Establishing Regeneration Systems And Studies On Agrobacterium-mediated Transformation Of Bt In Hawthorn

Hawthorn is one of the most important fruits in the world, there is abundance of nutrient substance inits fruit, and it was a kind of tonic as food and medicine. Hawthorn also has been used as tree forlandscaping since its trim crown and flourishing flowers and leaves. Pests cause heavy losses in hawthornproductions. Most of pests of hawthorn are Lepidoptera insects, among of them contain budworm andcarpenter moth. Some good varieties are close to extinct since the seriously damage by pests, andretrogression. They not only infest fruit but also bite leaves and stem.Seldom sources of resistance have been found, which limited the progress of regular breeding oninsect resistance. With plant gene transformation technique has been used widely, so it appears promisingthat breeding new fruit cultivar with good traits by biotechnology. The tissue culture and genetictransformation techniques were used for hawthorn in this study. The culture systems have been established.Shoots were regenerated from the young leaves of 'liao-hong' hawthorn. Based on regeneration system, theconditions of Agrobacterium-mediated transformation of Bt gene were determined and the transgenic plantswere obtained. The results were summarized as follows:1 Acquirement of plant Tissues CulturePollution rate, browning rate and death rate was comparated at selection of explants, single bud is abetter explant than stem segment with bud. Single bud was sterilized for 7 min in 0.1% HgCl₂ with lowercontamination and higher rate of germination. Base on grown condition of explant on different media MS issuitable culture medium for the hawthorn growth in vitro. Studies on Concentration of hormone aboutelongation and multiplication from plantlet by orthogonal experiments, Suitable bud multiplicationmedium:MS+BA2.0+NAA0.02and subculture medium: MS+BA1.0mg/L+GA₃0.3 mg/L+NAA0.02 mg/Lwere determinated.2 Rooting of 'liao-hong' hawthorn in VitroRooting of plantlet in vitro was studied in medium and hormone the result shows: 1/2MS is superior to1/4MS and 1/8MS, IBA is superior to NAA, the combination of NAA and IBA was better than only one ofthem, and the best sucrose concentration was 20%. Suitable rooting-culture medium: 1/2MS+IBA0.6mg/L+NAA0.2 mg/L+20g/L sucrose+7g/Lagar.3 Establishment of Regeneration System of 'liao-hong' hawthorn leaf3.1 Research on Callus Induction.Callus induction was affected by the days of darkness, genotype, hormones, media, on leaves. There were significant morphological from difference genotype callus. Among the three cultivars, 'liao-hong'showed the relatively higher regeneration capability, 'qiu-jinxing' lower, and no response for 'da-wang'.Dark treatment was necessary for callus induction of 'liao-ning' in the experiment, there were significantmorphological difference between 5～9d and 15～20d by the clays of darkness, 7d darkness is best. Calluswas induced on MS with BA and NAA or TDZ and NAA. The callus from medium with BA and NAA werehard, dark-green, and most of callus became brown and died after 20d. Callus from medium with TDZ andNAA were light-green and without browning.MS medium is more suitable for callus induction of'liao-hong' leave sthan WPM. The suitable medium was seclected for callus induction:MS+TDZ1.0mg/L+NAA0.05 mg/L.3.2 Bud regeneration system from callus.Studies on bud regeneration from two kind of callus (MS and WPM), the suitable medium was seclectedfor bud regeneration WPM+BA2.0mg/L+KT0.5mg/L+NAA0.02 mg/L. The effect of Cef on callusinduction and shoot regeneration of 'liao-hong' hawthorn and the effect of Kan on callus induction androoting in vitro were investigated. The optimal concentration of Cef was 500 mg/L, Kan were 5mg/L forcallus and 40mg/L for rooting.4 Agrobacterium-mediated gene transformate in hawthornAbout 100 pieces of callus of 'liao-hong' were infected by Agrobacterium tumefaciens, Agrobacteriumconcertation: OD₆₀₀=0.6; Infection time: 7min, but the shoots with resistance have not been obtained yet aslow regeneration rate.