CN111837934A - Breeding method of broccoli ball and seed variety with high glucoraphanin content - Google Patents

Abstract

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.

Description

Breeding method of broccoli ball and seed variety with high glucoraphanin content

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.

Example 1

As shown in fig. 1, this embodiment specifically provides a breeding method of a variety 'zhe qing 803' with high glucoraphanin content of cauliflower balls and grains, which comprises the following steps:

1. female parent breeding

1.1 through identification, the broccoli inbred line 'IL 609' is a high glucoraphanin germplasm and a high glucoraphanin grain material.

1.2 microspore culture of 'IL 609' to obtain 22 DH strains, and identifying to obtain a high sulforaphane material which is named as 'DH 803'.

1.3 hybrid pollination is carried out by taking cytoplasmic male sterile broccoli variety 'excellent' with excellent character as female parent and high glucoraphanin 'DH 803' as male parent to obtain first hybrid generation (F) ₁)。

1.4 taking high sulforaphane 'DH 803' as a recurrent parent, carrying out multi-generation backcross transformation, and screening out excellent sterile single plants through field agronomic character selection and identification.

1.5 continuous transformation backcross for 6 generations (BC)₆) The sterile line with stable male sterility and the content and the character of glucoraphanin and the like basically consistent with the recurrent parent 'DH 803' is obtained and named as 'DH 803A'.

2. Male parent breeding

2.1 identification of 60 parts of germplasm material without the alkenylated glucosinolates.

2.2 the 60 germplasm materials are mainly used for investigating commodity characters such as flower ball shape, color, yield and the like, and an inbred line '1404' with excellent characters is obtained.

2.3 microspore culture of '1404', identification and screening to obtain DH strain without alkenyl glucosinolate, named 'DH 404'.

3. Breeding of hybrid Zhejiang blue 803

3.1 obtaining a first hybrid generation using the obtained male sterile line 'DH 803A' as female parent and DH line 'DH 404' with excellent flower ball character as male parent.

The first filial generation of broccoli is medium-maturing and the ball is harvested after planting for about 80 days; the plant growth vigor is strong, the plant type is semi-vertical, and the number of branches is large; the leaves are feather leaves, and the color of the leaves is darker; the sitting ball is higher, the flower ball is flat, the bud color is deep, the flower is purple at low temperature, the flower stalk is longer, and the weight of a single ball is about 450 g.

3.2 the first filial generation seeds are observed in the field, compared in the plot and evaluated in the planting, the plants are neat and consistent, the plant types are semi-vertical, the sitting ball is high, the flower ball is oblate, the color is deep, the flower stalk is long, the commodity is good, the average single ball weight of the flower ball is about 450g, and the plant is named as 'Zhe Qing 803'.

TABLE 1 detection of glucosinolates in the broccoli 'Zhe Qing 803' curd (micromole/g, dry weight)

TABLE 2 detection of glucosinolates in broccoli 'Zhe Qing 803' seeds (micromole/g, fresh weight)

As can be seen from tables 1 and 2, the sulforaphane content in the 'Zhejiang green 803' flower ball and kernel was 12.8 micromoles/gram and 148.2 micromoles/gram, respectively, without any alkenylated glucosinolates.

The sulforaphane content in the 'Zhe green 803' curd is 12.8 micromoles/gram, which is 3.1 times and 4.2 times of 'excellent' and 'excellent cold resistance' of broccoli varieties respectively; the sulforaphane content in the seeds is 1.8 and 2.1 times of 'excellent' and 'excellent cold resistance' of common broccoli varieties; neither the ball nor the kernel contained the alkenyl glucosinolates.

The broccoli variety Zhe Qing 803 meets the germplasm standard of high-sulforaphane curd and the germplasm standard of high-sulforaphane seeds at the same time, and the curd has better curd commodity and is a high-sulforaphane variety for both curd and seeds.

EXAMPLE 2 seed production technique

1. Propagation of parents

Adopts a plastic greenhouse seed production method. The male parent directly adopts the seeds and is closed in the greenhouse, selfing and fructification. The female parent sterile line 'DH 803A' and the corresponding maintainer line 'DH 803' are planted according to the proportion of 2:1, bumblebees are released in the flowering phase to pollinate, the maintainer line is pulled out in the fruiting period, and the seeds received on the sterile line are the female parent seeds.

2. Hybrid seed production technology

The first generation hybrid seeds are produced in open air because of large production amount.

1) Strictly isolating the seed production base: the open-air seed production base generally adopts space isolation, namely, the seed production field can not plant other varieties or crops which can be hybridized with broccoli within 2 kilometers of square circle.

2) Adjusting the sowing period to ensure that the male parent and the female parent meet at the flowering period: the flowering phase of the female parent sterile line 'DH 803A' is generally 10-15 days earlier than that of the male parent 'DH 404', and the flowering phases of the male parent and the female parent are forced to meet by adjusting the sowing time according to local climate in the hybrid seed production process, so that the seed collection amount is improved.

3) Planting proportion: the male parent and the female parent are planted alternately according to the proportion of 1:2, and the female parent in two ridges and the male parent in one ridge are planted alternately.

4) Field management: in Hangzhou region, the female parent sterile line 'DH 803A' is generally sown in late 8 months, the male parent 'DH 404' is sown in late 9 months, the seedling stage is about 25 days, and the male parent is transplanted into the greenhouse, and the row spacing is 45 x 55 cm. In 3 months of the next year, the bumblebee flowers and releases to carry out supplementary pollination. Before blooming, the prevention and control of plant diseases and insect pests, the control of water and fertilizer and the pulling out of the plants are required to be enhanced. When the flower is blooming, the timely supplement of the fertilizer and water is noticed.

In Yunnan, the female parent sterile line 'DH 803A' is generally sown in the middle ten days of 8 months, the male parent 'DH 404' is sown in the late 8 months, the seedling stage is about 25 days, and the male parent is transplanted to an open-air base, and the row spacing is 45 multiplied by 55 cm. In 1 month, the bumblebee flowers in the second year, and supplementary pollination is released. Before blooming, the prevention and control of plant diseases and insect pests, the control of water and fertilizer and the pulling out of the plants are required to be enhanced. When the flower is blooming, the fertilizer and water are supplemented timely.

5) Timely harvesting seeds: when the horn fruit is yellow and ripe, the seeds harvested from the female parent sterile line are the first filial generation seeds.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (2)

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.

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