阅读说明：本技术 一种西瓜组培快繁方法 (Tissue culture and rapid propagation method for watermelons ) 是由 金炳奎 由守昌 王昌盛 杨猛 李宁 刘金宝 于 2021-07-05 设计创作，主要内容包括：本发明提供一种西瓜组培快繁方法,包括选取外植体、离体后处理、脱分化、分化培养、壮苗培养、嫁接、炼苗步骤；所述的选取外植体：外植体为茎尖,长度为1-1.5cm；所述分化培养：培养时间为20-24天；所述炼苗：采将组培嫁接苗移栽于培养基质中,覆膜,炼苗20天,前十天炼苗：光照强度为2800-2900Lx,光照时间为12-14h,控制温度：白天为27-28℃,夜间为17-20℃,湿度为67-70％；后十天炼苗：光照强度为3000-3100Lx,光照时间为10-11h,控制温度：白天为31-32℃,夜间为12-15℃,湿度为60-63％。本发明大大提高了组培苗的移栽成活率和嫁接成活率。(The invention provides a tissue culture and rapid propagation method of watermelons, which comprises the steps of explant selection, in-vitro post-treatment, dedifferentiation, differentiation culture, strong seedling culture, grafting and seedling hardening; selecting an explant: the explant is a stem tip, and the length of the explant is 1-1.5 cm; and (3) differentiation culture: the culture time is 20-24 days; hardening seedlings: transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2800-: 27-28 ℃ in the daytime, 17-20 ℃ at night and 67-70% in humidity; hardening seedlings in the last ten days: the illumination intensity is 3000-3100Lx, the illumination time is 10-11h, and the temperature is controlled as follows: 31-32 ℃ in the daytime, 12-15 ℃ at night and 60-63% in humidity. The invention greatly improves the transplanting survival rate and the grafting survival rate of the tissue culture seedlings.)

1. A tissue culture and rapid propagation method of watermelons is characterized in that: comprises the steps of explant selection, in vitro post-treatment, dedifferentiation, differentiation culture, strong seedling culture, grafting and seedling hardening; selecting an explant: the explant is a stem tip, and the length of the explant is 1-1.5 cm; and (3) differentiation culture: the culture time is 20-24 days; hardening seedlings: transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2800-: 27-28 ℃ in the daytime, 17-20 ℃ at night and 67-70% in humidity; hardening seedlings in the last ten days: the illumination intensity is 3000-3100Lx, the illumination time is 10-11h, and the temperature is controlled as follows: 31-32 ℃ in the daytime, 12-15 ℃ at night and 60-63% in humidity.

2. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: the culture medium of the seedling exercising step is prepared by the following method: coconut shell powder, chitin, wooden fish stone powder, river sand and chicken manure are mixed according to the weight ratio of 20-30: 5-8: 10-14: 9-13: 3-4, spraying deionized water to control the water content to be 34-36% to obtain a culture medium raw material; mixing the culture medium raw material with endo-cellulase, phytase, bacillus subtilis and bacillus mucilaginosus according to the ratio of 150-: 6-8:3-4 to obtain premix; fermenting the premix to obtain the culture medium.

3. The tissue culture and rapid propagation method of watermelon according to claim 2, which is characterized in that: the enzyme activity of the endo-cellulase is 15-18U/g; the enzyme activity of the phytase is 400-450U/g; the viable count of the bacillus subtilis is 8.2 multiplied by 10⁹-8.5×10⁹cfu/g, the viable count of the bacillus mucilaginosus is 2.5 multiplied by 10⁹-3×10⁹cfu/g。

4. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: grafting: the tissue culture seedling is a scion; the adopted stock is a calabash seedling with two leaves and one core; spraying growth promoting liquid every day from the second day to the fourth day after grafting, wherein the spraying time is 9:00-10:00 in the morning.

5. The tissue culture and rapid propagation method of watermelons according to claim 4, which is characterized in that: the growth promoting liquid is an aqueous solution prepared from 0.4-0.5mg/L of alginic acid, 0.25-0.31mg/L of chitosan, 0.12-0.15mg/L of xylitol, 0.02-0.04mg/L of brassinolide and 0.02-0.03mg/L of jasmonic acid.

6. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: the in vitro post-treatment comprises the following steps: transferring the explant to a tissue culture laboratory for in vitro post-treatment; the in vitro post-treatment comprises disinfection treatment and soaking treatment; the soaking treatment comprises the following steps: magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and sterile water are mixed according to the weight ratio of 2: 0.5: 1: 200 to obtain a mixed solution; placing the sterilized explant in the above mixed solution, and soaking at 20-25 deg.C for 2-2.5 min.

7. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: the dedifferentiation: the preparation method of the dedifferentiation culture solution comprises the following steps: the aqueous solution is prepared according to the following formula: KNO₃5.4g/L, 9.3g/L ammonium sulfate, 85mg/L, MgSO mg potassium dihydrogen phosphate₄·7H₂139mg/L of O, 20mg/L, KI 0.75.75 mg/L of zinc sulfate and 5.2mg/L, MnSO of boric acid₄·H₂O7.3 mg/L, sodium molybdate 12.5mg/L, Na2MnO 4.2H2O 9.25.25 mg/L, CuSO 4.5H2O 3.1.1 mg/L, CoCl₂·6H₂O 0.58mg/L、FeSO₄·7H₂7.2mg/L of O, 0.25mg/L of sodium chloride, 0.15mg/L of folic acid, 0.83mg/L of biotin, 0.01mg/L of choline, 0.06mg/L of indoleacetic acid and 0.06mg/L of 6-BA.

8. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: and (3) differentiation culture: transferring the callus onto a differentiation culture medium for culturing for 20-24 days, including first stage culture and second stage culture; a first-stage culture: the culture time is 10-12 days, the culture temperature is 26 ℃, the illumination intensity is 2400-; two-stage culture: the culture time is 10-12 days, the culture temperature is 26 ℃, the illumination intensity is 2200-.

9. The tissue culture and rapid propagation method of watermelon according to claim 8, which is characterized in that: the differentiation medium is as follows: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 8.5mg/L boric acid, 0.2mg/L inositol, 1.5mg/L casein hydrolysate, 0.01mg/L zeatin, 0.04 mg/L6-benzylaminopurine, 0.02mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, and 8g/L agar.

10. The tissue culture and rapid propagation method of watermelon according to claim 1, which is characterized in that: and (3) strong seedling culture: respectively inoculating the single cluster buds to a strong seedling culture medium, and culturing for 7-9 days at the illumination intensity of 2600-2800LX for 14-15 h/day and the culture temperature of 26-28 ℃; the strong seedling culture medium comprises: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 6.2mg/L boric acid, 0.35mg/L glutamic acid, 0.78mg/L cysteine, 0.15mg/L pantothenic acid, 0.08mg/L caroteneL, 0.01mg/L of indolebutyric acid, 0.03mg/L of indoleacetic acid, 0.01mg/L of 5-hydroxy-IAA, 0.02mg/L of 6-benzylaminopurine and 9.5g/L of agar.

Technical Field

The invention belongs to the field of plant tissue culture propagation, and particularly relates to a tissue culture rapid propagation method of watermelons.

Background

Watermelon, hermaphrodite. Both the male and female flowers are originated from the axilla of the leaf. Male flowers: the flower stalk is 3-4 cm long and is densely covered with yellow brown long and soft hair; the calyx tube is wide and bell-shaped, is densely covered with long velvets, has a needle shape with split calyx leaves, is nearly as long as the calyx tube and is 2-3 mm long; the corolla is yellowish, the diameter is 2.5-3 cm, the external surface is green, the quilt is long and soft, the splinters are oval and long, the length is 1-1.5cm, the width is 0.5-0.8 cm, the top end is blunt or slightly sharp, the pulse is yellowish brown, and the quilt is hairy; stamen 3, proximal to the birth, 1 chamber 1, 2 chambers 2, short filament, and chamber bent. Female flowers: the calyx and the corolla are the same as the male flower; ovaries are oval, 0.5-0.8 cm long, 0.4 cm wide, dense quilts are long and soft, style is 4-5 mm long, stigma 3, kidney shape.

The tissue culture propagation is a propagation method for culturing in vitro tissue cells into complete plants in an artificial culture medium. The research of the tissue culture propagation of fruit trees begins in the 40 th generation of the 20 th century. China starts late but develops faster. Tissue culture detoxification and seedling propagation work has been carried out on apple, grape, orange, strawberry, kiwi, pineapple, loquat and other trees.

Although the tissue culture technology is seemingly simple, in reality, different plants have different difficulties to really realize tissue propagation, and not all plants can be used in one set of technology. The early-spring Hongyu is a first-filial generation of extremely early-maturing small-sized red-pulp watermelon, the appearance of the variety is a long oval type, green bottom stripes are clear, and the growth vigor of plants is stable. The flesh color is fresh, the red meat is crisp, tender and tasty, the preservation time is long, and the marketability is good. The early spring red jade watermelon belongs to popular fruits and has high economic value, but the propagation of the early spring red jade watermelon in China is mainly based on seed propagation, and the development of a tissue culture propagation technology is immature.

The tissue culture propagation technology of early spring red watermelon has the following technical defects: the transplanting survival rate of the tissue culture seedlings is not high, and the grafting survival rate is low.

Disclosure of Invention

The invention aims to provide a tissue culture and rapid propagation method of watermelons, which has excellent transplanting survival rate and grafting survival rate.

In order to achieve the purpose, the invention adopts the following technical scheme:

a tissue culture and rapid propagation method of watermelons comprises the following steps:

(1) selecting explants

The early spring ruby watermelon is planted in a seed planting mode. Selecting strong and disease-free watermelon plant with main vine length of 1.5-1.8m, and cutting 1-1.5cm of stem tip as explant.

(2) In vitro post-treatment

And transferring the explants to a tissue culture laboratory for in vitro post-treatment. The in vitro post-treatment comprises disinfection treatment and soaking treatment. The disinfection treatment comprises the following steps: putting the explant into a wide-mouth bottle, adding mercuric chloride with the mass percentage concentration of 0.08% for disinfection for 40-50s, then washing with sterile water for 3 times, then adding alcohol with the mass percentage concentration of 75% for disinfection for 20s, and then washing with sterile water for 3 times. The soaking treatment comprises the following steps: magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and sterile water are mixed according to the weight ratio of 2: 0.5: 1: 200 to obtain a mixed solution. Placing the sterilized explant in the above mixed solution, and soaking at 20-25 deg.C for 2-2.5 min.

(3) Dedifferentiation

And (4) placing the soaked explants in a dedifferentiation culture solution for dedifferentiation to obtain callus. The preparation method of the dedifferentiation culture solution comprises the following steps:

the aqueous solution is prepared according to the following formula: KNO₃5.4g/L, 9.3g/L ammonium sulfate, 85mg/L, MgSO mg potassium dihydrogen phosphate₄·7H₂139mg/L of O, 20mg/L, KI 0.75.75 mg/L of zinc sulfate and 5.2mg/L, MnSO of boric acid₄·H₂O7.3 mg/L, sodium molybdate 12.5mg/L, Na2MnO 4.2H2O 9.25.25 mg/L, CuSO 4.5H2O 3.1.1 mg/L, CoCl₂·6H₂O 0.58mg/L、FeSO₄·7H₂7.2mg/L of O, 0.25mg/L of sodium chloride, 0.15mg/L of folic acid, 0.83mg/L of biotin, 0.01mg/L of choline, 0.06mg/L of indoleacetic acid and 0.06mg/L of 6-BA. Adjusting pH of the culture solution to 7.0, sterilizing the culture solution in a high pressure steam sterilizing pot for 30min, and cooling to 27 deg.C to obtain dedifferentiation culture solution.

The dedifferentiation: controlling the temperature of the dedifferentiation culture solution at 26-27 deg.C, and culturing in dark for 14-15 days.

(4) Differential culture

Transferring the callus onto a differentiation culture medium for culturing for 20-24 days, including first stage culture and second stage culture; a first-stage culture: the culture time is 10-12 days, the culture temperature is 26 ℃, the illumination intensity is 2400-; two-stage culture: the culture time is 10-12 days, the culture temperature is 26 ℃, the illumination intensity is 2200-. And (3) obtaining a large number of cluster buds through differentiation culture, and dispersing the single cluster buds for later use.

The differentiation medium is as follows: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 8.5mg/L boric acid, 0.2mg/L inositol, 1.5mg/L casein hydrolysate, 0.01mg/L zeatin, 0.04 mg/L6-benzylaminopurine, 0.02mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, and 8g/L agar. Adjusting pH of the culture solution to 6.8, sterilizing the culture solution in a high pressure steam sterilization pot for 30min, cooling to 25 deg.C, and standing for 4 hr to obtain differentiation culture medium.

(5) Strong seedling culture

Respectively inoculating the single cluster buds to a strong seedling culture medium, and culturing for 7-9 days at the illumination intensity of 2600-2800LX for 14-15 h/day and the culture temperature of 26-28 ℃; the strong seedling culture medium comprises: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 6.2mg/L boric acid, 0.35mg/L glutamic acid, 0.78mg/L cysteine, 0.15mg/L pantothenic acid, 0.08mg/L carotene, 0.01mg/L indolebutyric acid, 0.03mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, 0.02 mg/L6-benzylaminopurine and 9.5g/L agar. Adjusting the pH of the culture solution to 6.7, sterilizing the culture solution in a high-pressure steam sterilization pot for 30min, cooling to 25 ℃, and standing for 4h to obtain the strong seedling culture medium.

(6) Grafting

The tissue culture seedling after strong seedling culture is scion; the adopted stock is a calabash seedling with two leaves and one core.

When grafting, the true leaves and the growing points of the stock seedlings are firstly pinched off, and the tips of the bamboo sticks are obliquely inserted downwards from the base parts of the growing points at an angle of 60 degrees. The scion tissue culture seedlings are cut into wedges by a blade at a position 0.5cm below the growing point. And pulling out the bamboo stick, and obliquely inserting the cut scion into the inserting hole penetrated by the bamboo stick on the stock to obtain the tissue culture grafted seedling.

Sterile water is sprayed on the first day after grafting, and the sterile water is sprayed once at nine am and four pm.

Spraying the growth promoting liquid for 1 time every day from the second day to the fourth day after grafting, wherein the spraying time is 9:00-10:00 in the morning. The growth promoting liquid is an aqueous solution prepared from 0.4-0.5mg/L of alginic acid, 0.25-0.31mg/L of chitosan, 0.12-0.15mg/L of xylitol, 0.02-0.04mg/L of brassinolide and 0.02-0.03mg/L of jasmonic acid.

And 7 days after grafting, counting the grafting survival condition of the tissue culture seedling, and calculating to obtain the grafting survival rate of the tissue culture seedling.

(7) Hardening off seedlings

Transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2800-: 27-28 ℃ in the daytime, 17-20 ℃ at night and 67-70% in humidity; hardening seedlings in the last ten days: the illumination intensity is 3000-3100Lx, the illumination time is 10-11h, and the temperature is controlled as follows: 31-32 ℃ in the daytime, 12-15 ℃ at night and 60-63% in humidity.

The culture medium comprises: the preparation method comprises the following steps: coconut shell powder, chitin, wooden fish stone powder, river sand and chicken manure are mixed according to the weight ratio of 20-30: 5-8: 10-14: 9-13: 3-4, and spraying deionized water to control the water content to be 35% to obtain the culture medium raw material. Mixing the culture medium raw material with endo-cellulase, phytase, bacillus subtilis and bacillus mucilaginosus according to the ratio of 150-: 6-8:3-4 to obtain premix. Fermenting the premix for 72 hours at the temperature of 32-35 ℃, discharging and cooling to room temperature to obtain the culture medium.

The enzyme activity of the endo-cellulase is 15-18U/g; the enzyme activity of the phytase is 400-450U/g;

the viable count of the bacillus subtilis is 8.2 multiplied by 10⁹-8.5×10⁹cfu/g, the viable count of the bacillus mucilaginosus is 2.5 multiplied by 10⁹-3×10⁹cfu/g。

Transplanting the tissue culture grafted seedlings after hardening to a field, watering thoroughly once, counting the number of the survival watermelon seedlings after 10 days, and calculating to obtain the transplanting survival rate.

By adopting the technical scheme, the beneficial effects are that:

(1) the early spring Hongyu watermelon is bred by adopting the watermelon tissue culture rapid propagation method, the breeding rate is high, the trait inheritance is stable, the breeding rate is up to 70-80 times, and the incidence rate of virus diseases of tissue culture seedlings is zero.

(2) The tissue culture and rapid propagation method of the watermelon is adopted to propagate the early spring Hongyu watermelon, the grafting survival rate of the tissue culture seedling is 99.2-99.5%, and the transplanting survival rate is 99.7-99.9%.

Detailed Description

Example 1 tissue culture and rapid propagation method of watermelon

The method comprises the following steps:

(1) selecting explants

The early spring ruby watermelon is planted in a seed planting mode. Selecting strong and disease-free watermelon plants with the main vine length of 1.8m, and cutting 1.5cm of stem tips as explants.

(2) In vitro post-treatment

And transferring the explants to a tissue culture laboratory for in vitro post-treatment. The in vitro post-treatment comprises disinfection treatment and soaking treatment. The disinfection treatment comprises the following steps: putting the explant into a wide-mouth bottle, adding mercuric chloride with the mass percentage concentration of 0.08% for disinfection for 40s, then washing with sterile water for 3 times, then adding alcohol with the mass percentage concentration of 75% for disinfection for 20s, and then washing with sterile water for 3 times. The soaking treatment comprises the following steps: magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and sterile water are mixed according to the weight ratio of 2: 0.5: 1: 200 to obtain a mixed solution. And (3) placing the sterilized explants in the mixed solution, and soaking for 2.5min at 20 ℃.

(3) Dedifferentiation

And (4) placing the soaked explants in a dedifferentiation culture solution for dedifferentiation to obtain callus. The preparation method of the dedifferentiation culture solution comprises the following steps:

the aqueous solution is prepared according to the following formula: KNO₃5.4g/L, 9.3g/L ammonium sulfate, 85mg/L, MgSO mg potassium dihydrogen phosphate₄·7H₂139mg/L of O, 20mg/L, KI 0.75.75 mg/L of zinc sulfate and 5.2mg/L, MnSO of boric acid₄·H₂O7.3 mg/L, sodium molybdate 12.5mg/L, Na2MnO 4.2H2O 9.25.25 mg/L, CuSO 4.5H2O 3.1.1 mg/L, CoCl₂·6H₂O 0.58mg/L、FeSO₄·7H₂7.2mg/L of O, 0.25mg/L of sodium chloride, 0.15mg/L of folic acid, 0.83mg/L of biotin, 0.01mg/L of choline, 0.06mg/L of indoleacetic acid and 0.06mg/L of 6-BA. Adjusting pH of the culture solution to 7.0, sterilizing the culture solution in a high pressure steam sterilizing pot for 30min, and cooling to 27 deg.C to obtain dedifferentiation culture solution.

The dedifferentiation: the dedifferentiation culture solution was cultured at 27 ℃ for 14 days in the dark.

(4) Differential culture

Transferring the callus onto a differentiation culture medium for 24 days, including first-stage culture and second-stage culture; a first-stage culture: the culture time is 12 days, the culture temperature is 26 ℃, the illumination intensity is 2400LX, and the illumination duration is 9 h/day; two-stage culture: the culture time is 12 days, the culture temperature is 26 ℃, the illumination intensity is 2300LX, and the illumination time is 11 h/day. And (3) obtaining a large number of cluster buds through differentiation culture, and dispersing the single cluster buds for later use.

The differentiation medium is as follows: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 8.5mg/L boric acid, 0.2mg/L inositol, 1.5mg/L casein hydrolysate, 0.01mg/L zeatin, 0.04 mg/L6-benzylaminopurine, 0.02mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, and 8g/L agar. Adjusting pH of the culture solution to 6.8, sterilizing the culture solution in a high pressure steam sterilization pot for 30min, cooling to 25 deg.C, and standing for 4 hr to obtain differentiation culture medium.

(5) Strong seedling culture

Inoculating single cluster bud into strong budCulturing on a seedling culture medium for 7 days at the illumination intensity of 2800LX for 15 h/day and the culture temperature of 26 ℃; the strong seedling culture medium comprises: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 6.2mg/L boric acid, 0.35mg/L glutamic acid, 0.78mg/L cysteine, 0.15mg/L pantothenic acid, 0.08mg/L carotene, 0.01mg/L indolebutyric acid, 0.03mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, 0.02 mg/L6-benzylaminopurine and 9.5g/L agar. Adjusting the pH of the culture solution to 6.7, sterilizing the culture solution in a high-pressure steam sterilization pot for 30min, cooling to 25 ℃, and standing for 4h to obtain the strong seedling culture medium.

(6) Grafting

The tissue culture seedling after strong seedling culture is scion; the adopted stock is a calabash seedling with two leaves and one core.

When grafting, the true leaves and the growing points of the stock seedlings are firstly pinched off, and the tips of the bamboo sticks are obliquely inserted downwards from the base parts of the growing points at an angle of 60 degrees. The scion tissue culture seedlings are cut into wedges by a blade at a position 0.5cm below the growing point. And pulling out the bamboo stick, and obliquely inserting the cut scion into the inserting hole penetrated by the bamboo stick on the stock to obtain the tissue culture grafted seedling.

Sterile water is sprayed on the first day after grafting, and the sterile water is sprayed once at nine am and four pm.

Spraying the growth promoting liquid for 1 time every day from the second day to the fourth day after grafting, wherein the spraying time is 10:00 in the morning. The growth promoting liquid is an aqueous solution prepared from 0.4mg/L alginic acid, 0.31mg/L chitosan, 0.12mg/L xylitol, 0.04mg/L brassinolide and 0.02mg/L jasmonic acid.

And 7 days after grafting, counting the grafting survival condition of the tissue culture seedling, and calculating to obtain the grafting survival rate of the tissue culture seedling.

(7) Hardening off seedlings

Transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2800Lx, the illumination time is 12h, the temperature is controlled to be 28 ℃ in the daytime, 20 ℃ at night and the humidity is 70%; hardening seedlings in the last ten days: the illumination intensity is 3000Lx, the illumination time is 10h, the temperature is controlled to be 32 ℃ in the daytime, 15 ℃ at night and the humidity is 63%.

The culture medium comprises: the preparation method comprises the following steps: the feed additive is prepared from coconut shell powder, chitin, Muyu stone powder, river sand and chicken manure according to the proportion of 20: 8: 14: 9:3, and spraying deionized water to control the water content to be 35% to obtain the culture medium raw material. Mixing the culture medium raw material with endo-cellulase, phytase, bacillus subtilis and bacillus mucilaginosus according to the weight ratio of 160:1: 2.2: and 8:3 to obtain a premix. Fermenting the premix for 72 hours at 35 ℃, discharging and cooling to room temperature to obtain the culture medium.

The enzyme activity of the endo-cellulase is 18U/g; the enzyme activity of the phytase is 400U/g;

the viable count of the bacillus subtilis is 8.2 multiplied by 10⁹cfu/g, the viable count of the bacillus mucilaginosus is 2.5 multiplied by 10⁹cfu/g。

Transplanting the tissue culture grafted seedlings after hardening to a field, watering thoroughly once, counting the number of the survival watermelon seedlings after 10 days, and calculating to obtain the transplanting survival rate.

The tissue culture and rapid propagation method of the watermelon is adopted to propagate the early spring Hongyu watermelon, 2000 tissue culture seedlings are cultivated, and 1984 seedlings are grafted into a survival seedling plant, wherein the grafting survival rate is 99.2%; transplanting all the grafted and survived seedling plants into the field, wherein the transplanting survival rate of the transplanted and survived watermelon seedlings is 99.7 percent, and 1978 plants are the total plants; no virus-infected seedling strain is found in the whole culture process (from tissue culture to flowering and fruiting).

Example 2 tissue culture and rapid propagation method of watermelon

The method comprises the following steps:

(1) selecting explants

The early spring ruby watermelon is planted in a seed planting mode. Selecting strong and disease-free watermelon plants with the main vine length of 1.6m, and cutting 1.3cm of stem tips as explants.

(2) In vitro post-treatment

And transferring the explants to a tissue culture laboratory for in vitro post-treatment. The in vitro post-treatment comprises disinfection treatment and soaking treatment. The disinfection treatment comprises the following steps: putting the explant into a wide-mouth bottle, adding mercuric chloride with the mass percentage concentration of 0.08% for disinfection for 45s, then washing with sterile water for 3 times, then adding alcohol with the mass percentage concentration of 75% for disinfection for 20s, and then washing with sterile water for 3 times. The soaking treatment comprises the following steps: magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and sterile water are mixed according to the weight ratio of 2: 0.5: 1: 200 to obtain a mixed solution. And (3) placing the sterilized explants in the mixed solution, and soaking for 2.5min at 22 ℃.

(3) Dedifferentiation

And (4) placing the soaked explants in a dedifferentiation culture solution for dedifferentiation to obtain callus. The preparation method of the dedifferentiation culture solution comprises the following steps:

the aqueous solution is prepared according to the following formula: KNO₃5.4g/L, 9.3g/L ammonium sulfate, 85mg/L, MgSO mg potassium dihydrogen phosphate₄·7H₂139mg/L of O, 20mg/L, KI 0.75.75 mg/L of zinc sulfate and 5.2mg/L, MnSO of boric acid₄·H₂O7.3 mg/L, sodium molybdate 12.5mg/L, Na2MnO 4.2H2O 9.25.25 mg/L, CuSO 4.5H2O 3.1.1 mg/L, CoCl₂·6H₂O 0.58mg/L、FeSO₄·7H₂7.2mg/L of O, 0.25mg/L of sodium chloride, 0.15mg/L of folic acid, 0.83mg/L of biotin, 0.01mg/L of choline, 0.06mg/L of indoleacetic acid and 0.06mg/L of 6-BA. Adjusting pH of the culture solution to 7.0, sterilizing the culture solution in a high pressure steam sterilizing pot for 30min, and cooling to 27 deg.C to obtain dedifferentiation culture solution.

The dedifferentiation: controlling the temperature of the dedifferentiation culture solution at 26 ℃, and culturing for 15 days in the dark.

(4) Differential culture

Transferring the callus onto a differentiation culture medium for culturing for 22 days, wherein the culturing comprises first-stage culture and second-stage culture; a first-stage culture: the culture time is 10 days, the culture temperature is 26 ℃, the illumination intensity is 2500LX, and the illumination time is 9 h/day; two-stage culture: the culture time is 12 days, the culture temperature is 26 ℃, the illumination intensity is 2300LX, and the illumination time is 11 h/day. And (3) obtaining a large number of cluster buds through differentiation culture, and dispersing the single cluster buds for later use.

The differentiation medium is as follows: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate,0.37g/L of magnesium sulfate, 0.44g/L of calcium chloride, 8.5mg/L of boric acid, 0.2mg/L of inositol, 1.5mg/L of casein hydrolysate, 0.01mg/L of zeatin, 0.04mg/L of 6-benzylaminopurine, 0.02mg/L of indoleacetic acid, 0.01mg/L of 5-hydroxy-IAA and 8g/L of agar. Adjusting pH of the culture solution to 6.8, sterilizing the culture solution in a high pressure steam sterilization pot for 30min, cooling to 25 deg.C, and standing for 4 hr to obtain differentiation culture medium.

(5) Strong seedling culture

Respectively inoculating the single cluster buds to a strong seedling culture medium, and culturing for 8 days at the illumination intensity of 2700LX, the illumination time of 15 h/day and the culture temperature of 27 ℃; the strong seedling culture medium comprises: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 6.2mg/L boric acid, 0.35mg/L glutamic acid, 0.78mg/L cysteine, 0.15mg/L pantothenic acid, 0.08mg/L carotene, 0.01mg/L indolebutyric acid, 0.03mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, 0.02 mg/L6-benzylaminopurine and 9.5g/L agar. Adjusting the pH of the culture solution to 6.7, sterilizing the culture solution in a high-pressure steam sterilization pot for 30min, cooling to 25 ℃, and standing for 4h to obtain the strong seedling culture medium.

(6) Grafting

The tissue culture seedling after strong seedling culture is scion; the adopted stock is a calabash seedling with two leaves and one core.

When grafting, the true leaves and the growing points of the stock seedlings are firstly pinched off, and the tips of the bamboo sticks are obliquely inserted downwards from the base parts of the growing points at an angle of 60 degrees. The scion tissue culture seedlings are cut into wedges by a blade at a position 0.5cm below the growing point. And pulling out the bamboo stick, and obliquely inserting the cut scion into the inserting hole penetrated by the bamboo stick on the stock to obtain the tissue culture grafted seedling.

Sterile water is sprayed on the first day after grafting, and the sterile water is sprayed once at nine am and four pm.

And spraying the growth promoting liquid for 1 time every day from the second day to the fourth day after grafting, wherein the spraying time is 9:30 in the morning. The growth promoting liquid is an aqueous solution prepared from 0.45mg/L alginic acid, 0.28mg/L chitosan, 0.14mg/L xylitol, 0.03mg/L brassinolide and 0.024mg/L jasmonic acid.

And 7 days after grafting, counting the grafting survival condition of the tissue culture seedling, and calculating to obtain the grafting survival rate of the tissue culture seedling.

(7) Hardening off seedlings

Transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2900Lx, the illumination time is 14h, the temperature is controlled: 27 ℃ in the daytime, 17 ℃ at night and 68% humidity; hardening seedlings in the last ten days: the illumination intensity is 3100Lx, the illumination time is 11h, and the temperature is controlled as follows: the day was 32 deg.C, night was 12 deg.C, and humidity was 62%.

The culture medium comprises: the preparation method comprises the following steps: the feed additive is prepared from coconut shell powder, chitin, Muyu stone powder, river sand and chicken manure according to the weight ratio of 25: 6: 13: 11: 3.5, spraying deionized water to control the water content to be 35 percent, and obtaining the culture medium raw material. Mixing the culture medium raw material with endo-cellulase, phytase, bacillus subtilis and bacillus mucilaginosus according to a ratio of 155:1:2:7:3.5 to obtain a premix. Fermenting the premix for 72 hours at 34 ℃, discharging and cooling to room temperature to obtain the culture medium.

The enzyme activity of the endo-cellulase is 16U/g; the enzyme activity of the phytase is 430U/g;

the viable count of the bacillus subtilis is 8.4 multiplied by 10⁹cfu/g, the viable count of the bacillus mucilaginosus is 2.8 multiplied by 10⁹cfu/g。

The tissue culture and rapid propagation method of the watermelon is adopted to propagate the early spring Hongyu watermelon, 2000 tissue culture seedlings are cultivated, 1990 tissue culture seedlings are grafted to survival seedlings, and the grafting survival rate is 99.5%; transplanting all the grafted and survived seedling plants into the field, wherein the transplanting survival rate of the transplanted and survived watermelon seedlings is 99.9 percent, and the total number of the transplanted and survived watermelon seedlings is 1988; no virus-infected seedling strain is found in the whole culture process (from tissue culture to flowering and fruiting).

Example 3 tissue culture and rapid propagation method of watermelon

The method comprises the following steps:

(1) selecting explants

The early spring ruby watermelon is planted in a seed planting mode. Selecting strong and disease-free watermelon plants with the main vine length of 1.5m, and cutting 1cm of stem tips as explants.

(2) In vitro post-treatment

And transferring the explants to a tissue culture laboratory for in vitro post-treatment. The in vitro post-treatment comprises disinfection treatment and soaking treatment. The disinfection treatment comprises the following steps: putting the explant into a wide-mouth bottle, adding mercuric chloride with the mass percentage concentration of 0.08% for disinfection for 50s, then washing with sterile water for 3 times, then adding alcohol with the mass percentage concentration of 75% for disinfection for 20s, and then washing with sterile water for 3 times. The soaking treatment comprises the following steps: magnesium sulfate, potassium dihydrogen phosphate, calcium chloride and sterile water are mixed according to the weight ratio of 2: 0.5: 1: 200 to obtain a mixed solution. And (3) placing the sterilized explants in the mixed solution, and soaking for 2min at 25 ℃.

(3) Dedifferentiation

And (4) placing the soaked explants in a dedifferentiation culture solution for dedifferentiation to obtain callus. The preparation method of the dedifferentiation culture solution comprises the following steps:

the aqueous solution is prepared according to the following formula: KNO₃5.4g/L, 9.3g/L ammonium sulfate, 85mg/L, MgSO mg potassium dihydrogen phosphate₄·7H₂139mg/L of O, 20mg/L, KI 0.75.75 mg/L of zinc sulfate and 5.2mg/L, MnSO of boric acid₄·H₂O7.3 mg/L, sodium molybdate 12.5mg/L, Na2MnO 4.2H2O 9.25.25 mg/L, CuSO 4.5H2O 3.1.1 mg/L, CoCl₂·6H₂O 0.58mg/L、FeSO₄·7H₂7.2mg/L of O, 0.25mg/L of sodium chloride, 0.15mg/L of folic acid, 0.83mg/L of biotin, 0.01mg/L of choline, 0.06mg/L of indoleacetic acid and 0.06mg/L of 6-BA. Adjusting pH of the culture solution to 7.0, sterilizing the culture solution in a high pressure steam sterilizing pot for 30min, and cooling to 27 deg.C to obtain dedifferentiation culture solution.

The dedifferentiation: controlling the temperature of the dedifferentiation culture solution at 26 ℃, and culturing for 15 days in the dark.

(4) Differential culture

Transferring the callus onto a differentiation culture medium for culturing for 20 days, wherein the culturing comprises first-stage culture and second-stage culture; a first-stage culture: the culture time is 10 days, the culture temperature is 26 ℃, the illumination intensity is 2600LX, and the illumination time is 9 h/day; two-stage culture: the culture time is 10 days, the culture temperature is 26 ℃, the illumination intensity is 2200LX, and the illumination time is 11 h/day. And (3) obtaining a large number of cluster buds through differentiation culture, and dispersing the single cluster buds for later use.

The differentiation medium is as follows: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 8.5mg/L boric acid, 0.2mg/L inositol, 1.5mg/L casein hydrolysate, 0.01mg/L zeatin, 0.04 mg/L6-benzylaminopurine, 0.02mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, and 8g/L agar. Adjusting pH of the culture solution to 6.8, sterilizing the culture solution in a high pressure steam sterilization pot for 30min, cooling to 25 deg.C, and standing for 4 hr to obtain differentiation culture medium.

(5) Strong seedling culture

Respectively inoculating single cluster buds to a strong seedling culture medium, and culturing for 9 days at the illumination intensity of 2600LX for 14 h/day and the culture temperature of 28 ℃; the strong seedling culture medium comprises: the aqueous solution is prepared according to the following formula: KNO₃1.9g/L, 1.65g/L ammonium nitrate, 0.17g/L potassium dihydrogen phosphate, 0.37g/L magnesium sulfate, 0.44g/L calcium chloride, 6.2mg/L boric acid, 0.35mg/L glutamic acid, 0.78mg/L cysteine, 0.15mg/L pantothenic acid, 0.08mg/L carotene, 0.01mg/L indolebutyric acid, 0.03mg/L indoleacetic acid, 0.01 mg/L5-hydroxy-IAA, 0.02 mg/L6-benzylaminopurine and 9.5g/L agar. Adjusting the pH of the culture solution to 6.7, sterilizing the culture solution in a high-pressure steam sterilization pot for 30min, cooling to 25 ℃, and standing for 4h to obtain the strong seedling culture medium.

(6) Grafting

The tissue culture seedling after strong seedling culture is scion; the adopted stock is a calabash seedling with two leaves and one core.

When grafting, the true leaves and the growing points of the stock seedlings are firstly pinched off, and the tips of the bamboo sticks are obliquely inserted downwards from the base parts of the growing points at an angle of 60 degrees. The scion tissue culture seedlings are cut into wedges by a blade at a position 0.5cm below the growing point. And pulling out the bamboo stick, and obliquely inserting the cut scion into the inserting hole penetrated by the bamboo stick on the stock to obtain the tissue culture grafted seedling.

Sterile water is sprayed on the first day after grafting, and the sterile water is sprayed once at nine am and four pm.

Spraying the growth promoting liquid for 1 time every day from the second day to the fourth day after grafting, wherein the spraying time is 9:00 in the morning. The growth promoting liquid is an aqueous solution prepared from 0.5mg/L alginic acid, 0.25mg/L chitosan, 0.15mg/L xylitol, 0.02mg/L brassinolide and 0.03mg/L jasmonic acid.

And 7 days after grafting, counting the grafting survival condition of the tissue culture seedling, and calculating to obtain the grafting survival rate of the tissue culture seedling.

(7) Hardening off seedlings

Transplanting the tissue culture grafted seedlings into a culture medium, laminating, hardening seedlings for 20 days, and hardening seedlings in the first ten days: the illumination intensity is 2900Lx, the illumination time is 13h, the temperature is controlled: 28 ℃ in the daytime, 18 ℃ at night and 68% humidity; hardening seedlings in the last ten days: the illumination intensity is 3000Lx, the illumination time is 10h, the temperature is controlled as follows: day 32 ℃, night 14 ℃ and humidity 63%.

The culture medium comprises: the preparation method comprises the following steps: the feed additive is prepared from coconut shell powder, chitin, Muyu stone powder, river sand and chicken manure according to the proportion of 30: 5: 10: 13: 4, and spraying deionized water to control the water content to be 35% to obtain the culture medium raw material. Mixing the culture medium raw material with endo-cellulase, phytase, bacillus subtilis and bacillus mucilaginosus according to the weight ratio of 150:2: 1.5: and 6:4, mixing to obtain the premix. Fermenting the premix for 72 hours at 32 ℃, discharging and cooling to room temperature to obtain the culture medium.

The enzyme activity of the endo-cellulase is 15U/g; the enzyme activity of the phytase is 450U/g;

the viable count of the bacillus subtilis is 8.5 multiplied by 10⁹cfu/g, the viable count of the bacillus mucilaginosus is 3 multiplied by 10⁹cfu/g。

Transplanting the tissue culture grafted seedlings after hardening to a field, watering thoroughly once, counting the number of the survival watermelon seedlings after 10 days, and calculating to obtain the transplanting survival rate.

The tissue culture and rapid propagation method of the watermelon is adopted to propagate the early spring Hongyu watermelon, 2000 tissue culture seedlings are cultivated, and 1986 seedlings are grafted into survival seedlings, wherein the grafting survival rate is 99.3%; transplanting all the grafted and survived seedling plants into the field, wherein the transplanting survival rate of the transplanted and survived watermelon seedlings is 99.8 percent, and the total number of the transplanted and survived watermelon seedlings is 1982; no virus-infected seedling strain is found in the whole culture process (from tissue culture to flowering and fruiting).

Except for special description, the proportions are mass ratios, and the percentages are mass percentages.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.