| Gene engineer and other high technology breeding techniques were based on the foundation of high frequency of regeneration system.In this paper,base on the establishment high-frequency of regeneration system in broccoli,we use the colchicines induced tetraploid broccoli via tissue culture,as well as studied the frequency of shoot regeneration of autotetraploid broccoli.1 The high frequency of shoot regeneration in broccoli of cotyledons with petiole as explants was studied.The result showed that the 04J-20 was the highest frequency of 100% to the cotyledons with petiole,when the explants were pre-cultured for 8 days and then played on the MS medium supplemented with 6.0 mg·L-16-BA,0.1mg·L-1 NAA,0.8%Agar and 3%Sucrose.Frequent subcultures of existing shoots and shoot clusters to medium containing 0.2 mg·L-1NAA and 2.0 mg·L-1 6-BA promoted rapid shoot multiplication (proliferate about eight to nine times).The use of 0.1 mg·L-1 NAA in the rooting medium increased the number of healthy roots per rooted plant(root ratio was 100%).Rooted plantlets were transferred to soil-less mixture in pots.Applying this protocol,rooted plantlets proliferating 200 times were obtained.It was also studied on the effects of different concentration of plant hormones,seedling ages and genotypes on shoot regeneration.Leaf as explants were cultured on MS media with different combinations of growth regulator.The effects of growth regulator combinations on induction of shoots were evaluated.The optimal media for inducting shoots were MS media containing 0.8%Agar and 3%Sucrose supplemented with 4.0 mg·L-1 6-BA,0.1 mg·L-1NAA.The shoot induction percentage was 54.2%,and the number of buds per explants with shoots was 2.01.Then each shoot was transplanted on the MS medium containing 0.2 mg·L-1 NAA and 2.0 mg·L-1 6-BA proliferating 5 to 6 times.The use of 0.1 mg·L-1 NAA in the rooting medium increased the number of healthy roots per rooted plant(root ratio was 100%).Rooted plantlets were transferred to soil-less mixture in pots.Applying this protocol,we can obtain rooted plantlets.2 Variation of tetraploidy was observed among the regenerate when the cotyledons with petiole of broccoli were cultured on MS+0.01mg·L-1 NAA+9.0 mg·L-1 6-BA+0.7% Agar+3%Sucrose.The results showed that tetraploid plants exhibited significant differences in morphology of plantlets,leaves,stomata,pollens,shape and size of fruits compared with the diploid.Chromosome of autotetraploid in pollen grain cell was n=2x=18.In addition,the vitamin C,soluble protein and soluble sugar of tetraploid were more than the diploid.Tissue culture was a new and efficient way to obtain tetraploid broccoli.3 In this study,the tube plantlets of broccoli were treated with different concentration of colchicines and treatment time to introduce tetraploid via tissue culture.Tetraploid plants were selected and identified based on morphological,anatomic,agronomic,cytological observation and flow cytometry.The results showed that the treatment with 200mg·L-1colchicines for 48 hours yielded doubled chromosomes at highest rates,the number of shoots per explants was 2.1,the variation efficiency was 83.33%,and the rate of tetraploid was 79.17%.Compared with the diploid,the tetraploid plants possessed gigantism in leaves,stomata of plantlets.The flow cytometry was used to identify the ploidy of induced plants.The result suggested that the relative DNA content of CK was 200, and tetraploid relative DNA content was 400.The identified result of flow cytometry according to the chromosome analysis implied the regeneration plantlets were tetraploid.4 The frequency of shoot regeneration in autotetraploid broccoli was studied.Cotyledons with petiole as explants were cultured on MS media with different combinations of growth regulator.The effects of growth regulator combinations on induction of shoots were evaluated.The optimal media for inducting shoots were MS media containing 0.8%agar and 3%sucrose supplemented with 3.0 mg·L-1 6-BA,0.05 mg·L-1 NAA.The shoot induction percentage was 63.3%.Then each shoot was transplanted on the MS medium containing 0.04 mg·L-1 NAA and 4.0 mg·L-1 6-BA proliferating about 6 times.The use of 0.1 mg·L-1 NAA in the rooting medium increased the number of healthy roots per rooted plant(root ratio was 100%).In this study,we established high-efficient shoot regeneration system of autotetraploid broccoli and analyzed the ploidy stability of regenerates using flow cytometry.The result showed that the tetraploid relative DNA content was about two times of control.The identified result of flow cytometry according to the chromosome analysis implied the regeneration plantlets keep the original ploidy.
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