Method for creating luffa double haploid by distant hybridization

Abstract

The application discloses a method for creating luffa double haploid by distant hybridization, which comprises the following steps: applying brassinolide water solution to the pistil of luffa female flower which will bloom the next day; the next day, pollinating the luffa female flower to which the brassinolide water solution is applied by using the male flower of winter melon, and at the same time, applying thidiazuron water solution to the ovary and pistil of the luffa female flower; after the pollination, bagging and isolating the female flower; 3-6 hours and 20-26 hours after the pollination, applying thidiazuron solution to the ovary and the base of the fruit stalk; after harvesting the seed melon, taking the relatively full seeds to germinate and sow to obtain regenerated plants; training and transplanting the regenerated plants, and then normally cultivating and managing the plants. The thidiazuron and the brassinolide are combined to be used for distant hybridization induction, stimulate the embryo development, and improve the fruit setting rate; through the multiple thidiazuron treatment, the chromosome doubling is induced, the luffa double haploid is directly obtained, and the problem of low efficiency of the luffa double haploid is solved.

Description

Technical Field

[0001] This invention relates to a method for obtaining new plants, and more particularly to a method for inducing double haploids of loofah through distant hybridization. Background Technology

[0002] Luffa cylindrica 2n=26 is an annual climbing plant belonging to the Cucurbitaceae family, native to tropical and subtropical regions. Through long-term evolution and selection, it has developed strong adaptability and is widely cultivated in various regions of my country. Traditional breeding methods for selecting homozygous parents are time-consuming and labor-intensive, while using haploid / diploid breeding can quickly obtain homozygous parents, greatly shortening the breeding cycle and improving breeding efficiency.

[0003] Methods for obtaining haploids / double haploids in cucurbitaceous crops mainly include: anther culture, in vitro culture of unfertilized ovaries, and radiation pollination. Radiation pollination is the most effective method for haploid induction in cucurbitaceous crops, but the haploids formed through radiation pollination are not stable enough, and the efficiency of forming stable double haploids through chromosome doubling is low. Currently, double haploids in sponge gourd have not been successfully created. Chinese patent CN113080055A discloses a method for directly creating double haploids in melon by inducing embryo development through distant hybridization, using a combination of chlorpyrifos and thidiazuron for distant hybridization in melon, which is also in the cucurbitaceous family. However, this method has not been effective in creating double haploids in sponge gourd. Summary of the Invention

[0004] To address the aforementioned deficiencies in the prior art, the present invention aims to provide a method for inducing double haploids of loofah through distant hybridization, with the goal of improving the stability and efficiency of double haploid creation in loofah.

[0005] The technical solution of this invention is as follows: A method for inducing double haploids of loofah through distant hybridization, comprising the following steps:

[0006] Step 1: Apply brassinolide solution to the ovary of the female flower of the loofah that will bloom the next day;

[0007] Step 2: On the second day, take the male flowers of winter melon and pollinate the female flowers of loofah that have been treated with brassinolide solution. At the same time, apply thiamethoxam solution to the ovary and pistil of loofah. After pollination, bag the female flowers for isolation.

[0008] Step 3: Apply thidiazuron solution to the ovary and the base of the fruit stalk 3–6 h and 20–26 h after pollination;

[0009] Step 4: After harvesting the seed melons, take relatively plump seeds, germinate them, and sow them to obtain regenerated plants;

[0010] Step 5: Harden off the regenerated plants, transplant them, and then cultivate and manage them normally.

[0011] Furthermore, the concentration of the brassinolide aqueous solution is 0.03–0.05 mg / L.

[0012] Furthermore, the concentration of the thiamethoxam solution in step 2 is 10–15 mg / L.

[0013] Furthermore, in step 3, the concentration of the thiamethoxam solution used 3–6 h after pollination is 4–6 mg / L, and the concentration of the thiamethoxam solution used 20–26 h after pollination is 10–15 mg / L.

[0014] Furthermore, the loofah is either tube-shaped loofah or Xulv No. 1, and the winter melon is Baoyu fruit winter melon.

[0015] Furthermore, in step 1, the female flowers of the loofah that have been treated with brassinolide aqueous solution are bagged and isolated, and the male flowers of the winter melon that are to bloom the next day are also bagged and isolated.

[0016] Furthermore, the application of brassinolide aqueous solution and the application of thidiazuron solution are performed by smearing or spraying.

[0017] Furthermore, step 4, germination and sowing, involves sowing the seeds in a seedling substrate after they have sprouted white. Step 5 includes hardening off the seedlings when they reach the two-leaf-one-heart stage, and then transplanting them to the field after growing them at room temperature for 5-7 days.

[0018] Furthermore, the seedling substrate comprises peat moss and perlite.

[0019] The advantages of this invention compared to the prior art are:

[0020] The combined use of brassinolide and thidiazuron for distant hybridization induction promotes embryo development, ensures fruit development, and increases fruit set rate. Through multiple treatments with thidiazuron, chromosome doubling is induced, directly obtaining double haploids of loofah. The induction efficiency can reach 2% to 5%, solving the problems of insufficient stability of haploids and difficulty in chromosome doubling.

[0021] Pollination with distant pollen induces direct development into double haploid seeds, eliminating the need for tissue culture. The transplanted plants have a high survival rate, and the paternal lineage is widely available. The cultivated double haploid plants can be self-pollinated and used as breeding materials after field screening, and can be applied to the breeding of loofah varieties. Attached Figure Description

[0022] Figure 1 These are photographs of two regenerated plants obtained in Example 1.

[0023] Figure 2 The image shows the cell flow cytometer identification obtained in Example 1.

[0024] Figure 3 The image shows an SSR marker electrophoresis diagram of the first generation of self-pollinated regenerated plants obtained in Example 1.

[0025] Figure 4 The image shows the cell flow cytometer identification obtained in Example 2.

[0026] Figure 5 This is an electrophoresis image of the first generation of self-pollinated regenerated plants obtained in Example 2, labeled with SSR markers.

[0027] Figure 6 The image shows the cell flow cytometer identification obtained in Example 3.

[0028] Figure 7 This is an electrophoresis image of the first generation of self-pollinated SSR markers obtained in Example 3. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are used to explain the invention, but are not intended to limit the invention.

[0030] Example 1: A method for inducing double haploids of loofah through distant hybridization, using Xulv 1 as the female parent and Baoyu fruit melon as the male parent. The specific steps are as follows:

[0031] Step 1: Apply a 0.03 mg / L brassinolide solution to the ovary of the female flower of the loofah that will bloom the next day, and then cover it with a bag for isolation. At the same time, cover the male flower of the winter melon that will bloom the next day with a bag for isolation.

[0032] Step 2: Pollinate the male flowers of winter melon and the female flowers of loofah. At the same time, apply a 10mg / L thiamethoxam solution to the ovary and pistil of the loofah once. After pollination, cover the female flowers with bags for isolation.

[0033] Step 3: 3 hours after pollination, spray the ovary and the base of the fruit stalk with a 5 mg / L thiazuron solution once, and 20 hours after pollination, spray the ovary and the base of the fruit stalk with a 10 mg / L thiazuron solution once.

[0034] Step 4: Harvest the seed melons 6-7 weeks after pollination. Take relatively plump seeds and sow them in nutrient pots containing a seedling substrate after they have sprouted white hairs. The seedling substrate is a 2:1 volume mixture of peat moss and perlite. Figure 1 As shown, take 0.5–0.8 cm of seedling leaf. 2 A single-cell suspension was prepared with a concentration of 1.0 × 10⁻⁶. 5 ~1.0×10 7 The concentration of cells / mL was measured using flow cytometry, with *Gnaphalium affine* leaves as a control. The results showed that both seedlings were diploid plants. (See results below.) Figure 2 As shown.

[0035] Step 5: When the plants grow to the two-leaf-one-heart stage, begin hardening them off. After growing at room temperature for 5-7 days, transplant them to the field for management and self-pollination for seed saving.

[0036] Leaves were taken from first-generation seedlings, and DNA was extracted from the leaves using the CTAB method. SSR markers were used for identification, and the homozygosity of gene loci in the regenerated plants was detected. The results are as follows: Figure 3 As shown, the test results indicate that the diploid loofah plant has homozygous gene loci, meaning the plant is a double haploid.

[0037] Observations of the double haploids obtained above throughout their entire growth period revealed that the plants exhibited typical double haploid characteristics in botanical traits such as smaller leaves, shorter internodes, and slow early growth.

[0038] Example 2: A method for inducing double haploids of loofah through distant hybridization, using *Luffa cylindrica* as the female parent and *Melon hainanensis* as the male parent. The specific steps are as follows:

[0039] Step 1: Apply a 0.05 mg / L brassinolide solution to the ovary of the female flower of the loofah that will bloom the next day, and then cover it with a bag for isolation. At the same time, cover the male flower of the winter melon that will bloom the next day with a bag for isolation.

[0040] Step 2: Pollinate the male flowers of winter melon and the female flowers of loofah. At the same time, apply a 15mg / L thiamethoxam solution to the ovary and pistil of the loofah once. After pollination, cover the female flowers with bags for isolation.

[0041] Step 3: 6 hours after pollination, spray the ovary and the base of the fruit stalk with a 6 mg / L thiazuron solution once, and 26 hours after pollination, spray the ovary and the base of the fruit stalk with a 15 mg / L thiazuron solution once.

[0042] Step 4: Harvest the seed melons 6-7 weeks after pollination. Take relatively plump seeds and sow them in nutrient pots containing a seedling substrate after they show signs of sprouting. The seedling substrate is a 2:1 volume mixture of peat moss and perlite. Take 0.5-0.8cm of seedling leaves. 2 A single-cell suspension was prepared with a concentration of 1.0 × 10⁻⁶. 5 ~1.0×10 7 The concentration of cells / mL was determined using flow cytometry, with *Gnaphalium affine* leaves as a control. The results showed that the seedlings were diploid plants. Figure 4 As shown.

[0043] Step 5: When the plants grow to the two-leaf-one-heart stage, begin hardening them off. After growing at room temperature for 5-7 days, transplant them to the field for management and self-pollination for seed saving.

[0044] Leaves were taken from first-generation seedlings, and DNA was extracted from the leaves using the CTAB method. SSR markers were used for identification, and the homozygosity of gene loci in the regenerated plants was detected. The results are as follows: Figure 5 As shown, the test results indicate that the diploid loofah plant has homozygous gene loci, meaning the plant is a double haploid.

[0045] Observations of the double haploids obtained above throughout their entire growth period revealed that the plants exhibited typical double haploid characteristics in botanical traits such as smaller leaves, shorter internodes, and slow early growth.

[0046] Example 3: A method for inducing double haploids of loofah through distant hybridization, using *Luffa cylindrica* as the female parent and *Melon hainanensis* as the male parent. The specific steps are as follows:

[0047] Step 1: Spray the ovaries of the female flowers of the loofah that will bloom the next day with a 0.04 mg / L aqueous solution of brassinolide and isolate them by bagging. At the same time, isolate the male flowers of the winter melon that will bloom the next day by bagging.

[0048] Step 2: Pollinate the male flowers of winter melon and the female flowers of loofah. At the same time, spray the ovary and pistil of loofah with a 12mg / L thiamethoxam solution once. After pollination, bag the female flowers for isolation.

[0049] Step 3: 5 hours after pollination, spray the ovary and the base of the fruit stalk with a 4 mg / L thiazuron solution once, and 24 hours after pollination, spray the ovary and the base of the fruit stalk with a 12 mg / L thiazuron solution once.

[0050] Step 4: Harvest the seed melons 6-7 weeks after pollination. Take relatively plump seeds and sow them in nutrient pots containing a seedling substrate after they show signs of sprouting. The seedling substrate is a 2:1 volume mixture of peat moss and perlite. Take 0.5-0.8cm of seedling leaves. 2 A single-cell suspension was prepared with a concentration of 1.0 × 10⁻⁶. 5 ~1.0×10 7 The concentration of cells / mL was determined using flow cytometry, with *Gnaphalium affine* leaves as a control. The results showed that the seedlings were diploid plants. Figure 6 As shown.

[0051] Step 5: When the plants grow to the two-leaf-one-heart stage, begin hardening them off. After growing at room temperature for 5-7 days, transplant them to the field for management and self-pollination for seed saving.

[0052] Leaves were taken from first-generation seedlings, and DNA was extracted from the leaves using the CTAB method. SSR markers were used for identification, and the homozygosity of gene loci in the regenerated plants was detected. The results are as follows: Figure 7 As shown, the test results indicate that the diploid loofah plant has homozygous gene loci, indicating that the plant obtained in this example is a double haploid.

[0053] Observations of the double haploids obtained above throughout their entire growth period revealed that the plants exhibited typical double haploid characteristics in botanical traits such as smaller leaves, shorter internodes, and slow early growth.

[0054] Comparative Example 1

[0055] In step 1 of Example 1, brassinolide aqueous solution was not sprayed, and the remaining steps were the same as in Example 1, pollinating 30 female flowers of loofah.

[0056] Comparative Example 2

[0057] In step 2 of Example 1, thiabendazole solution was not sprayed during pollination, and the remaining steps were the same as in Example 1, pollinating 30 female flowers of loofah.

[0058] Comparative Example 3

[0059] In step 3 of Example 1, a 10 mg / L thiabendazole solution was sprayed once on the ovary and the base of the fruit stalk 20 hours after pollination. The remaining steps were the same as in Example 1. 30 female flowers of loofah were pollinated.

[0060] Comparative Example 4

[0061] In step 3 of Example 1, a 5 mg / L thiabendazole solution was sprayed once on the ovary and the base of the fruit stalk 3 hours after pollination. The remaining steps were the same as in Example 1. 30 female flowers of loofah were pollinated.

[0062] The results of the above embodiments and comparative examples are as follows: Comparative Example 1: 11 fruits set, 1 plump seed harvested, but no seed germinated; Comparative Example 2: 4 fruits set, but no plump seed was obtained; Comparative Example 3: 13 fruits set, but no plump seed was obtained; Comparative Example 4: 13 fruits set, 1 plump seed harvested, but no seed germinated. In Example 1, 50 female flowers of loofah were treated, resulting in 41 fruits set and 2 plump seeds obtained; in Example 2, 50 female flowers of loofah were treated, resulting in 38 fruits set and 1 plump seed obtained; in Example 3, 50 female flowers of loofah were treated, resulting in 40 fruits set and 1 plump seed obtained. All of these germinated normally, resulting in regenerated plants. The loofah double haploid induction efficiency can reach 2% to 5%.

Claims

1. A method for inducing double haploids of loofah through distant hybridization, characterized in that, Includes the following steps: Step 1: Apply brassinolide solution to the ovary of the female flower of the loofah that will bloom the next day; Step 2: On the second day, take the male flowers of winter melon and pollinate the female flowers of loofah that have been treated with brassinolide solution. At the same time, apply thiamethoxam solution to the ovary and pistil of loofah. After pollination, bag the female flowers for isolation. Step 3: Apply thidiazuron solution to the ovary and the base of the fruit stalk 3–6 h and 20–26 h after pollination; Step 4: After harvesting the seed melons, take relatively plump seeds, germinate them, and sow them to obtain regenerated plants; Step 5: Harden off the regenerated plants, transplant them, and then cultivate and manage them normally. The concentration of the brassinolide aqueous solution is 0.03–0.05 mg / L, the concentration of the thiazuron solution in step 2 is 10–15 mg / L, the concentration of the thiazuron solution used in step 3 at 3–6 h after pollination is 4–6 mg / L, and the concentration of the thiazuron solution used at 20–26 h after pollination is 10–15 mg / L.

2. The method for inducing double haploids of loofah through distant hybridization according to claim 1, characterized in that, The loofah in question is either the tube-shaped loofah or the Xulv No. 1 loofah.

3. The method for inducing double haploids of loofah through distant hybridization according to claim 1, characterized in that, In step 1, the female flowers of the loofah that have been treated with brassinolide solution are bagged and isolated, and the male flowers of the winter melon that are to bloom the next day are also bagged and isolated.

4. The method for inducing double haploids of loofah through distant hybridization according to claim 1, characterized in that, The application of brassinolide aqueous solution and the application of thidiazuron solution are performed by smearing or spraying.

5. The method for inducing double haploids of loofah through distant hybridization according to claim 1, characterized in that, Step 4, germination and sowing, involves sowing the seeds in a seedling substrate after they have sprouted white. Step 5 includes hardening off the seedlings when they reach the two-leaf-one-heart stage, and then transplanting them to the field after 5-7 days of growth at room temperature.

6. The method for inducing double haploids of loofah through distant hybridization according to claim 1, characterized in that, The seedling substrate contains peat moss and perlite.

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