| Several pear scion cultivars 'Dangshansu','Bayuehong', 'Starkrimson', 'Zaosu'and 'Suisho' were chosen as materials. Some factors that influence adventitious bud regeneration from leave, petiole, and internode segment, and Agrobacterium tumefaciens-mediated genetic transformation of pear were examined. The regeneration system of different explants and genetic transformation of pear were developed. 'Dangshansu', and 'Bayuehong' were transformed with P-l,3-glucanase and chitinase genes, and transgenic plants were obtained. Transformation with p-l,3-glucanase and chitinase genes were confirmed by PCR and Southern blots analyses.The main results were as fellows.1. Adventitious buds were regenerated from leave of five pear cultivars in vitro. It was for the first time that we obtained adventitious buds regeneration from leave of 'Dangshansu','Bayuehong', and 'Zaosu' pear cultivars. The efficient adventitious bud regeneration system were developed for 'Bayuehong', 'Suisho', and 'Dangshansu' pear cultivars.Effect of some factors on adventitious bud regeneration from leave were studied systemically, and efficient adventitious bud regeneration system were set up. Under optimal conditions, the regeneration frequency of 'Bayuehong', 'Suisho' , and 'Dangshansu' were 100%, 77.8%, and 83.3%, with the mean number of bud per leave were 5.78, 3.17, and 1.58, respectively.The genotype was a key factor on bud regeneration from leave of pear. The regeneration efficiency of five cultivars were different, and the regeneration capacities of 'Bayuehong' was more higher than others. The basal medium has an important effect on bud regeneration from leave of pear. NN69 medium is the suitable basal medium for bud regeneration from leave of pear. Some factors which significantly enhanced the regeneration frequency of pear consist of using young leave from upper part of shoots for explant, plating leave with abaxial downside on medium, adding up sucrose 40 g·L-1 , adjusting pH value of medium for 5.6, and dark period of 20 days at the beginning of cuture. Type and concentration of hormone was also a key factor on bud regeneration from leave of pear. The type and concentration of hormone were different for different pear cultivars. TDZ was suitable for bud regeneration from leave of 'Bayuehong'and 'Suisho', 6-BA was suitable for bud regeneration from leave of 'Dangshansu', and 'Starkrimson', but KT and ZT had no effect on bud regeneration from leave of pear. The type of auxins may significantly affect the regeneration frequency of pear. 1BA wassuitable for bud regeneration from leave of 'Bayuehong';IAA was suitable for bud regeneration from leave of 'Suisho'. Adding up AgNC>3 0.5 mg-L"1 in medium significantly enhanced the regeneration frequency of 'Bayuehong'.The basal medium type and 6-BA concentration were main factors on proliferation of regenerated bud from leave of'Dangshansu'pear, and the suitable shoot proliferation medium was MS + 6-BA 1.0 mg-L"1+ IBA 0.2 mg-L'1.The basal medium, IBA, NAA, sucrose, and the dark culture periods were used to compared their effects on rooting of the regenerated plants from leave of 'Bayuehong' and 'Dangshansu' pear by using the orthogonal experiment design. Results showed that the best combination for 'Dangshansu' pear was 1/4MS + IBA0.5 mg-L1 + sucrose 20 g-L"1, and dark cultured for 5d;for 'Bayuehong' was 1/4MS + IBA0.3 mg-L'1 + sucrose 30 g-L"1, and dark cultured for 7d.2. It is difficult that adventitious bud is regenerated from petiole of pear, but adventitious bud was regenerated from petiole of'Bayuehong'and 'Suisho' for the first time.The hormone had an important effect on bud regeneration from petiole of pear. TDZ was suitable for bud regeneration from petiole of pear, ZT had no effect on bud regeneration from petiole of pear. The combination of TDZ and IBA, IAA, or NAA greatly affected bud regeneration from petiole of pear. AgNO3 (0.1 1.5 mg-L"1) enhanced the regeneration frequency of petiole of pear. On the medium NN69+TDZ 1.0 mg-L"'+IBA 0.5 mg-L"1, the regeneration frequency and mean number of bud per petiole of 'Bayuehong' were 45.5% and 0.79, respectively. The regeneration frequency and mean number of bud per petiole of 'Bayuehong' were 42.4% and 1.0 on the medium NN69+TDZ 1.0 mg-L"'+IAA0.5 mg-L"', respectively.3. About regeneration of Intemode segment is not reported at present. For the first time, adventitious bud was regenerated from intemode segment of'Bayuehong' and 'Dangshansu'.Adventitious bud was regenerated from intemode segment of'Bayuehong' and 'Dangshansu'The regenerated shoot from intemode segment of 'Dangshansu'had rooted on rooting medium. The regenerated bud was formed from callus. The regeneration frequency and mean number of bud per intemode segment of 'Dangshansu' were 53.3% and 0.80 on the medium NN69+6-BA 5.0 mg-L"'+IBA 0.3 mg-L'1, respectively. The regeneration frequency and mean number of bud per intemode segment of 'Bayuehong' were 66.7% and 1.33 on the medium NN69+6-BA 10.0 mg-L'+IBA 0.3 mg-L"1, respectively.4. Studying effect of different antibiotic on the regeneration of pear leave, ensured antibiotic type and selecting pressure.On the basis of the regeneration system of pear leave, effect of Km, Nm, and Cef on the regeneration of pear leave were studied. The results showed , Km was not suitable for select reagent ofleave select culture, 40 mgL1 of Nm and 300 mgL1 of Cef were used as the concentration of effective for selection of bud and inhibition Agrobacterium growth.5. Establishing the genetic transformation system and transferring P-l,3-glucanase and chitinase genes into pear. For the first time, the transgenic plant of 'Bayuehong' and 'Dangshansu' were obtained.In this experiments, leave of 'Dangshansu','Bayuehong', and 'Starkrimson' pear were used as material, bud induction rate was treated as assessing index, some factors which influence Agrobacterium transfering pear including time of infection , Agrobacterium concentration, time of co-culture, medium type of co-culture, and selecting way were examined and an efficient gene transformation system for pear was established. The contents of this transformation system are as follows: using young leave, adding up AS 0.1 mmol ? L1 in Agrobacterium , leave should be infected 10 minutes in Agrobacterium with 0.30.50D6oo> co-cultivated 3days on solid medium in dark conditions, delayed to select 3days on leave regeneration medium adding up Cef 300 mg-L'1, then leave was selected 14days on leave regeneration medium adding up Cef 300 mgL"1 and Nm 40 mgL"1 in dark conditions, and was transferred to light conditions.The induction adventitious bud was transferred to subculture medium adding up Km 10 mgL'1 and Cef 300 mgL"', the non-transformed shoot were removed, the selected resistant shoots were proliferated and rooted.PCR analysis of 36 resistant shoots, 11 resistant shoots showed the same positive band as P-l,3-glucanase and chitinase genes. Together with subsequent southern-dot blot analysis of PCR positive shoots indicated the integration of 0-1,3-glucanase and chitinase genes into the genome of pear. The transgenic plant of 'Bayuehong' and 'Dangshansu' were obtained.For the first time, internode segment of pear was chosen as materials for genetic transformation. We also obtained resistant shoots from internode segment of 'Dangshansu' and 'Bayuehong' by Agrobacterium tumefaciens transformation. The resistant shoots are proliferated on subculture medium...
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