Researches On Tissue Culture Of Juglans Regia L.

Juglans regia L.is one of the most important economic tree species in China.In recent years,Chinese and foreign researchers have made certain progress in walnut in vitro culture,but several problems have not been solved hitherto,such as the high pollution rate during primary culture,the browning problem of in vitro culture,low shoot proliferation,the difficulty in organogenesis.In this paper,using walnut leaves,petioles,stem segments as explants,a series researches were carried out on several important factors which involved in walnut tissue culture.Main results were as follows:1.The best sterilization for stems with axillary buds was 0.1%HgCl2+Tween 80 for 5min 2 times.Under this treatment the survival rate was 75.27%and the pollution rate was 5.38%.For leaf explants,the best sterilize treatment was 0.1%HgCl2 for 8min,and the survival rate was 79.87%,the pollution rate was 3.23%.2.The best steps for browning inhibition were as follows:1)keep the explants in antioxidant solution(50mg/LAC+200mg/LVC+100mg/LPVP)after collected;2)after rinsing,soak the explants in antioxidant solution;3)cut explants and inoculated to DKW medium containing 100mg/L PVP-40.The browning rate was 7.29%.3.Effects of different basic culture media on germination rate and the growth status of axillary bud were different.DKW was the best medium for axillary buds germination induction,in which initiation rate and effective shoots rate reached 92.54%and 72.06%.4.CTK and auxin used alone or in combination all had significant influence on proliferation of axillary bud.The best medium for primarily culture of stems with axillary buds was DKW+6-BA 1.0mg/L+NAA0.05mg/L,in which proliferation ratio and average shoot height reached 5.07 and 1cm.5.Both sucrose content and NAA content had significant effects on shoots strength culture.The best medium was DKW+NAA2.0mg/L+sucrose 40g/L,in which the average shoot height and diameter reached 2.27cm and 0.28cm.6.Two-step rooting was applied in rooting culture.The first step:explants were cultured in l/4DKW+IBA5.0mg/L for 12d in dark;the second step:seedling were transferred to 1/2DKW medium without growth regulator and cultured under light,adventitious roots appeared in some plants after 20d.7.Best callus induction could be obtained by using petioles as explants.Effects of three growth regulators on callus induction was NAA>TDZ>6-BA.When the medium was DKW+NAA0.5mg/L+TDZ1.0mg/L+6-BA0.5mg/L,the callus induction rate of leaf and petiole explants reached 70%or more,and callus were yellowish green,loose and granular.While no adventitious shoots differentiated from callus.