CN111528100B

CN111528100B - Method for obtaining distant hybridization progeny of broccoli and cabbage type rape

Info

Publication number: CN111528100B
Application number: CN202010548207.8A
Authority: CN
Inventors: 刘春晴; 谢祝捷; 姚雪琴; 李光庆; 黄雷
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2020-06-16
Filing date: 2020-06-16
Publication date: 2022-07-26
Anticipated expiration: 2040-06-16
Also published as: CN111528100A

Abstract

The invention discloses a method for obtaining distant hybridization progeny of broccoli and brassica napus, which comprises the following steps: selecting broccoli male sterile line cultivated species as female parent, and cabbage type rape containing Ogura CMS restoring line cultivated species as male parent, and carrying out artificial pollination; after artificial pollination, performing in vitro culture, stripping ovules germinated in an in vitro culture ovary, inoculating to a differentiation culture medium, and culturing until lateral buds grow out; stripping off lateral buds, and inoculating the lateral buds to a rooting culture medium to obtain aseptic seedlings; and (4) hardening the seedlings when the seedlings grow to 4-5 leaves, transplanting the seedlings into nutrient soil for culturing, transferring the seedlings into a greenhouse, and performing conventional cultivation management. According to the invention, through the method of carrying out tissue culture on the ovary after artificial repeated pollination in the bud period, the problem of embryo abortion in the process of forming distant hybrid seeds is solved, the hybrid yield is improved, and then the restoring gene in the cabbage type rape is transferred into cauliflowers, the fertility of the cauliflowers is restored, and the utilization efficiency of the germplasm resources of the cauliflowers is improved.

Description

Method for obtaining distant hybridization progeny of broccoli and cabbage type rape

Technical Field

The invention relates to the technical field of biological breeding, in particular to a method for obtaining distant hybridization progeny of broccoli and brassica napus.

Background

The broccoli is rich in nutrient substances, is a variety of the cabbage, and belongs to the cruciferous brassica cross-pollinated crop. The heterosis is very obvious, and the utilization of the heterosis can not only improve the yield, but also improve the disease resistance and optimize the germplasm resources. Ogura cytoplasmic male sterility (Ogura CMS) is stable in sterility and easy to transform, is one of the main ways for heterosis utilization of cruciferous crops, and is increasingly used for production of cauliflower hybrid seeds in recent ten years. Ogura cytoplasmic male sterility is maternal inheritance, all descendants are male sterility, cannot be subjected to self-crossing separation, and is not beneficial to innovation and utilization of germplasm resources. An effective solution to this problem is to mine or create Ogura CMS restorer lines. However, to date, the Ogura CMS restorer line has not been found in cabbage vegetables.

Brassica napus, an important oil crop, contains abundant disease, insect, weed and fertility genes, and the Ogura CMS restorer line has been successfully created and used for hybrid production. In order to widen the genetic basis of cauliflower germplasm resources and create new excellent germplasm, research on the interspecific relationship of brassica is utilized, and fertility restorer genes and stress-resistant genes of brassica napus are introduced into cauliflowers through distant hybridization, so that the genetic breeding gene bank of the cauliflowers is widened, the resistance of the cauliflowers is improved, and the importance is increased.

However, distant hybridization has reproductive isolation, and thus is prone to have problems of hybrid incompatibility, embryo abortion, complex progeny genetic variation and the like after pollination. At present, although a small amount of different varieties of cabbages and cabbage type rape interspecific hybrids are formed, almost no cases exist for realizing interspecific hybridization of the cauliflowers and the cabbage type rape and obtaining progeny plants by utilizing an embryo rescue in vitro culture technology, the culture period is long, the hybrid obtaining rate is low, and the utilization of hybrid vigor is greatly limited.

Disclosure of Invention

The invention provides a method for obtaining distant hybridization progeny of broccoli and brassica napus to solve the technical problem.

The invention relates to a method for obtaining distant hybridization offspring of broccoli and brassica napus, which comprises the following steps:

(1) selecting broccoli Ogura cytoplasmic male sterile line cultivated species as a female parent, and carrying out artificial pollination on hybrid cabbage type rape containing Ogura CMS restoring line as a male parent;

(2) after artificial pollination is carried out for 10-15 days, cutting off an ovary, sterilizing the ovary, and putting the ovary into a culture medium for in vitro culture, wherein the culture medium is an MS basal culture medium;

(3) stripping ovules germinated in the ovaries cultured in vitro in the step (2) for 30-60 days, inoculating the ovules to a differentiation medium, and culturing until lateral buds grow;

(4) stripping the lateral buds in the step (3), and inoculating the lateral buds to a rooting culture medium for 20-30 days to obtain aseptic seedlings;

(5) and (5) after the seedlings grow to 4-5 leaves in the step (4), rooting, opening a cover of a culture bottle to train the seedlings, washing off the culture medium, transplanting the seedlings into nutrient soil to culture for 2 weeks, transferring the seedlings into a greenhouse, and performing conventional cultivation and management.

Further, in the step (2), the culture medium further comprises 30g/L of sucrose and 6g/L of agarose, and the pH value is 5.8-6.02.

Further, in the step (3), the differentiation medium is MS as a basic medium, and further comprises 30g/L of sucrose, 0.1mg/L of NAA, 1mg/L of 6BA and 6g/L of agarose, and the pH value is 5.8-6.0.

Further, in the step (4), the rooting medium takes MS as a basic medium, and also comprises 10g/L of sucrose and 9g/L of agarose, and the pH value is 5.8-6.0.

Further, in the step (1), the female parent is selected as cytoplasmic male sterile lines B14 and 137, and the male parent is selected as Rfo-1.

Further, in the step (1), the artificial pollination method comprises the following steps: stripping the female parent, respectively pollinating in the morning and at night, and smearing the fresh pollen of the male parent on the stigma of the female parent.

Further, the method for sterilizing the greenhouse in the step (2) comprises the following steps: sterilizing with 75% alcohol for 30s, sterilizing with 8% NaClO for 10min, washing with sterile water for 3 times, and sucking off water.

Further, in the step (5), the nutrient soil is a mixture of a substrate and vermiculite, and the volume ratio of the substrate to the vermiculite is 3: 1.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for obtaining distant hybridization progeny of cauliflowers and brassica napus, which is characterized in that after artificial repeated pollination is carried out for 10-15 days in a bud period, a method for carrying out tissue culture on a ovary is adopted, the problem of embryo abortion in the process of forming distant hybridization seeds is solved, the yield of hybrids is improved, then a restoring gene in the brassica napus is transferred into the cauliflowers, the fertility of the cauliflowers is restored, and the utilization efficiency of germplasm resources of the cauliflowers is improved; meanwhile, the heterogeous triploid hybrid AAC obtained by the invention is respectively from cauliflower and cabbage type rape and is used as a bridge species, so that the excellent characters in the cabbage type rape are favorably transferred to the cauliflower, and the breeding process of the cauliflower is improved.

Drawings

FIG. 1 is an isolated view of a hybrid pollination sleeve of the present invention;

FIG. 2 is a diagram showing the culture of ovary tissue in example 1 of the present invention;

FIG. 3 is a diagram of tissue culture seedling formation in example 1 of the present invention;

FIG. 4 is a graph showing the rooting of tissue culture seedlings in example 1 of the present invention;

FIG. 5 is a diagram of tissue culture seedling hardening in example 1 of the present invention;

FIG. 6 is a seedling morphology map of cauliflower-brassica napus parents and the resulting hybrid plants of example 1 of the present invention;

FIG. 7 is a flowering phase morphology of hybrid plants obtained from the cauliflower-brassica napus cross of example 1 of the present invention;

FIG. 8 is a graph showing the leaf patterns of plants of the parent generation and the hybrid generation in example 1 of the present invention;

FIG. 9 is a comparison of flower morphology of plants of parent and hybrid generations in example 1;

FIG. 10 is a graph showing ploidy analysis (relative DNA content) of a cauliflower-brassica napus hybrid according to example 1 of the present invention;

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

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 disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

The invention provides a method for obtaining distant hybridization progeny of broccoli and brassica napus, which comprises the following steps:

(1) selecting broccoli Ogura cytoplasmic male sterile line cultivated species as a female parent and a hybrid cabbage type rape containing an Ogura CMS restoring line as a male parent, and carrying out artificial pollination;

the bagging isolation is carried out after artificial pollination, and the bagging isolation is carried out after pollination, so that the pollution of pollen outside the male parent can be strictly controlled. The bagging method is not particularly limited in the invention, and the effect after pollination and bagging is shown in figure 1.

(2) After artificial pollination is carried out for 10-15 days, cutting off an ovary, sterilizing the ovary, and putting the ovary into a culture medium for in vitro culture, wherein the culture medium is an MS basal culture medium; the purpose of the isolated culture is to reduce the influence of the self of the mother strain on the development of ovules in ovaries after hybridization and simultaneously prevent the ovules from being degenerated.

(3) Peeling ovules germinated in the ovary cultured in vitro in the step (2) for 30-60 days, inoculating the ovules to a differentiation medium, and culturing until lateral buds grow;

(5) and (4) after the seedlings grow to 4-5 leaves in the step (4), opening a cover of a culture bottle to train the seedlings after rooting, washing off the culture medium, transplanting the seedlings into nutrient soil to culture for 2 weeks, transferring the seedlings into a greenhouse, and performing conventional cultivation management.

Preferably, in the step (2), the culture medium further comprises 30g/L of sucrose and 6g/L of agarose, and the pH value is 5.8-6.02.

The embryo in the in vitro tissue culture lacks the autotrophic ability, needs the exogenous carbon source sucrose to provide the carbon skeleton and energy required by the growth and development of the embryo, and simultaneously maintains the osmotic pressure of the culture medium; the addition of agarose at an appropriate concentration can promote the solidification of the culture medium at normal temperature to form a solid culture medium.

Preferably, in the step (3), the differentiation medium is MS-based medium, and further comprises 30g/L of sucrose, 0.1mg/L of NAA, 1mg/L of 6BA and 6g/L of agarose, and the pH value is 5.8-6.0.

The addition of 6BA with proper concentration can promote the ovary enlargement and the embryo differentiation; the low-concentration NAA promotes the ovary to expand, stimulates cell division and tissue differentiation and simultaneously promotes the plant to root;

preferably, in the step (4), the rooting medium takes MS as a basic medium, and further comprises 10g/L of sucrose and 9g/L of agarose, and the pH value is 5.8-6.0.

Preferably, in step (1), the female parents are B14 and 137, and the male parent is Rfo-1.

Preferably, in step (1), the artificial pollination method comprises the following steps: stripping the female parent, respectively pollinating in the morning and at night, and smearing the fresh pollen of the male parent on the stigma of the female parent.

Preferably, the method for sterilizing the neutron chamber in the step (2) comprises the following steps: sterilizing with 75% alcohol for 30s, sterilizing with 8% -10% NaClO for 10-15min, washing with sterile water for 3 times, and sucking off water.

Preferably, the nutrient soil in the step (5) is a mixture of a substrate and vermiculite, and the volume ratio of the substrate to the vermiculite is 3: 1.

Example 1

1. Materials and methods

Cytoplasmic male sterility (Ogura CMS) broccoli B14, 137 are used as female parent, and cabbage type rape RFO-1 containing Rfo restoring gene is used as male parent. The broccoli experimental material provides cultivated species for gardening of Shanghai agricultural academy, and the cabbage type rape material provides hybrid species for Yanliyong teachers of Shanghai agricultural academy crops.

2. Experimental method

2.1 bagging and hanging brand

And (4) bagging the broccoli and the cabbage type rape after bolting, and picking off flowers if the flowers are opened before bagging. The male sterile lines B14 and 137 of broccoli are separately bagged, the branding number is marked, each plant has 6 plants, the average value of the 6 plants is taken from all data, the cabbage type rape is not divided into individual plants, and the individual plants are mixed to pollinate the broccoli.

2.2 pollination

After bagging, artificial pollination is carried out when the broccoli and the cabbage type rape bloom (figure 1). Pollination is typically performed at ten am and one pm, where the effect is best. The pollination method comprises the following steps: 1) before pollination, hands are washed clean with water and then disinfected with alcohol. 2) And opening the gauze bag, picking the flowers of the cabbage type rape which are already full, and immediately sealing the gauze bag after picking. 3) Then the gauze bag of the broccoli plant is opened, the flower buds with proper size on the pollination branches in the bud period are stripped, the fresh pollen of the cabbage type rape is pollinated, and the bag is immediately sleeved and isolated after pollination to prevent pollen pollution. In order to increase the affinity of pollen to stigma, we used repeated pollination, which was repeated 2 times a day in the morning and evening.

2.3 embryo rescue

Because broccoli and brassica napus belong to different species, reproductive isolation exists. Embryo abortion easily occurs after pollination, and embryo rescue is needed to increase survival rate. There are three general methods for embryo rescue: ovary culture, ovule culture and embryo culture. The second method, ovary culture, was used in this experiment (FIG. 2). The method comprises the following specific steps:

taking the pod after pollination in the test field, and taking the pod 10-15 days after pollination. The pods are washed once with sterile water, then disinfected with 70% alcohol for 30 seconds, then disinfected with 0.8% sodium hypochlorite for 10min, and finally washed 3 times with sterile water, 5 minutes each time. The sterilized pods were placed in sterilized petri dishes with filter paper, the water on the pods was blotted, then the stems of the pods were carefully cut off with a sterilized knife and inoculated onto petri dishes containing germination medium (table 2), handling was done with sterility to avoid contamination, typically 10-12 pods were inoculated into one flask (fig. 2). After inoculation, the mouth and the cover of the culture bottle are burnt by an alcohol burner to sterilize and cover the bottle, and the bottle is placed into an incubator to be cultured under the conditions of 25 ℃, illumination of 16h.d-1 and illumination intensity of 20001 x. Observing the growth condition of the ovary on the culture medium and recording. Culturing for 30-60 days, transferring the grown cotyledon seedling (figure 3) to differentiation medium (figure 4) (table 2) to obtain test-tube seedling. After the seedlings grow to 4-5 leaves, the cover of the culture bottle is opened to train the seedlings after rooting, the culture medium is washed off, the seedlings are transplanted into nutrient soil to be cultured for 2 weeks (figure 5), the greenhouse culture condition is 25 ℃, and the illumination is 16h.d ^-1 And the illumination intensity is 20001x, transferring the greenhouse to a conventional cultivation management. The developed Rfo-specific marker (RFO-2F/RFO-NEW-R) was used to screen the distant hybrid population F1, and the number of positive individuals of Rfo was calculated.

During the embryo rescue period, the following data were observed and counted:

(1) the emergence rate of the hybrid ovary culture is observed and counted and is shown in table 1;

TABLE 1

As can be seen from Table 1, the seedling rate of B14 is 0.95%, the seedling rate of Rfo positive single plants is 0.26%, the seedling rate of 137 is 0.58%, and the seedling rate of Rfo positive single plants is 0.29%, which indicates that the distant hybridization seedling rates of different genotypes are different.

(2) Observing and counting ploidy of plants

And performing ploidy identification on the obtained interspecific hybrid F1 plant by using a flow cytometry method. Taking the relative content of the broccoli 2C DNA as a reference, the corresponding peak value of the G0/G1 stage is 150 channels, the cabbage type rape is 300 channels, and the ploidy of the hybrid progeny is judged according to the position of the peak value of the hybrid progeny G0/G1 stage. The ploidy identification result in FIG. 10 shows that the peak value of G0/G1 of the F1 hybrid plant is between 200 and 300, is between the broccoli and the Brassica napus, and is similar to triploid.

(3) Culture medium formula for hybrid ovary culture table 2

TABLE 2

(4) Morphological feature observation

Morphological evaluation showed that interspecific hybrids grew more vigorously than the parents (FIGS. 6 and 7), with leaf shapes, waxiness and flower colors between the parents, but some traits were more favorable to the rape parents (FIGS. 6-9). When the base of the petiole of the leaf has less splinter wax powder, the leaf color is light green mostly, and the leaf edge is in a sawtooth shape (figure 8); some flowers are larger than parents; there was a small amount of pollen on the stamens (FIG. 9). In conclusion, it can be seen that interspecific hybrids have a good genetic effect on the parental traits. At flowering, most of the positive individuals carrying RFO of the interspecific hybrids F1 have pollen, and some of the interspecific hybrids contain the RFO gene, but do not produce pollen, and only produce defective stamens, probably due to the heterologous abnormality of chromosome doubling. Interspecific hybrids that do not carry Rfo have no pollen during flowering.

Claims

1. A method for obtaining distant hybridization offspring of broccoli and brassica napus is characterized by comprising the following steps:

(2) after artificial pollination is carried out for 10-15 days, cutting off ovaries, disinfecting the ovaries, and putting the ovaries into a culture medium for in vitro culture, wherein the culture medium comprises an MS basal culture medium, 30g/L sucrose and 6g/L agarose, and the pH value is 5.8-6.02;

(5) after the seedlings grow to 4-5 leaves in the step (4), rooting, opening a cover of a culture bottle to harden the seedlings, washing off a culture medium, transplanting the seedlings into nutrient soil to culture for 2 weeks, transferring the seedlings into a greenhouse, and performing conventional cultivation and management;

in the step (3), the differentiation medium takes MS as a basic medium, and also comprises 30g/L of sucrose, 0.1mg/L of NAA, 1mg/L of 6-BA and 6g/L of agarose, and the pH value is 5.8-6.0;

in the step (4), the rooting culture medium takes MS as a basic culture medium, and also comprises 10g/L of cane sugar and 9g/L of agarose, and the pH value is 5.8-6.0;

in the step (1), the artificial pollination method comprises the following steps: stripping the female parent, respectively pollinating in the morning and at night, and smearing the fresh pollen of the male parent on the stigma of the female parent.

2. The method for obtaining progeny of distant hybridization of broccoli and brassica napus according to claim 1, wherein the method for sterilizing the ovary in step (2) comprises the following steps: sterilizing with 75% ethanol for 30s, sterilizing with 8% NaClO for 10min, washing with sterile water for 3 times, and sucking off water.