CN115589948B - Rhizoma bletillae non-symbiotic germination medium and propagation method - Google Patents

Abstract

The application belongs to the technical field of plant non-symbiotic germination breeding, and particularly discloses a rhizoma bletillae non-symbiotic germination culture medium and a breeding method, wherein the culture medium is an improved KC culture medium, and the improved KC culture medium is as follows: 10000mg l of the yeast is added on the basis of KC basic culture medium ^‑1 Coconut powder, 500mg l ^‑1 The propagation method for improving the activated carbon powder comprises the following steps: hua Bai and pod sterilization and seed treatment; hua Bai and non-symbiotic germination and culture: sowing Hua Bai and seeds on a modified KC culture medium for germination; hua Bai and seedling subculture: subculturing Hua Bai and germinated and cultured non-browned seedlings when the seedlings grow to 3-4cm high; hua Bai and seedling transplanting and planting: hua Bai and seedlings can be acclimatized and transplanted when growing to the top of a culture bottle cover, and the germination rate reaches 98% in an improved KC culture medium by adopting the technical scheme of the application, the seedlings can germinate 15 days after sowing seeds, and the phenomena of brown dead seedlings do not appear in the protocorm after differentiation, growth and subculture.

Description

Rhizoma bletillae non-symbiotic germination medium and propagation method

Technical Field

The application belongs to the technical field of non-symbiotic germination and breeding of plants, and particularly relates to a rhizoma bletillae non-symbiotic germination culture medium and a breeding method.

Background

Hua Bai and (Bletilla sinensis (Rolfe) Schltr.) are perennial herbaceous plants of the genus Bletilla (Bletilla) of the family Orientae, which have been listed as endangered species by the world natural protection Association (IUCN) red directory, as annex II species by the International trade convention for endangered wild animal and plant species (CITES), and as endangered grades in the China biological diversity red directory-higher plant rolls published in 2013, with very few wild populations and living numbers. Hua Bai, flower type is peculiar, artificial digging is serious, living environment is seriously damaged, the number of wild living bodies is reduced year by year due to population degradation, the wild living bodies are endangered, and Hua Bai and rescuing and conservation are not sustained.

Because the orchid seeds are tiny like dust and have no endosperm, the orchid seeds can not provide nutrients necessary for germination and seedling formation, the germination rate is extremely low in the field, and the natural updating of the population is slow. The artificial seedling breeding is an extremely effective measure for the conservation and the industrialization development and utilization of orchid plants. The methods commonly adopted for artificial seedling breeding of orchid plants are separated plant breeding and seed sterile (non-symbiotic) breeding, and the separated plant breeding is simple in operation, but has extremely low breeding efficiency in view of extremely small number of wild living bodies. The research on Hua Bai and non-symbiotic breeding methods can provide technical support for the regression, rejuvenation, reconstruction and conservation of rhizoma bletillae in the wild and industrialized large-scale seedling culture, and a large number of rhizoma bletillae seedlings bred by the method can reserve genes of the species and relieve endangered conditions.

The non-symbiotic germination method of seeds is a mode for rapid breeding of seedlings at present, but different culture mediums are used, and the variety and content of chemical components contained in the culture mediums can cause certain difference in germination rate and germination time of the seeds. Meanwhile, different culture mediums have different actions on seedlings in the process of protocorm differentiation and seedling growth, and some of the culture mediums can lead to browning, albinism and final death of the seedlings. Therefore, a large number of experiments are used for screening out the culture medium which has high seed germination rate, can normally differentiate protocorms into leaves, does not brown or whiten seedlings and can normally grow and grow the seedlings finally is key to Hua Bai and non-symbiotic germination propagation methods.

Disclosure of Invention

The application mainly aims to provide a high-efficiency bletilla striata seedling breeding method for solving the problems in the prior art of bletilla striata seedling breeding.

In order to achieve the above object, the present application provides the following technical solutions:

a rhizoma bletilla non-symbiotic germination medium, the medium being a modified KC medium, the modified KC medium being: based on KC basic culture medium, add10000mg l ^-1 Coconut powder, 500mg l ^-1 The activated carbon powder is improved.

The application also provides a high-efficiency breeding method of the rhizoma bletillae seedlings, which comprises the following steps:

(1) Hua Bai and pod sterilization and seed treatment;

(2) Hua Bai and non-symbiotic germination and culture: hua Bai and seeds were sown onto a modified KC medium for germination, which was: 10000mg l of the yeast is added on the basis of KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 Improving the activated carbon powder;

(3) Hua Bai and seedling subculture: subculturing Hua Bai and germinated and cultured non-browned seedlings when the seedlings grow to 3-4cm high;

(4) Hua Bai and seedling transplanting and planting: hua Bai and when the seedlings grow to the top of the culture bottle cover, hardening the seedlings and taking out the bottle for transplanting.

Preferably, the method for sterilizing the bletilla striata pods comprises the following steps: soaking 0.1% mercuric chloride for 10 min, and washing with a large amount of sterilized water for 5 times; soaking in 5% sodium hypochlorite solution for 10 min, and washing with a large amount of sterilized water for 5 times; sucking the surface water with sterile filter paper; the pods were flash baked in an alcohol burner for 10 seconds.

Preferably, the non-symbiotic germination and culture conditions of rhizoma bletillae are: the culture is carried out in a culture room at 25+/-1 ℃ under the illumination with the illumination intensity of 1000Lx and the illumination time of 4 hours/day.

Further, the subculture medium used for the subculture is: the amount of activated carbon in the modified KC medium was changed to 1000mg l ^-1 As a secondary medium.

Preferably, the subculture conditions are: the temperature is 25+/-1 ℃, the illumination intensity is 1000Lx, and the illumination time is 14 h/day.

Furthermore, the application also provides a specific method for transplanting and planting rhizoma bletillae after non-symbiotic culture, which comprises the following steps: s1: hardening seedlings; s2: root sterilization: taking out the seedlings from the culture flask after hardening, and sterilizing by using a bactericide after removing the root culture medium; s3: transplanting the sterilized seedlings into a culture medium, and controlling environmental conditions: temperature: 18-25 ℃, relative humidity 60-80%, illumination intensity: 1000-1500Lx.

Preferably, in the step S2, the specific method for sterilization and disinfection is as follows: the solution is soaked for 15min by using 2000 times of agricultural streptomycin sulfate.

In step S3, the cultivation substrate is a mixed substrate of bark (diameter: 9-12 mm), peat soil and vesuvianite (diameter: 1-2 cm) in a volume ratio of 1:1:1.

Preferably, the cultivation substrate is sterilized with 2000 times of potassium permanganate solution before use.

The technical scheme of the application achieves the following effects:

hua Bai and the culture medium adopted by the application have higher germination rate, and the germination rate is more than 90%. In the improved KC culture medium screened by the method, the germination rate reaches 98%, the seeds can germinate after being sown for 15 days, and the phenomena of browning and dead seedling of protocorms do not appear after differentiation, seedling growth and subculture. The non-symbiotic germination method is simple to operate, does not need to add hormone or additives, is low in cost, can realize bottle planting after 7 months of sowing, and greatly shortens the breeding period. By adopting the method and the culture medium formula, a large number of bletilla striata seedlings can be obtained in a short period, and a foundation is laid for subsequent Hua Bai and conservation work and industrialized development and utilization.

Drawings

Fig. 1 Hua Bai and pod picking state diagram;

FIG. 2 Hua Bai shows the view after pod sterilization treatment;

FIG. 3 is a diagram showing the non-symbiotic germination culture of rhizoma bletillae;

FIG. 4 is a view showing the browning state of rhizoma bletilla in a culture medium;

FIG. 5 Hua Bai and a normal growth shoot status plot without browning in the modified KC medium;

FIG. 6 Hua Bai and seedling subculture status diagram;

FIG. 7 Hua Bai is a graph showing rooting during seedling cultivation;

FIG. 8 Hua Bai is a graph showing the growth of seedlings during the seedling cultivation process;

fig. 9-Hua Bai and a seedling out-of-bottle transplanting planting state diagram.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. In order to avoid unnecessary detail, known techniques will not be described in detail in the following embodiments. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The reagents used in the practice are carried out under conventional conditions or conditions recommended by the manufacturer, if not specified, and the reagents or apparatus used are commercially available from conventional products.

Hua Bai and ground-on protection

The orchid conservation team of the academy of sciences of forestry and grassland in Yunnan province developed an investigation of the orchid resources of Yunnan province, 9 months 2021, found a field population of Hua Bai and Bletilla sinensis in Zhenkang county in Yunnan province, hua Bai after 122 years and reappeared in front of the world. Hua Bai and the population found in Zhenkang county at this time are not more than 300, only 2 seeds are found, and the natural setting rate is quite low. In order to carry out conservation and breeding research on the endangered species, a living plant with fruit pods and 2 plants without fruit pods are collected according to the technical specification and the requirements of a national important wild plant germplasm resource library and transplanted to an orchid greenhouse of a national important wild plant germplasm resource cymbidaceae plant preservation center for field protection. The seeds used in the subsequent propagation research are the seeds which are protected by the field and naturally set in the field.

Example 1

1. Hua Bai and on-site protection effect and pod harvesting

3 bletilla striata plants (fruit bearing of one plant) are collected from the herd of Hua Bai and Bletilla sinensis found by tranquilization in 9 of 2021 and transplanted into an orchid greenhouse of an orchid preservation center to serve as seed sources for site-moving protection, and after three months of management, the 3 plants survive, and the plant state is good. The ripe and uncracked pods were picked (the state of the clamps during picking is shown in fig. 1) for subsequent Hua Bai and seedling breeding studies (Hua Bai and flowering time of 6-7 months, natural fruiting pods during flowering, picking at 11 months, and about 4-5 months after pollination, i.e. 120-150 days).

2. Hua Bai pod and seed treatment

The picked fruit pods are washed by a large amount of tap water, brushed by a brush and washed by sterilized water in an ultra-clean workbench. Sterilizing the pods according to the following procedure; soaking 0.1% mercuric chloride for 10 min, and washing with a large amount of sterilized water for 5 times; soaking in 5% sodium hypochlorite solution for 10 min, and washing with a large amount of sterilized water for 5 times; sucking the surface water with sterile filter paper; the pods were flash baked in an alcohol burner for 10 seconds. The processed fruit pod (figure 2) is put into a sterile culture dish for temporary preservation, and the fruit pod is transversely sheared into two sections by using sterile scissors during sowing, and seeds are exposed out and can be used for sowing.

3. Hua Bai and seed non-symbiotic breeding culture medium formula

Improvement of B5 medium: 10000mg l of the culture medium is added on the basis of the B5 basic culture medium ^-1 Coconut powder, 500mg l ^-1 The activated carbon powder is improved. The formula of the basic culture medium B5 is as follows: 113.24mg l ^-1 Anhydrous calcium chloride, 150mg l ^-1 Sodium dihydrogen phosphate, 2500mg l ^-1 Potassium nitrate, 134mg l ^-1 Ammonium sulfate, 122.09mg l ^-1 Anhydrous magnesium sulfate, 27.8mg l ^-1 Ferrous sulfate, 37.3mg l ^-1 Disodium ethylenediamine tetraacetate, 10.0mg l ^-1 Manganese sulfate, 2.0mg l ^-1 Zinc sulfate, 0.25mg l ^-1 Sodium molybdate, 0.025mg l ^-1 Cobalt chloride, 3.0mg l ^-1 Boric acid, 0.75mg l ^-1 Potassium iodide, 0.025mg l ^-1 Copper sulfate, 10.0mg l ^-1 Vitamin B1, 1.0mg l ^-1 Vitamin B6, 1.0mg l ^-1 Nicotinic acid, 100.0mg l ^-1 Inositol 20000mg l ^-1 Sucrose, 7000mg l ^-1 Agar.

Improved KC medium: 10000mg l of the yeast is added on the basis of KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 The activated carbon powder is improved. The KC minimal medium formulation is as follows: 1000mg l ^-1 Calcium nitrate tetrahydrate, 250mg l ^-1 Potassium dihydrogen phosphate, 500mg l ^-1 Ammonium sulfate, 250mg l ^-1 Magnesium sulphate heptahydrate, 25mg l ^-1 Ferrous sulfate heptahydrate, 7.5mg l ^-1 Manganese sulfate tetrahydrate 20000mg l ^-1 Sucrose, 7000mg l ^-1 Agar.

Modified 1/4MS medium: 10000 mg/l of the basic culture medium of 1/4MS is added ^-1 Coconut powder, 500mg l ^-1 The activated carbon powder is improved. The 1/4MS minimal medium formulation is as follows: 475mg l ^-1 Potassium nitrate, 412.5mg l ^-1 Ammonium nitrate, 0.11mg l ^-1 Calcium chloride, 0.2075mg l ^-1 Potassium iodide, 2.15mg l ^-1 Zinc sulfate, 0.00625mg l ^-1 Cobalt chloride, 6.95mg l ^-1 Ferrous sulfate, 0.5mg l ^-1 Glycine, 0.125mg l ^-1 Nicotinic acid, 25mg l ^-1 Inositol, 42.5mg l ^-1 Potassium dihydrogen phosphate, 92.5mg l ^-1 Magnesium sulfate, 1.55mg l ^-1 Boric acid, 5.575mg l ^-1 Manganese sulfate, 0.0625mg l ^-1 Sodium molybdate, 0.00625mg l ^-1 Copper sulfate, 0.125mg l ^-1 Pyridoxine hydrochloride, 0.025mg l ^-1 Thiamine hydrochloride 9.325mg l ^-1 Disodium ethylenediamine tetraacetate 20000mg l ^-1 Sucrose, 7000mg l ^-1 Agar.

Improved Huabao No. 1 culture medium: at 1000mg l ^-1 20000mg l of Huabao No. 1 (N: P: K=7:6:19) solution was added ^-1 Sucrose, 7000mg l ^-1 Agar, 10000mg l ^-1 Coconut powder, 500mg l ^-1 Activated carbon powder.

Modified Huabao No. 2 culture medium: at 1000mg l ^-1 20000mg l of Huabao No. 2 (N: P: K=20:20:20) solution was added ^-1 Sucrose, 7000mg l ^-1 Agar, 10000mg l ^-1 Coconut powder, 500mg l ^-1 Activated carbon powder.

The pH of the prepared culture medium is adjusted to 5.6-5.8, and the culture medium is autoclaved for 20 minutes at 121 ℃.

5. Hua Bai non-symbiotic germination and culture

Hua Bai and seeds were spread evenly onto the surface of 5 media (FIG. 3), about 50 seeds per bottle, 5 bottles per media. The culture flask is placed in a culture room at 25+/-1 ℃ for illumination culture, the illumination intensity is 1000Lx, the illumination time is 4 hours/day, and the seed germination time and the germination rate are recorded. The breaking of the seed coat by the expanded embryo of the seed was confirmed as the germination of the seed, and the germination rate was divided by the total number of seeds to be germinated and multiplied by 100. (sowing time: 2021, 11, 24 days)

TABLE 1 germination time of bletilla striata seeds in different media

Hua Bai and the seed sowing are started until the 9 th day, so that the seed coat breaks through successively and the 5 kinds of culture mediums can germinate within 20 days basically. Taking the 30 th day after sowing as the time for counting the germination rate of seeds (table 2), the germination rates of rhizoma bletillae in 5 culture mediums are not greatly different and are basically higher than 90% of germination rate, and the highest germination rate reaches 98%. According to the actual germination time and germination rate, rhizoma bletillae seeds are easy to germinate in a non-symbiotic culture medium under study.

TABLE 2 germination Rate of bletilla striata seeds in different media (30D after sowing)

Hua Bai and seeds can differentiate cotyledons and grow leaves after germination in 5 culture mediums. At 60 days after sowing, seedlings were grown as milbema and the seedlings were brown and dead in different media (fig. 4). Seedlings were all brown and dead in the modified B5 medium, the modified 1/4MS medium and the modified Huabao No. 2 medium, half yellow and dead in the modified Huabao No. 1 medium, and all brown and dead after ten days of continuous culture. In the improved KC culture medium, seedlings are grown very robustly, and no browning and yellowing withering phenomenon occurs (figure 5), which shows that the improved KC culture medium is suitable for the growth of the Chinese white seedlings, and excessive or insufficient nutrient substances can cause the browning and withering of the Chinese white seedlings.

TABLE 3 browning and death of bletilla striata seedlings in different media (60D after sowing)

6. Hua Bai and seedling subculture

When the non-browned seedlings grow to 3-4cm high, hua Bai seedlings are subjected to subculture. The culture conditions are as follows: the temperature is 25+ -1deg.C, and 1000Lx is illuminated for 14 h/day. The dosage of the activated carbon in the modified KC culture medium is changed to 1000mg l ^-1 As a medium for the subculture. The bulbs continuously grow into a long shape in the secondary culture process, and when the seedlings grow to the top of the culture medium (shown in figures 6, 7 and 8), the seedlings can be acclimatized and transplanted out of the bottles. The non-symbiotic germination method is adopted, and the planting can be carried out in the improved KC culture medium from about 7 months after sowing, hua Bai and seedlings (the planting time of the first bottle-out in the study is 2022, 6 months and 27 days).

7. Hua Bai and seedling transplanting planting

Hua Bai and when the seedlings grow to the top of the culture medium, hardening off and transplanting. The seedling hardening process comprises the following steps: taking the culture bottle capable of emerging from a culture room to an orchid greenhouse for 3-5 days, unscrewing the bottle cap, placing for 1-2 days, opening half of the bottle cap, placing for 2-3 days, completely opening the bottle cap, and placing for 1-2 days to take the seedling out of the culture bottle. Removing culture medium on seedling root, washing with a large amount of water, soaking in 2000 times of agricultural streptomycin sulfate for 15min for sterilization and disinfection, air drying, and planting seedling into a prepared culture medium. Hua Bai and culture medium are mixed matrix of bark (diameter: 9-12 mm), peat soil and vesuvianite (diameter: 1-2 cm) at volume ratio of 1:1:1 (sterilized with 2000 times potassium permanganate solution). Hua Bai and planting conditions are: temperature: 18-25 ℃, relative humidity 60-80%, illumination intensity: 1000-1500Lx. The water and fertilizer management is carried out after the Hua Bai and the water and fertilizer management are carried out according to the prior slow release fertilizer and matrix whitening water spraying, the planting condition is shown in figure 9, and the survival rate of the seed balls is 99% after the bletilla striata seedlings are transplanted for 90 days.

It is apparent that the above-described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.

Claims (6)

1. The non-symbiotic germination propagation method for rhizoma bletillae is characterized by comprising the following steps of:

(1) Hua Bai and pod sterilization and seed treatment;

(2) Hua Bai and non-symbiotic germination and culture: hua Bai and seeds were sown onto modified KC medium for germination under the following culture conditions: culturing in a culture room at 25+ -1deg.C under illumination with illumination intensity of 1000Lx and illumination time of 4 hr/day;

(3) Hua Bai and seedling subculture: subculturing Hua Bai and the seedlings which are not brown after germination culture until the heights are 3-4cm, wherein the culture conditions are as follows: the temperature is 25+/-1 ℃, the illumination intensity is 1000Lx, and the illumination time is 14 h/day;

(4) Hua Bai and seedling transplanting and planting: hua Bai and when seedlings grow to the top of the culture bottle cover, hardening the seedlings and taking out the seedlings from the bottle for transplanting;

the improved KC medium is: 1000 mg.l ^-1 Calcium nitrate tetrahydrate, 250 mg.l ^-1 Monopotassium phosphate, 500 mg.l ^-1 Ammonium sulfate, 250 mg.l ^-1 Magnesium sulfate heptahydrate, 25 mg.l ^-1 Ferrous sulfate heptahydrate, 7.5 mg.l ^-1 Manganese sulfate tetrahydrate 20000mg l ^-1 Sucrose, 7000 mg.l ^-1 Agar, 10000 mg.l ^-1 Coconut powder, 500 mg.l ^-1 Activated carbon powder;

the secondary culture medium is as follows: the amount of activated carbon in the modified KC medium is changed to be 1000 mg.l ^-1 As a secondary medium.

2. The method according to claim 1, wherein in the step (1), the method for sterilizing the bletilla striata pods comprises: soaking 0.1% mercuric chloride for 10 min, and washing with a large amount of sterilized water for 5 times; soaking in 5% sodium hypochlorite solution for 10 min, and washing with a large amount of sterilized water for 5 times; sucking the surface water with sterile filter paper; the pods were flash baked in an alcohol burner for 10 seconds.

3. The method according to claim 1, wherein in the step (4), the step of transplanting rhizoma bletillae is:

s1: hardening seedlings;

s2: root sterilization: taking out the seedlings from the culture flask after hardening, and sterilizing by using a bactericide after removing the root culture medium;

s3: transplanting the sterilized seedlings into a culture medium, and controlling environmental conditions: temperature: 18-25 ℃, relative humidity 60-80%, illumination intensity: 1000-1500Lx.

4. A method according to claim 3, wherein in the step S2, the specific method of sterilization is: the solution is soaked for 15min by using 2000 times of agricultural streptomycin sulfate.

5. A method according to claim 3, wherein in the step S3, the cultivation substrate is a mixed substrate of bark, peat soil and vesuvianite in a volume ratio of 1:1:1, the bark has a diameter of 9-12 d mm, and the vesuvianite has a diameter of: 1-2 cm.

6. The method according to claim 4 or 5, wherein the cultivation substrate is sterilized with 2000 times potassium permanganate solution before use.