CN107047295A - A kind of sunflower tissue culture method - Google Patents

Abstract

The invention belongs to the field of plant biotechnology and cultivation technology, and specifically relates to a method for cultivating seedlings of sunflower groups. Multi-branch varieties are selected as tissue culture materials, and axillary buds of plants whose top flowers are about to open or have opened are selected as explants. Body culture, induction and differentiation, strong seedling culture, rooting culture, tissue culture seedling domestication, transplanting to obtain high-purity sunflower tissue culture seedlings, overcame the difficulties in seed propagation of double-petal sunflower, low seed setting rate, difficult to stabilize offspring traits, separation Serious problem, the purity of tissue culture seedlings is high, the frequency of its induced budding is high, and the propagation speed is fast; Since sunflower is a cross-pollinated plant, it is easy to produce various sunflower mutants with high ornamental value in the offspring. The preservation of quality resources is of great significance.

Description

A kind of method for cultivating seedlings of sunflower group

technical field

The invention belongs to the field of plant biotechnology and cultivation technology, and in particular relates to a method for cultivating sunflower seedlings.

Background technique

Sunflower (Helianthus anuus L.) is a cultivar of the genus Helianthus of the family Compositae, which originated in the southwestern part of North America. Sunflower was introduced into my country at the end of the 16th century or the beginning of the 17th century. As a dried fruit, it has gradually become one of the main sources of edible oil in the diet structure of our people since the 1990s. In the early 1880s, sunflower began to be used as an ornamental plant in Europe. It has a cultivation history of more than 100 years. From the early 1980s, people began to study sunflower tissue culture. Until now, it is still very difficult to regenerate sunflower plants in vitro. Even if it can regenerate, the repeatability is very poor. The regeneration pathway of sunflower is mainly through organogenesis and embryogenesis. The organ culture of sunflower has obtained regenerated plants in varying degrees from the cotyledons to the hypocotyls, true leaves, and shoot tips. These types of explants are more likely to induce regenerated plants from the cotyledons, hypocotyls, and shoot tips , but the overall frequency of induced budding is not high, generally 2.5% to 12%, and affected by various factors, buds are not easy to grow into plants. Sunflower embryo culture can overcome the incompatibility of distant hybridization, shorten the generation cycle of hybrid breeding, and speed up the breeding process.

In the article "Occurrence of Adventitious Buds in Sunflower Immature Embryo Culture" in "Journal of North China Agricultural Sciences" in Volume 8, No. 2, 1993, the research on the adventitious buds of sunflower immature embryos showed that the inbred lines, hybrid first generation and maintainer lines Regenerates more frequently than normal varieties. In "Journal of Inner Mongolia University for Nationalities (Natural Science Edition)" 2003, Volume 18, No. 5, "Studies on Tissue Culture and Regenerated Buds of Immature Embryos of Sunflower" on germination of immature embryos, induction of callus, differentiation and regeneration Studies on the cultivation and transplanting of seedlings have shown that the rate of embryo differentiation into seedlings 10-12 days after pollination can reach more than 73%.

The Jiangsu Institute of Agricultural Sciences in Coastal Areas has been engaged in research on the breeding of new sunflower varieties since 2011. After years of breeding, several new varieties of ornamental sunflowers with different characteristics have been cultivated. Practice has shown that single-petal sunflowers with multiple heads are easy to propagate by seeds Breed inbred lines with stable traits, but for double-petalled sunflowers (yellow single-petalled sunflowers, purple double-petalled sunflowers, white double-petalled sunflowers), seed propagation is used, the seed setting rate is low, the offspring have difficulty in stabilizing traits, and separation is serious .

Contents of the invention

In order to overcome the problems of multi-petal multi-head sunflower varieties (yellow double-petal multi-head sunflower, purple double-petal multi-head sunflower, white double-petal multi-head sunflower) seed propagation difficulties, low seed setting rate, difficult to stabilize offspring traits, and serious separation problems, the present invention proposes a A method for cultivating sunflower tissue culture seedlings with axillary buds as explants, and a sunflower regeneration system in vitro.

In order to solve the above problems, the technical solution of the present invention is as follows:

The invention discloses a method for cultivating seedlings of sunflower groups. The axillary buds of sunflowers are used as explants to cultivate sunflower seedlings through tissue culture.

Preferably, the group seedling cultivation method comprises the following steps:

A. the selection of explant: gather the axillary bud in the leaf axil below the top flower, the size of axillary bud is no more than 1cm;

B. Explant preparation: the explants are cleaned and sterilized, and then the tops of axillary buds with a size of about 5 mm are cut and inoculated on the differentiation medium as explants;

C. Induction and differentiation: the explants continue to be cultured and directly differentiate into adventitious buds;

D. Strong seedling cultivation: when the adventitious buds grow to 2-3cm, they are transferred to the strong seedling medium for cultivation;

E. Rooting culture: when the adventitious buds grow to 4-5cm, they are transferred to the rooting medium for cultivation;

F. Domestication of tissue-cultured seedlings: When the tissue-cultured seedlings have grown to 4-5 leaves, and 5-6 white and thick young roots are about 1-2 cm long, they are transplanted out of the bottle.

Preferably, in the group seedling cultivation method, the top flowers in step A are selected from varieties with ornamental value and many branches.

Preferably, in the group seedling cultivation method, the axillary buds in step A are selected from the top flowers that are about to open or have already opened, and it is easy to identify plants with consistent flower patterns, slow growth of axillary buds, and consistent other traits.

Preferably, the group seedling cultivation method,

In the step B, the explants are washed with water, and then rinsed with water for 0.5h. On the ultra-clean workbench, soak in 70% ethanol for 30-60s, and then use 10% hydrogen peroxide or 3% The sodium hypochlorite solution was disinfected for 15-20 minutes, and washed 3-4 times with sterile water; the differentiation medium was: MS + 0.6-1.5 mg/L 6-BA + 0.03mg/L NAA medium; among them, 6-BA The optimal concentration is 1.2 mg/L, and the differentiation rate is over 83.5%;

In the step C, the explants are directly differentiated into adventitious buds after being cultivated for 25-30 days, and then transferred to the strong seedling medium when the adventitious buds grow to 2-3 cm in 10-15 days; the cultivation of the explants The conditions are: culture in a tissue culture room with a temperature of 25-28°C, a light of 1600lx, and a fluorescent lamp for 12 hours a day. The solid medium contains 7.5g/L agar, and the pH is adjusted to 5.8-6.0 before sterilization;

In the step D, the medium for strong seedlings is: MS + 0.3-0.8 mg/L 6-BA + 0.03 mg/L NAA medium; wherein, the concentration of 6-BA is 0.6 mg/L is the optimal; The cycle is 25-30 days;

In the step E, the rooting medium is: 1/2 MS + 0.5 mg/L IBA medium; the culture period is 25 to 30 days;

In the step F, because the longer roots are easily broken and damaged during the cleaning process, the survival rate of transplanting is reduced, so before transplanting, an incomplete opening of the bottle is first carried out under the condition of sufficient sunlight indoors for 1 day. The water droplets on the cap of the culture bottle are completely dry, and then completely open the bottle for 2 days; the time of opening the bottle should not exceed 3 days, because the agar medium exposed to the air is undoubtedly a breeding ground for bacteria and other microorganisms, which is easy to infect and damage the plants and reduce the growth rate of tissue culture seedlings. survival rate.

Preferably, the transplanting described in step F of the method for cultivating seedlings of the group includes the following steps: gradually adapt the seedlings through domestication to external conditions, carefully take out the seedlings from the culture bottle with plastic tweezers, and try not to damage the young roots. If the medium is too dry, it can be soaked in tap water for 1-2 minutes before taking it out. The taken out tissue-cultured seedlings were soaked in 0.1% potassium permanganate for 1-2 minutes for disinfection, and the aged leaves on the tissue-cultured seedlings were removed, and then the young roots were lightly rubbed with absorbent cotton under running water. Rinse the root agar clean to prevent the agar residue from causing bacteria to breed and infect the plants. Before transplanting, the cultivation medium should be watered thoroughly in advance. Use a glass rod to make a small hole in the packed peat soil + perlite mixed matrix, and plant four-fifths of the underground part of the tissue culture seedlings in a moist matrix. Note that planting too deep or too shallow will affect the plant type , It is not conducive to the fixed growth and respiration of plants. Finally, water thoroughly and put the transplanted seedlings in a clean, clean, sunny and well-ventilated greenhouse or plastic greenhouse.

Preferably, in the group seedling cultivation method, the transplanting substrate is thoroughly sterilized, loose and permeable perlite, river sand or vermiculite plus peat soil.

Preferably, in the group seedling cultivation method, after transplanting, the cultivation temperature is maintained at 20-25°C, the humidity is 75%-85%, and a short-term 30% shade treatment is performed to promote rooting of the underground part. When the roots formed by plant callus have grown root hairs, functional new roots have grown slowly, which improves the water absorption capacity, and can be directly sprayed with thin fertilizer on the leaves.

Sunflower seedling diseases are mainly botrytis cinerea, root rot and stem rot, etc., which are easy to cause dead seedlings and rotten seedlings, and when serious, it is easy to cause huge losses; preferably, it can be effectively controlled:

1) Strengthen the management of the seedbed to prevent excessive humidity in the seedbed, control watering, the relative humidity of the air in the seedbed is 60-70%; properly heat the greenhouse at night; control the temperature of the seedbed at 25-30°C; increase the light intensity, and ventilation;

2) Chemical treatment, spray 70% chlorothalonil 500 times solution or 50% carbendazim 500 times solution or 200 times equivalent Bordeaux solution before the onset of the disease, and use 70% thiophanate-methyl solution or dilute 1 000 times of 50% dysonium medicinal liquid for spraying or spraying, or 50% dysonium 800-1000 times liquid for spraying or watering.

Preferably, in the method for cultivating seedlings of the group, the sunflower seedlings raised in plug trays can be transplanted to the field. The transplanting method is: water the transplanted seedlings thoroughly, dig them out together with the substrate, gently hold the soil mass, and The seedling roots are lightly placed in the holes opened in advance in the field, and the soil is filled and compacted.

Compared with the prior art, the advantages of the present invention are as follows:

The present invention proposes a method for cultivating sunflower tissue culture seedlings with axillary buds as explants. Compared with using cotyledons, hypocotyls, and shoot tips as explants, the frequency of induced budding is high, reaching more than 68%, and can be directly obtained from The explants induce adventitious buds, and each explant can increase in value by 3 to 5 times;

Fast propagation speed: Since the present invention selects multi-branch varieties as tissue culture materials, each individual plant can collect 15-20 axillary buds at a time, and some individual plants may have more, which can greatly improve the tissue culture propagation speed and shorten the establishment of isolated buds. The time to cultivate the regenerative system;

High purity of tissue culture seedlings: As the explants are selected as explants, the axillary buds of plants whose top flowers are about to open or have already opened can effectively improve the purity of tissue culture seedlings, quickly establish a homozygous in vitro culture regeneration system, and overcome the problem of using seeds for double-petal sunflowers. Difficulty in reproduction, low seed setting rate, unstable traits of offspring, and serious separation problems;

Since sunflower is a cross-pollinated plant, various sunflower mutants with high ornamental value are easy to be produced in offspring, and the invention has great significance for the preservation of sunflower specific planting resources.

Description of drawings

Fig. 1 Sunflower variety 1 suitable for patent tissue culture of the present invention——yellow common sunflower (single petal);

Fig. 2 Sunflower variety 2 applicable to the tissue culture of the patent of the present invention——common sunflower with purple core (single petal);

Fig. 3 Sunflower variety 3 suitable for tissue culture of the patent of the present invention - white ornamental sunflower (single petal);

Fig. 4 is suitable for sunflower variety 4 of the patent tissue culture of the present invention——yellow chrysanthemum sunflower (double petal);

Fig. 5 Sunflower variety 5 suitable for patent tissue culture of the present invention——yellow chrysanthemum sunflower (double petal);

Fig. 6 Sunflower variety 6 suitable for tissue culture of the patent of the present invention——yellow chrysanthemum sunflower (double petals);

Fig. 7 Sunflower variety 7 suitable for tissue culture of the patent of the present invention——yellow chrysanthemum sunflower (double petals)

Fig. 8 Sunflower variety 8 suitable for tissue culture of the patent of the present invention—color ornamental sunflower (double petals);

Fig. 9 Sunflower variety 9 suitable for tissue culture of the patent of the present invention—color ornamental sunflower (double petals);

Fig. 10 Sunflower variety 10 suitable for tissue culture of the patent of the present invention - white chrysanthemum sunflower (double petals);

Fig. 11 Sunflower variety 11 suitable for tissue culture of the patent of the present invention - white chrysanthemum sunflower (double petals);

The collection of Fig. 12 explant axillary bud of the present invention;

Fig. 13 The explant of the present invention is inoculated into the culture medium;

Figure 14 Differentiation of buds on explants (1);

Figure 15 Differentiation of buds on explants (2);

Figure 16 Differentiation of adventitious buds (1);

Figure 17 Differentiation of adventitious buds (2);

Figure 18 rooting culture (1);

Figure 19 Rooting culture (2).

detailed description

Example 1

Selection of sunflower varieties - choose varieties with ornamental value and many branches, such as Figure 1-11.

Selection of explants - select the top flower that is about to open or has already opened, and it is easy to identify plants with the same flower type, slow growth of axillary buds, and other consistent traits, and collect the axillary buds in each leaf axil below the top flower (see Figure 12). The size does not exceed 1cm.

Explant preparation - the collected explants were washed with clean water, and then rinsed with water for 0.5h, and then soaked in 70% ethanol for 30-60s on the ultra-clean workbench, and then soaked in 10% peroxide Disinfect with hydrogen or 3% sodium hypochlorite solution for 15 to 20 minutes, and wash with sterile water for 3 to 4 times. Then cut out the top of the axillary buds with a size of about 5 mm as explants and inoculate them on the differentiation medium (see Figure 13). The differentiation medium is: MS + 0.6-1.5 mg/L 6-BA + 0.03 mg/L NAA medium; , the optimal concentration of 6-BA was 1.2 mg/L, and the differentiation rate reached over 83.5%.

Induction and differentiation—the explants are cultured for 25-30 days and directly differentiate into adventitious buds. See Figure 14-15 for the differentiation of buds on the explants. Continue to culture for 10-15 days. When the adventitious buds grow to 2-3cm, transfer to Seedling culture medium; culture conditions are: temperature 25-28C, light 1600lx, daylight 12h per day in a tissue culture room to cultivate solid medium containing 7.5g/L agar, and adjust pH to 5.8-6.0 before sterilization.

Strong seedling cultivation——when the adventitious buds grow to 2-3cm, transfer the adventitious buds to the strong seedling medium and cultivate them for 25-30 days; see Figure 16-17 for the differentiation of adventitious buds; the strong seedling medium is: MS + 0.3 ～0.8 mg/L 6-BA + 0.03 mg/LNAA medium; among them, 6-BA concentration of 0.6mg/L is the best.

Rooting culture - when the adventitious buds grow to 4-5cm, they are transferred to rooting medium and cultured for 25-30 days; see Figure 18-19 for rooting culture; rooting medium is: 1/2 MS + 0.5 mg/L IBA medium.

Domestication of tissue-cultured seedlings——When the sunflower tissue-cultured seedlings grow up and produce 4-5 leaves, and 5-6 white and thick young roots are about 1-2 cm long, choose a suitable time to take out the bottle for training and transplanting. Because the longer roots are easy to be broken and damaged during the cleaning process, which reduces the survival rate of transplanting, before transplanting, carry out incomplete opening of the bottle for 1 day under the condition of sufficient sunlight indoors, and wait for the water droplets on the tissue culture bottle cap to dry. After fully opening the bottle and exercising for 2 days; the time of opening the bottle should not exceed 3 days, because the agar medium exposed to the air is undoubtedly a breeding ground for bacteria and other microorganisms, which are easy to infect and damage plants and reduce the survival rate of tissue culture seedlings.

Preparation before transplanting—the seedlings that have undergone domestication and exercise gradually adapt to the external conditions. Use plastic tweezers to carefully remove the seedlings from the culture bottle, and try not to damage the young roots. If the medium is too dry, it can be soaked in tap water for 1-2 minutes before taking it out. The taken out tissue cultured seedlings were soaked in 0.1% potassium permanganate for 1 to 2 minutes for disinfection, and the aged leaves on the tissue cultured seedlings were removed, and then the young roots were lightly rubbed with absorbent cotton under running water. Rinse the root agar clean to prevent the agar residue from causing bacteria to breed and infect the plants.

Transplanting substrate—the cultivation substrate for seedling cultivation requires large ventilation and porosity, and it is advisable to use perlite, river sand or vermiculite plus peat soil that is thoroughly sterilized, loose, and has good water permeability.

Transplanting - Transplanting should be thoroughly watered in the cultivation medium in advance. Use a glass rod to make a small hole in the packed peat soil + perlite mixed matrix, and plant four-fifths of the underground part of the tissue culture seedlings in a moist matrix. Note that planting too deep or too shallow will affect the plant type , It is not conducive to the fixed growth and respiration of plants. Finally, water thoroughly and put the transplanted seedlings in a clean, clean, sunny and well-ventilated greenhouse or plastic greenhouse.

Management after transplanting—the temperature should be maintained at about 20-25°C, and the humidity should be about 75%-85%. At the same time, short-term 30% shading measures should be taken to promote the rooting of the underground part. When the roots formed by plant callus have grown root hairs, functional new roots have grown slowly, which improves the water absorption capacity, and can be directly sprayed with thin fertilizer on the leaves.

Disease prevention and control after transplanting——Sunflower seedling diseases are mainly botrytis cinerea, root rot and stem rot, etc., which can easily cause dead seedlings and rotten seedlings, and in severe cases, it is easy to cause huge losses. Effective control measures mainly include: (1) prevent excessive humidity in the seedbed, control watering, the relative humidity of the air in the seedbed is 60-70%; properly heat the greenhouse at night; control the temperature of the seedbed at 25-30°C; Light intensity, and ventilation; the photosynthesis and respiration of the seedlings are strong, the growth is strong, and the disease resistance is enhanced. (2) Chemical treatment, spray 70% chlorothalonil 500 times solution or 50% carbendazim 500 times solution or 200 times equivalent Bordeaux mixture before the onset of the disease, or use 70% thiophanate-methyl solution after the onset Or dilute 1000 times of 50% Zineb solution for spraying or spray 50% Zineb 800-1000 times solution for spraying or watering.

Secondary transplanting——the sunflower seedlings raised in plug trays can be transplanted to the field. When transplanting, first water the transplanted seedlings thoroughly, dig out the transplanted seedlings together with the substrate, and hold the soil mass gently. Gently put the seedling roots into the holes opened in advance in the field, and fill and compact the soil.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not used to limit the protection scope of the present invention. Equivalent replacements or substitutions made on the above-mentioned basis all belong to the protection scope of the present invention.

Claims (10)

1. a kind of sunflower tissue culture method, it is characterised in that using the axillary bud of sunflower as explant, certain herbaceous plants with big flowers is cultivated by tissue cultures Flower seedling.

2. sunflower tissue culture method as claimed in claim 1, it is characterised in that the tissue culture method includes following step Suddenly：

A, explant selection：The axillary bud in the following axil of poppyhead is gathered, the size of axillary bud is no more than 1cm；

B, explant prepare：Explant is cleaned and sterilization, the top for then cutting the axillary bud of 5mm or so sizes is explant Body is inoculated on differential medium；

C, induction and differentiation：Explant, which continues to cultivate, is directly divided into adventitious bud；

D, strong seedling culture：Adventitious bud is transferred on strong seedling culture base when growing into 2~3cm and cultivated；

E, culture of rootage：Adventitious bud is transferred on root media when growing into 4~5cm and cultivated；

F, tissue-cultured seedling domestication：Tissue-cultured seedling is grown up 4~5 leaves of generation, and the sturdy young root length of 5~6 whites is of about 1~2 cm When, bottle outlet, which is taken exercise, transplants.

3. sunflower tissue culture method as claimed in claim 2, it is characterised in that poppyhead described in step A is selected to have and viewed and admired Kind more than the branch of value.

4. sunflower tissue culture method as claimed in claim 3, it is characterised in that axillary bud will selected from poppyhead described in step A Plant that is open or having opened.

5. sunflower tissue culture method as claimed in claim 4, it is characterised in that

In the step B, explant is washed with detergent, then 0.5h is rinsed with clear water, on superclean bench, first with 70% Ethanol soak 30~60s, then with 10% hydrogen peroxide or 3% liquor natrii hypochloritis sterilization 15~20min, use sterilized water Cleaning 3~4 times；The differential medium is：MS+0.6~1.5mg/L 6-BA+0.03mg/L NAA culture mediums；

In the step C, explant is directly divided into adventitious bud for 25~30 days in culture, continues to cultivate and works as adventitious bud in 10~15 days Strong seedling culture base is transferred to when growing into 2~3cm；The condition of culture of the explant is：In 25~28 DEG C of temperature, illumination Cultivated in 1600lx, the daily illumination 12h of fluorescent lamp tissue culture room, solid medium agar containing 7.5g/L, pH is adjusted before sterilizing For 5.8~6.0；

In the step D, the strong seedling culture base is：The mg/L NAA of MS+0.3~0.8 mg/L 6-BA+0.03 are cultivated Base；Cultivation cycle is 25~30 days；

In the step E, the root media is：The mg/L IBA culture mediums of 1/2 MS+0.5；Cultivation cycle is 25~30 My god；

In the step F, it is first sunny indoors before transplanting under the conditions of first carry out not exclusively corkage and take exercise 1 day, treat tissue culture bottle The lid globule is parched, then corkage is taken exercise 2~3 days completely.

6. sunflower tissue culture method as claimed in claim 5, it is characterised in that the transplanting described in step F includes following step Suddenly：The seedling taken exercise by domestication is taken out from blake bottle, soaking 1~2 min with 0.1% potassium permanganate carries out disinfection, clearly Except the blade of aging on tissue-cultured seedling, root agar is rinsed well；Transplanting medium is poured permeable, by five points of tissue-cultured seedling under ground portion Plant everywhere in moistening matrix, filling of watering is saturating, and transplanted seedling is put into greenhouse or vinyl house.

7. sunflower tissue culture method as claimed in claim 6, it is characterised in that the transplanting medium selects perlite or river Husky or vermiculite adds turfy soil.

8. sunflower tissue culture method as claimed in claim 7, it is characterised in that after transplanting, cultivate temperature maintain 20~ 25 DEG C, humidity be 75%~85%, while to carry out short-term 30% shelter from heat or light measure processing；The root of plant callus formation is grown During root hair, directly applied fertilizer in foliage spray.

9. sunflower tissue culture method as claimed in claim 8, it is characterised in that the method for disease control includes after transplanting：

Relative air humidity is 60~70% in seedbed；Nursery bed temperature is controlled at 25~30 °C；Increase intensity of illumination, and ventilation is changed Gas；

Chemicals treatment, premorbid sprays 500 times of liquid of 70% Bravo, 500 times of liquid or 50% carbendazim or 200 times of equivalent formula bohrs Many liquid preventing and treatings, carry out spray with 50% ambam decoction of 1000 times of 70% thiophanate methyl decoction or dilution after morbidity and control or spray Spill, or control or pour using 50% zineb, 800~1000 times of liquid sprays.

10. sunflower tissue culture method as claimed in claim 9, it is characterised in that sunflower seedling can be transplanted to crop field, transplanting side Method is：Transplanted seedling water is irrigated, dug out together with matrix, seedling root is planted into crop field.