Isolation and characterization of intergeneric somatic hybrids in the Apocynaceae family

Abstract

Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.

Introgression of the heterologous nuclear DNAs and efficacious compositions from Swertia tetraptera Maxim. into Bupleurum scorzonerifolium Willd. via somatic hybridization

“…For example, the level of raucaffricine produced in hybrids between Rauwolfia serpentina and Vinca minor reached tenfold of those achieved in R. serpentina (Kostenyuk et al 1991). Finally, the yield of ginsenoside in a somatic hybrid between American ginseng and carrot was higher than that in the donor American ginseng (Han et al 2009).…”

Intergeneric somatic hybridization and its application to crop genetic improvement

“…However, in a number of somatic hybrids derived from symmetric fusions, full nuclear complements are not present. Firstly, rearrangements and recombinations have been detected in many hybrids (Hoffmann and Adachi, 1981;Kostenyuk et al, 1991;De Jong et al, 1993;Begum et al, 1995;Vlahova et al, 1997;Escalante et al, 1998;Szarka et al, 2002). Secondly, spontaneous chromosome elimination of either or both parents has been extensively reported, leading to regeneration of asymmetric hybrids or cybrids (Gleba et al, 1988;Babiychuk et al, 1992;Gilissen et al, 1992;Ahuja et al, 1993;Nakano et al, 1996;Kisaka et al, 1997;Navra´tilova´et al, 1997;Hu et al, 2002a).…”

“…Helianthus Schnabl et al (1999) Vinca Catharanthus Kostenyuk et al (1991) used for chromosomal fragmentation (Ramulu et al, 1994;Forsberg et al, 1998b). Treatment of the donor protoplasts with bromodeoxyuridine prior to UV and gamma irradiation could promote formation of highly asymmetric hybrids (Trick and Bates, 1996).…”

“…But major differences in chromosome morphology and size between the fusion parents could facilitate hybrid verification based on karyotype analysis, which can also reveal parental chromosome contribution to the hybrids. Verification of hybrids via chromosome morphology difference has been used in characterizing a number of somatic hybrids (Kao, 1977;Binding and Nehls, 1978;Chien et al, 1982;Gleba et al, 1982Gleba et al, , 1988Gupta et al, 1984;Kushnir et al, 1987;Endo et al, 1988;Hinnisdaels et al, 1991;Kostenyuk et al, 1991;Babiychuk et al, 1992;Ahuja et al, 1993;Wang et al, 1993;Skarzhinskaya et al, 1996;Kisaka et al, 1998;Xing et al, 2001;Szarka et al, 2002).…”

“…Protoplasts of the fusion parents have been treated with ionizing radiation or metabolic inhibitors that prevent the division of the unfused or homofused protoplasts Yamagishi et al, 2002). The heterokaryons could grow smoothly and finally develop into hybrid callus or plants, due to genetic or physiological complementation (Kostenyuk et al, 1991;Wang et al, 1993). In some cases low or no regeneration capacity of parental protoplasts could be used for hybrid selection because the morphogenic potential of the fusants could be restored due to complementation (O'Neill et al, 1996;Xia et al, , 2003Hansen, 1998;Zhou et al, 2001a, b;Hu et al, 2002a;Xu et al, 2003).…”