CN104920212A - Siraitia grosvenorii tissue culture seedling propagation method - Google Patents

Abstract

The invention discloses a method for breeding Luo Han Guo tissue-cultured seedlings. The breeding method comprises the following steps: (1) collecting propagation materials, (2) disinfection treatment, (3) detoxification culture of stem tips, (4) segmented subculture, ( 5) induced rooting culture, (6) transplanting, (7) growing seedlings in greenhouses, (8) going out of sheds; the Luo Han Guo tissue culture seedling breeding method of the present invention cultivates tissue culture seedlings by cutting technology under sterile conditions. The planting of tissue cultured seedlings has the characteristics of high yield, fast propagation with less disease, wide adaptability, high yield and high yield in the same year of planting. The previous seed potato planting is gradually being replaced by tissue cultured seedlings.

Description

A kind of Momordica grosvenori tissue culture seedling breeding method

technical field

The invention relates to the technical field of fruit tree cultivation, in particular to a method for growing Luo Han Guo tissue cultured seedlings.

Background technique

The cultivation of Luo Han Guo has a long history in my country. In recent years, studies have found that the use value of Luo Han Guo is very high. People are more and more optimistic about the market prospect of Luo Han Guo, and the planting scale continues to expand. However, the traditional cultivation of Luo Han Guo is planted with seed potatoes. The disadvantages of seed potato planting are low yield, more virus accumulation, variety degradation, and the seedlings cannot meet the needs of mass planting. In recent years, people have carried out in-depth research on a series of technologies such as cultivating improved varieties of Luo Han Guo, standardized planting and demonstration, especially in the rapid propagation and detoxification of fine varieties through tissue culture. The planting of tissue culture seedlings has the great advantages of high yield and few diseases. The previous seed potato planting is gradually being replaced by tissue culture seedlings.

Contents of the invention

For the above shortcoming of the prior art, the present invention provides a kind of a large amount of rapid propagation Luo Han Guo tissue cultured seedlings, the Luo Han Guo tissue cultured seedling breeding method that satisfies the needs of large-area planting development on production, it is characterized in that: this breeding method comprises the following steps:

(1) Collection of propagation materials:

Choose to collect branches from varieties and individual plants that grow robustly, come to flowers in a timely manner, are easy to bear fruit, and have large and neat fruit;

(2) Disinfection treatment:

Cut young shoots of about 50 cm, rinse them with sterile water on the workbench of the tissue culture room, immerse them in 75% alcohol solution for 5 seconds, then immerse them in 0.1% mercuric solution for 10 minutes, take them out and rinse them with sterile water ;

(3) Virus-free cultivation of shoot tips:

Prepare the basic medium with MS+BA1.0mg/L+IAA0.1mg/L+agar 0.35%+sucrose 3%, adjust the pH value to 6, divide the prepared medium, each bottle is about 2cm high, Tighten the cap tightly, let it cool naturally after autoclaving;

Cutting out the shoot apical meristem, cutting out the shoot apical meristem of 0.5-1mm, and then inoculating it into the culture medium for culturing;

(4) Section subculture:

MS+BA0.5mg/L+IAA0.1mg/L+IBA0.5mg/L is used for the subculture medium of the preparation medium;

Segmented subculture When the seedlings grown from the shoot apical meristem grow to a height of 6 cm, the shoots are cut into segments, each segment has an internode of about 0.5 cm, and are inoculated on the subculture medium for subculture. Repeated subculture for 5-6 times in this way, and then transferred to the rooting medium to induce rooting. The temperature of the culture room is 24-29°C, the light intensity is 800-1200Lx, and the light is 12 hours a day;

(5) induced rooting culture:

Prepare the medium to induce rooting medium with MS+BA0.03mg/L+NAA0.3mg/L+0.01% activated carbon;

For inoculation, select bottle seedlings that have been cultured for more than 20 days, grow vigorously, and are non-polluting as seedlings for rooting culture. Cut off the leaves, and then cut off one section of the bud, requiring the base to be longer than the top, and then insert them into the culture medium of different culture bottles evenly according to the base, stem section, and top bud for cultivation, and each bottle receives 10 plants;

Early cultivation of rooted seedlings: 25°C, dark culture for 5 days, humidity 85%; middle stage: 28°C, light 1800LX, light 12h per day, humidity 85%; late stage: 25°C, light 1500LX, light 12h per day, humidity 85%. All can take root successively after 10d;

(6) seedling hardening:

After the tissue-cultured rooted seedlings grow about 0.5cm long, move them to the greenhouse for seedling training; 2 days before moving to the greenhouse for the first time, do not let the seedlings be directly exposed to strong light; after 2 to 3 days, open the bottle cap, and spray water to moisturize immediately after opening the cap. To prevent wilting, cover with damp newspaper after spraying with water. After 2 days, open the newspaper properly, but still avoid direct sunlight, and transplant after 10 days;

(7) Transplanting

① The ground of the greenhouse is disinfected with quicklime powder, and the walls and roof of the greenhouse are sprayed with a mixed solution of imidacloprid, chrysanthemum pesticides, and thiophanate-methyl;

②Nutritional soil cups use pond mud as a culture substrate, put it into a 7cm×6cm nutrient cup, and then use 500 times of carbendazim solution to drench and disinfect;

③Wash seedlings Put the selected normal seedlings in water, wash the culture medium adhered to the base, soak the washed seedlings in 500 times carbendazim solution for 5 seconds, then soak in 800 times rooting powder solution for 15 minutes .

④ After planting, use a spoon to dig up the soil, put the treated seedling base into the pit, cover the soil back, immediately drench the water, and then cover it with a film to form a small shed to keep warm and keep moisture.

(8) Greenhouse seedling cultivation

① Sterilize the seedlings before sterilizing and surviving, and spray the carbendazim aqueous solution every morning to sterilize the seedlings and replenish water to inhibit the growth of miscellaneous bacteria;

②Insulation In February, the outdoor temperature is low, so greenhouses are needed to keep them warm, and a small shed should be built in the greenhouses to keep them warm;

③Illumination The sunlight is not very strong in February, but the seedlings cannot stand the strong sunlight. When the sun is out, cover the sunshade net to prevent the seedlings from being burned. The initial management should be particularly careful and conscientious, observe the changes of the weather and the growth of the seedlings at any time, and take appropriate measures in time;

④After the seedlings with the film removed survive, they will enter the mid-growth stage. During the day when the temperature is relatively stable, the film can be uncovered to allow them to gradually adapt to the environment in the greenhouse, and then covered with the film at night; when the weather is warmer, there is no need to cover the film at night;

⑤For fertilization, use Duodefulu, potassium dihydrogen phosphate, compound fertilizer and other fertilizers for top dressing;

(9) out of shed

The tissue-cultured seedlings can start to emerge from the nursery when the leaves are dark green, the stems are thick, and the height is more than 6 cm; they are shipped in hard bamboo or plastic baskets.

Beneficial effects of the present invention: by using the technical method, a large number of Luo Han Guo tissue-cultured seedlings can be rapidly propagated to meet the needs of large-area planting and development in production. The tissue culture seedling breeding method of Luo Han Guo of the present invention is under sterile conditions, and the tissue culture seedlings cultivated by cutting technology have the characteristics of less disease, fast reproduction, wide adaptability, harvest in the same year and high yield.

specific embodiment

A kind of Luo Han Guo tissue culture seedling breeding method, this breeding method comprises the following steps:

(1) Collection of propagation materials:

Choose to collect branches from varieties and individual plants that grow robustly, come to flowers in a timely manner, are easy to bear fruit, and have large and neat fruit;

(2) Disinfection treatment:

Cut young shoots of about 50 cm, rinse them with sterile water on the workbench of the tissue culture room, immerse them in 75% alcohol solution for 5 seconds, then immerse them in 0.1% mercuric solution for 10 minutes, take them out and rinse them with sterile water ;

(3) Virus-free cultivation of shoot tips:

Prepare the basic medium with MS+BA1.0mg/L+IAA0.1mg/L+agar 0.35%+sucrose 3%, adjust the pH value to 6, divide the prepared medium, each bottle is about 2cm high, Tighten the cap tightly, let it cool naturally after autoclaving;

Cutting out the shoot apical meristem, cutting out the shoot apical meristem of 0.5-1mm, and then inoculating it into the culture medium for culturing;

(4) Section subculture:

MS+BA0.5mg/L+IAA0.1mg/L+IBA0.5mg/L is used for the subculture medium of the preparation medium;

Segmented subculture When the seedlings grown from the shoot apical meristem grow to a height of 6 cm, the shoots are cut into segments, each segment has an internode of about 0.5 cm, and are inoculated on the subculture medium for subculture. Repeated subculture for 5-6 times in this way, and then transferred to the rooting medium to induce rooting. The temperature of the culture room is 24-29°C, the light intensity is 800-1200Lx, and the light is 12 hours a day;

(5) induced rooting culture:

Prepare the medium to induce rooting medium with MS+BA0.03mg/L+NAA0.3mg/L+0.01% activated carbon;

For inoculation, select bottle seedlings that have been cultured for more than 20 days, grow vigorously, and are non-polluting as seedlings for rooting culture. Cut off the leaves, and then cut off one section of the bud, requiring the base to be longer than the top, and then insert them into the culture medium of different culture bottles evenly according to the base, stem section, and top bud for cultivation, and each bottle receives 10 plants;

Early cultivation of rooted seedlings: 25°C, dark culture for 5 days, humidity 85%; middle stage: 28°C, light 1800LX, light 12h per day, humidity 85%; late stage: 25°C, light 1500LX, light 12h per day, humidity 85%. All can take root successively after 10d;

(6) seedling hardening:

After the tissue-cultured rooted seedlings grow about 0.5cm long, move them to the greenhouse for seedling training; 2 days before moving to the greenhouse for the first time, do not let the seedlings be directly exposed to strong light; after 2 to 3 days, open the bottle cap, and spray water to moisturize immediately after opening the cap. To prevent wilting, cover with damp newspaper after spraying with water. After 2 days, open the newspaper properly, but still avoid direct sunlight, and transplant after 10 days;

(7) Transplanting

① The ground of the greenhouse is disinfected with quicklime powder, and the walls and roof of the greenhouse are sprayed with a mixed solution of imidacloprid, chrysanthemum pesticides, and thiophanate-methyl;

②Nutritional soil cups use pond mud as a culture substrate, put it into a 7cm×6cm nutrient cup, and then use 500 times of carbendazim solution to drench and disinfect;

③Wash seedlings Put the selected normal seedlings in water, wash the culture medium adhered to the base, soak the washed seedlings in 500 times carbendazim solution for 5 seconds, then soak in 800 times rooting powder solution for 15 minutes .

④ After planting, use a spoon to dig up the soil, put the treated seedling base into the pit, cover the soil back, immediately drench the water, and then cover it with a film to form a small shed to keep warm and keep moisture.

(8) Greenhouse seedling cultivation

① Sterilize the seedlings before sterilizing and surviving, and spray the carbendazim aqueous solution every morning to sterilize the seedlings and replenish water to inhibit the growth of miscellaneous bacteria;

②Insulation In February, the outdoor temperature is low, so greenhouses are needed to keep them warm, and a small shed should be built in the greenhouses to keep them warm;

③Illumination The sunlight is not very strong in February, but the seedlings cannot stand the strong sunlight. When the sun is out, cover the sunshade net to prevent the seedlings from being burned. The initial management should be particularly careful and conscientious, observe the changes of the weather and the growth of the seedlings at any time, and take appropriate measures in time;

④After the seedlings with the film removed survive, they will enter the mid-growth stage. During the day when the temperature is relatively stable, the film can be uncovered to allow them to gradually adapt to the environment in the greenhouse, and then covered with the film at night; when the weather is warmer, there is no need to cover the film at night;

⑤For fertilization, use Duodefulu, potassium dihydrogen phosphate, compound fertilizer and other fertilizers for top dressing;

(9) out of shed

The tissue-cultured seedlings can start to emerge from the nursery when the leaves are dark green, the stems are thick, and the height is more than 6 cm; they are shipped in hard bamboo or plastic baskets.

Experiments have proved that the tissue cultured seedlings cultivated by the cutting technique under aseptic conditions in the method for breeding Luo Han Guo tissue cultured seedlings of the present invention have the characteristics of less disease, fast reproduction, wide adaptability, harvest in the same year and high yield .

Claims (1)

1. a Luohanguo With Plantlets of Tissue Culture mating system, is characterized in that: this mating system comprises the following steps:

(1) propagating materials is gathered:

Select robust growth, spend bear fruit in time, easily, fruit kind neat greatly and individual plant on gather branch;

(2) disinfect:

Clip is about the tender tip of 50cm, on group training room workbench with aseptic water washing after immerse in 75% alcoholic solution the 5s that sterilizes, then immerse in 0.1% mercuric chloride solution the 10min that sterilizes, clean with aseptic water washing again after taking-up;

(3) stem apex detoxify is cultivated:

Preparation medium minimal medium MS+BA1.0mg/L+IAA0.1mg/L+ agar 0.35%+ sucrose 3%, pH value is adjusted to 6, and the medium prepared is carried out packing, and often bottled about 2cm is high, tightens lid, autoclaving relief its naturally cool for subsequent use;

Cut the shoot apical meristem that shoot apical meristem cuts 0.5 ~ 1mm, be then inoculated in medium and cultivate;

(4) segment squamous subculture:

Preparation medium subculture medium MS+BA0.5mg/L+IAA0.1mg/L+IBA0.5mg/L;

Segment squamous subculture carries out segment when the seedling that stem-apex Meristem culture goes out grows to 6cm height, and every length of tape internode is about 0.5cm, is inoculated on subculture medium and carries out squamous subculture.Subculture 5 ~ 6 times, then proceeds to root induction on root media so repeatedly.Culturing room's temperature is 24 ~ 29 DEG C, intensity of illumination 800 ~ 1200Lx, illumination every day 12h;

(5) root induction is cultivated:

Preparation medium root induction medium MS+BA0.03mg/L+NAA0.3mg/L+0.01% active carbon;

Inoculation select cultivated more than 20d, robust growth, free of contamination squamous subculture bottle seedling as the seedling of culture of rootage, be put on aseptic lead pad plate and vise callus with taking the photograph son, with scissors, the leaf on seedling is cut, cut off by a bud one section again, require that base portion is longer than top, then be evenly inserted into respectively according to base portion, stem section, terminal bud in the medium of different blake bottle and cultivate, the strain of every bottle graft 10;

Take root seedling cultivation early stage: 25 DEG C, light culture 5d, humidity 85%; Mid-term: 28 DEG C, illumination 1800LX, illumination every day 12h, humidity 85%; Later stage: 25 DEG C, illumination 1500LX, illumination every day 12h, humidity 85%.All can take root successively after 10d;

(6) hardening:

Group training take root seedling grow about 0.5cm long after, move in booth and carry out practicing seedling; Before just moving booth to, 2d can not allow high light direct projection seedling; After 2 ~ 3d, bottle cap is opened, after uncapping, water spray moisturizing is at once in case wilt, and covers after water spray with wet newspaper.After 2d, newspaper is suitably raised, but still high light direct projection will be avoided, can transplant after 10d;

(7) transplant

1. booth adopts quicklime powder to carry out ground sterilization, and booth wall and ceiling adopt Imidacloprid, chrysanthemum lipid agricultural chemicals, thiophanate methyl three kinds of pestsides synthesis spray solutions;

2. Nutrition Soil dress cup pond sludge makes culture matrix, loads in the nutrition cup of 7cm × 6cm, then to drench sterilization with the carbendazim solution of 500 times;

3. washing seedling is put in water by the field run plant elected, and cleaning sticks to the medium of base portion, washed seedling is put in the carbendazol of 500 times and soaks 5s, then steep 15min by 800 times of root-inducing powder immersions.

4. after plantation spoon digs earth, the seedling base portion handled well is put into hole and again earth lid is returned, water of drenching immediately, then cover film and become Small plastic shed with heat and moisture preserving.

(8) raise seedling in greenhouse

1. sterilization will carry out sterilization to seedling before surviving, and takes off film each morning and sprays the carbendazim aqueous solution, to seedling sterilization moisturizing, grow to suppress miscellaneous bacteria;

2. insulation outside air temperature in February is low, needs booth thermal insulation, takes Small plastic shed insulation in booth again;

3. illumination sunlight in February is not very strong, but seedling can't stand the acute irradiation of sunlight, time sunny will lid sunshade net in case seedling of burning.Initial management is careful especially, conscientious, and observe the change of weather and the growth condition shape of seedling at any time, take appropriate measures management in time;

4. take off and just to enter growth mid-term after film seedling survives, temperature metastable daytime, film can be opened and allow it progressively adapt to booth environment, membrane cover gets up by evening again; And time warmer to weather evening also do not need epiphragma.

5. apply fertilizer and use many get Fu Erlu, potassium dihydrogen phosphate, the fertilizer such as composite fertilizer topdress.

(9) canopy is gone out

Plantlet in vitro leaf look dark green, stem is sturdy, high more than 6cm time just can start garden; With hard bamboo basket or plastic crate shipment.