| The two ways that are living induction and in vitro induction were used to create autotetraploid broccoli whose ploid were identificated. The main agronomic characters and nutritional qualities were analyzed whose materials were the diploid and tetraploid of the broccoli breed’HuLv’. The article explored the best way to induce autotetraploid which could provide theoretical basis for the genetic breeding of autotetraploid, at the same time, this could make genetic resources center more abundant.1. In this study, the effects of colchicine on the broccoli had been conducted. The stem tips of cotyledon-stage seedlings of broccoli were treated with colchicine. Different colchicine concentration and various treated times were tested in order to induce polyploid, and morphological trait, stoma, chromosome number and flow cytometry techniques were made full use of ploid identification. The results show that:(1) The treatment with 0.1% colchicine for four times was the most effective. In this treatment, the survival rate of plants was 100%, while the induction rate of tetraploid was 5.63%; (2) Compared with diploid, tetraploid leaves revealed bigger and rounder features; floral organ was observed and comparison which exhibited augment in different degrees, the size of buds and petal、pistil length and the size of sepal increased by 8.56%× 17.00%,27.59%× 25.30%,14.94% and 37.69%X 10.19%, which had significant difference; the size of siliqueN beak length and seed diameter increased by 31.63%× 21.79%,24.57%and 33.53%in the pod bearing period, which performed significant difference except silique width and seak length; (3) The stoma of tetraploid had significant difference in size compared with diploid, that of tetraploid increased by 41.18% and 54.35%, while the stoma density of tetraploid lowered 21.98%; (4) Ploid was identified by the flow cytometry techniques, which found that the raletive DNA content of was 400 in tetraploid while that of diploid was 200. The number of chromosome analysis suggested that the chromosome number of tetraploid was 2n=4x=36.2. In this study, agronomic characters and nutrient qualities of diploid and tetraploid broccoli have been conducted. The result found that:(1) Tetraploid agronomic characters including plant height, developing degree, the size of leaf, leaf number and branch number increased in different degrees compared with diploid, plant height and the biggest leaf stalk length of tetraploid had extremely significant difference, meanwhile spear thickness and leaf number of tetraploid had significant difference; (2) Tetraploid flower head showed gigantism, and flower head diameter increase by 9.16%, which had significant difference, while single head weight increased by 20.88%; (3) Soluble protein, oluble sugar, nitrate and chlorophy in the tetraploid broccoli increased, however, dry matter content declined; (4) The content of glucosinolate and sulforaphen was the most in flower head stem than others, which was more abundant in flower bud than leaf; compared with diploid, the content of glucosinolate in tetraploid increased by 20.12%,14.20%and 56.65%, which had significant difference, while the content of sulforaphen increased by 48.91%、82.32% and 27.50%.3. The effects of the double-deck induction with different concentration of colchicine and treatment time duration in three species of broccoli have been conducted in this study, thereby in vitro double-deck induction system of broccoli was established, and these results contribute to provide a new way for the ploidy breeding, meanwhile it is also significant to increase the diversity of germplasm resources. In this study, diploidy cotyledon keeping 0.4-0.6 cm hypocoty in the cotyledon stage of the broccoli were treated as explants. After explants were pre-cultured two days, it would be transferred to the double-deck media which combined liquid medium including different colchicine concentrations with solid media without colchicines to induce autotetraploids. During the process, the colchicine concentration was 100,300 and 450 mg/L,at the same time, treatment time duration was 24 h,48 h and 96 h. The results showed that:(1) Treating with 300 mg/L colchicines for 48 h could get the best results, explants from LQ-2, S139-2 and LQ-23 had high survival rate, which was achieved to 100%, and coefficient propagation were 2,2.33 and 2.67, meanwhile the Rate of induced tetraploids could reach 66.67%,71.42%and 62.5%; (2) Compared with the diploid, tetraploid leaves exhibited larger, rounder and thicker. The size of tetraploid leaves for LQ-2, LQ-23 and S139-2 was 43.33 ×23.83 cm2,40.40× 25.17 cm2 and 39.13×22.33 cm2.Plant height, Ball-flower diameter and Single head weight of tetraploid increased in various degrees, Plant height, Ball-flower diameter and Single head weight of tetraploid for LQ-2 had highly significant differences, which increased by 12.42%,9.59% and 5.75%. Stoma size and density has a very significant difference. The size of tetraploid pollen grain for LQ-2、S139-2 and LQ-23 were 21.0 12 μm2、20.0 × 10.3 μm2 and 20.3 × 11.3 μm2, which increased by 18.85%×63.71%,25%×34.68% and 3.36%×61.86%; (3) The number of chromosome was 2n=4x=36. Analysis of the ploidy using flow cytometry indicated that the relative DNA content of diploidy was 300 while that of the tetraploid was 600. Based on a large number of experiments, the research found that the optimal colchicine concentration and treatment time duration for broccoli in the double-deck induction and the double-deck induction could reduce toxicity of colchicines, which made death rate lower and ensured inductivity. |
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