阅读说明：本技术 一种青花菜花球和籽粒高萝卜硫苷含量品种的选育方法 (Breeding method of broccoli ball and seed variety with high glucoraphanin content ) 是由 王建升 顾宏辉 虞慧芳 赵振卿 盛小光 沈钰森 于 2020-07-01 设计创作，主要内容包括：本发明公开了一种青花菜花球和籽粒高萝卜硫苷含量品种的选育方法,以小孢子培养获得的青花菜双单倍体(DH)系为父本,以多代回交转育获得的高萝卜硫苷青花菜品种的细胞质雄性不育系为母本,有性杂交获得的高萝卜硫苷青花菜新品种。本发明选育获得的高萝卜硫苷青花菜品种花球中萝卜硫苷含量为普通品种(市场上最高含量品种)的2.5倍以上,籽粒中萝卜硫苷含量为107微摩尔/克以上,且不含烯基化芥子油苷组分。(The invention discloses a method for breeding broccoli ball and seed variety with high glucoraphanin content, which uses a Double Haploid (DH) of broccoli obtained by microspore culture as a male parent, uses a cytoplasmic male sterile line of the broccoli variety with high glucoraphanin obtained by multi-generation backcross transformation as a female parent, and obtains a new variety of the broccoli with high glucoraphanin by sexual hybridization. The sulforaphane content in the high-sulforaphane broccoli variety curd obtained by the breeding method is more than 2.5 times of that of a common variety (the highest-content variety in the market), the sulforaphane content in grains is more than 107 micromoles/gram, and the high-sulforaphane broccoli variety curd does not contain alkenyl glucosinolate components.)

1. A method for selectively breeding the variety with high glucoraphanin content in broccoli balls and seeds is characterized in that a new variety of the broccoli with high glucoraphanin content is obtained by sexual hybridization by taking a double haploid line of the broccoli obtained by microspore culture as a male parent and taking a cytoplasmic male sterile line of the broccoli with high glucoraphanin obtained by multi-generation backcross transformation as a female parent.

2. The method for breeding the variety with high glucoraphanin content in broccoli bulbs and grains according to claim 1, which is characterized by comprising the following steps:

s1 female parent breeding

S11 culturing microspores with high sulforaphane broccoli ball and seed material as donor to obtain DH line;

s12 hybridizing the cytoplasmic male sterile broccoli material as female parent and the obtained high glucoraphanin broccoli DH line as male parent to obtain the first filial generation containing sterile source;

s13 using high sulforaphane broccoli DH line as recurrent parent, carrying out multiple generation backcross transformation to screen out excellent sterile single plant, continuously backcrossing to obtain genetic stable cytoplasmic male sterile line as female parent;

s2 male parent breeding

Selecting broccoli material without alkenyl glucosinolate component germplasm, carrying out microspore culture on individual plants with excellent curd character, and screening out DH strains with high glucoraphanin content, namely male parents;

s3 crossbreeding

And hybridizing the obtained female parent and the male parent, and screening out a first hybrid generation which has high sulforaphane content in the curd and the seeds and has excellent curd character, namely the dual-purpose high sulforaphane variety of the curd and the seeds.

Technical Field

The invention belongs to the technical field of plant breeding, and particularly relates to a method for breeding a variety with high glucoraphanin content in broccoli bulbs and grains.

Background

Broccoli (Brassica oleracea L.var. italica) also known as broccoli and cauliflower is a vegetable crop in Brassica species of Brassicaceae. Scientific research finds that the isothiocyanates generated by degrading some glucosinolates in broccoli have beneficial biological activity of human bodies, particularly the isothiocyanates generated by degrading sulforaphane (the scientific name is 4-methyl-sulfinylbutyl glucosinolate), namely sulforaphane (also called sulforaphane), can reduce the occurrence of various cancers, such as colon cancer, pancreatic cancer, bladder cancer and the like, and can also reduce the occurrence of cardiovascular diseases. As early as 2020, the official website of the U.S. clinical trial has been a clinical trial involving glucoraphanin or sulforaphane (https:// www.clin icaltrials. gov /).

Recently, the Saint Nerns corporation, USA, introduced a variety of glucosinolates of high radish

The sulforaphane content in the flower ball of the variety is more than 2 times of that of the common broccoli variety, and the flower ball is obtained by breeding a wild species Brassica villosa by a conventional breeding means by Richard F. Researches show that the content of the sulforaphane in the broccoli seeds (seeds) and the sprouts is about 10 times of that in the curd, so that the seeds or the sprouts germinated from the seeds are generally adopted as raw materials internationally to extract the sulforaphane for product research and development and production. In recent years, various health products, foods, and the like developed based on sulforaphane have been produced. However, various glucosinolate components are detectable in broccoli germplasm, sulfohydrocarbyl glucosinolates such as glucoraphanin are generally considered health beneficial components, while the efficacy of the alkenylated glucosinolate component is limited, particularly the 2-hydroxy-3-butenyl glucosinolate hydrolysate oxazolidine-2-thione, can cause goiter in some mammals. Thus, manufacturers have established requirements for the glucosinolate content and the amount of glucosinolate in seeds as a raw material for a variety or product of glucosinolates, such as seeds having a glucosinolate content of greater than 4.0% (about 91.5. mu. mol/g) and not having an alkenyl glucosinolate component such as 2-propenyl glucosinolate.

Disclosure of Invention

The invention aims to provide a method for breeding a variety with high glucoraphanin content in broccoli bulbs and seeds, and a novel variety with the same type of the broccoli bulbs and the seeds and the high glucoraphanin content and with independent intellectual property rights is bred. The method is characterized in that a homozygous breeding parent is quickly obtained by utilizing a microspore culture technology, and a new flower ball and grain dual-purpose variety which is high in glucoraphanin content and does not contain alkenyl glucosinolate is bred by combining a cytoplasmic male sterility approach.

The invention is realized by the following technical scheme:

a method for selectively breeding the variety with high glucoraphanin content in broccoli ball and seed includes such steps as culturing microspore to obtain Double Haploid (DH) of broccoli as male parent, backcrossing for multiple generations to obtain cytoplasmic male sterile line as female parent, and sexual hybridization to obtain the new variety of broccoli with high glucoraphanin content. The method specifically comprises the following steps:

s1 female parent breeding

S11 culturing microspore with high sulforaphane broccoli ball and seed material as donor to obtain DH line (double haploid);

s12 hybridizing cytoplasmic male sterile broccoli material as female parent and the obtained high glucoraphanin DH line as male parent to obtain the first filial generation containing sterile source (F) ₁)；

S13 using high sulforaphane broccoli DH line as recurrent parent, carrying out multiple generation backcross transformation to screen out excellent sterile single plant, continuous backcross to obtain genetic stable cytoplasmic male sterile line, namely female parent.

S2 male parent breeding

Selecting broccoli material without alkenyl glucosinolate component germplasm, carrying out microspore culture on individual plants with excellent curd character, and screening out DH strains with high glucoraphanin content, namely male parents.

S3 crossbreeding

And hybridizing the obtained female parent and the male parent, and screening out a first hybrid generation which has high sulforaphane content in the curd and the seeds and has excellent curd character, namely the dual-purpose high sulforaphane variety of the curd and the seeds.

The invention has the beneficial effects that:

1) the sulforaphane content in the high-sulforaphane broccoli variety curd is more than 2.5 times of that of the common variety (the highest-content variety in the market), and the sulforaphane content in grains is more than 107 micromoles/gram.

2) The average single ball weight of the flower ball is more than 400 g, the commodity is good, and the market requirement of the common flower ball can be met.

3) Because the male parent and the female parent are obtained by microspore culture, the degree of purity is high, the first filial generation of broccoli is uniform and consistent, the harvesting periods of the ball and seeds are concentrated, and the method is suitable for unified mechanical harvesting.

4) The obtained broccoli variety is prepared from a cytoplasmic male sterile line, can be pollinated by bees for reproduction of hybrid seeds and original stock seeds, and greatly reduces the cost of seed production.

5) The bulbs and seeds of the high-glucoraphanin broccoli variety do not contain alkenyl glucosinolate components.

Drawings

FIG. 1 is a flow chart of the breeding method of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a method for breeding broccoli ball and seed variety with high glucoraphanin content, which uses a Double Haploid (DH) line of broccoli obtained by microspore culture as a male parent, uses a cytoplasmic male sterile line of the broccoli variety with high glucoraphanin obtained by multi-generation backcross transformation as a female parent, and obtains a new variety of the broccoli with high glucoraphanin by sexual hybridization. The method mainly comprises the following steps:

1. Female parent breeding

1) And (4) carrying out glucoraphanin identification on the balls and grains of the introduced broccoli material respectively.

2) And (3) carrying out microspore culture by taking the identified high-sulforaphane material as a donor, and identifying the obtained sulforaphane of the DH line (double haploid) again.

3) Hybridizing the cytoplasmic male sterile broccoli material as female parent and the obtained high glucoraphanin broccoli DH line as male parent to obtain the first filial generation (F) containing sterile source₁)。

4) High-sulforaphane broccoli DH line is used as recurrent parent, multi-generation backcross transformation is carried out, and excellent sterile single plants are screened out through field agronomic character selection and identification.

5) After 6 generations of continuous Backcross (BC)₆) And obtaining the genetic stable cytoplasmic male sterile line, namely the female parent.

2. Male parent breeding

1) The introduced and created broccoli material is respectively subjected to glucoraphanin identification of the bulbs and grains, and germplasm materials which do not contain alkenyl glucosinolate components are screened.

2) Selecting excellent germplasm with a flower ball character from germplasm materials without alkenyl glucosinolate components, and mainly investigating important commodity characters such as the flower ball shape, the flower ball yield and the like.

3) Microspore culture is carried out on the individual plant with excellent flower ball character, and a DH strain with high glucoraphanin is identified and screened out, namely the male parent.

3. Hybrid seed selection

1) The genetically stable high sulforaphane anthocyanin broccoli cytoplasmic male sterile line obtained by transformation is taken as a female parent, broccoli DH line with excellent characters is taken as a male parent, and the first filial generation seed is obtained by hybridization combination preparation.

2) And performing field observation, cell comparison and planting evaluation on the obtained first-filial generation seeds to obtain a variety with excellent properties.

3) And (4) identifying the broccoli glucoraphanin by referring to a related patent method for the hybrid first-generation flower balls and seeds.

4) And screening the first filial generation with high sulforaphane content and excellent flower bulb character in the flower bulb and the seed, namely the flower bulb and seed dual-purpose high sulforaphane variety.