| Kiwifruit(Actinidia spp.)is one of the most important fruits in the world.The Actinidia genus contains 54 species and 75 taxa which originated from China and its neighboring countries.A.deliciosa(green-fleshed kiwifruit)and A.chinensis(gold-fleshed kiwifruit)are two mainly commercially cultivated species.A.arguta(‘baby’ kiwifruit)is rarely cultivated,whose fruits with smooth edible skin have distinctive aroma from those of the major commercial cultivars,has become a commercially valuable kiwifruit in recent years.A.arguta ‘Tian Yuan Red’ with full red smooth edible skin and fruit could be cultivated for fresh eating and wine,which are appreciated by consumers for their distinctive flavor and value.In order to establish an effective regeneration system for propagation,leaves from in vitro shoots was used as explants,and analyzed effects of plant growth regulators on leaf callus regeneration.Kiwifruit bacterial canker,which has been causing global serious threat to the kiwifruit industry,is a devastating bacterial disease.Until now there is no effective method to control kiwifruit bacterial canker.One of the effective methods to avoid canker losses is to breed disease-resistant-cultivars through genetic engineering.In the present study,the transgenic Actinidia chinensis shoots with the antibacterial peptide D was used as the materials,and many kiwifruit plants were rooted and transplanted in greenhouse.The protective enzyme activities of kiwifruit plants treated with bacterial canker pathogens were analyzed and the disease resistance changes of transgenic plants were predicted.The main results were as follows:(1)The results indicated that different concentrations of 6-BA combined with NAA had different effects on the mean number of shoots per explant.Shoot multiply rates were generally improved with increasing 6-BA concentration.The maximum shoot multiplication rate of 3.29±0.03 was obtained on MS medium containing a combination of NAA 0.5 mg/L and 6-BA.3.0 mg/L.(2)The effect of different concentrations of ZT or 6-BA on callus formation rate of leaf explants.The results indicated that ZT or 6-BA had varying effects on callus formation for A.arguta ‘Tian Yuan Red’.When the media were supplemented with different concentrations of 6-BA,no callus was induced,and the browning rate of explants was high,whereas ZT can significantly induce the formation of callus,and the browning rate was low.(3)The results of ZT and IAA combination on leaf regeneration revealed that the highest frequencies of shoot regeneration was 45.00 % when the media was supplemented with 2.5 mg/L ZT and IAA 0.5 mg/L.(4)The leaf explants were initially placed under 15 d,20 d,25 d,and 30 d of culture in the darkness,and then transferred to culture in light.When leaf explants was cultured 30 days in darkness,the maximum shoot regeneration frequency reached 81.67% in MS medium supplied with ZT 2.5mg/L and IAA 0.5 mg/L.(5)The transgenic kiwifruit shoots with antibacterial peptide D was used as the materials,and many kiwifruit plants were obtained via root induction.According to the results of rooting,the best result was obtained with shoots treating for 15 s with 1.0 g/L IBA,where less callus and more roots were induced.(6)The PCR and RT-PCR analysis of transgenic kiwifruit plants indicated that antibacterial peptide D has been integrated and transcribed in the kiwifruit genome and expression of exogenous gene differed among three transgenic lines.(7)The activity analysis of catalase(CAT),peroxidase(POD),and phenylalanine ammonialyase(PAL)enzymes showed that the above three enzyme activities of the transgenic plants were not obviously influenced by over-expressing of antibacterial peptide D gene.In response to canker bacteria treatment,enzyme activities of CAT and POD in transgenic plants were greatly reduced,and PAL enzyme activity was enhanced compared with the controls. |
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