| Strawberry (Fragaria ananassa Duch.) is a major berry fruit crop in the horticulture science. In the recent years, strawberry cultivation in plastic tunnels has become a characteristic agricultural industry in many regions of China countryside. However, heat and wet resistant ability was weak in some strawberry cultivars. Moreover, diseases and insects occurred seriously, which declined strawberry economic benefit. Thus, how to improve the strawberry heat resistant ability is a key problem of industrialization. ALA has been suggested being ability to improve plant resistant. In animal and yeast, ALA-S is a rate-limiting enzyme for ALA synthesis pathway. In Arabidopsis, GluTR is encoded by HemAl, and HemAl is regulated by light. We introduced a recombinant gene Yheml, where the yeast ALA synthase gene (Heml) was the functional gene, which was controlled by Arabidopsis HemAl promoter, into strawberry leaves. Detection by Km, GUS, Southern blot, PCR, RT-PCR and endogenous ALA levels showed that three of the screened plant lines could biosynthesize more endogenous ALA and higher capacity of chlorophyll fast fluorescence characteristics under high temperature stress, which imply that Yheml might be used for breeding of strawberry with stress resistance. The main results are as follows.1. The conditions for adventitious shoot regeneration of leaf explants of'Benihonpe' strawberry were studied in the work. The results showed that the highest induction efficiency was obtained when explants were incubated on MS medium with 1.5mg/L TDZ +0.4 mg/L IBA+0.1mg/L AgNN3, which reached 82.5%. The leaves with weak green, tender, thick and 15-30 days old were suitable for regeneration. Darkness pretreatment for 1 to 2 weeks could significantly improve the shoot regeneration. And regenerated buds would root on MS medium supplemented with 0.5mg/L NAA.2. An agrobacterium transformation system with yeast Heml was studied in strawberry. The cut explants were infected in the bacterial liquid 5 minutes with OD600=0.4-0.5, then incubated in MS+1.5 mg/L TDZ+0.4 mg/L IBA+100μmol/L AS acetosyringone for 3d. After co-culture, the infected leaves were transferred into selection medium, which contained MS+1.5 mg/L TDZ+0.4 mg/L IBA+20 mg/L Km+250 mg/L Carb. After the adventitious buds were induced, the induced buds were propagated in the medium of 1/2MS+0.2 mg/L IBA+5mg/L Km+100mg/L Carb.3. Based on the optimized genetic transformation system of strawberry, we obtained six resistant plantlets against Km. After GUS, PCR, Southern blot and RT-PCR detection, three of them were found transgenic, who could biosynthesize more endogenous ALA than the wild type. And endogenous ALA levels of transgenic strawberry were 2 times more than that of type plants.4. To study effect of the Yheml gene transformation on heat tolerance of strawberry, the chlorophyll fluorescence characteristics were compared between the wild type and the transgenic plants. The results showed that 45℃high temperature for 30 min significantly depressed fluorescence yield of strawberry leaves, which could recovered after normal temperature (28℃) for 12 h. The fluorescence yield was higher in transgenic leaves than the wild type, under whether normal or high temperature condition. OJIP test showed that the leaf photosynthetic performance index (PIABS), the probability of a trapped exciton moves an electron into the electron transport chain beyond QA-(Ψo) and electron transport (ETo/CS) in transgenic strawberry were significantly increased compared with the wild type, while the amplitude of the K step (Wk) and the approximate initial slope of the fluorescence transient (Mo) were decreased compared with the wild type, which suggested that Yheml transformation could increase the ability of strawberry against heat stress. |
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