Enhancing shoot recovery from transgenic avocado somatic embryos

Abstract

Use of biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos with an acceptable germination capacity. Use of semi-permeable cellulose acetate membranes on top of maturation medium has improved the quality of obtained embryos and their germination rate; however, in the case of transgenic embryos the conversion rate is still rather low. In this investigation, a protocol for recovery of transgenic plants has been developed. Mature avocado somatic embryos, over cellulose acetate membranes, were obtained and induced to germinate following the protocol described in Palomo-Ríos (2012). Some transgenic embryos developed buds ≤ 2 mm, which failed to elongate. For shoot recovery, cotyledons were partially removed and the embryonic axis cultured over 4 weeks in MS medium supplemented with either 1 mg/l BA, 1 mg/l TDZ or 1 mg/l BA and 1 mg/l TDZ. Highest shoot recovery was obtained in medium supplemented with 1 mg/l BA and 1 mg/l TDZ, 53.6±6.7% versus 23.2±5,7% and 39.6±6.4% in media supplemented with 1 mg/l BA or 1 mg/l TDZ, respectively. Afterwards, sprouted embryos were cultured over 4 additional weeks in MS medium supplemented with 1 mg/l BA. In some cases, resulting shoots could be induced to proliferate in GD medium (Gamborg et al., 1968) supplemented with 0.3 mg/l BA, while in other cases, they had to be recovered through micrografting onto in vitro germinated seedlings as described in Pliego-Alfaro and Murashige (1987). MS medium supplemented with 1mg/l BA and solidified with 6 g/l Sigma A-1296 agar, was used for micrografts. Shoots derived from micrografts were either induced to proliferate in GD medium with 0.3 mg/l BA or further grafted and cultured in liquid MS medium supplemented with 0.1 mg/l BA, using perlite as substrate. After 8-12 weeks, micrografts could be transferred to ex vitro conditions. References: Gamborg O.L., Muller, R.A., Iojima, K., (1968). Nutrient requirements of suspension cultures of soybean roots cells. Exp. Cell. Res. 50:151-158. Palomo-Ríos, E., (2012). Thesis: Regeneración y Transformación Genética de Aguacate (Persea americana Mill.). Ph.D. Dissertation, University of Málaga, Málaga Pliego-Alfaro, F., Murashige, T., (1987). Possible rejuvenation of adult avocado by graftage onto juvenile root stocks in vitro. Hort Sci. 22:1321-1324.