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5
3.6.4
Selection and regeneration of transformants
3.6.5
Recovery of transgenic plantlets
3.7
Histochemical GUS Assay
3.8
Molecular Analysis of Transgenic Plants
3.8.1
DNA isolation
3.8.2
Isolation of total RNA
3.8.3
Spectrophotometric estimation of nucleic acids
3.8.4
PCR analysis of transformants
3.8.5
Southern blot analysis
3.8.6
Radiolabelling of DNA
3.8.7
RT-PCR analysis
3.9
Segregation Analysis
3.10
Raising T1 plantlets
3.11
Hardening of T1 transgenic plantlets
3.12
Screening of suitable Rhizobium strain for application
3.13
Measurement of Nitrogenase activity of nodules by Gas Chromatography
3.14
Application of Mycorrhiza
3.14.1
Assessment of root colonization by VAM fungi
Chapter 4: RESULT & DISCUSSIONS
41-60
4.1
Regeneration of Vigna mungo via Multiple Shoot Formation
4.1.1
Elongation of shoot initials
4.1.2
Rooting and establishment of plantlets
4.2
Establishment of the Agrobacterium Mediated Transformation System of
Vigna mungo
4.2.1
Explants preparation
4.2.2
The co-cultivation medium and duration
4.2.3
Selection of the transformants
4.2.4
Recovery of transgenic plants
4.3
Histochemical and Molecular Analyses of T0 Transgenic Plants of V.
mungo
4.3.1
Histochemical analysis
4.3.2
PCR analysis of T0 putative transgenics
4.4
Molecular Analysis of the T1 Transgenic Plants of V. mungo
4.4.1
Southern blot analysis
4.4.2
RT-PCR analysis
4.5
Segregation
Analysis
4.6
Hardening of T1 transgenics plantlets
4.7
Screening and application of Rhizobium
6
4.8
Application of Mycorrhiza
4.9
Assessment of root colonization
4.10
Nitrogenase activity
SUMMARY
AND
CONCLUSIONS 62-64
BIBLIOGRAPHY:
65-92
7
CCHAPTER 1
INTRODUCTION
8
Introduction
Pulses, also known as grain legumes, are the edible dry seed of leguminous plants grown as
field crops throughout the world. They are the major source of protein for over three billion
people worldwide. Nutritionally the proteins obtained from pulses provide balanced diet
together with cereals as the leguminous proteins are rich in lysine and deficient in sulphur
containing amino acid cysteine while cereals are rich in cysteine and deficient in lysine.
India is the largest pulse producing country which produces 27% of pulses produced across
world. The area under pulse crops is presently around 23 million ha, with a production of
around 17 million tonnes and productivity about 600 kg/ha (www.fao.org 2012). The
production of pulses in India and other legume producing countries is limited because of
heavy losses due to biotic and abiotic stresses. Soil salinity and drought are major abiotic
stresses that adversely affected the crop yield worldwide (Blumwald & Grover et al 2006).
Among various abiotic stresses, salinity is one of the world's most serious environmental
problems as plant agriculture faces a loss of $12 billion annually due to salinity stress
worldwide (FAO Stat 2006).
Blackgram, (Vigna mungo L. Hepper) also referred to as the urd bean, urd, urid or black
lentil is a bean grown in southern Asia. It is an important grain legume commonly known
as blackgram for its characteristic black seeds and is grown for its protein-rich edible dry
seeds in the tropical and subtropical regions of the Indian subcontinent. Blackgram
originated in India and is grown as a rain-fed crop in kharif (monsoon) season primarily
along with cereals. It is also grown on residual moisture in rabi (winter) season two-three
days before the harvest of rice crop.
The production of blackgram is greatly hampered due to the various stresses. Viral diseases
caused by geminivirus known as Vigna mungo Yellow Mosaic Virus (VMYMV) are highly
distructive. Fungal diseases such as powdery mildew caused by Erisyphe polygoni and
Cercospora leaf spot also destroy the blackgram crops. The insect pests, pod borers
(Maruca testulalis and Etiella zinckenella), seed borer (Callosobruchus sp.) affect the
storage of the seeds. Among the abiotic stresses, the crop is susceptible to terminal drought
and salinity where the seed germination as well as seedling growth is severely affected.
9
According to the Indian Agricultural Statistics Data (2009) released by Directorate of
Economics and Statistics (DES), the production of pulses decreased in the major legumes
producing states of India by ca. 18.5% due to salinity and drought. Thus, in order to
maintain adequate food supply, emphasis has been laid on increasing the crop yield by
making the crop plants salinity and drought tolerant. Classical breeding methods have been
used to develop better resistance in the plants but due to limited genetic variation available
in the germplasm of blackgram, the breeding programme is restricted. With the
advancement of gene transfer technologies, where the specific genes can be transferred into
the genome without disturbing the desirable genetic arrangement, the path has been paved
for the development of genetically modified (GM) crops that are revolutionizing agriculture
world wide.
In the present study, it was aimed to develop transgenic Vigna mungo (blackgram) by
introducing the glyoxalase1 (gly1) gene driven by constitutive promoters viz. Cestrum
yellow leaf curling virus promoter [(CmYLCV) Stavalone et al 2003]. The Glyoxalase
system is a two component system and consists of the enzymes Glyoxalase I and
Glyoxalase II, catalyzing two different reactions.
Plant tissue culture has become an important and advantageous tool for rapid propagation
of several plant species. Despite many successful applications of this technique, there are
still some problems that limits its widespread use. The transfer of micropropagated
plantlets to the field is often difficult because invitro produced plantlets are not well
adapted to the in vivo environment (Pierik 1988). Weak root system is one of the major
hindrances in the successful establishment of micropropagated plantlets in the field
conditions (Subhan et al 1998). In general, Mycorrhizal fungi helps in the development of a
stronger root system and the Rhizobium helps in providing important forms of the amino
acids which are helpful in development of stronger shoots, leaves and fruits or seeds
(Ponton et al 1982). The Rhizobium and Mycorrhiza were applied to in vitro raised plantlets
of blackgram during hardening as well as during establishment into the soil to alleviate the
transplantation shock. Therefore, it was intended to develop transgenic blackgram
overexpressing a gene which obviously helps in abiotic stress tolerance. The study was
10
focused with the application of rhizobium and mycorrhiza in helping the tissue culture
raised plant against transplantation shock as well as to study their association with the
transgenic plants. This study was also aimed to find out that if there is any change in
association with the transgenic plants in comparison to control plants with the rhizobium or
mirorrhiza which are the major plant growth promoting microorganism.
The present study was taken up with the following objectives:
Objectives
1.
Refinement of regeneration and transformation protocols for Vigna mungo.
2.
Transformation studies of Vigna mungo using the glyoxalase I (gly I) gene
constructs driven by constitutive promoters.
3.
Molecular and biochemical analysis of transgenic plants of Vigna mungo.
4.
Effect of Rhizobium and Mycorrhiza on the growth of invitro raised transgenic as
well as untransformed control Vigna mungo plants and their association studies.
11
CCHAPTER 2
REVIEW OF LITERATURE
12
2. Review of Literature:
2.1 Plant growth promoting Rhizobium
Nitrogen is an essential element for plant growth. Nitrogen deficiency restricts plant
growth, but often in a subtle manner that can only be assessed by comparison to plants
grown with an adequate nitrogen supply. Severe deficiency causes necrosis (tissue death)
starting at the tips of older leaves, with the tissue death. Microbial decomposition of plant
and animal residues in soil releases mineral nitrogen, nitrate and ammonium that plants
can absorb from the soil solution. Biological nitrogen fixation is an important process by
which atmospheric nitrogen is made available for incorporation into organic compounds.
Only certain bacteria are capable of carrying out this process, the genus Rhizobium being
the most common. Members of this genus are Gram negative aerobic rods that occur free-
living in soil or as micro-symbionts in root nodules of leguminous plants (Jordan 1984).
Rhizobium in root nodules are estimated to carry out 50-70% of the world biological
nitrogen fixation reducing approximately 26 MT of atmospheric nitrogen to ammonia
(FAO 2006). The group of prokaryotes that do this is large and diverse and contains both
Eubacteria and Archaea. Symbiotic nitrogen fixation is, for the most part, restricted to a
limited number of bacterial groups, including the genera Rhizobium, Mesorhizobium,
Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia)
and Frankia (Widmer et al 1999; Zehr et al 2003). Except Frankia, all are from the
proteobacterial Rhizobiaceae family and induce nodules on plants from the Leguminosae
family. Frankia is a filamentous gram positive actinomycete that induces nodules on a
variety of woody plants from the Betulaceae, Casuarinaceae, Myricaceae, Elaegnaceae,
Rhamnaceae, Rosaceae, Coriariaceae, and Datisticaceae families (Benson et al 1993,
2000). Interestingly, it has been recently discovered that bacteria from outside the
Rhizobiaceae can induce nodules on legumes. For example, a strain of Methylobacterium,
a proteobacterium, can nodulate Crotalaria, and Burkholderia can nodulate Machaerium
lunatum and Aspalathus carnosa (Moulin et al 2001). Apparently these species have
acquired, by horizontal gene transfer, plasmids that contain many of the same genes used
by typical Rhizobiaceae to induce nodule formation and catalyze nitrogen fixation
13
(Moulin et al 2001). Nitrogenase, the enzyme complex responsible for nitrogen reduction,
is irreversibly inactivated by oxygen; therefore, this process requires conditions that are
anoxic or nearly anoxic (Quispel 1974).
2.1.1 Nodule formation to the roots of leguminous plants
The process of nodule formation is a complex phenomenon which involves the activity of
the rhizobia as well as the plant roots. Some species of rhizobia form symbiotic
relationship with certain species of legumes. This specificity is based on chemical signal
molecules. In order to initiate a productive symbiosis, rhizobia must recognize and then
respond to the presence of host plant roots. During growth in the rhizosphere of a host
plant, rhizobia sense compounds such as flavonoids and betaines secreted by the host root
and respond by inducing nod genes (Cassab 1998; Cosgrove et al 1997, 2002). These nod
genes code approximately 25 different enzymes required for the bacterial cell wall
synthesis and other specific proteins referred to as Nod factor. Nod factor initiates many
of the developmental changes seen in the host plant early in the nodulation process,
including root hair deformation, membrane depolarization, intracellular calcium
oscillations, and the initiation of cell division in the root cortex, which establishes a
meristem and nodule primordium. Thus the first step is the infection of the bacteria on the
root cells. To do this, they grow and divide inside a tubule called an infection thread
(Bosch et al 1989; Workum et al 1998). Infection thread formation is most often initiated
when rhizobia become trapped between two root hair cell walls. These bacteria grow and
divide inside the thread, thereby keeping the tubule filled with bacteria. The thread filled
with bacteria is propagated further toward the root interior. Due to this growth of the
bacteria, the branching of the thread occurs. Bacteria inside the infection thread
eventually exit it and enter nodule cells (Callaham et al 1981). Once inside the nodule
cells, the bacteria continue to differentiate and synthesize proteins required for nitrogen
fixation and for the maintenance of the mutualistic partnership. The nodule formation on
plant root can be summarized in the following steps- (i) Attraction of Rhizobium to the
roots (ii) Attachment of Rhizobium to the root hairs (iii) Curling of the root hairs (iv)
14
Formation of infection thread (v) Cell division of the root cortical cells (vi) Invasion of
cortical cell by Rhizobium (vii) Growth of the nodules and (viii) fully functional nodules
2.1.2 Process of control of number of nodules to the roots
When the Rhizobia and plants get together in soil, they do not automatically cause the
formation of as many nodules on the roots as possible. Instead the plant has a tight
control over the bacteria; it only allows them to make nodules under certain conditions.
The plant need to nourish the bacteria with sugar to provide them enough energy for
nitrogen fixation. Because the plant can not easily make unlimited amount of sugar, the
symbiosis is not just a benefit to the plant but also cost, therefore, the plant needs a
mechanism to control nodule numbers on its roots. This mechanism is called
autoregulation of nodulation (Vasse et al 1993; Penmetsa et al 2003).This involves long
distance signaling between roots and shoot. The first signal starts when the bacteria first
infect the roots. At this stage the Nod factor induces the release of signal which
translocated to the leaf (Dohler et al 1988; Caetano-Anolles 1988, 1991 a). A receptor in
the leaf called Nodulation Autoregulation Receptor which is a protein kinase and
commonly referred to as NARK (Nodulation Autoregulation Receptor Kinase) receives
the signal. This receives the signal and produces a second signal which is referred as
Shoot Derived Inhibitor (SDI). The SDI moves down the plant roots and inhibit the
formation of new nodules and also ensures nodule number do not get out of control to the
detriment of the plant (Caetano-Anolles et al 1991 b, c; Jarvis et al 1992).
2.1.3 Mechanism of Biological Nitrogen Fixation
The biochemical mechanism of N
2
fixation can be written in simplified form as follows:
N
2
(a) ------------------ NH3 amnio acid
proteins
The above mechanism indicates that the enzyme nitrogenase is responsible for fixing free
nitrogen systems. Adenosine triphosphate (ATP) is the source of energy necessary for the
cleavage and reduction of N
2
into ammonia. In rhizobia, for instance, ATP results from
+ ATP
+ H+
Nitrogenase
15
oxidative degradation of sugars and related molecules The sugar molecules are
manufactured by the host-plant during photosynthesis and transferred to the nodules
(Vasse et al 1993). In general, for each gram of N
2
fixed by Rhizobium, the plant fixes
one to twenty grams carbon through photosynthesis (Delgado et al 1993). This is an
indication that symbiotic N
2
fixation requires additional energy. The extra energy cost of
N
2
fixation can, however safely be carried by most field-grown legumes with little or no
loss of production (Gibson et al 1985). It is usually accepted that N
2
fixing systems
require more Phosphorus (P) than non-N
2
-fixing systems. Phosphorus is needed for plant
growth, nodule formation and development, and ATP synthesis, each process being vital
for nitrogen fixation (Lynd et al 1984).
Nitrogen fixation, which involves the chemical reduction of N
2
to NH
3
or NH
4
, requires a
source of electrons. Sources of electrons for the nitrogenase activity vary with the
organism. They are all small proteins and highly reductive molecules such as ferredoxin,
nicotinamide, or adenine trinucleotide phosphate. Nitrogenase is also an oxygen sensitive
enzyme which inactivates nitrogenase activity (Becana et al 1987; Devries et al 1989;
Pararjasingham 1990). In order to prevent the oxygen found in the root's environment
from reaching the nitrogenase, the plant produces a special form of hemoglobin called
laghemoglobin. This protein has a high affinity to oxygen and it's binding to the oxygen
originated around the root prevents it from reaching the nitrogenase complex (Cote et al
1989). Since nitrogen fixing is a costly process for the plant (it takes 16 ATP's to fix a
single nitrogen molecule), the plant regulates the nitrogenase's activity and expression
according to reduced nitrogen availability and oxygen presence (Pararjasingham 1990).
2.1.4 Biological nitrogen fixation for sustainable agriculture
The economic and environmental costs of the heavy use of chemical nitrogen fertilizers
in agriculture are now global concern (FAO 2007). Biological nitrogen fixation, a
microbiological process which converts atmospheric nitrogen into a plant usable form,
offers this alternative. Nitrogen-fixing systems are economically attractive and
ecologically efficient means of reducing external inputs and improving internal resources.
Symbiotic systems such as that of legumes and Rhizobium can be a major source of
16
nitrogen in most cropping systems and that of Azolla and Anabaena can be of particular
value to flooded rice crop (Bohlool et al 1992). The Biological Nitrogen Fixation system
most important for agriculture is the legume-rhizobia symbiosis where the fixation of
atmospheric nitrogen occurs within root nodules after rhizobial penetration of the root.
Thus, using this biological nitrogen fixation system, many legumes can grow vigorously
and yield well under nitrogen-deficient conditions may contribute nitrogen to the
farming system in the vegetative residues after grain harvest, or more significantly as
green manure incorporated in the soil. Legumes are important sources of protein, mainly
for feed in the developed world and for food in the developing world. They have been
used as sources of nitrogen most notably in the agricultural system of entire world
(Merrick et al 1995).
2.2 The Mycorrhiza
Mycorrhiza are highly evolved, mutualistic associations between soil fungi and plant
roots. The partners in this association are members of the fungus kingdom
(Basidiomycetes, Ascomycetes and Zygomycetes) and most vascular plants (Harley &
Smith 1983; Kendrick 1990; Brundrett 1991). In the Mycorrhizal literature, the term
symbiosis is often used to describe these highly interdependent mutualistic relationships
where the host plant receives mineral nutrients while the fungus obtains
photosynthetically derived carbon compounds (Harley & Smith 1983). Mycorrhizal
associations involve 3-way interactions between host plants, mutualistic fungi and soil
factors. The benefits to plants from Mycorrhizal symbiosis can be characterized either
agronomically by increased growth and yield or ecologically by improved fitness (i.e.,
reproductive ability). Mycorrhizal fungi usually proliferate both in the root and in the
soil. The soilborne or extramatrical hyphae take up nutrients from the soil solution and
transport them to the root. By this mechanism, Mycorrhizae increase the effective
absorptive surface area of the plant. In nutrient-poor or moisture-deficient soils, nutrients
taken up by the extramatrical hyphae can lead to improved plant growth and
reproduction. As a result, Mycorrhizal plants are often more competitive and better able
17
to tolerate environmental stresses than are nonmycorrhizal plants (Brundrett et al 1988,
1990).
2.2.1 Types of Mycorrhiza
Mycorrhiza has been classified on the basis of types of associations with the host plants.
At least seven different types of Mycorrhizal associations have been recognized,
involving different groups of fungi and host plants and distinct morphology patterns.
These are Vesicular-Arbuscular Mycorrhizas (VAM), Ectomycorrhizas (ECM),
Ectoendomycorrhiza, Arbutoid, Monotropoid, Ericoid, and Orchid. The most important
and most studied associations that exist widely in nature (Mark Brundrett 2004) are
Vascicular Arbuscular Mycorrhiza (VAM or VA) and Ectomycorrhiza (ECM)
2.2.2 Vesicular-Arbuscular Mycorrhiza (VAM)
VAM are associations where Zygomycete fungi in the glomales produce arbuscules,
hyphae and vesicles within the roots. The spores are formed in soil or at the roots. These
associations are defined by the presence of arbuscules. Fungi within roots spread by
linear hyphae or coiled hyphae
.
2.2.3 Ectomycorrhiza (ECM)
ECM are associations where Basidiomycetes and other fungi form short swollen lateral
roots covered by mantle hyphae. These roots have net hyphae around cells in the
epidermis or cortex.
The potential for manipulating Mycorrhizal associations to increase productivity in
plantation forestry, or plant establishment during ecosystem recovery after severe
disturbance, are the focus of major research initiatives. There is also much interest in
their potential utilisation in agriculture and horticulture (Bergero et al 2000). However, it
could be argued that we do not know enough about the role of Mycorrhizal associations
in natural, disturbed, or managed ecosystems to evaluate their potential for applied use
(Piercey 2002).
2.2.4 Mechanism of action of Mycorrhiza
The demand for a particular mineral nutrient depends on plant's internal requirements,
while the supply of that nutrient primarily depends on its availability and mobility in soils
18
(Russell 1977; Marschner 1995). Mineral nutrients such as phosphorus have very limited
mobility in soils (Bhat Nye 1974). Thus to obtain more phosphorus, plants intend to
increase the surface area root system. The most important role of Mycorrhizal fungus
hyphae is to extend the surface area of roots (Russell 1977; Marschner 1994).
Mycorrhizal fungus hyphae are considered to function primarily by increasing the soil
volume from which available forms of phosphorus are absorbed and provided to roots
(Hayman 1983; Harley Smith 1983). Hyphae of VAM fungi can respond to localized
sources of soil nutrients more rapidly than roots (John et al 1983, Warner 1984) and to
produce fine highly-branched absorptive hyphae in decomposing organic substrates
(Mosse 1959; Nicholson 1959). Harley and Smith (1983) showed that hyphae of VAM
fungi utilized the same forms of nutrients as roots. However, there now is also an
evidence that these associations have a greater benefit when phosphorus is present in
less-soluble forms (Bolan 1991; Schweiger 1994; Tarafdar and Marschner 1994;
Kahiluoto and Vestberg 1998). ECM fungus hyphae also proliferate in decomposing soil
organic matter and help the roots in absorbing the available phosphorus (Harvey et al
1976; Reddell and Malajczuk 1984; Bending and Read 1995). Hyphae of ECM fungi can
utilize both inorganic and simple organic sources of nitrogen and phosphorus
(Abuzinadah and Read 1989; Hausling and Marschner 1989).
2.3 Blackgram (Vigna mungo)
Vigna mungo commonly known as Urd bean or blackgram, is one of the most important
pulse crops which are grown for its protein rich edible seeds. Blackgram is an important
grain legume grown in south and south-east Asia. Presently it is cultivated in India,
Pakistan, Myanmar, Bhutan, Bangladesh, Thailand, Malaysia, Philippines, Afghanistan,
Iran, Kenya, Malawi and the United States. India is the largest producer of all. The average
production of blackgram in India is 2.20 Mt annually from an area of 3.96 million ha.
(http//www.fao.org). The production in India and Bangladesh is almost entirely for
domestic use as food. Blackgram is basically a tropical crop but it is grown in both winters
and summers in India. It is primarily intercropped with sugarcane, cotton, groundnut,
sorghum and pigeonpea during the wet season, monocropped on residual moisture in winter
19
(in rice fallow) and in spring/summer season in between the two main crops (Singh 1997).
Since this crop is grown in various agroecological conditions and cropping systems with
diverse cultural practices, an extensive survey has been done to collect the germplasm of
blackgram for potential utilization in development of appropriate cropping systems in
tropical Asia. The Asian Vegetable Research Development Centre (AVRDC), Taiwan,
maintains a collection of nearly 200 accessions, while the National Bureau of Plant Genetic
Resources (NBPGR), New Delhi, India maintains about 2100 accessions of which the
Indian Institute of Pulses Research, Kanpur (U.P.), India holds 829 active collections
(Singh and Shukla 1994; Nautiyal and Shukla 1999).
Urd bean is known to be closely related to mung bean because of similarity in their growth
habit, adaptation and their utilization. Earlier, both of them were classified in the genus
Phaseolus, but were recognized to be separate species later. The progenitor of Urd bean is
Vigna mungo, var. sylvestris and that for Mung bean is Vigna radiata, var. sublobata
(Verdcourt 1970). This distinction has been re-emphasized and has been reconfirmed on
the basis of seed coat patterns, pod size, position of pods on peduncle, number of seeds per
pod and nature of hilum in seed coat (Sneath et al 1973). It was noted that Urd bean and
Mung bean are partially cross-fertile only when Mung bean is used as the female parent.
Additionally F1 plants of crosses between the species have a high proportion of sterility
suggesting that a partial incompatibility barrier separates these two species (Abdul et al
2003).
2.3.1 Plant characteristics
Blackgram is an annual, semi-erect to spreading herb, 25-110 cm tall. Stems have enough
branching and covered with long dense brown hairs. Leaves are trifoliate, hairy with large
ovate leaflets. Flowers are pale yellow, papillionaceous and are borne in clusters of 5-6 on
a short peduncle in axillary racemes. Flowers are self-fertile and self pollinated.
2.3.2 Seed characteristics
The pods are short, brown to black, hairy with 4-7 seeds. The seeds are small, weigh ca.
40 mg, oblong and dark brown to black. Germination of the seeds is epigeal. The protein
rich edible seeds of blackgram are favored for easy digestibility, provitamin A content and
20
abundant phosphoric acid (Wanjari 1988). Their high lysine, low methionine content is
used to supplement the cereals which have high methionine, low lysine content to provide a
balanced protein diet to the mainly vegetarian Indian population. The seeds are used to
prepare various dishes after boiling or grinding. The green and dried stalks and leaves are
used as fodder.
2.4 Status of Varietal Improvement of Blackgram
Blackgram varietal improvement was initiated at Pusa, Bihar in 1925. Breeders collected
many samples from different districts of India and Burma. Single plant selection was made
from these samples and pure lines were isolated during the following years. Out of 52
varieties released so far, 32 are the result of selection, 18 of hybridization and 4 through
mutation breeding (Acharya et al 1993). Though more than 50 varieties of blackgram have
been released, a narrow genetic base coupled to extensive self-fertilization has proved to be
the major hurdle for adequate improvement via traditional breeding methods. The
developmental procedures to regenerate plants from single cells and organized tissues and
the discovery of methodologies to transfer genes in to regenerable target plant cells forms
the basis of genetic engineering in crop improvement. Thus, agricultural biotechnology
promises to improve this important crop by selective transfer of genes, which are not
naturally available in the gene pool, and hence would be an ideal supplement to
conventional breeding methods.
2.5 Introduction to Other Vigna Species
The genus Vigna is comprised of eight subgenera and more than 100 species (Verdcourt
1970), most of which are distributed in Asia and Africa. The cultivated species include
Vigna mungo, Vigna radiata, Vigna aconitifolia, Vigna angularis, Vigna unguiculata,
Vigna umbellata and Vigna subterranean. The first five are widely grown in Asia while
latter two are commonly found in African region.
2.6 Regeneration Studies in Crop Legumes With Special Reference to Vigna Species
Regeneration in crop legumes, like in other plants have been reported to occur by three
modes (1) de novo shoot regeneration (organogenesis) from different explants with
subsequent development of complete plants (2) somatic embryogenesis leading to complete
21
plant regeneration and (3) axillary bud proliferation from areas surrounding a meristem. All
the three modes of plant regeneration have been reported in important leguminous crops
like soybean, peanut, chickpea and pigeonpea. Organogenesis can be induced by the
manipulation of exogenous phytohormones and can occur directly from cultured explants
or from callus. In pigeonpea, diverse explants have been shown to produce de novo shoot
regeneration (George and Eapen 1994). In peanut, plant regeneration can be induced from
leaf lamina (McKently et al 1991), epicotyl and petiole (Cheng et al 1992). Similarly,
somatic embryogenesis in peanut could be induced from a variety of explants such as
mature and immature embryonic axes and mature and immature cotyledons (Eapen et al
1993) and plantlets could be regenerated. However, frequency of plant development from
somatic embryos in crop legumes is too low for effective utilization in transgenic
experiments. In Glycine max, there are numerous reports on somatic embryo production
(Finer 1988; Parrott et al 1989, 1994). However, reports on utilization of somatic embryo
technology for production of soybean transgenic plants are very few, mainly due to the
long time period associated with selection and development of transgenic plants. The third
mode of regeneration, namely axillary bud proliferation from areas surrounding the
meristems has been extensively used for regeneration and transformation for crop legumes
(Hinchee et al 1988). Since axillary bud proliferation yields quick results compared to
somatic embryogenesis or de novo organogenesis and the plants regenerated are healthy
with improved survival on transplantation to soil, the use of axillary buds with meristematic
areas are the most acceptable source of regenerating cells for development in crop legumes.
Since organogenesis and somatic embryogenesis take long periods to yield regenerated
plants, the quick regeneration protocol using cotyledonary nodes and shoot meristems has
been the preferred mode for introduction of genes into crop legumes (Popelka et al 2004).
2.7 Regeneration of Blackgram (Vigna mungo)
Plant regeneration in vitro has been established from many forage legumes but there is little
success in regenerating plants from grain legumes. Vigna species which had been earlier
recalcitrant to regeneration have now responded favorably via direct or indirect
organogenesis or somatic embryogenesis. For in vitro regeneration, different explants from
22
diverse sources and number of hormones in different combination have been reported.
Regeneration has also been achieved from cells in suspension, anthers and protoplasts in
some of the Vigna species.
Similar to the regeneration of other legumes, in vitro regeneration in Vigna mungo deals
with 1) direct or indirect organogenesis and 2) somatic embryogenesis using various
explants.
2.7.1 Organogenesis
The first report of regeneration of Vigna mungo via direct organogenesis was of Gill et al
(1987 a) by using cotyledons with intact cotyledonary node. Maximum multiple shoots
were induced by BAP at a concentration of 2.0 mg l
-1
. Histological studies demonstrated
the presence of axillary meristematic tissues at the time of culture. The shoots were rooted
on MS medium supplemented with 1.0 mg l
-1
NAA. Ignacimuthu et al (1987) studied the
response of different explant types derived from germinating seeds on MS basal medium.
Multiple shoot formation was the major step in regeneration and it was notably affected by
the presence or absence of one or both cotyledons and also shoot tips. It was observed that
number of shoots produced was more in explants with both cotyledons intact as compared
to explants with single or no cotyledon. Multiple shoots were produced from the axis of the
attached cotyledon, while the other side of the node without cotyledon showed no
regeneration and formed only callus. Removal of shoot tips improved the shooting
efficiency of the explants. BAP was the most effective phytohormone at a concentration of
2.0 mg l
-1
.
Sen and Guha-Mukherjee (1998) observed that kinetin was more effective than BAP in the
induction of multiple shoots from nodal explants derived from 6-day old germinated
seedlings. Explants with shoot initials were sub-cultured on B5 medium supplemented with
0.1 mg l
-1
kinetin. These were further kept on rooting medium on B5 supplemented with 0.1
mg l
-1
IBA. Ignacimuthu and Franklin (1997) reported the generation of sexually mature
blackgram plant regenerated from cotyledon and embryonal axis. It was reported that the
micronutrient concentration of 4X and an initial exposure of explants to a dark period of 15
days, before transfer to 16 h photoperiod was an essential factor for obtaining maximum
23
shoot bud induction. MS medium supplemented with 13.3µM BAP and 0.15µM NAA gave
a maximum response with 6-7 shoots per cotyledon explant and 4-5 shoots per embryonal
axis explant. Rooting was achieved by transferring the shoots to IBA fortified medium.
Saini et al (2002) reported that explant age, polarity and explant orientation decide the fate
of morphogenesis in explants. Seeds were germinated for 3 days on MS medium
supplemented with BAP and epicotyl explants were taken. These explants exhibited
regeneration from the apical as well as the basal end depending on the orientation in which
they were incubated on culture medium. When incubated in vertically upright position
these explants showed regeneration from both the cut ends but the mode of the
morphogenic pathway differed at both ends. The basal end in contact with the medium
formed non-friable and green, nodular callus from the surface of which, shoots were
formed with significantly lesser number of adventitious shoots while the apical cut end
formed callus with lesser number of shoots.
Das et al (1998) reported regeneration from stem and petiole explants obtained from BAP
pre-conditioned seedlings. Following three days of seed germination on MS medium
supplemented with BAP 2.0 mg l
-1
or TDZ 0.5 mg l
-1
, the hypocoytls were cut off just
below the cotyledonary node and the seeds were further subcultured on the same medium
with the cut end inserted in to the medium. Axillary shoots developing at the cotyledonary
node were excised after 12 days and were inoculated on ½ MS medium with 0.1 mg l
-1
NAA. The pH of the medium and the type of the explants were of critical importance.
Maximum shoot buds were obtained at pH 5.5. The stem explants with the intact shoot
apex proved superior to those without the apex and petiole explants.
2.7.2 Somatic embryogenesis
There are few reports for the regeneration of complete plants via somatic embryogenesis.
Gill et al (1987 b) reported that the embryogenic calli could be induced from cotyledonary
node cultured on MS medium with picloram 1.0 mg l
-1
, zeatin 1.0 mg l
-1
and IAA 0.1 mg l
-
1
. These calli were then transferred to liquid medium with the same concentration of
hormones except the level of picloram was reduced to 0.30 mg l
-1
. Embryoid like structure
was seen in about 10-20 % of cultures. The embryoids were subcultured on semisolid MS
24
medium supplemented with 0.1 mg l
-1
ABA and 0.1 mg l
-1
GA
3
. Further maturation and
germination of these embryoids was unsuccessful as they gradually turned brown and
necrosed.
Recovery of complete plants derived from somatic embryos was reported by Geetha et al
(1997 b). Calli were induced using hypocotyls on MS medium supplemented with 2,4-D
3.0 mg l
-1
, NAA 10 mg l
-1
and kinetin 0.5 mg l
-1
. Cell suspensions were initiated by
transferring these calli to liquid medium supplemented with 0.5 mg l
-1
BAP. Embryo
development and maturation was reported to take place on the same medium after two
subcultures. These embryos reportedly germinated in to plantlets on transfer to semisolid
medium supplemented with 0.1 mg l
-1
BAP.
2.8 Genetic Transformation of Blackgram
Blackgram is one of the important crops grown in the tropical and subtropical regions of
the Indian subcontinent. There have been relatively insignificant improvements towards
the production and productivity of this important crop. This is due to a narrow genetic base,
limited natural variation available and several natural stresses to which the crop is
subjected. These stresses include biotic and abiotic stresses. The yellow mosaic disease
caused by Vigna mungo yellow mosaic virus, fungal disease caused by Colletotrichum and
Cercospora and other storage insect pests are the major cause of the biotic stresses. Of the
abiotic stresses, excessive sensitivity to salinity, drought and heavy metal causes
considerable reduction in productivity of the crop. The solution for overcoming many of
these problems lies in genetically engineering the plants for stress tolerance.
There are only few reports available for the transformation protocol of blackgram.
Transient expression of GUS gene was reported in meristematic zones and embryo axes of
V. mungo by Bhargava and Smigocki (1994).
Karthikeyan et al (1996) were successful in
getting transgenic calli of V. mungo by cocultivating primary leaves with Agrobacterium.
Sarin et al (2004) reported engineering of salt tolerance in V. mungo by the overexpression
of the glyoxalase I (gly 1) gene
.
Saini et al (2003, 2005) reported the production of
morphologically normal and fertile transgenic plants from cotyledonary node and apical
meristem explants inoculated with Agrobacterium tumefaciens carrying binary vector
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