Search

The production of transgenic watermelon through Agrobacterium tumefaciens-mediated transformation with in vitro regeneration system is a time-consuming, labor-intensive and genotype-dependent process. To acquire large number of transgenic watermelons in a shorter period of time, the half-seed explants were infected with Agrobacterium strain EHA 105 harboring pCAMBIA1301 with bar and gus genes. The factors affecting in planta transformation efficiency, such as co-cultivation duration, acetosyringone concentration, sonication duration and vacuum infiltration were assessed in the present study. The half-seed explants were sonicated for 3 min and 2 min vacuum infiltrated in Agrobacterium suspension and co-cultivated for 3 days with 100 µM acetosyringone showed maximum transformation efficiency. The transformed watermelon plants were selected against BASTA® and GUS, PCR, Southern hybridization analysis confirmed the transgene integration. The amenability of this established protocol was analyzed on four genotypes, in which the response of all genotypes was positive, whereas Arka manik showed the higher transformation efficiency of 17%. The transformation protocol developed in the present study is efficient, economical and expeditious without the taking part of any tissue culture phases and produce a large number of transgenic lines within a short period of 56 days.

l-Dopa is the most potent and gold standard drug for the treatment of Parkinson disease. The present finding reports the effect of elicitors on enhanced production of l-Dopa from adventitious root cultures of H. enneaspermus. Adventitious root culture was established from leaf explant of H. enneaspermus. Highest root induction frequency 70.66% with 17.76 roots per explant were recorded in explants inoculated on MS medium supplemented with Indole-3-butyric acid (IBA) 0.5 mg/l. The influence of elicitors on the biosynthesis of l-Dopa content was studied by exposure of adventitious roots to different duration and concentrations of salicylic acid (SA), yeast extract (YE), methyl jasmonate (MeJA) and silver nitrate (AgNO3). Among the different elicitors tested, exposure to SA at 100 µM dosage for 6 h was found to enhance l-Dopa yield 12.64 mg/g dry weight (DW) when compared to control culture. The present findings reveal that elicitation experiment could improve the production of l-Dopa in adventitious root cultures of H. enneaspermus and the current study may be helpful for commercial production of these essential secondary metabolites in the future for clinical applications. This is the first report on adventitious root cultures and elicitation strategy on the significant improvement in l-Dopa production from H. enneaspermus.

The influence of exogenous polyamines (PAs) on somatic embryogenesis from immature leaf roll explants and Agrobacterium tumefaciens-mediated transformation of embryogenic callus of Saccharum spp. (sugarcane) ‘Co 86032’ was examined. Immature leaf roll-derived embryogenic callus was obtained on Murashige and Skoog with Gamborg B5 vitamins (MSB5) medium containing 3 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Various concentrations of PAs along with 2 mg L−1 2,4-D and 0.5 mg L−1 kinetin (Kin) were tested for somatic embryo induction. A total of 106 somatic embryos per 250 mg of callus (96.3% responsive explants) were obtained on medium supplemented with 20 mg L−1 putrescine (PUT) and 92.0% of the somatic embryos matured and produced 98 shoots per 250 mg of callus. Somatic embryo induction and maturation was increased more than two- and threefold, respectively, on PUT-supplemented medium compared to control cultures. Histomorphological analyses of various developmental stages verified somatic embryogenesis from immature leaf roll explants. The rooted plantlets were successfully hardened and exhibited normal growth. The efficiency of A. tumefaciens-mediated transformation of embryogenic callus using various concentrations of PAs in the infection, co-cultivation, and regeneration media was also assessed. Putrescine at 20 mg L−1 showed the highest regeneration (54.4%) and transformation (35.8%) efficiencies, which were more than twofold higher than the control treatment. These results demonstrate that exogenously supplied PAs improve plant regeneration using somatic embryogenesis and A. tumefaciens-mediated transformation of embryogenic callus of sugarcane ‘Co 86032’.

A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity.