Establishment of a suitable regeneration protocol is a pre-requisite to carry out transformation study in L. (sesame). In this paper, different parameters of regeneration were standardised to develop an efficient protocol for in vitro plant regeneration via direct adventitious shoot organogenesis using de-embryonated cotyledons of sesame as explants. Among the various treatments of MS medium supplemented with 6-benzylaminopurine, thidiazuron and indole-3-acetic acid, maximum regeneration frequency (25.93  ±  2.21%) was obtained in BTI 4 medium (MS supplemented with 33.33 µM BAP with 2.85 µM IAA) within 6 weeks of culture. Regeneration frequency increased further (50.37 ± 2.49%) by fortifying BTI 4 with 29.43 µM silver nitrate (AG 3 medium). Pre-culture of cotyledon explants in AB 3 medium (AG 3 supplemented with 3.78 µM abscisic acid) for 14 days followed by sub-culture in AG 3 medium further improved the regeneration frequency (68.15 ± 2.68%). The highest rate of shoot regeneration (94.82 ± 1.34%) was obtained by pre-culturing 4-day-old cotyledon in a vertical position in AB 3 medium for 14 days and sub-culturing in AG 3 medium for 4 weeks. Regenerated shoots proliferated in MS medium supplemented with 4.44 μM BAP and 1.44 μM gibberelic acid (GA). The highest frequency (65.33 ± 3.78%) of root induction was achieved by culturing the elongated shoots in MS medium supplemented with 2.69 μM α-naphthalene acetic acid (NAA) for 6 weeks. Rooted plants were acclimatised in soilrite and transferred to soil after 6-8 weeks. The rate of acclimatisation of plants was 76%.

Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) dramatically improved Agrobacterium infection efficiency in Sesamum indicum (sesame) cotyledon. Different parameters of this method such as the duration of sonication, Agrobacterium concentration and acetosyringone concentration during co-cultivation was standardized to develop a balanced protocol that gave optimum β-glucuronidase (GUS) expression with minimal tissue damage. Nine seconds was identified as the optimum duration for sonication. Agrobacterium concentration having OD600 nm 1.0 was found not to induce tissue damage. Usage of 100 μM acetosyringone, which facilitates T-DNA transfer by activating the vir gene in Agrobacterium, increased the transient GUS activity. The optimized protocol facilitated 85.56 ± 0.35% transient GUS activity. Mimicking SAAT by increasing the concentration of acetosyringone during the co-cultivation period to 400 μM in the conventional Agrobacterium-mediated transformation gave only 32.22 ± 1.53% GUS activity. The results suggest that not only acetosyringone but also other compounds were released during formation of micro-wound by sonication that increased the infection efficiency.

No abstract

No abstract