| Kiwifurit (Actinidia) has high nutritional value and special flavor. It is known as the king of fruit. Jujube (Zizyphus jujuba Mill.) is the first major dry fruit tree in China. However, kiwifruit is a dioecious species with a complicated genetic background, and jujube has smaller buds, lower natural fruit setting rate and higher embryo abortion rate. Conventional breeding was resulted in slow progress owing to long growth and reproductive cycle. Modern biotechnology creates a new path for kiwifruit and jujube breeding.In this paper, differential red flesh Kiwifurit in Henan was used as experimental materials.An efficient regeneration and genetic transformation system were developed and transgenic plants expressing antibacterial peptide D gene were obtained.The PCR and Southern blot analysis showed that the APD foreign gene had been integrated into kiwifruit genomes. In our study, leaves were used as explants for genetic transformation of Zizyphus jujuba’Huizao’, and an efficient regeneration and genetic transformation system were established. The main results were as follow:1. Using leaves of Actinidia chinensis’Funiu 95-2’as explants, influences of different plant growth regulators on regeneration, proliferation and rooting of adventitious bud were considered, and an efficient regeneration system was developed. The results indicated that frequency (87.5%) of adventitious shoot induction was obtained when leaves were incubated on MS medium supplemented with 1.0 mg/L ZT and 0.3 mg/L NAA. The best result for shoot proliferation was obtained in MS medium supplemented with 2.0 mg/L 6-BA plus 0.5 mg/L NAA, on which the highest multiplication coefficient was 4.2.1/2 MS medium with 2.0 mg/LNAA was suitable for rooting, and the rate of rooting was 100%.2. Using leaves of Actinidia chinensis’Funiu 95-2’as explants, effects of antibiotics on explant growth were studied. The results showed both of differentiation rate of callus and adventitious shoots were zero when 20mg/L kanamycin was added to media, so the optimal concentration kanamycin was 20mg/L in the genetic transformation of leaf of Actinidia chinensis. Roots were not induced from media containing 30mg/L Kanamycin. Cefotaxime and Carbenicillin had negative effects on adventitious shoot differentiation, while inhabitation of Carbenicillin was stronger than that of Cefotaxime. According to our experiment results,400mg/L Cefotaxime was an optimum antibiotics to genetic transformation by Agrobacteriumin of Actinidia chinensis’Funiu 95-2’.3. The results of genetic transformation of leaves of Acitnidia chinensis’Funiu 95-2’showed that the highest transient expression frequency (92.2%) was obtained when leaf explants were pre-cultured for 3 days, and then infected for 10 min with Agrobacterium tumefaciens (OD600= 0.3) by vacuum infiltration, and finally co-cultured for 4 days. PCR detection and GUS histochemical staining initially proved that the foreign gene had been integrated into the Acitnidia chinensis genome.4. The selection protocl of resistant shoots after co-cultured was investigated, and the transgenic plants with APD gene were detected by molecular biology. The results showed frequency of resistant shoot regeneration in election media with gradually increased kanamycin concentration was 28.4% higher than that in the stable pressure. PCR and Southern blot analysis of partial Kan-resistant plants indicated that the foreign gene had been integrated into kiwifruit genomes.5. Using vegetative shoots of jujube as materials, the influences of antibiotics on controlling contamination and different plant growth regulators on propagation and rooting were studied. The results showed that contamination rate remained as high as 97.8% when explants were firstly sterilized in 70% alcohol for 30s and then in 0.1% mercury dichloride for 10-15 min. Contamination rate of explants was reduced to zero when media was supplemented with 150mg/L or 200mg/L cefotaxime. The similar result was obtained with streptomycin, while streptomycin could inhibit developing of axillary buds. In the initial culture phase, strong vegetative shoots of jujube were obtained from the MS media containing 2.0 mg/L KT and 0.1mg/L NAA. In the subculture phase, the plantlets with fully expanded and dark green leaves grew stronger in MS media including 2.0 mg/L KT plus 0.3 mg/L NAA.However, the plantlets were weaker when sub-cultured in MS medium supplemented 2.0 mg/L 6-BA and 0.3 mg/L NAA, and the multiplication coefficient was 2.7. The half MS media containing 0.5mg/L NAA was optimal for rooting with 95.56% rooting rate. 6. The effects of basal medium, leaf maturity, leaf position on the shoot, orientation of explant contacting medium, sucrose concentration, days of dark incubation, and ethylene inhibitors on leaf regeneration were investigated. The results indicated that WPM supplemented with 0.5mg/L TDZ plus 0.2mg/L NAA was effective for leaf explant regeneration of’Huizao’. The optimal explant was young, and thick expanded leaves from the intermediate shoot. More adventitious buds developed at the petiole portion of the midrib and surrounding area, and regeneration capacity increased with 59.74% from the tip towards the base of leaf. The frequency of regeneration of’Huizao’was 16.81% higher when the abaxial surface of the explants was in contact with the medium compared that the adaxial surface of the explants was in contact with the medium. When sucrose concentration was 40g/L in the medium, the hyperhydric degree of adventitious buds was reduced. A 10-day culture in the dark could increase the frequency of regeneration with 81.6%. The addition of 0.5 mg/L AgNO3 and 0.1mg/LSTS to the medium could pomote leaf regeneration, and the frequency of regeneration of leaf explants was 82.25%,91.26% respectively.7. Influence of kanamycin on regeneration of leaves, cefotaxime on inhibiting Agrobacterium, and days of co-culture on transformation frequency were investigated. The results showed that the differentiation rate of adventitious bud was zero when kanamycin concentration in the medium was increased to 40mg/L, so it was the critical concentration in the genetic transformation of’Huizao’. The effects of different concentrations of cefotaxime were divers, and higher concentration (500mg/L) of cefotaxime could inhibit Agrobacterium growth. The transformation rate of Z.jujuba’Huizao’was 21.1% after 2 days of co-culture.The veins of transgenic plantlets were stained blue after histochemical assay, which preliminarily proved that the foreign gene had been integrated into the genomes of’Huizao’... |
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