In vitro regeneration and Agrobacterium mediated transformation of soybean (Glycine max L.)

Abstract

Agrobacterium mediated transformation of soybean has successfully been achieved. However, the efficiency is usually low indicating that Agrobacterium mediated transformation of soybean demands optimization of more suitable conditions for transfer of T-DNA from plasmid to plant cell; beside these constraints; transformation is also dependent upon plant genotype, Agrobacterium strain and type of plasmid. Present investigation was aimed to find suitable regeneration protocol for two soybean cultivars, NARC-4 and NARC-7 and to standardize some transformation conditions. Based on optimized regeneration and transformation scheme, LFY gene, for early flowering and rol genes (A, B & C) for enhanced rooting were introduced in soybean genome. For transfer of LFY gene EHA105 harboring pROKIILFYGUSintnptII and for rol gene, LBA4404 harboring pLBR were used. Cotyledonary node method was found suitable for direct organogenesis of soybean NARC-4 and NARC-7 cultivars. However, presence of different plant growth regulators in media resulted in variation in number of shoot produced, shoot length and in percentage response. Statistical analysis reveals that BAP resulted in higher frequency of shoot regeneration and number of shoots per explant while mean shoot length was found higher when ZTR was used in the medium. Out of both soybean cultivars, NARC-4 showed better response than NARC-7. For standardization of transformation conditions, different parameters were studied. It was observed that explant cutting in Agro-suspension culture and infection for 1hr resulted in the highest GUS expression (48.3% and 55.9%, respectively). While 5 days co-cultivation resulted in 55.17% GUS response and washing for 2hr in washing medium containing 1g/L cefotaxime was better. It was found that 30mg/L kanamycin was sufficient in selection medium. At this concentration number of GUS positive shoots were maximum (63.6% response). The overall percentage transformation efficiency of both soybean cultivars NARC- 4 and NARC-7 was 24.16% and 15.71% respectively, when tested at best conditions. 11Soybean cultivar NARC-4 was used for transformation of LFY and rol genes. EHA 105 containing LFY gene along with GUS as reporter and nptII as selectable marker showed transformation efficiency 2.2%. Only 4 plants were recovered and confirmed for presence of LFY gene by PCR. These plants were highly dwarfed with small leaves and short nodal distance. Early flowering was observed by these transforments. The flowers turned brown and died after few days. Few of them turned into pods but no seed formation was observed. All the rol transforments produced enhanced rooting as compared to control plants. However, plants morphology varied depending upon rol gene. RolA transforments were small in size and mildly shrubby with ovate to elliptical leaf shape while rolC transforments were also dwarf with divided stem at the base with ovate to slightly globular leaf shape. Soybean rolB transformed plants showed variation in morphology. These plants were dwarf to shrubby with variation in leaf shape. The shrubby plants had reduced nodal distance with a little more zigzag pattern as compared to non-transformed plants. Rol transforments produced flowers in less time period as compared to control plants. These flowers converted into pods and set seeds. PCR analysis confirmed presence of respective gene in these plants. Southern blot analysis confirmed insertion of T-DNA in soybean genome as single copy number to multiple copies in rol gene transforments. In this study rol and LFY genes were efficiently introduced in soybean cultivar NARC-4 after optimization of regeneration and some transformation conditions.