羽衣甘蓝游离小孢子培养与再生体系构建

发布时间：2018-08-27 12:33

【摘要】：羽衣甘蓝(Brassica oleraceavar.acephala)含有丰富的维生素、矿物元素及一些生物活性成份,耐寒性极强,因而在严寒的冬季既可以作为蔬菜,又可以组装色彩艳丽的冬季花坛。羽衣甘蓝的繁殖方式主要为种子繁殖,是有杂种优势较为显著的作物之一,所以得到纯系更加重要。游离小孢子技术是获得纯系自交系的快速途径之一,因此,提高游离小孢子出胚率与提高胚状体直接再生率,能缩短羽衣甘蓝纯合自交系的获得时间,提高育种效率。无性系能够保持品种的优良性状,羽衣甘蓝离体再生体系的构建是扩大繁殖游离小孢子单株的有效方法,本研究以将游离小孢子培养后得到的纯合优良育种品系为试材,建立高效稳定的再生体系,获得大量的纯合无性系。本试验试材以19个羽衣甘蓝为材料,分析了在游离小孢子培养中影响胚胎发生5个因素,分别为:基因型、发育时期、蔗糖浓度、低温预处理、高温热激。探讨了胚状体直接再生成苗的影响,如何获得健壮植株,以及探究对芽诱导的因素,并建立了高效的羽衣甘蓝小孢子植株再生体系。获得的主要研究结果有:1.在羽衣甘蓝小孢子诱导的过程中,19份材料中有12份材料能够获得胚状体。其中产胚率最高为‘红孔雀',最低为‘6BZ',其中‘42红'、‘42白'、‘食用'、‘DHZ,、‘DFZ'、‘黑皱-粉'、‘D10×6BZ' 7种基因型经多次反复培养实验均未获得胚状体。因此本研究认为基因型是影响羽衣甘蓝小孢子胚胎发生的主要因素。2.花蕾大小可以反映小孢子发育进程,但不同基因型材料,小孢子发育时期存在很大的差异。当羽衣甘蓝花蕾长度在2.51～4.00mm时游离小孢子一般处在单核早期到单核晚、双核早期阶段,此时的游离小孢子具有较高的胚诱导率。3.不同基因型的羽衣甘蓝游离小孢子对低温处理的时间有不同的要求,总体上,各基因型在小孢子培养前将花蕾放置4℃低温预处理24～48h对胚产量均有提高,时间过长会抑制小孢子的诱导率。高温热激在33℃下热激24～48h对羽衣甘蓝小孢子胚状体的产胚率有一定促进作用。不同基因型对适宜的蔗糖浓度的需求量不同,总体上讲,适宜小孢子培养的蔗糖浓度为130g·L~（-1）和160g·L~(-1）。4.对子叶型胚状体诱导前,在4℃低温下预处理4天后转接到固体培养基,更有利于羽衣甘蓝胚状体直接再生植株。与其他发育时期的胚状体相比,子叶型胚状体诱导成功率较高,MS为最适宜子叶胚状体直接再生成苗的基本培养基,此时更有容易直接再生成苗。最适宜羽衣甘蓝胚状体再生的激素浓度配比为0.2mg L~（-1）6-BA+O.1 mg·L~（-1）NAA。在1/2MS培养基中添加适宜浓度的NAA对小孢子植株的长势,及其生根都有显著的促进作用。NAA浓度为0.05mg·L~（-1）和O.1mg·L~（-1）时有利于快速、高效的培养出健壮的小孢子植株。5.在对芽诱导再生的过程中,腋芽是理想的再生外植体材料,在培养基中添加NAA的基础上加入一定浓度的6-BA有助再生芽的健壮生长,激素配比为MS+1.Omg·L~（-1）6-BA+0.05mg·L~(-1) NAA时最适宜诱导腋芽再生。6.在培养基内添加适宜浓度的6-BA有助于再生芽的继代培养,当6-BA浓度为2.0mg·L~(-1)时继代增殖效果达到最佳。当NAA浓度为0.2mg·L~（-1）时,芽生长势最好,叶色油绿,叶片面积最大,茎粗壮,生长旺盛,为最佳壮苗培养基。7.根系长度为0.5～1.5cm最适合再生植株的生根移栽,后期小苗死亡率较低,植株生长状态最好。
[Abstract]:Brassica oleracea var. acephala is rich in vitamins, mineral elements and some biological active ingredients. It has strong cold resistance. Therefore, it can be used as a vegetable in cold winter and can assemble colorful winter flower beds. Free microspore technology is one of the fast ways to obtain pure inbred lines. Therefore, increasing the rate of free microspore embryogenesis and the rate of direct embryoid regeneration can shorten the time of obtaining homozygous inbred lines and improve the breeding efficiency. The construction of in vitro regeneration system of cabbage is an effective method for expanding the propagation of isolated microspore single plant. In this study, a high efficient and stable regeneration system was established and a large number of homozygous clones were obtained by using the excellent homozygous breeding strains obtained after free microspore culture. Five factors affecting embryogenesis in spore culture were genotype, developmental stage, sucrose concentration, low temperature pretreatment and high temperature heat shock. The effects of direct embryoid regeneration on seedling formation, how to obtain robust plants, and the factors affecting bud induction were discussed. The main results are as follows: 1. In the process of microspore induction, 12 of 19 materials were able to obtain embryoids. Among them, the highest embryogenic rate was'Red Peacock', and the lowest was'6BZ', among which'42 Red','42 White','edible','DHZ,'DFZ','black wrinkle-powder', and'D10 x 6BZ'seven genotypes were not obtained after repeated culture experiments. 2. Bud size can reflect the microspore development process, but the microspore development stages of different genotype materials are very different. When the bud length of kale is 2.51-4.00 mm, the free microspore is generally in the early stage of monocyte. The free microspore of different genotypes of Brassica oleracea had different requirements on the time of low temperature treatment. Generally speaking, the embryo yield of each genotype was increased when the bud was pretreated at low temperature for 24-48 hours before microspore culture. Heat shock at 33 C for 24 to 48 h promoted the embryogenesis of microspore embryoids in Brassica oleracea L. Different genotypes had different requirements for suitable sucrose concentration. Generally speaking, the sucrose concentration suitable for microspore culture was 130 g L ~(- 1) and 160 G (- 1). 4. Before induction, the cotyledon embryoids could be induced more successfully than other embryoids. MS was the most suitable medium for cotyledon embryoids to regenerate seedlings directly, and it was easier to regenerate seedlings directly. The best ratio of hormone concentration for embryoid regeneration of Brassica oleracea L. ~(-1) 6-BA + O.1 mg (-1) NAA was 0.2 mg L (-1) NAA. Adding appropriate concentration of NAA to 1/2 MS medium could significantly promote the growth and rooting of microspore plants. In the process of bud induction and regeneration, axillary bud is an ideal regeneration explant material. Adding a certain concentration of 6-BA on the basis of adding NAA in the medium can promote the healthy growth of regeneration bud. The optimum ratio of hormone is MS + 1.Omg (-1) 6-BA + 0.05mg (-1) NAA. 6. Adding suitable concentration of NAA in the medium is the best way to induce axillary bud regeneration. When the concentration of NAA was 0.2 mg (-1), the bud growth vigor was the best, the leaf color was oily green, the leaf area was the largest, the stem was strong, and the growth was vigorous. 7. Root length was 0.5-1.5 cm, which was the best medium for the regeneration of strong seedlings. In the later stage, the seedling mortality rate was low, and the plant growth condition was the best.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S681.9

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