Studies On Mechanism Of A High-efficient Regeneration System And Genetic Transformation Of Strawberry

Strawberry is one of most important fruit crop cultivated widely in world. In spite of its important, several factors such as its low resistance to chilling injury and its susceptibility to insect, disease and virus which limit the cultivation of strawberry fruit. Some progress has been made in strawberry yield and quality through conventional breeding techniques that has been limited due to several factors. Genetic limitations of strawberry associated with high heterozygosity and polyploidy are the main severe limiting factors that make it laborious, costly, and time-consuming to improve strawberry quality and yield by the conventional breeding techniques, which usually results in a low frequency. With the development of biotechnology, foreign genes could be incorporated into plant genome for desired agronomic traits by the new technologies of Agrobacterium-mediated transformation. An efficient regeneration method is one of the most important factors in determining the success of plants genetic transformation. Establishment of a high-efficient regeneration and transformation system in strawberry would make a significant contribution in the improvement of this fruit on gene engineering level. In this study, Fragaria ananassa Duch. cv. Toyonoka was used as source material, we studied the effects of plant growth regulator combination, plastic color film, AgNO3, dark treatment and antibiotics on shoot regeneration. Regeneration mechanism under color films and response of in vitro strawberry to AgNO3 were also studied. Using the EH 105 strain harboring pBI121-APD and pBI121-INS that we constructed respectively, leaf discs of strawberry were infected with the Agrobacterium, which carried the two genes. Kanamycin-resistant (Kan~R) shoots and calli were gotten from the explants. The results of PCR, Southern hybridization analysis showed that the APD gene was integrated into strawberry genomes. INS gene was integrated into strawberry genomes by PCR analysis. The results were summarized below:1. Establishment of a highly efficient system of shoot regeneration from leaf explants of strawberry1). Types and combinations of plant growth regulators had significant effects on shoot regeneration in strawberry. TDZ alone induced regeneration of adventitious buds. Maximum regeneration rate (47.45%) was achieved with higher concentration of TDZ (at 2.5 mg.L~-1), but it resulted in extended rates of vitrification. Therefore, TDZ above 1.5 mg.L~-1 on strawberry regeneration were not encouraging. 1.5 mg.L~-1 TDZ associated with 0.4 mg.L~-1 IBA significantly increased regeneration efficiency in terms of both percent of shoot regeneration and number of shoots per explant. The maximum percentage of shoot regeneration (76.67%) and the highest number of shoots per explant (5.78) were achieved by MS+1.5 mg.L~-1 TDZ+0.4 mg.L~-1 IBA. 2,4-D significantly decreased shoot regeneration with maximum browning and concentrations above 1.0 mg.L~-1 had adverse effect on shoot regeneration. 2). Significant difference was observed in percent shoot regeneration and number of shootsper explant when explants were exposed to AgNO3 for 10 d. With AgNC>3 in the medium, an earlier (5-7d) and stable shoot regeneration was observed, highest regeneration (87.38%) and numbers of shoots per explant (11.67) were achieved with the shoot regeneration media (MS+1.5 mg.L"1 TDZ+0.4 mg.L"1 IBA) containing 1.0 mg.L"1 AgNO3.3). Color films had significant effects on the shoot regeneration efficiency. Under red films, 100% shoot regeneration was achieved after 45 days in culture, closely followed by green (98.28%). Fluorescent light (Control) ranked the third (75. 81%), followed by blue (60.09%) and the lowest shoot regeneration was under yellow films (20.69%). Highest number of shoots (25.75) was recorded for green, followed by red (25.11) films. Calli under yellow plastic films started browning after 50 days in culture. Red and green films promoted while yellow inhibited regeneration of adventitious buds.4). Dark treatment could significantly affect shoot regeneration efficiency. Shoot regeneration percentage and number of shoots per explant were improved with increasing dark treatment weeks. 100% regeneration percentage and 9.05 shoots per explant were observed when a 4-week dark pretreatment was used. However, the regeneration percentage and number of shoots per explant declined when longer dark treatments were used.2. Regeneration mechanism of strawberry under different color plastic films1). A significant difference in the antioxidant enzyme activities was observed in culture of explants in vitro under color films. Changes in SOD, G-POD and CAT activities were positively correlated with shoot regeneration as compared to negative correlation between regeneration of adventitious bud and MDA. SOD, G-POD and CAT activities were highest under the red films, followed by green as compared to the lowest under yellow ones.2). Red and green plastic films induced a lower chlorophyll a/b ratio and a higher level of chlorophyll b in strawberry tissue culture. However, lowest content of chlorophyll highest chlorophyll a/b ratio was recorded for blue films.3). There was direct correlation between endogenous growth hormones contents and adventitious bud differentiation. After 15 days in culture, gibberellic acid (GA3) content was found highest under fluorescent light followed by red and green plastic films. Zeatin (ZT) and GA3 contents were highest under red plastic films; however, indole-3-acetic acid (IAA) content was highest under blue plastic films after 45 days in culture.3. Response of Toyonoka strawberry to AgN(>31). Half-strength MS containing 1.0 mg.L"1 AgNO3was an optimum medium for rooting. AgNO3 advanced root emergence and increased percent rooting, root length, dry weight and activity.2) AgNO3 had a significant stimulatory effect on chlorophyll, soluble protein contents and antioxidant enzyme activities. Chlorophyll and soluble protein contents, SOD, POD and CAT activities were increased in the presence of AgNO3 and reached maximum at 1.0 mg.L'1 AgNO3. Root water content, O2 ", MDA content, proline accumulation and...