Plant Science, 43 (1986) 2i9--222 219 Elsevier Scientific Publishers Ireland Ltd. EMBRYO RESCUE IN NICOTIANA HYBRIDS BY IN-VITRO CULTURE U. SUBHASHINI*, T. VENKATESWARLU and K. ANJANI Central Tobacco Research Institute, Rajahmundry, Andhra Pradesh (India) (Received August 26th, 1985) (Revision received October 23rd, 1985) (Accepted December 12th, 1985) Interspecific hybridization between Nicotiana benthamiana, a disease and pest resistant species, and N. tabacum was successfully obtained by in-vitro culture. The medium used was that of Nitsch (J.P. Nitsch, Phytomorphology, 19 (1969) 389). The hybrid nature was confirmed cytologically. Fertility was restored by colchicine treatment. Back-crosses were made for transferring the desirable characters. Key words: Nicotiana; embryo rescue; in-vitro culture; interspecific hybridization Introduction The in-vitro culture of hybrid embryos has recently enabled the induction of genetic variability in a number of crops which are otherwise incompatible. Embryo rescue by in-vitro culture was attempted in a number of genera like Arachis [1] Vigna [3] Gossy- pium [2] Brassica [5] and also in Nicotiana [9--11]. The species N. bentharniana is im- mune to the leaf eating caterpillar Spodo- ptera, moderately resistant to Tobacco Mosaic Virus and Tobacco Distortion Virus. This species is taken for hybridization pro- gramme, in order to transfer resistance to N. tabacum. As the cross is a failure by conventional techniques, in-vitro hybridiz- ation was taken up to rescue the embryo and the results are presented. Materials and methods The flower buds of N. benthamiana (2n = 38) were emasculated 1 day before anthesis and were cross pollinated with N. tabacum *To whom reprint requests should be sent. Abbreviations: A1, anaphase 1; BAP, benzyl amino purine; IAA, indole acetic acid; M1, metaphase 1; MS, Murashige and Skoog medium; NAA, naph- thalene acetic acid. (2n = 48) on the next day when the stigma is receptive. Immature fertilized ovules at the critical stage, i.e. 13th day after pollina- tion were aseptically excised and cultured on Nitsch [7] medium. Developing ovules from 18 capsules were inoculated for raising hybrids. All the manipulations were con- ducted under sterile conditions in a laminar flow chamber. For callusing Murashige and Skoog's medium (MS) [6] supplemented with 6 mg of naphthalene acetic acid (NAA) and 2 mg of benzyl amino purine (BAP)/1 was used. Shooting was induced on MS supplemented with 2 mg of indole acetic acid (IAA) and 2 mg of kinetin/l. They were transferred to a well illuminated and temp- erature controlled room at 25 ° + 2°C. Meiotic studies were made from pollen mother cells. Results The developing capsules of N. benthamiana × N. tabacum will wither away from the parent plant on 13th day after pollination. When these fertilized ovules were inoculated at the critical stage, only 8 seeds germinated (Fig. 1). In order to multiply the population, plants were regenerated from the explants by manipulating the growth hormones as described above. 0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland