CN111248083A

CN111248083A - Breeding method of hybrid broccoli

Info

Publication number: CN111248083A
Application number: CN202010236197.4A
Authority: CN
Inventors: 张振超; 戴忠良; 潘永飞; 姚悦梅; 秦文斌; 山溪; 吴国平; 孙春青; 马志虎; 孙国胜
Original assignee: Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Current assignee: Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-06-09

Abstract

The invention discloses a breeding method of hybrid broccoli. The breeding method comprises the following steps: selecting broccoli inbred lines with excellent comprehensive properties for mutual hybridization and inbreeding, and planting the inbred lines in a greenhouse for additional generations to obtain seeds of F2 generations and double-cross seeds H2 generations; and (3) separating and purifying the optimized single plant by adopting a microspore culture technology, doubling chromosomes, and screening to obtain a DH strain with excellent comprehensive properties. The selfing line 08103 and the cabbage cytoplasmic male sterile line CMS116 are continuously backcrossed for 6 generations to obtain the cytoplasmic male sterile line. The obtained DH strain and the cytoplasmic male sterile line are hybridized, prepared and combined, and the field quality ratio identification is carried out. The method adopts a conventional breeding means to polymerize excellent genes, combines a microspore culture technology, can quickly obtain a DH strain and a cytoplasmic male sterile line, and has important significance for improving the breeding efficiency and accelerating the breeding process.

Description

Breeding method of hybrid broccoli

Technical Field

The invention relates to a plant breeding method, in particular to a breeding method of hybrid broccoli.

Background

Broccoli (Brassica oleracea var. italica), also known as broccoli, is a variety of Brassica vegetables in Brassica of brassicaceae, and is known as "vegetable crown" because the nutritional ingredients such as protein, fat, dietary fiber, sugar, carotene, vitamin, calcium, phosphorus, iron, and the like, which are rich in the body, are in the head of the similar vegetables. In addition, the broccoli is a functional vegetable, and the anticancer active ingredient sulforaphane contained in the broccoli can obviously reduce the morbidity of various cancers, such as liver cancer, gastric cancer, lung cancer, breast cancer, colorectal cancer, prostatic cancer and the like, and has certain effects on preventing the occurrence of cardiovascular and cerebrovascular diseases, treating myopia, regenerating skin and the like. In recent years, with the gradual deepening of people's understanding of eating methods and nutritional values, the demand of broccoli in markets at home and abroad is increased year by year, the export and the domestic sales are both strong, the annual production area of vegetables is increased year by year in China, the cultivation benefit is prominent day by day, and the method has very important economic and social benefits.

Since the nineties of the last century, as the demand of the foreign market for broccoli increases, the business opportunity of exporting broccoli products to earn foreign exchange is gradually shown, and foreign varieties are introduced to carry out trial production in China. With the increase of planting benefits, the broccoli planting area is enlarged, and the work of introduction, innovation and new variety breeding of broccoli germplasm resources is gradually developed in part of colleges and universities and scientific research and breeding units in China. The broccoli is originated from the coast of the Mediterranean sea, the domestic germplasm resources are deficient, the seed price is high, the foreign excellent germplasm is difficult to obtain, and the adverse factors restrict the development of the domestic broccoli excellent variety breeding work. In view of this, domestic breeding units have been introduced with a large number of foreign different types of commercial varieties prepared from self-incompatible lines by cooperating with foreign seed companies, and have been bred into new varieties by obtaining self-incompatible lines with stable genetic traits through conventional breeding means. With the application of male sterility in broccoli breeding, commercial varieties in the market can not obtain inbred line breeding materials in a self-crossing separation mode, so that the further development of germplasm resource innovation is restricted, and the process of new variety breeding is hindered.

Application No. 201210361576.1 discloses a breeding method of early-maturing broccoli in generation-adding breeding, which is mainly characterized in that in a temperature-controlled greenhouse, at least 5 generations of collected broccoli breeding materials are subjected to continuous selfing and directional selection to obtain a favorable selfing line, a cytoplasmic male sterile line is bred through 5 generations of backcrossing selection, a hybrid combination is prepared with an excellent selfing line, and the hybrid combination with excellent comprehensive properties is obtained through field quality ratio identification. The method is feasible in practical application, but the conventional breeding means needs at least 10 generations of transformation process for breeding the inbred line and the cytoplasmic male sterile line with stable genetic characters, wastes time and labor and has lower efficiency.

Disclosure of Invention

Aiming at the problems of deficient broccoli breeding resources and low breeding efficiency, the breeding method of broccoli germplasm resource innovation, creation of cytoplasmic male sterile line materials, new variety breeding and quality ratio identification is provided, the breeding process is accelerated, the breeding efficiency is improved, and the production requirement is met. In order to meet market demands, a large number of homozygous stable inbred lines obtained by introducing broccoli germplasm resources at the early stage are taken as a basis, single hybrid lines and double hybrid lines are obtained by crossing among different types, then single plants with excellent comprehensive properties are preferably selected from separated offspring to culture isolated microspores, a large number of haploid pure lines are rapidly obtained, chromosomes are doubled to form DH plants, after the single plants with excellent comprehensive properties are preferably selected, a hybridization combination is prepared with cytoplasmic male sterile line materials obtained by transformation, and the combination with excellent comprehensive property performance is determined to be a new variety to be popularized and applied by cultivating and quality ratio under different cultivation conditions. The method improves the breeding efficiency, shortens the breeding time of new varieties and has wide market application potential.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a method for breeding hybrid broccoli comprises the following steps:

(1) selecting broccoli inbred lines 0812, 0836, 0858, 0887, 08103 and a common head cabbage cytoplasmic male sterile line CMS116 with excellent comprehensive properties, and hybridizing the inbred lines 0812 and 0836, 0858 and 0887 and 08103 and CMS116 under flowering phase and isolation conditions to obtain F1-1, F1-2 and F1-3 hybrid generation seeds;

(2) carrying out generation-adding planting on the F1-1 and F1-2 hybrid seeds, namely sowing and raising seedlings in a temperature-controlled greenhouse at 15-30 ℃, planting after seedling formation, and carrying out normal field management; during the whole growth period, according to the growth vigor and the agronomic characters of the plants, removing the plants with weak growth vigor, poor flower ball character and poor disease resistance, and selecting and reserving 3-5 broccoli plants with excellent comprehensive characters; in the flowering phase, F1-1 and F1-2 are selfed and then hybridized to obtain F2-1, F2-2 hybrid second generation seeds and double hybrid seeds H1; backcrossing an inbred line 08103 serving as a male parent and F1-3 serving as a female parent to obtain backcrossed first-generation seeds BC1, selecting a single plant with similar agronomic characters to the inbred line male parent 08103 from backcrossed progeny of BC1 as the female parent, backcrossing the plant with the inbred line male parent 08103 under flowering time and isolation conditions, sowing the obtained seeds, performing directional selection under generative planting conditions, and continuously backcrossing the seeds with the inbred line male parent 08103 for 6 generations to obtain a cytoplasmic male sterile CMS08103 with consistent genetic characters of the inbred male parent 08103;

(3) planting the H1 seeds obtained in the step (2) in a temperature-controlled greenhouse at 15-30 ℃, carrying out normal field management, selecting and reserving 3-4 plants which are robust in growth, free of plant diseases and insect pests, normal in development and round in flower ball according to plant growth vigor and agronomic system characters in the whole growth period, and carrying out selfing and seed reservation under the flowering period and isolation conditions to obtain H2 seeds;

(4) planting the obtained seeds of F2-1, F2-2 and H2 in a temperature-controlled greenhouse at 15-30 ℃, selecting and reserving single plants with excellent comprehensive properties during growth to culture free microspores to obtain a homozygous plant population, and observing and screening a large number of double haploid systems DH with excellent comprehensive properties;

(5) taking the broccoli cytoplasmic male sterile line CMS08103 obtained by the transformation in the step (3) as a female parent, taking a DH plant obtained by microspore culture as a male parent, and preparing a hybrid combination under an isolation condition to obtain a first generation hybrid;

(6) and (5) carrying out field planting and quality ratio identification on the first-generation hybrid seeds obtained in the step (5) to breed a new variety with excellent performance after hybridization.

As an improvement, the standard of excellent comprehensive properties in the step 1 is as follows: the plant is strong, the growth is vigorous, the black rot, the sclerotinia and the downy mildew are resisted, the cold resistance is good, the flower ball is round and compact, the bud grain is fine and uniform, the color of the surface of the flower ball is gray green, the development of the flower organ is normal, and the self-fertility is good.

As an improvement, the shape of the single plant selected from the BC1 backcross progeny in the step 2, which is similar to the shape of the self-bred line male parent 08103, refers to plant height, openness and/or flower ball character.

The improvement is that 5-10 excellent individual plants are respectively selected from F2-1, F2-2 and H2 separated offspring in the step 4 to culture isolated microspores, which is beneficial to reducing the separation range, and 20-30 parts of DH line breeding materials with stable heredity can be rapidly obtained after 0.1% -0.3% of the obtained regeneration plants are artificially doubled.

The improvement is that the excellent performance after hybridization in the step 5 refers to strong winterness, good cold resistance, strong plants, vigorous growth, moderate development degree, black rot, sclerotinia and downy mildew resistance, round and compact flower balls, fine and uniform bud grains, high content of wax powder on the surface of the flower balls, gray green color and male sterility.

Has the advantages that:

compared with the prior art, the breeding method of the hybrid broccoli has the advantages that:

1) by preferably selecting plants with excellent comprehensive characters from the separated progeny of the single cross hybrid F2 and the double cross hybrid H2, most of invalid character genes are eliminated to a certain extent, the number of the excellent character genes is reduced, and the excellent gene polymerization is facilitated;

2) the isolated microspore culture technology is adopted to culture the preferred single plants of F2 and H2, and the obtained regenerated plant population has higher single plant proportion with excellent comprehensive properties and performance, thereby improving the breeding efficiency and accelerating the breeding process;

3) by adopting the generation-adding planting method, flowering and seed setting twice a year can be realized, so that the breeding process is accelerated, and the breeding efficiency is improved;

4) the cytoplasmic male sterile line is obtained by backcross transformation of the inbred line with excellent comprehensive properties, the sterility is stable, the sterile plant rate and the sterility degree reach 100 percent, the flower organ is complete and normal in development, the flowering and fruiting performance is good, the growth vigor is vigorous, and the combining ability is strong;

5) f1 progeny prepared by using the cytoplasmic male sterile line as a female parent and using the obtained DH line as a male parent is also a cytoplasmic male sterile line, which is favorable for protecting the intellectual property of the variety; the hybrid seeds prepared by the sterility have high seed yield, low seed production cost and labor and time saving;

6) the new germplasm has excellent comprehensive characters, the disease resistance and stress resistance of filial generation are obvious, the yield is increased obviously, the efficiency is improved by more than 1 time compared with the traditional method, the breeding time of new varieties is shortened by 2-3 years, and the method has good market application prospect.

Drawings

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

Detailed Description

The present invention is further illustrated by the following figures and specific examples, which are to be construed as merely illustrative and not limitative of the remainder of the disclosure, and all changes and modifications that would be obvious to those skilled in the art are intended to be included within the scope of the present invention and the appended claims are intended to be embraced therein.

The agricultural scientific research institute of Zhenjiang in Jiangsu hilly area can provide all the seeds used in the embodiment of the invention.

The hybridization method mentioned in the invention is that the flowering phase is adjusted according to the growth habits of different inbred lines and sterile lines when sowing, when the flowering phase is not met, the main flower ball of the earlier-flowering plant is removed to promote the development of lateral branches, when the flowering phases meet, the opened flower and small flower buds on the mother plant are removed, the flower buds opened in the next day are manually stripped to remove the male parent pollen, and finally the whole plant is bagged and isolated;

the generation-adding planting method comprises planting all broccoli breeding materials in a greenhouse with controllable temperature, humidity, sufficient light, convenient fertilization and watering, the temperature can be controlled at 15-30 deg.C, and the broccoli breeding materials can be planted and bloomed for 2 times a year;

the double hybrid has 4 excellent parent genes polymerized, and may be used in obtaining breeding material with excellent comprehensive character and stable inheritance through multiple generation selfing separation and screening.

A method for breeding hybrid broccoli comprises the following steps:

step 1, selecting the plant height, the plant spread, the leaf blade and flower bulb surface wax powder content, the spherical shape and the size, the non-abortive bud of the flower bulb, the good fructification, the strong stress resistance and the strong disease resistance, and the vigorous growth of the broccoli inbred lines 0812, 0836, 0858, 0887, 08103 and the cabbage cytoplasmic male sterile line CMS116 as breeding materials from breeding materials, performing plug-seedling in a tray sowing and seedling culture in 8 months and 20 days, planting the seedlings in 9 months and 20 days to a parent garden in Jiangsu province sentence city, and performing normal field management. During the growth period, weak seedlings, abnormal plants and diseased plants are removed, and plants with typical characters of the inbred line are selected and reserved. In the next year, about one month before flowering, most of main flower balls are cut when the weather is fine, other flower branches are stimulated to be normally taken out, and carbendazim or thiophanate methyl solution is coated on the cut immediately after cutting to prevent plants from rotting. In the full bloom stage of 3-4 months, the inbred lines 0836 and 0887 and the sterile line CMS116 are used as female parents, 5-10 branches are reserved for each plant, 15-20 flower buds to be opened are reserved for each branch in the next day, manual bud stripping and emasculation are carried out, pollen of the inbred lines 0812, 0858 and 08103 (male parents) is respectively smeared on the emasculated stigma, the rest small flower buds and the opened flowers on the branches are removed and strictly marked, and then sulfuric acid paper bags are sleeved on the branches and tied for isolation. F1 generation hybrids were harvested in months 5-6, numbered F1-1, F1-2 and CMS-3, respectively.

And 2, carrying out additive planting on the F1-1, the F1-2, the CMS-3 and the 08103 in a greenhouse with the temperature controlled to be 15-30 ℃. In the middle of 6 months, the seeds are vernalized, sowed and grown in plug trays, and fixed planted in the middle of 7 months, the growth temperature is controlled at 18-25 ℃, and the field management is performed normally. Selecting 10 plants which grow vigorously, have good flower ball character and no plant diseases and insect pests in the last ten days of 9 months, cutting flower balls, and reserving 5-10 flower branches for each plant. When bolting and blooming in late 9 months, carrying out artificial bud period and flowering period selfing pollination on an F1-1 strain and an F1-2 strain under isolation conditions to obtain seeds of F2-1 and F2-2; then, a strain F1-1-1 is selected as a male parent and a strain F1-2-1 is selected as a female parent to be hybridized under the isolation condition, and the obtained hybrid seed is a double-cross seed which is marked as H1.

Meanwhile, CMS-3 is used as a female parent, 08103 is used as a male parent, backcross is carried out under strict isolation conditions, and the obtained seeds are backcross first-generation seeds and are marked as BC 1. Selecting a single plant which has the closest agronomic characters with the male parent 08103, including plant height, expansion degree, flower ball characters and the like, from BC1 backcross offspring as the female parent, backcrossing with the transferred male parent under the flowering phase and isolation conditions, sowing the obtained seeds, performing directional selection under the condition of generative planting, and continuously backcrossing with the transferred male parent for 6 generations to obtain the cytoplasmic male sterile CMS08103 with the genetic character consistent with that of the transferred male parent.

If the flowering period of each seeding material is not in contact, the main branch with earlier flowering can be removed, the lateral branch can be promoted to develop, and the flowering period can be adjusted. The seeds are mature from the beginning of 11 late months to 12 months, the seeds are harvested and tested for each plant according to the condition, and the obtained seeds are dried in the sun after being marked.

And 3, in the middle of 12 months, performing vernalization treatment on seeds of F2-1, F2-2 and H1, sowing and raising seedlings by using plug trays, and planting 300 plants of F2-1 and F2-2 and 50 plants of H1 in the middle and last ten days of the next 1 month. Controlling the temperature in the greenhouse at 18-25 ℃, and performing normal field management. During the period from field planting to flowering, weak seedlings and abnormal plants are pulled out according to the botanical traits and the agronomic traits, typical plants with strong growth vigor, round and round flower balls, gray green ball color, thin and uniform flower buds, good disease resistance and stress resistance and normal flower bud development are selected, and 5-10 plants are selected for each plant line. When flowering in 3-4 months, selecting buds from the late stage of excellent mononuclear to the early stage of binuclear for free microspore culture, and performing broccoli microspore culture and plant regeneration method (study on microspore culture and plant regeneration of cabbage plants, Zhang Zhen et al, nuclear agriculture report, 2013,27(7):0929 and 0937), and performing self-pollination on the selected single plants. And (5) harvesting mature seeds from a single plant at the beginning of 6 months according to the pollination condition. The seeds obtained by F2 individual plant self-crossing are marked as F3 generation seeds, and the seeds obtained by H1 self-crossing are marked as H2.

And 4, sowing the seeds obtained in the step 3 again in the middle of the month of 6, planting 150 seeds in the F3 generation and planting 200 seeds in the H2 generation respectively. When bolting and flowering in 9 months, the selected plants are subjected to artificial selfing pollination, and F3 and H2 generations are selected to optimize buds from late mononuclear stage to early binuclear stage of the single plants for free microspore culture and plant regeneration tests, and the method is the same as above, and the selected single plants are subjected to selfing pollination simultaneously. And (5) harvesting mature seeds of the single plant at the beginning of 12 months according to the pollination condition. The seeds obtained by F3 individual plant self-crossing are marked as F4 generation seeds, and the seeds obtained by H2 self-crossing are marked as H3.

And 5, in the third year, carrying out subculture and rooting culture on the regenerated plants obtained in the steps 3 and 4 by the isolated microspore culture technology in a laboratory. At the beginning of 8 months, all the tissue culture seedlings with strong root systems are carefully taken out from the tissue culture bottle after hardening in a laboratory for 1 week, treated by 0.1-0.3% colchicine solution and transplanted to a 72-hole plug tray filled with a special vegetable seedling culture substrate, and then covered with a black shading net for shading and moisturizing. After growing for 15 days, 6521 plants which are strong and have developed root systems are planted in the field at the beginning of 9 months. During the growth period, screening the microspore regeneration plant population of each line according to the standard, and selecting 73 individual plants with excellent comprehensive properties.

And 5, taking 73 microspore regeneration plants as male parents and CMS08103 as female parents, preparing hybridization combinations, carrying out field identification and screening in autumn, measuring the combining ability, and finally breeding to obtain 4 medium-early-maturing combinations with excellent comprehensive properties, namely CMS08103 multiplied by MD-18, CMS08103 multiplied by MD-26, CMS08103 multiplied by MD-45 and CMS08103 multiplied by MD-70.

Through the selection of the inbred line with excellent comprehensive characters, the excellent character genes are stably inherited to filial generations through a genetic breeding means, individual plants with parent genes polymerized are selected from F2 and H2 generations, the individual plants are quickly purified by adopting an efficient free microspore culture technology, so that 73 excellent DH strains are obtained in a short time, and a generation-adding planting method is adopted, so that flowering and seed setting twice a year can be realized, the breeding process is accelerated, and the breeding efficiency is improved; the inbred line with excellent comprehensive properties is transformed into a cytoplasmic male sterile line (table 1) as a female parent and a DH line as a male parent to prepare hybrid seeds, so that the seed yield is high, the seed production cost is low, the labor and the labor are saved, and the intellectual property of the variety is protected; the new germplasm has excellent comprehensive characters, the disease resistance and stress resistance of filial generation are obvious, the yield is increased obviously, and the new combination is tested in a plurality of places (Table 2).

TABLE 1 cytoplasmic Male sterile line CMS08103 of broccoli and major agronomic traits

Table 24 major agronomic traits for the combinations of crosses

On the basis of the patent of application No. 201210361576.1, the invention selects plants with excellent comprehensive characters from the progeny separated by single cross hybrid F2 and double cross hybrid H2, eliminates most invalid character genes to a certain extent, reduces the number of excellent character genes, is beneficial to excellent gene polymerization, and has higher proportion of single plants with excellent comprehensive characters in the obtained regeneration plant population after separation by adopting the isolated microspore culture technology, thereby improving the breeding efficiency and accelerating the breeding process.

Claims

1. A breeding method of hybrid broccoli is characterized by comprising the following steps:

(6) and 3, taking the broccoli cytoplasmic male sterile line CMS08103 obtained by the transformation in the step 3 as a female parent, taking a DH plant obtained by microspore culture as a male parent, and preparing a hybrid combination under an isolation condition to obtain a first-generation hybrid.

2. A breeding method of hybrid broccoli as claimed in claim 1, wherein the criteria for the superior comprehensive traits in step 1 are: the plant is strong, the growth is vigorous, the black rot, the sclerotinia and the downy mildew are resisted, the cold resistance is good, the flower ball is round and compact, the bud grain is fine and uniform, the color of the surface of the flower ball is gray green, the development of the flower organ is normal, and the self-fertility is good.

3. A method as claimed in claim 1, wherein the shape of the individual plant selected from the BC1 backcross progeny in step 2 is similar to the shape of the inbred line male parent 08103, and is characterized by plant height, flowering and/or curd character.

4. A breeding method of hybrid broccoli as claimed in claim 1, wherein in step 4, 5-10 excellent individual plants are selected from among F2-1, F2-2 and H2 segregation progeny respectively for free microspore culture, which is beneficial to reduce the segregation range, and 20-30 parts of genetically stable DH line breeding material can be obtained quickly after 0.1% -0.3% of the obtained regeneration plants are artificially doubled.

5. The method as claimed in claim 1, wherein the excellent performance after hybridization in step 5 refers to strong winterness, good cold resistance, robust plants, vigorous growth, moderate development degree, resistance to black rot, sclerotinia and downy mildew, round and compact ball, fine and uniform bud, high wax powder content on the surface of ball, gray green color, and male sterility.