Tissue Culture Technology Research On Xinjiang Thin-shelled Walnut

Walnuts are one of the main economic tree species in China,and have important value in nutrition,health care,and medicine.Xinjiang walnut has abundant germplasm resources and a wide cultivation area,occupying an important position in the domestic walnut market.With the increasing demand for walnut kernels in the walnut industry,high-quality and large-scale breeding is an inevitable trend.Through tissue culture technology,while achieving rapid seedling reproduction,it can also ensure the stable inheritance of excellent characteristics.Therefore,in this experiment,the stem segments with axillary buds and young embryos of Xinjiang characteristic thin shell walnut variety’Xinwen 185’were used as materials to compare the optimal disinfection and browning inhibition treatments.The stem explants were graded,and the rapid propagation system of’Xinwen 185’walnut stem segments and the rapid propagation system of’Xinlu’walnut embryo seedlings were preliminarily established by screening the optimal culture conditions during the initiation,subculture,and rooting culture processes,In addition,this study investigated the culture conditions for inducing callus production and proliferation in walnut leaves,and basically achieved stable callus production and proliferation culture.The main research results of this article are as follows:1.Preliminary establishment of a rapid propagation system for walnut stem segments with axillary buds in’Xinwen 185’When culturing mature semi lignified stem segments with axillary buds in vitro,the treatment effect of disinfectant Na Cl O was better than that of H₂O₂.Disinfection with 1.0%Na Cl O for 10～15 minutes had a better effect,with a pollution rate as low as 23.00%and a higher survival rate.Adsorbents（PVP,AC）have better effects on inhibiting stem browning and promoting axillary bud germination than antioxidants（Na₂SO₃,Vc）.As a specific adsorbent for phenolic substances,PVP has the lowest browning rate of12.73%and germination rate of 83.94%at a concentration of 0.2 g/L.The initiation effect of stem segments with axillary buds on DKW medium was better than that on MS and WPM medium.On DKW medium supplemented with 1.5～2.0 mg/L 6-BA+0.1 mg/L IBA,the initiation effect was good,with a seedling rate of 93.00%;The proliferation rate of the first generation of cultured shoots transferred to DKW proliferation medium supplemented with 1.0 mg/L 6-BA+0.01 mg/L IBA was3.01,and the adventitious buds had short and strong growth,small leaf spread,and light green leaf color.The average seedling height of adventitious bud seedlings cultured on DKW+0.5 mg/L 6-BA+0.05 mg/L IBA medium was 3.86 cm,with neat and robust growth and strong green leaves;Adventitious root could be induced on 1/4 DKW+3.0 mg/L IBA medium,but the rooting rate was only 24.44%.2.Preliminary Establishment of Rapid Propagation System for Embryo Seedling Formation of’Xinlu’WalnutThe young embryos of’Xinlu’walnut can germinate on MS,WPM,and DKW media.On 1/2DKW solid medium,it is beneficial for direct seedling formation,while on DKW medium,it is beneficial for lateral bud formation;The optimal medium for inducing adventitious buds is DKW+2.0 mg/L 6-BA,with a germination rate of 93.64%and a seedling rate of 89.63%.After removing the young embryos from the hypocotyl,on the proliferation medium of DKW+1.0 mg/L 6-BA+0.01 mg/L IBA,the shoots grew quickly,with moderate growth,light green leaves,and a multiplication multiple of 5.84.The adventitious buds after proliferation have the best seedling strengthening effect on DKW+0.5 mg/L 6-BA+0.05 mg/L IBA medium,with an average seedling height of 3.96 cm,robust seedling growth,and green leaves;Similarly,the culture medium for adventitious root was 1/4DKW+3.0 mg/L IBA,and the rooting rate was 24.44%.3.Callus induction and proliferation of walnut leavesAs the concentration and time of disinfectant increased,the pollution rate of mature field leaves gradually decreased while the browning rate gradually increased.Disinfection with 1%Na Cl O for 6～10minutes resulted in a pollution rate of 12.73～16.06%and a browning rate of 20.00～22.73%,respectively.After disinfection,the leaves were induced to differentiate on DKW medium of 1.0 mg/L 2,4-D+2.0 mg/L6-BA,and then transferred to medium of 1.0～2.0 mg/L TDZ+0.1 mg/L NAA.The leaves were cultured in a dark environment,and the leaf healing rate reached over 93.00%;The proliferation rate of callus tissue with good growth condition on DKW+1.0 mg/L TDZ+0.5 mg/L NAA medium was 3.32.The callus tissue grew quickly and appeared as light yellow loose particles,watery and almost without browning.