Studies On Protoplast Culture And In Vitro Chromosome Doubling With Leaf Explants Of Populus Spp. (Section Tacamahaca)

It is of great potential to focus on polyploidy breeding of triploid poplar, due to its outstanding performance in forestry production. Chromosome doubling for somatic cells and somatic hybridization via cell fusion are effective methods to create polyploidy. In the present paper, Popuhlus pseudo-simonii and P.×beijingensis were employed as study materials. Based on the studies of cell suspension culture protocol and in vitro culture system, protoplast isolation and culture protocol and in vitro chromosome doubling by treating leaf explants with colchicine were investigated, which provide experiences for polyploidy poplar induction via cell fusion and in vitro chromosome doubling. Results are as follows:1. Axillary buds culture and callus differentiation were studied in this paper, which provide experience for in vitro chromosome doubling and protoplast culture techniques. Axillary buds germinated in MS medium containing NAA 0.05 mg/L, and rooted 100% in 1/2 MS containing IBA 0.5 mg/L. Hypocotyl and stem cultured on MS containing 2,4-D 2 mg/L and BA 0.05-0.1 mg/L resulted in high cullus induction and calli appeared to be yellow and grainiess. The optimal concentration of PGRs for shoot induction from callus was BA 1.0mg/L with NAA 0.2mg/L.2. Cell suspension culture system of P. pseudo-simonii was established in order to provide materials for protoplast culture. Cells which derived from stem and cultured in MS containing 2,4-D 2 mg/L, BA 0.1mg/L, ME 500mg/L, glutamine 1500mg/L and 4% sucrose showed a 'S' type growth curve. Subculture cycle should be 12d. Long exposure time under high 2,4-D concentration for cell suspension may result in the lost of differentiation ability, so that modulation of 2,4-D and BA concentration seemed necessary. For longtime conservation of cell suspension, solid-liquid in turn culture were employed.3. Protoplast isolation protocols of P. pseudo-simonii and P.×beijingensis were investigated. The optimal protocol for mesophyll protoplast isolation was treating leaves cultured for 35 d with CPW solution supplemented with 3.0% Celluase R-10,0.5% Macerozyme R-10,0.1% Pectolase Y-23 and 0.6 M mannitol for 8 h. The yield and viability were up to 2.44×10' protoplasts/g and 78.7%, respectively. The suitable protoplast isolation protocols for cell suspension of P. pseudo-simonii and P.×beijingensis were similar. The cells at logarithmic growth after 6 d cultured were incubated in CPW solution supplemented with 1.0% Celluase RS,0.5% Macerozyme R-10 and 0.6 M mannitol for 6 h of Populus pseudo-simoni and for 4 h of P. xbeijingensis.4. Protoplast culture of P. pseudo-simonii and P. xbeijingensis was successfully conducted to obtain regeneration plants. Plant regeneration from protoplasts was closely related to protoplast sources. No calli were obtained from mesophyll protoplast. For protoplast culture of cell suspension culture from hypocotyls P. pseudo-simonii and P.×beijingensis, the highest plating efficiency achieved when protoplasts cultured in liquid MMS medium containing 2,4-D 2 mg/L, BA 0.2 mg/L and 0.6 M glucose at a density of 2×105 pp/mL. Protoplast-derived calli that subcultured for 1-2 times on proliferation medium were used for shoot induction. The highest shoot formation frequency were observed when calli were cultured on medium containing 1.0 mg/L BA and 0.1 mg/L NAA for P. pseudo-simonii and with BA 0.5 mg/L and NAA 0.1 mg/L for P. xbeijingensis. Regenerated plants rooted on 1/2 MS medium containing IBA 0.5 mg/L.5. Tetraploids of P. pseudo-simonii were obtained for the first time via colchicine induced in vitro chromosome doubling using leaf explants. The most suitable concentration of PGRs for shoot regeneration from leaf explants was BA 0.4 mg/L with NAA 0.2 mg/L and the shoot formation frequency was 75.2%. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that had pre-cultured for 6 d and then cultured in liquid MS with 30 mg/L colchicine for 3 d. Polyploidy levels of 36 tetraploid plants were stable after maintained in vitro for five cycles of subculture.