Studies On The Establishment Of Efficient Regeneration System And Genetic Transformation In Actinidia Latifolia

Kiwifruit with rich nourishment especially high content of Vitamin C finds favor in human's eyes in recent decades (Huang, 2000). China is rich in kiwifruit resources with more than 59 species out of total 66. Among these 59 species, most grow naturally and haven't been exploited, even some are at the brink of extinction. It is imperative to exploit and use the germplasm of these wild species while maintaining their genotypes. Actinidia latifolia generally known as "Duo Hua", grows naturally in Yunnan, Guangxi, Hunan, Sichuan and Guizhou provinces of China and bears edible fruits. Its fruits are best for making fruit juice and wine. Vitamin C content in its fruits of 939.8~2140mg/100g is higher than all other kiwifruit species (Cui, 1993). To our knowledge, this is the first study on an in vitro regeneration method and genetic transformation for A. latifolia. The results of this study will help facilitate the application of genetic engineering methods to improve the commercial traits in A. latifolia. The main results are summarized as follows:1. An efficient plant regeneration protocol in vitro for A. latifolia was achieved via organogenesis from leaf and petiole explants. In this paper, effects of explant types, medium formulation, different combinations of growth regulators, dark treatments and sucrose concentrations on the organogenesis are examined. For leaf explants, after 6 weeks of culture maximum shoot regeneration frequency of 91.7% with 6.9 shoots per explant was obtained on half-strength MS medium (Murashige and Skoog, 1962) containing 5μM Zeatin, 0.1μMα -naphthaleneacetic acid and 20g/L sucrose, incubated in the dark for 21 days then exposure to light. For petiole explants, after 5 weeks of culture maximum shoot regeneration frequency of 100% with 18.2 shoots per explant was obtained on half-strength MS medium containing 0.1μ Mα-naphthaleneacetic acid, 5μM Zeatin and 20g/L sucrose, incubated in the dark for 14 days then exposure to light. High concentration of 6-benzyladenine (10μM) inhibited shoot formation, and also resulted in vitrification. We proposed that light is essential for shoot regeneration for A. latifolia. Regenerated shoots were transfered on BW rooting media (Sugawara et al., 1994) supplemented with different combinations of auxins and 6-benzyladenine. The highest rate of root formation reached 100% associated with maximum roots per explant (17.5). Plantlets with good root system were successfully acclimatized to thefield conditions and produced healthy plants.2. Effects of antibiotics on in-vitro regeneration and organogenesis of A. latifolia from leaf and petiole explants were examined. Kanamycin inhibited significantly in-vitro regeneration and organogenesis. Callus and shoot formation from leaf explants was completely inhibited in presence of 20 mg/L kanamycin, and that from petiole explants was in presence of 25 mg/L kanamycin. Results also showed that cefotaxime is superior to carbenicillin for shoot differentiation. When 300mg/L cefotaxime was present in the regeneration medium, both shoot formation rate and mean number of shoots per explant reached the highest levels, 100% and 9.8 shoots/explant from leaf explants, 100% and 12.4 shoots/explant from petiole explants. However, a negative effect of cefotaxime (≥200mg/L) on rooting of regenerated shoots was also observed. Carbenicillin had no marked influence on rooting. In the treatments of different combinations or alone of kanamycin and carbenicillin on rooting, carbenicillin had no marked influence on rooting but kanamycin inhibited markedly rooting. With the increase of kanamycin concentration, the rooting rate decreased sharply. No root appeared on rooting medium with 50mg/L kanamycin. It was considered that cefotaxime 300mg/L was suitable for A. latifolia to elimination of Agrobacterium tumefaciens during transgenic shoot induction; kanamycin 20mg/L was suitable in the selection of transgenic versus false-positive shoots from leaf explants and 25mg/L was suitable for petiole explants; during rooting phase kanam...