| The disadvantages of traditional cross breeding were as follows:led to embryo abortion easily , brought cross breeding difficultly , and prolonged the breeding period.Sexual reproduction and bio-technology was combined in this trial,hybrid embryos were used to tissue culture,and the hybrid seedlings were obtained in the same year,new individual plant which had variation characters could be obtained in two years.This methods could not only overcome the difficulties of cross breeding,but also shorten the period of walnut breeding.There existed such problems of walnut embryo culture that were the high explants pollution , serious browning after inoculation , low propagation coefficient,and the lower survival rate.Aimed to the above problems,the reproductive characteristics of early fruit walnut,establishment of asepsis reproductive system,embryo culture technology of anti-browning,influence factors of walnut embryo culture,and the methods of rapid seedlings were studied from 2007 to 2008. The results were as follows. 1.Reproductive characteristics of flowering phenophase,the capacity of partheno genes and pollen shape and size of five early-bearing walnut cultivars such as'Lv ling'etc were studied,the results showed that:The accumulative temperatures from expanding to blossoming of female flower bud of the differents early-bearing walnut cultivars were'Lv zao'(130℃),'lv ling','Xiang ling','Liao 1'and'Liao 3'(360～410℃).The accumulative temperature from sprouting to florescence of female flower bud of'Lv zao'was lower than other cultivars.The male flowering period of protogynous cultivars'Lv ling','Xiang ling','Liao 3'and'Liao 1'(from the initial stage to final stage of pollen giving out)were13,17,18 and 13 days,generally was 13~18 days,the female flowering period (from the initial stage to final stage of female flower) were9,9,9 and 8 days, generally was 8～11 days;The intervals of male and female flower florescence were15,16,19 and 15 days.Enlargement stage of female bud of protandrous cultivar'Lv zao'was March 20th,there was 11 days from enlargement of female bud to flowering stage, the female florescence(from the initial stage to final stage of female flower) was about 11 days;enlargement stage of male bud of'Lv zao'was March 18th,the flowering stage was April 9th,the male florescence (from the initial stage to final stage of pollen giving out) was 15 days;the interval of male and female flower florescence was 8 days.The fruit setting stage and physiologydrops stage were 15 and 30 days after flowering.There existed apomictic capacity of both protogynous and protandrous walnut cultivars,and the apomictic capacity of protandrous walnut cultivars was lowerer than that of protandrous cultivars.There existed significant differences of apomictic rate among different early-bearing walnut cultivars.The apomictic rate of'Lv ling'was the highest that was 42.10% , next were'Xiang ling'and'Liao 1','Lv zao'was the lowest that was 11.64%.Pollen vitalities of'Lv zao'and'Liao 3'were more than 93%,the pollen vitality of'Xiang ling'was significantly lower than that of other varieties , that was only 62.72%.When the stigma presented the invert'eight'shape,the's receptivity of'Lv zao'was the highest,and it was the best pollination stage,from then on,the stigma began to wither gradually and lost the ability of pollination.The effective pollination period of'Lv zao'was 1~6 days after flowering,and the best pollination period was 2~5 days after flowering. 2.Natural hybrid walnuts of'Xiang ling'were used to embryo culture after storage,when inoculated for one week,contaminated rate of embryo with cotyledons which was disinfected for 5 min with HgCl2 was the lowest,only 7%,undisinfected embryo reached the highest contaminated rate for 87%; embryos with cotyledons which were disinfected with 25%,15% of NaClO and 75% alcohol were not polluted when the embryo culture carried on immediately after the fruits were collected. The optimal medium for walnut embryo culture in the four kinds of selected medium in this trial was MS +3% sucrose +0.6%~ 0.8% agar,and pH was 6.0~7.0,and the germination rate of hybrid embryos was the highest which was 90.2%,rootage rate was 89.6%;the optimal culture environments were as follows:light was 4000LX,temperature was 25~ 30℃;the best medium for tissue seedlings in the late period was DKW +2mg·L-16-BA +1.0mg·L-1IAA;the survival rate of rootage seedlings was the highest whichwas up to 83.3% when they transplanted in sand medium,next was in common soil which was 80%,the lowest was in vermiculite medium which was only 40%.The germination rate of hard-core hybrid embryos cultured in vitro was 86.6%,and the seedlings formation time was 40 days earlier than that of embryos cultured with mature embryos. Radicle elongation was promoted by stored the fruits under 4℃for 1 to 2 weeks,but it had no effects on the growth of stems and leaves in the latter period.3.Walnut embryo contained cotyledon became brown seriously in the different ways of embryo inoculation,browning occurred in the same day after inoculated,embryo became brown entirely 1 day after inoculation,brown area of culture medium was the largest;next was embryo with 1/2 cotyledons,embryo became brown entirely 1 day after inoculation also,brown area of culture medium was small;embryos without cotyledons did not became brown after inoculation.There had on effects of supplementing 300~500mg·L-1 Polyvinyl Pyrrolidone and 5.0～15.0 mg·L-1 vitamin C into the medium on the embryo with cotyledons becoming brown,medium color became dark brown.Embryo transfer could postpone medium brown , but could not lighten the brown of embryo with cotyledons.Embryos with cotyledon began turn to green , split and expand after four weeks.Embryos without cotyledon could became seedlings after four weeks culture,and transplant survival rate was up to 88.9%. |
PDF Full Text Request