Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

Abstract

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.

Keywords: Agrobacterium tumefaciens; Blackgram; Direct shoot regeneration; Primary leaf.

Fig. 1

a-f In vitro regeneration of multiple shoots from 4-day-old primary leaf explants of Vigna mungo cv. PS1 a Primary leaf explant with petiole excised from 4-day-old in vitro-raised seedlings on MSB medium containing 10.0 μM BA b Direct shoot regeneration from primary leaf petiole on MSB medium supplemented with 1.0 μM BA c Multiple shoot regeneration on MSB medium supplemented with 1.0 μM BA after 4 weeks of culture d Induction of roots on in vitro regenerated shoot on MSB basal medium supplemented with 2.5 μM IBA e Establishment of an in vitro regenerated plant in pot containing soil f A regenerated plant with flower and pods

Fig. 2

A schematic representation of the T-DNA of pCAMBIA2301 containing the uid A (GUS) and nptII genes (not drawn to scale). The position of HindIII is indicated on the T-DNA. No other HindIII sites are present on pCAMBIA2301 (total size 11.6 kb). LB/RB: left and right T-DNA border sequences

Fig. 3

a-b Transient GUS expression in primary leaf explants inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pCAMBIA2301 that contained uid A (GUS) and nptll genes a. The non-transformed (control) primary leaf with petiole explant showing no GUS activity b. Primary leaf explant with petiole showing transient GUS activity at the regeneration site after 3 days of co-cultivation with Agrobacterium tumefaciens EHA105 (pCAMBIA2301)

Fig. 4

Molecular analysis of primary transformants of Vigna mungo transformed with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCAMBIA2301 that contained uid A (GUS) and nptII genes a PCR analysis of putative transformants with primers specific to the coding region of nptII gene Lane M: DNA Molecular weight marker (1 kb) Lane C: DNA from non-transformed (control) plant Lane P: positive plasmid DNA Lanes 1 to 10: DNA from transformed plants (T₀) b. PCR analysis of putative transformants with primers specific to the coding region of vir A gene Lane M: DNA Molecular weight marker (1 kb) Lane C: DNA from non-transformed (control) plant Lane P: Plasmid DNA Lanes 1 to 9: DNA from transformed plants (T₀)

Fig. 5

Southern hybridization of genomic DNA from primary leaf explants with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pCAMBIA2301 that contained uid A (GUS) and nptII genes. a. Southern blot analysis of genomic DNA of transformed and non- transformed control plants. The DNA was digested with HindIII, and blot was probed with the PCR amplified fragment (680 bp) of nptII gene. Lane M: DNA Molecular weight marker (1 kb) Lane C: DNA from untransformed (control) plant Lanes 1–3: DNA from transformed plants Lane P: Plasmid DNA

Fig. 6

PCR analysis of T₁ plants using primers specific to nptII gene Lane M: DNA Molecular weight marker (1 kb) Lane C: DNA from non-transformed (control) plant Lane P: Positive plasmid DNA Lanes1 to 6: DNA from T₁ transformed plants Lane N: Negative control (water)