Studies On Regeneration And Transformation Of Eucalyptus Urophylla×E. Camaldulensis

This paper deals with regeneration and transformation of Eucalyptus urophylla×E. camaldulensis clone DH201-2. A system of high frequency regeneration and Agrobacterium-mediated transformation of E. urophylla×E. camaldulensis was established and some kanamycin-resistant plantlets of E. urophylla×E. camaldulensis were obtained successfully. The establishment of regeneration and Agrobacterium-mediated transformation of E. urophylla×E. camaldulensis can provide the important references for further research. The main conclusions of this study are as follows:1. By screening to experiment material, adjustment of hormone kind and proportion, selection of culture conditions, the optimal medium of regeneration are mMS + TDZ 0.05 mg.L^-1 + IBA 0.1 mg.L^-1 for leaf explants and mMS + TDZ 0.05 mg.L^-1 + NAA 0.5 mg.L^-1 for stem explants, with regeneration rates of 48.3% and 57.0% respectively. The proper concentration of calcium nitrate for the induction of adventitious shoots from leaves and stems explants are 0.706 g.L^-1. Leaf explants from plantlets with a rooting culture of 15 days have a high regeneration rate, while plantlets with a rooting culture of 30 days are the best for the collection of stem explant. Most of the explants can produce clump shoots. This offers solid foundation for genetic transformation.2. DH201-2 clone of Eucalyptus urophylla×E. camaldulensis has a relatively high kanamycin resistance. The kanamycin resistance of leaf and stem explants are 90 mg.L^-1 and 120 mg.L^-1 respectively and plantlets have a resistance of 150 mg.L^-1 kanamycin.3. Agrobacterium can be gotten rid of in three subcultures with 300 mg.L^-1 cefotamicine and regeneration rate can be kept the same as normal regeneration.4. Four Agrobacterium strains, namely GV3101, EHA105, LBA4404, C58C1 were tested by transient expression and GV3101 has the highest infection rate and transient expression rate. The transient expression rate of stem explants was 70% and that of leaf explants was 30%.5. It has the highest transient expression rate in pre-cultured 6 days, infected 30 minutes in solution of bacterial concentration OD600 0.4～0.5, added AS 100μM in co-culture medium and bacterial solution, adjusted pH 5.6 of co-culture medium and co-cultured 4 days in stem explants. It obtained the highest transient expression rate in pre-cultured 3 days, infected 30 minutes in solution of bacterial concentration OD600 0.4～0.5, added AS 100μM in co-culture medium, adjusted pH 5.6 of co-culture medium and co-cultured 3 days in leaf explants.6. The leaf explant is less fit for gene tranformation because it has lower infection rate and less transient expression rate. The stem explant is suited for transformation as it can produce high quality callus for shoot regeneration and has a high regeneration rate and high GUS gene expression rate.