| Populus spp.(Section Tacamahaca) is a very special populus which have many types in China, it is important to accelerate the process of Populus genetic improvement. Establishment suspension cell and culture protoplast are the foundation of germplasm innovation and breeding from cellular level. P.×Beijingensis、P. pseudo-simonii×P. nigra ’Zheyin3#’ and their hybrid as testing materials to build suspension regeneration system and P.×Beijingensis mesophyll protoplast.(1) Calli which were induced from various materials such as anther, ovary, seed and so on. Explants which included P.×Beijingensis anther, ’Zheyin3#’ ovary, and their hybrid offspring like seed, hypocotyl, cotyledon and root segment were used to induce loose callus (LC), Suitable culture conditions:anthers cultured in MS+4 mg/L 2,4-D+2 mg/L KT+200 g/L sucrose in dark for 4～5 w induced calli, then calli were transferred to MS+1 mg/L 2,4-D+1.5 g/L Gln+0.5 g/L ME+30 g/L sucrose for 3～5 times; ’Zheyin3#’ ovary cultured on MS+2 mg/L 2,4-D+30 g/L sucrose in dark for 40 d, calli cultured on the same media with 0.5 g/L CH and 0.05 g/L Arg for 30 d and transferred external calli to MMS+2 mg/L 2,4-D+0.1 mg/L 6-BA+30 g/L sucrose for 40 d; hybrids material cultured on MS+2 mg/L 2,4-D+1 mg/L KT+30 g/L sucrose for 60 d to LC. Related research laid the material foundation for building suspension cell lines.(2) A method for enriching embryogenic cell was proposed, and the technique of building and regenerating P. ×Beijingensis anther embryonic suspension cells line. Suspension cell line was made of round embryonic cell and irregular non-embryonic cell; cells were separated by standing and double cell filters screening, based on embryonic cells could be translate to non-embryonic cells in the medium with NH4+, embryonic cells cultured in 50-100 rpm shaky liquid medium without NH4+ induced Round Callus (RC), RC incubated plantlet by somatic embryogenesis with 0.1～0.2 mg/L CPPU, differentiation rates were 100%.554 plantlet were detected by flow cytometry,102 tetraploid were detected, the reason was chromosomes doubling during the course of incubating RCs.(3) A method to culture and fast direct regeneration for embryonic suspension cells of ’Zheyin3#’ without pollination ovary was proposed. Research results showed that ovary callus direct dispersion in liquid media (MS+0.1～1 mg/L ZT+0.2 mg/L NAA+0.1 mg/L CPPU+1.5 g/L Gln+40 g/L sucrose) to form embryonic suspension cell lines, white particles induced in the same media for 40 d and turned green after light culture for 15d. particles regenerated by somatic embryogenesis. Compared with the method of anther callus, the method saved more than 2 m time.(4) Establishment of multiple genotypes cell suspension lines of P. pseudo-simonii x P. nigra ’Zheyin3#’ culture and regeneration technology. Study found dissociated embryonic cells in the same compact size number had not significantly different among different genotypes of seeds and cotyledons. The problem that suspension cell PCV of different genotypes had differ significantly and the successive cycles were inconsistent were solved by separating embryonic cells to establish embryonic cells line.20 genotypes of seeds and cotyledon-derived embryogenic suspension cell lines were made; embryogenic suspension cells from same explants had no significant difference in calli proliferation rate and RC number. RC incubated plantlet on anther media. Study on calli differentiation rate and found from same sources had no significant difference in the number of plantlets, in which seed sources had 5.55 ± 0.11 plantlets per RC and cotyledon sources had 2.18 ± 0.15 plantlets per RC. Histological analysis showed seed RC regeneration by somatic embryogenesis and organogenesis, cotyledon RC regeneration by organogenesis.(5) P.×Beijingensis mesophyll protoplast culture and regeneration technology were established. Research results showed that aseptic seedling mesophyll protoplasts was better than the outdoor trees, the young leaves of less than 60 d were suitable for preparing protoplasts. The best enzymatic hydrolysis conditions was:3% cellulose R-10+0.2% macerozyme R-10+0.05% pectinase Y-23 at 27℃ standing for 3 h 45 min and 20 rpm oscillating for 15 min, protoplast yield was 2.13x107/g, protoplast viability>90%; Adding 0.2 g/L VC,0.2 g/L GSH,0.5 g/L putrescine or 0.5 g/L spermine effectively reduced intracellular reactive oxygen species levels, maintain the stability of plasma membrane; Adding 10 g/L d-Galactose promoted cell wall reconstruction and cell agglutination. A two-step of reduce osmotic pressure that adding 2 mL and 4 mL osmolarity regulator on the 3 d and 10 d effectively promoted cell lines growth and calli induced. Calli Differentiated adventitious buds in the media (1/2MS+(0.2、0.4、0.8) mg/L ZT+0.4 mg/L NAA+0.2 mg/L CPPU+2 g/L Gln+30 g/L sucrose). Adventitious buds grew into plants by elongating and rooting. |
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