Chapter – I
INTRODUCTION AND REVIEW OF LITERATURE
INTRODUCTION
The Western Ghats, extending along the west coast of India, covers
an area of 180,000 square kilometers. The Western Ghats comprises the
major portion of the Western Ghats and Sri Lanka Hotspot, one of 34
global biodiversity hotspots for conservation and one of the two on the
Indian subcontinent. The area is extraordinarily rich in biodiversity.
Although the total area is less than 6 percent of the land area of India, the
Western Ghats contains more than 30 percent of all plant, fish,
herpetofauna, bird, and mammal species found in India. Like other
hotspots, the Western Ghats has a high proportion of endemic species.
The region also has a spectacular assemblage of large mammals and is
home to several nationally significant wildlife sanctuaries, tiger reserves,
and national parks. The Western Ghats contains numerous medicinal
plants and important genetic resources such as the wild relatives of grains
(rice, barley, Eleucine coracana), fruits (mango, garcinias, banana,
jackfruit), and spices (black pepper, cinnamon, cardamom, and nutmeg).
Biodiversity in the Western Ghats is threatened by a variety of
human pressures. Of the approximately 180,000-square-kilometer area in
the Western Ghats region, only one-third is under natural vegetation.
Moreover, the existing forests are highly fragmented and facing the
prospect of increasing degradation.
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Vegetation in the Western Ghats
According to a recent study conducted by the Indian Institute of
Remote Sensing (IIRS), incorporating both field-based analysis of
vegetation communities as well as satellite image interpretation, there are
four major forest types in the Western Ghats: evergreen, semi-evergreen,
moist deciduous, and dry deciduous. Together the forests cover
approximately 20 percent of the total area of the Western Ghats. Among
the four broad vegetation types, moist deciduous forests occupy the
largest area followed by semi evergreen, dry deciduous, and finally
evergreen.
The majority of the area under moist forest types falls within the
southern states of Kerala and Karnataka. Together they account for 80
percent of the evergreen forest and 66 percent of the moist deciduous
forests in the Western Ghats (IIRS 2002).
Evergreen forests
The highest levels of endemism are found in the evergreen forests.
These forests occur within a 200-1,500-meter elevational range and, 500-
to 5,000-millimeter rainfall range. They vary widely along the length and
breadth of the Western Ghats. A broad distinction can be made between
the northern evergreen forests and the southern evergreen forests. The
Wayanad evergreen forests of Kerala represent a transition zone from the
moist Cullenia-dominated forests in the south Western Ghats to the
northern drier dipterocarp forests (Rodgers and Panwar 1988).
The habitat types of the southern Western Ghats tropical evergreen
forests also include the wet montane evergreen forests and shola-rassland
complexes in the higher elevations (1,900-2,200 meters). The montane
evergreen forests are diverse, multistoried and rich in epiphytes, with a
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low canopy at 15 to 20 meters (Puri et al. 1989; Ganesh et al. 1996).
More than half the tree species found in these forests are endemic,
especially among the families Dipterocarpaceae and Ebenaceae. The
majority of the fifty endemic plant genera are also monotypic. The
distribution of richness and endemism is not uniform within this forest
type, with some areas having higher concentrations of endemics than
others.
Semi-evergreen forests
Semi-evergreen forests occur primarily in the states of Maharashtra, Goa,
and Karnataka in the Western Ghats, within an elevational range of about
300-900 meters (IIRS 2002). This forest type includes secondary
evergreen dipterocarp forests, lateritic semievergreen forests, bamboo
brakes, and riparian forests as described by Champion and Seth (1968).
The structure and composition of these forests varies widely from north
to south and especially from east to west. The dominant species include:
Terminalia paniculata, Aporusa lindleyana, Olea dioica, Syzygium spp,
Mesua ferrea, Vateria indica, Elaeocarpus tuberculatus, Celtis
timorensis, Hopea parviflora, Lagerstroemia microcarpa, Holigarna
arnottiana, Hydnocarpus laurina, Memcylon umbellatum, and Careya
arborea. These forests also tend to have high levels of tree diversity and
endemism (IIRS 2002).
Moist deciduous forests
The moist deciduous forest type occupies the largest area within
the Western Ghats. It occurs within an elevational range of 500-900
meters in areas with mean annual rainfall of 2,500-3,500 millimeters. The
swath of moist deciduous forests is very narrow on the steeper, windward
side of the mountain range, where the southwest monsoon rains promote
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wet evergreen forests. On the less steep leeward side, the drier conditions
caused by the rain shadow result in a broader, uneven swath of moist
deciduous forests that extend further into the Deccan Plateau. Rainfall on
the leeward side is influenced by complex landforms, with some areas
receiving less than one-fifth of the 3,000 millimeters or more of annual
precipitation that is deposited higher in the mountains.
Dry deciduous forests
The dry deciduous forests occur on the leeward side of the Western
Ghats Mountain Range within an elevational range of 300-900 meters in
areas of 900-2,000 millimeters mean annual rainfall. They extend across
the southern Indian states of Karnataka and Tamil Nadu. The tall Western
Ghats mountain range intercepts the moisture from the southwest
monsoon, so that the eastern slopes and the Deccan Plateau receive
relatively little rainfall, from 900 to 1,500 millimeters. The undulating
hillsides have very shallow soils. Thorny plants become more common in
areas where grazing pressure is high Kamal et al. (2007).
Plant species in Western Ghats It is estimated that there are four thousand species of flowering
plants known from the Western Ghats and 1,500 (nearly 38 percent) of
these are endemic (Nair and Daniel 1986). Approximately 63 percent of
India’s woody evergreen taxa are endemic to the Western Ghats
(Johnsingh 2001). Of the nearly 650 tree species found in the Western
Ghats, 352 (54 percent) are endemic (Daniels, 2001). The tree genera
endemic to the Western Ghats include Blepharistemma, Erinocarpus,
Meteromyrtus, Otenophelium, Poeciloneuron, and Pseudoglochidion.
Other plant genera endemic to the Western Ghats include Adenoon,
Griffithella, Willisia, Meineckia, Baeolepis, Nanothamnus, Wagatea,
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Campbellia, and Calacanthus (Nair 1991). The grass family Gramineae
(Poaceae) has the highest number of endemic genera and the genus
Nilgirianthus has the maximum number of endemic species (20) across
all genera in this family (Nair 1991).
There are several centers of plant endemism and species richness
within the Western Ghats. For instance, of the 280 woody endemic
species found south of Karnataka, 70 species are endemic to the
southernmost Travancore region (Nair 1991). Herbaceous species
richness is the highest in the stretch of hills to the south of Kodagu
district in Karnataka (Nair 1991). The Nilgiri Mountains are one of the
most important centers of speciation for flowering plants in the Western
Ghats, with 82 species restricted to this area alone (Daniels 2001).
Several species are endemic to the Agastyamalai-Nilgiri Hills and
the Sri Lankan highlands, including Abarema subcoriacea, Biophytum
nudum, Chrysoglossum maculatum, Eugenia rotundata, Fahrenheitia
zeylanica, Filicium decipens or fern tree, Pavetta zeylanica, and Rubus
micropetalus or wild aspberry. The flora of the Agastyamalai Hills bears
a remarkable similarity to that of Sri Lanka’s southwestern wet zone not
just in terms of shared taxa, but also with respect to the remarkably high
incidence of highly localized “point” endemics (Ramesh & Pascal, 1997).
Tree species endemism is the highest in the southern Western Ghats,
while herb species endemism appears to be highest in the north (Daniels
2001).
The Western Ghats comprise the mountain range that runs along
the western coast of India, from the Vindhya-Satpura ranges in the north
to the southern tip. The ecosystems of the Western Ghats are located
mainly in the following regions, the tropical wet evergreen forests in
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Amboli and Radhanagari; the Montane evergreen forests in
Mahabaleshwar and Bhimashanker; moist deciduous forests in Mulsi and
the scrub forest in Mundunthurai.
There is a great variety of vegetation all along the Ghats, scrub
jungles, grassland along the lower altitudes, dry and moist deciduous
forests, and semi-evergreen and evergreen forests. There are two main
centres of diversity, the Agashyamalai hills and the Silent Valley. The
complex topography and the heavy rainfall have made certain areas
inaccessible and have helped the region retain its diversity.
Among the floristic diversity, medicinal plants are an important
source which have been used all over the world. India’s vast resource
base of medicinal plants is well known to the world over. In India, the
medicinal plants are widely used by all sections of the population, and
country is richly endowed with a wide variety of plants of medicinal
value, which represents the great national resource (Alok, 1991). It is
estimated that atleast 70.00 per cent of country’s population relay on
herbal medicines for primary health care in the country (Holley and
Williams, 1996). In India, different classical medicinal systems such as
Ayurveda, Siddha and Unani are being practiced in the country and in
addition to this; innumerable local folk medicinal traditions exist. In all
over 8000 plant species are in medicinal use. Across the country, this
constitutes 45.00 per cent of 17,500 known flowering plant species of
India (Ved et al., 2000). This rich medicinal wealth is mainly distributed
in two hot spots diversity that is north eastern region and western ghat.
The Western Ghat comprises of a hill range running about 1500
km long the western edge of Indian sub-continent. Although it covers a
mere 5.00 per cent of the country’s total land area in the country, it is
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believed to be more than 27.00 per cent of country’s plant species
remarkably high level of endemism ranging from 25.00 to 60.00 per cent
of recorded species in various taxa (Pascal, 1992). Sixty three per cent of
Indian ever green tree species are believed to be endemic to that area. In
addition to species richness, it is endemic of biodiversity of the regions.
Sixty per cent of amphibians, 50.00 per cent of reptiles and 25.00 per cent
of plants of Western Ghats are endemic (Vijay, 2006). Sixty three per
cent of India’s ever green tree sp. is believed to be endemic to this area.
Medicinal trees are important components of the biodiversity of the
Western Ghats. The high anthropogenic pressures and associated
Fragmentation of the landscapes has resulted in loss of habitat and
species. Medicinal plants are also under constant threat due to over
exploitation from natural habitats in the absence of cultivation.
Biogeographically, the Western Ghats have long been isolated from the
vast south-east Asian humid forest tract and thus protect a relict pocket of
evolutionarily distinct biota. Geology, soil and climate also contribute to
promote high biodiversity in three forests. This region so many palnt
species are critically endangered or extinct of the Western Ghats one of
the species conserves here Abutilon ranadei Woodr & Stapf. some
important information of the genus Abutioln and their family Malvaceae.
The genus Abutilon Mill. Belongs to family Malvaceae and is
represented by about 150 species. India is home to 12 species, 2
subspecies and 5 varieties Paul, (1993), Woodrow (1897) of these two
species and 4 varieties are endemic to India Tetali et al. (2004). The
genus is distributed mostly in the tropical or subtropical parts of the
world. Many species are commercially important as they are highly
ornamental.
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Family Malvaceae
Family Malvaceae is cosmopolitan in distribution. This plant family
comprises 88 genera and 2300 species. In Pakistan it is represented by 19
genera with 94 specific and intraspecific taxa e.g Abutilon, Hibiscus,
Abelmoscus, Gossypium etc. The members of this family are mostly
annual to perennial herbs to shrubs or small trees Paul, (1993).
Importance of the family Malvaceae (Bhattacharyya, 1971 & Singh,
1983). Popular plants of the family Malvaceae are generally valued for
commercial cotton, gorgeous spring blossoms and some for their
colourful foliage. Some plants are grown for decoration (Hibiscus).
Additionally they have medicinal value as well. Marsh mallow (Althaea
officinalis) has demulcent properties. Sometimes it is used to treat
inflammations and irritations of the mucous membranes such as the
alimentary canal, the urinary and the respiratory organs. The root is used
for peptic ulceration and gastritis. Hollyhock, (Althaea rosea) has uses
similar to marsh mallow. Its flowers are used medicinally for their
emollient, demulcent and diuretic properties. Roots of marsh mallow are
used medicinally for cough. Salmalia malabarica gum is good for curing
kidney troubles, leucorrhoea and tuberculosis. Flowers and barks of the
same plant have ability to cure conjunctivitis and cutaneous infections. It
is also expectorant, laxative and suppurative. Hibiscus rosa is
aphrodisiac. Leaves are good for curing boils. Flowers are laxative.
Abelmoschus esculentus improves and increases sperm count. Hibiscus
cannabinus treats guinea worm sores. Hibiscus sabdariffa is good to treat
high blood pressure. Economically Malvaceae is very important. The
commercial cotton is derived from the dense hairy seeds of Gossypium.
Cotton fibres have been traced to the Indus valley civilization. Gossypium
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hirsutum cultivated in the USA, yields long staple cotton, G. arboretum
and G. herbaceum which yield short staple cotton, are cultivated in Asia.
Cotton seeds are rich in fat and yield edible oil called the cotton seed oil.
It is also used in manufacturing soaps and lubricants; the oil cake is used
as a cattle feed. Hibiscus cannabinus is another plant which yields fiber,
used widely for cordage, ropes etc; fatty oil used in the manufacture of
linoleum, paints and varnishes and also as edible oil and oil cake is used
as a cattle feed. Other fiber yielding plants of minor importance are
Abutilon indicum, A. persicum and A. theophrasti (China jute), Sida
cordifolia, S. acuta, Urena lobata and many others. Hibiscus sabdariffa
(commonly called rosella) is a shrub, native to the West Indies; its
epicalyx and calyx are fleshy, rich in acids and pectin and used in
preparation of jellies and confectionery. Many species of Hibiscus such
as H. mutabilis, H. rosasinensis, H. schizopetalous are cultivated as
ornamentals; H. elatus (blue mahoe) is the national flower of Jamaica, H.
syriacus growing in eastern Mediterranean has large flowers of pinkish
colour and is commonly called ‘rose of sharon’.
The fruits of Abelmoscus esculentus or lady finger are used as a
vegetable. Roots of Althaea officinalis or marshmallow are used
medicinally, considered to be a very useful herbal remedy for cough. A.
rosea or holly hock is a cultivated garden ornamental. The mucilaginous
roots of Pavonia hirsua from Zimbabwe are added to milk to hasten
butter production Dutta, (1988). The mucilaginous substance obtained
from the stem of Kydia calycina is used for clarifying sugars.
Genus Abutilon Mill.
Annual or perennial herbs, undeshrubs or shrubs. Leaves simple,
entire or lobed, mostly cordate at base, acute or acuminate at apex,
9
palminerved without nectarines. Flowers axillary, solitary, sometimes in
lax panicles by reduction or dercrescence of upper leaves, rarely in lax
corymbose racemes; pedicels jointed in the upper half. Epicalyx absent.
Calyx usually campanulate; lobes 5, divided to the middle or below.
Corolla usually yellow, orange, whit or pink , rarely with a dark purple
centre, rotate, campanu-late. Staminal column shorter than petals, much
widened at base. Carpels and style-branches 5-40, styles filiform ;to
clavate, capitate stigmatose at apex. Schizocarps globular, campanulate,
rarely discoid; mericarps 50-40 , dehiscent, follicular, flattened-reniform,
round, acuminate or biaristate at apex, often falling leaving a slender
truncate, columella. Seeds 2-9 in each mericarp, reniform to subreniform,
upper ones ascending; lower ones pendulous or horizontal , finally falling
out of mericarp. In tropical and subtropical regions of the World , ca
150species; 12 in India. Abutilon Parker, (1921) is a large genus of about
150 species of broadleaf evergreens in the mallow family (Malvaceae).
The genus includes annuals, perennials, shrubs, and are conspicuous, with
five petals, mostly red, pink, orange, yellow or white. Small trees from 1-
10 m tall, and is found in the tropical and subtropical regions of all
continents.
Importance of genus Abutilon
Valuable fibers are drawn from different species of Abutilon e.g.
Abutilon indicum, A. polyandrum and A. asiaticum Nasir, (1979). the
fibers obtained from these plants are used for making ropes cordage’s ,
jute dyes, drugs, rugs , wrapping cloth, tissue papers, for making coarse
cloth, cigarette paper, rubber, tyre, fabrics, shoe polishes ,etc. Its leaves
are applied in ulcers and gonorrhoea. Leaves of A. indicum yields
mucilage used in pectorial troubles. Abutilon avicennae provides a useful
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fiber named Chinese jute. Seeds of Abutilon specie are laxative and
demulcent Sammia (2008).
Abutilon ranadei Woodr.& Stapf. was first collected by N.B.
Ranade, ex-keeper of the herbarium at the College of Science, Pune
Woodrow & Stapf. (1894) described it as a new species and named it
after Ranade. It is an endemic known so for only from four district of
Maharashtra State. According to Cooke (1901), it is a rare plant due to its
narrow range of distribution and extreme rarity the species has been
declared as endangered Nayar and Sastry, (1987), Venkanna & Das Das
(2000) or even presumed extinct Ahmedullah and Nayar, (1986).
However it was recollected from its type locality after a lapse of almost
95 years (Mistry& Almeida, 1989; Almeida, 1996; Walter and Gillett,
(1997). Since then the species has been collected from eight new
localities in Pune, Satara, Kolhapur and Ratnagiri district.
It is an interesting species of Abutilon, restricted to hill slopes in
main crest of Sahyadri ranging from Amba ghat in south to Junnar-
Harischandragad region in north. The individuals are sparsely distributed
in the region and known only form few localities showing (Table-1 and
Photo plate No. 1).
It is critically endangered species of Western Ghats. It has flowers
of ornamental value. The seed setting is very low and there is a need for
restoration of natural populations of these species. This species is intended
to be recovered by conventional and tissue culture method.
The immediate major is reintroduction at the historical sites of
occurrence. As per Primik (2004) 10,000 individuals in 20, 000 Sq. k.m.
are to be planted for viable population. Although it is a immediate measure
11
to save the species from being extinct, the real cause for its course of
extinction is to be figured out.
In view of this it is intended to study the species thoroughly which
is expected to pin point the cause of extinction in view of this following
parameters were studied viz.
a) Morphology
b) Anatomy
c) Polynology
d) Floral biology
e) Cytology
f) Seed germination and viability
g) Soil microflora
h) Phytochemistry
h) Tissue culture protocol
i) AFLP Marker
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REVIEW OF LITERATURE
Abutilon ranadei, commonly know as Shonghanta, is perhaps the
critically endangered species in Western Ghats. Endemic and Threatened
Flowering Plants of Maharashtra. Botanical Survey of India (Ahmedullah
& Nayar 1986; Nayar & Sastry, 1987; Mistry & Almeida, 1989 and
Mishra & Singh, 2001). Morphological studies of Abtuilon ranadei
reported in some Floras and articles (Woodrow, & Stapf, 1894; Cooke,
1901; Paul, 1993 Bachulkar & Yadav, 1997; and Punekar et al. 2001).
Taxonomical and some related work of other species of genus
Abutilon. Pollen morphology of Abutilon Mill., from Pakistan (Siddiqui,
et al. 1984). Effects of shade on reproduction and some morphological
characteristics of Abutilon theophrasti and Leaf margin serration and
taxonomy in the genus Abutilon Benvenuti and Macchia (1994); Bhat
(1999). Pollination of three species of Abutilon (Malvaceae) intermediate
between bat and hummingbird flower syndromes (Buzato, et al. 1994).
Circumscription of Malvaceae (Malvales) as determined by a preliminary
cladistic analysis of morphological, anatomical, palynological, and
chemical characters (Alter and Steven, 1997). Genus Abutilon reported
in anatomical and dermatological studies of some other species, Root,
Stem, Leaf of Abutilon theophrasti (Ysegul, et al. 2003). Intraspecific
variation in morphological characteristics and growth habit of newly and
accidentally introduced velvetleaf (Abutilon theophrasti Medik.) into
Japan. (Kurokawa et. al 2003b). Taxonomic Revision of Genus Abutilon
Mill. in Saudi Arabia General View of Malvaceae Juss. (Wafaa 2009).
Implication of foliar epidermal features in the taxonomy of Abutilon Mill.
(Malvaceae) and Pollen morphology of 14 species of Abutilon and
Hibiscus of the family Malvaceae (Nighat et al. 2009).
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The area under Abutilon ranadei is decreasing day by day due to its
critically endangered species in Western Ghats its potential for better
adaptation to diversified soil and climatic conditions. There is few
localities of the Maharashtra State because poor germination, slow
growth of rootstock seedlings, lack of information on season and age of
rootstocks for grafting and micropropagation protocol has rendered the
clonal multiplication process more difficult to produce large scale. The
available information on various aspects of seed dormancy, softwood
grafting and micropropagation in Abutilon ranadei is very meager, the
relevant literature on other Abutilon species, grown under similar
situations have been reviewed. For better understanding of the subject the
information is organized under the following headings. a. Different seed
treatments on breaking dormancy. Propagation of stem cutting by
softwood grafting. Micropropagation.
When Haberlandt attempted the first cell culture studies, his
intention was to develop a versatile tool to explore morphogenesis and to
demonstrate totipotency of plant cells (Haberlandt, 1902). The first report
of viable callus culture was reported by Gautheret (1939) and White
(1939) in tobacco and carrot, respectively. The relative concentration of
auxin and cytokinin decides the plant morphogenesis in vitro (Skoog and
Miller, 1957). Effect of Auxins and Kinetin on in-Vitro Regeneration in
Tissue Cultures of Abutilon-Indicum (Nataraja and Patil 1980).
Hardseededness and germinability of velvetleaf (Abutilon theophrasti) as
affected by temperature and moisture. (Horowitz and Taylorson 1984).
Abscisic Acid and Sucrose Control of Velvetleaf (Abutilon tbeopbrasti)
Ovule Development In Vitro (Laura et al. 1984). Abscisic acid and
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sucrose control of velvetleaf (Abutilon theophrasti) ovule development in
vitro and The in vitro culture of excised ovules from velvetleaf (Abutilon
theophrasti). (Thompson, et al. 1984). Responses of Isolated Floral Buds
and Anthers of Abutilon indicum in-Vitro. (Nataraja and Patil 1984).
Micropropagation has a great commercial potential due to the speed of
propagation, decreased production space requirement and the ability to
multiply elite clones exhibiting superior growth and enhanced stress
tolerance (Garton and Mosses, 1985; Kane et al., 1989).
Intrapopulation variation in Abutilon theophrasti seed mass and its
relationship to seed germinability (Baloch, et al. 2001). The most widely
used cytokinins are kinetin, BAP and 2-IP, auxins or cytokinins alone
cannot show organogenesis through cell division or callus in vitro. Since
many fruit crops are sensitive to photoperiod requirement these are
cultured in vitro under day night regime and a light cycle of 16 hrs
followed 8 hrs of darkness is the most preferred one (Conger, 1987). The
optimum growth of tissue cultures has been observed at a temperature
regime 24-26°C (Pierik, 1987). Establishment of culture explant Pierik
(1987) reported that genotype, plant age, physiological state (vegetative
or regenerative), stage and age of explants, general health of plant,
position of explant within plant, size of the explant, method of
inoculation etc. affects the growth of explant in vitro. Effect of shade on
velvetleaf (Abutilon theophrasti) growth, seed production, and dormancy
(Bello, et al., 1995). Temporal changes in velvetleaf (Abutilon
theophrasti) seed dormancy (Cardina and Sparrow, 1997).
Record of Abutilon ranadei Woodrow & Stapf in an area other
than the type locality. (Bachulkar & Yadav 1997). Showed that different
15
treatments like hot water treatment at 700C for 10 minutes followed by
concentrated H2SO4 scarification for five minutes was most effective in
breaking the dormancy which was imposed by seed coat in Abutilon
indicum. (Gupta et al. 2001). Techniques to remove hard seededness in
the wild medicinal plant Abutilon indicum. Journal of Medicinal and
Aromatic Plant Sciences (Gupta, 2001). Ecological and conservation
studies of Abutilon ranadei Woodr. et. Stapf. (Tetali et al. 2004).
Micropropagation also can be used to establish and maintain virus-free
plant stock. This is done by culturing the plant's apical meristem, which
typically is not virus-infected. Once new plants are developed from the
apical meristem, they can be maintained and sold as virus-free plants.
The major branch of biotechnology which has become
commercially viable is micropropagation. Micropropagation has been
defined as ‘in vitro’ regeneration of plants from organs, tissues, cells or
protoplasts (Beversdorf, 1990) and as ‘the true to type propagation of a
selected genotypes using in vitro culture techniques’ (Debergh and Read,
1991). True-totype propagation has advantages for heterozygous fruit
crops such as aonla, pomegranate, papaya etc. The concept of totipotency
is the basis for micropropagation. Micropropagation has been
successfully employed for rapid production of uniform and superior
quality planting material. Rapid and large scale clonal propagation of
many fruit crops is now possible tissue culture. The regeneration can take
place as extension or proliferation of shoot tip or axillary buds, direct
production of organs from explant i.e. organogenesis or through the
production of somatic embryos i.e. embryogenesis. An in vitro
propagation involving an unorganized callus phase may produce variant
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plants i.e. somaclonal variants, clonal in vitro propagation is achieved by
using meristems and axillary buds (Kumar and Kumar, 1998). In tissue
culture studies, one has to make use of plant growth regulators in addition
to the supply of other salts, mineral, vitamins and sugars to obtain the
desirable response to achieve the set of objectives. Auxins are a class of
plant growth regulators which cause cell elongation, apical dominance
and root initiation. The most frequently used auxins are 2, 4-D, NAA,
IAA and IBA of which IAA occurs naturally in plants. NAA and 2, 4-D
are the most effective auxins to initiate callus. Cytokinins are
phytohormones which promote cell division, proliferation of tissues.
While selecting a suitable explant for micropropagation, these factors
should be taken into consideration. Muralikrishna (1988) reported highest
percentage of shoot establishment in explants of 10 mm size followed by
7.5 mm. As the size of explant increased the shoot establishment
percentage of also increased upto 10 mm (56.3%) and later decreases as
size increased. He also stated that the rapid adventitious shoot initiation
was found only in axillary bud (9 shoots/culture) than shoot tip explants.
The later were able to produce fewer (2.17) shoots per culture in
pomegranate. In Jackfruit, Rajmohan and Mohankumaran (1988) studied
the influence of explant source on the in vitro propagation. They reported
maximum response from the seedling explants (17.4 shoots) followed by
the fresh stem sprouts from five year old trees (4.50 shoots). A drastic
reduction was observed from explants excised from ten to thirty year old
trees (2.80 and 2.09 shoots, respectively). Maximum number (15-20) of
shoots were induced in shoot tip closely followed by cotyledonary node
(10-15), nodal section (8-10) and hypocotyls (6-8) of ber (Mathur et al.,
1993). Nodal segment explants of 1-2 cm length when cultured from mid
17
portion of the indeterminate shoots of mature tree of aonla cv. NA-7 in
the month of April-July to August- November gave better bud induction
(Mishra et al., 1999). Singh et al. (2002) studied the in vitro propagation
of grapes. They obtained better culture establishment with nodal explants
than the shoot tips. Studies on in vitro propagation of papaya cv. CO-5
revealed that highest callusability per cent within 25 days on MS medium
supplemented with 1.0 mg/l IAA and 4.5 mg/l BA in case of shoot tip
culture than explants like leaf bits and nodal segments (Suthamathi et al.,
2002). Micropropagation studies of explants from mature trees Aonla
Verma and Kant (1996) cultured nodal segment explants of aonla on
modified MS medium supplemented with BA (3-5 mg/l) and NAA (0.5
mg/l) during February-April and August-October. The bud break
observed in only 8-10 per cent of explants. Leaching of phenolic
compounds resulted in the loss of almost 90 per cent in cultures. The
regenerated shoots were elongated on hormone free medium and
subsequently rooted on half strength MS medium supplemented with IBA
(3.0 mg/l) and sucrose (1.5%). In aonla cv. NA-7, Mishra et al. (1998)
reported heavy inborn contamination and phenol exudation were the
major problem in establishing the in vitro cultures of aonla. Dipping of
explants in bavistin (1%) and cholramphenicol (0.1%) for 60-90 minutes
followed by treatment with HgCl2 (0.1%) for 8 minutes significantly
reduced the explant contaminationn. The application of paraffin wax on
the cut end of explants reduced browning by 20 per cent and increased
the survival rate to 80 per cent as compared to non-waxed explants with
or without antioxidant. Non-waxed explants treated with PVP showed the
50.30 per cent survival and browning to an extent of 47.73 per cent. In
aonla, Kant et al. (1999) reported 3-4 multiple shoots from nodal explants
18
from mature tree on MS medium supplemented with BAP (5 mg/l) and
IAA (0.5 mg/l) along with antioxidants. These shoots were elongated on
hormone free MS medium. Rooting of the shoots was achieved on half
strength MS medium fortified with IBA (2 mg/l) and sucrose (1.5%).
Micropropagation studies in aonla were carried out from nodal
explants by Mishra et al. (1999) to develop the protocol for mass
multiplication of true-to-type plants. The better shoot proliferation (33-
37.5%) on modified MS medium supplemented with kin 0.4 mg/l + GA
1.0 mg/l than in WPM. Higher GA3 concentration (3 mg/l) caused
complete defoliation and dropping of determinate shoots. Regenerated
shoots from 3 week old cultures in MS media supplemented with growth
regulators and antioxidants failed to produce roots. Mishra and Pathak
(2001) studied the effect of nodal position and seasonal influence on in
vitro shoot proliferation of aonla. The highest bud induction rate
(76.40%) and longest indeterminate shoot (0.53 cm) was obtained with
explants excised from the 10th to the 15th node during August-November
in MS medium supplemented with kin (0.4 mg/l) and GA3 (0.4 mg/l).
Other fruit crops Amin and Jaiswal (1987) developed a rapid clonal
propagation through in vitro shoot proliferation from nodal explant of
guava. The best response was reported on MS medium supplemented
with 4.5 µM BA alone. The response of in vitro shoot nodal segments
was better in comparison to the field grown trees. Maximum response
with minimum contamination and browning of explant and medium was
obtained during April-June. Muralikrishna (1988) reported the strategies
of micropropagation of pomegranate. To control browning in culture
medium he claimed that the subsequent transfer of explants on fresh
19
medium as well as soaking of explants in 0.1 per cent ascorbic acid for 1-
2 hours in the solution. In micropropagation studies, the axillary buds and
shoot tip explants were regenerated on basal MS medium supplemented
with BAP 0.5 µM. Growth of axillary bud was slower than shoot tips, but
axillary bud produce about 9.5 buds per explant as compared to 2.33 buds
per explant by shoot tip. Rooting occurred when shootlets were soaked in
IBA 100 mg/l solution for one hour before subculture. In jackfruit,
Rajmohan and Mohankumaran (1988) reported that problem of
polyphenol interference was minimized by incorporation of activated
charcoal (1%) and GA3 1 mg/l in the establishment medium, insoluble
PVP in the proliferation medium and by frequent subculturing.
They recorded cent per cent survival and production of healthy,
growing cultures of shoot apices in MS medium supplemented with GA3
1 mg/l along with AC (1%). In woody plants, no tissue lacks phenolic
compounds including growing cell. Oxidation of phenolic compounds
released from the cut ends of explants by phenol oxidase, peroxidase
cause lethal browning of plant and culture medium. This problem can be
overcome by sealing the cut ends of explants with liquid paraffin wax
(Bhat and Chandel, 1991). Shoot organogenesis in cv. Nana, a dwarf
variety pomegranate was studied by Zhang and Stoltz (1991). Each
culture produced a mean of 5.2 shoots with BA 1 µm+ NAA 2 µM while
6.6 shoots produced with BA 2 µM + NAA 1 µM. Study indicated that
auxin (NAA) at 1 µM was optimum for in vitro shoot formation and that
BA levels more than 2 µM inhibits shoot formation. Amin and Jaiswal
(1993) described a method of shoot tip culture for rapid multiplication of
mature jackfruit trees. BA and kinetin (4.5 to 9 µM) either alone or in
20
combination, supported shoot proliferation. The ideal season for
collection of apical budswas during November to January. Shoots rooted
with 60 per cent to 80 per cent success using half strength MS salts and
10 µM IBA or NAA. In Zizypus mauritiana, Mathur et al. (1995)
reported that stem explants from mature tree when grown on modified
MS medium with 3800 mg/l potassium nitrate, 2475 mg/l ammonium
nitrate, 11 µM BA produced 15-20 shoots per inoculation. Rooting was
induced by pretreatment with 50 µM IBA or NAA for 24 hours followed
by transfer to auxin free whites medium. Roy and Roy (1996) reported
that maximum shoots proliferation from shoot tips and nodal segments
was induced in jackfruit on MS medium containing BA 2.5 mg/l and
NAA 0.5 mg/l, with an average of 10.3 shoot/culture after 28 days.
Rooting of excised shoots was achieved on half strength MS medium
containing NAA and IBA each at 1.0 mg/l. Seventy five per cent of the
plantlets survived after transplanting into the open field. Roy et al. (1996)
reported that multiple shoots were obtained from nodal explants of S.
cumini on MS medium supplemented with kinetin 2.5 mg/l. Repeated
subculture resulted in rapid shoot multiplication. Addition of 100 mg/l
urea to media increased number of shoots rooted in half strength MS
media containing 0.5 mg/l each of IBA, NAA and IAA. After
transplanting in field, 75 per cent plantlets were survived with uniform
growth, in tamarind Balakrishnamurthy and Ganga (1997) reported the
seasonal influence of explant collection on induction of multiple shoots
from axillary buds. The monthwise collection and culture of axillary buds
in MS medium fortified with six per cent sucrose, BA 0.5 mg/l and GA3
0.5 mg/l indicated that best results with respect to per cent bud break
(82.5%), number of days taken for bud break (9.33 days), per cent
21
response to multiple shoot induction (22.50%), number of shoots per
culture (2.17) and length of micro shoots (3.21 cm) were recorded by the
axillary buds collected and cultured during the month of May. The
axillary buds collected and cultured during the months December,
January, February, March, September and October did not record any
response. Kantharajah et al. (1998) observed high frequency adventitious
bud formation in pomegranate, when nodal cuttings of cv. Wonderful
were cultured on half MS medium supplemented with BAP 0.5 mg/l +
NAA 0.1 mg/l. NAA was most effective than BAP in root elongation and
the highest shoot length occurred on media containing NAA 0.1 mg/l.
Roots were induced after 12 days on WPM with NAA 2 mg/l. Mehta et
al. (2000) developed a protocol for in vitro regeneration of plants via
adventitious bud formation from mature embryo axis of tamarind.
Induction of adventitious shoot buds was achieved in the cut surface of
the axis of tamarind. Induction of adventitious shoot bud was achieved in
the cut surface of the axis when cultured on medium containing zeatin
0.91 µM, calcium panthothenats 0.41 µm and biotin 0.40 µM supported
the differentiation of buds to form elongated shoots. Rooting occurred in
half strength MS medium with 2 per cent sucrose following a 72 hrs
treatment with auxin mixture in dark. Suthamathi et al. (2002) carried out
in vitro studies on papaya cv. CO-5. They obtained highest callusability
in case of shoot tips cultured on MS medium supplemented with 1.0 mg/l
IAA and 4.5 mg/l BA. Development of shoots was observed from shoot
tip callus cultured on MS medium supplemented with BA 4-5 mg/l
compared to kinetin. Pattepur (2003) did the tissue culture studies in
tamarind. He obtained multiple shoots when cotyledonary explants were
cultured on MS medium supplemented with BAP 0.5 mg/l + coconut
22
water (CW) 10 per cent. Auxillary buds collected from mature tree gave
highest per cent bud break and response to multiple shoot induction on
medium containing BAP 2.0 mg/l + CW 10 per cent was noticed.
Biotechnology for Endangered Plant Conservation (Anca Paunescu
2009). In vitro Micropropagation of Abutilon indicum L. through Leaf
Explants. Plant Tissue (Jyoti et al. 2009). In Vitro Propagation for
Conservation of Rare and Threatened Plants of India –A Review (Vera,
2010), Biswas (2007).
Abutilon ranadei is most important parameter of the genetic
diversity because the extinct of the species survey of the literature related
to the genus or family. Using protein electrophoresis to investigate the
phylogeny of velvetleaf (Abutilon theophrasti) (Stegink and Spencer
1988). Localization of Abutilon mosaic virus DNA within leaf tissue by
in-situ hybridization (Horns and Jeske 1991).Abutilon mosaic
geminivirus double-stranded DNA is packed into minichromosomes
(Pilartz and Jeske 1992). Complementation and recombination between
mutants of complementary sense genes of DNA A of Abutilon mosaic
virus (Evans and Jeske 1993). DNA forms indicate rolling circle and
recombination dependent replication of Abutilon mosaic virus (Holger et
al. 2001). Molecular and morphological differentiation between the crop
and weedy types in velvetleaf (Abutilon theophrasti Medik.) using a
chloroplast DNA marker, seed source of the present invasive velvetleaf
(Kurokawa et al. 2004). AFLP mediated genetic diversity of malvaceae
species (Nighat et al. 2010).
Most important factor of the Chemical investigations on Abutilon
species were undertaken as early as the 1929; when Kosaka, (1929);
23
Zaheera, (1990); some chemicals phytochemical investigation are
reported, Pentacyclic Triterpene and phytochemical investigation of
Abutilon Pakistanicum Ahmed et al. (1990); Ahmed et al.(1991); Ahmed
et al. (1993); Ahmed et al. (2006); reported the presence of anthocyanins
in Abutilon avecenae, however real chemical research on Abutilon
species began only in 1956 with the isolation of Rutin from Abutilon
avicenae. Developments after 1960 progressed more rapidly mainly
because of the invention of more refined techniques of separation and
structure elucidation. Since then large variety of compounds have been
isolated from genus Abutilon and majorities of them are flavonoids,
terpenoids and tetraketones various author Yemets and Steblyuk, (1994);
Sauar Ali (2009), Mabry (1970), Bagi (1985).
This study will provide valuable information about basic
information and restoration of the species point of view. This is the initial
step towards conservation of species.
AIMS AND OBJECTIVES
The present work was initiated for the doctoral dissertation, which
entails the morphology, anatomy, floral biology, cytology, seed
germination, vegetative propagation, soil microflora, phytochemistry,
tissue culture and AFLP marker of Abutilon ranadei the critically
endangered species in Western Ghats of family Malvaceae. In order to
correlate the need of above parameters with the active ingredients, the
research plan was chalked out as detailed below.
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PLAN OF WORK
The plan of work of these studies had six phases.
Phase I: Collection for the minimum 10 localities
1. Collection of the plant materials (herbarium specimen, stem
cutting, floral buds, seeds and explants).
2. Preservation of the plant material for anatomical and other
experimental work.
Phase II: Morphology and Anatomy
1. Study of morphological characters like vegetative characters and floral
characters
2. Study of anatomical characters different parts like Root, Stem, Petiole
and Leaf
Phase III: Floral biology and Cytology
1. Study of floral biology
2. Chromosomal study (Meiosis)
Phase IV: Propagation by conventional methods
1. Vegetative propagation by stem cutting in different treatments
2. Seed Germination by various treatments
Phase V: Photochemistry
1. Preparation of plant material for the phytochemical study
2. Extraction of aerial parts and roots in different solvents.
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3. Preliminary phytochemical study like Tanin, Saponine, Akaloides,
Protein, Ash, Total Ash, Reducing sugar, non-reducing sugar,
carbohydrates etc.
4. Antibacterial activity
Phase VII: Micropropagation
1. In vitro studies in Imature seed
2. Callus Induction for different explants like (Root, Stem, Petiole, Leaf,
Floral buds and Anther).
3. Multiplication by nodal segments and immature seeds
Phase VIII: AFLP Marker
1. Study of the genetic diversity in minimum five localities by different
primers
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