Transformation Of Betaine-aldehyde Dehydrogenase Gene Into Stocks Of Apple Varieties

Apple is one of the most important fruit in the world.The efficiency oftraditional apple breeding is low because of long juvenility and complexgenetic backgroud.With the development of biotechnology, we can crone geneand transfer gene into plants in order to create transgenic plants. This make thegreat development for plant breeding.In this experiment,we have established the perfect regeneration system forluo 6 and zhumei and studied the inside and outside factors that affect theregeneration of leaves in vitro. We have found the combination that canproduces the best regeneration and get the high frequency.This regenerationsystem makes the best base for transformation. Through studying differentfactors that affect Genetic transformation mediated by Agrobactium,weoptimize the condition and find the best combination. Last we get thetransgenic plants.The main results are as following:1. Establish the perfect regeneration system(1)The effect of hormone on regeneration:Different cytokinin and auxins makeup different combination so that we can study the effect of hormone onregeneration.In this experiment we have found that luo 6 gets 85%when theconcentration of BA is 6.0 mg/L and NAA is 0.2 mg/L.ZhuMei gets 90% whenthe concentration of BA is 3.0 mg/L and NAA is 0.3 mg/L. Cytokinin isnecessary to the regeneration.With the concentration of BA rising,regenerationfrequency rise obviously when the concentration of BA is among 0~6.0mg/L.But the frequency will decline and plants can not grow well when theconcentration of BA is beyond 6.0mg/L.As far as ZhuMei is concerned, thebest combination is that the concentration of BA is 3.0 mg/L and NAA is 0.3.With the concentration of BA rising, regeneration frequency will rise andsubsequently decline. The extent of change is nallow and I think this maybehave relation with genotype.(2) The effect of dark on regeneration: Through study the effect of differenttime of dark treatment on regeneration, we have found that dark treatment isnecessary to regeneration and best time is 2 weeks. If dark time is too 4山东农业大学硕士学位论文long,buds can not strongly grow. If dark time is too short, leaves can hardlyregenerate buds.(3)The effect of operation mode on regeneration:In this experiment incisioncan obviously promote callus and the mode of placing also affectsregeneration.Results is that the effect of up-placing is better than that of down-facing.(4)The effect of medium on regeneration:Studying the effect of MS,N6,B5medium on regeneration,we have found that MS medium has the best results.(5) he effect of genotype on regeneration: Leaves with different genotype needdifferent combination. In this experiment we have found that luo 6 gets85%when the concentration of BA is 6.0 mg/L and NAA is 0.2 mg/L.ZhuMeigets 90% when the concentration of BA is 3.0 mg/L and NAA is 0.3 mg/L.Under the same conditions,they have different results:ZhuMei has betterresults than luo 6.(6)The effect of size and texture of leaves on regeneration:Under the sameconditions,different kinds of materials and physiology have different results.Moderate size of leaves have the better effect than others.2. Genic transformation mediated by Agrobactium(1)Sensitivity of plants to antibiotics: Apple is very sensitive to Km. If theconcentration of BA is beyond 5mg/L,the grow of buds will be restricted.Weuse two-step selection in the following experiment: During differentiation,theconcentration of Km is 5mg/L.When the height of is during 2~3cm,Km is40mg/L.results indicatethat the concentration of Cef is 300mg/L.(2)The effect of time of immersion by Agrobacterium on transformation:whenthe time of immersion by Agrobacterium is 10 minutes,the frequency oftransformation is the highest.(3)The effect of AS on transformation:AS can obviously promotetransformation in the experiment and the appropriate concentration is 10mg/L.(4)The effect of different modes of immersion on transformat...