Direct shoot organogenesis on hypocotyl explants with collar region from in vitro seedlings of Coffea canephora Pierre ex. Frohner cv. C × R and Agrobacterium tumefaciens-mediated transformation

Abstract

Direct differentiation of shoot buds from the collar region of hypocotyl segments of Coffea canephora was obtained on Murashige and Skoog (MS) medium supplemented with 40 lM silver nitrate (AgNO 3 ) and growth regulators indole-3-acetic acid (IAA) and N6 benzyladenine (BA). The highest response to shoot differentiation of 60% frequency and the maximum number of multiple shoots (2-3) per explant were obtained on MS medium containing 8.87 lM BA and 2.85 lM IAA. Apart from this, 70% of hypocotyl explants produced yellow friable embryogenic callus and also globular primary somatic embryos. Subsequent transfer onto the same medium induced secondary somatic embryogenesis. The micro-shoots, upon transfer to the same medium, in the following 6 weeks developed into 4-cm-long shoots with a single root. Further subculturing onto the same medium induced 4-5 roots in a 4-week period. The resulting plantlets were hardened and transferred to micro-pots containing sand:compost mixture (1:2), where 65% of them survived and resumed growth. By using optimal levels of AgNO 3 , it was possible to obtain effective direct organogenesis and embryogenesis. This system was used for genetic transformation using Agrobacterium tumefaciens. A stable transformation frequency of 2-5% was obtained when both types of explants, i.e., hypocotyl explants with collar region or hypocotyl explants without collar region, were co-cultivated with A. tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector. This is the first report of a hypocotyl collar region-based Agrobacterium-mediated transformation protocol for the economically important tropical plant C. canephora.

Effects of silver nitrate on shoot regeneration of Artemisia annua L.

“…In A. annua silver nitrate can increase not only its shoot induction frequency but also the regenerated shoot number per explants (Figure 1-A, Figure 1-B). Silver nitrate addition at a concentration above 6 mg l −1 slightly inhibited the shoot induction of A. annua, which was in concurrence with a similar response in other plants (Sridevi et al 2010;Zhang et al 2001), indicating that too high level of Ag + can be detrimental to its shoot induction. Silver nitrate can affect plant regeneration mode, thus capable of directly inducing shoot formation without intermediate callus phase (Sridevi and Giridhar 2013;Zhang and Hou 1996;Zhang and Ling 1995).…”

“…Furthermore, silver nitrate addition into culture medium can effectively rehabilitate decreased regeneration potentials of cultured cells and tissues incurring through their subcultures (Ogura and Shimada 1978). In the recent years, silver nitrate has been successfully employed to improve somatic embryogenesis and plant regeneration in tissue culture (AkasakaKennedy et al 2005;Al-Khayri and Al-Bahrany 2004;Fei et al 2000;Sridevi et al 2010;Zhang et al 2001). However, effect of silver nitrate on shoot regeneration of A. annua is still unknown.…”

“…Punica granatum (Naik and Chand 2003), Manihot esculenta (Zhang et al 2001), Coffea canephora (Sridevi et al 2010) and Malaxis acuminate (Meena et al 2010). Under certain circumstances, silver nitrate was shown to have negative effects on shoot regeneration of Saccharum spp.…”

Somatic Embryogenesis in Coffea spp.

“…canephora has also been transformed. A. tumefaciens LBA4404 (Leroy et al 1997(Leroy et al , 1999(Leroy et al , 2000, strain EHA101 harboring pIG121-Hm, containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransferase II genes (Hatanaka et al 1999), strain C58C1 harboring the binary vector pER10 W-35SRed without (Canché-Moor et al 2006) or with the gene WUSCHEL under an estradiol-inducible transcriptional control (Arroyo-Herrera et al 2008), and strain GV 3101 harboring pCAMBIA 1305.2 binary vector (Sridevi et al 2010) are among the different strains used for its transformation.…”

“…In other cases, the initial explant, such as hypocotyls (Sridevi et al 2010) or leaf explants (Canché-Moor et al 2006) are transformed prior to the induction of the SE process.…”