| Populus×Xiaohei CV.Baicheng is a hybrid species based on the above-ground part of the hybrid seedlings of Populus simonni×P.nigra in the Chinese Academy of Forestry,which is a fast-growing,cold-resistant,drought-resistant and excellent timber species in Northeast China.But the existing tissue culture and genetic transformation system of Populus simonni×P.nigra is not suitable for the P.×Xiaohei CV.Baicheng,which seriously limits the promotion and application of this excellent species.（1）In view of the above problems,this study was conducted to establish and optimize the tissue culture and genetic transformation system for P.×Xiaohei CV.Baicheng.Stems of P.×Xiaohei CV.Baicheng in vitro plants were selected as explants and MS medium was used to establish tissue culture system by adjusting the hormone concentrations of 6-BA,NAA,TDZ and IBA.Based on the tissue culture system,we confirmed the optimal concentrations of kanamycin and infection time to establish Agrobacterium tumefaciens-mediated genetic transformation system for P.×Xiaohei CV.Baicheng.The tissue culture system of P.×Xiaohei CV.Baicheng consisted of three stages,including adventitious bud differentiation induction(MS+6-BA 0.5 mg·L-1+NAA 0.1 mg·L-1+TDZ 0.001 mg·L-1,shoot differentiation rate 92.59%),stem induction(MS+6-BA 0.2 mg·L-1+NAA 0.05 mg·L-1,multiplication coefficient 6.50)and rooting induction(1/2 MS+IBA 0.4 mg·L-1,rooting percentage 100%).The most optimal kanamycin concentration for genetic transformation was 30 mg/L and the most suitable infection time was 20 minutes.The transgenic plants were obtained successfully after 30 days of adventitious bud differentiation induction,15-30 of stem induction and 25 of rooting induction with 2%transformation efficiency.（2）The drought tolerance gene PtrNAC006 was identified in a previous study in our laboratory,which enhanced the drought tolerance of the plants.In this study,we created and functionally resolved PtrNAC006 overexpression plants in P.×Xiaohei CV.Baicheng by applying this genetic transformation system.The growth phenotypes of the PtrNAC006overexpression plants and the wild-type plants were analyzed,and the transgenic plants showed no significant difference in plant height,reduced diameter and tenth stem node length,and increased stem node number.Morphological observations using paraffin sections revealed that the transgenic plants showed a later appearance of secondary growth and lignin deposition with smaller vessel cells and a significant increase in the number and area of vessels per unit area.The results of wood component analysis showed that the soluble lignin contents of transgenic plants increased significantly by up to 53.23%;total lignin contents increased significantly by up to 5.89%,and there were no significant changes in acid insoluble lignin;glucose contents decreased significantly by up to 2.96%;xylose contents decreased significantly by up to 9.51%;total carbohydrate contents were significantly reduced significantly by up to 4.06%;galactose and arabinose contents did not change significantly.（3）To further analyze the effect of the altered vessel cell morphology caused by PtrNAC006overexpression on drought tolerance of plants,drought stress experiments were conducted on PtrNAC006 overexpression plants and wild-type plants in this study,and it was found that the transgenic plants showed a significant drought tolerance phenotype with a maximum survival rate of 62.50%after rehydration,and higher net photosynthetic rate and water use efficiency under drought stress conditions;stomatal conductance and stomatal conductance and transpiration rate were reduced.In contrast,drought stress produced cavitation on the xylem of the plant to form embolism,and after measuring the hydraulic conductivity of the plants before drought stress and after heavy drought,it was found that the hydraulic conductivity of the transgenic plants decreased by 28.14%,which was significantly lower than that of the wild type.In summary,this study successfully established an efficient tissue regeneration system and Agrobacterium-mediated genetic transformation system in P.×Xiaohei CV.Baicheng.Using this system,we successfully created transgenic plants with the drought gene PtrNAC006 and analyzed the functions of this gene in terms of growth,wood formation,drought tolerance and photosynthesis,and proved that PtrNAC006 is an excellent candidate gene for drought tolerance in poplar.This study provides an efficient technical system for the breeding of new high-quality forest trees varieties and new clues for the analysis of the mechanism of drought stress response in forest trees,and the obtained PtrNAC006 transgenic plants are also excellent forest tree germplasm with both growth and drought tolerance. |
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