation and nitrogen fixation. The number of nodules
recovered by the increase of Ca concentration at any B level, but only nodules
developed at high B and high Ca concentration could fix nitrogen. Addition of
extra B and Ca during plant growth restored nodule organogenesis and
structure, which was absolutely damaged by high salt. Salinity also reduced the
content in plant of other nutrients important for plant development and
particularly for symbiotic nitrogen fixation as K and Fe. They observed that
balanced nutrition of B and Ca (55.8m ^M B, 2.72 mM Ca) was able to
counter-act the deficiency of these nutrients in salt-stressed plant, leading to a
huge increase in salinity tolerance of symbiotic pea plants.
Grewal and Williams (2003) conducted a farm experiment in Australia
to investigate the cultivar variation in alfalfa (lucerne) (Medicago sativa L.)
with respect to soil acidity and lime application. Ten cuUivars (Hunter River,
Hunter field, Seeptre, Aurora, Genesis, Aquarius, Venus, PL 90, PL 55 and
breeding line Y 8804) were tested at two levels of lime (0 and 2 tons per ha).
They reported that lime application significantly increased the root growth,
nodulation, leaf retention, leaf to stem ratio, herbage yield and crude protein
content and element composition of alfalfa shoots. Liming also improved the
Ca concentrations of shoots, while there was a little effect on P and Zn
concentration of alfalfa shoots. They further reported that effect of lime
43
application was greater in 'Y 8804' and 'Aurora' cultivars; however yield
increased by 32 % and 31 % respectively, while yield increase was 11-22 % in
other cultivars.
Murata et al. (2003) studied the effect of solution pH and calcium
concentration on seed germination, seedling survival and growth of groundnut
cv. "Falcon' in sand culture at Pretoria (South Africa). The treatment consisted
of solution pH values ranging from 3.0 to 6.0 and a factorial combination of pH
(3.5, 4.5 and 5.5) and Ca (0, 0.5, 1.0, 1.5 and 2.0 mM). They reported that
increasing the solution Ca concentration diminished the adverse effects of low
pH on germination and seedling survival. It was observed that increasing the
Ca concentration from 0 to 2.0 mM Ca increased root length by 140 % and
total root surface area by 95 % shoot and root dry mass increased with
increasing solution pH and Ca concentration. The adverse of low pH on
seedling growth were alleviated by high solution Ca concentrations.
Naeem et al. (2005) conducted a simple pot experiment at Aligarh (Uttar
Pradesh), with an aim to cultivate the Cassia tora L. {Cassia obtusifolia L.) on
scientific lines, to study the effect of five levels of calcium, viz. 0, 0.2, 0.4, 0.6
and 0.8 g Ca per pot on growth, yield and quality attributes. The source of
calcium was calcium chloride. Sampling was done at 120 days after sowing
(DAS) for growth parameters and plants were harvested at 150 DAS. They
reported that most of the parameters were significantly affected by 0.6 g Ca per
pot. For example, seed-yield and seed-protein content were enhanced by 31.4
% and 10.6 %, respectively by this treatment over their respective controls.
Thus, 0.6 g Ca per pot (or 0.12 g Ca per kg soil) may be recommended for best
yield and quality of this medicinal plant.
Naeem et al. (2005) conducted a simple pot experiment at Aligarh (Uttar
Pradesh), to study the effect of four levels of calcium (0, 15, 30 and 45 kg Ca
per ha) on mung bean (Vigna radiata L. Wilczek). Observations were recorded
on fresh and dry weights per plant, number and dry weight of nodules per plant,
nodule-nitrogen content and total chlorophyll and carotenoids contents. Among
44
the four levels of calcium, 45 kg Ca per ha proved best for all the parameters.
The application of calcium (45 kg Ca per ha) significantly enhanced fresh and
dr>' weights, number and dr}' weight of nodules per plant, nodule-nitrogen
content, total chlorophyll and total carotenoids content over their respective
controls.
2.5 Concluding Remarks
The above review of literature on the effect of phosphorus and calcium
on leguminous plants is exhaustive. It may be noted that there is hardly any
reference on the effect of these two minerals on hyacinth bean, senna sophera
and coffee senna. Therefore, it was justified to study the effect of phosphorus
and calcium on these plants under the local conditions of Aligarh, Uttar
Pradesh, India.
45
Materials and Methods
3.1
3.2
3.3
3.4
3.5
3.6
3.6.1
3.6.2
3.6.3
3.6.4
3.6.5
3.6.6
3.7
3.7.1
CONTENTS
Agro-climalic conditions
Preparation of pots
Soil analysis
Seeds
Rhizobium inoculation
Pot Experiments
Experiment 1
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Experiment 6
Sampling technique
Growth attributes
Page No.
46
47
47
47
48
48
48
49
50
50
51
51
52
52
3.7.2 Biochemical attributes 52
3.7.3 Yield and quality attributes 53
3.8 Total chlorophyll and carotenoids contents 53
3.9 Nitrate reductase activity (NRA) 54
3.9.1 Development of colour 55
3.9.2 Development of standard curve for NRA 55
3.10 Leaf-NPK and Ca contents 56
3.10.1 Digestion of leaf powder 56
3.10.2 Nitrogen 56
3.10.2.1 Standard curve for nitrogen 57
3.10.3 Phosphorus 57
3.10.3.1 Standard curve for phosphorus 57
3.10.4 Potassium 58
3.10.4.1 Standard curve for potassium 59
3.10.5 Calcium 59
3.10.5.1 Standard curve for calcium 59
3.11 Nodule-nitrogen content 59
3.12 Leghemoglobin content 59
3.13 Seed analysis 60
3.13.1 Seed-protein content 60
3.13.2 Standard curve for seed-protein 61
3.14 Total anthraquinone glycosides content 62
3.15 Statistical analyses 62
CHAPTER 3
MATERIALS AND METHODS
To achieve the aims and objectives highlighted in Chapter 1, six-pot
cxperinienls were conducted during the 'Rabi' (winter) seasons of 2002-2004
to investigate the effect of basal doses of phosphorus and calcium application
on three selected medicinally important leguminous plants. Experiments 1 and
2 were conducted on hyacinth bean {Lablab purpureas L.), while Experiments
3 and 4 were conducted on senna sophera {Cassia sophera L.) during 2002-
2003. Experiments 5 and 6 were conducted on coffee senna {Senna occidentalis
L.) during 2003-2004. All the six-pot experiments were conducted in the net
house of the Department of Botany, Aligarh Muslim University, Aligarh. The
details of agro-climatic conditions, soil analyses of the experimental pots and
the techniques and procedures employed in this regard are given below.
3.1 Agro-climatic conditions
Aligarh, is one of the seventy districts of Uttar Pradesh (North India),
with an area of 5,024 sq kms and is situated at 27° 52' N latitude, 78° 51' E
longitude, and 187.45 m altitude above sea level. It has semi-arid and
subtropical climate, with severest hot dry summers and intense cold winters.
The winter extends from the middle of October to the end of March. The mean
temperatures for December and January, the coldest months, are about 15°C
and 13°C, respectively. The extreme minimum recorded for any single day is
2°C and 0.5°C respectively. The summer season extends from April to June and
the average temperature for May is 34.5°C and 45.5°C, respectively. The
monsoon extends from the end of June to the middle of October 26°C to 30°C.
The mean annual rainfall is about 847.3 mm. More than 85 % of the total down
pour is delivered during a shon span of four months from June to September.
The remaining showers are received during winter. Winter rainfall is useful for
'Rabi' (winter) crops. The relative humidity (RH) of the winter season ranges
between 56 % and 77 % with an average of 66.5 %. In the summer, it ranges
between 37 % to 49 % with an average of 43 %. Whereas, in the monsoon
46
season, the R.H. ranges between 63 % and 73 % with an average of 68 %. The
meteorological data were recorded during the investigation period at the
Meteorological Obserx'atory, Department of Physics, Aligarh Muslim
University, Aligarh and are presented in Fig. 5, 6 and 7. Aligarh district has the
same soil composition and appearances as those found generally in the plains
of Uttar Pradesh. Different types of soils, such as sandy, loamy, sandy-loam
and clay-loam are found in the district.
3.2 Preparation of pots
For the experimental pots, the soil was brought from a fanner's field.
After removing weeds and undesirable particles, the soil was mixed unifomily
with the farmyard manure in the ratio of 5:1. Each pot (25 cm diameter x 25
cm height) was filled with 5 kg of this homogenous mixture. These filled pots
were arranged according to the simple randomized block design of the
experiment in the net house. Department of Botany, Aligarh Muslim
University, Aligarh. The soil was maintained at proper moisture to ensure
better germination of the seeds.
3.3 Soil analysis
Before sowing, soil samples were collected randomly from different
pots for the analysis of the soil characteristics. The samples were analyzed in
the Soil-Testing Laboratory, Government Agriculture Farm, Quarsi, Aligarh.
The physico-chemical properties of soil are given in Table 1.
3.4 Seeds
Authentic seeds of hyacinth bean {Lablab purpureas L.) and coffee
senna {Senna occidentalis L.) were received from United States, Department of
Agriculture, Agricultural Research Service, Plant Genetic Resources
Conservation Unit, University of Georgia, Griffin, GA, USA by the courtesy of
Dr. Brad Morris. The seeds of senna sophera {Cassia sophera L.) were
obtained from Regional Research Institute of Unani Medicine, Aligarh.
Healthy seeds of uniform size were selected and their viability was tested.
47
1 r A S 0 N D
MONTHS
Extreme Maximum & Minimum
yilil Mean Daily Maximum & Minimum
^ • ^ Mean Monthly
Yearly Mean Maximum
•• Yearly Mean Temperature
Yearly Mean Minimum
Fig. 5: Monthly temperature variation at Aligarh
J F M A M J J A S O N D
MONTHS
Fig. 6: Average monthly rainfall at Aligarh
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Additionally, they were surface sterilized with 95 % ethyl alcohol for five
minutes and washed thoroughly with distilled water before sowing.
3.5 Rhizobium inoculation
Healthy seeds of hyacinth bear, were surface sterilized with ethyl
alcohol followed by repeated washings with distilled water, and dried in shade
before applying the Rhizobium inoculum. Authentic and viable Rhizobium
culture {Rhizobium sp.), compatible for hyacinth bean, was obtained from
Culture Laboratory, Government Agriculture Farm, Quarsi, Aligarh. According
to the method given by Subba Rao (1972) for the preparation of culture, 200 g
of colourless gum Arabic (coating material) and 50 g of sugar were dissolved
in 500 mL warm water. After cooling, 100 g of Rhizobium culture was mixed
well with the cooling material, getting the inoculum finally in the form of a
muddy solution was obtained. Required amount of seeds were mixed
vigorously with the inoculum until they were evenly coated by the inoculum
mixture. The inoculated seeds were spread in a clean tray and dried in shade
prior to sowing.
3.6 Pot Experiments
Six-pot experiments were conducted on hyacinth bean, senna sophera
and coffee senna to determine the effect of phosphorus and calcium
application. The details of six-pot experiments are given separately below.
3.6.1 Experiment 1
Experiment 1 was conducted during 2002-2003, on hyacinth bean
(Lablab purpureus L.). The aim of this experiment was to investigate the
optimum basal dose of phosphorus on the basis of growth, yield and quality
attributes of hyacinth bean under local conditions. Biochemical attributes (total
chlorophyll content, total carotenoids content, nitrate reductase activity, leaf-
NPK and Ca contents, nodule-nitrogen content and leghemoglobin content)
were also recorded at various stages. The experiment was conducted according
to simple randomized block design using five levels of phosphorus, viz. 0, 25,
50, 75 and 100 mg P per kg soil (Po, Pi, Pi, P3 and P4, respectively). Phosphorus
48
was applied in the form of potassium dihydrogen orthophosphate (KH2PO4) at
the time of sowing (Table 2). Each treatment was repHcated three times.
However, being a leguminous crop, hyacinth bean was not supplied with
nitrogen. The requirement of nitrogen is fulfilled by the crop itself by virtue of
biological nitrogen fixation as Rhizobium was already inoculated. Sufficient
quantity of nitrogen and potassium was already present in the soil (Table 1). A
uniform basal dose of 120 mg Ca per kg soil was given. Healthy seeds of
uniform size were selected and surface sterilized with 95 % ethyl alcohol for
five minutes and then washed thoroughly with distilled water before sowing.
Thereafter, all the seeds were inoculated with Rhizobium as described earlier.
Initially, five seeds were sown in each pot. Later on, one healthy plant was
allowed to grow to maturity in each pot. The plants were kept free from weeds
and irrigated as and when required. In this experiment, three samplings were
done at vegetative stage (60 DAS), flowering stage (90 DAS) and fruiting stage
(120 DAS), respectively. Seeds were sown in the pot on 20'*' September 2002
and the yield attributes were recorded at the time of harvest (150 DAS).
3.6.2 Experiment 2
Experiment 2 was conducted during 2002-2003 on hyacinth bean
{Lablab purpureus L.). The aim of this experiment was to study the effect of
optimum dose of calcium on the basis of growth, yield and quality attiibutes of
hyacinth bean under local conditions. Biochemical attributes (total chlorophyll
content, total carotenoids content, nitrate reductase activity, leaf-NPK and Ca
contents, nodule-nitrogen content and leghemoglobin content) were also
recorded at various stages. The design of the experiment was simple
randomized with five levels of calcium, viz. 0, 40, 80, 120, 160 mg Ca per Kg
soil (Cao, Cai, Ca2, Cas and Ca4, respectively). Calcium was applied in the form
of calcium chloride (CaC^) at the time of sowing (Table 3). A uniform basal
dose of 10 mg P per kg soil was given. Three samplings were done during the
experiment at vegetative stage (60 DAS), flowering stage (90 DAS) and
fruiting stage (120 DAS), during the experiment. The seeds were sown on 20''*
September 2002. Yield attributes were recorded at the time of harvest (150
49
1.
2.
3.
4.
5.
Po
P.
P2
Ps
P4
Table 2. Summary of treatments for Experiments 1, 3 & 5 (Simple randomized block design).
S.No. Basal treatments Phosphorus
(mg P per kg soil)
0
25
50
75
100 NB: Before sowing a uniform basal dose of Ca (120 mg Ca per kg soil)
was applied.
Table 3. Summary of treatments for Experiments 2, 4 «& 6 (Simple randomized block design).
S.No. Basal treatments Calcium
(mg Ca per kg soil)
0
40
80
120
160
NB: Before sowing a uniform basal dose of P (10 mg P per kg soil) was applied.
1.
2.
3.
4.
5.
Cao
Ca,
Ca2
Ca3
Ca4
DAS). The cultural practices employed were the same as described in
Experiment 1.
3.6.3 Experiment 3
Experiment 3 was conducted during 2002-2003 on senna sophera
{Cassia sophera L.). This experiment was planned to determine the optimum
dose of phosphorus on growth, yield and quality attributes of senna sophera
under local conditions. Biochemical attributes (total chlorophyll content, total
carotenoids content, nitrate reductase activity and leaf-NPK and Ca contents)
were also recorded at various stages. Five basal levels of phosphorus, viz. 0,
25, 50, 75 and 100 mg P per kg soil (PQ, PI, P2, P3 and P4, respectively) were
applied in the form of potassium dihydrogen orthophosphate (KH2PO4) at the
time of sowing (Table 2). A uniform basal dose of 120 mg Ca per kg soil was
given. The plants were sampled at vegetative stage (120 DAS), flowering stage
(150 DAS) and fruiting stage (180 DAS), respectively. The seeds were sown on
20^ September 2002. Yield attributes were recorded at the time of harvest (210
DAS). The cultural practices employed were the same as described in
Experiment 1 excluding the Rhizobium application, as the plant does not fix
nitrogen.
3.6.4 Experiment 4
Experiment 4 was carried out during 2002-2003 on senna sophera
{Cassia sophera L.). The aim of this experiment was to establish the optimum
dose of calcium on the basis of growth, yield and quality attributes of senna
sophera under local conditions. Biochemical attributes (total chlorophyll
content, total carotenoids content, nitrate reductase activity and leaf-NPK and
Ca contents) were also recorded at various stages. Five basal levels of calcium,
viz. 0, 40, 80, 120, 160 mg Ca per kg soil (Cao, Ca,, Ca2, Ca3 and Ca4,
respectively) were applied in the form of calcium chloride (CaCl2) at the time
of sowing (Table 3). A uniform basal dose of 10 mg P per kg soil was given.
This experiment was conducted according to simple randomized block design.
Three samplings were carried out at vegetative stage (120 DAS), flowering
stage (150 DAS) and fruiting stage (180 DAS), respectively. The seeds were
50
sown on 20 '' September 2002. Yield attributes were recorded at the time of
harvest (210 DAS). All the other cultural practices were kept the same as
described in Experiment 3.
3.6.5 Experiment 5
Experiment 5 was conducted during 2003-2004 on coffee senna {Senna
occidentalis L.). The objective of this experiment was to establish the optimum
dose of phosphorus in terms of growth, yield and quality attributes of coffee
senna under local conditions. Biochemical attributes (total chlorophyll content,
total carotenoids content, nitrate reductase activity and leaf-NPK and Ca
contents) were also recorded at various stages. The experiment was conducted
according to simple ranuuiiiizcu block design, using five levels of phosphorus,
viz. 0, 25, 50, 75, 100 mg P per kg soil (PQ, PI , P2, P3 and P4, respectively).
Phosphorus was applied to the soil in the form of potassium dihydrogen
orthophosphate (KH2PO4) at the time of sowing (Table 2). A uniform basal
dose of 120 mg Ca per kg soil was applied. In this experiment three samplings
were carried out at vegetative stage (120 DAS), flowering stage (270 DAS) and
fruiting stage (300 DAS), respectively. Sowing date of the crop was 7" October
2003. Yield attributes were recorded at the time of harvest (330 DAS). All
other cultural practices were the same as in Experiment 3.
3.6.6 Experiment 6
This was the last pot experiment that was conducted during 2003-2004
on coffee senna {Senna occidentalis L.). The aim of this experiment was to
establish the optimum dose of calcium in terms of growth, yield and quality
attributes of coffee senna under local conditions. Biochemical attributes (total
chlorophyll content, total carotenoids content, nitrate reductase activity and
leaf-NPK and Ca contents) were also recorded at various stages. The
experiment was conducted according to simple randomized block design. Five
levels of calcium, viz. 0, 40, 80, 120, 160 mg Ca per kg soil (Ca©, Cai, Ca2, Cas
and Ca4, respectively) were applied in the fora^ of calcium chloride (CaCl2) at
the time of sowing (fable 3). A uniform basal dose of 10 mg P per kg soil was
applied. In this experiment samplings were done at vegetative stage (120
51
DAS), flowering stage (270 DAS) and fruiting stage (300 DAS). The crop was
sown on ?"' October 2003. Yield attributes were recorded at the time of harvest
(330 DAS). All other cultural practices were the same as in Experiment 3.
3.7 Sampling technique
Three plants from each treatment were sampled randomly to assess the
growth and biochemical attributes of hyacinth bean, senna sophera and coffee
senna at vegetative, flowering and fruiting stages and yield and quality
attributes were analyzed at the time of harvest. The attributes recorded are
listed below.
3.7.1 Growth attributes
The following growth attributes were recorded at vegetative, flowering
and fruiting stages using standard methods.
1. Fresh weight per plant
2. Dry weight per plant
3. Number of nodules per plant (in hyacinth bean only)
4. Dry weight of nodules per plant (in hyacinth bean only)
3.7.2 Biochemical attributes
The following biochemical attributes were studied at vegetative,
flowering and fruiting stages.
1. Total chlorophyll content
2. Total carotenoids content
3. Nitrate reductase activity (NRA) in fresh leaves
4. Leaf-NPK and Ca contents
5. Nodule-nitrogen content (in hyacinth bean only)
6. Leghemoglobin content in fresh nodules (in hyacinth bean only)
52
3.7,3 Yield and quality attributes
The following yield and quality attributes were analyzed at the time of
harvest.
1. Number of pods per plant
2. Number of seeds per pod
3. 100-seed weight
4. Seed-yield per plant
5. Seed-protein content
6. Total anthraquinone glycosides content (in senna sophera and coffee
senna only)
At all the stages, three plants from each treatment were uprooted taking
care of nodules and washed with running tap water to wipe off all adhering
foreign particles. They were soaked thereafter using blotting sheets. Later, the
fresh weight was recorded. Then the plants were dried in hot air oven at SOX
for 24 hours to recorded dry weight. After counting, the root nodules were kept
overnight in a hot-air oven at 80°C. Thereafter, the dry weight of the nodules
was recorded (Experiments 1& 2). For the yield attributes, six plants from each
treatment were collected at the time of harvest. Pods were threshed and
cleaned. The number of pods (per plant) was noted. The number of seeds per
pod was also recorded. Then the seeds were dried in the natural sunlight to
maintain a constant moisture content for recording the 100-seed weight and
seed-yield.
The biochemical attributes were studied, using the following methods.
3.8 Total chlorophyll and carotenoids contents
The total chlorophyll and carotenoids contents were estimated in fresh
leaves collected from each treatment at 60, 90 and 120 DAS (for hyacinth
bean), at 120, 150 and 180 DAS (for senna sophera) and at 120, 270 and 300
DAS (for coffee Senna).
53
The total chlorophyll and carotenoids contents in leaves were estimated
by the method of Mac Kinney (1941) and MaClachlan and Zalik (1963),
respectively. 100 mg of fresh tissue from interveinal areas of leaves was
ground with the help of mortar and pestle, using 5 mL of 80 % acetone. The
content tlltered with Whatman filter paper No. 1, was collected in a volumetric
flask the final volume 10 mL with 80 % acetone. The optical density (O.D.) of
the solution was read at 645 and 663 nm for chlorophyll estimation and at 480
and 510 nm for carotenoids estimation using a spectrophotometer (Spectronic
20D, Milton and Roy, USA). This equipment was used for all
spectrophotometeric analysis presented in this thesis. The total chlorophyll and
carotenoids contents (mg g"' FW) were calculated using the following formula:
Total chlorophyll content (mg g ' ) = 20.2 (O.D. 645) + 8.02 (O.D. 663) x ^ J^^^^
Carotenoids content (mg g'') = 7.6 (O.D. 480) - 1.49 (O.D. 510) x ^ >< w x lOOQ
Where, O.D = Optical density of the extract at the given wavelengths (viz. 645,
663, 480 and 510 nm.)
V = Final volume of chlorophyll extract in 80 % acetone
W = Fresh weight of leaf tissue (g)
d = Length of light path (1 cm)
3.9 Nitrate reductase activity (NRA)
The activity of nitrate reductase was estimated in fresh leaves collected
from each treatment in hyacinth bean at 60, 90 and 120 DAS, in senna sophera
at 120, 150 and 180 DAS and in coffee senna at 120, 270 and 300 DAS.
54
The enzyme activity was estimated by the intact tissue method of
Jaworski (1971), which is based on the reduction of nitrate to nitrite as per the
following biochemical reaction:
N0{ + NADH + H J % . NO2" + NAD^ + H2O
The nitrite formed was determined spectrophotometrically. 200 mg of
fresh chopped leaves were weighed and transferred to a plastic vial. To each
vial. 2.5 mL of O.IM phosphate buffer (having pH 7.5) and 0.5 mL of 0.2 M
potassium nitrate solution were added, followed by the addition of 2.5 mL of 5
% isopropanol (Appendix). Finally, two drops of chloramphenicol solution
were added to avoid bacterial growth in the medium. Eachc viair was incabat^
for two hours in the dark at 30°C. // f ^^ l-S^ ^ \
3.9.1 Development of colour ^
0.4 mL of the incubated mixture was taken in a test tube, t o it,-0.3 mL^
each of 1 % sulphanilamide and 0.02 % N-(l-7Vaphthyl)ethylenediamine
dihydrochloride (NED-HCL) was added (Appendix). The test tube was kept for
20 minutes at room temperature for colour development. The mixture was
diluted to 5 mL by adding sufficient amount of double distilled water (DDW).
The optical density was read at 540 nm. A blank was also run side by side.
3.9.2 Development of standard curve for NRA
30 mg sodium nitrite (NaN02) was dissolved in 100 mL DDW. Of this
solution, 0.8 mL was taken in a 100 mL flask. The volume was made upto 100
mL using DDW. From this diluted solutions, ten aliquots, measuring 0.2, 0.4,
0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mL were taken in separate test tubes. To
each of the test tubes, 0.3 mL each of 1 % sulphanilamide and 0.02 % NED-
HCL was added. The solution was diluted to 5 mL with DDW and the optical
density was read at 540 nm, using a blank. A standard curve was plotted, using
the selected concentrations of pure sodium nitrite versus O.D. of the solution.
The optical density of the sample was compared with a calibrated curve
and NRA was expressed as n MN02"g''FWh"\
/ /
55
3.10 Leaf-NPK and Ca contents
The leaf samples from all the plants were collected at their growth
stages (60, 90 and 120 DAS in hyacinth bean, at 120, 150 and 180 DAS in
senna sophera and at 120, 270 and 300 DAS in coffee senna) for assessing the
NPK and Ca contents. The leaves were dried in hot-air oven at 80° C for twenty
four hours. Dried leaves were powdered and the powder was meshed through a
72 mesh. The powder was labeled and stored in small polythene bags for the
chemical analysis. The same leaf-powder was used for the estimation of
nitrogen, phosphorus, potassium and calcium contents.
3.10.1 Digestion of leaf powder
100 mg of oven-dried leaf powder was carefully transferred to a
digestion tube and 2 mL of AR Grade concentrated sulphuric acid was added to
it. It was heated on a temperature controlled assembly for about two hours to
allow complete reduction of nitrates present in the plant material. After heating,
the contents of the tube turned black. It was cooled for about 15 minutes at
room temperature and then 0.5 mL 30 % hydrogen peroxide (H2O2) was added
drop by drop. The solution was heated again till the colour of the solution
changed from black to light yellow. Again after cooling for about 30 minutes,
additional 3 to 4 drops of 30 % H2O2 were added, followed by reheating for
another 15 minutes. The addition of H2O2 followed by heating was repeated
until the contents of the tube turned colourless. The peroxide-digested-material
was transferred from the tube to a 100 mL volumetric flask with three washings
of DDW. The volume of the volumetric flask was then made up to the mark
(100 mL) with DDW. This aliquot was used to estimate NPK and Ca contents.
The details of methods used for the analysis of these elements are given below
separately.
3.10.2 Nitrogen
Nitrogen was estimated according to the method of Lindner (1944). A
10 mL aliquot of the digested material was taken in a 50 mL volumetric flask.
To this, 2 mL of 2.5 N sodium hydroxide (Appendix) and I mL of 10 %
sodium silicate (Appendix) solutions were added to neutralize the excess of
56
acid and to prevent turbidity, respectively. The volume was made up to the
mark with DDW. In a 10 mL graduated test tube, 5 mL aliquot of this solution
was taken and 0.5 mL Nessler s reagent (Appendix) was added. The contents of
the test tubes were allowed to stand for 5 minutes for maximum colour
development. The O.D. of the solution was read at 525 nm, using the
spectrophotometer. The reading of each sample was compared with the
standard calibration curve to estimate the per cent nitrogen content on dry
weight basis.
3.10.2.1 Standard curve for nitrogen
50 mg of the ammonium sulphate was dissolved in 1 litre of DDW.
From this, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mL solution was
pipetted in ten test tubes, separately. The solution in each test tube was diluted
to 5 mL with DDW. To it, 0.5 mL Nessler's reagent was added. After 5
minutes, the O.D. was read at 525 nm, using a blank. A standard curve was
plotted using different concentrations of ammonium sulphate solution versus
O.D. of the solution.
3.10.3 Phosphorus
Phosphorus was estimated according to the method of Fiske and Subba
Row (1925). A 5 mL aliquot was taken in a 10 mL graduated test tube. To it, 1
mL molybdic acid (2.5 %) was added careftilly, followed by addition of 0.4 ml
l-amino-2-naphthol-4-sulphonic acid (Appendix). When the colour turned
blue, the volume was made up to 10 ml with the addition of DDW. The
solution was shaken for five minutes and then transferred to a
spectrophotometric tube. The O.D. of the solution was read at 620 nm, using a
blank.
3.10.3.1 Standard curve for phosphorus
351 mg pure potassium dihydrogen orthophosphate (KH2PO4) was
dissolved in sufficient DDW to which 10 mL of 10 N sulphuric acid was added
and the volume was made up to I litre with DDW. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9 and 1.0 mL of this stock solution were taken in ten test tubes.
57
separately. The solution in each tube was diluted to 10 mL with DDW. To it, 1
mL molybdic acid and 0.4 ml l-amino-2-naphthol-4-sulphonic acid were
added. After 5 minutes, the O.D. of the solution was read at 620 nm. A blank
was also run simultaneously. The standard curve was plotted using different
concentrations of potassium dihydrogen orthophosphate versus O.D. The
per cent phosphorus content was detennined on the dry weight basis.
3.10.4 Potassium
Potassium content in leaf was analyzed flame-photometrically (Hald,
1946). In the flame-photometer, the solution (peroxide digested material) is
discharged through an atomizer in the form of a fine mist into a chamber,
where it is drawn in to a flame. Combustion of the elements produces light of a
particular wavelength (X max for K^ 767 nm (violet). The light produced was
conducted through the appropriate filters to impinge upon a photoelectric cell
that activates a galvanometer.
The air was supplied through an air pump and LPG (Liquid Petroleum
Gas) was used for combustion. The chimney of the equipment was removed
and the gas was ignited using an electric lighter. The final pressure of the two
gases was adjusted to 15 pounds per inch. When the flame formed sharp blue
cones, the correct filter was set and DDW was introduced (using a beaker) and
the galvanometer was set to zero. Then the standard solution of the element
was sucked through a capillary tube and the galvanometer was adjusted to the
100 posifion by using the amplifier. Unless the 0 and 100 points are maintained
on successive readings, the gas pressure, air-pressure or both were adjusted to
bring about a stable position. Thereafter, intermediate standards (i.e. diluted
solutions of known concentrations between 0 and 100 per cent) were checked
and a graph was prepared. The relationship between the galvanometer reading
and the concentration do not appear in a straight line. Rather it appears in a
curvilinear fashion. Lastly, the samples were run and exact concentration of the
element was calculated using the graph.
58
3.10.4.1 Standard curve for potassium
1.91 g of potassium chloride was dissolved in 100 inL ot DDW. Of this
solution I mL was diluted to 1 litre. The resulting potassium solution was of 10
ppm. From this stock solution, 10 mL of each of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10
ppm potassium solution was prepared in ten vials separately, using DDW. The
solution of each vial was analyzed separately, using a flame-photometer. A
blank was also run with each set of determination. A standard curve was
prepared, using different dilutions of potassium chloride solution versus the
readings on the scale of the galvanometer. The per cent potassium content was
estimated on dry weight basis.
3,10.5 Calcium
Like potassium content, calcium content was also estimated using
tlame-photometer as stated above. After setting the filter for calcium, 10 mL
digested material was run. A blank was also run side by side.
3.10.5.1 Standard curve for calcium
2.5 g of CaCOs was dissolved in 5 mL of hydrochloric acid in a 1000
mL volumetric flask. After the reaction was over, the final volume was made
up to the mark with DDW. This stock solution contained 1 g of calcium per
litre (1000 ppm Ca). From this stock solution of Ca, 10, 20, 30, 40 and 50 ppm
Ca solution was prepared in five vials separately. The solution of each vial was
analyzed, setting the filter for Ca in position. A blank (DDW) was also run with
each set of determination. The calibration curve was plotted in the same way as
for potassium, and the per cent calcium content in the leaves was measured on
dry weight basis.
3.11 Nodule-nitrogen content
Nodule-nitrogen content (in only hyacinth bean) was estimated
similarly as that in plant leaves according to the method of Lindner (1944).
3.12 Leghenioglobin content
Leghemoglobin (Lb) content was estimated in fresh nodules only in
hyacinth bean {Lablab purpureus L.) at three stages (60, 90, and 120 DAS).
59
Leghemoglobin content was determined by the method as described in
Biochemical Methods (Sadasivam and Manickam, 1996).
The fresh nodules were mixed with 3 volumes of phosphate buffer and
macerated using a homogenizer. Thereafter, the homogenate was filtered
through two layers of cheesecloth. The nodule debris was discarded. The turbid
reddish brown filtrate was centrifuged at 10,000 rpm for 15 minutes, for the
clarification. To 3 mL of the extract an equal volume of alkaline pyridine
reagent was added and mixed. The solution became greenish-yellow due to the
formation of ferric hemochrome. The hemochrome was divided equally
between two test tubes. To one of the test tubes, 50 mg crystals of sodium
dithionite were added to reduce the hemochrome. The solution was stirred
without aeration and read at 556 nm after 5 minutes against a reagent blank. 50
mg crystals of potassium hexacyanoferrate were added to another test tube to
oxidize the hemochrome. The solution's O.D. was read 539 nm.
Lb content was calculated using following formula
Lb concentration (mM) = P.P. 556-O.D. 539 x 2D
23.4
Where O.D. 556 and O.D. 539 represent absorbance (O.D.) values read
at 556 and 539 nm, respectively and D is the initial dilution.
3.13 Seed analysis
The seeds were chemically analyzed for the seed-protein content and
total anthraquinone glycosides content. The seed samples of each treatment
were dried and ground to fine powder and meshed through a 72 mm sieve. The
dried powder was stored in the small polythene bags. It was labeled and kept
for analyses.
3.13.1 Seed-protein content
The protein content of seeds was estimated by the method of Lowry et
al. (1951). 50 mg oven dried seed powder was transferred to a glass centrifuge
tube, adding 1 mL of cold 5 % trichloroacetic acid. The solution was kept for
60
30 minutes at room temperature and shaked thoroughly for the complete
precipitation of the proteins. The material was centrifuged at 4,000 rpm for 10
minutes and the supernanent was discarded. 5 mL of 1 N NaOH was added to
the residue and mixed well. It was left for 30 minutes on water bath at SOX for
the complete precipitation of the proteins. After cooling for 15 minutes, the
mixture was centrifuged at 4,000 rpm for 15 minutes and the supernatant was
collected in 25 mL volumetric flask with three washings of 1 N NaOH. The
volume was made up to the mark with 1 N NaOH and used for the estimation
of proteins. 1 mL of NaOH-extract was transferred to 10 mL test tube, adding 5
mL of the reagent-B (Appendix). The solution was mixed well and allowed to
stand for 10 minutes at room temperature. To it, 0.5 mL of Folin phenol
reagent (Appendix) was added rapidly with immediate mixing. The content
started turning blue. It was left for thirty minutes for maximum colour
development. The absorbance of the solution was read at 660 nm. A blank was
run side by side. Seed- protein content was calculated by comparing the optical
density of each sample with calibration curve plotted by taking known graded
dilutions of standard solution of bovine serum albumin. The per cent seed-
protein content was expressed on dry weight basis.
3.13.2 Standard curve for seed-protein
50 mg bovine serum albumin was dissolved in 50 mL DDW. 10 mL of
this solution was again diluted to 50 mL. Thus, 1 mL of this solution contained
200 |4, g of protein. Of this, 0.2, 0.4, 0.6, 0.8 and 1.0 mL was transferred to 5
test tubes separately. The solution of each test tube was diluted to 1 mL with
DDW. A blank was also run with each set of determination. To each test tube,
including the blank, 5 mL reagent-B (Appendix) was added. The contents were
allowed to stand for 10 minutes. To this, 0.5 mL of Folin phenol reagent was
added, mixed well and inoculated at room temperature in the dark for 30
minutes. The O.D. of the blue colour solution, thus obtained was read at 660
nm.
61
3.14 Total anthraquinone glycosides content
Total anthraquinone glycosides content was estimated
spectrophotometrically according to the method given in Standard of ASEAN
Herbal Medicine (ASEAN Countries, 1993). 1.2 g of the seed powder was
taken and 30 mL of H2O was added. It was mixed, weighed and then refluxed
for 15 minutes. The aqueous extract, thus prepared, was allowed to cool and
weighed. The original weight was adjusted with H2O. The extract was
centrifuged at 4,000 rpm for 10 minutes. The residue was discarded and the
supernatant was collected. To 0.1 mL of 2 M HCl, 20 mL of supernatant was
added. The extraction was done for 3 times with 15 mL chloroform. Two
layers, each of chloroform and water appeared. The chloroform layer was
discarded and aqueous layer was collected. To this, 0.10 g of sodium carbonate
was added and the content was shaken thoroughly for 3 minutes. Thereafter, it
was centrifuged at 4,000 rpm for 10 min. Of the glycoside fraction, 10 mL was
taken. To it, 20 mL of 10.5 % (w/v) FeCl3.6H20 was added (Appendix). The
content was refluxed for 20 min. Then 1 mL of the concentrated HCl was
added to it, refluxing finally for 20 minutes. Aglycone fraction was extracted
with ether forming two layers -aqueous layer and ether layer. Aqueous layer
was discarded and ether layer was washed with 15 mL of H2O for two times.
The volume was adjusted to 100 mL with 25 mL of ether. The ether layer was
evaporated to dryness and the residue was dissolved in 10 mL of 0.5 % (w/v)
magnesium acetate in MeOH (Appendix). Consequently, a pink colour was
developed. The O.D. of the pink solution was read at 515 nm. A blank was also
run side by side with each set of determination. The reading of each sample
was compared with the standard calibration curve of sennoside. The total
per cent anthraquinone glycosides content was expressed on dry weight basis.
3.15 Statistical analyses
The experimental data were statistically analyzed using the analyses of
variance techniques according to Gomez and Gomez (1984). In applying the
T ' test, the error due to replicates was also determined. When 'F ' value was
found to be significant at the 5 % level of probability, the least significant
62
difference (LSD) was calculated. The model of analyses of variance (ANOVA)
for each experiment is given in Table 4. Ihe correlation of leaf-phosphorus and
calcium contents, seed-yield and seed-protein content were also determined
with various attributes (Tables 31-36).
63
Table 4. Model of the 'analysis of variance' (ANOVA) for Experiments 1-6
Experiments 1-6 (Simple randomized block design)
Source of variation DF SS MSS F value
Replications
Treatments
Error
2
4
8
Total 14
Tjq^erimentaC suCts
CONTENTS
Page No. *t5
4.1 EXPERIMENT 1 64
4.1.1 Growth attributes 64
4.1.1.1 Fresh weight per plant 64
4.1.1.2 Dry weight per plant 65
4.1.1.3 Number of nodules per plant 65
4.1.1.4 Dry weight of nodules per plant 65
4.1.2 Biochemical attributes 65
4.1.2.1 Total chlorophyll content 65
4.1.2.2 Total carotenoids content 65
4.1.2.3 Nitrate reductase activity 66
4.1.2.4 Leaf-nitrogen content 66
4.1.2.5 Leaf-phosphorus content 66
4.1.2.6 Leaf-potassium content 66
4.1.2.7 Leaf-calcium content 66
4.1.2.8 Nodule-nitrogen content 67
4.1.2.9 Leghemoglobin content 67
4.1.3 Yield and quality attributes 67
4.1.3.1 Number of pods per plant 67
4.1.3.2 Number of seeds per pod 67
4.1.3.3 100-seed weight per plant 67
4.1.3.4 Seed-yield per plant 67
4.1.3.5 Seed-protein content 68
4.2 EXPERIMENT 2 68
4.2.1 Growth attributes 68
4.2.1.1 Fresh weight per plant 68
4.2.1.2 Diy weight per plant 69
4.2.1.3 Number of nodules per plant 69
4.2.1.4 Dry weight of nodules per plant 69
4.2.2 Biochemical attributes 69
4.2.2.1 Total chlorophyll content 69
4.2.2.2 Total carotenoids content 70
4.2.2.3 Nitrate reductase activity 70
4.2.2.4 Leaf-nitrogen content 70
4.2.2.5 Leaf-phosphorus content 70
4.2.2.6 Leaf-potassium content 70
4.2.2.7 Leaf-calcium content 71
4.2.2.8 Nodule-nitrogen content 71
4.2.2.9 Leghemoglobin content 71
4.3 Yield and quality attributes 71
4.3.1 Number of pods per plant 71
4.3.2 Number of seeds per pod 71
4.3.3 100-seed weight per plant 72
4.3.4 Seed-yield per plant 72
4.3.5 Seed-protein content 72
4.3 EXPERIMENT 3 72
4.3.1 Growth attributes 72
4.3.1.1 Fresh weight per plant 73
4.3.1.2 Dry weight per plant 73
4.4 Biochemical attributes 73
4.4.1 Total chlorophyll content 73
4.4.2 Total carotenoids content 73
4.4.3 Nitrate reductase activity 74
4.4.4 Leaf-nitrogen content 74
4.4.5 Leaf-phosphorus content 74
4.4.6 Leaf-potassium content 74
4.4.7 Leaf-calcium content 74
4.5 Yield and quality attributes 75
4.5.1 Number of pods per plant 75
4.5.2 Number of seeds per pod 75
4.5.3 100-seed weight 75
4.5.4 Seed-yield per plant 75
4.5.5 Seed-protein content 75
4.5.6 Total anthraquinone glycosides content 75
4.4 EXPERIMENT 4 76
4.4.1 Growth attributes 76
4.4.1.1 Fresh weight per plant 76
4.4.1.2 Dry weight per plant 76
4.4.2 Biochemical attributes 77
4.4.2.1 Total chlorophyll content 77
4.4.2.2 Total carotenoids content 77
4.4.2.3 Nitrate reductase activity 77
4.4.2.4 Leaf-nitrogen content 77
4.4.2.5 Leaf-phosphorus content 77
4.4.2.6 Leaf-potassium content 78
4.4.2.7 Leaf-calcium content 78
4.4.3 Yield and quality attributes 78
4.4.3.1 Number of pods per plant 78
4.4.3.2 Number of seeds per pod 78
4.4.3.3 100-seed weight 78
4.4.3.4 Seed-yield per plant 79
4.4.3.5 Seed-protein content 79
4.4.3.6 Total anthraquinone glycosides content 79
4.5 EXPERIMENT 5 79
4.5.1 Growth attributes 80
4.5.1.1 Fresh weight per plant 80
4.5.1.2 Dry weight per plant 80
4.5.2 Biochemical attributes 80
4.5.2.1 Total chlorophyll content 80
4.5.2.2 Total carotenoids content 80
4.5.2.3 Nitrate reductase activity 81
4.5.2.4 Leaf-nitrogen content 81
4.5.2.5 Leaf-phosphorus content 81
4.5.2.6 Leaf-potassium content 81
4.5.2.7 Leaf-calcium content 81
4.5.3 Yield and quality attributes 82
4.5.3.1 Number of pods per plant 82
4.5.3.2 Number ofseeds per pod 82
4.5.3.3 100-seed weight 82
4.5.3.4 Seed-yield per plant 82
4.5.3.5 Seed-protein content 82
4.5.3.6 Total anthraquinone glycosides content 83
4.6 EXPERIMENT 6 83
4.6.1 Growth attributes 83
4.6.1.1 Fresh weight per plant 83
4.6.1.2 Dry weight per plant 83
4.6.2 Biochemical attributes 84
4.6.2.1 Total chlorophyll content 84
4.6.2.2 Total carotenoids content 84
4.6.2.3 Nitrate reductase activity 84
4.6.2.4 Leaf-nitrogen content 84
4.6.2.5 Leaf-phosphorus content 85
4.6.2.6 Leaf-potassium content 85
4.6.2.7 Leaf-calcium content 85
4.6.3 Yield and quality attributes 85
4.6.3.1 Number of pods per plant 85
4.6.3.2 Number of seeds per pod 86
4.6.3.3 100-seed weight 86
4.6.3.4 Seed-yield per plant 86
4.6.3.5 Seed-protein content 86
4.6.3.6 Total anthraquinone glycosides content 86
CHAPTER 4
EXPERIMENTAL RESULTS
1 he important results of six-pot experiments (Tables 5-30) are deseribed brielly in this chapter.
4.1 EXPERIMENT 1
The effect of five levels of phosphorus, viz. 0, 25, 50, 75 and 100 mg P
per kg soil (PQ, PI, P2, P3 and P4 respectively) on the basis of growth, yield and
quality attributes of hyacinth bean {Lablab purpureus L.) was studied in a
simple randomized block design pot experiment. Growth and biochemical
attributes were analyzed at 60, 90 and 120 DAS and yield and quality attributes
were studied at the time of harvest (150 DAS). The effect of phosphorus
application on all the attributes was found significant at all the stages except
leghemoglobin content at 120 DAS and number of seeds per pod and 100-seed
weight at harvest (150 DAS). The dose P3 proved optimum for most of the
attributes studied. However, the effect of P4 was equaled with that of P3
treatment at all stages. For convenience, the details of the data are briefly
described below and summarized in Tables 5-9.
4.L1 Growth attributes
The growth attributes, namely fresh and dry weights per plant, number
of nodules and dry weight of nodules per plant recorded at 60, 90 and 120
DAS, were found to be affected significantly by the phosphorus application.
The salient features of data (Table 5) are given below.
4.L1.1 Fresh weight per plant
The effect of phosphorus was found significant on fresh weight per plant
at 60, 90 and 120 DAS over the Control. Treatment P3 significantly increased
fresh weight per plant by 27.8, 37.1 and 35.1 % at 60, 90 and 120 DAS,
respectively over the Control (Table 5).
64
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4.1.1.2 Dry weight per plant
riic application of phosphorus on dr\ weight per plant was noted to be
significant at all stages. Treatment P3 signitlcantly increased dv\' weight per
plant by 46.5, 55.8 and 52.8 % at 60, 90 and 120 DAS, respectively over the
Control (Table 5). Treatment Pj and P2 proved equally effective at all the three
stages.
4.1.1.3 Number of nodules per plant
Application of phosphorus increased the number of nodules per plant
over the Control at all stages. P3 level of phosphorus enhanced number of
nodules per plant by 42.8, 50.4 and 40.4 % at 60, 90 and 120 DAS, respectively
over the Control (Table 5). The Control showed poorest performance.
4.1.1.4 Dry weight of nodules per plant
All phosphorus levels gave maximum value of dry weight of nodules in
comparison to the Control. P3 significantly increased dry weight of nodules by
43.0, 51.5 and 39.6 % at 60, 90 and 120 DAS, respectively over Po (Table 5).
Treatments Pi and P2 were found at par with each other at all stages.
4.1.2 Biochemical attributes
Effect of basal application of phosphorus on all biochemical attributes
was found significant at 60, 90 and 120 DAS. However, leghemoglobin content
at 120 DAS was not affected by the phosphorus application. The details of the
data (Tables 6-8) are in the following pages.
4.1.2.1 Total chlorophyll content
Among the phosphorus levels, P3 proved optimum and significantly
increased total chlorophyll content by 8.3, 10.0 and 7.9 % at 60, 90 and 120
DAS, respectively (Table 6). The value of Control recorded was minimum at
all stages.
4.1.2.2 Total carotenoids content
The total carotenoids content was also increased by P3 level of
phosphorus over Po by 8.8, 7.0 and 5.4 %, at 60, 90 and 120 DAS, respectively.
However, the effect of P4 was at par with that of P3 treatment at all stages. The
65
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elTect of PQ, PI and P2 were found equal with each other at 120 DAS. The
Control gave lowest value that was equaled by Pj at 60 and 90 DAS (Table 6).
4.1.2.3 Nitrate reductase activity
Phosphorus application affected nitrate reductase activity significantly at
all stages. P3 level of phosphorus gave maximum nitrate reductase activity by
30.2, 28.3 and 24.4 % at 60, 90 and 120 DAS, respectively over the Control
(Table 6). The Control gave lowest value at all stages.
4.1.2.4 Leaf-nitrogen content
Application of phosphorus on leaf-nitrogen content was found effective
over Po at all stages. Treatment P3 gave maximum effect and significantly
increased nitrogen content by 20.6, 18.7 and 16.3 % at 60, 90 and 120 DAS,
respectively over PQ. The Control registered lowest value (Table 7).
4.1.2.5 Leaf-phosphorus content
A progressive increase in leaf-phosphorus content was found with
increasing levels of phosphorus at all stages. With regard to phosphorus
application, it was found that the treatment P3 significantly increased the
phosphorus content by 20.8, 24.0 and 17.1 %, respectively over the Control at
all stages (Table 7). Treatments Pi and P2 gave equal effect with each other at
all stages.
4.1.2.6 Leaf-potassium content
Like leaf-nitrogen and phosphorus contents, the application of
phosphorus gave maximum value of potassium content at all stages. P3
increased potassium content by 18.4, 14.1 and 17.8 % at 60, 90 and 120 DAS,
respectively over the Control. The control gave lowest value (Table 7).
4.1.2.7 Leaf-calcium content
Like leaf-nitrogen, phosphorus and potassium contents, the phosphorus
application on calcium content was found effective at 60, 90 and 120 DAS. P3
significantly increased calcium content by 10.3, 13.2 and 8.5 % at 60, 90 and
120 DAS, respectively over PQ. Treatments Pi and P2 showed equal effect with
each other at all stages (Table 7).
66
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4.1.2.8 Nodule-nitrogen content
Treatment P3 significantly enhanced nodule-nitrogen content and
maximum increase (recorded for P3) was 8.2, 6.4 and 5.3 % at 60, 90 and 120
DAS, respectively over PQ. Control (PQ) gave the minimum value (Table 8).
4.1.2.9 Leghemoglobin content
The etYect of phosphorus on leghemoglobin content in fresh root
nodules was found significant at 60 and 90 DAS, respectively. However, its
effect was found non- significant at 120 DAS. P3 level of phosphorus gave
higher value than the Control by 40.8 and 27.4 % at 60 and 90 DAS,
respectively (Table 8).
4.1.3 Yield and quality attributes
All yield and quality attributes studied at harvest (150 DAS), were found
significant except, number of seeds per pod and 100-seed weight and the data
are described below (Table 9).
4.1.3.1 Number of pods per plant
Application of phosphorus significantly increased number of pods per
plant over the Control. Treatment P3 produced 38.3 % higher pods than PQ.
However, effect of P4 level of phosphorus showed equal effect with that of
treatment P3 (Table 9).
4.1.3.2 Number of seeds per pod
Number of seeds per pod did not significantly affect by phosphorus
treatments and result was found non-significant (Table 9).
4.1.3.3 100-seed weight per plant
The effect of phosphorus was found non-significant on this attribute
(Table 9).
4.1.3.4 Seed-yield per plant
All Phosphorus levels affected seed-yield significantly and there was a
linear increase upto P3 treatment. However, P3 significantly enhanced seed-
67
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) ield by 38.3 % over the Control (Table 9). Treatment P4 gave equal value with
that of P3.
4.1.3.5 Seed-protein content
Application of phosphorus significantly enhanced seed-protein over the
Control and the maximum increase was recorded for P3. It was 14.9 % higher
than that of Control (Table 9).
4.2 EXPERIMENT 2
Experiment 2 was conducted according to simple randomized block
design, to study the effect of five levels of calcium, viz. 0, 40, 80, 120 and 160
mg Ca per kg soil (Cao, Cai, Ca2, Cas and Ca4, respectively) in terms of growth,
yield and quality attributes of hyacinth bean (Lablab purpureas L.). Growth
and biochemical attributes were determined at 60, 90 and 120 DAS. Yield and
quality attributes were studied at the time of harvest (150 DAS). The effect of
calcium application on all the attributes was found significant at all the stages.
However, the effect was found non-significant on number of seeds per pod and
100-seed weight, respectively. The Ca3 treatment proved optimum for most of
the attributes studied. The effect of Ca4 was at par with that of Ca3 at all stages.
The data are described below and summarized in Tables 10-14.
4.2.1 Growth attributes
The effect of calcium application on all growth attributes, namely fresh
and dry weights per plant, number of nodules and dry weight of nodules per
plant was found significant at 60, 90 and 120 DAS. The description of data
(Table 10) is given below.
4.2.1.1 Fresh weight per plant
Treatment Ca3 proved superior to the other levels of calcium and gave
maximum fresh weight at 60, 90 and 120 DAS, respectively. Ca3 gave
maximum fresh weight by 25.3, 31.6 and 29.2 % at 60, 90 and 120 DAS,
respectively over the Control. The Cao showed poorest perfonnance (Table 10).
68
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4.2.1.2 Dry weight per plant
All treatments gave significant value of dry weight than the Control
(Cao). Ca3 enhanced dry weight per plant by 40.6, 54.2 and 35.3 % over the
Control at 60, 90 and 120 DAS, respectively (Table 10).
4.2.1.3 Number of nodules per plant
Treatment Cas gave maximum number of nodules over the Control at all
stages. The optimal treatment (Caa) increased number of nodules per plant by
41.3, 43.8 and 38.3 % at 60, 90 and 120 DAS, respectively over the Control
(Table 10). However, the effect of Ca4 was found equal with that of Ca2 at 120
DAS and with Ca^ at all stages.
4.2.1.4 Dry weight of nodules per plant
Like number of nodules, dry weight of nodules was found significant to
calcium application at all stages over the Control. The maximum dry weight of
nodules was given by Cas and the increase was 27.6, 35.3 and 32.2 %, at 60, 90
and 120 DAS, respectively. Control gave the minimum value (Table 10). At
120 DAS, Ca4 showed equal effect with that of Ca2 treatment and with Ca3 at
all stages.
4.2.2 Biochemical attributes
Biochemical attributes namely, total chlorophyll content, total
carotenoids content, nitrate reductase activity, leaf-NPK and Ca contents,
nodule-nitrogen and leghemoglobin contents were studied at 60, 90 and 120
DAS, were found to be significantly increased by calcium application. The
description of the data (Tables 11-13) is given below.
4.2.2.1 Total chlorophyll content
The total chlorophyll content was found maximum in Caa treated plants
over the Control at all stages. Treatment Ca^ increased total chlorophyll content
by 14.3, 19.4 and 17.6 % at 60, 90 and 120 DAS, respectively than the Control
(Table 11). The Control registered lowest value at all the three stages.
69
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4.2.2.2 Total carotenoids content
Like lolal chlorophyll, total carotenoids content was maximally affected
by calcium treatment at all stages. Cas gave higher value in comparison to
Control. The maximum increase was noted in Ca^, by 9.5, 15.6 and 14.3 % at
60. 90 and 120 DAS, respectively over the Control. The value given by Cai
was found equal with that of Control at 120 DAS (Table 11).
4.2.2.3 Nitrate reductase activity
Treatment Ca^ surpassed the other treatments and proved best. The
maximum nitrate reductase activity was recorded in the treatment Ca3, by 41.1,
36.7 and 36.1 % at 60, 90 and 120 DAS, respectively over Cao. However, the
value given by Ca4 was found equal with that of Ca3 at all stages. The Control
(Cao) showed the poorest performance (Table 11).
4.2.2.4 Leaf-nitrogen content
Calcium treatments showed positive response to leaf-nitrogen content at
all stages over the Control (Table 12). Treatment Cas significantly increased
leaf-nitrogen content by 24.3, 19.2 and 17.3 % at 60, 90 and 120 DAS,
respectively over the Control.
4.2.2.5 Leaf-phosphorus content
Like leaf-nitrogen content, the effect of calcium on leaf-phosphorus
content was noted the same at all stages. The maximum increase in phosphorus
content was given by Cas (17.2, 24.2 and 22.6 %, respectively) at 60, 90 and
120 DAS and proved best. Control gave the lowest value at all stages (Table
12).
4.2.2.6 Leaf-potassium content
Like leaf-nitrogen and phosphorus contents, the maximum leaf-
potassium content was noted in Ca^ at all stages. Ca3 significantly increased
potassium content by 23.5, 20.1 and 19.2 % at 60, 90 and 120 DAS,
respectively over Cao. The Control gave the minimum value at all stages
(Table 12).
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4.2.2.7 Leaf-calcium content
The calcium content was also enhanced by Cas level of calcium by 29.4,
16.8 and 26.7 % at 60, 90 and 120 DAS, respectively than the Control. Cao
gave the minimum value at all stages. However, treatment Ca4 was found at par
with that of Ca. at 90 DAS and Ca3 at all stages (Table 12).
4.2.2.8 Nodule-nitrogen content
A progressive increase in nodule-nitrogen content was observed by the
application of calcium. The maximum value (recorded for Caj) was 6.7, 4.3
and 4.8 % at 60, 90 and 120 DAS, respectively over the Control (Table 13).
4.2.2.9 Leghemoglobin content
The application of calcium on leghemoglobin content was found
positive at all stages over the Control. The highest leghemoglobin content was
found in Ca3 level. Cas caused an increase of 22.4, 20.4 and 16.9 % at 60, 90
and 120 DAS, respectively over the Control (Table 13). Treatments Cao and
Cai were found equal with each other in their effects at 60, 90 and 120 DAS.
However, Ca4 showed parity with that of Ca2 at 120 DAS and with Ca^ at all
stages.
4.3 Yield and quality attributes
All yield and quality attributes studied at the time of harvest (150 DAS),
were found significant except, number of seeds per pod and 100-seed weight
and the data (Table 14) are briefly described below.
4.3.1 Number of pods per plant
Calcium treatments significantly enhanced number of pods per plant.
Ca3 gave maximum number of pods (28.7 % than Cao) and proved superior.
However, the effect of Ca4 showed parity with that of Cas (Table 14). The
Control gave minimum value.
4.3.2 Number of seeds per pod
Number of seeds per pod did not significantly affected by calcium and
the effect was found non-significant (Table 14).
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4.3.3 100-seed weight per plant
The effect of calcium was also found non-significant on 100-seed
weight (Table 14).
4.3.4 Seed-yield per plant
Seed-yield was significantly affected by calcium levels and a
progressive increase was observed upto Caa (Table 14). The maximum seed-
yield was recorded by Ca^ (30.3 % higher over the Control). The Control gave
minimum seed-yield.
4.3.5 Seed-protein content
Among the all levels of calcium, treatment Ca3 proved best and gave
11.6 % higher seed-protein content over the Control (Table 14). However, the
effect of Ca4 was found equal with that of Cas.
4.3 EXPERIMENTS
The effect of five levels of phosphorus, viz. 0, 25, 50, 75 and 100 mg P
per kg soil (PQ, PI , P2, P3 and P4, respectively) on various attributes of senna
sophera (Cassia sophera L.) was studied in a simple randomized block design
pot experiment. Plants were sampled at 120, 150 and 180 DAS. Yield and
quality attributes were studied at time of harvest (210 DAS). The effect of
phosphorus on all the attributes was found significant at all the stages except
leaf-calcium content at 180 DAS, number of seeds per pod, 100-seed weight
and total anthraquinone glycosides content at harvest. The treatment P3 proved
optimum for most of the attributes studied. However, the effect of P4 was found
equal with that of P3 at all stages. The data (Tables 15-18) are described below.
4.3.1 Growth attributes
The growth attributes, namely fresh and dry weights per plant were
recorded at 120, 150 and 180 DAS. The response of phosphorus application
was found to be significant on these attributes at all stages. A brief description
of the data (Table 15) is given in the below text.
72
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4.3.1.1 Fresh weight per plant
P3 level of phosphorus gave maximum fresh weight (32.1, 38.5 and 30.2
% higher over the Control at 120, 150 and 180 DAS, respectively). The
minimum fresh weight was recorded in PQ and the value was found at par with
that of P, at 120 DAS (Table 15).
4.3.1.2 Dry weight per plant
Out of five levels of phosphorus, P3 treatment proved superior in
comparison to other treatments and significantly gave maximum value of dry
weight at all stages. An increase of 38.9, 49.2 and 46.3 % was observed in P3
level of phosphorus over PQ at 120, 150 and 180 DAS, respectively (Table 15).
4.4 Biochemical attributes
All biochemical attributes were affected significantly at all stages except
leaf-calcium content at 180 DAS. The data (Tables 16-17) are described in the
following pages.
4.4.1 Total chlorophyll content
It is evident from Table 16 that P3 gave significantly maximum total
chlorophyll content, while PQ gave the lowest value. P3 level of phosphorus
gave highest total chlorophyll content (7.0, 7.7 and 4,4 % higher at 120, 150
and 180 DAS, respectively over the Control. The effect of treatment P] was
found equal with that of P2 at 120 DAS.
4.4.2 Total carotenoids content
All phosphorus levels gave positive response to carotenoids content at
all stages. P3 proved optimum and increased the total carotenoids content over
the Control at all stages. The per cent increase in total carotenoids content
resulted from the application of P3 over PQ at 120, 150 and 180 DAS was 6.3,
9.0 and 6.0 %, respectively. The treatments Po, Pi and P2 found equal with each
other in their effects at 120 DAS (Table 16).
73
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4.4.3 Nitrate reductase activity
The maximum nitrate reductase activity was recorded in treatment P3 by
25.3. 21.3 and 20.5 % at 120, 150 and 180 DAS, respectively than in Po (Table
16). The Control (Po) gave the lowest value, but its effect was at par with that
of Pi at 180 DAS. However, the effect of Ca4 was found at par with that of Ca3
at all stages.
4.4.4 Leaf-nitrogen content
The application of phosphorus increased leaf-nitrogen content over the
Control at all stages. P3 level gave the highest nitrogen content compared with
that of Control at 120, 150 and 180 DAS (Table 17). The highest nitrogen
content was recorded by PQ. This treatment increased leaf-N content by 15.4,
19.7 and 14.5 % at 120, 150 and 180 DAS, respectively over the Control.
4.4.5 Leaf-phosphorus content
Maximum phosphorus content was recorded in treatment P3 at all stages.
It was 20.6, 14.0 and 15.1 % higher than the value for Po at 120, 150 and 180
DAS respectively. Treatments Pi and P2 were equaled with each other in their
effects at 120, 150 and 180 DAS (Table 17). The Control gave minimum value.
4.4.6 Leaf-potassium content
All phosphorus levels showed positive response to leaf-potassium
content at all stages. Out of five phosphorus levels, P3 proved optimum and
gave maximum higher value of potassium content. P3 gave 19.4, 14.9 and 19.1
%, respectively value for potassium content than PQ at 120, 150 and 180 DAS
(Table 17). The Control gave the lowest value,
4.4.7 Leaf-calcium content
Like nitrogen, phosphorus and potassium content, the effect of
phosphorus on leaf-calcium content was found significant at 120 and 150 (but
not on 180) DAS (Table 17). P3 significantly increased calcium content by 6.9
and 8.6 % at 120 and 150 DAS, respectively over the respective Control. The
Control registered lowest value at both stages.
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4.5 Yield and quality attributes
All yield and quality attributes studied at the time of harvest (210 DAS)
and significantly affected by the phosphorus application except number of
seeds per pod, 100-seed weight and total anthraquinone glycosides content,
respectively. The data are briefly described below (Table 18).
4.5.1 Number of pods per plant
The effect of P3 treatment on number of pods per plant significantlx'
increased 31.0 % over the Control. However, the effect of P4 was found equal
with that of P3. The Control showed poorest performance (Table 18).
4.5.2 Number of seeds per pod
Table 18 reveals that the effect of phosphorus on number of seeds per
pod was found non-significant.
4.5.3 100-seed weight
lOO-seed weight was not significantly affected by phosphorus levels and
results found were non-significant (Table 18).
4.5.4 Seed-yield per plant
Application of phosphorus significantly increased seed-yield over the
Control. The maximum increase was recorded for P3 that was 30.1 % higher
than the value for the Control. However, P4 showed parity with that of P3. The
Control gave minimum value (Table 18).
4.5.5 Seed-protein content
P3 level of phosphorus gave higher seed-protein content (13.6 %) than
the Control. However, the effect of P4 was found equal with that of P3. The
Control gave lowest value (Table 18).
4.5.6 Total anthraquinone glycosides content
The effect of phosphorus levels on total anthraquinone glycosides
content in seeds was found statistically non-significant (Table 18).
75
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4.4 EXPERIMENT 4
This experiment was planned according to simple randomized block
design, to study the effect of five levels of calcium, viz. 0, 40, 80, 120 and 160
mg Ca per kg soil (Cao, Ca|, CRI. Ca3 and Ca4, respectively) on the basis of
growth, yield and quality attributes of senna sophera {Cassia sophera L.) and
samplings were carried out at 120, 150 and 180 DAS. Yield and quality
attributes were studied at the time of har\'est (210 DAS). The effect of calcium
application on all the attributes was found significant at all the stages except
number of seeds per pod, 100-seed weight and total anthraquinone glycosides
content which were found non-significant. The Ca3 level of calcium proved
optimum for most of the attributes studied. However, the effect of Ca4 was
found equal with that of Cas at all stages. The data are described below and
summarized in Tables 19-22.
4.4.1 Growth attributes
The growth attributes, namely fresh and dry weights per plant recorded
at 120, 150 and 180 DAS, were found to be affected significantly by the
calcium application. The salient features of data (Table 19) are given below.
4.4.1.1 Fresh weight per plant
The fresh weight was maximally affected by the application of calcium
and Ca3 proved superior over the Control at 120, 150 and 180 DAS. Cas
significantly enhanced the fresh weight by 22.3, 29.3 and 23.2 % Cao at 120,
150 and 180 DAS, respectively. The value given by Caj was at par with that of
Cao at 120 DAS (Table 19).
4.4.1.2 Dry weight per plant
The effect of Ca on dry weight per plant was similar to that noted for
fresh weight i.e. Ca gave maximum dry weight by 37.6, 46.8 and 50.1 % at
120, 150 and 180 DAS. respectively over Cao- The Control (Cao) gave
minimum value at 120, 150 and 180 DAS (Table 19).
76
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4.4.2 Biochemical attributes V ^ jj
Application of calcium was found significant ^on all hiochemic^i'
attributes studied at all stages. The description of results is given'b?tDW'(Tables
20-21).
4.4.2.1 Total chlorophyll content
The maximum total chlorophyll content was given by Cas and the
per cent increase was 10.4, 15.2 and 12.6 at 120, 150 and 180 DAS,
respectively than that of Control. The Control gave minimum value at all stages
(Table 20).
4.4.2.2 Total carotenoids content
Like chlorophyll content, the total carotenoids content was also
increased by Ca3 level of calcium. Cas proved optimum and recorded 15.3, 15.8
and 13.4 % higher value at 120, 150 and 180 DAS, respectively than the Cao.
The Control gave lowest value (Table 20).
4.4.2.3 Nitrate reductase activity
Nitrate reductase activity was found significantly affected by calcium
level at all stages. A progressive increase was observed in Cas by 40.6, 38.2
and 30.5 %, at respective stages over the Control. Ca© recorded minimum value
at all stages (Table 20).
4.4.2.4 Leaf-nitrogen content
The effect of calcium levels on nitrogen content was found maximum at
all stages. Cas treatment proved optimum and gave 21.9, 16.9 and 20.6 %
higher value at 120, 150 and 180 DAS, respectively over the Control
(Table 21).
4.4.2.5 Leaf-phosphorus content
Table 21 indicates that Ca3 proved optimum and increased the
phosphorus content by 26.3, 19.6 and 14.3 % at 120, 150 and 180 DAS,
respectively over the Control. However, the value given by Cao, Cai and Ca2
was at pur at 180 DAS (Table 21).
77
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4.4.2.6 Leaf-potassium content
Leaf-potassium content was also affected significantly by the calcium
levels. Cu:, ireaUnenl proved superior to the rest of the treatments and recorded
highest value for potassium content by 16.8, 22.6 and 18.2 % at 120, 150 and
180 DAS, respectively over Cao (Table 21). The Control gave minimum value.
4.4.2.7 Leaf-calcium content
Like leaf-nitrogen, phosphorus and potassium contents, the effect of
calcium was also found the same on calcium content at all stages. Ca3
significantly enhanced calcium content by 24.7, 26.4 and 20.8 % at 120, 150
and 180 DAS, respectively over the Control. Cao showed poorest effect (Table
21).
4.4.3 Yield and quality attributes
The plants supplied with calcium exhibited a considerable response to
the most of the yield and quality attributes. The results are briefly described
below (Table 22).
4.4.3.1 Number of pods per plant
The effect of calcium on number of pods per plant was found significant
over the Control. Ca3 significantly enhanced 23.9 % higher number of pods
over the Cao, which gave the least value (Table 22). However, the effect of Ca4
showed parity with that of Ca3.
4.4.3.2 Number of seeds per pod
Number of seeds was not significantly affected by calcium levels and
result was found non-significant (Table 22).
4.4.3.3 100-seed weight
The effect of calcium application on 100-seed weight was found non
significant (Table 22).
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4.4.3.4 Seed-yield per plant
As expected, Cas gave highest value for seed-yield (23.7 % higher than
Control). However, the effect of Ca4 was equaled with that of Ca.v The Control
gave minimum value (Table 22).
4.4.3.5 Seed-protein content
Seed-protein content was found significantly affected by Ca^. Ca^
proved superior to other treatments and gave maximum seed-protein content by
12.8 % over the Control (Table 22). However, the effect of Ca4 was equaled
with that of Ca^. Seed protein content was found minimum in no-calcium
plants (Control).
4.4.3.6 Total anthraquinone glycosides content
The effect of calcium application on total anthraquinone glycosides
content in seeds was found non-significant (Table 22).
4.5 EXPERIMENT 5
The effect of five levels of phosphorus viz. 0, 25, 50, 75 and 100 mg P
per kg soil (PQ, PI, P2, P3 and P4 respectively) on the basis of growth, yield and
quality attributes of coffee senna {Senna occidentalis L.) was studied in a
simple randomized block design pot experiment. Growth and biochemical
attributes were determined at 120, 270 and 300 DAS and yield and quality
attributes were studied at the time of harvest (330 DAS). The effect of
phosphorus on all the attributes was found significant at all the stages, except
number of seeds per pod, 100-seed weight and total anthraquinone glycosides
content that was not affected by the phosphorus application. The P3 level of
phosphoms proved optimum for most of the attributes studied. However, the
effect of P4 showed parity with that of P3 at all the three stages. The details
of the data are briefly described in the following pages and summarized in
Tables 23-26.
79
4.5.1 Growth attributes
Growth attributes (fresh and dry weights per plant) were found affected
by the application of phosphorus at 120, 270 and 300 DAS. The data are
described below (Table 23).
4.5.1.1 Fresh weight per plant
The effect of phosphorus treatment on fresh weight per plant was found
maximum at 120, 270 and 300 DAS than that of Control. Maximum fresh
weight was recorded in plants receiving P3 treatment and increase was 25.3,
40.5 and 29.7 % at 120, 270 and 300 DAS, respectively over the Control
(Table 23).
4.5.1.2 Dry weight per plant
Like fresh weight, the effect of phosphorus on dry weight was noted the
same at all stages. Out of the five levels, P3 proved optimum and enhanced the
dry weight by 43.5, 52.2 and 46.3 % at 120, 270 and 300 DAS, respectively
than that of Control (Po) that gave minimum value (Table 23).
4.5.2 Biochemical attributes
Effect of phosphorus levels was found significant on all biochemical
attributes studied at 120, 270 and 300 DAS. The results (Tables 24-25) are
briefly described in the below text.
4.5.2.1 Total chlorophyll content
The total chlorophyll content was found maximum in phosphorus
treated plants over the Control at all stages. P3 increased total chlorophyll
content by 6.4, 8.5 and 5.4 % at 120, 270 and 300 DAS, respectively. Control
gave minimum value (Table 24).
4.5.2.2 Total carotenoids content
Like total chlorophyll content, total carotonoids content was also
increased by phosphorus levels. P3 proved superior and gave higher value for
total carotenoids content by 4.8, 8.4 and 4.6 % at 120, 270 and 300 DAS,
respectively than that of Control. However, the value of P4 was equaled by P3
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at all stages. The value given by PQ, PI and P2 was found at par at 120 and 300
DAS. The Control gave the minimum carotenoids content (Table 24).
4.5.2.3 Nitrate reductase activity
The maximum nitrate reductase activity was recorded in treatment P3.
The value was 27.6, 20.8 and 26.1 % higher at 120, 270 and 300 DAS,
respectively over PQ. The value of P4 was at par with that of P3. The Control
(Po) gave the lowest value (Table 24).
4.5.2.4 Leaf-nitrogen content
The maximum leaf-nitrogen content was given by P3 and the increase
was 17.1, 15.3 and 14.8 % at 120, 270 and 300 DAS, respectively over the
respective Control. The Control registered lowest value (Table 25).
4.5.2.5 Leaf-phosphorus content
The effect of basal phosphorus application on leaf-phosphorus content
was noted significant. Obviously, P3 increased the phosphorus content at all
stages over the Control by 28.4, 23.2 and 19.2 % at 120, 270 and 300 DAS,
respectively. The effect of treatments Pi and P2 was found at par at all stages
(Table 25).
4.5.2.6 Leaf-potassium content
Like leaf-nitrogen and phosphorus contents, the effect of P3 on leaf-
potassium content was found significant at all stages. P3 proved optimum and
registered 11.2, 15.1 and 12.3 % higher value over PQ at 120, 270 and 300
DAS, respectively. The Control gave the lowest value at all stages (Table 25).
4.5.2.7 Leaf-calcium content
Table 25 reveals that calcium content was found maximum in
phosphorus treated plants in comparison to no phosphorus. P3 produced leaf-
calcium content by 11.4, 7.2 and 10.1 % higher over PQ at 120, 270 and 300
DAS, respectively (Table 25).
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4.5.3 Yield and quality attributes
Most of the yield and quality attributes were found affected significantly
by the phosphorus application and a brief description of the data (Table 26) is
given below.
4.5.3.1 Number of pods per plant
The effect of phosphorus levels on number of pods per plant was found
to be significant over the Control. P3 proved optimum and gave 41.2 % higher
number of pods over the PQ. However, the effect of P4 level of phosphorus was
found equal with that of P3. The Control gave least value (Table 26).
4.5.3.2 Number of seeds per pod
Effect of phosphorus levels on number of seeds per pod was found non
significant (Table 26).
4.5.3.3 100-seed weight
The effect of phosphorus levels on 100-seed weight was found to be
non-significant (Table 26).
4.5.3.4 Seed-yield per plant
Seed-yield is a cumulative performance of number of pods per plant,
number of seeds per pod and 100-seed weight. Although, the later two
attributes were non-significant, however the number of pods played pivotal role
considerably in increasing seed-yield. Thus, phosphorus significantly increased
seed-yield and a maximum increase was recorded by 35.5 % in P3 over the
Control. However, the yield given by P4 was equaled with that of P3. The
Control (Po) gave lowest value (Table 26).
4.5.3.5 Seed-protein content
Among different levels of phosphorus, treatment P3 gave highest seed-
protein content and Control gave the minimum. P3 increased seed-protein
content by 12.8 % over the Control. However, the effect of P4 showed parity
with that ofPj (Table 26).
82
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4.5.3.6 Total anthraquinone glycosides content
Effect of phosphorus application on total anthraquinone glycosides
content in seeds was found non-significant (Table 26).
4.6 EXPERIMENT 6
The effect of five levels of calcium, viz. 0, 40, 60, 120 and 160 mg Ca
per kg soil (Cao, Cai, Ca2, Caj and Ca4, respectively) on the basis of growth,
yield and quality attributes of coffee senna (Senna occidentalis L.) was studied
in a simple randomized block design pot experiment. The samplings of growth
and biochemical attributes were determined at 120, 270 and 300 DAS. Yield
and quality attributes were studied at the time of harvest (330 DAS). The effect
of calcium levels on all the attributes was found to be significant at all the
stages. The exceptions were found in phosphorus content at 300 DAS, number
of seeds per pod, 100-seed weight and total anthraquinone glycosides content
which were non-significant. Cas proved opdmum for most of the attributes
studied. However, the effect of Ca4 was equaled with that of Cas treatment. The
data are described briefly in the following pages and summarized in Tables 27-
30.
4.6.1 Growth attributes
Calcium treated plants gave significant response to fresh and dry
weights per plant over the Control at 120, 270 and 300 DAS. The data are
described below (Table 27).
4.6.1.1 Fresh weight per plant
Effect of calcium application on fresh weight per plant was found
statistically significant. Cas proved optimum at all the stages, and gave
maximum fresh weight of plant that was 30.2, 33.2, and 25.6 % higher over the
Control at 120, 270 and 300 DAS, respectively. The Control gave minimum
value (Table 27).
4.6.1.2 Dry weight per plant
Like fresh weight, dry weight was also significantly affected by calcium
application at all stages. Ca3 increased dry weight of plants 43.6, 44.7 and 39.7
83
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Dry weight was minimum in the Control plants at all stages.
4.6.2 Biochemical attributes
Effect of basal application of calcium on all biochemical attributes was
found to be significant at 120, 270 and 300 DAS except leaf-phosphorus
content at 300 DAS. The details of the data are given below (Tables 28-29).
4.6.2.1 Total chlorophyll content
Table 28 indicates that calcium application showed the best response to
enhance the total chlorophyll content in comparison to no calcium treated
plants (Control) at all stages. Cas gave maximum total chlorophyll content
(12.5, 17.4 and 13.3 % higher, respectively) at 120, 270 and 300 DAS over no
calcium. However, the effect of Ca4 was found equal with that of Ca3. The
Control gave the minimum value.
4.6.2.2 Total carotenoids content
Like total chlorophyll content, total carotenoids content was maximally
affected by the Ca3 treatment at all stages. Cas proved optimum and gave
maximum value that was 12.3, 15.4 and 10.6 higher % at 120, 270 and 300
DAS, respectively over Cao. However, the effect of Ca4 was at par with that of
Ca3 at all stages (Table 28). Treatments Cai and Ca2 were found equal in their
effects at all stages.
4.6.2.3 Nitrate reductase activity
All levels of calcium produced maximum nitrate reductase activity over
the Control at all stages. However, the response of Ca4 was found equal with
that of Ca3. The maximum nitrate reductase activity was observed in treatment
Ca3. The value was 40.4, 31.7 and 29.7 higher % at 120, 270 and 300 DAS,
respectively. The Control (Cao) registered lowest value at all stages (Table 28).
4.6.2.4 Leaf-nitrogen content
The leaf-nitrogen content was found maximum in treatment Ca3 at all
stages. Ca3 enhanced leaf-nitrogen content by 20.2, 25.2 and 20.8 % at 120,
270 and 300 DAS, respectively (Table 29). However, the value given by Ca4
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OS O SO
O
o so
so O
oo so o O
in
o —I ^ CM
OS
vn
o so Csl
o
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o
so
as 00
O OS m
OS 00
so
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i n 0 0 so o
^ ON (N
r4
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m i n
r«-i <N i n CN
o l > o CO
00 i n
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OS m <N
rr m ^ en
so i n
f—«
m oo csi
i n m r~; r*S
f — 1
so
so * - H
00 rsi
• *
»- r-; r^
o so
o
o m o o
(N i n o c5
CZ!
so CO
O m
O vn
oo OS
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d
OS
d
o oo
00 m so
CS csl
so 0 0 CS)
d
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i n OS
vq en
s u
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o
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m
Q
DO
o
C/3
was found equal with that of Ca3 at all stages The Control gave minimum
value at all stages.
4.6.2.5 Leaf-phosphorus content
The effect of basal application of calcium was found significant on leaf-
phosphorus content at all stages except 300 DAS. Like nitrogen content, a
progressive increase was found from Cao to Cas at 120 and 270 DAS. Cas gave
21.0 and 16.2 % higher leaf-phosphorus content over Cao at 120 and 270 DAS,
respectively. The value given by Control was minimum (Table 29).
4.6.2.6 Leaf-potassium content
Like nitrogen and phosphorus contents, the potassium content was
affected by calcium treatments at all stages. The maximum potassium content
recorded in Ca3 treated plants that were 23.8, 23.4 and 13.6 % at 120, 270 and
300 DAS, respectively over the Control (Table 29). The Control showed
poorest performance.
4.6.2.7 Leaf-calcium content
The response of calcium was found to be significant at all stages over
the Control. As expected, Ca3 significantly enhanced the calcium content by
27.3, 22.3 and 19.4 %, at 120, 270, and 300 DAS, respectively than that of Cao.
The Control (Ca©) gave lowest value (Table 29).
4.6.3 Yield and quality attributes
Application of calcium was found significant on most of the yield and
quality attributes studied at harvest (330 DAS). The results are described below
(Table 30).
4.6.3.1 Number of pods per plant
The influence of calcium on number of pods per plant was found
significant over the Control. Ca^ proved best and registered 27.0 % higher
number of pods as compared with Control. However, the effect of Ca4 was at
par with that of Cas. The Control gave minimum value (Table 30).
85
t
J
Q R K
CO c c u u
o o
1/5 U
• * - »
i
3 cr •o c CO 12 .2 '> c o H
_3
[5 o o V)
"S >
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+- Q <-> Z : ,4) O
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-"
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• ^
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( N ON VO
OO
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« Q 00 -J
c3
c C efl (/5
c o Z II
C/5
4.6.3.2 Number of seeds per pod
Different calcium levels did not significantly affect the number of seeds
per pod and thus, result was found non-significant (Table 30).
4.6.3.3 100-seed weight
The effect of calcium on 100-seed was found non-significant (Table 30).
4.6.3.4 Seed-yield per plant
Table 30 shows that seed-yield was found significant to calcium levels
over the Control. The data also confirm that the seed-yield was solely
contributed by the number of pods per plant as number of seeds per pod and
100-seed weight were non-significant. Cas proved optimum and gave
maximum seed-yield being 27.6 % higher over the Cao- The effect of Ca4 was
found equal with that of Ca3. The Control (Cao) registered lowest value (Table
30).
4.6.3.5 Seed-protein content
Seed-protein content was found maximum in calcium treated plants
over the Control. Ca3 significantly increased seed-protein content by 10.6 %
over the Control. However, the response of treatment Ca4 was found equal with
that of Ca3 (Table 30). The minimum seed-protein content was found in the
Control.
4.6.3.6 Total anthraquinone glycosides content
The effect of calcium levels on total anthraquinone glycosides content
in the seeds was found non-significant (Table 30).
86
(Discussion
CONTENTS
Page No. • t .
5.1 Introduction 87
5.2 Growth attributes 88
5.2.1 Effect of phosphorus application 88
5.2.2 Effect of calcium application 90
5.3 Biochemical attributes 92
5.3.1 Effect of phosphorus application 92
5.3.2 Effect of calcium application 96
5.4 Yield attributes 99
5.4.1 Effect of phosphorus application 99
5.4.2 Effect of calcium application 100
5.5 Quality attributes 101
5.5.1 Effect of phosphorus application 101
5.5.2 Effect of calcium application 101
5.6 Conclusion 102
CHAPTER 5
DISCUSSION
5.1 Introduction
Medicinal herbs are the most important source of therapeutic agents
used in modern as well as traditional systems of medicine and constitute the
important natural wealth of the country. Medicinal herbs are used to combat
illness and support the body's own defense to regain good health. However, the
supply of these medicines lags behind their demand in the market. One of the
best solution to this problem is the cultivation of these plants on scientific lines.
This would augment the yield and quality of these herbs ensuring their steady
supply in the market.
Therefore, it is essential to conduct studies regarding mineral nutrition
and other agricultural requirements of at least those medicinal plants whose
supply is not keeping pace with increasing demand (I.C.A.R, 2002; Farooqi
and Sreeramu, 2003; Khan and Mohammad, 2006). In fact, mineral nutrients
applied basally or through foliar application, enhance the plant growth and
productivity and could be used as tools to ameliorate the quality of the herbal-
medicines.
The genetic make up of a plant is primarily responsible for its growth,
development and yield. The productivity as well as quality of a crop is,
however, also controlled by environmental factors. Of which, the uptake and
utilization of mineral nutrients from the soil, or by the fertilizers applied to the
plants, is of prime importance. Over the last 30 years, additional nutrients
applied as fertilizers have been responsible for 55 per cent increase in the yield
in developing countries (FAO, 1998).
It is common view that the absorption of nutrients continues
throughout the life of plants. However, their nutrient requirements may
increase with the development of sink. When plants grow old, the absorption
process of the nutrients may become slow due to the weakening of the root
87
system or unavailability of nutrients through leaching, fixation and
decomposition of nutrients leading to a lowering of nutrient status of plants,
particularly at crucial growth stages. Under such conditions, the ready
availability of adequate quantities of nutrients via basal application and/or.
supplemental top-dressing/foliar spray is required. Additionally, the efficient
absorption and utilization of the nutrients is also highly desirable to ensure the
full exploitation of the genetic potential of plants. Understandably, this has
proved a highly successful technique for profitable cultivation of a number of
crop plants.
Keeping this in mind, six-pot experiments were conducted to study the
growth, biochemical, yield and quality attributes of three medicinally important
leguminous plants namely, hyacinth bean, senna sophera and coffee senna. The
results, presented in the previous chapter, are discussed in the following pages.
5.2 Growth attributes
Growth is a manifestation of the interplay of the meristematic and
metabolic processes. These processes are controlled by a balanced supply of
mineral nutrients (Moorby and Besford, 1983). Thus, high agricultural yields
depend strongly on the adequate application of mineral nutrients (Russell,
1950; Salisbury and Ross, 1992; Marschner, 2002). The effects of the mineral
nutrient studies, undertaken regarding the present research work, are discussed
below.
5.2.1 Effect of phosphorus application
The effect of phosphorus application on growth attributes (fresh and
dry weights per plant) of hyacinth bean, senna sophera and coffee senna, was
significant at all the growth stages (Tables 5, 15 & 23,).
Tables 5, 15 and 23 indicate that the values of the growth attributes
significantly increased as a result of basal application of phosphorus. Among
the phosphorus treatments, P3 (75 mg P per kg soil) proved opiimum for
growth attributes of hyacinth bean, senna sophera and coffee senna. It is well
established that phosphorus is required for several physiological processes. It is
88
also required for cell division and cell elongation as well as for bud growth
(Hewitt, 1963; Raghothaina, 1999; Marschner, 2002). Thus, on the basis of the
metabolic roles played by phosphorus, such an increase in plant fresh and dry-
weights could be expected as a result of phosphorus application. In fact,
phosphorus plays an important role in photosynthesis, respiration and
regulation of a number of enzymes (Devlin and Witham, 1986; Raghothama,
1999; Marschner, 2002), through the energy rich bonds of ATP and similar
compounds. Additionally, "phosphorus is involved practically in every
synthetic reaction of the cell" (Hewitt, 1963). Beneficial effect of phosphorus
on the growth attributes (fresh and dry weights per plant) of various plants has
also been reported by a number of workers, including Khan (1985), Yahiya
(1993), Yahiya and Samiullah (1995), Rana et al. (1998), Sheoran et al. (1999),
Khan et al. (2000), Reddy and Swamy (2000), Khan and Samiullah (2002),
Samiullah and Khan (2003), Azam (2002), Naeem and Khan (2005) and
Siddiqui (2005).
Table 5 indicates that the effect of application of phosphorus (P3) was
significant on the number and dry weight of nodules. Phosphorus has been
reported to affect nodulation and nodule growth much more than that it affects
root or shoot gro>vth (Sonoboir and Sarawgi, 2000). Actually, legumes require
high amount of phosphorus for their growth, nodule formation and N2-fixation.
In fact, leguminous crops have comparatively a higher inherent potential of
phosphorus utilization because of their higher requirement during nodulation
(Carling et al, 1978) and N2-fixation (Israel, 1987; Sonoboir and Sarawgi,
2000). The observed enhancement in number and dry weight of nodules due to
phosphorus application could be due to an additional fixation of molecular
nitrogen leading to an improvement in nitrogen metabolism in case of hyacinth
bean (Table 5). The improvement in nodulation due to phosphorus application
might expectedly be through its role in the proliferation of roots which provides
a larger surface area for bacterial infection (Al-Niemi et al., 1998). Diener
(1950) reported phosphorus enhanced nodulation in legumes through
favourable effect of phosphorus on N2-fixing bacteria. The beneficial effect of
89
applied phosphorus on nodulation could also be due to phosphorus-mediated
suftlcient production and supply of photosynthates to various plant parts.
Similar positive effects of phosphorus on number and dry weight of nodules
have also been reported by Dadson and Acquaah (1984), Sankar et al. (1984),
Rao and Singh (1985), Israel (1987), Prasad and Singh (1987), Pereira and
Bliss (1987), Raju et al. (1991), Yahiya (1993), Yahiya and Samiullah (1995),
Rana et al. (1998), Arya et al. (1997), Kumar et al. (2000), Khan and
Samiullah (2002), Sharma et al. (2002) and Singh and Chauhan (2004) in case
of various crops.
In the present study, the number and dry weight of nodules increased
upto 90 DAS in hyacinth bean (Table 5) and thereafter the values decreased
sharply. This is due to the fact that initially the competition for photosynthates
was confined to root, nodules and aerial vegetative organs. However, at 90
DAS, flowering and fruit setting provided strong sinks for the utilization of
photosynthates. This created a shortage of photosynthates supply to the
nodules, leading to the nodule degeneration as advised by Samiullah and Khan
(2003).
It is noteworthy to add in this cormection that senna sophera and coffee
senna are among the exceptional members of Leguminosae (Fabaceae) family,
lack of root nodules. This makes them incapable of biological nitrogen-fixation
with the help of rhizobia (Leonard, 1925; Mckee, 1962; Faria etal, 1989).
5.2.2 Effect of calcium application
Application of calcium significantly enhanced growth attributes (fresh
and dry weights per plant) of hyacinth bean, senna sophera and coffee seena at
all the growth stages (Experiments 2, 4 and 6). Cas level of calcium (120 mg
Ca per kg soil) proved optimum and significantly increased fresh and dry
weights per plant over the respective controls at all the stages (Tables 10, 19
&27).
Among the growth attributes, dry weight is considered as the most
important parameter as all physiological and biochemical activities lead to the
90
production of dry matter. Utilization of internal calcium for different
physiological and metabolic processes contributes to the biomass production
(Dieter et al., 1984). The growth of a plant depends on the availability of
calcium in the soils and that controls a wide variety of physiological and
cellular processes (Nayyar, 2003). Calcium facilitates rapid translocation of
photosynthates from the plant parts thus leading to an increase in yield (Mengel
and Kirkby, 1987). Further, calcium regulates many physiological functions
such as, cell elongation, cell division and differentiation, stabilization of cell
wall and plasma membrane, membrane stability and cell integrity,
polymerization of proteins, regulation of enzymes and fruit development
(Burstrom, 1968; Loneragan and Snowball, 1969; Epstein, 1972; Marschner,
1974; Hanson, 1983; Kirkby and Pilbeam, 1984; Hepler and Wayne, 1985;
Kauss, 1987; Mengal and Kirkby, 1987; Marschner, 2002; Ng and McAnish,
2003; Nayyar, 2003; White and Broadley, 2003; Hirschi, 2004). The most
important function of cytoplasmic calcium is related with the calcium-binding
protein (CaM). Calcium is sequestered by CaM (Cheung, 1980; White and
Broadley, 2003) and plays a crucial role in plant growth and development by
regulating cell division and modulating the enzyme activities of the cells
(Rasmussen and Means, 1989). The ameliorating effect of calcium on fresh and
dry weights of these medicinal plants used in Experiments 2, 4 and 6,
respectively, corroborates the findings of Savitliramma (2002, 2004), Naeem et
al. (2005), Pintro and Taylor (2005) and Khan and Naeem (2006) in case of
various plants.
The effect of calcium on number and dry weight of nodules per plant
was significant (Experiment 2) in hyacinth bean at 60, 90 and 120 DAS over
the Control. Among the calcium treatments, Cas proved best and exhibited a
considerable increase in the number and dry weight of nodules at all the growth
stages. Both number and dry weight of nodules were maximum at 90 DAS and
minimum at 120 DAS as observed in Experiment 1 (Table 10).
91
The favourable response of plants to applied calcium regarding
number and dry weight of nodules observed in the present study was most
probably due to sufficient production and supply of photosynthates from the
source to sink sites. Generally, legumes require a higher amount of calcium,
especially when they depend upon symbiotic nitrogen fixation alone. Soil-Ca
may increase nodulation on the basis of the current understanding of the role of
calcium in nodulation process (Purcino and Lynd, 1986; Brauer, 2002). It is
reported that a higher amount of calcium is needed for the formation of nodules
than for nitrogen fixation and plant growth in case of legumes (Loneragan and
Dowling, 1958; Andrew, 1976; Lie, 1974; Lov^her and Loneragan, 1968;
Munns, 1978). It is well known that calcium increases the number of nodules
required at the early stages of infection events (Lowther and Lonergan, 1968;
Munns, 1970, 1978). Favourable effect of calcium application on number and
dry weight of nodules has also been reported by Munns (1970, 1978), Bell et
al. (1989b), Pijnenborg and Lei (1990), Brauer et al. (2002), El-Hamdaoei et
al. (2003), Grewal and Williams (2003), Naeem et al. (2005) and Khan and
Naeem (2006).
5.3 Biochemical attributes
5.3.1 Effect of phosphorus application
Phosphorus levels increased total chlorophyll and carotenoids contents
significantly at all growth stages in case of hyacinth bean, senna sophera and
coffee seena (Tables 6, 16 & 24). Phosphorus is an integral component of
important compounds of plant cells, including the sugar-phosphate
intermediates of respiration and photosynthesis, and the phospholipids that
constitute the plant membranes. It is also an important component of ATP
(used in energy metabolism), DNA and RNA (Taiz and Zeiger, 2002). It
promotes regulation of ribulose-l,5-bisphosphate (Rao and Terry, 1989;
Fredeen et al., 1990), biosynthesis of ribulose-l,5-bisphosphate carboxylase
and adenosine triphosphate (Dietz and Foyer, 1986) and assimilation of carbon
dioxide (Longstreth and Nobel, 1980). Thus, the phosphorus is directly or
92
indirectly helpful in enhancing the photosynthetic process of plants. A
significant increase in chlorophyll content due to phosphorus application has
also been observed by Thapar et al. (1990), Lopez-Cantarero et al. (1994) and
Shubhra et al. (2003, 2004). Similarly, Naeem and Khan (2005) have reported
a positive effect on total chlorophyll and carotenoids contents in case of Cassia
tora. The above facts get support by the significant and positive correlations
between total chlorophyll and carotenoids contents on the one hand and leaf-P
and Ca contents on the other at all the growth stages of these plants (Tables 31,
33 & 35) suggest that the biosynthesis of the pigment molecules was dependent
on the uptake of P and Ca.
Total chlorophyll and carotenoids contents were maximum at 90
DAS, 150 DAS and 270 DAS in case of hyacinth bean, senna sophera and
coffee serma, respectively. The chlorophyll content declined invariably at later
growth stages (Tables 6, 16 & 24). Reduction in total chlorophyll and
carotenoids contents might be due to the accelerated leaf-senescence as a result
of ageing effect.
The effect of phosphorus application on nitrate reductase activity was
significant in case of hyacinth bean, serma sophera and coffee senna over the
Control at all growth stages (Tables 6, 16 & 24). Nitrate reductase activity in
plants is influenced by different growth conditions including not only the
environmental factors such as light and temperature (Lillo, 1994; Campbell,
1999), but also the application of phosphorus fertilizers (Oaks, 1985). Leidi and
Rodriguez-Navarro (2000) observed that when high P was supplied to the
plants, an increase in the concentration of nitrogen led to an increased capacity
of the plant for nitrate assimilation. The presence of phosphorus in the nutrient
solution has been reported to induce higher assimilation of NO3 in case of com
(De Magalhaes et al., 1998) and bean (Gniazdowaska et al., 1999). Huber and
Huber (1995) reported two important effects of phosphorus application on NR
activity in spinach-darkened leaves: (i) phosphate facilitates NR
dephosphotylation, (ii) phosphate increases NR activity in a
93
dephosphorylation-independent and time dependent process. The enhancement
in NR activity as a result of phosphorus application has also been confirmed by
ihe findings of Leidi and Rodriguez-Navarro (2000), Camacho-Cristobal et al.
(2002), Villora et al. (2002) and Naeem and Khan (2005) in case of various
leguminous and non-leguminous crops. .
Nitrate reductase activity was maximum at 60 DAS in hyacinth bean.
It was highest at 120 DAS in senna sophera and coffee serma as shown in
Tables 6, 16 & 24. Further, it decreased with increasing age of the plants,
comparatively more slowly from vegetative to flowering stage and more
rapidly from flowering to fruiting stage in case of all the plants employed for
study (Tables 6, 16&24).
Experiments 1, 3 and 5 clearly showed that application of phosphorus
significantly increased the leaf-NPK and Ca contents almost invariably (Tables
7, 17 & 25). The observed beneficial effect of phosphorus application on leaf-
NPK and Ca contents could seemingly be ascribed to the increase in over all
growth of the plant (Tables 5, 6, 15, 16, 23 & 24). The increased uptake of
other nutrients with increasing levels of phosphorus could presumably be
attributed to the beneficial effect of phosphorus on over all growth of the plant
owing to the balanced plant nutrition that accordingly resulted into a better crop
yield. Moreover, maximum enhancement in leaf-NPK and Ca contents was
observed with basally applied phosphorus that might have resulted in
absorption of other essential nutrients from the soil.
Enhancement in leaf-NPK and Ca contents by phosphorus application
has also been reported by several workers (Andrew and Robins, 1969; Chahal
et al., 1983; Akhtar, 1985; Khan, 1985; Akhtar et al., 1987; Singh and Ram,
1992; Parmar et al., 1999; Alloush et al, 2000; Araujo et al, 2000; Khan et al,
2000; Azam, 2002; Khiriya and Singh, 2003; Shrivastava et al., 2003; Singh
and Verma, 2002; Siddiqui, 2005; Naeem and Khan, 2005). Correlation of leaf-
P and Ca content (Tables 31, 33 & 35) with various growth attributes was
positively significant at all growth stages of these plants. Considering NPK and
94
Ca contents of leaf at 90 and 120 DAS in hyacinth bean, at 120 and 180 DAS
in senna sophera and at 270 and 300 DAS in coffee senna, a decrease in content
of all these nutrients was noted, with the increase in the age of the plant (Tables
7, 12, 17, 21, 25 & 29). Such a decrease in NPK and Ca contents of leaf may
be due to continuous utilization of these nutrients by the developing pods (sink)
and their translocation from vegetative parts (source). Rapid decline in N
concentration could be due to exponential increase in plant growth (weight and
volume). As a result of such dilution effect, any increase in nutrient
concentrations is nullified and even higher quantities of nutrients appear to be
less when expressed on per unit basis (Moorby and Besford, 1983). Such a
decline in leaf-P concentration has been reported by Gomide et al. (1969).
They argued that the decline in nutrient content was due to dilution factor
because of increased growth and/or redistribution of these nutrients to younger
plant parts. Similarly, rapid decline in leaf-K concentration has been reported
by Rhykerd and Overdahl (1972). Declining trend in leaf-Ca content from
vegetative to fruiting stage in hyacinth bean, senna sophere and coffee senna
has been observed in this study and clearly shown in Tables 7, 12, 17, 21, 25 &
29, respectively.
In Experiment 1, the effect of phosphorus application on nodule
nitrogen (at 60, 90 and 120 DAS) and ieghemoglobin contents (at 60 and 90
DAS) was significant (Table 8). It is noteworthy that phosphorus has dramatic
effect on legumes when it is applied to the soils low in phosphorus. In fact,
leguminous crops show excellent response to phosphatic fertilizers because
phosphorus helps the leguminous plants in dinitrogen fixation and efficient
partitioning of photosynthates from vegetative to reproductive parts (Sonoboir
and Sarawgi, 2000; Abo-Shetia and Soheir, 2001). Phosphorus treated plants
are, thus, expected to have more amounts of photoassimilates, enhanced nodule
dry weight and much more growth than that of the growth of roots or shoots. In
fact, it is not surprising that legumes have particularly a high phosphorus
requirement for optimum growth, nodule formation and N2-fixation. The afore
mentioned facts clearly indicate that phosphorus plays an important role in
95
symbiotic biological N2-fixation in legumes. However, very little attention
seems to have been paid on this aspect in hyacinth bean, particularly regarding
nodule-nitrogen and leghemoglobin contents. The present work also gets
support from the positive correlation of leaf-P content with nodule-nitrogen and
leghemoglobin contents (Table 31). Vyas and Desai (1953), De Mooy and
Pesek (1966), Graham and Rosas (1979), Dadson and Acquaah (1984), Sankar
et al (1984), Rao and Singh (1985) and Santhaguru and Hariram (1998) have
noted similar significant effects of phosphorus on nodulation and
leghemaglobin content in case of pea, soybean, bean, soybean, groundnut,
chickpea and lablab bean, respectively.
In the present study, nodule-nitrogen and leghemoglobin contents
declined with the age of the crop. It is logical to state that bacteria in the
nodules depend upon the plants for their energy source. Therefore, prior to
flowering, nodules can compete as a carbohydrate sink. However, once the
plant enters reproductive phase, the seeds act as a stronger sink for
carbohydrates than nodules, consequently the latter show comparatively a
decreased dry weight of nodules and lower nitrogen-fixing capacity.
5.3.2 Effect of calcium application
Total chlorophyll and carotenoids contents were maximally exhibited
by these plants in response to calcium application at all the growth stages
(Experiments 2, 4 and 6). Ca3 proved the best compared to the control plants.
Total chlorophyll and carotenoids contents were enhanced in plants treated
with Ca3 at all the growth stages (Tables 11, 20 & 28). These results are in line
with the findings of Savithramma (2004), Naeem et al. (2005) and Khan and
Naeem (2006) in case of various non-leguminous and leguminous crops. As far
as total chlorophyll and carotenoids contents in leaf of these plants are
concerned, the maximum content was found at flowering stage and minimum at
fruiting stage as observed in Experiments 1, 3 & 5 and shown in Tables 11, 20
&28.
96
Calcium is very important for plant metabolism in general and for
photosynthetic process in particular (Ramalho et ai, 1995). Calcium plays a
dual function in photosynthesis. On the one hand; Ca"* ions, bound to
photosystem II (PS II), are involved in the oxygen evolution and. on the other,
they play a structural role in the peripheral antenna assembly (Ramalho, 1995).
It is established that influx of external calcium is required for signalling of
chloroplast mechano-relocation (Sato et al., 2001). Moreover, calcium is
effective in protection of chlorophylls and proteins, as well as the functional
ability of PS-II (Katoh and Han, 1993; Swamy et ai, 1995; Savithramma,
2004).
The effect of calcium application on nitrate reductase (NR) activity of
these plants was found significant at all growth stages (Experiments 2, 4 and
6). NR activity was significantly enhanced as a result of basal supply of
calcium (Caa) compared to the Control.
The reduction of nitrate in the leaves depends upon the presence of
calcium ions and a metabolic connection exists between nitrate assimilation
and Ca content of leaves (Dekock et al., 1979). However, the role of calcium in
nitrogen metabolism depends upon the nitrogen source. In case, the N source is
NH4 , the application of calcium increases NH4' uptake and improves N
utilization in the plant, resulting in increased production of dry matter (Fenn et
al., 1994). The role of Ca as an activator for nitrate reductase activity in leaves
has been reported by Sane et al. (1987), Fenn et al. (1994) and Ruiz et al.
(1999). The above results are further supported by a strong positive correlation
between NR activity and leaf-Ca content in the present study (Tables 32, 34 &
36). In addition, the present study revealed the declining trend of NR activity
from vegetative to fruiting stage as observed in the Experiments 1, 3 & 5.
The effect of calcium on leaf-NPK and Ca contents was found
positively significant at all stages of these plants except at 180 DAS in case of
senna sophera (Tables 12, 21 & 29). The control plants which were not
supplied with nutrients had to depend totally on the nutrients present in low
97
concentration in the soil. On the other hand, calcium suppHed plants got an
adequate supply of it, ensuring continuous absorption by roots followed by
smooth translocation to the foliage. The increased accumulation of NPK and
Ca due to calcium supply may be because of enhanced dry matter
accumulation, effective nodulation, synthesis of higher amounts of chlorophyll
and carotenoids and increased activity of NR (Tables 10, 11 «fe 13 for hyacinth
bean, 19 & 20 for senna sophera and 27 & 28 for coffee senna). The effect of
soil Ca concentration on nutrient absorption rates might presumably play an
important role in nutrient uptake from soil by these legume crops (Andrew and
Johnson, 1976; Bell et ai, 1989 a). In fact, increasing supply of calcium
concentrations has been reported to markedly augment the absorption of NPK
and Ca contents in legumes (Bell et al., 1989a). The beneficial effect of
calcium application on NPK and Ca contents has also been reported by Andrew
and Johnson (1976), Bell et al. (1989 a, b), Ruiz et al (1999), Grewal and
Williams (2003) and Savithramma (2002, 2004) in various plants.
Leaf-NPK and Ca contents decreased with increasing age of the
plants, as observed in Experiments 1, 3 and 5. It is noteworthy that no work has
been reported on this aspect in these plants. Therefore, the present findings
provide some new and valuable information about leaf-NPK and Ca contents in
these medicinally important leguminous plants under different levels of
calcium application.
Experiment 2 indicates the enhancing effect of calcium application in
comparison to control plants, on nodule-nitrogen and leghemoglobin contents
in hyacinth bean (Table 13) was at 60, 90 and 120 DAS. The present study
reveals that calcium treated plants produce maximum nodule-nitrogen and
leghemoglobin contents compared to control plants. Calcium might have
increased soil pH, so as to result in increased nodulation in legumes through
calcium binding protein Rhicadhesin (Smit et al., 1989; Kiramnayee et al.,
1995). The findings of Suttan et al. (1994) indicated that one nodulation protein
{NodO) appears to be Ca binding protein. This suggests that calcium could be a
98
regulator agent in N-fixation in nodules. The results of the present study are
also supported by the findings of Kiranmayee et al. (1995), Carpena et al.
(2000), El-Hamdaoui (2003), Naeem et al. (2005) and Khan and Naeem (2006)
in case of various leguminous crops. Additionally this study reveals that
nodule-nitrogen and leghemoglobin contents decreased slowly from 60 to 90
DAS and sharply from 90 to 120 DAS (Table 13) as observed in the
Experiment 1.
5.4 Yield attributes
Yield is the final manifestation of several complex morphological and
physiological attributes. These attributes are, in turn dependent upon various
factors, including absorption and proper balance of essential nutrients and their
efficient utilization thereafter. The following discussion reveals the effect of
phosphorus and calcium on yield attributes of these plants studied.
5.4.1 Effect of phosphorus application
Yield attributes (number of pods per plant and seed-yield) were
significantly affected by the application of phosphorus. Of the five levels of
phosphorus, P3 proved optimum for the yield contributing attributes (Tables 9,
18 & 26). The maximum yield in phosphorus-supplied plants was found mainly
due to increase in number of pods (Tables 9, 18 & 26).
The enhanced values of yield attributes with phosphorus fertilization is
not surprising as phosphorus plays pivotal role in root development, energy
translocation and other metabolic processes of a plant. As a result of which
there might have occurred increased translocation of photosynthates towards
the sink. Moreover, the significant increase in seed-yield of the plants included
in the study might be due to the cumulative manifestation of the beneficial
effect of phosphorus application on growth, nutrient status, enzyme activity and
yield contributing attributes. The optimum supply of phosphorus in the early
stage of the plant growth is expected to cause rapid cell division and cell
elongation in the meristematic regions, leading to full development of seeds
and increased seed-yield (Spencer and Chan, 1991; Turk et al., 2003).
99
Moreover, the positive correlation of leaf-NPK and Ca contents as
well as of growth and yield attributes with seed-yield depict their contribution
to seed-yield (Tables 31, 33 & 35). The beneficial effect of phosphorus
application on yield attributes of hyacinth bean (Experiment 1), senna sophera
(Experiment 3) and coffee senna (Experiment 5) is in line with the findings of
other workers in this regard, (Verma, 1975; Wasiuddin et ai, 1982; Samiullah
et oL, 1984; Bhati, 1993; Yahiya, 1993; Patel et ai, 1994 a & b, 1995;
Kumawat et ai, 1998; Sinha et ai, 2000; Menna et ai, 2001; Ram and Vemia,
2001; Azam, 2002; Khan and Samiullah, 2002; Sharma et ai, 2002; Samiullah
and Khan, 2003; Naeem and Khan, 2005).
5.4.2 Effect of calcium application
The effect of calcium application was found significant on yield
attributes (number of pods per plant and seed-yield). The higher seed-yield of
calcium treated plants was mainly due to increased number of pods (Tables 14,
22 & 30) of hyacinth bean, serma sophera and coffee senna. The role of
calcium in increasing seed-yield can possibly be ascribed to its involvement in
the processes of photosynthesis and translocation of carbohydrates to young
pods (Sawan et ai, 2001). It clearly indicates that availability of calcium at an
earl)' stage of growth helps in the active growth and metabolism, which
ultimately leads to an increase in the yield. Our results are in accordance with
the findings of others in this regard (Khan, 1995; Devkumar and Giri, 1998;
Geethalakshi and Lourduraj, 1998; Khan et ai, 2001; Rao and Shaktawat,
2001; Sarkar and Malik, 2001; Adhikari et al., 2003; Naeem et al, 2005). The
results of the present study are also supported by correlation studies where
seed-yield was found to be significantly and positively related to most of the
attributes studied at various stages of growth of these plants. Additionally seed-
yield was significantly correlated with fresh and dry weights, total chlorophyll
and carotenoids contents (fables 32, 34 & 36), leaf-NPK and Ca contents, NR
activity and yield attributes (Tables 32, 34 & 36).
100
5.5 Quality attributes
5.5.1 Effect of phosphorus application
The effect of phosphorus appHcation was found beneficial on seed-
protein content in Experiments 1, 3 and 5. Phosphorus level (P3) proved most
effective and gave maximum seed-protein content of hyacinth bean, senna
sophera and coffee senna in comparison to no phosphorus application (Tables
9, 18 & 26). Phosphorus proved effective due to its assured availability and
continuous utilization in carbon skeleton and amino acid synthesis as well as in
the synthesis of energy rich molecules such as ATP. The enhancement in leaf-
NPK content was observed due to phosphorus application. This enhancement
was perhaps responsible for the enhanced synthesis of protein during seed
development. It is obvious that nitrogen is a part of the protein molecules. Its
increase by phosphorus application would have enhanced the protein synthesis.
Furthennore, phosphorus is an integral part of energy-rich molecule i.e. ATP
(Taiz and Zeiger, 2002) and potassium activates several enzymes involved in
protein synthesis (Evans and Sorger, 1966; Tamhane et ai, 1970). Favourable
effect of phosphorus application on seed-protein content of various plants has
earlier been reported by Gupta and Singh (1982), Dadson and Acquaah (1984),
Jain et al. (1990), Patel et al. (1994), Yahiya (1993), Meena et al. (2001), Khan
and Samiullah (2003) and Naeem and Khan (2005). The significantly positive
correlation of seed-protein content with leaf-NPK and Ca contents at various
stages of growth (Tables 31, 33 & 35) further confirms the involvement of
these nutrients in the synthesis of protein.
5.5.2 Effect of calcium application
Like leaf-nutrients and yield attributes, seed- protein content was also
significantly enhanced by the application of calcium (Experiments 2, 4 and 6).
Ca3 proved the best and enhanced seed-protein content over no calcium
application (Tables 14 for hyacinth bean, 22 for senna sophera and 30 for
coffee senna). It is an established fact that calcium binds certain proteins
known as calmodulins (CaM) that are directly stimulated by calcium. These
proteins phosphorylate several enzymes to activate them (Marschner, 2002).
Moreover, calcium increased accumulation of NPK and Ca contents that might
have been responsible for the enhanced synthesis of proteins during seed
development. A favourable effect of application of calcium on seed-protein
content has also been reported by Rao and Shaktawat (2001), Savithramma
(2004) and Naeem et al. (2005). The strong positive correlation of leaf-NPK
and Ca contents with seed-protein content at various growth stages found in the
present study further supports this view (Tables 32, 34 & 36).
5.6 Conclusion
From the aforesaid discussion it may be concluded that basal application
of phosphorus (75 mg P per kg soil) and calcium (120 mg Ca per kg soil)
proved the best for most of the growth, biochemical, yield and quality attributes
of hyacinth bean, serma sophera and coffee senna. It may form the basis for
undertaking field trials and select economic dose to augment the productivity
and quality of these plants.
The meticulous information gathered by the author and presented in this
thesis is hereby claimed to be an original contribution in the field of cultivation
of medicinal plants in the following respects:
1. The optimum basal doses of phosphorus and calcium for the hyacinth
bean, senna sophera and coffee senna under the agro-climatic conditions
of Aligarh (Western Uttar Pradesh) have been determined with
precision in pot culture.
2. Important biochemical and quality attributes, like nodule-nitrogen
content, leghemoglobin content, nitrate reductase activity,
anthraquinone glycosides content, have been studied for the first time in
these plants.
3. The correlation studies of various physiological and biochemical
attributes with leaf-P and -Ca contents, seed-yield and seed-protein
were also undertaken. From these studies, one point has emerged that
the seed-yield and seed-protein content were significantly and positively
correlated with (i) fresh and dry weights per plant (ii) total chlorophyll
and carotenoids contents (iii) leaf-NPK and Ca contents and (iv) nitrate
102
reductase activity at various growth stages. One interesting conclusion
is also worthy of mention here, i.e. one could predict the seed-yield and
quality of these plants at even vegetative stage by analyzing the plants
leaf-NPK or determining its growth attributes. In case of low values of
above parameters, immediate corrective measures such as top-dressing
or foliar application of NPK and Ca nutrients, could be adopted that
would increase the nutrient levels in plants as a consequence and since
there is positive correlation between the nutrient content and
yield/quality, the desired productivity and quality may be achieved.
4. Plausible explanations, based mainly on physiological consideration,
have been given at appropriate places in the above discussion regarding
the entire data on these plants.
5. The findings of the present work also answers the hypothesis in
affirmation that low phosphorus and calcium was the main reason of
low productivity and quality of these plants in this region. Thus the
same could be ameliorated by the application of P and Ca nutrients.
The present author wishes to claim, with all the modesty at his
command, that he has been able to enrich the scientific literature on the three
selected medicinally important leguminous plants by contributing the above
new findings.
103
Table 31. Correlation coefficient values (r) of various attributes with leaf-phosphorus and calcium contents (60 DAS), seed-yield and seed-protein content (150 DAS) of hyacinth bean (Experiment I).
Attributes
Fresh weight per plant
Dry weight per plant
Number of nodules per plant
Dry weight of nodules per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf-nitrogen content
Leaf-phosphorus content
Leaf-potassium content
Leaf-calcium content
Nodule-nitrogen content
Leghemoglobin content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield per plant Seed-protein content
DAS
60 90 120 .60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 150 150 150 150 150
Leaf-phosphorus
content 0.997** 0.983** 0.993** 0.998** 0.957* 0.999** 0.997** 0.956* 0.994** 0.998** 0.984** 0.995** 0.997** 0.957* 0.993** 0.992** 0.953* 0.957* 0.999** 0.949* 0.993** 0.970** 0.997** 0.996**
-0.984** 0.997** 0.978** 0.961** 0.992** 0.985** 0.999** 0.996** 0.988** 0.989** 0.986** 0.988** 0.979** 0.868^^^ 0.994** 0.672^^ 0.900* 0.998** 0.981**
Leaf-calcium content 0.970** 0.999** 0.997** 0.971** 0.991** 0.997** 0.977** 0.991** 0.983** 0.978** 0.998** 0.996** 0.978** 0.991** 0.985** 0.995** 0.970** 0.955* 0.984** 0.986** 0.981** 0.987** 0.989** 0.994** 0.985** 0.999** 0.971** 0.998** 0.992** 0.982**
-0.987** 0.972** 0.989** 0.984** 0.988** 0.953* 0.975** 0.827^^* 0.997** 0.657^^^ 0.916* 0.992** 0.944*
Seed-yield
0.993** 0.997** 0.984** 0.991** 0.985** 0.993** 0.996** 0.984** 0.985** 0.993** 0.998** 0.978** 0.996** 0.985** 0.997** 0.993** 0.964** 0.981** 0.997** 0.980** 0.984** 0.985** 0.991** 0.997** 0.998** 0.992** 0.991** 0.985** 0.987** 0.995** 0.992** 0.998** 0.988** 0.996** 0.991** 0.964** 0.980** 0.983** 0.864*^^ 0.999** 0.696^^ 0.924*
-0.974**
Seed-protein content 0.984** 0.978** 0.967** 0.982** 0.981** 0.978** 0.979** 0.979** 0.980** 0.974** 0.976** 0.971** 0.975** 0.983** 0.975** 0.965** 0.900* 0.914* 0.975** 0.978** 0.978** 0.935* 0.947* 0.972** 0.981** 0.947* 0.982** 0.933* 0.978** 0.974** 0.944* 0.974** 0.983** 0.960** 0.949* 0.951* 0.969** 0.933* 0.858^^^ 0.961** 0.638^^ 0.860^^ 0.974**
-
* Significant at F>0.05 (0.878); DAS = Days after sowing
** Significant at P>0.0\ (0.959); NS = Non-significant
Table 32. Correlation coefficient values (r) of various attributes with leaf-phosphorus and calcium contents (60 DAS), seed-yield and seed-protein content (150 DAS) of hyacinth bean (Experiment 2).
Attributes
Fresh weight per plant
Dry weight per plant
Number of nodules per plant
Dry weight of nodules per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf-nitrogen content
Leaf-phosphorus content
Leaf-potassium content
Leaf-calcium content
Nodule-nitrogen content
Leghemoglobin content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield per plant Seed-protein content
DAS
60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 60 90 120 150 150 150 150 150
Leaf-phosphorus
content 0.994** 0.992** 0.991** 0.981** 0.995** 0.996** 0.996** 0.993** 0.994** 0.997** 0.988** 0.971** 0.988** 0.988** 0.998** 0.993** 0.981** 0.965** 0.984** 0.993** 0.983** 0.950* 0.995** 0.999**
— 0.970** 0.965** 0.962** 0.992** 0.947* 0.967** 0.975** 0.995** 0.981** 0.937* 0.957* 0.962** 0.967** 0.914* 0.947* Q397NS 0.917* 0.962** 0.970**
Leaf-calcium content 0.974** 0.984** 0.989** 0.968** 0.948* 0.984** 0.984** 0.942* 0.971** 0.965** 0.931* 0.909* 0.919* 0.926* 0.970** 0.974** 0.998** 0.996** 0.997** 0.990** 0.959* 0.985** 0.943* 0.976** 0.967** 0.980** 0.947* 0.975** 0.991** 0.977**
-0.895* 0.981** 0.988** 0.953* 0.957* 0.998** 0.982** 0.921* 0.997** 0.298' ^ 0.871^^ 0.998** 0.999**
Seed-yield
0.973** 0.982** 0.983** 0.970** 0.946* 0.981** 0.979**. 0.939* 0.972** 0.957* 0.922* 0.914* 0.911* 0.913* 0.967** 0.971** 0.996** 0.996** 0.994** 0.987** 0.961** 0.991** 0.932* 0.969** 0.962** 0.987** 0.957* 0.981** 0.988** 0.986** 0.998** 0.887* 0.974** 0.989** 0.967** 0.968** 0.999** 0.990** 0.939* 0.997** 0.341^^ 0.886*
-0.999**
Seed-protein content 0.977** 0.985** 0.988** 0.970** 0.954* 0.986** 0.984** 0.946* 0.976** 0.966** 0.936* 0.919* 0.923* 0.928* 0.973** 0.979** 0.999** 0.998** 0.997** 0.992** 0.962** 0.986** 0.945* 0.977** 0.970** 0.984** 0.954* 0.977** 0.992** 0.979** 0.999** 0.899* 0.983** 0.989** 0.961** 0.963** 0.999** 0.988** 0.933* 0.996** 0.326^^ 0.887* 0.999**
-
* Significant at P>0.05 (0.878); DAS = Days after sowing
• * Significant at P>0.01 (0.959); NS = Non-significant
Table 33. Correlation coefficient values (r) of various attributes with leaf phospiiorus and calcium contents (120 DAS), seed-yield and seed-protein content (210 DAS) of senna sophera (Experiment 3).
Attributes
Fresh weight per plant
Dry weight per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf-nitrogen content
Leaf-phosphorus content
Leaf-potassium content
Leaf-calcium content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield per plant Seed-protein content Total Anthraquinone glycosides content
DAS
120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 210 210 210 210 210 210
Leaf-phosphorus
0.980** 0.952* 0.992** 0.961** 0.989** 0.980** 0.997** 0.932* 0.967** 0.933* 0.965** 0.965** 0.993** 0.998** 0.982** 0.987** 0.988** 0.986**
-0.993** 0.969** 0.991** 0.986** 0.999** 0.997** 0.991** 0.731*^^ 0.996** 0.557^^ 0.597^^ 0.991** 0.996** 0.954*
Leaf-calcium
0.985** 0.968** 0.993** 0.956* 0.981** 0.970** 0.998** 0.925* 0.962** 0.952* 0.972** 0.963** 0.995** 0.999** 0.974** 0.993** 0.987** 0.988** 0.997** 0.994** 0.965** 0.992** 0.989** 0.998**
-0.989** 0.674^^ 0.992** 0.516^^^ 0.553^^ 0.991** 0.996** 0.960**
Seed-yield
0.995** 0.968** 0.999** 0.930* 0.990** 0.966** 0.989** 0.967** 0.989** 0.949* 0.991** 0.989** 0.999** 0.990** 0.992** 0.993** 0.999** 0.998** 0.991** 0.982** 0.949* 0.999** 0.998** 0.994** 0.991** 0.987** 0.707^^ 0.994** 0.586^^ 0.588^^
-0.996** 0.980**
Seed-protein content 0.986** 0.958* 0.996** 0.957* 0.993** 0.980** 0.998** 0.945* 0.974** 0.936* 0.978** 0.981** 0.998** 0.997** 0.989** 0.989** 0.993** 0.990** 0.996** 0.994** 0.971** 0.995** 0.991** 0.998** 0.996** 0.996** 0.701'^^ 0.999** 0.533^^ 0.551^^ 0.996**
-0.961**
* Significant at P>0.05 (0.878); DAS = Days after sowing
** Significant at P>0.01 (0.959); NS = Non-significant
Table 34. Correlation coefficient values (r) of various attributes with leaf-phosphorus and calcium contents (120 DAS), seed-yield and seed-protein content (210 DAS) of senna sophera (Experiment 4).
Attributes
Fresh weight per plant
Dry weight per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf-nitrogen content
Leaf-phosphorus content
Leaf-potassium content
Leaf-calcium content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield per plant Seed-protein content Total anthraquinone glycosides content
DAS
120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 120 150 180 210 210 210 210 210 210
Leaf-phosphorus
content 0.991** 0.996** 0.999** 0.995** 0.988** 0.984** 0.994** 0.978** 0.984** 0.996** 0.994** 0.985** 0.975** 0.987** 0.991** 0.926* 0.981** 0.979**
-0.993** 0.958* 0.999** 0.990** 0.989** 0.995** 0.981** 0.959** 0.996** 0.966** 0.581^^ 0.998** 0.995** 0.794^^
Leaf-calcium content 0.985** 0.982** 0.992** 0.992** 0.990** 0.986** 0.987** 0.978** 0.995** 0.994** 0.979** 0.971** 0.976** 0.978** 0.990** 0.932* 0.970** 0.970** 0.995** 0.981** 0.926* 0.998** 0.985** 0.987**
-0.979** 0.967** 0.992** 0.973** 0.604^ ^ 0.992** 0.983** 0.836*^
Seed-yield
0.997** 0.994** 0.999** 0.998** 0.993** 0.991** 0.999** 0.987** 0.979** 0.990** 0.993** 0.992** 0.985** 0.995** 0.996** 0.944* 0.990** 0.989** 0.998** 0.995** 0.957* 0.996** 0.997** 0.995** 0.992** 0.990** 0.971** 0.998** 0.954* 0.552^^
-0.996** 0.796* ^
Seed-protein content 0.987** 0.998** 0.998** 0.988** 0.977** 0.977** 0.995** 0.974** 0.963** 0.989** 0.997** 0.990** 0.970** 0.991** 0.987** 0.923* 0.986** 0.984** 0.995** 0.991** 0.971** 0.991** 0.990** 0.986** 0.983** 0.980** 0.949* 0.989** 0.938* 0.512^^^ 0.996**
-
^nyf^
* Significant at /^0.05 (0.878); DAS = Days after sowing
** Significant at/^O.Ol (0.959); NS = Non-significant
Table 35. Correlation coefficient values (r) of various attributes with leaf-phosphorus and calcium contents (120 DAS), seed-yield and seed-protein content (330 DAS) of coffee senna (Experiment 5).
Attributes
Fresh weight per plant
Dry weight per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf nitrogen content
Leaf phosphorus content
Leaf potassium content
Leaf calcium content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield per plant Seed-protein content Total anthraquinone glycosides content
DAS
120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 330 330 330 330 330 330
Leaf-phosphorus
content 0.999** 0.999** 0.996** 0.960** 0.996** 0.981** 0.978** 0.985** 0.998** 0.990** 0.987** 0.968** 0.993** 0.941* 0.967** 0.996** 0.996** 0.997**
-0.999** 0.999** 0.999** 0.999** 0.998** 0.998** 0.978** 0.987** 0.997** 0.373"^ 0.465''^ 0.987** 0.998** 0.955*
Leaf-calcium content 0.996** 0.996** 0.997** 0.959* 0.998** 0.984** 0.981** 0.980** 0.998** 0.989** 0.984** 0.964** 0.994** 0.947* 0.972** 0.993** 0.993** 0.999** 0.998** 0.996** 0.996** 0.998** 0.999** 0.999**
-0.982** 0.992** 0.992** 0.425^^ 0.518^^^ 0.986** 0.998** 0.964**
Seed-yield
0.987** 0.992** 0.996** 0.992** 0.985** 0.995** 0.996** 0.996** 0.994** 0.983** 0.997** 0.920* 0.998** 0.979** 0.991** 0.968** 0.995** 0.980** 0.987** 0.993** 0.985** 0.982** 0.990** 0.993** 0.986** 0.961** 0.982** 0.976** 0.416* ^ 0.468" ^
-0.995** 0.927*
Seed-protein content 0.996** 0.997** 0.999** 0.975** 0.997** 0.992** 0.990** 0.989** 0.999** 0.988** 0.992** 0.954* 0.999** 0.962** 0.983** 0.987** 0.997** 0.995** 0.998** 0.999** 0.997** 0.995** 0.999** 0.999** 0.998** 0.979** 0.992** 0.990** 0.407 ^ 0.486" ^ 0.995**
-0.954*
i
* Significant at P>0.05 (0.878); DAS = Days after sowing
Significant at P>0.01 (0.959); NS = Non-significant
Table 36. Correlation coefficient values (r) of various attributes with leaf-phosphorus and calcium contents (120 DAS), seed-yield and seed-protein content (330 DAS) of coffee senna (Experiment 6),
Attributes
Fresh weight per plant
Dry weight per plant
Total chlorophyll content
Total carotenoids content
Nitrate reductase activity
Leaf-nitrogen content
Leaf-phosphorus content
Leaf-potassium content
Leaf-calcium content
Number of pods per plant Number of seeds per pod 100-seed weight Seed-yield Seed-protein content Total anthraquinone glycosides content
DAS
120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 120 270 300 330 330 330 330 330 330
Leaf-phosphorus
content 0.917* 0.976** 0.984** 0.990** 0.973** 0.952* 0.964** 0.967** 0.958* 0.976** 0.953* 0.961** 0.964** 0.958* 0.924* 0.981** 0.927* 0.955*
-0.972** 0.819"^ 0.990** 0.950* 0.988** 0.994** 0.960** 0.997** 0.990** 0.894* 0.537 " 0.957* 0.994** 0.970**
Leaf-calcium content 0.936* 0.981** 0.986** 0.999** 0.985** 0.952* 0.965** 0.971** 0.961** 0.986** 0.971** 0.974** 0.960** 0.967** 0.929* 0.963** 0.943* 0.965** 0.994** 0.976** 0.869^^^ 0.976** 0.964** 0.994**
-0.961** 0.989** 0.993** 0.922* 0.514^^ 0.963** 0.999** 0.945*
Seed-yield
0.988** 0.997** 0.992** 0.973** 0.994** 0.998** 0.998** 0.999** 0.998** 0.990** 0.991** 0.973** 0.996** 0.999** 0.994** 0.960** 0.993** 0.997** 0.957* 0.972** 0.915* 0.959** 0.997** 0.983** 0.963** 0.999** 0.971** 0.974** 0.899* 0.526''^
-0.972** 0.880*
Seed-protein content 0.944* 0.987** 0.992** 0.999** 0.990** 0.964** 0.975** 0.979** 0.972** 0.992** 0.978** 0.982** 0.971** 0.974** 0.943* 0.971** 0.951* 0.971** 0.994** 0.985** 0.872^ ^ 0.983** 0.971** 0.998** 0.999** 0.971** 0.993** 0.997** 0.930* 0.543''^ 0.972**
-0.947*
* Significant at P>0.05 (0.878); DAS = Days after sowing
** Significant at P>0.0\ (0.959); NS = Non-significant
Summary
CHAPTER 6
SUMMARY
The present thesis comprises six chapters. In chapter 1 (Introduction),
the importance of the problem (Effect of phosphorus and calcium on selected
medicinally important leguminous plants) has been discussed briefly.
Justification and need for undertaking the present study has been put forward.
Chapter 2 (Review of Literature) exhibits scientific literature pertaining
to the general description of hyacinth bean, senna sophera and coffee senna, a
brief history of inorganic plant nutrition, physiological roles of NPK and Ca in
plants and relevant available references regarding individual effect of
phosphorus and calcium on leguminous plants with special emphasis on
medicinal legumes.
Chapter 3 (Materials and Methods) deals with the details of research
materials used and the techniques employed for the six-pot experiments
conducted during the present study. The relevant information on
meteorological and edaphic data has been given in this chapter.
Chapter 4 (Experimental Results) includes the results of experiments
conducted showing significance of the data at P < 0.05. The more salient points
regarding the results obtained are summarized below.
Experiment 1 (2002-2003) was conducted according to simple
randomized block design, to study the effect of phosphorus application on
hyacinth bean with regard to (i) growth attributes (fresh weight per plant and
dry weight per plant, number of nodules per plant and dry weight of nodules
per plant), (ii) biochemical attributes (total chlorophyll and carotenoids
contents, nitrate reductase activity, leaf-NPK and Ca contents and nodule-
nitrogen and leghemoglobin contents), (iii) yield and quality attributes (number
of pods per plant, number of seeds per pod, 100-seed weight, seed-yield per
plant and seed-protein content). The aim of this experiment was to find out the
104
optimum dose, out of five levels of phosphorus viz. 0, 25, 50, 75 and 100 mg P
per kg soil (PQ, PI, P2, P3 and P4, respectively) so as to get the best performance
of hyacinth bean using the above attributes. All the growth and biochemical
attributes were studied at 60, 90 and 120 days after sowing (DAS). Yield and
quality attributes were studied at the time of harvest (150 DAS).
Most of the attributes studied in this experiment showed significant
response to phosphorus application. P3 (75 mg P per kg soil) proved optimimi,
giving maximum values of all the growth, biochemical, yield and quality
attributes except the leghemoglobin content at 120 DAS, and number of seeds
per pod and 100-seed weight at harvest. P3 level of phosphorus significantly
increased seed-yield and seed-protein content by 38.3 and 14.9 %, respectively
over the Control.
Experiment 2 (2002-2003) was conducted according to simple
randomized block design. The aim of this experiment was to find out the best
dose of calcium among the five calcium levels viz. 0, 40, 80, 120 and 160 mg
Ca per kg soil (Cao, Caj, Cai, Cas and Ca^, respectively) for achieving the
maximal potential of hyacinth bean in terms of the same attributes recorded at
the same stages as in the Experiment 1. The values of all the growth,
biochemical, yield and quality attributes studied in this experiment (except
number of seeds per pod and 100-seed weight) were significantly enhanced as a
result of basal application of calcium Among the basal levels of calcium, Caj
proved the best that increased seed-yield and seed-protein content by 30.3 and
11.6 %, respectively over the Control.
Experiment 3 (2002-2003) was also conducted according to simple
randomized block design. The aim of this experiment was to work out the best
dose of phosphorus to get the optimum response of senna sophera in terms of
growth attributes (fresh weight per plant and dry weight per plant), biochemical
attributes (total chlorophyll and carotenoids contents, nitrate reductase activity
and leaf-NPK and Ca contents) and yield and quality attributes (number of
pods per plant, number of seeds per pod, 100-seed weight, seed-yield per plant,
105
seed-protein content and total anthraquinone glycosides content). Five basal
levels of phosphorus viz. 0, 25, 50, 75 and 1.00 mg P per kg soil (PQ, PI, P2, P3
and P4. respectively) were applied at the time of sowing. All the growth and
biochemical attributes were studied at 120, 150 and 180 DAS. Yield and
quality attributes were analyzed at the time of harvest (210 DAS). Of the five
levels of phosphorus, treatment P3 proved best that enhanced all the growth,
biochemical, yield and quality attributes except number of seeds per pod, 100-
seed weight and total anthraquinone glycosides content which were found non
significant. This treatment significantly increased seed-yield and seed-protein
content by 30.1 and 13.6 %, respectively.
Experiment 4 (2002-2003) was conducted to establish the optimum dose
of calcium for senna sophera on the basis of grovs^, biochemical, yield and
quality attributes. This experiment was planned according to simple
randomized block design. Calcium was applied at 0, 40, 80, 120 and 160 mg
Ca per kg soil (Cao, Cai, Ca2, Cas and Ca4, respectively) at the time of sowing.
The attributes studied and the sampling stages were the same as in the
Experiment 3. C&2 treatment proved the best among the doses of calcium tested
for all the growth, biochemical, yield and quality attributes except number of
seeds per pod, 100-seed weight and total anthraquinone glycosides content,
respectively that were not affected by the calcium application. This treatment
significantly increased seed-yield and seed-protein content by 23.7 and 12.8 %
compared to the Control.
Experiment 5 (2003-2004) was conducted on coffee senna according to
the simple randomized block design, to study the effect of phosphorus levels
viz. 0, 25, 50, 75 and 100 mg P per kg soil (PQ, Pi, P2, P3 and P4, respectively).
The aim of this experiment was to find out the best dose of phosphorus for the
optimum response of coffee senna. The crop performance was determined by
the same attributes as mentioned in case of Experiments 3 and 4. Growth and
biochemical attributes were studied at 120, 270 and 300 DAS. Yield and
quality attributes were determined at harvest (330 DAS). Among phosphorus
106
levels, Pj proved optimum for all the growth, biochemical, yield and quality
attributes studied, except number of seeds per pod, 100-seed weight and total
anthraquinone glycosides content, respectively which were found non
significant. This treatment gave 35.5 and 12.8 % higher value of seed-yield and
seed-protein content, respectively compared to the Control.
Experiment 6 (2003-2004) was conducted on coffee senna according to
simple randomized block design, to investigate the effect of five levels of
calcium viz., 0, 40, 80, 120 and 160 mg Ca per kg soil (Cao, Caj, Ca , Ca3 and
Ca4, respectively) in terms of growth, biochemical, yield and quality attributes.
The aim of this experiment was to establish the best dose of calcium for the
optimum response of coffee senna. In this experiment, all the attributes studied
and sampling stages employed were the same as in Experiment 5. The
application of calcium increased most of the growth, biochemical, yield and
quality attributes studied at all the three stages. Among the calcium levels, Ca^
proved the best and significantly increased all the attributes except number of
seeds per pod, 100-seed weight and total anthraquinone glycosides content,
respectively that were not affected by the calcium application. This treatment
(Cas) increased seed-yield and seed-protein content by 27.6 and 10.6 %,
respectively over the Cao (Control).
In Chapter 5 (Discussion), the important results of the present study
have been discussed in the light of earlier findings published by other workers
in this regard.
The present chapter 6 (Summary) is the resume of the present thesis. It
is followed by upto-date references cited in the text. Lastly, an appendix,
containing the various fonnulations employed for chemical analysis has been
given at the end.
107
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Jippencfv(^
APPENDIX
PREPARATION OF REAGENTS
The reagents for various biochemical determinations were prepared according to the following methods:
1. Reagents for NRA estimation:
(i) Isopropanol (5 %) 5 mL isopropanol was mixed in 95 mL DDW.
(ii) Naphtiiylethylenediamine dihydrochloride (NED-HCL) solution (0.02 %)
20 mg naphthylethylenediamine dihydrochloride was dissolved in sufficient DDW and final volume was maintained upto 100 mL with DDW.
(iii) Phosphate buffer (O.IM) for pH 7 (a) 13.6 g potassium dihydrogen orthophosphate was
dissolved in sufficient DDW and final volume was made upto 1000 mL with DDW.
(b) 17.42 g dipotassium hydrogen orthophosphate was dissolved in sufficient DDW and final volume was made upto 1000 mL with DDW. 160 mL of solution (a) and 840 mL of solution (b) was mixed for getting phosphate buffer pH 7.5.
(iv) Potassium nitrate solution (0.02 M) 2.02 g potassium nitrate was dissolved in sufficient DDW and
final volume was made upto 1000 mL with DDW.
(v) Sulphanilamide solution (1 %) 1 g sulphanilamide was dissolved in 3 N hydrochloric acid
and final volume was made upto 100 mL with 3 N hydrochloric acid.
2. Reagents for leaf-NPK estimation:
(i) Nessler's reagent 3.5 g of potassium iodide was dissolved in 100 mL DDW in
which 4 % mercuric chloride solution was added with stirring until a slight red precipitate remains. Therefore, 120 g of sodium
hydroxide with 250 mL distilled water were mixed. The volume was made upto 1000 mL with distilled water. The mixture was decanted and kept in amber coloured bottle.
(ii) Molybdic acid reagent (2.5 %) 6.25 g of ammonium molybdate was dissolved in 175 niL
distilled water to which 75 mL of 10 N-sulphuric acid was added.
(iii) l-ainio-2-naphthoIe-4-sulphuric acid 0.5 g l-amio-2-naphthole-4-sulphuric acid was dissolved in
195 mL of 15 % sodium bisulphide solution to which 5 mL of 20 % sodium sulphate solution was added. The solution was kept in amber coloured bottle.
(iv) Sodium hydroxide solution (2.5 N) 100 g NaoH dissolved in sufficient DDW and final volume
was maintained up to 1000 mL with DDW.
(iv) Sodium silicate solution (10 %) 10 g sodium silicate dissolved in sufficient DDW and final
volume was maintained up to 100 mL with DDW.
3. Reagents for protein measurement
(a) Reagent A 0.5 % copper sulphate solution and 1 % sodium
sulphate solution were mixed in equal volumes.
(b) Reagent B 50 mL of 2 % sodium carbonate solution was mixed
with 1 mL of reagent A.
(i) Folin phenol reagent 100 g of sodium tungstate and 25 g of sodium molybdate
were dissolved in 700 mL of DDW to which 50 mL of 85 % phosphoric acid and 100 mL of concentrated hydrochloric acid were added. The solution was reflected on a heating mantle for 10 h. At the end, 150 g of lithium sulphate, 50 mL of DDW and 3-4 drops of liquid bromine were added. The reflex for 15 minutes to remove excess bromine cooled and diluted upto 1000 mL. The strength of this acidic solution was adjusted to 1 N by titrating it with 1 N-sodium hydroxide solution.
4. Reagents for total anthraquinone glycosides content estimation
(i) Ferric chloride (10.5 %) 10.5 g ferric chloride dissolved in 100 tnL DDW.
(ii) Magnesium acetate (0.5 %) 0.5 g magnesium acetate dissolved in 100 mL methanol.
in
