CN111528100B - Method for obtaining distant hybridization progeny of broccoli and cabbage type rape - Google Patents
Method for obtaining distant hybridization progeny of broccoli and cabbage type rape Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及生物育种技术领域,具体涉及一种获得青花菜与甘蓝型油菜远缘杂交后代的方法。The invention relates to the technical field of biological breeding, in particular to a method for obtaining distant hybrid progeny of broccoli and Brassica napus.
背景技术Background technique
青花菜,营养物质丰富,是甘蓝的一个变种,属于十字花科芸苔属异花授粉作物。其杂种优势十分明显,利用杂种优势不仅能提高产量,还能提高抗病性,优化种质资源。Ogura细胞质雄性不育(Ogura CMS)的不育性稳定,转育容易,是十字花科作物杂种优势利用的主要途径之一,近十年来也越来越多地用于花菜杂交种种子的生产。Ogura细胞质雄性不育是母性遗传,所有后代均是雄性不育,无法自交分离,不利于种质资源的创新、利用。解决这一问题的有效方法是挖掘或创制Ogura CMS恢复系。然而,迄今为止,尚未发现在甘蓝类蔬菜中存在Ogura CMS恢复系。Broccoli, rich in nutrients, is a variant of cabbage that belongs to the Brassica family of cruciferous cross-pollinated crops. Its heterosis is very obvious. The use of heterosis can not only improve yield, but also improve disease resistance and optimize germplasm resources. Ogura cytoplasmic male sterility (Ogura CMS) has stable sterility and easy transfer. It is one of the main ways to utilize the heterosis of cruciferous crops, and it has been increasingly used in the production of cauliflower hybrid seeds in the past decade. . Ogura cytoplasmic male sterility is a maternal inheritance, all offspring are male sterile and cannot be self-separated, which is not conducive to the innovation and utilization of germplasm resources. An effective way to solve this problem is to dig or create the Ogura CMS restorer system. However, to date, no Ogura CMS restorer line has been found in cabbage vegetables.
甘蓝型油菜作为一种重要的油料作物,含有丰富的抗病、虫、草害以及育性基因,且Ogura CMS恢复系已经成功创制并用于杂交种生产。为了拓宽花菜种质资源的遗传基础,创造新的优异种质,利用芸薹属种间关系研究,通过远缘杂交将甘蓝型油菜的育性恢复基因、抗逆基因导入花菜中,从而拓宽花菜的遗传育种基因库,提高花菜的抗性,越发显得重要。Brassica napus, as an important oil crop, is rich in disease resistance, pest resistance, weed resistance and fertility genes, and the Ogura CMS restorer line has been successfully created and used for hybrid production. In order to broaden the genetic basis of cauliflower germplasm resources and create new excellent germplasms, the research on the relationship between Brassica species was used to introduce the fertility restoration gene and stress resistance gene of Brassica napus into cauliflower through distant hybridization, so as to broaden the cauliflower. It is more and more important to improve the resistance of cauliflower by developing the genetic and breeding gene pool.
然而,远缘杂交由于存在生殖隔离,容易在授粉后出现杂交不亲、胚败育以及后代遗传变异复杂等问题。目前虽有少量甘蓝不同变种与甘蓝型油菜种间杂种成,几乎没有利用胚挽救离体培养技术实现花菜与甘蓝型油菜种间杂交并获得后代的植株的案例,并且培养周期长、杂种获得率较低,大大限制了杂种优势的利用。However, due to the existence of reproductive isolation, distant hybridization is prone to problems such as hybrid incompatibility, embryo abortion and complex genetic variation of offspring after pollination. Although there are a small number of interspecific hybrids between different varieties of cabbage and Brassica napus, there are almost no cases of interspecific hybridization between Brassica oleracea and Brassica napus using embryo rescue in vitro culture technology to obtain offspring plants, and the culture period is long and the rate of hybrid acquisition lower, which greatly limits the utilization of heterosis.
发明内容SUMMARY OF THE INVENTION
本发明提供一种获得青花菜与甘蓝型油菜远缘杂交后代的方法以解决上述技术问题。The present invention provides a method for obtaining distant hybrid progeny of broccoli and Brassica napus to solve the above-mentioned technical problems.
本发明所述的一种获得青花菜与甘蓝型油菜远缘杂交后代的方法,包括以下步骤:A method for obtaining the distant hybrid offspring of broccoli and Brassica napus according to the present invention, comprises the following steps:
(1)选择青花菜Ogura细胞质雄性不育系栽培种为母本,含有Ogura CMS恢复系杂交种甘蓝型油菜为父本,进行人工授粉;(1) selecting the broccoli Ogura cytoplasmic male sterile line cultivar as the female parent, and containing the Ogura CMS restorer line hybrid Brassica napus as the male parent, and artificially pollinated;
(2)在人工授粉10-15天后,将子房剪下,进行子房消毒,置于培养基中离体培养,所述培养基为MS基础培养基;(2) after 10-15 days of artificial pollination, the ovary is cut off, and the ovary is sterilized, and placed in a culture medium for in vitro culture, and the medium is MS basal medium;
(3)剥离步骤(2)中离体培养30~60天子房里萌发的胚珠,接种于分化培养基,培养至长出侧芽;(3) in the stripping step (2), the ovules germinated in the ovary are cultured in vitro for 30 to 60 days, inoculated in a differentiation medium, and cultured until lateral buds grow;
(4)剥离步骤(3)中的侧芽,接种于生根培养基20-30天,得无菌苗;(4) the lateral buds in the stripping step (3) are inoculated in the rooting medium for 20-30 days to obtain sterile seedlings;
(5)待步骤(4)中幼苗长至4~5片叶,生根后打开培养瓶的盖子练苗,洗去培养基,移栽至营养土中培养2周后,转入大棚,常规栽培管理。(5) wait for the seedling to grow to 4~5 leaves in step (4), after rooting, open the lid of the culture bottle to practice seedlings, wash off the culture medium, transplant it into the nutrient soil and cultivate for 2 weeks, then transfer to a greenhouse for conventional cultivation manage.
进一步地,步骤(2)中,所述培养基还包括30g/L蔗糖、6g/L琼脂糖,PH值5.8~6.02。Further, in step (2), the culture medium further includes 30 g/L sucrose, 6 g/L agarose, and has a pH value of 5.8-6.02.
进一步地,步骤(3)中,分化培养基为以MS为基础培养基,还包括30g/L蔗糖、0.1mg/L的NAA、1mg/L的6BA、6g/L琼脂糖,PH值5.8~6.0。Further, in step (3), the differentiation medium is MS-based medium, and also includes 30g/L sucrose, 0.1mg/L NAA, 1mg/L 6BA, 6g/L agarose, pH value 5.8~ 6.0.
进一步地,步骤(4)中,生根培养基以MS为基础培养基,还包括10g/L蔗糖、9g/L琼脂糖,PH值5.8~6.0。Further, in step (4), the rooting medium uses MS as the basal medium, also includes 10 g/L sucrose, 9 g/L agarose, and has a pH value of 5.8-6.0.
进一步地,步骤(1)中,选取的母本为细胞质雄性不育系B14和137,父本为Rfo-1。Further, in step (1), the selected female parent is cytoplasmic male sterile lines B14 and 137, and the male parent is Rfo-1.
进一步地,步骤(1)中,人工授粉的方法包括以下步骤:对母本进行剥蕾,分别在早上和晚上采取重复授粉,将父本新鲜花粉涂抹与母本柱头。Further, in step (1), the method for artificial pollination includes the following steps: peeling off the buds of the female parent, repeating pollination in the morning and evening respectively, and smearing the fresh pollen of the male parent on the stigma of the female parent.
进一步地,步骤(2)中子房消毒的方法包括以下步骤:75%酒精消毒30s,8%的NaClO消毒10min,无菌水冲洗3次,吸干水分。Further, the method for sterilizing the ovary in step (2) includes the following steps: sterilizing with 75% alcohol for 30 seconds, sterilizing with 8% NaClO for 10 minutes, rinsing with sterile water for 3 times, and absorbing water.
进一步地,步骤(5)中所述营养土为基质和蛭石混合物,基质和蛭石体积比为3∶1。Further, in step (5), the nutrient soil is a mixture of matrix and vermiculite, and the volume ratio of matrix and vermiculite is 3:1.
与现有技术相比,本发明具有以下有益效果:Compared with the prior art, the present invention has the following beneficial effects:
本发明提供了一种获得花菜与甘蓝型油菜远缘杂交后代的方法,通过蕾期人工重复授粉10~15天后,对子房进行组织培养的方法,克服远缘杂交种子形成过程中胚败育的问题,提高杂种获得率,进而将甘蓝型油菜中的恢复基因转到花菜中,恢复花菜的育性,提高花菜的种质资源利用效率;同时,本发明获得的异缘三倍体杂种AAC,分别来自于花菜和甘蓝型油菜,作为桥梁物种,有利于将甘蓝型油菜中的优良性状转移到花菜中,提高花菜的育种进程。The invention provides a method for obtaining the distant hybrid progeny of cauliflower and Brassica napus, which overcomes embryo abortion during the formation of distant hybrid seeds by performing tissue culture on the ovary after 10 to 15 days of artificial repeated pollination at the bud stage. The problem is to improve the hybrid acquisition rate, and then transfer the restorer gene in Brassica napus to the cauliflower, restore the fertility of the cauliflower, and improve the utilization efficiency of the germplasm resources of the cauliflower; at the same time, the allotriploid hybrid AAC obtained by the present invention , respectively from cauliflower and Brassica napus. As a bridge species, it is beneficial to transfer the excellent traits in Brassica napus to cauliflower and improve the breeding process of cauliflower.
附图说明Description of drawings
图1是本发明杂交授粉套袋隔离图;Fig. 1 is the isolation diagram of hybrid pollination bagging of the present invention;
图2是本发明实施例1子房组织培养图;Fig. 2 is the ovary tissue culture diagram of
图3是本发明实施例1组培成苗图;Fig. 3 is the embodiment of the
图4是本发明实施例1组培苗生根图;Fig. 4 is the rooting diagram of tissue culture seedlings in Example 1 of the present invention;
图5是本发明实施例1组培炼苗图;Fig. 5 is the embodiment of the
图6是本发明实施例1花菜-甘蓝型油菜亲本及获得的杂交植株的幼苗形态图;Fig. 6 is the seedling morphology diagram of the cauliflower-brassica napus parent and the obtained hybrid plant in Example 1 of the present invention;
图7是本发明实施例1花菜-甘蓝型油菜杂交获得的杂交植株的花期形态图;Fig. 7 is the morphological diagram of the flowering stage of the hybrid plant obtained by the hybridization of cauliflower-Brassica napus in Example 1 of the present invention;
图8是本发明实施例1亲本与杂交种代植株叶型对比图;Fig. 8 is the comparison diagram of leaf shape of parent and hybrid generation plant in Example 1 of the present invention;
图9是本发明实施例1亲本与杂交种代植株花形态对比图;Fig. 9 is the flower morphology comparison diagram of parent and hybrid generation plant in Example 1 of the present invention;
图10是本发明实施例1花菜-甘蓝型油菜杂交种的倍性分析(DNA相对含量)图;Fig. 10 is the ploidy analysis (DNA relative content) figure of the cauliflower-brassica napus hybrid in Example 1 of the present invention;
具体实施方式Detailed ways
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which the documents are referred. In the event of conflict with any incorporated document, the contents of this specification shall control.
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present invention are exemplary only.
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.
本发明提供一种获得青花菜与甘蓝型油菜远缘杂交后代的方法,包括以下步骤:The invention provides a method for obtaining the distant hybrid progeny of broccoli and Brassica napus, comprising the following steps:
(1)选择青花菜Ogura细胞质雄性不育系栽培种为母本,含有Ogura CMS恢复系杂交种甘蓝型油菜为父本,进行人工授粉;(1) selecting the broccoli Ogura cytoplasmic male sterile line cultivar as the female parent, and containing the Ogura CMS restorer line hybrid Brassica napus as the male parent, and artificially pollinated;
人工授粉后进行套袋隔离,授粉后套袋隔离,可严格控制父本以外的花粉污染。本发明对所述套袋的方法没有特殊限定,授粉套袋后效果如图1所示。Bag isolation after manual pollination, and bag isolation after pollination, can strictly control pollen pollution other than the male parent. The method of bagging is not particularly limited in the present invention, and the effect of bagging after pollination is shown in FIG. 1 .
(2)在人工授粉10-15天后,将子房剪下,进行子房消毒,置于培养基中离体培养,所述培养基为MS基础培养基;离体培养的目的是减少母株自体对杂交后子房内胚珠发育的影响,同时防止胚珠退化。(2) after 10-15 days of artificial pollination, cut off the ovary, carry out ovary disinfection, and place it in a medium for in vitro culture, and the medium is MS basal medium; the purpose of in vitro culture is to reduce the number of mother plants. The effect of autologous on ovule development in the ovary after hybridization while preventing ovule degeneration.
(3)剥离步骤(2)中离体培养30~60天子房里萌发的胚珠,接种于分化培养基,培养至长出侧芽;(3) in the stripping step (2), the ovules germinated in the ovary are cultured in vitro for 30 to 60 days, inoculated in a differentiation medium, and cultured until lateral buds grow;
(4)剥离步骤(3)中的侧芽,接种于生根培养基20-30天,得无菌苗;(4) the lateral buds in the stripping step (3) are inoculated in the rooting medium for 20-30 days to obtain sterile seedlings;
(5)待步骤(4)中幼苗长至4~5片叶,生根后打开培养瓶的盖子练苗,洗去培养基,移栽至营养土中培养2周后,转入大棚,常规栽培管理。(5) wait for the seedling to grow to 4~5 leaves in step (4), after rooting, open the lid of the culture bottle to practice seedlings, wash off the culture medium, transplant it into the nutrient soil and cultivate for 2 weeks, then transfer to a greenhouse for conventional cultivation manage.
优选的,步骤(2)中,所述培养基还包括30g/L蔗糖、6g/L琼脂糖,PH值5.8~6.02。Preferably, in step (2), the medium further comprises 30 g/L sucrose, 6 g/L agarose, and has a pH value of 5.8-6.02.
离体组织培养中种胚缺乏自养能力,需要外源碳源蔗糖提供其生长发育所需的碳骨架和能量,同时维持培养基的渗透压;添加适宜浓度的琼脂糖能促进培养基在常温下固化形成固体培养基。The embryo lacks autotrophic ability in in vitro tissue culture, and needs exogenous carbon source sucrose to provide the carbon skeleton and energy required for its growth and development, while maintaining the osmotic pressure of the medium; adding a suitable concentration of agarose can promote the medium at room temperature. solidified to form a solid medium.
优选的,步骤(3)中,分化培养基为以MS为基础培养基,还包括30g/L蔗糖、0.1mg/L的NAA、1mg/L的6BA、6g/L琼脂糖,PH值5.8~6.0。Preferably, in step (3), the differentiation medium is MS-based medium, and also includes 30g/L sucrose, 0.1mg/L NAA, 1mg/L 6BA, 6g/L agarose, pH value 5.8~ 6.0.
添加适宜浓度6BA有促进子房膨大和幼胚分化的作用;低浓度的NAA促进子房膨大、刺激细胞分裂和组织分化的同时还促进植物生根;Adding appropriate concentration of 6BA can promote ovary enlargement and immature embryo differentiation; low concentration of NAA promotes ovary enlargement, stimulates cell division and tissue differentiation, and also promotes plant rooting;
优选的,步骤(4)中,生根培养基以MS为基础培养基,还包括10g/L蔗糖、9g/L琼脂糖,PH值5.8~6.0。Preferably, in step (4), the rooting medium uses MS as the basic medium, and also includes 10 g/L sucrose and 9 g/L agarose, with a pH value of 5.8-6.0.
优选的,步骤(1)中,选取的母本为B14和137,父本为Rfo-1。Preferably, in step (1), the selected female parent is B14 and 137, and the male parent is Rfo-1.
优选的,步骤(1)中,人工授粉的方法包括以下步骤:对母本进行剥蕾,分别在早上和晚上采取重复授粉,将父本新鲜花粉涂抹与母本柱头。Preferably, in step (1), the method for artificial pollination includes the following steps: budding the female parent, repeating pollination in the morning and evening respectively, and smearing fresh pollen from the male parent on the stigma of the female parent.
优选的,步骤(2)中子房消毒的方法包括以下步骤:75%酒精消毒30s,8%-10%的NaClO消毒10-15min,无菌水冲洗3次,吸干水分。Preferably, the method for sterilizing the ovary in step (2) includes the following steps: sterilizing with 75% alcohol for 30 seconds, sterilizing with 8%-10% NaClO for 10-15 minutes, rinsing with sterile water for 3 times, and absorbing water.
优选的,步骤(5)中所述营养土为基质和蛭石混合物,基质和蛭石体积比为3∶1。Preferably, in step (5), the nutrient soil is a mixture of matrix and vermiculite, and the volume ratio of matrix and vermiculite is 3:1.
实施例1Example 1
1.材料与方法1. Materials and methods
以细胞质雄性不育(Ogura CMS)青花菜B14、137为母本,以含有Rfo恢复基因的甘蓝型油菜RFO-1为父本。青花菜实验材料为上海市农科院园艺所提供栽培种,甘蓝型油菜材料由上海农科院作物所杨立勇老师提供杂交种。The cytoplasmic male sterile (Ogura CMS) broccoli B14 and 137 were used as the female parent, and the Brassica napus RFO-1 containing the Rfo restorer gene was used as the male parent. The experimental materials of broccoli were cultivars provided by the Horticulture Institute of Shanghai Academy of Agricultural Sciences, and the materials of Brassica napus were provided by Yang Liyong from the Crop Institute of Shanghai Academy of Agricultural Sciences.
2.实验方法2. Experimental method
2.1套袋、挂牌2.1 Bagging, listing
青花菜和甘蓝型油菜抽薹后套袋,如果套袋之前已有开放的花,要将花摘掉。青花菜雄性不育系B14、137单独套袋,挂牌编号标记,各6个单株,所有的数据取6个单株的平均值,甘蓝型油菜不分单株,混合给青花菜授粉。Broccoli and Brassica napus are bolted and bagged, and if there are already open flowers before bagging, remove the flowers. The broccoli male sterile lines B14 and 137 were individually bagged, marked with a serial number, and each had 6 individual plants. All data were taken as the average of the 6 individual plants.
2.2授粉2.2 Pollination
套袋以后,青花菜与甘蓝型油菜开花时,进行人工授粉(图1)。授粉一般在早上十点和下午一点进行,此时效果最佳。授粉的方法:1)在授粉之前首先用水将手洗干净,然后再用酒精消毒。2)打开纱网袋,摘下已经盛开的甘蓝型油菜的花朵,摘完以后立即将纱袋封好。3)再打开青花菜植株的纱网袋,将蕾期授粉枝上大小适合的花蕾剥开,授以甘蓝型油菜的新鲜花粉,授粉后立即套袋隔离防止花粉污染。为了增加花粉与柱头的亲和性,我们采用的是重复授粉法,每天早晚重复授粉2次。After bagging, broccoli and Brassica napus were artificially pollinated when they bloomed (Figure 1). Pollination is generally carried out at ten o'clock in the morning and one o'clock in the afternoon, when the best results are obtained. Methods of pollination: 1) Wash your hands with water before pollination, and then disinfect them with alcohol. 2) Open the gauze bag, pick off the flowers of rapeseed that have been in full bloom, and seal the gauze bag immediately after picking. 3) Open the gauze bag of the broccoli plant again, peel off the flower buds of suitable size on the pollinating branches at the bud stage, and give fresh pollen of the cabbage type rapeseed. In order to increase the affinity between pollen and stigma, we used the repeated pollination method, repeating pollination twice a day in the morning and evening.
2.3胚胎抢救2.3 Embryo rescue
由于青花菜和甘蓝型油菜属于不同的种,存在生殖隔离。授粉后容易发生胚败育,为了增加成活率,需要进行胚抢救。胚胎抢救的常规方法有三种:子房培养、胚珠培养、胚培养。本试验采用的是第二种方法-子房培养(图2)。具体步骤如下:Because broccoli and Brassica napus belong to different species, there is reproductive isolation. Embryo abortion is prone to occur after pollination. In order to increase the survival rate, embryo rescue is required. There are three conventional methods for embryo rescue: ovary culture, ovule culture, and embryo culture. This experiment used the second method - ovary culture (Figure 2). Specific steps are as follows:
在试验田取授粉以后的荚果,取授粉后第10-15天的荚果。将荚果先用无菌水冲洗一遍,然后用70%的酒精消毒30秒,再用0.8%的次氯酸钠消毒10min,最后用无菌水清洗3遍,每遍清洗5分钟。将消毒好的荚果放到已经灭菌的带有滤纸的培养皿中,把荚果上的水分吸干,然后小心的用灭菌的小刀把荚果的果柄切掉,接种到装有生芽培养基(表2)的培养皿上,操作时要注意无菌、以免污染,一般一个培养瓶中接种10-12个果荚(图2)。接种完以后把培养瓶瓶口、盖子用酒精灯烧一下灭菌盖好,放到培养箱中培养,培养条件是25℃,光照16h.d-1,光照强度20001x。观察子房在培养基上的生长情况,做好记载。培养30-60天,将已经长出的子叶苗(图3)转接到分化培养基(图4)(表2)上,获得试管苗。待苗长至4~5片叶,生根后打开培养瓶的盖子练苗,洗去培养基,移栽至营养土中培养2周后(图5),温室培养条件是25℃,光照16h.d-1,光照强度20001x,转入大棚,常规栽培管理。利用已开发的Rfo特异标记(RFO-2F/RFO-NEW-R)对远缘杂种群体F1进行筛选,计算Rfo的阳性单株数。Take the pods after pollination in the experimental field, and take the pods on the 10th to 15th day after pollination. The pods were first rinsed with sterile water, then sterilized with 70% alcohol for 30 seconds, then sterilized with 0.8% sodium hypochlorite for 10 minutes, and finally washed with sterile water 3 times for 5 minutes each. Put the sterilized pods into a sterilized petri dish with filter paper, absorb the water on the pods, and then carefully cut off the stalks of the pods with a sterilized knife, and inoculate them into the pods containing buds. On the petri dish of the base (Table 2), attention should be paid to sterility during operation to avoid contamination. Generally, 10-12 fruit pods are inoculated in one culture bottle (Figure 2). After inoculation, burn the bottle mouth and lid of the culture bottle with an alcohol lamp to sterilize and cover it, and put it in an incubator for cultivation. Observe the growth of the ovary on the medium and make a record. After culturing for 30-60 days, the grown cotyledon seedlings (Fig. 3) were transferred to differentiation medium (Fig. 4) (Table 2) to obtain test-tube seedlings. When the seedlings grow to 4 to 5 leaves, open the lid of the culture bottle to practice seedlings after rooting, wash off the medium, and transplant to nutrient soil for 2 weeks (Figure 5). d -1 , the light intensity was 20001x, and it was transferred to a greenhouse for routine cultivation and management. The distant hybrid population F1 was screened using the developed Rfo-specific marker (RFO-2F/RFO-NEW-R), and the number of Rfo-positive individual plants was calculated.
胚胎抢救期间,观察统计如下数据:During the embryo rescue, the following statistics were observed:
(1)观察并统计杂种子房培养的出苗率见表1;(1) observe and count the emergence rate of hybrid seed house cultivation and see Table 1;
表1Table 1
由表1可以看出,B14的成苗率为0.95%,Rfo阳性单株成苗率为0.26%,137的成苗率为0.58%,Rfo阳性单株成苗率为0.29%,说明不同基因型的远缘杂交成苗率有差异。It can be seen from Table 1 that the seedling rate of B14 is 0.95%, the rate of Rfo-positive single plant is 0.26%, the seedling rate of 137 is 0.58%, and the rate of Rfo-positive single plant is 0.29%, indicating that different genes There are differences in the seedling rates of distant hybrids.
(2)观察并统计植物倍性(2) Observe and count plant ploidy
利用流式细胞仪对获得的种间杂种F1植株采用流式细胞术方法进行倍性鉴定。以青花菜2C DNA相对含量为参照,其G0/G1期对应峰值为150通道处,甘蓝型油菜为300通道处,根据杂种后代G0/G1期峰值的位置,判断其倍性。由图10倍性鉴定结果表明,F1杂种植株G0/G1期峰值在200-300之间,介于青花菜和甘蓝型油菜之间,近于三倍体。Flow cytometry was used to identify the ploidy of the obtained interspecific hybrid F1 plants by flow cytometry. Taking the relative content of 2C DNA in broccoli as a reference, the corresponding peak of the G0/G1 phase is at channel 150, and the 300 channel of rapeseed. The results of ploidy identification in Figure 10 show that the peak value of F1 hybrid plants in G0/G1 stage is between 200 and 300, which is between broccoli and Brassica napus, and is close to triploid.
(3)杂种子房培养的培养基配方表2(3) the medium formula table 2 of hybrid seed house cultivation
表2Table 2
(4)形态学特征观察(4) Observation of morphological features
形态学评价表明,种间杂交种比双亲生长旺盛(图6,7),叶形、蜡质和花色介于双亲之间,但有些性状更倾向于油菜亲本(图6-9)。叶片在叶柄的基部有较小的裂片蜡粉较少时,叶色多为淡绿色,叶缘呈锯齿状(图8);有些花比双亲大;雄蕊上有少量花粉(图9)。综上所述,可以看出,种间杂种对双亲性状都有较好的遗传效果。在开花期,大多数种间杂种F1携带RFO的阳性个体都有花粉,有些种间杂种含有RFO基因,但没有产生花粉,只产生了有缺陷的雄蕊,这可能是由于染色体加倍的异源异常所致。未携带Rfo的种间杂交种在开花期间没有花粉。Morphological evaluation showed that the interspecific hybrids grew more vigorously than their parents (Figures 6, 7), and the leaf shape, waxiness and flower color were intermediate between the parents, but some traits were more inclined to the rape parents (Figures 6-9). The leaves have smaller lobes at the base of the petiole. When the wax powder is less, the leaf color is mostly light green, and the leaf margin is serrated (Figure 8); some flowers are larger than the parents; there is a small amount of pollen on the stamens (Figure 9). To sum up, it can be seen that interspecific hybrids have good genetic effects on both parental traits. During flowering, most of the F1-positive RFO-carrying individuals of interspecific hybrids had pollen, and some interspecific hybrids contained the RFO gene, but did not produce pollen, only defective stamens, probably due to heterologous abnormalities in chromosome doubling caused. Interspecific hybrids that do not carry Rfo have no pollen during flowering.
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