Pulses are the climate resilient crops as they promote sustainable agriculture, decrease green house gases, fix atmospheric nitrogen, improve soil fertility and use less water compared to other crops (
Singh, 2016). Moreover, they are the richest source of protein for vegans as they are highly nutritious and aid in weight management. India has attained self-sufficiency in pulse production by achieving an all-time high production of 23 million tonnes during 2016-17 and more than 24 million tonnes during 2017-18 (
Anonymous 2019). The maximum share in pulse production is taken care of by chickpea followed by pigeonpea. Green gram and blackgram then offer a fair share of ca. 10-12% in total pulse production. Amongst the different pulses grown and consumed, blackgram is relatively less popular and a lesser explored crop even after being a highly nutritious legume crop and also enriched with medicinal values.
Blackgram is grown through out the year and in all seasons in tropical country like India. People in south region of country use it as staple food along with rice. To improve production of crop, biotic and abiotic stresses need to be addressed. Genetic improvement of crop is need of the hour and in this context various regeneration protocols and
Agrobacterium mediated transformation methods have been worked on and reported. Various studies reported can be utilised for exploring wider research opportunities in blackgram. Reports suggest
in vitro regeneration of blackgram has been possible through direct organogenesis, indirect organogenesis and somatic embryogenesis although latter had meagre response compared to other two modes. This review reports regeneration protocols and genetic transformation methods in blackgram, highlights their regeneration potency, genetic advancement and scope of research to address serious concerns of crop including biotic and abiotic stresses and crop productivity.
In vitro regeneration by direct organogenesis
In direct organogenesis, shoots are induced directly from explants on addition of cytokinins and rooting in these shoots is induced using auxins. There are various studies on
in vitro regeneration by direct organogenesis and in most of them multiple shoots inductions are reported
(Das et al., 1998; Geetha et al., 1998; Ignacimuthu et al., 1997; Franklin and Ignacimuthu, 2000;
Mony et al., 2010; Ignacimuthu and Franklin, 1999;
Sen and Guha-Mukherjee, 1998;
Saini et al., 2003; Sen et al., 2002; Das et al., 2002; Saini and Jaiwal, 2002). Successful plant development from multiple shoots obtained via
in vitro regeneration by direct organogenesis were reported by
Ignacimuthu et al., (1997), Mony et al., (2008); Agnihotri et al., (2001); Saini and Jaiwal (2002);
Srilatha (2014),
Muruganantham et al., (2014), Guru
Prasad et al., (2014); Sainger et al., (2015); Adlinge et al., (2014); Roy et al., (2007), Saha et al., (2017). All type of explants were explored for the purpose including hypocotyl, epicotyl, cotyledon, immature and mature cotyledonary node, petiole, leaf, axillary shoots, axillary bud, shoot tip, root tip, stem and embryonic axis. Survival percentage of regenerated plants was above 70% in most of the studies in which plants produced was reported. Almost 45-60 days time period was required for
in vitro development of plants.
Srilatha (2014) developed protocol for direct regeneration of blackgram using cotyledonary nodes as explants in
cv Vamban 1 using cytokinins 6-Benzyl amino purine (BAP), Thidiazuron (TDZ) and kinetin supplemented in Murashige and Skoog’s (MS) media. Optimum concentration of 4.0 mg/L TDZ gave better response to shoot induction and produced maximum 4.3 shoots per cotyledonary explant. Shoots were elongated on the same media and transferred to Indole-3-butyric acid (IBA) supplemented media for rooting. 100% rooting was observed on ½ MS media supplemented with 0.5 mg/L IBA. The survival rate of plants developed was significantly high (appx 80%).
Another study of same group reported increase in number of shoots on adding Indole 3-acetic acid (IAA) to media.When nodal explants of blackgram
cv Vamban 1 were cultured on MS media containing varying concentration of TDZ, BAP and Kinetin (0.5-3.0 mg/L) alone and in combination with IAA (0.5 mg/L), shoot induction and proliferation improved. Maximum number of shoot bud proliferation was observed at 0.5 mg/L IAA and 2.0 mg/L TDZ whereas higher frequency of shoots were induced at 0.5 mg/L IAA + 2.0 mg/L TDZ and 0.5 mg/L IAA and 2.5 mg/L kinetin. It has also been observed that high cytokinin to auxin ratio promotes shoot formation in blackgram. On supplementing IAA/IBA to MS media, roots were produced in 96% of plants with ca. 14.3 roots per explant. It was recorded that maximum shoots were obtained on TDZ supplemented MS media
i.e. 7 shoots per explant corresponding to 62% shooting efficiency
(Srilatha et al., 2014). Details of
in vitro regeneration in blackgram via direct shoot organogenesis are shown in Table 1.
In vitro regeneration by indirect organogenesis
Regeneration of
Vigna mungo by indirect organogenesis
i.e. shoot induction from callus originated from various explants. Researchers have worked on all possible explants and various hormonel combinations that resulted in formation of callus and eventually shoots emerged from them that developed into plant. In one such study, when primary leaf explant of cultivar Co5 was placed on MS media supplemented with different concentration of 2,4- Dichlorophenoxyacetic acid (2,4-D), BAP, 2-Isopentyl adenine (2iP) and kinetin, led to calli formation that produced shoots and plant
(Karthikey et al., 1996). In another report, many explants were selected to initiate regeneration including cotyledonary node, hypocotyl, epicotyl, axillary bud and immature leaf of blackgram
cv LBG. They were cultured on MSB media with phytohormones 16.1 µM 1-Napthalene acetic acid (NAA) and 2.2 µM BAP that produced callus but a plant was not regenerated
(Geetha et al., 1997a). Goel et al., (1983) showed plant development from
in vitro cultured shoot tips that developed into mature plant. Later on
Sen and Guha-Mukherjee (1998) studied regeneration potency of two explants hypocotyl and cotyledon of various cultivars of blackgram
i.e. PS-1, TM-9, PU-19, PU-35. Explants were cultured on MSB supplemented with 0.045 µM TDZ produced callus and shoots.
Das et al., (1998) showed callus formation and shoot bud development when axillary shoot of
cv PS-1 were cultured on half strength MS media supplemented with phytohormones 0.54 µM BAP and 2.2 µM NAA. They further reported use of stem and petiole derived from axillary shoots as explants of various cultivars including T9, Pusa1, Pusa 2 and cultured these explants on half strength MSB5 media supplemented with 0.1 mg/L NAA. It produced callus and shoot buds in 5-6 weeks time period
(Das et al., 1998). Avenido and Hattori (1999) reported regeneration of blackgram using cotyledonary node of
cv silvestris on MSB media with 1.0 mg/L BAP. Survival rate of plants developed was reported as 50-100%.
Varalaxmi et al., (2007) used cotyledon of T9 cultivar and cultured on MSB media supplemented with 4.0 mg/L BAP. It produced callus and multiple shoots in around 5 weeks time period. Plants developed had survival rate of 90%.
Harisaranraj et al., (2008) used half seed as explant of blackgram
cv silvestris and cultured on B5 media supplemented with 13.3 µM BAP and 13.5 µM 2,4-D. It took about 5 weeks for callus and multiple shoots production.
Srivastava and Pandey (2011) reported callus induction and regeneration of blackgram
cv silvestris from leaf explants on MS media supplemented with varying concentration of 2,4-D (0.5 µM-72.5 µM ) and NAA (0.5 µM-72.5 µM ). Callus induction occured in all combinations and proliferation was seen maximum in media with 2,4-D. Shoot induction was observed on 6 µM 2,4-D at 6 weeks after transferring the callus with 86.67% growth. Media with 6 µM 2,4-D and 3% sucrose improved frequency of callus induction, maturation and further development.
Varalaxmi et al., (2013) showed regeneration of plant blackgram cultivar T9 in 5-6 weeks. They used cotyledon with embryonic axis as an explant and cultured on MSB media supplemented with 4.0 mg/L BAP.
Roy et al., (2003) reported callusing from hypocotyl and roots of 4-7 days old seedlings on culturing in modified MS media supplemented with 2,4-D followed by plants development. Detail of regeneration of blackgram via indirect organogenesis has been shown in Table 2.
In vitro regeneration via somatic embryogenesis
In vitro regeneration protocol via somatic embryogenesis has been worked on but there is meagre response for entire plant development from somatic embryos
(Gill et al., 1987; Eapen and George, 1990;
Geetha et al., 1997a; Geetha et al., 1997b; Sen et al., 2002 and
Muruganantham et al., 2010). Detail of regeneration of blackgram via somatic embryogenesis has been shown in Table 3.
Gill et al., (1987) used callus culture obtained from cotyledonary node as explant and grown on MSL media supplemented with picloram, zeatin and IAA. It lead to development of embryoids.
Eapen and George (1990) showed somatic embryos and shoots developed when immature cotyledon of
cv no. 55 was cultured on L-6 liquid media supplemented with NAA, 2iP, BAP, kinetin and zeatin. When such explants were cultured on MS media supplemented with NAA, 2,4-D and picloram, only callus was obtained that did not form somatic embryos.
Geetha et al., (1997a) showed callus formation when cotyledonary node, hypocotyl, epicotyl, axillary bud and immature leaf of cultivar LBG were used as explant and placed on MSB media supplemented with NAA and BAP. But embryos or plants could not be formed. Later on
Geetha et al., (1997b) used hypocotyl of cultivar LBG placed on MSB media supplemented with 2,4 D, NAA and kinetin. But it also did not lead to formation of somatic embryos, only callus was formed. When callus obtained from hypocotyl were placed on MSL supplemented with BAP and 2,4-D, it lead to formation of somatic embryos.
Sen et al., (2002) used cotyledon explants of
cv PS-1 cultured on MSB media supplemented with picloram and proline. It lead to formation of somatic embryos.
Muruganantham et al., (2010) used primary leaf of
cv Vamban3 as an explant. They used MS and MSL media supplemented with varying concentration of 2,4-D to yield callus, somatic embryos and plants.
Agrobacterium mediated genetic transformation in blackgram
Genetic enhancement of
Vigna mungo has been attempted with various genes capable of improving biotic and abiotic stresses tolerance. Various methods can be used to introgress novel genes in crops but
Agrobacterium mediated transformation is preferred over others due to higher transformation efficiency. Various strains of
Agrobacterium have been used such as
LBA4404,
EHA105,
GV3101 etc for the purpose but
LBA4404 is reported to have higher transformation efficiency that is 23% whereas for
EHA105 it was 10%. Moreover, traits of economic importance, insect and disease resistance, and adaptation to changing climate must be introduced. There are very few reports on genetic improvement of blackgram with very few cultivars explored such as T9, Co5, Vamban1, Vamban3, PS-1, Pusa2 and LBG20. Most of the regeneration protocols used were amenable to
Agrobacterium mediated transformation except the few such as
Karthikey et al., (1996) and
Nukula and Anandan (2018) in which transgenic plants could not be produced or presence of transgene could not be confirmed.
pCAMBIA 2301 has been the preferred vector and in nearly all reports, confirmation of transgene
i.e. development of protocol was main objective. Details of
Agrobacterium mediated transformation in blackgram have been compiled in Table 4.
Transgenic
Vigna mungo were regenerated mostly on MS media except for few modification on strength such as MS full strength, MS half strength
etc. ½ MS is preferred for root induction whereas 1/3 MS has also been used in one report. It has been recorded that in all transformation reports, BAP is preferred phytohormone to induce callus or shoots for organogenesis and few other phytohormones used are in combination with BAP. Transformation frequency varied from 1.0 % to 10.2% depending on the different protocols followed.
Transgenic lines of
Vigna mungo were developed by
Agrobacterium mediated transformation using immature cotyledonary node and shoot tip as explants
(Muruganantham et al., 2007). It was an approach to develop and demonstrate method of regeneration amenable to
Agrobacterium mediated transformation for production of transformants and transgenics with desired biotic and abiotic stress related genes. They developed Basta (herbicide) tolerant transformants using phosphinothricin as selection agent. Transformant shoots were regenerated on MS basal media supplemented with 4.44 µM Benzyl adenine (BA), 0.91 µM TDZ and adenine sulphate in 6 weeks. Shoots regenerated were kept on MS media supplemented with 7.36 µM IBA for rooting. All plants developed were reported to be fertile and followed mendelian genetics.
Insect pests, pathogens and diseases are the biotic stresses causing loss of quantity and quality of yield of crop. Barley chitinase and ribosome inactivating protein (RIP) were used to transform and develop transgenic blackgram effective against fungal disease Corynespora leaf spot (
Chopra and Saini, 2014).
Balaji et al., (2003) used primary leaf segment as explant of blackgram
cv Co5 according to regeneration protocol of
Karthikey et al., (1996). Plasmids
pTiBo542 virB and virG were used and electroprated in
Agrobacterium tumefaciens strain
LBA4404 to produce transgenic plants.Transformants were evaluated on basis of GUS staining in stably transformed callii. The octopine vir helper strain
LBA4404 harbouring
pBAL2, the plasmid
pBAL3 with 3’ end of virB and complete virG of
pTiBo542 increased transformation efficiency. Multiple copies of 3’ end of virB and virG of
pTiBo542 enhanced
Agrobacterium tumefacien mediated stable transformation efficiency of blackgram.
Varalaxmi et al., (2013) used cotyledon derived calli of
cv T9 for transformation.
Agrobacterium tumefacien strain
LBA4404 for genetic transformation in
Vigna mungo and regenerated on MSB5 media supplemented with 4mg/L BAP in 5-6 weeks time with 3.8% transformation efficiency. Presence of transgene in plants was confirmed by PCR of
nptII gene. Transient GUS expression studies revealed that a cell density of 10
8 cells/ml, 100 µM acetosyringone and 330 µM cysteine were effective in increasing the transformation frequency and obtaining stable transformants with a 3.8% transformation efficiency. IBA pulse treatment was effective in root induction of kanamycin selected putative transformants.
Karthikey et al., (1996) transformed
Vigna mungo, calli were obtained by co-cultivation of segment of primary leaf with
Agrobacterium tumefacien strain
LBA4404 harbouring the binary vector
pGA472 was used for the purpose. Transgene was confirmed by neomycin phosphotransferase assay. Stable integration of transferred DNA into
Vigna mungo genome was confirmed by southern blot analysis. Regeneration was standardised with 45 µM 2,4-D, 0.015 µM BAP, 0.015 µM 2µP, 0.015 µM kinetin but transgenic plants were not advanced.
Saini et al., (2003) showed regeneration using cotyledonary node in cultivar PS-1 of blackgram.
Agrobacterium strain
LBA4404 was used for genetic transformation. MSB supplemented with 5 µM BAP was used to develop transgenics in 6-8 weeks time with 1% transformation efficiency.
Saini and Jaiwal (2005) used shoot apex of
cv s.PS-1, Pusa2, Vamban1, Co-5, T9 for transformation.
Agrobacterium strain
EHA105 harbouring binary vector
pCAMBIA 2301 was used for genetic transformation. Regeneration was done on MSB media supplemented with 10 µM BAP. It lead to development of transgenic plants.
Nukula and Anandan (2018) utilised
pBinAR harbouring
CryIAcF gene to genetically transform de-embryonated cotyledon of
cv Vamban3. It was later regenerated on MS media supplemented with 3.0 mg/L BAP and rooting was induced on 0.5 mg/L NAA. It took 7 weeks to develop plants but were not confirmed transgenics.
Saini and Jaiwal (2007) used
Agrobacterium tumefacien strain
EHA105 harbouring
pCAMBIA 2301 to transform cotyledonary nodes of
cv PS-1 and regeneration was continued on MS media supplemented with 0.5 µM BAP for shoot induction and 2.5 µM IBA for rooting and produced transgenic plants in 6-8 weeks. Transformation efficiency reported was 4.3%.
Bhomkar et al., (2008) used
Agrobacterium strain
GV3101 and binary vector
pCAMBIA 2301 to transform cotyledonary node of embryonic axis of
cv LBG-20. Plants were regenerated on MS media supplemented with 1.0 mg/L BAP in 10-11 weeks time period. Transformation efficiency of regenerated transgenic plants was estimated as 2.25%.
Unexplored areas in blackgram and future prospects
Development of transgenic blackgram with different novel genes that are effective in controlling infestation of chewing and sucking insect pest is need of the hour. In the regime of climate change, viral diseases in blackgram are a big problem. These viral diseases are majorly transmitted through pests like whitefly, aphids
etc. Though improved crop lines flow in continuously to give tolerance to the diseases, they are not completely resistant. Hence, resistance imparting genes must be explored and incorporated in transgenic lines that are effective against biological vectors like whitefly and eventually controls transmission of virus and viral diseases.
