Establishment of an indirect organogenesis protocol for Eucalyptus grandis species and hybrids.

Abstract

The prospect of integrating transgenic eucalypts with conventional breeding programmes is of value to the Plantation Forestry and Forest Products Industries. However, significant progress in this regard has still to be reported, one constraint is the lack of appropriate high yielding regeneration culture methods for clonal material. Such was the main aim of the present study. The strategy was to develop a suitable protocol using in vitro shoots of an E. grandis x E. urophy/la clone (GU185) and thereafter to test its applicability to other clones. Explants from greenhouseestablished cuttings provided the in vitro shoots, which were multiplied via axillary bud proliferation either on semi-solid medium or using a RIT A system. To determine the best conditions for callus and shoot regeneration, parameters such as vessels (petri dishes and tubes) and types and levels of plant growth regulators were tested. The best callus production (100%) and shoot regeneration (78.9 - 100% callus with shoots) for GU185 occurred on MS, 30 g rl sucrose, 4 g rl Gelrite, 5 mg rl IAA and 0.25 mg rl BAP. Parameters tested to identify the most suitable explants for indirect organogenesis were the age of parent plants, different systems to generate in vitro shoots, elongation status of explants, 1 sI and 2nd generation in vitro shoots and the use of hyperhydric shoots. Of these, the most suitable explants for indirect organogenesis were shoots from axillary bud multiplication of 3-month-old parent plants using the semi-solid system (33 shoots/dish). Up to 90% rooting was achieved on 1f4 MS (Murashige and Skoog, 1962), 15 g rl sucrose, 0.1 mg rl biotin, 0.1 mg rl calcium pantothenate, 4 g rl Gelrite and mA. The highest rooting was obtained when regenerated shoots were first multiplied and then placed on medium without plant growth regulators for one week, before transfer to root induction medium containing 0.1 - 0.5 mg rl mA. Acclimatization success was 95% when rooted shoots were placed in pots with a rooting mix (2 perlite: 1 coir) enclosed in plastic bags and the humidity was gradually reduced over four weeks. The developed indirect organogenesis protocol appeared to have a broad general application, although the tested clones exhibited a genotype-dependent response, with GU180, GUI77 and TAG31 producing fewer shoots (9, 6 and 7 shoots/dish) than ZG14 and GU185 (24 and 18 shoots/dish). Similarly high levels of rooting were obtained for TAG3l (93.8%) and ZG14 (90%) and for hardening-off (90.7% for TAG31 and 91.4% for ZG14).