Post on 17-Apr-2018
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II. Review of Literature Saffron is classified into Magnoliophyta Division, Class Liliopsida and Order
Asparagales. It is a member of the Iridaceae family and the Crocus L. genus.
Crocus consists of 9 species viz, Crocus cartwrightianus and its derivatives,
Crocus sativus, Crocus moabiticus, Crocus oreocreticus, Crocus pallasii, Crocus
thomasii, Crocus badriaticus, Crocus asumaniae and Crocus mathewii. Some
archeological and historical studies have indicated that domestication of saffron
dates back to 2,000-1,500 BC (Tammaro, 1987; Negbi, 1999). However, the sites
where the first saffron plants appeared differ according to the opinion of various
authors. Vavilov (1951) placed saffron into IV plant Centre of Origin, Middle East
(Minor Asia, Turkestan) whereas more recent reports indicate that the process of
saffron domestication has to be identified on Crete during the last Bronze Age
(Negbi, 1999). According to Negbi (1999), wild C. cartwrightianus herb was
harvested and used, then its mutant C. sativus was observed, selected and
domesticated on the Crete. C. cartwrightianus grows on a volcanic ash near
Akrotiri, Santorini and is harvested for local consumption by the villagers
(Mathew, 1999). However, C. thomasii Ten, or C. pallasii Herb, have also been
identified as possible saffron ancestors (Brighton, 1977; Chichiricco, 1989a;
Tammaro, 1990). The most recent taxonomic publications (Mathew, 1999) and
most of the former classifications (Tutin, 1980; Mathew, 1982) arranged C.sativus
and C. cartwrightianus to each other either as C.sativus to be a sub-species of C.
cartwrightianus or as a variety or a mutated derivative. Experiments of cross
pollinations turned C. thomasii out to produce some seeds with C.sativus
(Chichiricco, 1989a; Grilli Caiola, 2004). Further studies also defined C.
cartwrightianus and C. thomasii to be closest to saffron (Grilli Caiola and
Brandizzi, 1998). Mathew (1999) put forth taxonomy of C. sativus and its allies as
under:
Genus Crocus
I. Subgenus Crocus. Type species: C. sativus L.
A. Section Crocus. Type species: C. sativus L.
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(a) Series Verni Mathew. Type species: C. sativus L.
(b) Series Scardici Mathew. Type species: C. vernus Hill.
(c) Series Versicolores Mathew. Type species: C. scardicus Kos.
(d) Series Longiflori Mathew. Type species: C. longiflorus Raf.
(e) Series Kotschyani Mathew. Type species: C. kotschyanus Koch.
(f) Series Crocus. Type species: C. sativus L.
B. Section Nudiscapus Mathew. Type species: C.reticulatus Stev. Ex Adams
(g) Series reticulati Mathew. Type species: C. reticulatus Stev. ex Adams
(h) Series Biflori Mathew. Type species: C. biflorus Mill
(i) Series Orientales Mathew. Type species: C. korolkovii Regel ex Maw
(j) Series Flavi Mathew. Type species: C. flavus Weston
(k) Series Aleppici Mathew. Type species: C. aleppicus Baker
(l) Series Carpetani Mathew. Type species: C. Carpetanus Bioss. & Reut
(m) Series Intertexti (Maw) Mathew. Type species: C. fleischeri Gay
(n) Series Speciosi Mathew. Type species: C. speciosus M. Beib.
(o) Series Laevigati Mathew. Type species: C. laevigatus Bory & Chaub
I. Subgenus Crociris (Shur) Mathew. Type species: C. banaticus Gay
Series Crocus includes Crocus sativus and its allies.
Species comprising series Crocus:
1. C. cartwrightianus
2. C. sativus
3. C. moabiticus Bornm. & Dinsm
4. C. oreocreticus B.L.Burtt
5. C. pallasii Gold
6. C. thomasii Ten
7. C. hadriaticus Herbert
8. C. asumaniae B. Mathew & T.Baytop
9. C. mathewii Kerndorff & Pasche
Cytofluemetry analysis of nuclear DNA amount and base composition of
C. sativus, C. cartwrightianus and C. thomasii compared to spring diploid
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C.biflorun (Brandizzi and Grilli Caiola, 1998) have revealed that C. sativus is a
genomic clone of C. cartwrightianus from which it could have originated by
mutation or out-cross process. RAPD technique (Zanier, 2000) was used to
discriminate nuclear DNA compliment in autumnal flowering Crocus belonging to
C. sativus group. Because of complex genetic make up which includes triploid
nature, not much improvement has been brought about in saffron through
conventional breeding procedures.
Saffron with sub-hysteranthous behavior is a perennial herbaceous plant attaining
a height of 25-40 cm. Corm, foliar structure and floral organs constitute the main
parts of saffron plants (Mathew and Brighton, 1977; De Hertogh and Nard, 1993;
Nehvi et al., 2010a). Corms that are 3-5 cm in diameter covered by tunic consist of
nodes and are internally made up of starch-containing parenchymatous cells.
Apical, subapical and axillary buds protected by dark reddish scales are found in
internodes. As their diameter increases, they tend to group together, so that the
majority can be found in one, two or three internodes (Perez, 1997). Roots emerge
radically at the height of the third basal internodes. They are thin white in color,
numerous and variable in length. At the base of the daughter corm, a much thicker
root than the absorbent roots may arise, which is known as contractile root
(Khalesi et al., 2004). Each corm produces five to eleven green leaves or
monophylls. The leaves known as bristles are 1.5 to 2.5 mm wide per sprout and
can measure upto 50 cm (Dhar and Mir, 1997; Lucceno, 1999). The photosynthetic
activity of the leaves during the early winter and spring months contribute to the
formation of replacement corms at the base of the shoots. Corms that are covered
by tunic are dormant during summer and sprout in autumn producing 1 to 4
flowers in cataphyll with linear leaves. Cataphylls not only protect and strengthen
the stem in course of its appearance on the surface (Botella et al., 2002) but also
protect the corms once formed from degradation and possible lesions (Lopez,
1989). Propagation occurs every year via formation of daughter corms from the
parent after flower harvesting (Negbi et al., 1989; Negbi, 1990; De Hertogh and
Nard, 1993).The flower has an underground ovary, a style (5 to 9 cm long),
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dividing at the top into three red trumpet like stigmas (2 to 3 cm long) which when
dried form the commercial spice-the saffron (Sampathu et al., 1984; Tammaro,
1990). Flowers with six stigmas have also been observed in the saffron fields.
However this kind of variation doesnot persist in the next flowering season and are
generally termed as freaks (Nehvi et al., 2004). The corm is a vegetative organ of
saffron. After flowering, the base of the stem enlarges producing a daughter corm
that propagates the plant (Tammaro, 1990; Jirage et al., 1994). The number of scar
like buds covered by scaly leaf present on the surface of mother corm vary from
2 to 20 depending on the corm size (1.0-6.0 cm).
Triploid nature of the species allows for vegetative multiplication but not for
regular sexual reproduction. This is because triploid meiosis and gamete
development are irregular, resulting in many anomalies in sporogenesis and
gametophyte development (Chichiricco, 1984). Saffron infertility is mainly related
to the male gametophyte (Chichiricco, 1989a, 1990; Grilli Caiola and Chichiricco,
1991). It does not produce viable seeds; therefore corms are indispensable for its
propagation. The karyotype of Crocus sativus L. was the subject of research by
many authors for genetic differentiation in relation to chromosome number, arm
ratio, arm size and centromeric position while the other species of Crocus sativus
aggregated in this respect remain poorly studied (Himmelbauer, 1926; Karasawa,
1933; Rzakuliyev, 1945; Brighton et al., 1973; Brighton, 1977; Chichiricco, 1984;
Dhar et al., 1988; Agayev, 2002; Agayev et al., 2010). Saffron triploidy has been
demonstrated by numerous authors (Karasawa, 1933; Brington, 1977). Studies on
micro and megasporogenesis in saffron confirmed that meiosis occurs in an
anomalous manner with irregular chromosome pairing, division and distribution in
the derived nuclei (Chichiricco and Grilli Caiola, 1984; Chichiricco, 1987, 1989b;
Grilli Caiola and Chichiricco, 1991). Due to triploidy, meiosis in C. sativus is
highly erratic, forming 8 trivalents and genetically unbalanced gametes which lead
to formation of sterile gametes, hence no seed formation. No fertilizable gametes
having 8 (n) or 16 (2n) chromosomes were found in saffron (Ghaffari, 1986) and
further self incompatibility has been observed (Chichiricco and Grilli Caiola,
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1986, 1987). In rare case of fertilization there is some embryonic and endosperm
development but it terminates at an early stage (Chichiricco, 1987). In saffron, on
the basis of overall length and centromeric position, the somatic chromosomes are
assembled in seven triplets (3 metacentric, 2 submetacentric and 2 telocentric) one
pair (submetacentric) and one single chromosome (metacentric). Saffron from
Kashmir is identical in structure of karyotype with usual saffron (Crocus sativus
L.). However, some significant distinctions are present: 1) All three satellites at
three chromosomes of the second triploid are identical in size 2) The total length
of all chromosomes of triploid set 2n=3x=24 in Kashmir saffron is authentically
more than in usual Crocus sativus. In triplet IV, the homologues 1 and 2 have an
arm ratio equal to 1.50 whereas the homologue III have an arm ratio of 1.2. The
difference is significant being absent in usual Crocus sativus (Agayev et al.,
2010).
Saffron is valued for its color, taste and aroma. The compounds that give it these
properties are what define its quality. Saffron predominantly contains chemical
constituents such as crocin, picrocrocin and safranal responsible for its color,
flavor and aroma, respectively. Crocetin glycosyl esters are responsible for its
characteristic color and these compounds are found in extremely important
proportion in stigmas (Sampathu et al., 1984; Tarantilis et al., 1995; Dufresne et
al., 1997)-the part being the storehouse of the caretenoids which form the major
share. The caretenoids present in minor quantities include alpha and beta carotene,
lycopene and zeaxanthin as well as a conjugated xanthocarotenoid. Saffron
contains flavonoids and one of the general characteristic defining this extensive
group of compounds is bitterness. The first researchers to identify a flavonoid in
saffron extracts through mass spectrometry were Tarantilis et al., 1995. The
characteristic bitter taste of saffron has been postulated due to the presence of a
glycoside namely picrocrocin whose structure was established by Khun and
Winterstein (1934). Picrocrocin is a precursor of safranal, the major compound
responsible for saffron aroma. The study of saffron aroma began around the first
quarter of the 20th century with the isolation and identification of safranal, the
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major aromatic compound. It is generated from crocetin esters and was obtained
for the first time in 1922 by Winterstein and Teleczky by means of alkaline or acid
hydrolysis of picrocrocin. Carotenoid degradation either by thermal treatment or
enzyme activity gives rise to small compounds that contribute to aroma and flavor.
Saffron is also a rich source of proteins, vitamins (riboflavin and thiamine),
potassium, iron, copper, zinc, sodium and manganese thus imparting antioxidant
property to it together with the status of functional food (Delgado et al., 2006).
Records on ancient cultures established in Mesopotamia, depict use of saffron
principally as a condiment in religious rites and celebrations, and also as a dye for
their clothes (Perez, 1995). Egyptians and Hebrews used it to carry out ablutions
in temples and sacred places (Capel and Girbes, 1988). Saffron, its extracts and
tinctures have been used in traditional medicine as an antispasmodic, eupeptic,
sedative, carminative, diaphoretic, expectorant, stomachic, stimulant, aphrodisiac,
emenagogue and abortive agents (Basker and Negbi, 1983; Rios et al., 1996). It
has also been used for the treatment of ocular and cutaneous conditions (Xuon et
al., 1999), lowering blood pressure (Abe, et al., 1999; Soeda et al., 2001), for
wounds, fractures and joint pain; to prevent plague and other epidemics; to cure
anaemia, migranes and insomnia, promoting and regulating menstrual periods
(Akhondzadh et al., 2004), sores and as a cardiotonic (Verma and Bordia,1998;
Schmidth et al., 2007; Bathaie and Mousavi, 2010) and treatment of respiratory
disorders (Xiag et al., 2006; Xi, 2007). It is known for its antigastric effects (Al-
Mofleh et al., 2006), antidiabetic activity (Liu et al., 2005; Shen and Quin, 2006),
anticonvulsion and antidepressant remedy (Zhang et al., 1994), anti-inflammatory
effect (Hosseinzadh and Sadeghnia, 2007), antigenotoxic effect (Prem kumar
et al., 2003), antioxidant activity (Chatterjee et al., 2005), antitumoural and
anticarcinogenic activity and its cytotoxic and antimutagenic effects have also
been reported (Abdullaev, 2002). It is also used as a tonic and promoter of
defenses in Ayurvedic medicine, for some disorders of the central nervous system
in Chinese medicine and for homeopathic preparations.
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Saffron finds its use in the perfumery and cosmetic industry, besides, the most
important current use in food industry. This spice forms a part of some of the best
known traditional dishes. It is used to dye high textiles manufactured with silk,
cotton or wool (Takaoka et al., 1992; Tsatsaroni and Eleftheriadis, 1994;
Liakopoulou-Kyriakides et al., 1998; Tsatsaroni et al., 1998). As a dye, it is also
utilized in combination with hematoxylin, erythrosine and others to achieve
human and animal histological staining (Desmettre et al., 2001; Rostoker et al.,
2001; Alyahya et al., 2002; Edston, 2002). In Kashmir, saffron has a long history
of being used in culinary (Kashmiri cusine, wazwaan) and Kashmiri tea (Kehwa).
It is also widely used in confectionary, alcoholic and non alcoholic beverages,
colouring agent for sausages, oleomargarines, dairy products such as butter, cheese
and icecream for color and flavour improvement (Hosseini, 2000).
Taking into view the objectives of present investigation, the available literature
relevant to various aspects of study is reviewed in present chapter.
II.1 Micro-propagation
In 1838, Schwann and Scheilden put forward the so-called totipotency theory
which states that cells are autonomic and in principle are capable of regeneration
so as to give rise to a complete plant. Their theory was in fact the foundation of
plant cell and tissue culture (Pierik, 1999). Two concepts, plasticity and
totipotency, are central to understanding of plant cell culture and regeneration.
Particularly important aspects of this adaptation as far as plant tissue culture and
regeneration are concerned are the abilities to initiate cell division from almost any
tissue of the plant and to regenerate lost organs or undergo different
developmental pathways in response to particular stimuli. When plant cells and
tissues are cultured in vitro they generally exhibit a very high degree of plasticity
which allows one type of tissue or organ to be differentiated from another type. In
this way, whole plants can be subsequently regenerated. This regeneration of
whole organisms depends upon the concept that all plant cells, given the correct
stimuli, express the total genetic potential of the parent plant (Razdan, 2000).
Trecul (1853) pointed out that establishment of tissue culture involves the process
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of cell dedifferentiation. Buvat (1944, 1945) devised a scheme according to which
dedifferentiation involves two steps: 1) regression to the cambial stage and 2)
return to the cytological structure of primary meristematic cells. The first stage
affects highly differentiated cells while the second is accompanied by the
organization of bud or root meristems. Regeneration of whole plants in tissue
cultures may occur via shoot or root differentiation. Alternatively, the cells may
undergo embrogenic development to give rise to bipolar embryos also called
embryoids.
Within the last few decades, an increasing number of bulbous and cormous
monocotyledons have been successfully cultured. Tissue culture technology was
greatly influenced by the demand and rapid multiplication and clonal propagation
of slow growing monocots. Schenk and Hildebrandt (1972) reported the
importance of medium composition and techniques for induction and growth of
monocotyledonous and dicotyledonous plants in cell culture. They found that a
high level of auxin-type growth regulating substance generally favoured cell
cultures of monocotyledonous plants while low levels of cytokinins were essential
for most dicotyledonous cell structures. Several economically important monocot
species constituting nutritional, medicinal or ornamental groups of plants were
used for in vitro clonal propagation (Sutter, 1986) and production of secondary
metabolites (Aslanyants et al., 1988).
In vitro asexual multiplication of plant tissues into new plants has been
successfully used to speed up the initial stages of saffron corm production
programmes by supplementing them with transplants obtained from pathogen free
micropropagated material. Approach offers the capability to produce large
quantities of propagating material in short time as well as the production of
commercially important chemical constituents like crocin, picrocrocin and safranal
(Mushtaq et al., 2014). Micropropagation protocols are presently underway as an
alternative route for propagation. Explants from corm tissues, lateral or apical
buds, leaf or nodal tissues and different floral parts have been used for in vitro
regeneration of saffron (Homes et al., 1987; Huang, 1987; Illahi et al., 1987; Isa
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Ogasawara, 1988; Ahuja et al., 1993; Dhar and Sapru, 1993; Karamian et al.,
2004; Koraoglu et al., 2007).
Ding et al. (1979, 1981) was the first to report the successful tissue culture of
Crocus. They successfully regenerated callus and intact plantlets from corm
explants on culture media containing indole-3-acetic acid (IAA) and 2,4-D.
Similar results have also been reported by Illahi et al. (1987) using low levels of
2,4-D (0.1 mg l-1) and NAA (2 mg l-1).
Laneri and Lucretti (1983a) reported regeneration of shoots from young, dormant
and germinating corms. Several morphogenetic events occurred according to
explants and growth regulators used. The development of dormant axillary bud
were observed with incorporation of BAP in the culture medium alone or in
combination with NAA while the induction of adventitious shoots were observed
from young leaves and floral primordial with higher levels of BAP and NAA.
Young flower buds or corm tissues were also reported to induce cormels in liquid
medium with better response under dark conditions. Laneri and Lucretti (1983b)
reported callus formation on medium supplemented with 10 µM NAA with
subsequent formation of numerous tiny corm like structures originating from
compact callus on liquid medium containing 30 µM BAP and 2.5 µM NAA.
Sutter (1986) reported that axillay bud proliferation in presence of BAP can reduce
the formation of callus which may result in ploidy changes as is the case in other
bulb or corm producing species such as gladiolus. On the other hand, it is critical
to use the lowest possible concentration of 2,4-D to minimize the generation of
somaclonal variation in the cultures for multiplication purposes.
Infections caused by viruses, mycoplasma, bacteria, and fungi can be eliminated
using meristem-tip culture, which is one of the most useful applications of tissue
culture applied to many vegetatively propagated crops (Boxus and Druart, 1986;
Jones, 1986; Pierik, 1987; Slack and Tufford, 1995). It is based on the fact that the
extreme tip of the meristem (0.1-0.5 mm) is normally free from internal bacterial,
fungal and particularly viral contamination and plants regenerated from this zone
are usually pathogen-free (Collin and Edwards, 1998).
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Sano and Himeno (1987) used excised young intact stigmas plus ovaries of Crocus
sativus L. for production of stigma like structures (SLS). MS medium
supplemented with either cytokinins or auxins or in combinations favoured SLS
production. Benzyladenine and kinetin at concentrations of 0.1, 1.0 and 5.0 mg l-1
supported growth together with in vitro biosynthesis of crocin in stigma. Auxins
had little effect. LS medium supplemented with kinetin at concentration of 1 or 5
mg l-1 and NAA or IBA at a concentration of 10 mg l-1 in combinations recorded
stigma like structures directly or indirectly through meristematic tissues.
Minicorm development in saffron was reported by Homes et al. (1987) after
culturing explants on combination of MS or B5 minerals and organic media
supplemented with 1 mg l-1 2,4-D and 0.1 mg l-1 kinetin and subsequent transfer of
callus to the same basic media containing 2 mg l-1 2,4-D. Similar reports of callus
formation on N6 medium (Chu, 1978) with 2 mg l-1 2,4-D and 0.5 mg l-1 BAP
were reported by Huang (1987). After transferring on MS medium supplemented
with 0.5 mg l-1 BAP and 0.2 mg l-1 NAA, calli differentiated into buds that
germinated with high frequency in half strength MS media with 1 mg l-1 NAA
after 8 months at 150C. Ilahi et al. (1987) cultured saffron corms on half strength
MS medium supplemented with different combinations of growth regulators viz.
auxin, cytokinins, and coconut milk. Callus was induced on medium containing
0.5 mg l-1 each of 2,4-D and BAP and 2% coconut milk. Increase in 2,4-D
enhanced callus formation but suppressed shoot-bud formation. Transfer of callus
to MS medium containing 0.5 mg l-1 NAA, 0.1 mg l-1 of either BAP or kinetin and
2% coconut milk gave rise to roots after 4 weeks of culture with subsequent
suppression of the shoot development.
Gui et al. (1988) reported corm development on MS medium supplemented with
2.5 mg l-1 BAP and 5 mg l-1 IAA using corm explants with dormant buds. Isa and
Ogasawara (1987, 1988) reported two efficient methods for regenerating shoots
from corm callus. One was based on the subculturing of calli induced by 0.5 mg l-1
2,4-D in combination with 0.3 mg l-1 zeatin at 250C in dark in liquid MS medium
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with 1 mg l-1 2,4-D. Globular structures were formed which in presence of 1 or 3
mg l-1 BAP and 1 mg l-1 NAA differentiated into shoots after 3 months.
Fakhrai and Evans (1989) used explants of leaves, basal plates, petals, anthers and
ovaries of young growing corms of Crocus chrysanthus var E.P Bowlers to study
morphogenic potential. No major change was observed except on ovary explants
after sub-culturing of explants on MS basal medium with 20 different
combinations of either kinetin and NAA or BAP and 2,4-D in the dark. Corm
formation and shoot regeneration was obtained from the callus when the ovary
explants were cultured on media containing 50 mg l-1 and 10 mg l-1 BAP and 0.5
mg l-1 2,4-D. Increasing the level of 2,4-D markedly reduced the number of shoots
produced per explants.
Plessner et al. (1990) reported in vitro corm production in saffron using smaller
corms about 1 cm in diameter and apical buds isolated from larger corms as
explants using MS medium (Murashige and Skoog, 1962) supplemented with
sucrose (3%), nicotinic acid (5 mg l-1), pyrodoxine-HCL (1 mg l-1), thiamine-HCl
(0.5 mg l-1), myo-inositol (100 mg l-1), adenine sulphate (160 mg l-1) and casein
hydrolysate (500 mg l-1) and growth hormones viz. 1 mg l-1 2,4-D, 3-12 mg l-1
kinetin and 3 mg l-1 zeatin. Study revealed that cytokinin, particularly zeatin, and
auxin namely 2,4-D were essential for regular bud development in vitro. Study on
effects of ethylene and ethapon on organogenesis revealed that ethylene and
ethaphon pretreatments inhibited leaf development and induced corm production
and dormancy.
A study on in vitro production of stigma like structures from stigma explants of
Crocus sativus L. by Sarma et al. (1990) revealed that SLS were produced from
stigma explants of Crocus sativus L. under defined conditions. MS medium
supplemented with NAA (10 mg dm-3) + BAP (1.0 mg dm-3) induced the optimum
response. NAA was found to be an important addendum to achieve good response.
A culture temperature of 200C seemed better than 25oC.
George et al. (1992) obtained callus initiation from meristematic regions of corms
of Crocus sativus L. on Murashige and Skoog's (1962) medium (MS)
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supplemented with 2,4-D (2 mg l-1) and kinetin (0.5 mg l-1). Somatic
embryogenesis was obtained on transfer of callus to MS medium supplemented
with indole-3-acetic acid (2 mg l-1), kinetin (2 mg l-1) and ascorbic acid (100 mg
l-1). The globular embryos on 1/2 strength MS liquid medium with 1 mg l-1
abscisic acid showed further differentiation. Adventitious shoots were obtained
from callus on MS medium with NAA (2 mg l-1) and kinetin (4 mg l-1). Plantlet
formation was obtained from globular callus cultured using filter paper support in
liquid medium containing MS basal salts with indole-3-acetic acid (2 mg l-1),
kinetin (2 mg l-1) and ascorbic acid (100 mg l-1).
Dhar and Sapru (1993) studied in vitro production of corm and shoot like
structures using 0.1% mercuric chloride for sterilization and MS medium
containing 2% sucrose supplemented with NAA (1-3 mg l-1), 2,4-D (2-5 mg l-1)
and kinetin (1-5 mg l-1). Results revealed that floral apices used as explants
responded to callus production on MS medium supplemented with 2 mg l-1 each of
2,4-D and kinetin. Kinetin (2 mg l-1) in combination with NAA (1 mg l-1) helped
in callus proliferation on MS medium.
Aguero et al. (1994) described two main phases for effective saffron
micropropagataion namely shoot multiplication and bulbification. Multiplication
phase was achieved through scission of lateral buds of adult corm on Murashige
and Skoog medium (MS) half strengthened in nitrogen supplemented with 1.5 mg
l-1 BAP and 30 g l-1 sucrose. After every 40 days the mass of newly developed
buds were divided into two explants which were transferred to the same medium.
The bulbification phase was achieved when explants from the multiplication phase
were cultured on the same medium but deprived of growth regulators and
supplemented with 60 g l-1 sucrose. Short day conditions (8h) under the
photosynthetic photon flux (PPF) of 50 μmolm-2s-1 and moderate temperature
(150C) produced big sized minicorms after 120-150 days of in vitro culture
showing the possibility to harvest 60-70 minicorms with a mean fresh weight of
0.5-1.25 g after one year of culture.
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Ahuja et al. (1993) reported corm differentiation and development at the base of
excised shoots proliferated from callus cultures. Induction from cultured bulblets
was achieved on half-strength Murashige and Skoog (MS) basal medium
containing BAP (5×10-6 M) and NAA (5×10-6 M) + 2% activated charcoal
incubated at 15±10C. Microsurgery of the apical meristematic bud in corms prior
to culture increased the induction of cormogenic nodules. High concentrations of
BAP (2 mgcnt.dotl-1) and low levels of 2,4-D (0.1 mgcnt.dotl-1) were found to be
essential for the development and proliferation of cormogenic nodules. The
application of paclobutrazol and imazalil increased the induction rate of
adventitious shoots in the nodular cormogenic calli and the growth of microcorms.
Somatic embryogenesis was initiated in Crocus sativus L. from shoot meristem on
LS medium containing BAP (2x10-5 M) + NAA (2x10-5 M) by Ahuja et al. (1994).
Various stages of somatic embryogenesis were observed in the same medium and
the development was asynchronous. Somatic embryo development preceded
through well recognized sequence (globular to embryoids) with clearly
discremible bipolar regions. Matured embryos germinated on half strength MS
medium containing GA3 (20 mg l-1). Complete plantlets with well developed root
system and corm formation were obtained on transferring germinated embryos on
half strength MS supplemented with BAP (5x10-6 M) + 2% activated charcoal.
Root tip squashes of plantlets regenerated from somatic embryos had normal
(2n=3x=24) chromosomes number.
Igarashi et al. (1994) reported that nodular calli was induced with NAA in
combination with cytokinin, especially BAP. Appropriate concentrations for the
induction of nodular calli were 0.5-50 µM NAA and 0.5-50 µM BAP. Frequency
of callus induction was 10-15%. Poor callus induction and necrosis were observed
in 2,4-D containing medium with or without cytokinin. Vegetative shoots were
formed 20-40 days after transplantation at the regeneration frequency of 20-50%
in the wide range of NAA and BAP concentrations with maximal regeneration
frequency (50%) with 2.5 shoots per callus observed with 0.5-50 µM NAA in
combination with 0.5-5 50 µM BAP.
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Piqueras et al. (1995) from their studies on micropropagation of saffron (Crocus
sativus L.) by microcorm regeneration reported 50% reduction in explant
contamination by using ultrasonic bath. Basal medium supplemented with 0.1 mg
l-1 2,4-D + 0.5 mg l-1 BAP developed nodular calli with some adventitious shoots
after 8 weeks which on transfer to basal medium developed into green plantlets in
6 weeks. The results are partially in agreement with Ilahi et al. (1987) and Isa and
Ogasawara (1988).
Direct adventitious shoot regeneration from ovary explants of Crocus sativus L.
was revealed by Bhagyalakshmi (1999) and emphasized the role of media
components, incubation conditions and age of the explants on regeneration
potential. Full strength MS medium supplemented with 0.54 μM NAA and 2.22
μM BA produced the best shoot response both in terms of leaf length and number.
Ovaries of different growth stages having stigmas of pale yellow, pale orange and
bright orange regenerated a maximum mean number of shoots per ovary. Further
development of ovary-derived shoots was influenced by the composition of basal
salts in the culture medium where full strength MS salts gave the best response of
those tested. Regenerated shoots produced normal photosynthetic leaves and
corms.
In vitro micropropagation studies by Koul et al. (1999) revealed possibility to
regenerate saffron plantlets and in vitro corm development through somatic
embryogenesis/organogenesis. Bipolar somatic embryos germinated on MS
medium containing GA3 which on transfer to half strength MS medium
supplemented with BA + NAA resulted into formation of complete plantlets. The
shoots/plantlets regenerated via organogenesis/somatic embryogenesis when
excised and incubated in medium containing BAP + NAA at 150C in dark
developed corms at the base.
Loskutov et al. (1999) conducted studies to optimize the in vitro production of
stigma like structures (SLS) that produced important biochemical constituents
responsible for colour, taste and aroma naturally found in the stigmas of autumn
Crocus. The optimum proliferation of SLS was developed on B5 basal medium
20
containing NAA (5.4 µM), BAP (44.4 µM), MS organics, casein hydrolysate
(0.05%) and L-alanine (11.2 mM) after inoculation. Some explants formed other
structures (root, corms, petals, leaves), the growth and development of which
substantially reduced the development of SLS. Removal of brown tissues and
other tissues during subculture allowed continuous culture of half ovary explants
for 9-10 months. Activated charcoal (1%) added to B5 basal medium containing
NAA (5.4 µM), BA (44.4 µM) and sucrose (3%) was found to be helpful
addendum to prevent browning of explants and to accelerate the initiation, growth
and development of SLS.
Piqueras et al. (1999) while studying the development of cormogenic nodules and
microcorms by tissue culture reported that microsurgery of the apical meristematic
bud in corms prior to culture increased the induction of cormogenic nodules.
Positive effect of microsurgery is related to an increase in ethylene production
upon wounding caused by removal of the meristems since the application of this
plant growth regulator to isolated buds have been shown to induce the
development of both axillary and adventitious buds in several species as iris (Perl,
1985), hyacinth (Pierik and Steegmans, 1975), potatoes (Palmer and Barker, 1973)
and lilium (Van Aartrijk and Bloom-Barnhoorn, 1986). High concentration of
BAP (2 mg l-1) and low of 2,4-D (0.1 mg l-1) were found to be essential for
development and proliferation of cormogenic nodules. A high cytokinin/auxin
ratio has been considered necessary for shoot induction and development in plant
tissue culture (Skoog and Miller, 1957; Hussey, 1975). The application of
paclobutrazol and imazalil increased the induction rate of adventitious shoots in
nodular cormogenic calli and the growth of microcorms. The corms with
adventitious shoots were rooted in medium without growth regulators and were
able to generate dormant microcorms in vitro.
Sharma et al. (1999) reported three modes of regeneration system which include
direct organogenesis of shoot from corm upper segment, callus through shoot bud
organogenesis with corm middle segment and via callus through somatic
embryogenesis with bulblet explants induced respectively on B-5 and MS medium
21
supplemented with various levels of BAP and NAA. Complete plantlets with
slight base swelling were obtained on transferring germinated embryos and shoots
to 1/2 strength MS medium with high sucrose level (6% w/v) fortified with NAA.
A study on comparative effect of BAP and TDZ on multiplication of
micropropagated saffron (Crocus sativus L.) corms by Blazquez (2001) revealed
that TDZ (0.1 mg l-1) was significantly more efficient for production of
microcorms with fully developed leaf primordial than BAP (2 mg l-1).
Study on somatic embryogenesis in saffron (Crocus sativus L.), morphological
differentiation and the role of antioxidant enzymatic system by Blazquez et al.
(2004a) revealed that development of embryoids was characterized by the
emergence of shoot apical meristem and cotyledon (monopolar stage) and
subsequent differentiation of minicorm in the basal part of the embryo (dipolar
stage). The asynchronous mode of development observed in the embryogenic
callus of saffron has been previously described in different monocots during
somatic embryogenesis (Wang et al., 1999; Fereol et al., 2002). During
morphological differentiation, changes in the antioxidant enzymatic system of
somatic embryos were detected with increased ASOD and catalase activity during
the initial stages of the process which were in agreement with the sequence of
embryogenic differentiation reported in several cases of monocots (Ho and Vasil,
1983; Fransz and Schell, 1991; Samaj et al., 2003).
Blazquez et al. (2004b) from their studies on somatic embryogenesis in saffron:
optimization through temporary immersion and polyamine metabolism reported
that saffron embryogenic calli increased fresh weight four times when cultured in
temporary immersion system compared to those cultured on solid medium. 1 mg l-
1 paclobutrazol was effective in reducing hyperhydricity in the explants. The
development of somatic embryos was improved on solid medium supplemented
with 0.5 mg l-1 jasmonic acid (JA). Plant regeneration via somatic embryogenesis
was obtained after eight weeks of treatment with combination of JA and sucrose.
Jasmonates are involved and enhance the development of tubers, bulbs and corms
of several geophytes owing to their positive influence of carbohydrate
22
accumulation (Ravnikar et al., 1993; Koda, 1997; Santos and Salema, 2000).
Increased level of polyamines were found during embryo development.
Karamain (2004) from his studies on plantlet regeneration via somatic
embryogenesis in four species of Crocus viz. C. sativus, C. cancellatus, C.
michelsonii and C. caspicus reported that somatic embryogenesis was initiated
using shoot meristem culture on LS medium containing 4 mg l-1 NAA and 4 mg l-1
BAP or 1 mg l-1 2,4-D and 4 mg l-1 kinetin. Somatic embryogenesis was
asynchronous in all the four species and various stages of somatic embryo
development were observed when embryogenic calli with globular somatic
embryos were transferred to half strength MS medium containing 1 mg l-1 abscisic
acid. Complete plantlets were obtained by transferring germinated embryos to half
strength MS medium supplemented with 1 mg l-1 NAA and 1 mg l-1 BAP at 200C
under 16/8 hr (light/dark) cycle which are in agreement with the earlier reports of
this genus (Ebrahimzadeh et al., 2000). Many aspects can affect the maturation
and germination process such as temperature and light conditions, age of explants
and concentration of growth regulators (Firoozabday and DeBoer, 1993).
Zhigang et al. (2005) reported enhancement of cell growth in suspension cultures
by investigating the relationship between morphological transformation and cell
growth in callus and suspension cultures of saffron cells belonging to the cell line
C96 induced from Crocus sativus L. An unbalanced osmotic pressure between the
intra and extra cell regions induced large morphological transformation which
affected normal division of the saffron cells. An increase in osmotic pressure
caused by the addition of sucrose inhibited the vacuolation and shrinkage of
cytoplasm in the cells. As the sucrose concentration increased, the total amount of
accumulated biomass also increased. Besides sucrose concentration, increased
ionic strength and inoculation ratio also restrained to a large extent the vacuolation
and shrinkage of the cytoplasm in the suspended cells which resulted in increased
biomass.
To optimize an in vitro protocol for propagation of saffron through somatic
embryogenesis, effect of various concentrations of 2,4-D (0, 0.25, 0.5, 1, 2, 4 and
23
8 mg l-1) in combination with BAP (0, 0.25, 0.5, 1, 2, 4 and 8 mg l-1) were studied
by Saboora et al. (2006). Study revealed that 2.0 mg l-1 2,4-D + 1 mg l-1 BAP were
effective for induction of embryos. Comparative effect of different concentrations
of plant growth regulators in micropropagation of saffron by Taghizadeh et al.
(2006) revealed highest percentage of microcorm production with 4.5 mg l-1 2,4-D
in absence of kinetin. However, kinetin in lower concentrations induced callus
production.
Study on the effect of different hormone treatments on non-embryogenic and
embryogenic callus induction and time-term enzyme treatment on number and
viability of isolated protoplasts derived from embryogenic callus on saffron by
Darvishi et al. (2007) revealed that culturing of apical meristem of young corms
after sterilization on solid LS medium supplemented with 2 mg l-1 NAA and BAP
exhibited best effect on induction of non-embryogenic callus whereas 1 mg l-1 2,4-
D and BAP exhibited best effect on induction of embryogenic callus.
Jun (2007) reported that explant’s age according to style length, culture conditions
containing temperature and light and growth regulator were important factors
influencing in vitro flowering from styles of saffron. Style incised from 55-70 mm
length floral buds with light blue parienths as explants cultured on media
supplemented with 26.8 µM NAA and 31.1 µM BAP in dark at 200C were optimal
conditions for in vitro flowering.
Karaoglu et al. (2007) while studying various explants revealed that an
insignificant quantity of corms could be obtained by sterilization with 100%
commercial bleach for 20 minutes. Treatment with sulphuric acid or high
temperature treatments (47.50C) were damaging. Spores or dominant
microorganisms occurring as endogenic contaminants were resistant to
disinfectants under any condition of sterilization. These findings are partially in
line with Ozel et al. (2006) and Taylor et al. (1998). Stigma or leaf explant failed
to develop into cormlets even after a long period of culture. However, cormlet
regeneration via somatic embryogenesis was achieved from mature floral bases on
MS medium containing BAP (1 mg l-1) and NAA (1 mg l-1). After 3 weeks of
24
culture, the whole corms did not show any development. However the explants
consisting of eye buds showed development of single shoots with leggy
appearance after 6 week of culture which on subculturing on the same medium
showed regeneration of one or two cormlets from the main explants after 24 weeks
of culture.
To prevent low and heterogeneous morphogenic potential of meristems, a study on
enhanced plantlet regeneration from cultured meristems in sprouting buds of
saffron corms by Majourhat et al. (2007) revealed clear differences after culturing
of explants on different cytokinin types and concentrations (BAP, 2ip and TDZ)
for six weeks for number of new shoots per initial explants, their length and
quality. Higher multiplication rate (5.6) was observed with 5 mg l-1 BAP.
Raja et al. (2007) reported in vitro microcorm production by culturing leaf
segments of saffron on MS medium containing BAP (4.0 mg l-1) + NAA (0.50 mg
l-1) + 9% sucrose. Maximum explant survival was observed when leaf was used as
an explants source (48.90%) and incubated on MS medium supplemented with
BAP (1.0 mg l-1) and 2,4-D (1.0 mg l-1). Maximum proliferation of established
cultures (56.30) was obtained with BAP (2.0 mg l-1) + NAA (0.50 mg l-1). Plant
regeneration through somatic embryogenesis using regenerable embryogenic calli
was also obtained from leaf explants cultured on MS medium containing BAP (1.0
mg l-1) and 2,4-D (1.0 mg l-1). Somatic embryo formation was attained to the tune
of 8.66 embryos per culture by using MS medium supplemented with BAP (2.25
mg l-1 ) and 2,4-D (0.10 mg l-1). Matured embryos germinated after incubation on
MS medium containing GA3 (20.0 mg l-1) in combination with ABA (2.0 mg l-1)
after five days. An average of 10.82 shoots per explant of proliferated culture were
obtained when MS medium was supplemented with BAP (2.0 mg l-1) + NAA
(0.50 mg l-1). In vitro induction of embryogenesis in iridaceous genera was
reported earlier by Wang et al. (1990), Bach (1992), Kim and Kang (1992) and
Jehan et al. (1994).
Study on induction of somatic embryogenesis in saffron by Sheibani et al. (2007)
using different concentrations of TDZ (0, 0.1, 0.25 and 0.5 mg l-1) and 5 different
25
types of corm explants (terminal or axillary buds, upper or lower parts of the corm
tissue and terminal buds from pre-treated corms at 40C for 2 weeks) revealed that
TDZ concentrations affected the induction of somatic embryogenesis significantly
while different types of corm explants showed no significant effect on this process.
Among TDZ concentrations, 0.5 mg l-1 was the most effective treatment for
embryogenesis induction. Embryogenic calli (globular stage) proliferated well
when subcultured on MS medium supplemented with 0.25 mg l-1 TDZ before
transferring to hormone-free MS medium containing 6% sucrose for maturation
(scutellar or horn-shape stage). Matured embryos were transferred to half strength
MS medium without growth regulators which resulted in microcorm formation
from basal part after 3 months. Most of the matured embryos cultured on 1/2
strength MS medium without growth regulators produced plantlets with
microcorm and occasionally roots within 3 months whereas the ones cultured on
medium containing NAA (1 mg l-1) + BAP (1 mg l-1) returned to globular stage
and produced more globular embryos and callus.
Bhatti et al. (2009) reported successful ex vitro shoot regeneration from terminal
and lateral buds of saffron corms pulse treated with gibberelic acid (GA3), indole-
3-acetic acid (IAA) and thidiazuron (TDZ) at various concentrations and time
durations. Karamian and Ebrahimzadeh (2009) reported somatic embryogenesis
from shoot meristem culture on LS medium containing kinetin (4 mg l-1) and 2,4-
D (1 mg l-1). To initiate morphogenesis, embryogenic calli were transferred to
either half strength MS medium without growth regulators or with medium
supplemented with 1 mg l-1 abscisic acid. Sub-culturing of germinated embryos on
half strength MS media supplemented with 0.1 mg l-1 NAA and 1 mg l-1 BA lead
to plant regeneration. SLS were also obtained.
MalekZadeh et al. (2009) studied two culture media namely LS and MS in liquid
and solidified form supplemented with different concentrations of 2,4-D (2 mg l-1),
NAA (2 mg l-1) as auxins and BAP (1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg l-1), kinetin
(1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mg l-1) as cytokinins using apical and lateral
meristems of spring and autumn corms for regeneration ability of saffron.
26
Solidified LS medium supplemented with 4 mg l-1 BAP and 2 mg l-1 NAA showed
an adequate response for producing corm and shoots for cryopreservation of apical
and lateral corm meristems.
Mathe et al. (2009) conducted a study with an aim to investigate the tissue culture
potential of several members of Crocus genus within their natural habitat in
Carpathian Basin. Tissue culture was successfully established in case of Crocus
heuffelianus and Crocus scepusiensis. Embryogenic calli were induced through
sub-culturing of shoot tip meristems on MS medium supplemented with 2% (w/v)
sucrose, Gamborg’s vitamins, 0.8% (w/v) bacto-agar and the proper
auxin/cytokinin concentrations.
In vitro production of callus and microcorm formation in saffron (Crocus sativus
L.) was studied by Sharaf-Eldin et al. (2009). Protocol for callus induction and
microcorms production were established. Three media were compared using basal
MS medium containing 3% (w/v) sucrose supplemented with NAA (0.5 mg l-1) +
BAP (2 mg l-1); MS1, NAA (0.5 mg l-1); MS2 and BAP (3.5 mg l-1); MS3.
Various stages of somatic embryogenesis were observed in the same medium and
the development was asynchronous. Maximum explant response was observed
when sprouts or corms were used as explants and incubated on MS1 or MS2. The
highest percentage of callus induction or corm formation were obtained when
corm were used as explants and incubated on MS1 or MS2.
Sharifi and Ebrahimzadeh (2009) studied globular embryo like structures and high
efficiency multiple shoot formation in saffron (Crocus sativus L.) induced by
thidiazuron. To find out suitable conditions for shoot formation from corms, the
effect of cytokinins: N6-benzyladenine and N-phenyl-1,2,3 thidiazol-5-yl urea
known as thidiazuron were compared at different concentrations. Thidiazuron at
4.54 µM induced shooting in all corm explant with an average of 39.5±5.1
(shoots/corm) whereas benzyladenine induced multiple shoot formation in only
3.6-11.4% of corm explants. To optimize further plant regeneration from the
induced adventitious shoots obtained from thidiazuron treatment, they were
transferred to MS and B5 media supplemented with different concentration and
27
combinations of NAA and BAP. Highest rate of plant regeneration was observed
on G5 media supplemented with NAA and BAP at a concentration of 2.22 µM and
2.68 µM respectively. Upon optimized hormonal condition an average of
19.55±5.75 shoots and 3.18±1.5 roots per explants were obtained.
Vatankhah et al. (2009) studied the effect of various hormonal combinations on
regeneration of shoots and roots from meristems of Crocus sativus L. The most
efficient regeneration occurred with NAA (1 mg dm-3) + thidiazuron (1 mg dm-3)
and NAA (1 mg dm-3) + kinetin (2 mg dm-3). For sprouting, regenerated shoots
were sub cultured on MS medium containing NAA (1 mg dm-3) + BAP (1mg dm-
3).
Zaffar et al. (2009a) reported that saffron calli were induced from leaf explants on
MS medium supplemented with BAP and 2,4-D at concentration of 1 mg l-1 each.
After subculturing the calli two or three times, two types of calli, a nodular one
and a friable one were induced. The nodular calli had high regeneration ability and
resulted in development of shoots after incubation on MS medium supplemented
with different concentrations of BAP and NAA. Regeneration frequency was
optimal with BAP (2.0 mg l-1) + NAA (0.50 mg l-1). Altering the concentration of
ammonical (NH4+) and nitrate (NO3
-) forms of nitrogen resulted in rooting of
proliferated cultures and both the rooting frequency and the average number of
roots per proliferated culture showed maximum values when incubated on MS
medium containing 40 mM NH4 + 20 mM NO3..
Studies on effect of media composition and growth conditions on in vitro
microcorm formation in saffron (Crocus sativus L.) by Zaffar et al. (2009b)
revealed that higher concentration of BAP and paclobutrazol in MS media resulted
in formation of bigger microcorms. Low temperature (150C) and incubation under
light appeared to be optimum for microcorm formation resulting in thickening of
the basal parts of the shoots suggesting synergistic interactions between low
temperature and light. Microcorm formation synchronized with in vivo
developmental cycle. The microcorms had a high survival rate upon transferring
28
directly into soil after preconditioning at temperature of 300C for 1 month and
storage upto planting time at 50C.
Devi et al. (2010) used Murashige & Skoog (1962) medium containing BAP and
2,4-D. Irrespective of the corm size, bud sprouting was season dependent.
Maximum bud sprouting occurred in the months from November to December and
minimum from May to August. Buds sprouted from the months of March to May
regained further growth only in the next growing phase i.e., from September to
December, indicating that the in vitro propagules followed the same natural
rhythm for bud sprouting. Young leaves from these bud sprouts were used as
explants for initiation of somatic embryos using TDZ and picloram. Lumps of
somatic embryos proliferated further to form secondary somatic embryos.
Different treatments of PGRs (ABA, GA3, BAP, 2,4-D, IAA, NAA) of varying
medium strengths (¼, ½, ¾) and sucrose concentrations (3, 6, 9, 12%) were used
for the conversion of these embryos into plantlets. Cormlets developed at the base
of these plantlets on medium supplemented with growth retardants (paclobutrazol
& CCC). These in vitro produced cormlets were transferred to green house
conditions for further growth evaluation.
Mir et al. (2010a) obtained a corm size (1.3 g) from eye bud explants cultured on
LS media supplemented with 21.6 µM NAA and 22.2 µM BAP. Microcorm
formation was influenced by external BAP concentration; also growth of corm was
improved with increased period of inoculation. Regenerated corms were kept at
5oC for 5 weeks and then transplanted to a potting mixture where the germination
percentage was very low (4%) which may be due to small corm size.
In another study by Mir et al. (2010b) for development of stigma like structures
under in vitro conditions, various explants were cultured on LS, MS, and G5
media supplemented with different combinations of phytohormones. Stigma-like
structures appeared on cultured half ovaries and the best response (60%) was
observed with half ovaries cultured on G5 media supplemented with 27 µM NAA
and 44.4 µM BAP followed by 55% on LS media supplemented with 27 µM NAA
and 44.4 µM BAP. The length of SLS ranged from 4 to 6 cm and number of SLS
29
varied from 8-12. The accumulation of pigments like safranal, crocin and
picocrocin has been confirmed by GCMS and spectrophotometrically.
Field evaluation of 8200 in vitro corms weighing <5 g and 5-7 g at Pampore,
Kashmir by Nehvi et al. (2010c) confirmed survival of above 60% among in vitro
corms weighing 5-7 g. Flowering percentage was recorded to be less than 1% in
the first year. Corm yield m-2 doubled after one year of plantation. Corm number
increased three times in 5-7 g category with an average corm number of 2.81
corms/mother corm whereas for <5 g category it was 1.5 times with 0.88
corms/mother corm.
In vitro studies on application of biotechnological tools for saffron propagation by
Parray et al., (2010) revealed maximum number of cormlets (70±3) from corm
slices cultured on half strength MS medium supplemented with thidiazuron (TDZ)
20 µM + indole-3- acetic acid (IAA) 10 µM + sucrose 40 g l-1. The prominent
increase in corm size with a weight range of 1.9-2.1 g was recorded on thidiazuron
(TDZ) 15 µM + indole-3-acetic acid (IAA) 12.5 µM + sucrose 30 g l-1 in 40% of
in vitro raised minicormlets via callus. Apical vegetative buds of actively growing
corms were cultured for cormlet development and corms of size 2.5 g were
developed on MS medium with BAP (20 µM) + NAA (15 µM) + 30 g l-1 sucrose.
50% recorded most prominent increase in corm size with a weight range of 1.5-2.5
g. Vegetative growth in in vitro corms was observed in above 90% corms
transferred in cups containing soil collected from collection sites under green
house conditions at a temperature of 200C.
Study on the effect of paclobutrazol on in vitro corm formation and enlargement in
saffron (Crocus sativus) by Zaffar et al. (2010) reported that in vitro propagation
of saffron through cormogenesis can be an efficient alternative method for large
scale propagation of pathogen free corms if a reproducible and efficient in vitro
micro-propagation protocol is available. Effects of different concentrations of
cytokinins in combination with paclobutrazol were investigated. In vitro
regenerated shoots from callus derived from corm sections were cultured on
Murashige and Skoog (MS) medium supplemented with (0.2-1.5 mg l-1) BAP,
30
paclobutrazol (2-10 mg l-1) and sucrose (3-12%). Higher concentrations of PAC (5
mg l-1) along with BAP (0.25 mg l-1) and 9% sucrose resulted in formation of
relatively larger microcorms. There was a significant interaction between
paclobutrozol and sucrose for microcorm weight.
A study on in vitro cormlet production and growth evaluation under greenhouse
conditions in saffron (Crocus sativus L.) – a commercially important crop reported
by Devi et al. (2011) described the effects of season on initial bud sprouting, direct
shoot regeneration from the base of the sprouted bud and cormlet production from
multiple shoots and provided growth evaluation of in vitro produced cormlets
under greenhouse conditions. Initial sprouting of buds from corm segments was
previously described in medium supplemented with 2,4-Dichlorophenoxyacetic
acid (2,4-D, 9.05 μM) and 6-benzylaminopurine (BAP, 26.64 μM). Maximum bud
sprouting (90%) was observed during November and December. Direct multiple
shoot primordia were initiated from the base of these sprouted buds on BAP (26.64
μM). Multiplication of shoots was achieved in BAP (26.64 μM) and α-naphthalene
acetic acid (1.0 and 5.0 μM). Growth retardants (chlorocholine chloride and
paclobutrazol) were used for cormlet production from multiple shoots and
paclobutrazol (1.7 μM) evinced maximum cormlet production (86.07%). Growth
of these in vitro produced cormlets was evaluated under greenhouse conditions and
91.66% sprouting was observed. An increase in cormlet weight (66.88%) was also
observed under in vivo conditions.
Devi et al. (2012) while studying the genome size in tissue culture raised plants of
saffron (Crocus sativus L.) revealed that genome size of tissue raised plants
remained stable. Directly regenerated shoots, somatic embryo derived plants and
mother plants i.e. plants produced under field conditions were assessed by
flowcytometery for genome size stability. Estimation of nuclear DNA content with
emphasis on nuclei preparation and testing of inhibitors for PI binding was carried
out. 3C nuclear DNA of somatic embryo derived and directly formed shoots was
9.74±0.02 and 9.73 ± 0.04 pg and 1C genome size was 4.81±0.01 x 109 bp and
31
4.80±0.01 x 109 bp, respectively. 3C nuclear DNA and IC genome size of mother
plant was 9.77±0.02 and 4.82±0.01 x 109 bp, respectively.
Study on CsSERK gene expression associated with shoot organogenesis
competence in saffron (Crocus sativus L.) by Vatankhah et al. (2012) was
conducted to study the expression of SERK gene inorganic (shooting) and non-
organic nodular calli using RT-PCR. Two hormonal treatments were used for
induction of shooting nodular calli (MS media containing NAA, TDZ and IBA,
TDZ) and two culture media namely MS and LS supplemented with 2,4-D and
kinetin for induction of non-organic nodular calli. Sequence analysis of CsSERK1
revealed high levels of similarity (85%) to AcSERK (Areca catechu SERK) gene.
Analysis of SERK expression showed that it could be detected in shooting nodular
calli before shoot development. In contrast, it was not detected in non-organic calli
thus suggesting that CsSERK expression is associated with induction of shoot
organogenesis and that it could be a potential marker for cells competent to form
shoot in saffron tissues cultured in vitro. Also SERK gene may have a broader role
in morphogenesis in cultured tissue rather than being specific to somatic
embryogenesis.
A study on influence of 2,4-D and kinetin combinations on callus induction in
saffron (Crocus sativus L.) was carried by Vahedi et al. (2012) to develop a
protocol for callus induction exploiting the influence of 2,4-D and kinetin in
different combinations. Lateral and terminal meristem of plants were collected and
inoculated on MS media supplemented with 2,4-D (1, 2 and 4 mg l-1) and kinetin
(0.5, 1, 4 and 8 mg l-1) in different combinations with 3% sucrose for callus
induction after thorough surface sterilization. The first callus was induced after 35
days of inoculation from terminal meristem explants. However, lateral meristem
was observed to be less responsive. The highest frequency of callus induction was
achieved on MS medium supplemented with 2,4-D (2 mg l-1) and kinetin (0.5 mg
l-1).
Mir et al. (2012) reported production of stigma like structures (SLS) under in vitro
conditions. Highest response was observed with half ovaries on G5 media
32
supplemented with different combinations of phytohormones. The relative
quantification through RT-PCR for expression of apocarotenoid genes like CsLYC,
CsZCD, CsBCH and CsGT-2 revealed that there was an increase in expression
from callus to SLS development. Expression pattern of CsLYC, CsZCD, CsBCH
and CsGT-2 was also studied in different flower parts and highest expression was
found in stigma followed by style and petal. Expression of the regulatory genes
responsible for biosynthesis of apocarotenoids viz. crocin, picrocrocin and safranal
was upregulated in SLS in saffron revealing that these structures are
developmentally closely related to natural stigma.
Parray et al. (2012) studied the influence of plant growth promoting rhizobacteria
(PGPR) on the size of cormlets of Crocus sativus Kashmirianus under in vitro
conditions. The study was conducted to observe the growth of bacterised tissue
cultured cormlets under in vitro conditions after 4-6 weeks of growth on MS
medium supplemented with BAP (20 µM) + NAA (15 µM). The study revealed
the effects of three plant growth promoting rhizobacteria (PGPR) i.e.
Pseudomonas sps., Bacillus subtilis and Acintobacter sps isolated from saffron
rhizosphere soil on increasing the size of cormlets under in vitro conditions. The
minicorms were subjected to three treatments; in the first treatment they were co-
cultivated with individual bacterial strains, in the second treatment co-cultivated
with combinations of three strains and in the last treatment cultured without strains
(control). The results revealed that the combination of Bacillus subtilis and
Acintobacter sps proved useful in enhancing the size of cormlets upto 8 g
compared to the control (2-4 g). Introduction of rhizobacteria to saffron cells
during in vitro micropropagation process established an early associative
interaction between the plant cells and bacteria. In the association, the
rhizobacteria provided the host plants with phytohormones and other stimulators.
To develop a protocol for in vitro microcorm formation in saffron, effects of
different concentrations of cytokinins in combination with paclobutrazol were
investigated by Zaffar et al. (2012). Embryogenic callus derived by culturing leaf
segments/sprouted apical buds/corm sections on Murashige and Skoog (MS)
33
medium supplemented with 2,4-D (1.0 mg l-1) and BAP (1 mg l -1). Shoots were
regenerated from callus by subculturing on MS medium fortified with BAP (1.0
mg l-1) and NAA (1.0 mg l-1). In vitro regenerated shoots from callus were
cultured on Murashige and Skoog (MS) medium supplemented with BAP (0.2-1.5
mg l-1), paclobutrazol (PAC) 2-10 mg 1-1 and sucrose (3-12%). Higher
concentrations of PAC (5 mg l-1) along with BAP (0.25 mg l-1) and 9% sucrose
resulted in the formation of relatively larger microcorms. There was a significant
interaction between paclobutrazol and sucrose for microcorm weight.
Ziaratnia et al. (2012) studied in vitro callus induction and production of stigma-
like structures of saffron (Crocus sativus). Callus induction was carried out by
application of different combinations of plant growth regulators to saffron explants
grown on two types of medium, MS and B5. Results showed that B5 medium
supplemented with 2,4-D (2 mg l-1) and kinetin (4 mg l-1) and MS with NAA (8
mg l-1) and BAP (1 mg l-1) exhibited better callus induction. In another
experiment, production of stigma-like structure was tested from different types of
saffron explants including stigma, style, ovary and half ovary. For that purpose,
MS medium was supplemented with several levels of auxins (2,4-D and NAA)
and cytokinins (kinetin and BAP). Results showed that all types of explants from
saffron flowers were able to produce SLS except those from flower buds. It was
also found that among different types of explants, intact ovaries were more
suitable for production of direct SLS than others while the induction of indirect
SLS was higher on styles than intact and half ovaries. The best hormonal
combination for induction of direct and indirect SLS was kinetin (2 mg l-1), NAA
(8 mg l-1) and NAA (20 mg l-1), BAP (1 mg l-1) respectively. HPLC comparison of
natural stigma and SLS showed that all the three saffron constituents are present in
SLS derived in the study but at lower levels compared to natural ones.
Zeybek et al. (2012) developed an efficient in vitro tissue culture system for
saffron (Crocus sativus L.) complete with roots and corms. In indirect
organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, ascorbic
acid (100 mg l-1), and the combination of 2,4-D (0.25 mg l-1) and BAP (1 mg l-1)
34
were best for callus initiation and growth while BAP (1.5 mg l-1) was excellent for
high rate of adventitious shoot formation. Indole-3-butyric acid (1 mg l-1) was
more preferable for adventitious corm and root initiation as well as growth.
Overall, 64% rooting and 33% corm production rates were achieved in indirect
organogenesis. In direct organogenesis, MS medium supplemented with 3%
sucrose, ascorbic acid (100 mg l−1) and BAP (1 mg l−1) was optimum for shoot
growth. While IBA (1 mg l−1) was best for adventitious corm formation, IBA
(2 mg l−1) promoted adventitious root initiation and growth. Overall, 36% and 57%
of explants had corms and contractile roots respectively.
Simona et al. (2013) observed that 2,4-D (1 mg l-1) and BAP (1 mg l-1)
combination was favorable for direct organogenesis and 2,4-D (0.25 mg l-1) and
BAP (1 mg l-1) combination was superior for indirect organogenesis. Cavusoglu et
al. (2013) achieved in vitro plant regeneration and daughter corm formation of
saffron (Crocus sativus L.) from corm parts consisting of meristematic region via
direct organogenesis. For direct shoot regeneration and foliation, sterile
meristematic node-containing corm explants were cultured on 1/2 strength
Murashige and Skoog (MS) Medium or MS medium supplemented with BAP.
Maximum shoot initiation to the tune of 96.7% and maximum foliation rate
(93.3%) was observed using MS medium supplemented with BAP (6 mg l-1) while
MS + BAP (1 mg l-1) gave the least result in shoot initiation (16.7%); MS + BAP
(1 mg l-1) and MS + BAP (10 mg l-1) gave the least result in foliation from the
initiated shoots in 90 days. However, daughter corm formation and rooting were
achieved on MS supplemented with IBA or IAA. They showed that MS + IAA (1
mg l-1) have the best results on daughter corm formation rate (76.7%) and daughter
corm number (1.74 corms/corm). On the other hand, rooting rate (46.7%) and root
number (1.5) were highest on MS with IBA (2 mg l-1) in 120 days.
Mir et al. (2014) studied regeneration of microcorms under in vitro conditions.
Apical bud explants were cultured on different nutrient media supplemented with
various concentrations of growth regulators. Microcorm formation was observed
on all media combinations. Maximum number (10) and weight (1.54 g) of
35
microcorms developed were observed on MS media supplemented with BAP
(2 mg l-1) + NAA (0.5 mg l-1) + paclobutrazol (1.5 mg l-1). Shoot and root
regeneration was observed in the microcorms developed under in vitro conditions.
Maximum number of shoots (11.6) and shoot length (11.4 cm) was also observed
on MS media supplemented with NAA (2.16 µM) + BAP (22.2 µM). Maximum
number of roots (11) and length of roots (11.4 cm) were obtained on G5 media
containing NAA (21.6 µM) + BAP (22.2 µM).
II.2 Mutation
Genetic variations are the basic tools to develop new cultivars with better traits like
tolerance against various environmental stresses, resistance against pests and
diseases and improved yield and quality. Thus, mutagenesis technology has been
applied to plant breeding comprehensively which allow crops to produce beneficial
varieties with good traits (Maluszynski et al., 1995; Gu et al., 2003). Although
naturally occurring mutagenesis is very simple and requires no tools to be brought
about, it occurs at very low frequency to rely alone on for accelerated plant
breeding and is in most instances very lethal to plants and thus selection is
cumbersome. Besides spontaneous mutations, it is possible to induce them
artificially using mutagens. The only option left with the interested plant breeder is
to fully utilize the “induced-mutagenesis” technique in order to feed the
burgeoning population of the world and to wage war against the alarming
environmental stresses in the current century. The usefulness of any mutagen in
plant breeding depends not only on its mutagenic effectiveness but also on its
mutagenic efficiency, efficient mutagenesis being the product of the maximum
desirable changes accompanied by the least possible undesirable changes (Tamina
and Tabash, 2010). Effectiveness and efficiency are two distinct properties of
mutagens that have been extensively discussed elsewhere (Kawai, 1969, 1975;
Shah et al., 2008; Girija and Dhanavel, 2009). Effectiveness usually means the rate
of point mutations relative to dose whereas efficiency refers to the rate of point
mutations relative to other biological effects induced by the mutagen and is
considered a measure of damage (Konzak et al., 1965). Thus, the two agents may
36
be equal in mutagenic effectiveness because at a given dose, they induce a
mutation with the same frequency. However, when they diverge in their ability to
produce undesirable changes such as sterility and lethality, they may be said to
differ in mutagenic efficiency (Tamina and Tabash, 2010). Induced mutation using
physical and chemical mutagen is one method to create genetic variation resulting
into new varieties with better characteristic.
Historically, the use of mutagenesis in breeding has involved forward genetic
screens and selection of individual mutants with improved traits and their
incorporation into breeding programmes. Over the past 70 years, more than 2500
varieties derived from mutagenesis programmes have been released, as listed in the
IAEA/FAO mutant variety database, including 534 rice lines, 205 wheat lines, and
71 maize lines. Although this approach has clearly proved very successful, there
are limitations imposed by, for example, the difficulty of identifying a small
number of individuals with novel phenotypes within a large population or by the
genetic redundancy present in many plant species as a result of gene duplication
and polyploidy, such that many mutations have no detectable effect on the plant.
Recently, reverse genetic approaches have permitted the silencing or interruption
of individual candidate genes, providing the opportunity to investigate gene
function and to relate sequence information to traits. However, these approaches
have disadvantages: methods based on post-transcriptional gene silencing such as
RNAi have variable success rates and rely on time-consuming vector construction
and plant transformation. T-DNA insertional mutagenesis is also dependent on
efficient plant transformation while insertional mutagenesis by endogenous
transposons is only available in a small number of crops, notably maize, although
there has been some success in transferring these into other species such as rice
(Kolesnik et al., 2004). In any case, these insertional methods are likely to result in
complete disruption of gene function rather than in generating allelic series of
mutants with partial loss-of-function and thus will not produce the range of
mutation strengths necessary for crop improvement. Furthermore, the insertion
sites within the genome may not be distributed randomly, increasing the number of
37
insertion lines required for full genome coverage to unrealistic levels (Zhang et al.,
2007).
Mutations may be gross, resulting in large-scale deletions of DNA or only involve
point mutations. Induced mutation is one of the most widely used techniques for
creating additional variability in flower character. Exploiting natural or induced
genetic diversity is a proven strategy for improvement of all major food crops and
the use of mutagenesis to create novel variation is particularly valuable in crops
with restricted genetic variability due to inherent problem of reproductive sterility.
It is a technique being utilized both by nature and human beings in order to
improve the qualitative and quantitative traits in plants against various biotic and
abiotic stresses Chemical or physical mutagenesis have a number of advantages
over such approaches owing to the fact that mutagens introduce random changes
throughout the genome generating a wide variety of mutations in all target genes
and a single plant can contain a large number of different mutations resulting in
manageable population sizes (Parry et al., 2009).
Mutation can be induced by irradiation with non-ionizing (e.g. UV) or ionizing
radiation (e.g. X and gamma rays, alpha and beta rays, fast and slow neutrons);
such physical mutagens often result in large scale deletion of DNA and changes in
chromosome structure. While ionizing radiation still remains the most suitable
means for inducing variability (Wit et al., 1985; Brunner, 1995; Yang, 1995;
Hayashi et al., 1996; Xu et al., 1998; Bhatia et al., 2001; Zhang et al., 2002; Irfaq
and Nawab, 2003; Chen et al., 2004; Joseph et al., 2004; Lao et al., 2004; Sangsiri
et al., 2005; Sun et al., 2005; Tah, 2006), a number of chemicals have been found
to be equally and even many times more effective and efficient mutagens (Thakur
and Sethi, 1995; Kharkwal, 1998; Solanki, 2005; Rekha and Langer, 2007; Basu et
al., 2008, Dhanavel et al., 2008; Ganapathy et al., 2008; Thilagavathi and
Mullainathan, 2009; Wani, 2009). Ionizing radiations such as γ-rays are highly
effective in inducing chromosomal aberrations (Nilan and Konzak, 1961). Gamma
rays are the most energetic form of electromagnetic radiation, possesses the energy
level from 10 keV to several hundred kilo electron volts and are considered to be
38
most penetrating in comparison to other radiation such as alpha and beta rays
(Kovacs and Keresztes, 2002). Gamma rays belong to ionizing radiation and
interact on atoms or molecules to produce free radicals in cells. These radicals can
damage or modify important components of plant cells and have been reported to
affect differentially the morphology, anatomy, biochemistry and physiology of
plants depending on the irradiation level. Chemical mutagens usually cause point
mutation but loss of a chromosome segment or deletion can also occur
(Thilagavathi and Mullainathan, 2011). Mutagens such as EMS act primarily on
base pairs of the DNA molecule and yield a higher number of gene mutations.
Because of the basic mechanistic difference between these two groups of
mutagens, chemical mutagens are generally considered to be superior to physical
mutagens for induction of mutation (Nilan and Konzak, 1961). By contrast,
chemical mutagens most often only affect single nucleotide pairs. For plants, some
of the more widely used mutagens include hydrogen fluoride (HF), sodium azide,
N-methyl-N-nitrosourea (MNU), hydroxylamine and alkylating agents like ethyl
methane sulphonate (EMS) and methyl methane sulphonate (MMS). The alkyl
group of the chemical mutagens reacts with DNA which may change the
nucleotide sequence and cause a point mutation (Broertijes and Van Harten, 1988).
The choice of chemical mutagen influences the maximum permissible mutation
rate achievable: EMS creates a larger proportion of non-sense mutations involving
the introduction of novel stop codons than a mutagen such as MNU due to the
specificity of EMS in creating mainly G–A and C–T transitions and any individual
mutations is therefore more likely to have a phenotypic effect (Parry et al., 2009).
Mutagenesis plays an important role in creating new varieties in clonally
propagated plants because mutations in such plants may easily get stabilized and
can be manipulated. Critically, mutations in important traits or genes (e.g. in
nutritional quality, resource use efficiency, architecture or phenology) can be
readily exploited by plant breeders without the legislative restrictions, licensing
costs, and societal opposition applied to GM approaches. This is despite the fact
that transcriptomic analyses have shown that large-scale plant mutagenesis may
39
induce greater changes in gene expression patterns than transgene insertion (Batista
et al., 2008).
Though induced mutations are a valuable tool in crop improvement, work related
to saffron in this aspect is limited. Hence, research work done on mutation
breeding of other vegetatively propagated crops are also reviewed here under.
Gamma rays induced mutants in Gladiolus studied by Raghava et al. (1988)
revealed that percentage of sprouting was affected significantly at 10 and 15 Krad.
LD50 was found to be between 10 and 15 Krad. Doses of 10 Krad and above
proved to be detrimental for vegetative and floral traits. Plants treated with 10
Krad did not produce flower spikes whereas the plants in 15 Krad treatment
although sprouted did not survive afterwards. When treated with 10 Krad, plant
height, leaf number and leaf-size were reduced significantly and leaves became
narrow and leathery. Flowering was delayed significantly at 5 Krad. Radiation
treatments caused decrease in spike length, number of florets per spike and floret
size. A desirable and stable mutant with shell pink floret colour was isolated from
the variety 'Wild Rose' in 1 Krad treament and it has been released as 'Shobha'.
Khan (1999) conducted studies on gamma rays induced mutations in saffron
(Crocus sativus L.) with an aim to develop mutants of saffron which can flower
regularly by irradiating the corms with 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 Krad dose of
gamma rays. Lower doses (0.5, 1.5 Krad) resulted in increased plant height with a
corresponding negative effect on plant height with higher doses (1.0, 1.5 and 2.0
Krad). Some petal mutations viz. serrated petals and 3-petal flowers were
observed.
Colchicine was applied to the emerging apical buds of saffron corms at 0.05, 0.1,
0.2 and 0.4% aqueous concentration employing cotton plugs for 48 hrs (12 hours
for four consecutive days) by Zaffar et al. (2004). Studies of C0 and C1 generation
showed that the colchiploids exhibited delayed flower and leaf emergence. The
leaves were thicker, shorter somewhat flat-ridgeless, coarser in feature and dark
green in colour with reduction in number of leaves per plant. Stomatal studies
revealed decrease in number with a concomitant increase in size of stomata in C1
40
generation. There was reduction in the number of flowers per plant, floral variants
observed included smaller sized flowers, irregularly shaped/reduced tepal number,
lobed and dentate tepals, flowers with deep pigmentation in stigmas extending to
style region and orange red pigmented anthers.
Study on induced mutagenic variability in saffron (Crocus sativus L.) by Khan
(2004) revealed delayed sprouting, slow growth in higher dose, increase and
decrease of plant height in lower and higher doses respectively is due to the
amount of auxin synthesis. Formation of serrated petal is the result of somatic gene
mutations whereas 3-petal mutant is the result of alternate inhibition of apical cell
division of petal at initial stage of flower development caused by irradiation. But
surprisingly these mutations failed to appear in VM2 generation. Results are in
confirmation with earlier reports of Gordon (1957). Stunted growth, reduction in
survival and reduced fertility was also attributed to genetic loss due to
chromosomal aberrations and gene mutations (Sparrow et al., 1961; Datta and
Gupta, 1980)
Rastegari et al. (2006) studies induced mutation in saffron (Crocus sativus L.) and
revealed that induced mutation is highly effective for enhancement of natural
genetic resources and have assisted in developing improved cultivars of many
crops. Exposure of saffron corms of different weights (6, 8, 15, 12 and 14 g) to
different doses of gamma irradiation (2.5, 5, 7.5, 10, 12 and 15 Gy) revealed
number of variations in size and colour of petals of saffron flowers. In M1V1, non-
significant differences were observed for yield. Cytogenetic studies showed
chromosomal abnormality i.e chromosome fragments, translocation and chromatid
bridges with 8-10 g of corms and 7.5-10 Gy gamma radiation. It was concluded
that the best dose of gamma radiation were 6 to 10 Gy. Higher irradiation dose (>
10 Gy) prevented corm bud sprouting.
Effects of 0~25Gy 60Co γ-rays irradiation on the development of Crocus sativus L.
corms (12~14 g weight) were investigated by Jun et al. (2007). The results showed
that irradiation at the dose of 5~10 Gy can stimulate flowering of saffron and can
improve the harvest ratio of offspring. The harvested cormlet had flowering ability
41
only after 5 and 10 Gy irradiation treatment. It was concluded that 5~10 Gy
irradiation was suitable mutation dose for corms of 12-14 g weight.
A study on development of high yielding saffron mutants by Khan (2007) revealed
variability in respect of sprouting time, plant height, flower induction, petal shape
and number of filaments in stigma. Five fid stigmas with an average length of 2.70
cm and average dry weight of 6.950 mg were observed which may arise as a result
of gene mutations.
Nehvi et al. (2007) made an attempt to create new saffron variants for economic
characters using gamma irradiation from 60Co source at 0.25, 0.50, 0.75 and 1
Krad doses. Delayed sprouting was observed at higher dose (1 Krad). Irradiation
doses had significant effect on plant height, sprouting, survival, number of
flowering plants, number of daughter corms, corm yield, number of flowers and
pistil recovery/100 pistils. Induction of early sprouting in 0.25 Krad and 0.5 Krad
and delayed sprouting at higher doses was noted. Radiation at lower doses (0.25
Krad) enhanced plant biomass whereas a reverse reaction was observed at higher
doses. M1 mutants with 0.25 Krad radiation dose showed enhanced corm yield
and saffron yield by a margin of 53.44% and 84.28%, respectively. Increased
saffron recovery resulted from more number of flowers/plant. Plant survival was
reduced to 79% at 1.0 Krad. Radiation treatment at higher doses did not reveal any
significant effect on number of daughter corms, corm yield, number of flowers and
saffron yield. The results are in general agreement with other reports (Akhund-
Zade and Mazaferova, 1975; Laneri and Lucretti, 1983a; Khan, 2004). Superiority
of mutants in terms of saffron recovery was maintained to the tune of 47.36%.
Elite mutants also revealed superiority in terms of pistil length and increased
number of flowers/spathe. Tamina and Tabash (2010) reported higher average
effectiveness of EMS than γ-rays. Studies in wheat (Gaul and Aastveit, 1966),
Arabidopsis thaliana (Brock, 1971) and cowpea (Girija and Dhanavel, 2009) have
also shown that EMS is more effective than radiation in inducing polygenic
variability.
42
Abdullah et al. (2009) revealed that mean survival rate fell sharply from 63% at 20
Gy to 7% at 30 Gy. This decreasing trend was followed by 2% survival at 40 Gy.
Radiosensitivity test (LD50) for the Curcuma alismatifolia was approximately at
25 Gy. Gamma irradiation affected the survival rate of rhizomes, extension of days
to shoot emergence, plant height, leaves and shoot number as well as
modifications in plant morphology and flower development.
Study of possibility of mutation induction in saffron (Crocus sativus L.) was
carried out by Kamali et al. (2009). Effect of oryzalin on different saffron explants
including corm segments, callus and somatic embryos at globular stage for
mutation induction and increasing ploidy level was studied. Results showed that
different concentrations of oryzalin on corm segments prior to establishment in
culture medium resulted in decreasing the explant viability. Treatment with 12.5
µM oryzalin for 3 days caused soft and undifferentiated callus which was not able
to regenerate. By using 12.5 µM oryzalin for 1 day, deformed shoots were
obtained. Flowcytometry examination did not show any ploidy change. Treatment
with 10 µM oryzalin for 14 days on embryogenic calluses showed mixoploidy
forms. Different concentration of oryzalin on embryogenic calli at globular stage
were less effective and there were slight morphological changes in colour and
shape.
Corms at different stages of growth rate were subjected to different doses of
physical and chemical mutagens by Nehvi et al. (2010b). On the basis of
morphological and anatomical attributes, 11 variants were identified in M2
generation. Among physical mutagens, gamma radiation (0.1 and 0.2 Krad) when
given on 1st
and 15th
June induced more number of stomata ranging from 20-24 per
field. Radiation with 0.5 Krad resulted in narrow leaves exhibiting only one
stomata. Enhanced stomatal number was associated with thicker and broader
leaves. Stomata size increased with 0.2 Krad as compared to 0.1 Krad which was
comparable with control. Among chemical mutagens, similar results were
observed with application of ethyl methane sulphonate (0.1%), ethidium bromide
(0.2%) and colchicine (0.05%) when applied on 15th
September and 1st
June,
43
respectively. Treatments induced more number of stomata (>20). Colchicine
variant recorded narrow leaves (0.7 mm) with maximum leaf thickness (56.95
mm). Similar results for leaf width were also observed for ethyl methane
sulphonate (0.2%) when applied on 1st
September. Morphological variants with
increased number of stomata also recorded maximum saffron recovery on account
of increased number of flowers per corm except for colchicines that was
responsible for increased pistil length. More stomata number associated with
thicker and wider leaves evident from gamma radiation (0.2 Krad), ethidium
bromide (0.2%) and ethyl methane sulphonate (0.1%) were also associated with
increased number of heavier flowers and saffron recovery in terms of fresh pistil
weight. Study confirmed that mutagenesis made the plants photosynthetically
more efficient thus contributing to increased saffron recovery.
Tamina and Tapash (2010) reported that lower doses of mutagens were effective
and efficient in causing polygenic variability in various quantitative characters with
a negative relationship between effectiveness and mutagen dose in sesame. Similar
findings were reported by Roy Chowdhury et al. (2004) in mungbean, Dhanavel et
al. (2008) in cowpea, Ganapathy et al. (2008) in little millet and Thilagavathi and
Mullainathan (2009) in black gram.
Tiwari et al. (2010) reported reduction in survival rate, root length, number of
spike/plant, number of floret/spike, days to flower, shelf life, vase life, floret size,
number of corms/plant and weight of corms/plant with an increase in exposure of
gamma rays in gladiolus. They observed morphological abnormalities in leaf and
plants showing an increase in the number of abnormal leaves with an increase in
the gamma radiation dose.
An attempt was made by Khan et al. (2011) to create new variants for increasing
corm production per planting cycle through induction of mutations using physical
(gamma rays in Kilo-Roentgen) and chemical (ethyl methane sulphonate,
colchicine, ethidium bromide) mutagens at different growth stages of saffron using
fortnight treatments (Ist June, 15th June, 1st July, 15th July, 1st August, 15th August,
1st September, 15th September). Initially, 44 plants were selected on the basis of
44
their higher yield performances. Evaluation of elite mutant lines identified
treatment-D2T6 (15th June treatments of corms with 0.1% EMS) and D8T6 (15th
June treatment of corms with 0.05% colchicine) both producing highest number of
daughter corms (15) per mother corm followed by D2T2 (15th June treatment of
corms with 0.2 Krad) producing 12 daughter corms per mother corm as compared
to control (natural population) producing only 5 daughter corms per mother corm.
15th June was proposed as an ideal time for treatment of saffron corms in order to
induce increased number of daughter corms per mother corm. Further, 0.2 Krad
showed positive effect on number of daughter corms per mother corm.
Study on colchicine induced variability by Gowhar et al. (2012a) revealed that
corms treated with colchicines @ 1% induced morphological and anatomical
variants associated with thicker and broader leaves with maximum chlorophyll
content and stomatal size associated with reduction in stomatal number and
leaves/plant.
Chatterjee et al. (2012) reported that induced mutations often produce
abnormalities which cause morphological alterations in external form of plants
including color, shape, size etc. Study on physical (gamma rays), chemical (EMS
alone + EMS combined with gamma rays) mutagenesis to determine the effective
dose of mutagens to create plant deformities and chlorophyll (chl) mutations in
opium poppy revealed that the frequency of chl mutations were maximum for 50
Krad (0.42%) followed by 40 Krad (0.24%) in NBR1-1 while frequency was
highest for 50 Krad in combination with 0.8% EMS (0.86%) in NBR1-5. Among
the chlorophyll mutations, albino was the most frequently screened followed by
xantha type at all doses.
Variability for flower colour mutants in gladiolus induced by ethyl methane
sulphonate (EMS) for fifteen quantitative traits was studied in M1V2 generation of
gladiolus by Bhajantri and Patil (2013). A significant shift in mean values in the
positive direction was observed for all the 15 quantitative traits studied. Corm
weight and corm diameter recorded high phenotypic and genotypic coefficient of
variation at 0.50% EMS treated population in both Ethyl Cav Cole and White
45
Prosperity genotypes. The highest variability for days to spike initiation in both
the genotypes was recorded at 0.50% EMS. The moderate PCV and GCV values
for the characters like days to sprouting, days to first floret to show colour, days to
full spike emergence, spike length, rachis length, spike girth and number of florets
per spike was observed in all treated populations in both genotypes. Plant height
and spike length had moderate variability at 0.50% EMS treated populations of
White prosperity. Low PCV and GCV was recorded for characters like, leaf width,
inter floret length and floret diameter in all treated populations of Ethyl Cav Cole.
High heritability estimates along with moderate genetic advance (GA) and genetic
advance over mean (GAM) was found for most of the characters viz. number of
leaves per plant, leaf width, days to full spike emergence, days to first floret to
show colour, spike length, rachis length, florets per spike, inter floret length and
floret diameter in all treated mutant populations.
Study on induction of mutation in commercial varieties of gladiolus using physical
mutagen (60Co) by Patil (2014) revealed that percentage of sprouting and survival
was affected significantly at 1 Krad to 4 Krad. LD50 was found to be beyond 7
Krad for both sprouting and survival. 4 Krad and above proved to be detrimental
for various vegetative and floral traits. Plants treated with 6 Krad and 7 Krad did
not produce flower spikes in cv. Nova Lux and Eurovision whereas cv. American
Beauty produced few flower spikes. When corms were treated with higher doses,
plant height, leaf number and leaf size were reduced significantly and leaves
became narrow and leathery. Colour variations in florets and whole spike were
also increased with increase in dose rate along with increase or decrease in number
of floral organs. Radiation treatments at higher doses caused delay in spike
initiation with decrease in spike length, number and size of florets, vase life and
yield per plot while lower doses responded positvely. The desirable mutants with
light colours were isolated from all the three varieties with 5 Krad whereas one
mutant had bifurcated spike at 6 Krad from cv. American Beauty.
Induction of mutations in Browallia speciosa using sodium azide and
identification of genetic variation by El-Mokadem and Mostafa (2014) revealed
46
that 800 ppm sodium azide increased the number of branches and leaf, chlorophyll
content, fresh weight of vegetative growth and roots, dry weight of vegetative
growth and roots and root length in M1 and M2. All the concentration of sodium
azide produced changes in flower colour, flower shape and leaf form in both the
generations.
II.3 Components of variability
Biological characteristics of saffron makes its breeding significantly complicated.
In crop improvement, the selection of plants is made on the basis of their
phenotype and the effectiveness of selection would largely depend on the
proportion of phenotype due to the genotype which is heritable. Classification of
total variability (existing or induced) into its heritable and non-heritable
components such as phenotypic genetic advance is of paramount importance in
understanding the genetic make-up of any breeding material under improvement.
Statistically, the amount of variation is measured and expressed as variance.
Genotypic variance is a pre-requisite for an effective breeding programme.
Genetic coefficient of variation is used to measure the range of genetic variability
present in a particular character. For effective utilization of germplasm resources,
it is important to understand the amount of genetic diversity present in such
germplasm resources. Also, it is important to assess the relative magnitude of
components of variability in order to use such information together with other
selection parameters for improvement of the plant type through adoption of
effective breeding methods (Johnson et al., 1955; Hanson et al., 1956; Williams,
1964; Briggs and Knowels, 1967). It is necessary to divide the total phenotypic
variance of the entire characters into its components as these are the basis for
genetic analysis and the dimensions of these components dictate the breeding
behavior of the populations. Such selection parameters, particularly, genetic
variability helps to choose a potential genotype whereas heritability (h2) along
with genetic advance as percentage of mean (GA%) are more useful in predicting
the resultant effect from selection of best genotypes. The knowledge on the extent
of variation and diversity in the yield and quality components of germplasm
47
resources and identification of a good number of genotypes as potential donors in
yield and quality improvement programme is essential.
For most efficient mobilization of available germplasm resources, it is vital to
have better understanding of nature and magnitude of genetic variability.
Divergence studies are of paramount importance in understanding the extent of
variability and possibilities of its future utilization in subsequent crop
improvement programmes because corm multiplication in saffron does not induce
genome variation with the exception of some natural mutation that in triploid
saffron population are not easily detectable. Therefore, one of the important
considerations in the formulation of efficient selection programme is the
knowledge regarding the relative contribution of genes in the expression of a
particular trait. Genetic advance, i.e. the improvement in genotypic value of the
new population as compared to the base population depends among other things
on the magnitude of differences among genotypic values of individuals in the base
population and non-heritable agencies. Success in changing the characteristics of
population therefore depends upon the correspondence between phenotypic and
genotypic values.
Quantitative measures which provides information about choice between
genotypic variance and phenotypic variance is called heritability. The concept was
originally presented by Lush (1945) to describe the ratio between genotypic and
phenotypic variance and is now known as broad sense heritability. However, mean
genotypic value of the progeny is determined by the average effects of genes
transmitted by the parents in question. In other words, it is breeding value of the
parents which determines the genetic properties of the progeny. Hence it is the
proportion of phenotypic variance that is made up of variation attributable to the
breeding values (known as additive genetic variance) which is of considerable
practical interest to the breeder.
New variants of saffron with increased number of stigmas maintaining 2n=24 have
been reported by Estalai (1978) with a frequency 1.2 x 10-6 of a rare type flowers.
Morphological differences with flowers having higher number of stigma branches
48
and stamens have been described by Piccioli (1932) from saffron cultivation at L.
Aquali (Italy). In addition different commercial products are known suggesting the
existence of different saffron ecotypes (Tammaro, 1987) or commercial varieties
(Di Crecchio, 1960).
Munshi (1992) from his studies on saffron reported wide range of variability.
Information on coefficients of variation, heritability and genetic advance was
derived from data on 10 yield components in 11 diverse saffron genotypes (mostly
from Jammu and Kashmir with some exotic varieties) grown during kharif 1986-
89. Significant genotypic differences were observed for all characters except for
days to 100% flowering. The coefficients of variation was highest for number of
daughter corms/mother corm and number of flowers per spathe. High values of
heritability and genetic advance were observed for number of daughter
corms/mother corm and yield/plant.
Lattoo (1997) reported appreciable differences in the coefficient of variation for all
floral characters at different locations. Observations on floral characters (fresh
flower weight and size, stigma length, stigma fresh weight, dry weight and saffron
percentage) were recorded from 200 flowers collected from 4 locations in Kashmir
Valley (Chrar-Sharief, Sanatnagar, Pampore and Malabagh) during October-
November 1988. Due to clonal propagation in saffron, the population is expected
to be genetically similar. However, the variability observed appeared to be due to
varied microclimates at different sites.
Survey conducted in Pampore kerawa by Gohil (1999) revealed some interesting
and promising variants. The promising variants were recommended to be
multiplied through clonal selection. Latto and Dhar (1999) studied temporal
saffron populations of Kashmir for six floral characters of saffron viz. number of
flowers per spathe, fresh flower weight, flower size (perianth area), stigma length,
fresh stigma weight and dry stigma weight. Appreciable differences in coefficient
of variation for all the characters were observed. Maximum coefficient of variation
was recorded for flowers per spathe (59.15) whereas minimum coefficient of
variations (0.42) was recorded for stigma length. For fresh flower weight, flower
49
size, fresh stigma weight and dry stigma weight, coefficient of variation ranged
from 12.16-127.46, 13.32-31.22, 17.36-51.03 and 23.27-36.17 at four different
locations respectively. Studies on status of saffron cultivation in Kashmir and
strategies for improving productivity through crop improvement by Zargar (1999)
revealed that genetic variability existing in the natural population of saffron in
Kashmir has been an outcome of spontaneous mutations that have occurred over
centuries and need to be collected, evaluated and propagated.
In order to verify the possible phenotypic and genotypic variation in saffron,
corms from different countries (Italy, Israel, Spain and Holland) have been
examined (Grilli Caiola et al., 2001). Analysis of phenotypic variation revealed
differences in flower size, tepal shape and colour intensity with lobbed tepal in
plant from Israel and more intense colour of tepal in plant from Sardinia (Italy).
Cytofluorimetric analysis on nuclear DNA was carried to detect genome size and
base pair composition and results revealed no differences in DNA content and
composition in saffron corms from different countries (Brandizzi and Grilli Caiola,
1998).
Zargar (2001) from the studies on genetic variation in saffron and importance of
seed corm reported that significant variability exhibited for floral traits in eleven
populations of saffron (including 3 exotic genotypes from Spain, Iran and
Netherlands). Co-heritability estimates indicated that perianth size, number of
flowers per perianth and saffron recovery per flower had high co-heritability and
would help as a selection index for increasing the saffron yield. Mean fresh and
dry weight of 100 flowers in the temporal sub-population of district Budgam and
Srinagar (new plantation) was 29.03 and 4.39 g, respectively, yielding
approximate 8.01 dg of lacha grade saffron. The range and phenotypic coefficient
of variation was high. The analysis of sub-population of main saffron growing area
(district Pulwama) revealed that the fresh and dry weight of 100 flowers was only
23.28 and 3.29 g, respectively, which represented about 19.0 and 9.0 percent
decrease, respectively as compared to mean of the sub-populations of district
Budgam. The recovery of lacha grade saffron was 7.34 dg/100 flowers, which was
50
about 8.36% less as compared to the mean recovery from sub-populations of
district Budgam. The possible reason was attributed to the fact that the crop fields
in district Pulwama were exhausted as compared to new plantations in district
Budgam. The magnitude of phenotypic variability and its coefficients of
variability were comparable. On 100 g flower basis (sub-population of district
Pulwama), the mongra and lacha grade saffron recovery was 18.04 and 25.26 dg,
respectively. Thus 1dg fresh flower produced approximately 18 and 25 g of dried
mongra and lacha saffron grade, respectively.
Nehvi (2003) and Nehvi et al. (2004 and 2006) reported wide spectrum of
variability for floral and corm attributes in temporal subpopulation of Kashmir and
the results implied a great scope for saffron improvement. Perianth size, number of
flowers per spathe and saffron recovery per flower were identified as selection
index criteria for increasing saffron yield. Temporal subpopulation of district
Pulwama exhibited highest range of variability. Flowers completely devoid of
style and anthers, freaks with 4 and 5 stigmas were observed from natural
population. The presence of more number of stigmas per flower (4-6) were due to
physiological and or developmental irregularities. However, the detection of
flowers with increased number of stigmas in natural population is a universal
phenomenon. Magnitude of variability revealed that the mean fresh and dry weight
of 100 flowers in temporal sub-population of Jammu and Kashmir was 23.46 and
3.82 g, respectively, yielding 0.75 g of lacha grade saffron. Stigma length ranged
from 2.41-3.87 cm with a mean value of 2.5 cm. Number of flowers per spathe
ranged from 0.65-5.58 with fresh flower weight ranging from 172-355 mg.
Economic product on fresh basis ranged from 14.37-68.42 mg whereas on dry
weight basis, it ranged from 6.0-14.60 mg. Greater magnitude of phenotypic
variance than corresponding genotypic variance was observed with low values of
broad sense heritability for all the traits except for fresh flower weight, fresh
perianth weight and stigma length. Genetic gain as percent of mean was high for
number of flowers per spathe, fresh flower weight, fresh pistil weight and crocin
content, whereas, it was medium for dry pistil weight, stigma length and number
51
of daughter corms and low for other studied traits.
Makhdoomi (2006) studied components of phenotypic variability after taking
genotype x environment interaction into account and indicated that a wide range
of variability existed for number of flowers per corm (0.21-5.59), fresh flower
weight per corm (154.7-529.5 mg), fresh perianth weight per corm (100.00-468.3
mg), fresh pistil weight per corm (11.63-40.35 mg), fresh stigma weight per corm
(5.9-33.98 mg), fresh style weight per corm (1.85-7.80 mg), fresh stamen weight
per corm (11.28-47.9 mg), stigma length (1.7-3.8 cm), style length (1.23-1.3 cm),
pistil length (3.48-7.68 cm), dry flower weight per corm (33.88-78.25 mg), dry
perianth weight per corm (33.58-52.00 mg), dry pistil weight per corm (4.15-15.30
mg), dry stigma weight per corm (2.5-13.5 mg), dry stamen weight per corm (4.73-
16.1 mg), number of daughter cormels per mothercorm (7.5-50.2), average weight
of daughter cormels per mother corm (18.8- 41.4 mg), leaf length (255.5-1487.3
cm), number of radical leaves per corm (2.38-10.81) and dry leaf weight per corm
(1.55-12.54 mg). Similar results of wide range of phenotypic variability were also
reported in gladiolus by Lal et al. (1985) and Khanna and Arora (1986). High
value of heritability accompanied with high genotypic and phenotypic variation
were reported for weight of cormels per corm, total number of florets per spike,
spike length and spike weight, whereas, plant height, number of leaves and days to
flower exhibited heritability with low genetic advance. High heritability and high
expected genetic gain was observed for number of cormels. Similar results in
gladiolus for number of flowers, spike length, spike weight, weight of daughter
corms and number of daughter corms were reported by Misra and Saini (1988),
Soorianathasundaram and Nambisan (1991), Mahanta and Paswan (1993),
Ashwath and Parthasarthy (1994), Prasad et al. (1994), Sarangi et al. (1994) and
Sheikh et al. (1995) whereas Gowda (1989) and Anuradha and Gowda (1994)
reported low heritability with low value of genetic gain for plant height, days to
flowering and spike length. Sharief-ud-Din et al. (2000) reported medium value
for corm weight and corm size.
52
Studies on productivity, growth and qualitative attributes of 10 Iranian saffron
accessions under climatic conditions of Charra Mahal Bakhtiari, Central Iran by
Parviz et al. (2004) revealed superior performance of 3 accessions including
Shahr-Kord, Birjand and Ghaen with productivity level of 3.26, 2.67 and 2.66/ha
respectively in terms of dry stigma yield. Data collected for different traits in the
first year, in general and for stigma yield in particular were highly variable.
Treatment effects were statistically significant for stigma yield, total dry matter,
corm number and weight. For most of traits, majority of variation due to the
treatment effect arose from the differences of the Birj and Ghaen and Shahr-Kord
with the rest of accessions.
Agayev (2006) reported that metaphase chromosomes of saffron can be arranged
according to their size and morphological features in 8 triplets ranging in size from
11.58 ± 0.13µm (triplet 1) to 4.57 ± 0.13µm (triplet 8). Triplet 1 and 2 consist of
subacrocentric, 3, 4 and 8 metacentric and 6 and 7 submetacentric chromosomes.
Three chromosomes in the specified triplet, as a rule are similar although in some
triplets one of them is slightly distinguishable from the other two. Triplet 5 shows
an extreme difference so that it always contain 2 kinds of chromosomes:
chromosome 5(1) and chromosome 5(2,3). Chromosome 5(1) is metacentric
(r=1.49) and 6.04±0.13µm in length but chromosome 5(2,3) are subacrocentric
(r=3.49) and noticeably smaller (5.41±0.09µm). Application of C-banding
technique revealed heterochromatin segments: the sharpest, sharp and weak. New
specific and sharpest heterochromatin segment using acto-iron-hematoxylin stain
was revealed on satellite chromosomes (triplet 2) on the proximal part of the long
arm. It was assumed that the species C. sativus is obviously a clone of one triploid
plant originated spontaneously in nature through crossing between 2 closely
related species with participation of n and 2n gametes.
Genetic variability studies by Verma et al. (2006a) revealed that mean squares
were highly significant for all the characters viz. flower weight, fresh pistil weight,
dry flower weight, dry pistil weight, parienth length, parienth breadth, stigma
length, style length and total length of stigma and style indicating enough
53
variability for all the traits. Fresh flower weight ranged from 0.60-0.85 g and total
length of stigma and style varied from 39.09 to 97.10 mm which showed a good
range of variation between the genotypes. Style length exhibited highest values of
genotypic coefficient of variation (18.56) and phenotypic coefficient of variation
(24.77). Stigma length and total length of style + stigma showed highest estimate
of broad sense heritability. High estimate of heritability (broad sense), genotypic
coefficient of variation and genetic advance was observed for total length of style
+ stigma which showed possibility of improvement through selection.
A study on exploration of Kashmir (Budgam & Srinagar) valley for the selection
of superior genotypes of saffron by Verma et al. (2006b) revealed a high degree of
variability in the plant age (1 to 30 years), number of cormels/mother corm (1.22-
11.8), average weight of cormels (1.65-10.16 g), average length of cormels (1.40-
1.92 cm), corm girth (1.27-2.66 cm) and corm size (1.426-5.107 cm). Floral
characters of 141 samples also revealed marked variability in fresh and dry weight
of flower, fresh and dry weight of pistil, parienth length and breadth and length of
stigma and style.
Agayev et al. (2007) showed that within each weight group of corms, number of
flowers were differing in large extent in different clusters (holes). Roughly
35-50% of clusters had no flowers at all. 20-25% had one flower, 13-15% had two,
5-10% had three, 2-7% had four, 1-2% had five flowers, etc. Unique clusters had
8-10 and even up to 12-13 flowers. Results revealed heterogeneity of experimental
populations.
Comparison of saffron clones, each grown from one corm of the same weight,
resulted in the identification of ‘‘superior’’ clones in terms of exceptionally large
numbers of flowers and large (≥ 10g) corms (Agayev et al., 2009). Based on the
number of flowers and number of large corms-the two most economically
important attributes of saffron, the clones were classified as extraordinary,
superior, ordinary, inferior and declining clones. The first two classifications of
clones, which had the highest numbers of flowers and largest corms have been
chosen for use in a saffron breeding program aimed at developing new high
54
yielding cultivars of saffron. Those clones have been found to be suitable for
facilitating the mechanization of saffron agriculture in terms of the lifting, sorting,
corm plantation, weeding and flower harvesting. Clone 27F-4 had the highest
number of flowers (27) with an initial corm weight of 8 g. It was characterized by
the highest parameters of corm set: total weight of corms increased upto 304.6 g;
‘‘Big corm index’’ was 92.3%, number of large corms was very high (16); average
weight of one big corm was desirable (17.6 g); ‘‘Flower creating index’’ was
satisfactory (11.3 g) and ‘‘Multiplication index’’ was high (38.19). On the whole,
the clone unified high positive qualities.
A preliminary characterization of saffron germplasm from the CROCUS BANK
collection was done by De-Los-Mozos-Pascual et al. (2009) for phenology (date
of sprouting, flowering, duration of flowering), floral morphology (tepals length
and width and length of stamen filament) and saffron production (number of
flowering corms, number of flowers/corm, saffron spice weight/flower). Stamen
filament length was the only measured parameter identical in all the accessions.
Concerning other parameters, there were significant differences with proportion of
the total variance due to differences among accessions ranging from 53% for the
duration of the flowering period to 24% for the number of flowers per corm. For
some characters like precosity, a part of the observed variation was due to small
differences in the initial weight of the mother corms. The variation in other
characters like dry saffron weight per flower or the duration of the flowering
period were independent of this factor and was of interest for selection and
breeding.
Study on genetic difference among wild Greek Crocus taxa and cultivated forms
(Crocus sativus L.) currently being maintained in ex situ conservation at the
Balkan Botanic Garden of Kroussia (Greece) by Tsoktouridis et al. (2009)
reported significant variation for the wild accessions, identifying several
transitions, transversions and indels whereas no nucleotide difference was
observed for 10 accessions of C. sativus L.
55
Khan et al. (2010) reported that utilization of heterogenity in the natural
population which is due to genetic and environmental factors offers a tremendous
scope for saffron improvement. Germplasm conservation and evaluation is
underway for identification of elite clones with distinct yield superiority.
Evaluation of 24 genotypes with distinct superiority over natural population for
yield stability recorded superiority of SMD-87, SMD-61, SMD-9, SMD-102,
SMD-98 and SMD-170 with average saffron yield ranging from10.41 to 12.00
kg/ha.
Study on genetic variability in saffron (Crocus sativus L.) was undertaken by
Makhdoomi et al. (2010) to generate information on the nature and magnitude of
components of phenotypic variability including heritability, genetic gain, nature of
interrelationship among components of economic worth, contribution of different
morphological and yield component traits in 240 saffron populations collected
from natural saffron growing areas of Kashmir. Significant variations among
populations were observed for all the traits indicating presence of high level of
variability. G x E interaction was also observed to be significant for all the traits
indicating differential behaviour of populations over years. Wide range of
variability was observed for all the traits implying considerable scope for saffron
improvement through clonal selection. Range of variability was observed to be
high in second year estimates and phenotypic variances were observed to be
higher than the corresponding estimates of genotypic variances. Estimates of
phenotypic and genotypic coefficients of variation demonstrated similar trend. For
most of the traits, genotypic coefficient of variation ranged from 8.76 to 32.24,
whereas, style length, pistil length, dry flower weight and dry perianth weight
recorded medium to high values of genotypic coefficient of variation (GCV).
Heritability in broad sense was observed to be high for all traits in both the years.
However, on the basis of pooled analysis over years, it was medium for all the
traits except fresh flower weight, fresh perianth weight, fresh pistil weight, fresh
stigma weight, stigma length and average weight of daughter corms revealing a
strong influence of environment on the performance of populations. Expected
56
genetic gain (% of mean) ranged from 5.15-40.68 and was observed to be high for
all the traits except plant height, pistil length, dry perianth weight and style length.
Study on stability analysis in saffron (Crocus sativus L.) by Nehvi et al. (2010d)
revealed that significant G x E interaction was observed for all the traits except
days to 50% sprouting and 50% flowering thereby revealing that genotypes
perform differently for traits under study at different locations. Highly significant
mean squares for environments except for flowering traits indicated that
environment selected were random and were different in agro climatic conditions.
Genotypes observed average stability for all the characters except for number of
radical leaves and corm yield per plot.
Agayev et al. (2012) gave new specific scientific concepts of clonal selection of
saffron (Crocus sativus L.) viz. initial corm weight, corm set formulae, big corm
index, ultra-extraordinary clone, extraordinary clone, superior clone, selective
(elite) clone, low grade clones, ordinary clone, inferior clone, declining clone,
flower creating index and multiplication index.
The study of clonal selection of saffron was undertaken with an aim of searching,
identification and separation of genetically superior clones of saffron from existing
population with higher number of flowers and higher quantity of large sized corms
(Gowhar et al., 2012b). Samples of saffron population from most ancient
cultivating regions of Kashmir were collected from 500 sites for the study with >
2000 corms comprising the sampled population from each region. The corms were
divided into two weight groups viz. < 8 gm and > 8 gm and then planted into pits
separately with pit to pit distance of 50 cm. Superior clones in terms of production
of large number of daughter corms both in terms of weight and number associated
with maximum number of radical leaves with increased plant height were
identified.
Nehvi et al. (2012) carried out genetic resource management to explore and utilize
available genetic variability present in natural sub-populations of Jammu and
Kashmir. About 2500 saffron accessions collected from saffron areas of Jammu
and Kashmir during 2002-2011 were subjected to variability and divergence
57
studies at genetic and molecular level. Selected lines from natural sub-population
exhibited a wide range of variability for economic, morphological and corm
attributing traits indicating considerable scope for saffron improvement through
clonal selection.
Morphological characterization of saffron clones by Sheikh et al. (2012) revealed
significant genotypic differences for number of flowers per corm, fresh pistil
weight per corm, pistil length, stigma length, number of daughter corms, average
weight of daughter corms, plant height, number of radical leaves per plant,
stomatal size, stomatal frequency and chlorophyll content. Components of
variability indicated a wide range of variability for the traits under study.
Estimates of phenotypic variance were higher than corresponding genotypic
variance thereby indicating influence of environment in the expressions of these
traits. High values of heritability were recorded for all the traits. Estimates of GCV
were higher in magnitude though similar in direction for all the traits.
II.4 Genetic divergence
Geographic diversity among parents as an index of genetic diversity has been
acclaimed and disclaimed in numerous published reports. Murty and Arunachalam
(1965) hypothesized the Mahalanobis (1928) generalized distance a measure of
metric distance between population centroids that can be a very useful multivariate
statistical tool for effective discrimination among parents on the basis of genetic
diversity. Precise information about genetic divergence is critical for productive
breeding programme as genetically diverse parents are known to produce high
heterotic effects consequently increasing yield in desirable segregants. High
yielding parents with greater genetic diversity are required to develop productive
hybrids. For identifying genetically diverse parents for hybridization, multivariate
analysis (Mahalanobis D2 statistics, 1936) has been used in almost all crop species.
D2 statistics gives a result based on the magnitude of divergence dependent on the
sample size. Genetic diversity in biological populations has been found to occur
due to several causes. Human selection has led to quite a big array of varieties
grown for the same end product and thus effected their diversity, whereas, stress
58
conditions, natural selection and genetic drift maintained divergence (Ram and
Panwar, 1970; Das and Borthakur, 1973). Studies in a number of crop species with
different breeding systems by means of D2 statistics suggested that genetic
diversity need not be directly related to geographical diversity (Murty and
Arunachalam, 1965, 1966). Experimental evidences in Drosophila (Brunica, 1954;
Wallse, 1955) have demonstrated that crosses of strains of diverse origin exhibited
greater heterotic response than crosses of strains of the same origin.
Nature and magnitude of genetic divergence was assessed in different genotypes
and species of gladiolus using D2 statistics by several workers (Avishai and
Zohary 1980; De and Misra 1993; Arya et al., 1999; Desh and Misra 1999;
Nimbalkar et al., 2002). These workers have grouped the genotypes into different
clusters. D2 statistics revealed that clustering behavior and mean performance of
genotypes of individual clusters were not consistent over the environments
because of large genotype x environment interactions. Number and weight of
corms and cormels plant-1, number of florets plant-1, and plant height contributed
considerably to divergence.
Brandizzi and Grilli Caiola (1998) while studying DNA content of saffron corm
received from Italy, Israel, Spain and Holland reported no difference in DNA
content and composition of saffron corms. Zanier (2000) while analyzing nuclear
DNA by RAPD technique revealed that saffron corms from different cultivation
areas did not identify genomic redundant differences. There were no differences in
DNA present in 10 corms from saffron in L-Aquila. The RAPD data were in
accordance with the DNA content and base pair composition among different
Crocus sativus cultivars as revealed by the previous cytofluorimetric studies.
Pardo et al. (2004) investigated the distinctive variability of Crocus sativus from
several geographical areas (Italy, Israel, Greece and Spain) using 38 random
amplified polymorphic DNA (RAPD) markers. Low percentage of primers were
suitable for differentiating four saffron samples from different origin. However
other studies with triploid species with apomictic reproduction offered a large
genetic variation in the genetic structure of populations.
59
Grilli Caiola et al. (2004) carried out Random Amplified Polymorphic DNA
(RAPD) analysis on DNAs from 5 Crocus sativus (saffron) accessions cultivated
in different countries (Italy, Israel, Spain and Holland) and on 6 closely related
Crocus species (C. asumaniae, C. cartwrightianus, C. hadriaticus, C.
oreocreticus, C. pallasii and C. thomasii) to determine whether cultivated saffron
has maintained a constant genomic organization and to clarify its relationship with
possible ancestor species. For the 15 primers, which produced positive results, the
DNA of saffron corms from different accessions presented the same amplification
pattern in accordance with the similar DNA content and base composition pointed
out in previous studies. The amplification of the DNA of the 7 Crocus species
with 21 primers provided 217 repeatable and interpretable fragments which were
scored for presence/absence of band and employed for cluster analysis. The results
indicated that C. sativus is very closely related to C. cartwrightianus and is similar
to C. thomasii. This result concurring with part of the previous evidence rules out
the hypothesis of close relationships between C. sativus and C. pallasii. Genetic
relationships among populations of Crocus hyemalis Boiss. and Blanche collected
from different regions of Jordan were studied using Random Amplified
Polymorphic DNA analysis. C. hyemalis was compared to the cultivated species
C. sativus and C. vernus. RAPD and cluster analysis indicated high degree of inter
and intra-population variation within the wild C. hyemalis population. High
genetic association was found among some wild populations originating from the
same collection sites.
DNA molecular comparison using RAPD markers on six species (C. pallasii
subsp. Hausknechtii, C. cancellatus, C. speciosus, C. caspius, C. michelsonii, C.
sativus) by Tarazi and Rashid Mohassel (2006) revealed that the origin of
cultivated saffron in Iran is Western Iran possibly Zagros area.
Taghizadeh (2006) conducted a study on molecular genetic assessments in Crocus
sativus L. and reported that DNA polymorphism based AFLP method confirmed
close relationship between species of Crocus sativus L. from several geographical
areas using random amplified polymorphic DNA (RAPD) markers.
60
Soheilivand et al. (2007) while studying diversity in flowering rate of two saffron
(Crocus sativus) populations of Iran revealed that in cluster analysis diagram,
populations planted with different weight of corms, based on 5 traits related to
flowering rate, constituted 3 groups at a distance of 10 units. Gonabad population
with 3, 4, 5, 6 and 7 g corm weight were placed in group I. The result showed that
cluster analysis could distinguish plants with lower maternal corm weight of one
specific population from the other specific population. The results also showed
that the maternal corms with 3 to 7 g had non-significant effect on flowering rate.
Gonabad populations with 8 and 9 g corm weight and Ghaen population with 3, 4,
5 and 6 g corm weight were placed in group II thereby showing different
flowering rates between corm derived from Gonabad and Ghaen. Two populations
of 10 g weight of Gonabad and 7 g weight of Ghaen were placed in cluster III. The
results showed that cluster analysis could completely distinguish the corms of
different weight. It also depicted the difference in the number of flowers produced
by heavy and light corms and emphasized the fact that the heavier maternal corms
compared to the lighter corms produce more flowers. The results were in
consistent with results of other investigators (Pandey et al., 1979; De-mastro et al.,
1993; Omidbeigi et al., 2003).
Studies in relation to molecular variability in saffron by Imran et al. (2007)
revealed considerable amount of genetic diversity among tested genotypes.
Similarity index based on Jaccards coefficient ranged from 0.375 to 0.834 with
maximum similarity coefficient (0.834) observed between the genotype SMD-45
and SMD-79. The dendrogram based on molecular data divided the tested
genotypes in two clusters. However, genotypes viz. SMD-3, SMD-45, SMD-79
and SMD-68 formed cluster I and genotypes viz. SMD-11, SMD-52, SMD-81,
SMD-211, SMD-224 and control formed cluster II at similarity coefficient of 44%
which showed a high level of genetic diversity between two clusters containing
different genotypes. A different level of variability was observed among different
genotypes within each cluster.
61
Fluch et al. (2009) reported that out of the 6803 available saffron ESTs, 200
sequences proved to contain SSR region. Out of these, for 38 sequences, primers
could be developed and tested on 29 different saffron accessions. In addition to the
nuclear SSRs, 8cp regions were investigated. None of the region revealed any
genetic difference among the investigated probes. Sequencing of 5 genes was
conducted in order to identify single base pair mutations in the respective genomic
areas.
Henna et al. (2009) studied genetic divergence in saffron under temperate
conditions of Kashmir. Cluster analysis revealed sixteen clusters in pooled
analysis, three in year 1 and two in year 2. Highest intercluster distance was
observed between cluster XV and cluster XVI followed by cluster V and XVI
respectively. The characters viz. fresh stamen weight, plant height, fresh flower
weight, pistil length and fresh pistil weight contributed maximum towards
divergence.
Genetic divergence in saffron studied by Maqhdoomi et al. (2009) grouped all the
genotypes in eleven clusters in pooled analysis with majority of genotypes (229) in
cluster I. All other clusters were monogenotypic except cluster II. Percent
contribution of different floral, morphological, corm and quality attributes towards
divergence in saffron germplasm lines revealed strong influence of fresh pistil
weight, stigma length, and crocin content. Hence, these characters qualify to be a
selection criteria for identification of divergent lines.
Nemati et al. (2009) studied genetic diversity of 51 accessions of Iranian saffron.
56 novel polymorphic microsatellite loci were isolated and characterized.
Preliminary analyses revealed that the number of polymorphic alleles ranged from
3 to 6 and heterozygosity was determined from 0.17 to 0.39. Genetic variation was
observed among studied accessions and the isolated microsatellites markers can
provide an efficient tool for diversity assessment in saffron.
Fougat et al. (2012) carried investigation to assess genetic diversity among
commercially available brands of saffron through DNA based SSR markers. Study
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points to conclusion that SSR markers could be successfully utilized to assess
genetic diversity and to check clonal fidelity of various saffron cultivars.
Study of Gaikwad et al. (2012) on molecular diversity analysis in saffron (Crocus
sativus) using AFLP markers demonstrates the efficacy of AFLP markers in
identification of polymorphism. Genetic variability among 13 different clonal
accessions of saffron collected from different areas of Jammu and Kashmir was
determined by employing nine AFLP primer pairs. A total of 154 amplicons were
generated, 98.7% of which were polymorphic. No two accessions analysed in the
study were found to be identical using AFLP markers. The average heterozygosity
revealed was 0.33% and values of polymorphism information content (PIC) varied
from 0.09 to 0.43.
Izadpanah et al. (2012) studied variations in saffron (Crocus sativus L.) accessions
and wild species by RAPD analysis. Five accessions of cultivated saffron from
five areas in Khorasan and Esfahan, namely, Gon-Abad, Ferdows, Ghaen,
Estahbanat and Golpaygan were used. Out of nine species of saffron naturally
growing in Iran, two wild species (C. caspius & C. speciosus) from the north of
Iran (Gilan province) were selected for study. RAPD markers were used to classify
these species and to find the relationship between them. In the results of the study,
cluster analysis showed 2 main groups. Also, maximum similarity value was seen
between wild (C. caspius & C. speciosus) genotypes (0.82) and minimum was
between Estahbanat, Ferdows accessions and wild (C. speciosus) genotype (0.33).
Abedi et al. (2012) conducted a study to assess and classify 65 different saffron
accessions for morphological attributes at reproductive and vegetative stages. The
accessions were collected from various climatic areas of Khorasan, Razavi and
South Khorasan provinces. UPGMA cluster analysis using morphological traits at
vegetative stage grouped the accessions into four distinct groups. The third and
fourth clusters contained the accessions with high mean for leaf number and leaf
length. Grouping the accessions using morphological traits at reproductive stages
produced different classification.
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Laura et al. 2013 demonstrated that saffron corms of different origins grown in the
same experimental field produced daughter corms with different dimensions and
produce stigma samples with different pigment profiles. Furthermore, daughter
corm dimensions and pigment profile even more so could be related to the origin
of the sample and therefore pigments can be used as chemotaxonomic markers.