CN103329793A - Breeding, multiplying and seed production methods for non-heading cabbage cytoplasmic male sterility line - Google Patents

Abstract

The invention discloses a breeding method for a non-heading cabbage cytoplasmic male sterility line. The breeding method comprises the following steps of: 1) performing distant hybridization on brassicajancea rape cytoplasmic male sterile material hau CMS serving as a female parent and non-heading cabbage self-incompatible line 'aijiaohuang' as a male parent, performing duplicitas saving culture on separated hybrid strains, and transplanting the separated hybrid strains into a large field to obtain interspecific hybrids F1; and 2) performing continuous backcross for six generations by taking sterile strains in the interspecific hybrids F1 as a female parent and the 'aijiaohuang' as a recurrent parent to obtain the non-heading cabbage cytoplasmic male sterility line. The non-heading cabbage cytoplasmic male sterility line obtained by the breeding method has the characteristics of stable sterility, no influence caused by the environment and the genetic background and the like; and due to continuous 5-year 10-generation planting and sterility authentication, the sterility and the sterile strain rate of the sterility line are 100 percent.

Description

The seed selection of Chinese cabbage cytoplasmic male sterile line, breeding and producing method for seed

Technical field

The invention belongs to the vegetable breeding technical field, be specifically related to a kind of seed selection, breeding and producing method for seed of Chinese cabbage cytoplasmic male sterile line.

Background technology

The main path of brassicaceous vegetable heterosis utilization is self-incompatibillity and male sterility, wherein cytoplasmic male sterility is desirable pollination control system. the brassicaceous vegetable first generation of hybrid utilizes work to carry out widely, Asian countries particularly, as China, Japan and Korea S etc., adopt at present the method for self incompatible line or self compatible line to carry out the first generation of hybrid and produce in the majorityly, the decline of parents' vitality easily takes place and occur problem such as pseudostationary easily but this method exists selfing many generations.Utilize the cytoplasmic male sterile line production of hybrid seeds, can obtain high-quality filial generation.Produce the not balling cytoplasmic male sterile line type that goes up utilization at present and be mainly rape pol cytoplasmic male sterility (pol cms) (pol CMS) and male sterile with radish cytoplasm (ogu CMS), but there is the unsettled shortcoming of sterility in Pol CMS cytoplasmic male sterile line; There are shortcomings such as low temperature yellow and nectary degeneration in ogu CMS, therefore excavates and formulate new cytoplasmic male sterility type, solves the single problem of cytoplasm, widens existing germ plasm resource, has great importance.

Pol CMS is the Fu Ting of Hua Zhong Agriculture University discovery in 1972, in rape and brassicaceous vegetable, extensively utilize, Ke Guilan etc. are female parent with the Ma Xibaozhibuyuyuan of Pori, are male parent with Chinese cabbage 3411-7, have bred allo-plasm of Chinese cabbage male sterile line CMS3411-7.Since Ogura1968 found ogu CMS, Chinese scholars had been carried out a large amount of backcross transformations to this sterile material, at present transformation in brassicaceous vegetables such as rape, wild cabbage, Chinese cabbage, broccoli.Fang Zhiyuan 1998 introduces Ogu CMS material after 6 parts of new improvement such as CMS625, CMS629 from the U.S., and a part broccoli inbred line is carried out transformation surplus part wild cabbage inbred line and 20 surplus 30, advantages such as the not yellow of backcross progeny performance low temperature lower blade, the sterile flower that CMS625, CMS629 transformation come opens have application promise in clinical practice.

Summary of the invention

Purpose of the present invention is exactly the weak point that overcomes existing Chinese cabbage cytoplasmic male sterile line, the selection of the stable Chinese cabbage cytoplasmic male sterile line of a kind of new sterility is provided, and the breeding of this Chinese cabbage cytoplasmic male sterile line, producing method for seed, for the vegetables Hybrid provides new germ plasm resource.

The present invention such as utilizes the applicant disclosed mustard type rape cytoplasmic male sterility material hau CMS and sterility thereof has been stable, thorough and not affected by environment at advantage, by with Chinese cabbage " short pin Huang " distant hybridization and back cross breeding, obtain the stable Chinese cabbage cytoplasmic male sterile line of sterility, breed more Chinese cabbage cytoplasmic male sterile line under the isolation condition in the fall, and with it and " Shanghai green grass or young crops " hybridization, obtain highly purified Chinese cabbage male sterile Hybrid.

The present invention is achieved by the following technical programs:

A kind of selection of Chinese cabbage cytoplasmic male sterile line may further comprise the steps:

(1) utilize mustard type rape cytoplasmic male sterility material hau CMS to be female parent, " " short pin Huang " is male parent; carry out distant hybridization; separate the embryo of hybrid strain and place the MS solid culture medium; place then in 25 ℃ of culturing room; shine 14h every day with 1000 ~ 1500lx light, cultivates and transplants acquisition species hybrid F in land for growing field crops after 30 days with the Chinese cabbage self incompatible line ₁

(2) select species hybrid F ₁In sterile strain be maternal, be 6 generations of recurrent parent continuous backcross with " short pin Huang ", obtain Chinese cabbage cytoplasmic male sterile line.

Breeding, the producing method for seed of described Chinese cabbage cytoplasmic male sterile line are: under autumn sowing isolation condition, it is maternal adopting Chinese cabbage cytoplasmic male sterile line, be male parent with " short pin Huang ", according to male parent: maternal row is bred described Chinese cabbage cytoplasmic male sterile line than the ratio that is 2: 4 ~ 6;

The Chinese cabbage cytoplasmic male sterile line of not receiving of 100% sterile degree that obtains with breeding is maternal, be male parent with " Shanghai green grass or young crops ", under the isolation condition in the fall, according to male parent: maternal row is than the ratio that is 2: 4 ~ 6, carry out hybrid seeding, obtain highly purified Chinese cabbage male sterile Hybrid.

The invention has the beneficial effects as follows:

1, Chinese cabbage cytoplasmic male sterile line of the present invention has the stable characteristics of sterility, and be not subjected to the influence of environment (as low temperature or high temperature) and genetic background, continuous 5 years 10 generations planted and carried out fertility and identify that sterile degree and the sterile plant rate of this male sterile line are 100% in Lanzhou, Gansu and Wuhan, Hubei.

2, overcome that parent's (male sterile line) purity that exists in the prior art is not high, the yellow in seedling stage, nectary is undeveloped and defective such as trace-pollen.

3, adopt the Chinese cabbage cytoplasmic male sterile line hybrid seeding program simple (artificial pollination and chemical treatment) of seed selection of the present invention, Hybrid purity height (the male sterile line sterile degree of utilization is 100%), with the obvious advantage.

Description of drawings

Fig. 1: be technology path figure of the present invention.

Fig. 2: the floral organ figure of mustard type rape cytoplasmic male sterile line hau CMS and the Chinese cabbage cytoplasmic male sterile line 98-1A of seed selection of the present invention, Chinese cabbage keeps system's " short pin Huang ", and species hybrid F ₁Photo.Among the figure: A, B are mustard type rape cytoplasmic male sterile line hau CMS floral organ figure, C, are the floral organ figure of the Chinese cabbage cytoplasmic male sterile line 98-1A of seed selection of the present invention; D is that Chinese cabbage keeps " short pin Huang " floral organ figure of system; E is the individual plant figure of Chinese cabbage cytoplasmic male sterile line 98-1A; F saves the species hybrid F that the back obtains through embryo ₁Tissue culturing seedling.

Fig. 3: the anther development cytological observation of Chinese cabbage cytoplasmic male sterile line 98-1A and species hybrid F ₁(n=28) chromosome is identified.Among the figure: A is the anther development cytological observation of Chinese cabbage cytoplasmic male sterile line 98-1A; B is species hybrid F ₁Chromosome identify.

Embodiment

The present invention is described in detail below in conjunction with embodiment.

The seed selection of embodiment 1 Chinese cabbage cytoplasmic male sterile line

(1) be that 200510086942.7 Chinese invention patent is open at application number with mustard type rape cytoplasmic male sterility material hau CMS(, this seed on November 12nd, 2005 in China's typical culture collection center preservation, preserving number is: CCTCC NO:P200504) do maternal, keep system's " short pin Huang " (seed of this breeding material is public offering before the applying date) to do the male parent distant hybridization, adopt the embryo rescue method to cultivate, transplant the land for growing field crops and obtain species hybrid F ₁

(2) the species hybrid F that obtains with step (1) ₁Making female parent is Chinese cabbage " short pin Huang " hybridization with maintenance, obtains Chinese cabbage BC1F from embryo culture ₁

(3) the Chinese cabbage BC1F that obtains with step (2) ₁Doing maternal is " short pin Huang " hybridization with keeping, and obtains Chinese cabbage BC2F from embryo culture ₁

(4) the Chinese cabbage BC2F that obtains with step (3) ₁Do maternally, do four generations of recurrent parent continuous backcross to keep system's " short pin Huang ", obtain Chinese cabbage cytoplasmic male sterile line 98-1A.

The embryo rescue method:

1) being maternal with mustard type rape cytoplasmic male sterile line hau CMS, is that male parent is carried out distant hybridization with male parent " short pin Huang ", obtains species hybrid F ₁

2) from the species hybrid F of growth after 15-20 days ₁Get angle fruit on the plant, with the ovary of protuberance in 4 ℃ of preservations;

3) at superclean bench with 70% ethanolic solution processing angle fruit 5 minutes;

4) use the mercuric chloride solution processing angle fruit 15 minutes of 0.1 % again;

5) distilled water with sterilization washs the angle fruit three times, each about 5 minutes;

6) peel off embryo with the dissecting needle or the tweezers that disinfect;

7) embryo that disinfects is placed in the MS solid culture medium of preparation in advance, place in 25 ℃ of culturing room intensity of illumination 1000-1500lx, illumination every day 14h, namely obtain tissue culturing seedling through cultivation in about 30 days, the group that will obtain at last training seedling is transplanted to big Tanaka in good time.

The MS culture medium preparation:

In the triangular pyramidal bottle, adorn following medium: MS minimal medium (Murashige T, skoog F, 1962, A revised medium for rapid growth and bioassays with tobacco tissue culture cultures physiol plant 15:473-479), additional 6-BA1.5 mg/L, NAA0.05 mg/L, sucrose 30.0g/L, agar 7.0g/L adds water to 1L, transfers pH5.9-6.0.(121 ℃, high pressure steam sterilization 30 minutes) sterilization according to a conventional method, the triangular pyramidal bottle is taken out in the sterilization back from autoclave, lie in to make its cooled and solidified standby on the experimental bench.

This sterile source is novel mustard type rape male sterile material hau CMS, does not report both at home and abroad.

Breeding, the production of hybrid seeds of embodiment 2 Chinese cabbage cytoplasmic male sterile line 98-1A

Under Chinese Hubei Wuhan autumn sowing (annual September) or Chinese Gansu Lanzhou summer sowing (annual May) isolation condition, it is maternal adopting Chinese cabbage cytoplasmic male sterile line 98-1A, " short pin Huang " is male parent, press male parent: female parent is that the row of 2:4 is bred described Chinese cabbage cytoplasmic male sterile line 98-1A than (the quantity ratio of row), and other cultivation managements are identical with the breeding of common Chinese cabbage.

To breed back Chinese cabbage cytoplasmic male sterile line 98-1A and " Shanghai green grass or young crops " hybridization, obtain Chinese cabbage cytoplasmic male sterility Hybrid.

The phenotype of table 1 Chinese cabbage cytoplasmic male sterile line 98-1A and novel leaf mustard hau CMS relatively

The cytoplasmic male sterility type	Novel leaf mustard hau CMS	Chinese cabbage male sterile line 98-1A
			Investigation strain number (strain)	1900	3500
Sterile degree (%)	100％	100%
			Sterile strain (strain)	1900	3500
Flower bud loses phenomenon	Do not have	Do not have

Claims (2)

1. the selection of a Chinese cabbage cytoplasmic male sterile line is characterized in that may further comprise the steps:

(1) utilize mustard type rape cytoplasmic male sterility material hau CMS to be female parent, be male parent with Chinese cabbage self incompatible line " short pin Huang ", carry out distant hybridization, separate the embryo of hybrid strain and place the MS solid culture medium, place then in 25 ℃ of culturing room, shine 14h every day with 1000 ~ 1500lx light, cultivate and transplant acquisition species hybrid F in land for growing field crops after 30 days ₁

(2) select species hybrid F ₁In sterile strain be maternal, be 6 generations of recurrent parent continuous backcross with " short pin Huang ", obtain Chinese cabbage cytoplasmic male sterile line.

2. the breeding of a Chinese cabbage cytoplasmic male sterile line, producing method for seed, it is characterized in that: under autumn sowing isolation condition, it is maternal adopting Chinese cabbage cytoplasmic male sterile line, " short pin Huang " is male parent, according to male parent: maternal row is bred described Chinese cabbage cytoplasmic male sterile line than the ratio that is 2: 4 ~ 6;

The Chinese cabbage cytoplasmic male sterile line of not receiving of 100% sterile degree that obtains with breeding is maternal, be male parent with " Shanghai green grass or young crops ", under the isolation condition in the fall, according to male parent: maternal row is than the ratio that is 2: 4 ~ 6, carry out hybrid seeding, obtain highly purified Chinese cabbage male sterile Hybrid

Wherein, described Chinese cabbage cytoplasmic male sterile line obtains according to the following steps seed selection:

(1) utilize mustard type rape cytoplasmic male sterility material hau CMS to be female parent, be male parent with Chinese cabbage self incompatible line " short pin Huang ", carry out distant hybridization, separate the embryo of hybrid strain and place the MS solid culture medium, place then in 25 ℃ of culturing room, shine 14h every day with 1000 ~ 1500lx light, cultivate and transplant acquisition species hybrid F in land for growing field crops after 30 days ₁

(2) select species hybrid F ₁In sterile strain be maternal, be 6 generations of recurrent parent continuous backcross with " short pin Huang ", obtain Chinese cabbage cytoplasmic male sterile line.

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