Plant Signaling & Behavior

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AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper Muruganantham Mookkan & Ganapathi Andy To cite this article: Muruganantham Mookkan & Ganapathi Andy (2014) AgNO3 boosted highfrequency shoot regeneration in Vigna mungo (L.) Hepper, Plant Signaling & Behavior, 9:10, e972284, DOI: 10.4161/psb.32165 To link to this article: http://dx.doi.org/10.4161/psb.32165

Accepted online: 31 Oct 2014.

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SHORT COMMUNICATION Plant Signaling & Behavior 9:10, e972284; October 1, 2014 ; © 2014 Taylor & Francis Group, LLC

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper Muruganantham Mookkan1,2,3,4,* and Ganapathi Andy1 1

Department of Biotechnology and Genetic Engineering; School of Biotechnology; Bharathidasan University; Tiruchirappalli, Tamil Nadu, India; 2The S. Tolkowsky Laboratory; Department of Plant Pathology-The Virology Unit; Agricultural Research Organization; Volcani Center; Bet Dagan, Israel; 3Department of Horticulture; University of Georgia Tifton Campus; Tifton, GA USA; 4Division of Plant Sciences; University of Missouri; Columbia, MO USA

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Keywords: black gram, direct shoot regeneration, thidiazuron, silver nitrate, subculture

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO3 on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BACTDZCAdsCAgNO3 for multiple shoot induction. AgNO3 influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO3 in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA3 (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.

Introduction Black gram [Vigna mungo (L.) Hepper] is an important leguminous source of protein for a large segment of the vegetarian population in the developing countries of Asia, Africa and Latin America,1 also plays in important role in sustaining soil fertility by improving soil physical properties and fixing atmospheric nitrogen. Although better agriculture and breeding practices have significantly improve the yield of this pulse over the last decade, productivity has been greatly limited by several viral and bacterial diseases. Since conventional breeding has several constrains,2 in vitro culture methods would serve as platform to produce black gram cultivars with desirable characters by genetic engineering techniques. The seeds of black gram contain 78–80% nitrogen in the form of albumin and globulin3 and the dry seeds are also good source of phosphorus. This crop is subjected to various biotic and abiotic stresses which limit its productivity.4 Severe yield losses in black gram corps, caused by a high incidence of viral disease and fungal pathogens5 have spurred research into the development of disease-resistant cultivars by genetic transformation. Shoot tip,6 cotyledons,7 cotyledonary nodes8,9 and immature cotyldeonary nodes10 derived from in vitro-raised seedlings have all been used to regenerate shoots in in vitro. Silver nitrate is well-known to increased regeneration in many crop plants such as peanut,11 cowpea,12 Brassica species,13,14 Capscicum species,15 watermelon,16 Coffea canephora,17 Cucumber,18 Pomegranate,19 White marrygold,20 Cassava,21 Petunia22 and P. vulgaris.23 In the previous study reported from immature cotyldonary nodes.10 Although immature cotyledonary nodes are

highly responsive for in vitro propagation, continuous maintenance of stock plants is tedious and waste of time. In this paper, using mature seed and we first reported the addition of AgNO3 to shoot regeneration media to obtain high frequency plant regeneration as well as increase in number of shoots through direct shoot regeneration form shoot tip and cotyledonary node explants of Vinga mungo cultivar Vamban 3.

Results and Discussion In the present study, multiple shoots were enhanced from the cotyledonary node explants. Shoot induction varied with the type of growth regulator and their combinations used. In legumes, multiple shoot induction through mature cotyledonary nodes has been widely used for regenerating and for transformation. Axillary meristems of the cotyledonary node explants possess cell that are competent for regeneration and hence are considered as target tissue for gene delivery in legumes.24 BA is the most widely used and most effective in cytokinin in vigna mungo.8,5,10 Requirement of BA for induction of multiple shoots has already been reported in other legumes, such as mungbean,25, Chickpea.26 The factorial combination of BA with auxins produced basal callusing followed by rhizogenesis and no shoots emerged from the explants (data not shown). Similarly, callusing response was also observed in the presence of NAA (0.1mg/l) in mungbean.27 TDZ was tested individually as well as in combination with BA for enhanced multiple shoot induction. Among the different

*Correspondence to: Muruganantham Mookkan; Email: [email protected] Submitted: 07/09/2014; Accepted: 07/27/2014 http://dx.doi.org/10.4161/psb.32165

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concentrations of TDZ examined, TDZ at 0.1 mg/l produced 9 shoots/explant at the end of second subculture. A maximum of 12 shoots per cotyledonary node was observed in MS B5 medium fortified with a combination of BA (1.0mg/l) and TDZ (0.1mg/l) at the end of second subculture. TDZ has been reported to be the best cytokinin to induce maximum number of shoots in peanut,28 pigeon pea29 and mungbean.30 Although TDZ at 0.1mg/l induced higher number of multiple shoots, the shoots exhibited stunted growth. To enhance the number of shoots still further with normal shoot growth, different concentration of AdS were employed along with BA and TDZ. A combination of AdS (15mg/l), BA (1mg/l) and TDZ (0.1mg/l) significant increase the shoot number at the end of first subculture. Although those combination to making minimal basal callusing formation and browning of the explants (data not shown). To avoid this to add the AgNO3 (1mg/l) containing MSB5 with BA, TDZ and AdS. Based on the previous results we initially added AgNO3 with combination with BA the shoot number was increased significantly in the end of first subculture. Further we tested individually to all the hormones i.e., TDZ, AdS, then tested with combination with AgNO3, BA, TDZ and AdS. Visibly see the tiny shoot buds newly formed and maximum 39 shoots/explants observed at the end of second subculture. In our work, the use of AgNO3 with TDZ had a key effect of enhanced shoot regeneration in V. mungo. When TDZ use alone only 9 shoots developed whereas when it is applied combination with AgNO3 the shoot (10-12/explant) increased. The shoots produced in this combination of plant growth regulators showed normal green and healthy growth. Similar concept was reported23 in P. vulgaries when they applied BA combination with AgNO3 100% of shoots developed. Recently reported31 to induce the shoot regeneration, elongation and also significantly reduce the callus formation in Artemisia annua.32 reported the positive effect of AgNO3 on adventitious shoots formation and from cotyledonary nods of

Table 1. Effect of various growth regulators with 15mg/l AdS on multiple shoot production from cotyledonary node explants Growth Regulator (mg/L)

BAP 0 0.5 1 1.5 2 2.5 TDZ 0 0.1 0.5 1 1.5 2 BACTDZ 1.0C0.1mg/l

Explants with shoots %

Mean No. of shoots/explants

ST

CN

ST

CN

0 96 100 98 90 90

0 97 100 97 95 98

0j 4 d, e 7c 4.2 d 2.5 f, g 1

0k 6.2 d 8c 6 d,e 3h 2i

0 100 90 80 75 65 100

0 100 92 90 79 70 100

0j 8b 4.5 f 2h 1i 1i 16 a

0k 10 b 4.8 f 4g 1j 1j 28 a

M. sativa and also shoot organogenesis have been reported.33,34 AgNO3 as an additive has generally been used in association with other cytokinins such as TDZ.35,34 Among the five AgNO3 concentrations, 1mg/l initiated significantly better response to shoot regeneration and induced higher number of shoots of per explants. However, above 1mg/l the shoot induction are reduced further no development (Fig 1). Periodic excision of shoots from explants as and when they attained a length of 1cm greatly enhanced the production of multiple shoots in subsequent subcultures from pre-existing meristems of explants. In earlier studies, repeated transfer of explants to fresh medium was found to increase regeneration from explant tissue and activation and conducting of meristems, and this has been supported by the present study (Table 1, Fig. 1). Subculture was terminated at the end of 7–8 wk as there was no further increase in the number of shoots (data not shown). MSCB5 medium containing BA and TDZ did not favor shoot elongation. Therefore, shoots were subjected to shoot elongation in MS medium containing GA3. Gibberellic acid at 0.6mg/l promoted maximum elongation shoot tip (7.6 cm) cotyledonary node (7.9 cm) of the shoots (Table 2). Similar observation also made Vicia faba. However, shoots did not elongate in MS medium devoid of GA3 (data not shown). Elongated shoots produced roots in half strength MS medium supplemented with IBA 1.0mg/l to produce a maximum number of roots in shoot tip (9.1cm) cotyledonary node (9.3cm). IBA played a major role in the root formation (Table 3) and also other Vigna species, such as V. radiata. Well rooted plants Figure 1. Effect of AgNO3 with combination of BA, TDZ and AdS to improve the shoot multipliwere transferred to small plastic cups filled cation from shoot tip and cotyledonary node explants after second subculture. with sterilized garden soil and sand (1:1v/v)

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Table 2. Effect of GA3 on elongation of shoot regenerated from shoot tip and cotyledonary node explants of V. mungo cv vamban 3 GA3 mg/l

0.2 0.4 0.6 0.8 1.0

Shoot tip

Cotyledonary node

% of response

No. of internode/shoots

Shoot length

% of response

No. of internode/shoots

Shoot length

34 c 52 b 81 a 32 d 26 e

3c 4b 7a 3c 2d

3.1 d 4.3 b 7.6 a 3.9 c 2.6 e

36 c 53 b 84 a 35 d 26 e

3c 4b 8a 3c 2d

3.2 d 4.4 b 7.9 a 3.8 c 2.6 e

Table 3. Effect of IBA on rooting of shoot regenerated from shoot tip and cotyledonary node explants of V. mungo cv vamban 3

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IBA mg/l

0.5 1.0 1.5 2.0

Shoot tip

Cotyledonary node

% of response

No. of roots/shoots

Root length

% of response

No. of roots/shoots

Root length

39 d 79 a 56 b 42 c

4c 8a 6b 4c

5.1 c 9.1 a 5.6 b 3.6 d

38 d 80 a 55 b 40 c

4c 8a 5b 4c

4.9 c 9.3 a 5.6 b 3.4 d

for acclimatization. After 2 wk acclimatized plants were transferred to the field. In conclusion, present study to improve the multiple shoot formation from the shoot tip and cotyledonary node explants using AgNO3 boosted higher frequency than the previous reports in black gram using other explants. In the presence of BA, TDZ, AdS and AgNO3 combinations for 2 consecutive subcultures enhanced regeneration frequency significantly. The entire procedure starting from germinating seeds to establishing a plant under greenhouse conditions took maximum 55–70 d. (Figure 2). This improved protocol increases the opportunity for black gram breeding by gene transfer, particularly in cultivars or genotypes.

Materials and Methods Seed material: Seeds of V. mungo cv Vamban 3 were obtained from the National Pulses Research Center, Tamil Nadu, India. Surface sterilization was performed using 0.1% aqueous mercuric chloride (7 min). The sterilant was removed and the seeds were washed five times with sterile distilled water. Seeds were germinated on MS (Murashige and Skoog, 1962) basal medium, with MS salts36 and B5 vitamins,37 3% (w/v) sucrose and 0.8% agar (HiMedia,

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Figure 2. Shoot bud formation/multiplication scheme for V. mungo

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Mumbai, India) and maintained under the white fluorescent light (Phillips India) at a photon flux of 24 mmol m¡2 S¡2 for 16/8 h light/dark photoperiod at 25 § 2  C. All media were sterilized at 120  C for 20 min. Explant preparation: Cotyledons and root initials were removed from the 3–5-d-old germinated seedlings. Cotyledonary-node (10 mm) and shoot-tip (5 mm) explants were excised and cultured on MS salts and B5 vitamins supplemented with individual concentration and combinations of benzyl aminopurine (0.5mg-2mg/l) and thidiazuron (0.1mg-0.5mg/l) Ads at different concentrations (5, 10. 15, 20, 25mg/l) AgNO3 at different concentrations (0, 0.1, 1, 2, 3 and 4 mg/l) was used to studied its effect on in vitro multiple regeneration. Different concentrations of auxins (NAA/IAA) individually and in combination with BA were also tested for their shoot induction efficiency. Explants with emerging shoots were subcultured in the same medium (BACTDZCADSCAgNO3) twice at the interval of 2 wk each. Shoots that attained 1 cm length were excised from explants and transferred to the elongation medium [MS salts, B5 vitamins with Gibrallic acid GA3 0.6mg/l. The elongated shoots (5–7cm) were rooted in half strength MS medium, B5 vitamins with IBA 1mg/l. Cultures were maintained under the white fluorescent light (Phillips India) at a photon flux of 24 mmol m¡2 S¡2 for 16/8 h light/dark photoperiod at 25 § 2  C. All media were sterilized at 120  C for 20 min. The pH of the medium was adjusted to 5.8, and the cultures were maintained in culture tubes (25 £ 100 mm, Borosil, Chennai, India). TDZ, AgNO3, and References 1. Muruganantham M, Amutha S, Ganapathi A. Somatic embryo production by liquid shake culture of embryogenic calluses in Vigna mungo (L.) Hepper. In Vitro Cell Dev Biol Plant 2010; 46:34-40; http://dx.doi.org/ 10.1007/s11627-009-9224-8 2. Jaiwal PK, Gulati A. Current status and feature strategies of in vitro culture techniques for genetic improvement of mungbean [Vigna radiata (L.) Wilczek.]. Euphytica 1995; 86:167-81 3. Das DK, Siva Prakash N, Bhalla-Sarin N. An efficient regeneration system of black gram (Vigna mungo L.) through organogenesis. Plant Sci 1998; 134:199-206; http://dx.doi.org/10.1016/S0168-9452(98)00044-2 4. Eapen S. Advances in development of transgenic pulse crops. Biotechnol Adv 2008; 26:162-8; PMID:18055156; http://dx.doi.org/10.1016/j.biotechadv.2007.11.001 5. Sahoo L, Sugla T, Jaiwal PK. In vitro regeneration and genetic transformation of Vigna species. In: Jaiwal PK, Singh RP. eds. Biotechnology for the improvement of legumes. Netherlands: Kluwer. 2002; 1-40. 6. Goel S, Mudgal AK, Gupta SC. Development of plants from in vitro cultured shoot tips of Vigna mungo and V. radiate. Trop Plant Sci Res 1983; 1:31-5 7. Gill R, Eapen S, Rao PS. Morphogenic studies of cultured cotyledons of urd bean (Vigna mungo L. Hepper). J Plant Physiol 1987; 139:1-5; http://dx.doi.org/ 10.1016/S0176-1617(87)80295-X 8. Ignacimuthu S, Franklin G, Melchias G. Multiple shoot formation and in vitro fruiting of Vigna mungo L. Hepper Curr Sci 1997; 73:733-5 9. Saini R, Sonia, Jaiwal PK. Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens. Plant Cell Rep 2003; 21:851-9; PMID: 12789502 10. Muruganantham M, Ganapathi A, Amutha S, Vengadesan G, Selvaraj N. Shoot regeneration from

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gibberellic acid (GA3) were obtained from Sigma, St Louis, USA and were added to warm autoclaved media after filter sterilization (0.2mm, Pall Gelman Sciences, Mumbai, India). Cultures were incubated in the growth room described above. Visible shoot regeneration (> 2 mm) from explants was observed after 15 d culture and recorded. Only regenerating explants were transferred to fresh medium. Each growth regulator treatment consisted of 50 explants and was repeated five times. Shoot regeneration was scored at the end of every transfer and refers only to regenerating shoots. At the end of initial culture and every transfer, regenerating explants (at least 10) with shoots were removed from the culture, and the shoots separated manually (destructively) from the regenerating area and counted for data analysis. A complete randomized design was used in all experiments and analysis of variance and mean separations were performed using Duncan’s Multiple Range Test (DMRT). Statistical significance was determined at 5% level.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

Authors thank Department of Biotechnology (DBT), Ministry of Science and Technology, Govt. of India for providing financial support for conducting the research reported here.

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AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper.

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO3 on this process was investigated in this s...
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