CN114287339A

CN114287339A - Cultivation method of green cauliflower rich in beneficial glucosinolates and low in useless glucosinolates

Info

Publication number: CN114287339A
Application number: CN202111566694.1A
Authority: CN
Inventors: 丁云花; 刘光敏; 宋曙辉; 赵学志; 何洪巨
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-04-08
Anticipated expiration: 2041-12-20
Also published as: CN114287339B

Abstract

The invention discloses a cultivation method of a green cauliflower rich in beneficial glucosinolates and low in useless glucosinolates. The method disclosed by the invention comprises the following steps: selfing Luhui to obtain a selfing line, selecting a selfing compatible line as a recurrent parent to carry out backcross transformation, and obtaining a cytoplasmic male sterile line; and (3) taking a strain with the RAA content higher than 90 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 95 mu mol/gDW and the PRO content lower than 1 mu mol/gDW in the inbred line as a male parent, and taking a strain with the RAA content higher than 80 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 85 mu mol/gDW and the PRO content lower than 1 mu mol/gDW in the cytoplasmic male sterile line as a female parent to carry out hybridization, thereby finishing the cultivation of the target cauliflower. The method of the invention is utilized to breed the new green cauliflower variety with high content of beneficial glucosinolate and low content of non-beneficial glucosinolate.

Description

Cultivation method of green cauliflower rich in beneficial glucosinolates and low in useless glucosinolates

Technical Field

The invention relates to a cultivation method of green cauliflowers rich in beneficial glucosinolates and low in useless glucosinolates, belonging to the field of plant breeding.

Background

The big health industry is an emerging industry developed in recent years and is one of the most concerned hot spots of people at present. In the vegetable health industry, broccoli (green broccoli) is a very important vegetable, which contains not only nutrients such as vitamin C, dietary fibers, proteins, soluble sugars, mineral elements, etc. possessed by other vegetables, but also Glucosinolates (Glucosinolates, abbreviated as Glucosinolates). The varieties of glucosinolates are various, and 3-methyl sulfur oxygen allyl glucosinolate (IBE), 2-hydroxy-3-butenyl glucosinolate (PROGOITRIN, PRO for short), 2-propenyl glucosinolate (Sinigrin, SIN), 4-methyl sulfur oxygen butyl glucosinolate (Glucoraphanin, RAA for short) and 3-butenyl glucosinolate (Gluconapin, NAP for short) mainly exist in green cauliflower; 4-hydroxyindolyl-3-methylthioglucosinolate (4-hydroxyglucopyranoside, abbreviated as 4OH), 4-methylthiobutenyl glucosinolate Glucoerucin, ERU), 3-methylindolyl glucosinolate (Gluconrasicin, abbreviated as GBC), 4-methoxyindolyl-3-methylthioglucosinolate (4-methoxyglucopyranoside, abbreviated as 4ME), 1-methoxyindolyl-3-methylthioglucosinolate (Neoglucobrasicin, abbreviated as NEO). At present, the known glucosinolates beneficial to human bodies comprise RAA, 4OH, GBC, 4ME, NEO and the like, and some bioactive substances generated by hydrolysis have the effects of resisting oxidation, delaying senescence, preventing cancers, resisting cancers and the like and have important effects on the health of the human bodies; thioglycosides, such as PRO, which are undesirable for human health, are precursors of goitrogen 5-ethenazolidine-2-thione. RAA is the most main component of glucosinolate in the cauliflower, and the RAA content occupies the first place in various vegetables, so the cauliflower has the reputation of 'the king of anticancer vegetables'. However, the RAA content of the green cauliflower has larger difference among different varieties, and the variation range can be more than ten times. Researches show that the content of glucosinolates in the cauliflower can be changed through genetic improvement, the glucosinolate content of the same variety has correlation in different periods of seeds, sprouts, curd and the like, and the seeds and the sprouts have the highest content and the curd is used for the second time. With the deep understanding of people on glucosinolates, the demand and market for extracting beneficial glucosinolates by utilizing the cauliflower seeds are continuously expanded, and the cultivation of cauliflower varieties rich in beneficial glucosinolates and low in useless glucosinolates is more and more important.

Disclosure of Invention

The invention aims to solve the technical problem of how to cultivate the green cauliflower rich in beneficial glucosinolate and low in useless glucosinolate, in particular to how to cultivate the green cauliflower rich in beneficial glucosinolate and low in useless glucosinolate in seeds.

In order to solve the technical problems, the invention firstly provides a method for cultivating green cauliflowers, which comprises the following steps:

1) carrying out continuous multi-generation selfing on the green cauliflower variety green glow to obtain a genetically stable selfing line; measuring the self-compatibility index through pollination in the flowering phase to obtain a self-compatibility line with the flowering phase self-compatibility index more than 2;

2) carrying out continuous multi-generation backcross on the recurrent parent by using the self-compatible line obtained in the step 1) and a cytoplasmic male sterile source of the green vegetable flower to obtain a cytoplasmic male sterile line with the morphological characters (such as plant and ball morphology) same as or similar to those of the recurrent parent except fertility;

3) detecting the content of glucosinolate in the self-bred line obtained in the step 1) and the cytoplasmic male sterile line obtained in the step 2), selecting a line with the RAA content higher than 90 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 95 mu mol/gDW and the PRO content lower than 1 mu mol/gDW in the self-bred line as a male parent, and selecting a line with the RAA content higher than 80 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 85 mu mol/gDW and the PRO content lower than 1 mu mol/gDW in the cytoplasmic male sterile line as a female parent for hybridization, wherein the first filial generation is a target green vegetable flower line, and the cultivation of the target green vegetable flower is realized.

The self-compatibility index of the flowering phase is the number of seeds/pollinated flowers.

In the above method, the cytoplasmic male sterile source of brassica campestris can be verdure No. 2.

In the above process, the thioglucoside may be IBE, PRO, SIN, RAA, NAP, 4OH, ERU, GBC, 4ME and/or NEO.

In the above method, the backcross in step 2) may be five backcross generations.

And selecting a single plant with biased plant and ball morphology or similar to the recurrent parent and pollen abortion as a female parent in each generation of backcross.

In the method, the selfing in the step 1) is carried out in a single-seed transmission mode.

The number of selfing generations in step 1) may be determined as appropriate, as long as the conditions are satisfied to select a selfing line satisfying the conditions in step 1). In one embodiment of the present invention, the number of generations of the selfing is six.

In the method, the seeds of the cultivated target cauliflower can be directly obtained.

The application of the method for cultivating the green cauliflower rich in the beneficial glucosinolates in cultivating the green cauliflower also belongs to the protection scope of the invention.

The application of the method for cultivating the green cauliflower with low content of the useless glucosinolates also belongs to the protection scope of the invention.

The application of the method for cultivating the green cauliflower rich in the beneficial glucosinolates and low in the non-beneficial glucosinolates also belongs to the protection scope of the invention.

In the present invention, the beneficial thioglycoside may be RAA, GBC, NEO, 4OH and/or 4 ME. The non-beneficiary thioglycoside may be PRO.

The invention provides a method for cultivating healthy functional cauliflower with beneficial glucosinolates improved and beneficial glucosinolates reduced, and a new variety of cauliflower with high content of seed beneficial glucosinolates RAA, GBC, NEO, 4OH and 4ME and low content of non-beneficial glucosinolates PRO is bred by using the method.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

HPLC detection of glucosinolate (IBE, PRO, SIN, RAA, NAP, 4OH, ERU, GBC, 4ME, NEO) content in broccoli is carried out as follows:

(1) extraction of glucosinolates

The seeds to be tested were dried with a vacuum freeze dryer, and 0.2g of the pulverized freeze-dried sample was weighed and placed in a 15mL plastic tube. Adding 0.25mL of internal standard TRO (benzyl thioglycoside), quickly adding 100% preheated methanol, bathing for 20min at 80 ℃, and vortexing once every 4-5 min. Centrifuging at 3000r/min for 10 min, taking supernatant, pouring into a 15mL plastic tube, and placing in an ice basin. Extracting the precipitate twice with 70% methanol, treating as above, and mixing the supernatant to obtain sample solution. A disposable syringe is taken, glass wool is added into the syringe and the syringe is compacted to 0.5ml, and the syringe is placed on a test tube. 2mL of DEAE gel solution was added, washed twice with 1mL of double distilled water, and then 2mL of the sample solution was added. When the sample solution was not dropped any more, 0.02M NaAc solution was added. After no more liquid was dropped, the syringe was transferred to another tube and 75 μ L of the sulfatase solution was added and sealed overnight.

The DEAE gel solution preparation method comprises the following steps: weighing 10g of DEAE Sephadex A25 Sephadex gel in a volumetric flask, adding 80ml of 2mol/L acetic acid solution, soaking for 24h, adding 2mol/L acetic acid solution with the same volume, sealing and storing in a refrigerator at 4 ℃ for later use. The preparation method of the sulfatase comprises the following steps: helix pomatia H1 type (EC3.1.6.1), the activity unit of sulfatase per milliliter is not less than 0.5, and the sulfatase solution is ready to use.

The overnight syringe was washed 3 times with 0.5mL portions of double distilled water. The syringe was squeezed with the syringe tip to transfer the liquid as far as possible into the test tube. Transferring the liquid in the test tube into a small glass bottle through a filter membrane of 0.45 mu m, and freezing and storing to obtain the solution to be tested.

(2) Analysis of glucosinolates

And (3) carrying out HPLC analysis on the solution to be detected obtained in the step (1). Conditions for HPLC analysis: waters C18 column, 3.9 × 150mm, 5 μm; the detection wavelength is 229 nm; column temperature: 25 ℃; the sample volume is 10 mu L; the flow rate of the mobile phase is 1.0 mL/min^-1The gradient elution was as follows:

0-1min (including 1 min): a mobile phase A;

1-21min (excluding 1min, including 21 min): the volume percentage content of the mobile phase A is linearly reduced from 100% to 0, and the volume percentage content of the mobile phase B is linearly increased from 0 to 100%;

21-26min (excluding 21min, including 26 min): the volume percentage content of the mobile phase B is linearly reduced from 100% to 0, and the volume percentage content of the mobile phase A is linearly increased from 0 to 100%;

26-31min (excluding 26min, including 31 min): mobile phase B.

Wherein, the mobile phase A: 1g of tetramethylammonium chloride (TMACL) was dissolved in 2L of double distilled water, mixed well and filtered.

Mobile phase B: dissolving 1g tetramethylammonium chloride (TMACL) in 1.6L double distilled water, adding 400mL chromatographically pure acetonitrile, mixing, and filtering.

The benzyl thioglycoside is adopted as an internal standard, and the thioglycoside component is quantitatively determined according to retention time and peak area. The content of thioglucoside was calculated in μmol/gDW using internal standards and response factors.

Example 1 cultivation of healthy functional Cauliflower with high-content of beneficial and low-content of undesirable glucosinolates

The cultivation process is as follows:

1. selfing, separating and purifying the green cauliflower variety green brille in a single seed transmission mode, and performing 6-generation single plant selfing to obtain 11 genetically stable selfing lines LH-1, LH-2, LH-3, LH-4, LH-5, LH-6, LH-7, LH-8, LH-9, LH-10 and LH-11 through separation.

2. And (2) carrying out self-compatibility identification on the 11 self-compatible lines obtained in the step (1) to obtain 4 self-compatible lines LH-1, LH-2, LH-3 and LH-6, wherein the 4 self-compatible lines meet the following requirements: the flowering phase self-compatibility index is more than 2, wherein the flowering phase self-compatibility index is the seed setting number/pollinated flower number.

3. Respectively taking LH-1, LH-2, LH-3 and LH-6 selected in the step 2 as recurrent parents, carrying out backcross transformation with the broccoli cytoplasmic male sterile source verve No. 2 (Beijing research and agriculture, Beijing) Setaria science and technology limited), and obtaining 4 broccoli cytoplasmic male sterile lines CMSLH-1, CMSLH-2, CMSLH-3 and CMSLH-6 after five generations of backcross. The backcross aims to transform the inbred line into a cytoplasmic male sterile line, each generation of backcross selects a plant and a backcross progeny single plant with biased ball morphology or similar recurrent parent and pollen abortion as a female parent, and backcross pollination is carried out on the backcross progeny single plant and the corresponding recurrent parent to obtain backcross progeny. After five-generation backcross selection, the sterile line CMSLH-1 and the inbred line LH-1 have basically the same properties except the difference in pollen fertility. Similarly, sterile line CMSLH-2 and inbred line LH-2, sterile line CMSLH-3 and inbred line LH-3 and sterile line CMSLH-6 and inbred line LH-6 are the same, except for differences in pollen fertility, and the morphological traits are essentially the same.

4. Taking seeds as samples to be detected, respectively detecting the contents of glucosinolates (IBE, PRO, SIN, RAA, NAP, 4OH, ERU, GBC, 4ME and NEO) in the green cauliflowers by adopting HPLC, wherein the detected objects are as follows: sterile line CMSLH-1, CMSLH-2, CMSLH-3 and CMSLH-6, and inbred line LH-1-LH-11. The results are shown in Table 1.

TABLE 1 results of the detection of glucosinolates of different strains (unit:. mu. mol/gDW)

In Table 1, TOL represents the total glucosinolate content, and TOL1 represents the total content of RAA, 4OH, GBC, 4ME and NEO.

5. Taking a sterile line CMSLH-3 with the RAA content higher than 80 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 85 mu mol/gDW and the PRO content lower than 1 mu mol/gDW as a female parent, and hybridizing with an inbred line LH-5 with the RAA content higher than 90 mu mol/gDW, the total content of RAA, 4OH, GBC, 4ME and NEO higher than 95 mu mol/gDW and the PRO content lower than 1 mu mol/gDW to obtain F1 generation seeds of CMSLH-3 multiplied by LH-5;

hybridizing a sterile line CMSLH-3 serving as a female parent with inbred lines LH-6, LH-8 and LH-10 respectively to obtain F1 generation seeds of CMSLH-3 × LH-6, CMSLH-3 × LH-8 and CMSLH-3 × LH-10 serving as a control; sterile line CMSLH-6 is used as female parent, and is respectively hybridized with inbred lines LH-3, LH-5, LH-8 and LH-10 to obtain F1 generation seeds of CMSLH-6 x LH-3, CMSLH-6 x LH-5, CMSLH-6 x LH-8 and CMSLH-6 x LH-10 as a control.

6. The thioglucoside content of each F1 generation seed obtained in step 5 was measured by HPLC, and the results are shown in table 2.

The total content of beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of the F1 generation seeds of the combined CMSLH-3 XLH-5 (124.147. mu. mol/gDW) is 41.5 percent and 28.3 percent higher than that of the parent CMSLH-3 (87.749. mu. mol/gDW) and LH-5 (96.749. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (0.271. mu. mol/gDW) is 64.3 percent lower and 70.7 percent lower than that of the parent CMSLH-3 (0.759. mu. mol/gDW) and LH-5 (0.926. mu. mol/gDW) respectively. The improvement of the beneficial glucosinolate content and the reduction of the useless glucosinolate content of the seeds of the generation F1 reach a very significant difference level.

The total content (117.647. mu. mol/gDW) of the F1 generation seed-beneficial glucosinolates RAA, 4OH, GBC, 4ME, NEO of the combined CMSLH-3 XLH-6 was 42.4% and 63.1% higher than that of the parent CMSLH-3 (87.749. mu. mol/gDW) and LH-6 (72.151. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (1.347 mu mol/gDW) is 77.5 percent higher than that of the female parent CMSLH-3(0.759 mu mol/gDW) and 98.2 percent lower than that of the male parent LH-6(76.545 mu mol/gDW). The improvement of the beneficial glucosinolate content of the F1 generation seeds and the improvement of the non-beneficial glucosinolate content of the F1 generation seeds compared with the female parent and the reduction compared with the male parent reach extremely obvious difference levels.

The total content (99.366. mu. mol/gDW) of the F1 generation seed-beneficial glucosinolates RAA, 4OH, GBC, 4ME, NEO of the combined CMSLH-3 XLH-8 was 11.62% and 34.1% higher than that of the parent CMSLH-3 (87.749. mu. mol/gDW) and LH-8 (74.090. mu. mol/gDW), respectively; the content of the unprofitable thioglycoside PRO (0.124 mu mol/gDW) is 83.7 percent and 99.8 percent lower than that of the parent CMSLH-3(0.759 mu mol/gDW) and LH-8(55.439 mu mol/gDW) respectively. The improvement of the beneficial glucosinolate content and the reduction of the useless glucosinolate content of the seeds of the generation F1 reach a very significant difference level.

The total content (84.519 mu mol/gDW) of the beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of the F1 generation seeds of the combined CMSLH-3 x LH-10 is lower than that of the female parent CMSLH-3(87.749 mu mol/gDW) by 3.7 percent and is higher than that of the male parent LH-10(58.406 mu mol/gDW) by 44.7 percent; the content of the unprofitable thioglycoside PRO (0.284 mu mol/gDW) is 62.6 percent lower and 88.0 percent lower than that of the parent CMSLH-3(0.759 mu mol/gDW) and LH-10(2.371 mu mol/gDW) respectively. The content of the beneficial glucosinolates of the F1 generation seeds is slightly reduced compared with that of the female parent, but the difference is not obvious, and the improvement of the beneficial glucosinolates of the F1 generation seeds compared with that of the male parent reaches a very obvious level; the reduction of the content of the useless glucosinolates compared with that of the parents reaches a very obvious difference level.

The total content of the F1 generation seed beneficial glucosinolates RAA, 4OH, GBC, 4ME, NEO of the combined CMSLH-6 XLH-3 (40.143. mu. mol/gDW) was 42.1% and 54.7% lower than that of the parent CMSLH-6 (69.313. mu. mol/gDW) and LH-3 (88.566. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (68.707 mu mol/gDW) is 13.3 percent lower than that of the female parent CMSLH-6(79.292 mu mol/gDW) and 95.5 percent higher than that of the male parent LH-3(0.712 mu mol/gDW). The reduction of the beneficial glucosinolate content and the reduction of the non-beneficial glucosinolate content of the F1 generation seeds compared with the female parent reach the extremely obvious difference level, and the improvement of the non-beneficial glucosinolate content compared with the male parent also reaches the extremely obvious difference level.

The total content of beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of the F1 generation seeds of the combined CMSLH-6 XLH-5 (35.976. mu. mol/gDW) is 48.1 percent and 62.8 percent lower than that of the parent CMSLH-6 (69.313. mu. mol/gDW) and LH-5 (96.749. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (59.423 mu mol/gDW) is 25.1 percent lower than that of the female parent CMSLH-6(79.292 mu mol/gDW) and 63.2 percent higher than that of the male parent LH-5(0.926 mu mol/gDW). The reduction of the beneficial glucosinolate content and the reduction of the non-beneficial glucosinolate content of the F1 generation seeds compared with the female parent reach the extremely obvious difference level, and the improvement of the non-beneficial glucosinolate content compared with the male parent also reaches the extremely obvious difference level.

The total content of beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of the F1 generation seeds of the combined CMSLH-6 XLH-8 (42.379. mu. mol/gDW) is 38.9 percent and 42.8 percent lower than that of the parent CMSLH-6 (69.313. mu. mol/gDW) and LH-8 (74.090. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (98.022 mu mol/gDW) is 23.6 percent and 76.8 percent higher than that of the parent CMSLH-6(79.292 mu mol/gDW) and LH-8(55.439 mu mol/gDW) respectively. The reduction of the beneficial glucosinolate content and the improvement of the useless glucosinolate content of the seeds of the generation F1 reach a very significant difference level.

The total content of beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of the F1 generation seeds of the combined CMSLH-6 XLH-10 (29.227. mu. mol/gDW) is 57.8 percent lower and 50.0 percent lower than that of the parent CMSLH-6 (69.313. mu. mol/gDW) and LH-10 (58.406. mu. mol/gDW), respectively; the content of unprofitable thioglycoside PRO (54.488 mu mol/gDW) is 31.3 percent lower than that of the female parent CMSLH-6(79.292 mu mol/gDW) and 22.0 percent higher than that of the male parent LH-10(2.371 mu mol/gDW). The reduction of the beneficial glucosinolate content and the reduction of the non-beneficial glucosinolate content of the F1 generation seeds compared with the female parent reach the extremely obvious difference level, and the improvement of the non-beneficial glucosinolate content compared with the male parent also reaches the extremely obvious difference level.

According to the results, the total content of the beneficial glucosinolates RAA, 4OH, GBC, 4ME and NEO of F1 generation seeds combining CMSLH-3 xLH-5 is greatly improved compared with that of parents, the absolute value is higher than 120 mu mol/gDW, the content of the unprofitable glucosinolates is greatly reduced compared with that of parents, the absolute value is lower than 1 mu mol/gDW, and other combinations can not realize the aim, which indicates that the sterile lines with the RAA content higher than 80 mu mol/gDW, the total content of the RAA, 4OH, GBC, 4ME and NEO higher than 85 mu mol/gDW and the PRO content lower than 1 mu mol/gDW are selected during hybridization, the female parent lines with the RAA content higher than 90 mu mol/g, the RAA, 4OH, GBC, 4ME and NEO total content higher than 95 mu mol/gDW and the PRO content lower than 1 mu mol/gDW are selected, the male parent lines with the RAA content higher than 90 mu mol/gDW and the male lines with the PRO content lower than 1 mu mol/gDW are successfully hybridized to obtain the male parent lines with greatly improved content and greatly reduced beneficial glucosinolates, the method has good application prospect in breeding, and can be used for producing green cauliflowers with high content of beneficial glucosinolates and low content of useless glucosinolates.

TABLE 2 results of detection of thioglucoside F1 (unit: μmol/gDW)

In Table 2, TOL represents the total glucosinolate content, and TOL1 represents the total content of RAA, 4OH, GBC, 4ME and NEO.

Specifically, the preparation method of F1 generation seeds of CMSLH-3 and LH-5 is shown in step 7.

7. Production of F1 generation seed of Cauliflower CMSLH-3 × LH-5

(1) Propagation of female parent CMSLH-3

Seed collection is carried out by utilizing a sunlight greenhouse or a plastic greenhouse, and the sterile line CMSLH-3 and the corresponding maintainer line LH-3 are mixed according to the ratio of 2: 1 row ratio, and the plant row spacing is 45 cm square. The greenhouse or the shed is isolated from the outside by a gauze. And placing a box of bees in the greenhouse or the shed for pollination after blooming. After the florescence is finished, the maintenance line LH-3 is pulled out. The female parent CMSLH-3 seed is obtained from the sterile CMSLH-3 plant.

(2) Reproduction of male parent LH-5

Seeds are collected in a sunlight greenhouse or a plastic greenhouse, LH-5 is planted according to the plant and the row spacing of 45 cm, and the greenhouse or the plastic greenhouse is isolated from the outside by a gauze. And placing a box of bees in the greenhouse or the shed for pollination after blooming. Spraying 4-5% saline water on stigma at flowering stage, 9-10 am, and spraying once every other day without spraying leaves. Collecting seeds of the male parent LH-5.

(3) Production of CMSLH-3 × LH-5 in the F1 generation

A seed production base with good isolation conditions is selected to produce F1 generation seeds of CMSLH-3 x LH-5, and cruciferous vegetables, especially cabbage vegetables such as green cauliflower, white cauliflower, cabbage and the like are not required to be planted in the seed production field for more than 2 kilometers. The growth periods of the male parent LH-5 and the female parent CMSLH-3 are similar, and the male parent LH-5 and the female parent CMSLH-3 can be sowed and planted at the same period. Female parent: the male parent is 2: planting at a ratio of 1, wherein the plant and row spacing is 45 cm. Releasing bees for pollination in the flowering period. After flowering, the parent plant is pulled out, and only the parent plant is left. Before blooming, paying attention to water and fertilizer control, and timely pulling out the seedlings; and (5) enhancing the disease and insect pest management in the field before and after blooming. And harvesting the seeds from the female parent strain after the flowering phase is finished, namely F1 generation seeds of CMSLH-3 xLH-5.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. A method for cultivating green vegetable flowers, comprising:

1) selfing a green cauliflower variety under the conditions of green brightness to obtain a selfing line, and determining a selfing affinity index through pollination in a flowering phase to obtain a selfing affinity line with the selfing affinity index in the flowering phase being more than 2;

2) backcrossing the self-compatible line obtained in the step 1) with a recurrent parent and a cytoplasmic male sterile source of green vegetable flowers to obtain a cytoplasmic male sterile line with morphological characters the same as those of the recurrent parent except fertility;

2. The method of claim 1, wherein: the cytoplasmic male sterile source of the green cauliflower is verdure No. 2.

3. The method according to claim 1 or 2, characterized in that: the thioglucoside is IBE, PRO, SIN, RAA, NAP, 4OH, ERU, GBC, 4ME and/or NEO.

4. A method according to any one of claims 1-3, characterized in that: in the step 2), backcrossing is carried out for five backcrossing generations.

5. The method according to any one of claims 1-4, wherein: the selfing in the step 1) is carried out in a single seed transmission mode.

6. Use of the method according to any one of claims 1 to 5 for cultivating flowers of Brassica oleracea which are rich in advantageous glucosinolates.

7. Use of the method of any one of claims 1 to 5 for growing low-content, non-beneficiated glucosinolates brassica rapa.

8. Use of the method according to any one of claims 1 to 5 for cultivating green cauliflower rich in beneficial glucosinolates and low in non-beneficial glucosinolates.