High frequency regeneration via direct somatic embryogenesis and efficientAgrobacterium- mediated genetic transformation of tobacco

Abstract

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.

Coexpression of <i>WUSCHEL related homeobox</i> (<i>WOX</i>) <i>2</i> with <i>WOX8</i> or <i>WOX9</i> promotes regeneration from leaf segments and free cells in <i>Nicotiana tabacum</i> L.

“…Interestingly, it is well known that the two species show different regeneration behaviors in vitro; the explants from the former easily develop adventitious shoots in response to cytokinin, but rarely show somatic embryogenesis, whereas those from the latter easily develop embryogenic calluses in response to auxin, but rarely develop adventitious shoots. There are some reports regarding somatic embryogenesis in tobacco tissue culture (Gill and Saxena 1993;Guo et al 2013;Pathi et al 2013;Srinivasan et al 2007); however, the researchers may have observed organogenesis but not embryogenesis, judging from their use of shoot inducers (cytokinin) in the culture and morphological aspect and greenish color shown in the figures in their reports. Previously, in tobacco tissue and cell culture, cytokinin was essential for regeneration with the exception of pollen embryogenesis (Kyo et al 2014).…”

In vitro ROOTING OF TENERA HYBRID OIL PALM (Elaeis guineensis Jacq.) PLANTS1

“…They also allow large-scale production of synthetic plants and seeds, which have the advantages of ease of handling and transport, long-term storage potential, uniform production, and the potential for automating the entire production process (Pathi et al, 2013).…”

Somatic Embryogenesis in Annatto (Bixa orellana L.)

“…The bipolarity linked to the single cell origin of the embryos may reduce the development of chimeras or mixoploidy in the regenerants (Pathi et al 2013). Somatic embryogenesis may occur directly or in an indirect way.…”