Multiple Shoot Regeneration from Detached Embryonic Axis in Greengram (Vigna radiata) cv.SML 668

A short duration crop with low input requirements and outstanding nutritional value of easily digestible protein (Itoh et al., 2006; Gan et al., 2017), the grain legume mungbean [Vigna radiata (L.) Wildzek] belonging to the family Leguminoseae (Fabaceae), is grown globally in an area of about 7.3 million ha with a global production of 5.3 million tons (2015-17) (https://avrdc.org/intl-mungbean-network). India and Myanmar account for 30%, China for 16% and Indonesia for 5% of the total global produce of mungbean. Yet, the average seed yield of the crop in major growing countries is low (aprox. 0.5 to 1.5 t ha^-1) (Nair and Schreinemachers, 2020) due to loss in yields from both biotic and abiotic stress(es).

Amongst viral diseases, Yellow Mosaic Disease (YMD) is a major constraint not only in India but also globally (Kulkarni et al. 2019; Mishra et al. 2020). Conventional integrated pest management strategies desperately need an update especially in the prevailing regime of changing climate. Improved mungbean bred by conventional means tend to lose their resistance to disease and face problems of narrow genetic base in pulses. Further genetic improvement in mungbean now requires implementation of modern biotechnological tools like translational genomics, genome editing and transgenic (s) (Kang et al., 2014, 2015; Mishra et al., 2020). In this context, it is essential to have an efficient in vitro regeneration system for mungbean in desired genotype for further research on genetic transformation or genome editing. Earlier reports on Agrobacterium mediated transformation have shown low frequencies of regeneration and recovery of transformants despite several optimizations in protocols (Gulati and Jaiwal, 1990; Khatun et al., 2008; Sahoo et al., 2016). Recalcitrance of mungbean towards regeneration and transformation (Atif et al., 2013; Chandra and Pental, 2003; Chaudhary et al., 2010) still exists and is also genotype and explant dependent (Anandan et al., 2019).

With limited reports on popular Indian mungbean cultivars in this context, this study was initiated with the sole objective to evaluate the impact of different plant growth regulators on in vitro shoot organogenesis from double cotyledonary node and embryonic axis explants to establish an optimal regeneration system.

Plant material

The cultivar SML668, having a high demand in Indian market (Mehandi et al. 2019) and derived through selection from NM 94 was selected for this study. Being YMD sensitive (Suman et al., 2015) this cultivar has huge scope of improvement through in vitro studies. Pure seeds of Breeder’s grade were obtained from ICAR-IIPR, Kanpur. The entire experiment was conducted in Division of Plant Biotechnology, ICAR-Indian Institute of Pulses Research, Kanpur during the years 2019-2020.

Seed sterilization

Handpicked healthy and uniform seeds were surface cleaned with Tween 20 for 15 mins and rinsed under running tap water. Thereafter seeds were treated with the 0.2% HgCl₂ solution for 3 mins, 70% ethanol for 1 min and rinsed thrice with sterile distilled water. Sterilized seeds were placed on sterilized blotting sheets for further use.

Explant preparation

Surface sterilized seeds were aseptically germinated on MS (Murashige and Skoog, 1962) media with B5 vitamins (Gamborg et al., 1968) (MSB5) devoid of any phytohormones, 3% (w/v) sucrose solidified with 0.8% (w/v) agar and with pH 5.8.

Double cotyledonary nodes were excised from 4 old seedlings by removing the seed coat and cut on both the sides covering both hypocotyl and epicotyl along with the apical portion of cotyledons.

Embryonic axis excised from sterilized and soaked seeds in sterile distilled water for 24 h in dark were split open to detach both the cotyledons exposing 3-5 mm embryonic axis.

Inoculation and shoot multiplication

Aseptically prepared explants were inoculated in shoot bud induction media i.e. MSB5 fortified with different concentration of phytohormones viz. BAP alone (0.5 mg l^-1, 1 mg l^-1 and 2 mg l^-1) or in combination with other phytohormones (NAA, IAA, TDZ). Amino acid (glutamine, proline and cysteine) supplementation in basal media was also tried in effort to enhance shoot proliferation. On emergence of primary shoots, they were excised and transferred to MSB5 media devoid of phytohormones for further elongation. Sub culturing was done every fortnight. With emergence of new shoots, they were excised and placed on fresh media for elongation. Callus appearing at base of explant was intermittently removed.

In each experiment, MS medium was used for culture and pH of the medium was maintained at 5.8±0.02. Cultures were maintained at 25±2°C under a 16:8 h (light, dark) photoperiod with light intensity 25 μmol m^-2 s^-1 provided by cool white fluorescent tube lights.

Rhizogenesis and hardening of plantlets

In vitro regenerated shoots with a length more than 2 -3 cm were individually transferred onto root initiation media: MSB5 supplemented with 1.0 mg l-1 of IAA or IBA or NAA for DCN explant. EA derived shoots were rooted in MSB5 media without any phytohormone. Shoots with a developed rooting system were then gently removed from media, washed with tap water to remove agar entangled in roots, initially hardened in small pots with soilrite and covered with poly bags having pores for 2 weeks in culture room. The bags were then removed and established plants were transferred to a mixture of soil, sand and vermiculite kept in greenhouse for further development. Prevailing temperature recorded was 27±2°C with relative humidity of ca. 30-35%. 

Data recorded

Each treatment had 15 explants in triplicates. Visual observations of increase in shoot number, length and callus were taken every week and data for shoot multiplication were recorded as frequency of shoot differentiation, numbers of shoots regenerated per explant and shoot length, 4 weeks post inoculation. The root length, percentage of shoots with rooting and days to rooting were recorded 2-3 weeks post inoculation in rooting media.

In vitro shoot regeneration

DCN as explant

Basal culture medium viz. MSB5, devoid of any phytohormones, did not regenerate multiple shoots. Assessment of number of shoots regenerated in vitro using different phytohormones individually and in combination along with amino acids was hence done (Table 1). Cytokinins are known to be a prerequisite for legume in vitro regeneration, as in ground nut (Venkatachalam and Jayabalan, 1997); lentil (Fratini and Ruiz, 2002) and urdbean (Adlinge et al., 2014) and amongst many cytokinins, BAP has been the most responsive, especially with Vigna species (Chandra and Pal, 1995). Hence, BAP was chosen as the cytokinin for this study. In comparison to basal media alone, higher number of shoots regenerated in presence of BAP with a maximum of 3.1±0.08 shoots per explant in presence of 1.0 mg l^-1 BAP (Fig 1).





Though per cent regeneration was not statistically different in media supplemented with BAP at 0.5 mg l^-1 and 1.0 mg l^-1, number of shoots regenerated were significantly higher at 1.0 mg l^-1 BAP. Reports indicate that combination of BAP and auxin aid in axillary shoot induction and elongation (Rasool et al., 2009; Yadav et al. 2010, Singh et al., 2014) across different crops; hence influence of BAP in combination with auxin on shoot regeneration was also tested. It was observed that shoot regeneration in media supplemented with BAP (1.0 mg l^-1) in combination with NAA (0.1 mg l^-1) was at par with BAP 1.0 mg l^-1 alone but there was a significant decrease in per cent regeneration. Mahalaxmi et al., (2003) had reported a positive effect of amino acid supplements on in vitro regeneration of cotyledonary node explant.  On a similar line,  basal media supplemented with  BAP and NAA and amino acid (s), (L-Cysteine, Proline and Glutamine 50 mg l^-1 each) was tested for multiple shoot regeneration in mungbean to find that the combination regenerated lesser number of shoots in comparison to  media supplemented with NAA alone or BAP alone.  Among all the treatments tested, media with 1.0 mg l^-1 BAP + 0.1 mg l^-1 NAA responded better for number of shoots per explant  (2.86±0.19) followed by the media 1.0 mg l^-1 BAP + 0.1 mg l^-1 NAA + 50 mg l^-1 Glutamine (2.34±0.19) (Fig 2). A decrease in per cent regeneration was also documented.

Role of TDZ as a shoot multiplication hormone (Kumar et al., 2003; Amutha et al., 2006) for mungbean cultivars was tested but failed to reproduce the results. TDZ induced multiple shoot node formation rapidly and effectively in comparison to BAP, but these formations remain stunted and converted into callus. Even in combination with BAP, TDZ regenerated single shoot with callusing that ultimately died due to tissue necrosis.

Although there are number of published optimized in vitro regeneration protocols for mungbean reporting regeneration of more than 104 shoots per explant using TDZ (Amutha et al. 2006); 27 shoots per explant using combination of BAP and NAA (Yadav et al. 2010) however, they were not reproducible in the cultivar SML 668. The phytohormone BAP (1.0 mg l^-1) was found most efficient for in vitro multiple shoot induction in mungbean cultivar SML668. The maximum average shoot length recorded was 4.15±0.75 with 1.0 mg l^-1 BAP (Table 2). This study highlighted that SML 668 is not much responsive towards tissue culture while using DCN as choice explant.



Embryonic axis as explant

Cotyledonary node segments produce shoots from  nodal sections and remain the first choice for shoot differentiation via organogenesis amongst legumes (Chandra and Pental, 2003), but embryonic axis as an explant can also be utilized for tissue culture due to presence of fully exposed and broad preexisting meristems that have predetermined fate of regeneration. Embryonic axis (EA) has been explored for establishing efficient regeneration and transformation systems in other crops such as Cicer (Krishnamurthy et al., 2000; Aasim et al., 2011, Shukla et al., 2015); Glycine spp. (McCabe et al., 1988), Phaseolus vulgaris (Gatica Aria et al., 2010) and several Vigna sps. (Bhargava and Smigocki,1994; Acharjee et al., 2012; Ivo et al., 2008), but no such report  was retrieved for Vigna radiata.

In this study, per cent regeneration and number of shoots regenerated per explant were assessed in basal media supplemented with different concentrations of BAP alone. Maximum shoot proliferation (4.02±0.19) was recorded in MSB5 supplemented with 1.0 mg l^-1 BAP (as with DCN (Table 3; Fig 3). This is the first ever report of EA as an explant in cv. SML668 for direct organogenesis in mungbean along with comparative analysis with DCN.

A single shoot regenerated in media without any phyto hormone using EA as explant and was similar to that observed while using DCN as explant. Embryonic axis was more responsive than DCN and that too for all doses of BAP, as evident by the per cent regeneration response as given in Table 3. Maximum number of shoots regenerated per explant (4.02±0.19) was found in media supplemented with 1.0 mg l^-1 BAP and was significantly higher than other doses of BAP. It was also significantly higher to the number of shoots regenerated (3.1±0.08) in same dose of BAP using DCN as explant. 




 
Effect of duration of exposure of explant to BAP on shoot regeneration  
 
Significant increase in shoot multiplication was observed in explants (both DCN and EA) exposed to BAP for 20 days and then kept in basal media devoid of any hormone (Table 4). Exposure to phytohormones BAP for more than 20 days reduced the number of shoots produced and gradually caused necrosis and shoot deterioration. Further elongation in shoots was obtained on basal medium itself.

               
 
Rhizogenesis
 
Root formation was successfully obtained in 1.0 mg l-1 IAA within 2 weeks from DCN explant. Root initiation frequency at same concentration of IBA (77.70%) and NAA (61.70%) revealed growth of an improper and less natural root system. The roots developed were shorter and thicker on use of NAA and response on use of IBA was delayed (by ca. 5-10 days) in comparison to IAA (Table 5). This result is in accordance with those of (Gulati and Jaiwal, 1994) and (Mahalakshmi et al., 2003) who reported that IAA was best for inducing (Khatun et al., 2008) reported NAA and (Yadav et al., 2010) and (Patra et al., 2018) reported IBA as best rooting hormone for mungbean.



EA regenerated shoots successfully rooted (rooting frequency ca. 96.30%) in MSB5 medium without any exogenous growth regulator giving an average root length of 8.70±0.31 cm (Fig 4). This self-induction might be the results of pluripotent cells present at hypocotyl region that aid in endogenous induction of rooting. The in vitro multiplied plantlets were hardened and acclimatized in a mixture of sand, soil and vermiculite with a survival rate of 85-90%. The hardened shoots were transferred to the greenhouse where they developed pods and set viable seeds. 

Efficient in vitro regeneration system in Vigna radiata using BAP as choice phytohormone has been reported by several researchers, however, when tested in cv. SML 668, the number of shoots regenerated per explant was found to be low. Understanding that response of genotype is a crucial parameter to develop an efficient and reproducible in vitro regeneration system, equally important is the choice of explant and its response. In this context, efforts were made to study the effect of phytohormones and explant on mungbean genotype SML668. Embryonic axis was a better responding explant that gave higher shoot number per explant in comparison to the cotyledonary node with 1.0 mg l^-1 BAP. Thus, we conclude that EA carries equal potential for direct multiple shoot regeneration as the explant DCN and this is the first report in mungbean SML668 where embryonic axis could be successfully used for in vitro regeneration. Further studies with use of supplements and different phytohormones and explants may pave way for efficient regeneration in mungbean in this cultivar and many others.

Authors are thankful to ICAR-Indian Institute of Pulses Research, Kanpur for supporting this study. They are also thankful to Dr. Aditya Pratap, IIPR, Kanpur for providing seeds of SML668.