Study On The Establishment Of Regeneration System For In Vitro Leaves Of Zizyphus Jujuba 'Huizao'

Jujube (Zizyphus jujuba Mill.) is one of the most important and representative fruit trees in China.'Huizao'is a cultivated variety of jujube in China that has the longest planting history, the largest orchard area, and highest annual production. Since'Huizao'has small flowers, artificial emasculation is difficult. High embryo abortion rate and the low rate of the fruit set in this perennial woody fruit tree make improvement of quality by conventional inter-specific cross breeding quite difficult. Leaves of regeneration technology is the basis of mutation breeding on the level of cell, mutation screening, and isolation of chimeric gene transfer and so on. From then on, shoot regeneration systems from leaf explants have been achieved in several Z. jujuba cultivars, such as'Mingqinxiazao','Zhanhuadongzao','Junzao'and'Maoyezao'. As of yet there is no reliable regeneration procedure applicable to'Huizao', one of the most prominent high quality varieties of jujube.Critical factors affect the frequency of leaf regeneration in jujube, such as basal medium, concentration and combination of plant growth regulators, leaf maturity, leaf position on the shoot, orientation of explant contacting medium, and incubation conditions. These factors are investigated in this study, and our objective was to obtain an efficient and stable regeneration system using leaf segments from'Huizao'.The main conclusions are as follows:(1)The leaves of tissue culture plantlets were the optimal explant source for callus induction of Z. jujuba'Huizao'. The callus induction frequency of leaves from the central stem of plantlet is higher than the ones which from the lower part and the upper part. The optimal explant is the leaves on the three to five of the stem, which are flat, large and thick. Cut 3-5 knives vertical to the main vein not to hurt the leaf margin before vaccination, the callus induction frequency can be increased to 65.44% compared to parallel to the main vein and no cuts. Effect of explant orientation on callus induction frequency was not significant. Incubation length of pre-culture in darkness can accelerate the process of callus formation, appropriate time in darkness is 14 days. The optimal callus induction medium is 1/2MS supplemented with 0.5mg/L 6-BA and 2.0 mg/L 2,4-D, 0.5mg/L AgNO3, callus induction frequency can achieve to 96.67%. Only the yellow-green, dense callus cultured in differentiation medium on the 3rd generation can differentiate regeneration shoots. Cut them into 0.5cm2 size and then incubate on the differentiation medium. The differentiation frequency of callus on MS supplemented with 6-BA 3.0 mg/L and IBA 0.2mg/L is 13.77%, and one callus can only differentiate one shoot. The differentiation frequency of callus on MS supplemented with ZT 2.0mg/L and IBA 0.1mg/L was 37.34%.(2)WPM with 0.5mg/L TDZ plus 0.2mg/L NAA was effective for leaf explant regeneration of'Huizao'. The optimal explant is the young leaves on the middle of the stem, which are flat, large and thick. While more adventitious buds initials developed at the petiole portion of the midrib and surrounding area, regeneration capacity increased 59.74% from the tip towards the base of the leaf. The frequency of regeneration of'Huizao'was 16.81% higher when the adaxial surface of the explants was in contact with the medium compared to the abaxial surface of the explants was in contact with the medium. The concentration of sucrose in 40g/L can reduce the hyperhydric degree of adventitious buds. A 10-day dark of inoculated can increase the frequency of regeneration. The frequency of regeneration of leaf explants increased 14.14% with the addition of 0.5 mg/L AgNO3 to the medium and the average number of shoots per explant can reach to 2.64 piece when compared to that of the control without AgNO3.(3)1/2MS supplemented with 0.3mg/L NAA and 2.0mg/L IBA was effective for adventitious roots induced by leaf explant of'Huizao', the frequency can reach to 92.95%, the adventitious roots induced are white, thick. Cut adventitious roots into 0.5cm,then inoculate on WPM supplemented with 1.0mg/L TDZ and 0.2mg/L NAA, the differentiation frequency can reach to 44.51%, average number of adventitious buds were 2.08 piece.(4)Inoculate the adventitious buds through three ways above on the proliferation medium which contain MS plus 2.0mg/L 6-BA, 0.5mg/L IBA and 0.5mg/L KT, cultured for 35 days, multiplication coefficient were about 3.0.(5)Cut the proliferation of the regeneration buds into 2.0cm,then inoculate on the root induction medium, the best rooting medium was 1/2MS plus 0.5mg/L NAA. After 40 days culture, rooting frequence can achieve to 95.56%. Only a small number of callus at the base of the plantlet, the occurrence of the root were thick, had good quality.