| Dwarf and compact culture have become a general tendency of fruit tree, especially in apple production development in the world , because which have some characters with early fruit,high yield. At the present,in apple production, primary approach of making tree dwarf was obtained by selective breeding of dwarfing cultivars and dwarfing rootstocks,breeding dwarfing rootstocks is the best method. GM256 dwarfing rootstocks have many traits ,such as dwarfing ,cold resistant,drought resistant,salination resistant. Because it is very difficult to root ,so it was used as the intermediate rootstocks,the trait of drought resistant,salination resistant was lost. Genetic transformation technology applied a modern tool for improving apple intermediate rootstocks,. which will have a high potencial to get a new rootstocks cultivar of cold resistance, dwarfing, drought resistant,salination resistant and being apt to root.Because apple tissue regeneration is very difficult ,it restrict apple genetic improvement In this experiment ,tissue culture system of apple rootstocks M9 and GM256 was first studied , genetic transformation system was optimized , for establishing genetic transformation system of Gm256 and M9.The main results are as shown:1. Establishment of a stable and efficient axillary bud proliferation systemIn this experiment, firstly, aseptic system was established and the condition which affect the incipience culture was optimized. Before new shoot growth, it was found that the best time of bud inoculation is the lateral buds have four or five extended leaves; the period of the new stem increase quickly(4-5 month) is the best inoculation period; Several methods have been adapted to prevent from browning ,such as 0.1% AC was added into incipience media, and the explant was cultured in dark with continuous transfer to new medium , these methods effective prevented browning which often exists in woody plant tissue culture.It was found that GM256 growing point straight growth is very difficult. M9 in the media of BN2 (MS+BA0.5mg/L+NAA0.05mg/L ) can come into being the plant which have obvious stipital. The stem having one bud or two buds was used as the explant in the orthogonal experiment. The medium of FH8 (MS+BA5.0mg/L+NAA0.02mg/L) is the best differentiation medium. Stems were cultured for seven days in FH8 medium and weretransfered to the medium without hormone so that it promoted the bud growth. The highest rate of differentiation is 98%, the times of proliferation is 5.1. Big shoot were cut and the rest part were cultured in medium. There was a lot of buds differentiated from the rest part, the times of proliferation was gone up, which was proved to be a very good proliferation method. 2. Regeneration system establishment(1) Effect of hormone on regeneration: Cytokinin is necessary to the regeneration, it is also an important factor which affect regeneration frequency. Different combination of cytokinin and auxins were made so that we can study the effect of hormone on regeneration. It was found that the combination of BA and NAA had no effect on leaf regeneration and leaves in the media generate more callus. In the medium of KT and NAA combination, leaves could not regenerate but got enlarged, fragile with no callus.Leaves in the media of BK3(MS+BA1 .Omg/L+KTl .5mg/L+NAA0.8mg/L+IBA0.5mg/L) have high regeneration rate of 16.7%. TDZ is good for apple leaves regeneration. In the fitting medium of MT3 (MS+TDZ3.0mg/L+NAA0.5mg/L), leaves acquire high regeneration frequency of 37.6%. GM256 formed no adventitious bud in the media. We presumed it might be related with its genotype. In the medium, adventitious bud straight growth was difficult and the morpha of leaves had a little change, when it was transfered into BA medium, the adventitious buds can recovery.(2) Effect of sugar source on regeneration: Sugar sourse in tissue culture are both carbon source and energy resource, it is also the important osmotic pressure coordination. Sorbitol and sucrose were used in this experiment. The suitable concentration of sorbitol and sucrose was 30g/L. At the concentration, regeneration frequency is not different to the leaves in the medium.Leaves in the medium with sorbitol can be preserved for a long time. Number of per-leaves regenerating buds is big. But the bud from the medium was little, the speed of increase is slow. It is concluded that sorbitol is good for the adventitious shoot induction, sucrose is good for the adventitious shoot growth.(3) Others factors on regeneration: When study the effect of operation mode on leaves regeneration, results indicated that up-placing and down-facing have no dissimilarity, adventitious shoot is often issue from incision of leaf vein and the bottom of stalk, up-placing is good for the shoot growing;The incision fashion also has effect on regeneration,the best way is cutting three times by the scalpel on vertical vein, leaves were cut three parts did not increase regeneration frequency , leaves generated more callus, leaves from adventitious budswhich regenerate from shoot in vitro had high frequency of regeneration. 3. Acquire rooting transgenic plant(1) Antibiotics sensitivity experiment : It was found that the leaves was sensitive to Kan, the concentration of 5mg/L inhibit the regeneration of leaf; The medium containing 40mg/L of Kan inhibited the shoots of 2-3 cm in length growing in vitro.Ths total plant become yellow and the tip of plant got withered.(2) The no-root plants of 2~3cm in length were cultured in the medium which have auxin IBA0.2mg/L for three days, the bottom of the plant were effaced Agrobacterium C58C1 which have rolB gene. Agrobacterium was drop on the surface of medium, the plants were inserted over there and were co-cultivated for three days, and the plants were transferred into the medium for removing Agrobacterium and continuous culture, the rate of rooting is 20%. But the rate of rooting is 5% with the plant did not were cultured in the medium of having auxin IBA. The roots of five rooting plants were used to extract DNA, two plants were proved to be rolB-transgenic root by PCR amplification.
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