CN115589948A

CN115589948A - Bletilla striata non-symbiotic germination culture medium and propagation method

Info

Publication number: CN115589948A
Application number: CN202211312312.7A
Authority: CN
Inventors: 华梅; 孔继君; 蒋宏
Original assignee: Yunnan Academy of Forestry and Grassland Sciences
Current assignee: Yunnan Academy of Forestry and Grassland Sciences
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2023-01-13
Anticipated expiration: 2042-10-25
Also published as: CN115589948B

Abstract

The invention belongs to the technical field of non-symbiotic germination and breeding of plants, and particularly discloses a bletilla striata non-symbiotic germination culture medium and a breeding method, wherein the culture medium is an improved KC culture medium which is: adding 10000mg l on KC basic culture medium ^‑1 Coconut powder, 500mg l ^‑1 The active carbon powder is improved, and the propagation method comprises the following steps: sterilizing and treating seeds of rhizoma bletillae; hua Baiji non-symbiotic germination and culture: mixing Hua BaiSowing seeds on an improved KC culture medium for germination; 5363 and subculturing the seedling Hua Baiji: subculturing when the bletilla striata seedlings which are not browned grow to 3-4cm high after germination culture; hua Baiji seedling transplanting and planting: hua Baiji when the seedlings grow to the tops of culture bottles, the seedlings can be hardened and taken out of the bottles for transplantation, by adopting the technical scheme of the invention, the germination rate of rhizoma bletillae in an improved KC culture medium reaches 98%, the rhizoma bletillae can germinate 15 days after seed sowing, and the phenomenon of browning and seedling death of protocorms after differentiation, seedling growth and subculture does not occur.

Description

Bletilla striata non-symbiotic germination culture medium and propagation method

Technical Field

The invention belongs to the technical field of non-symbiotic germination and breeding of plants, and particularly relates to a bletilla striata non-symbiotic germination culture medium and a propagation method.

Background

Hua Baiji (Bletilla sinensis (Rolfe) Schltr.) is a perennial herb of Bletilla genus (Bletilla) of the family Orchidaceae, has been listed as an endangered species by the red record of the world natural protection alliance (IUCN), is listed as an annex II species by the International trade convention for endangered wild animal species (CITES), and is evaluated as an endangered grade in the Chinese biodiversity red record-higher plant volume published in 2013, and the wild species and the number of living bodies are extremely small. Hua Baiji has peculiar flower type, serious artificial digging, serious damage to habitat and population degradation, causes the number of field living bodies to be reduced year by year, is endangered to extinction, and is not suitable for rescue and conservation of rhizoma bletillae.

The seeds of the orchidaceae plants are fine, dusty and free of endosperm, so that the orchidaceae plants cannot provide nutrients necessary for the germination and seedling formation of the seeds, the germination rate in the wild is extremely low, and the population naturally renews slowly. The artificial seedling breeding is an effective measure for the conservation and the industrial development and utilization of the orchids. The method commonly adopted for the artificial seedling breeding of the orchidaceae plants comprises the plant division breeding and the sterile (non-symbiotic) seed breeding, and although the operation of the plant division breeding is simple, the breeding efficiency is extremely low in view of the extremely small number of field living bodies. The research on the Hua Baiji non-symbiotic breeding method can provide technical support for wild regression, rejuvenation, reconstruction and conservation and industrialized large-scale seedling culture of rhizoma bletillae, and a large amount of rhizoma bletillae seedlings bred by the method can store the genes of the species and relieve endangered conditions.

The non-symbiotic seed germination method is one mode of fast breeding seed seedling, and has different kinds and content of chemical components in different culture mediums, so that the seed germination rate and germination time are different. Meanwhile, different culture media have different effects on seedlings in protocorm differentiation and seedling growth, and some culture media can lead the seedlings to brown and whiten and finally die. Therefore, a culture medium which is screened out through a large number of experiments and has high seed germination rate, normal differentiation of protocorms to leaves, no browning and albinism of seedlings and normal growth and growth of seedlings is the key of the Hua Baiji non-symbiotic germination propagation method.

Disclosure of Invention

The invention mainly aims to provide a bletilla striata germchit efficient breeding method to solve the problems in the background technology of bletilla striata germchit breeding.

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

a bletilla striata non-symbiotic germination culture medium is an improved KC culture medium, and the improved KC culture medium is as follows: adding 10000mg l on KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 The active carbon powder is improved.

The invention also provides a rhizoma bletillae seedling high-efficiency breeding method, which comprises the following steps:

(1) Sterilizing and treating seeds of the rhizoma bletillae;

(2) Hua Baiji non-symbiotic germination and culture: sowing bletilla striata seeds on an improved KC culture medium for germination, wherein the improved KC culture medium is as follows: adding 10000mg l on KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 Improving activated carbon powder;

(3) 5363 and subculturing the seedling Hua Baiji: subculturing when the bletilla striata seedlings which are not browned grow to 3-4cm high after germination culture;

(4) Hua Baiji seedling transplanting and planting: and (3) hardening the seedlings, taking out of bottles and transplanting when the Hua Baiji seedlings grow to the tops of culture bottles.

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

Preferably, the non-symbiotic germination and culture conditions of rhizoma bletillae are as follows: culturing at 25 + -1 deg.C under illumination with illumination intensity of 1000Lx for 4 hr/day.

Further, in the above-mentioned case,the subculture medium used for subculture was: the dosage of the active carbon in the improved KC culture medium is changed to 1000mg l ^-1 As a subculture medium.

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

Further, the invention 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 bottle after hardening, removing the root culture medium, and sterilizing by using a bactericide; s3: transplanting the disinfected seedlings into a culture medium, and controlling the environmental conditions: temperature: 18-25 ℃, relative humidity 60-80%, illumination intensity: 1000-1500Lx.

Preferably, in step S2, the specific method of sterilization includes: soaking for 15min by using 2000 times of agricultural streptomycin sulfate.

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

Preferably, the culture medium is sterilized by 2000 times of potassium permanganate solution before use.

The technical scheme of the invention achieves the following effects:

the bletilla striata culture medium adopted by the invention has high germination rate, and the germination rate is over 90 percent. In the screened improved KC culture medium, the germination rate reaches 98%, the seeds can germinate 15 days after sowing, and the phenomenon of browning and death of seedlings does not occur in the protocorm after differentiation, seedling growing and subculture. The non-symbiotic germination method is simple to operate, does not need to add hormones and additives, is low in cost, can realize bottle-out planting after 7-month sowing, and greatly shortens the breeding period. By adopting the method and the culture medium formula, a large amount of bletilla striata seedlings can be obtained in a short period, and a foundation is laid for subsequent conservation work and industrial development and utilization of bletilla striata.

Drawings

FIG. 1 is a diagram of the plucking status of bletilla striata and fruit pods;

FIG. 2 is a schematic representation of a rhizoma Bletillae pod after sterilization treatment;

FIG. 3 shows a schematic representation of the cultivation of bletilla striata non-symbiotic germination;

FIG. 4 is a diagram of the browning state of bletilla striata in the culture medium;

FIG. 5 is a diagram of the normal growth of bletilla striata without browning in modified KC medium;

FIG. 6 is a state diagram of bletilla hyacinthina seedling subculture;

FIG. 7 is a diagram of rooting during bletilla hyacinthina seedling culture;

FIG. 8 is a diagram showing the growth of seedlings in the cultivation process of bletilla striata;

FIG. 9 is a diagram of the transplanting state of bletilla striata seedlings out of bottles.

Detailed Description

The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. To avoid unnecessary detail, the following examples will not describe the known art in detail. 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 invention belongs.

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

Huabai earth protection

In 2021, 9 months, orchids conservation teams of forestry and grassland academy of sciences in Yunnan province carry out resource investigation of orchids in Yunnan province, and a field population of Hua Baiji Bletilla sinensis is found in Zhenkang county in Yunnan province, which means that Bletilla hybrida appears before the world again after 122 years of time. The Hua Baiji population found in Zhenkang county investigation at this time has the population number not more than 300, only 2 seeds are found, and the natural maturing rate is quite low. In order to carry out conservation 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 requirements of a national important wild plant germplasm resource bank and transplanted to an orchid greenhouse of an orchid conservation center of the national important wild plant germplasm resource bank for ex-situ protection. The seeds used for the subsequent propagation research are the seeds which are protected in the ex-situ and naturally fruit in the field.

Example 1

1. Rhizoma bletillae ex situ protection effect and fruit pod harvesting

3 Bletilla striata plants (one plant bearing fruit) collected from Hua Baiji Bletilla sinensis population discovered by Zhenkang in 9 months of 2021 are transplanted into an orchid greenhouse of an orchid preservation center to be taken as seed sources for ex-situ protection, and after three months of management and protection, the 3 plants survive and are in good plant states. And (3) picking mature and uncracked pods at 23/11 of 2021 (the state of the pod clamp is shown in figure 1 during picking) for subsequent study on bletilla striata seedling breeding (bletilla striata blossoms for 6-7 months, and naturally fruited pods in the flowering period are picked at 11 months, and the time is about 4-5 months after pollination, namely 120-150 days).

2. Hua Baiji fruit pod and seed treatment

Washing picked fruit pods with a large amount of tap water, brushing with a brush, and washing with sterilized water in a superclean bench. Sterilizing the fruit pods according to the following procedures; soaking in 0.1% mercuric chloride for 10 min, washing with a large amount of sterilized water for 5 times; soaking in 5% sodium hypochlorite solution for 10 min, washing with a large amount of sterilized water for 5 times; sucking up surface water by using sterile filter paper; the pods were flash baked in an alcohol lamp for 10 seconds. And (3) placing the processed fruit pods (shown in figure 2) into a sterile culture dish for temporary storage, transversely cutting the fruit pods into two sections by using sterile scissors during sowing, and exposing the seeds for sowing.

3. Bletilla striata seed non-symbiotic breeding culture medium formula

Modified B5 Medium: 10000mg l of basic culture medium B5 ^-1 Coconut powder, 500mg l ^-1 The active carbon powder is improved. The formula of the B5 minimal medium 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 Ethylenediaminetetraacetic acid, 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.

Modified KC medium: adding 10000mg l on KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 The active carbon powder is improved. The KC basic culture medium has the following formula: 1000mg l ^-1 Calcium nitrate tetrahydrate, 250mg l ^-1 Potassium dihydrogen phosphate 500mg l ^-1 Ammonium sulfate, 250mg l ^-1 Magnesium sulfate 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 culture medium: 10000mg l of the culture medium is added on the basis of 1/4MS minimal medium ^-1 Coconut powder, 500mg l ^-1 The active carbon powder is improved. The formula of the 1/4MS minimal medium 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 Ethylenediaminetetraacetic acid disodium salt 20000mg l ^-1 Sucrose, 7000mg l ^-1 Agar.

Improving a Huabao No. 1 culture medium: at 1000mg l ^-1 To the Huabao No. 1 (N: P: K = 7) ^-1 Sucrose, 7000mg l ^-1 Agar, 10000mg l ^-1 Coconut powder, 500mg l ^-1 Activated carbon powder.

Improved Huabao No. 2 culture medium: at 1000mg l ^-1 To the Huabao No. 2 (N: P: K = 20) solution was added 20000mg l ^-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 sterilized by autoclaving at 121 ℃ for 20 minutes.

5. Hua Baiji non-symbiotic germination and culture

Bletilla striata seeds were sown evenly onto the surface of 5 media (fig. 3), approximately 50 seeds per bottle, 5 bottles per media. Placing the culture bottle in a culture room at 25 +/-1 ℃ for illumination culture, wherein the illumination intensity is 1000Lx, the illumination time is 4 hours/day, and recording the seed germination time and the seed germination rate. The seed swollen embryo breaks through the seed coat and is confirmed as seed germination, and the germination rate is obtained by dividing the number of germinated seeds by the total number of seeds and multiplying the total number by 100. (seeding time: 2021 year, 11 months and 24 days)

TABLE 1 Geranium sibiricum seeds Germination time in different media

Hua Baiji seeds start to germinate by breaking through seed coats after being sowed by 9 days, 5 culture mediums have different germination time and basically can germinate within 20 days after being sowed. The time for counting the germination rate of the seeds (table 2) is 30 days after sowing, the germination rate of rhizoma bletillae in 5 culture media is not different, is basically higher than 90% and reaches 98% to the maximum. According to the actual germination time and the actual germination rate, the bletilla striata seeds are easy to germinate in the non-symbiotic culture medium under study.

TABLE 2 Germination rates of bletilla striata seeds in different media (30D after sowing)

5363 the seeds of Hua Baiji can differentiate cotyledon and grow leaf after germinating in 5 kinds of culture medium. At 60 days after sowing, the seedlings grew into dense hemp, and the seedlings died due to browning in different media (fig. 4). Seedlings in the improved B5 culture medium, the improved 1/4MS culture medium and the improved Huabao 2 culture medium are all browned and withered, half of seedlings in the improved Huabao 1 culture medium are browned and withered, and all seedlings are browned and withered after continuous culture for more than ten days. In the modified KC culture medium, seedlings grow robustly all the time, and no phenomena of browning and withering from yellowing occur (figure 5), which shows that the modified KC culture medium is more suitable for the growth of the Huabai seedlings, and excessive or insufficient nutrient substances can cause the Huabai seedlings to brown and wither from browning.

TABLE 3 Brown and withered condition of bletilla hyacinthina seedlings in different media (60D after sowing)

6. Hua Baiji seedling subculture

When the non-browned seedlings grow to 3-4cm high, subculturing the bletilla striata seedlings. The culture conditions were: the temperature is 25 +/-1 ℃, and the illumination is 1000Lx, 14h/day. The dosage of the active carbon in the modified KC culture medium is changed to 1000mg l ^-1 As a medium for subculture. The bulbs continuously grow into a long shape in the process of subculture, and when the seedlings grow to the top of the culture medium (as shown in figures 6, 7 and 8), the seedlings can be hardened and taken out of bottles for transplantation. Adopting a non-symbiotic germination method, and in an improved KC culture medium, the bletilla striata seedlings can be grown in bottles from about 7 months after sowing (in the research, the first bottle growing time is 2022 years, 6 months and 27 days).

7. 5363 transplanting seedling of Hua Baiji

Hua Baiji when the seedling grows to the top of the culture medium, the seedling can be hardened and taken out of a bottle for transplantation. The seedling exercising process comprises the following steps: taking a culture bottle capable of seedling emergence from a culture room to an orchid greenhouse for 3-5 days, unscrewing a bottle cap, placing for 1-2 days, opening a 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 large amount of water, soaking in 2000 times of agricultural streptomycin sulfate for 15min for sterilization, air drying, and planting the seedling in the prepared culture medium. Hua Baiji culture medium is a mixed substrate of bark (diameter: 9-12 mm), peat soil and vesuvianite (diameter: 1-2 cm) in a volume ratio of 1. The planting conditions of rhizoma bletillae are as follows: temperature: 18-25 ℃, relative humidity 60-80%, illumination intensity: 1000-1500Lx. The planted rhizoma bletillae is subjected to water and fertilizer management by putting slow release fertilizer and matrix whitening water spray in advance, the planting condition is shown in figure 9, and the survival rate of seed balls is 99% after the rhizoma bletillae seedlings are transplanted for 90 days.

Obviously, the embodiments described above are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.

Claims

1. The bletilla striata non-symbiotic germination culture medium is characterized in that the culture medium is an improved KC culture medium, and the improved KC culture medium is as follows: adding 10000mg l on KC basic culture medium ^-1 Coconut powder, 500mg l ^-1 The active carbon powder is improved.

2. The bletilla striata non-symbiotic germination propagation method is characterized by comprising the following steps:

(1) Sterilizing and treating seeds of rhizoma bletillae;

(2) Hua Baiji non-symbiotic germination and culture: seeding bletilla striata seeds onto the modified KC medium of claim 1 for germination;

(4) Hua Baiji seedling transplanting and planting: hua Baiji when the seedlings grow to the top of the culture bottle, the seedlings can be acclimatized, taken out of the bottle and transplanted.

3. The efficient bletilla striata seedling breeding method according to claim 2, wherein in the step (1), the bletilla striata pod disinfection method comprises the following steps: soaking in 0.1% mercuric chloride for 10 min, washing with a large amount of sterilized water for 5 times; soaking in 5% sodium hypochlorite solution for 10 min, washing with large amount of sterilized water for 5 times; sucking up surface water by using sterile filter paper; the pods were flash baked for 10 seconds outside the alcohol burner.

4. The efficient bletilla striata seedling breeding method according to claim 2, wherein in the step (2), the non-symbiotic germination and culture conditions of bletilla striata are as follows: culturing at 25 + -1 deg.C under illumination with illumination intensity of 1000Lx for 4 hr/day.

5. The efficient bletilla striata seedling breeding method according to claim 2, wherein in the step (3), the subculture medium used for subculture is: the dosage of the active carbon in the improved KC culture medium is changed to 1000mg l ^-1 As a subculture medium.

6. The efficient bletilla striata seedling breeding method according to claim 2 or 5, wherein subculture conditions are as follows: the temperature is 25 +/-1 ℃, the illumination intensity is 1000Lx, and the illumination time is 14 h/day.

7. The efficient bletilla striata seedling breeding method as claimed in claim 2, wherein in the step (4), the bletilla striata transplanting and planting step is as follows:

s1: hardening seedlings;

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

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

8. The efficient bletilla striata seedling breeding method as claimed in claim 7, wherein in the step S2, the specific sterilization and disinfection method comprises the following steps: soaking for 15min by using 2000 times of agricultural streptomycin sulfate.

9. The efficient bletilla striata seedling breeding method as claimed in claim 7, wherein in the step S3, the cultivation substrate is a mixed substrate of bark, peat soil and vesuvianite, wherein the mixed substrate is prepared from the bark, the peat soil and the vesuvianite according to a volume ratio of 1: 1-2cm.

10. The efficient bletilla striata seedling breeding method as claimed in claim 8 or 9, wherein the culture medium is sterilized by 2000 times of potassium permanganate solution before use.