CN111990254A

CN111990254A - Dendrobium nobile culture medium and application thereof

Info

Publication number: CN111990254A
Application number: CN202010893867.XA
Authority: CN
Inventors: 向增旭; 王将; 张子璇; 罗丽娜
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-11-27

Abstract

The invention belongs to the technical field of dendrobe culture, and particularly relates to a dendrobe culture medium and application thereof. The dendrobium culture medium comprises a liquid culture medium, wherein the liquid culture medium takes an N6 culture medium as a basic culture medium, and further comprises NAA 0-0.5 mg/L and sucrose 30g/L, and the pH of the culture medium is 5.8-6.0. According to the liquid culture medium disclosed by the invention, the germination rate of the dendrobium seeds is improved, the dendrobium seeds can turn green, and protocorms can be seen by naked eyes after the dendrobium seeds turn green, so that the problem that the dendrobium seeds are unevenly distributed in a tissue culture bottle due to low germination rate, and further the dendrobium grows unevenly is solved.

Description

Dendrobium nobile culture medium and application thereof

Technical Field

The invention belongs to the technical field of dendrobe culture, and particularly relates to a dendrobe culture medium and application thereof.

Background

Herba Dendrobii (Dendrobium) is a perennial epiphytic herb of Dendrobium of Orchidaceae, has effects of nourishing yin, clearing heat, benefiting stomach and promoting fluid production by using fresh or dry stem section as medicine, is a traditional and famous Chinese medicinal material, and has reputation of "gold in medicine", "climbing groundsel" and "Mesona chinensis". Modern pharmacological research shows that dendrobium polysaccharide, stilbenes, flavonoids, alkaloids and volatile components are main drug effect substances of the dendrobium polysaccharide, stilbenes, flavonoids, alkaloids and volatile components, and the dendrobium polysaccharide and stilbenes have multiple effects of enhancing immunity, resisting tumors, resisting oxidation, resisting bacteria, resisting inflammation, reducing blood sugar, protecting liver and stomach and the like.

The dendrobium nobile has extremely small seeds, incomplete embryonic development, only embryos, no endosperm and extremely low natural fertility because the dendrobium nobile can germinate under the condition of symbiosis with fungi in the wild environment. In recent years, the attention on medicinal value and ornamental value of dendrobium is continuously increased, so that wild resources are nearly exhausted and are in an endangered state, and the technical problems of artificial planting and seedling production are urgently needed to be solved and broken through.

In the nineties of the last century, researchers began to explore tissue culture technology to establish a dendrobe regeneration system, through continuous exploration and research of predecessors, the current dendrobe tissue culture industrialized seedling system is mature, a large number of dendrobe seedlings with stable heredity and uniform characters can be obtained in a short time by applying the tissue culture technology, and the method is the most widely used method for dendrobium industrialized seedling at present.

However, 3-4 ten thousand seeds exist in one dendrobium capsule, the germination rate of the culture medium seeds in the traditional three-step seedling method is low, and a lot of seeds are wasted in the transfer process from germination to strong seedlings; and moreover, because the seed germination rate is low, seedlings are unevenly distributed in the tissue culture bottle, and further the dendrobium grows unevenly.

Disclosure of Invention

The culture medium not only improves the germination rate of seeds, but also solves the problem that seedlings are unevenly distributed in a tissue culture bottle due to low germination rate of dendrobium seeds, so that dendrobium grows unevenly.

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

the invention provides a dendrobium culture medium which comprises a liquid culture medium, wherein the liquid culture medium takes an N6 culture medium as a basic culture medium and further comprises 0-0.5 mg/L of NAA and 28-32 g/L of sucrose, and the pH value of the culture medium is 5.8-6.0.

Preferably, the culture medium further comprises a solid culture medium, wherein the solid culture medium comprises 80-120 g/L of potatoes, 40-60 g/L of bananas, 0.3-0.5 mg/L of NAA, an MS culture medium and/or Huabao No. 1, 28-32 g/L of cane sugar and 6-7 g/L of agar, and the pH value of the culture medium is 5.8-6.0;

the content of the Huabao No. 1 is 0.5-1.5 g/L.

The invention also provides application of the dendrobium culture medium in the scheme in improving germination rate of dendrobium seeds and high-efficiency seedling culture.

Preferably, the application comprises the following steps:

(1) adding seeds in a dendrobe capsule into a liquid culture medium in the dendrobe culture medium, performing dark culture and then performing illumination culture, and obtaining protocorm liquid after the seeds turn green;

(2) diluting the protocorm liquid, dibbling the diluted protocorm liquid into a solid culture medium containing the dendrobium culture medium, and carrying out illumination culture to obtain a test-tube plantlet;

(3) and (4) hardening, washing and transplanting the test-tube plantlets to obtain dendrobium seedlings.

Preferably, the dibble seeding quantity is determined according to the area of the solid culture medium, and is specifically 0.9-1.25 granules/cm²。

Preferably, the dark culture conditions include: the temperature is 23-25 ℃, the humidity is 75-85%, and the time is 5-7 d;

the conditions of the light culture comprise: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 2000-2500 Lx, and the illumination time is 12-14 h/d.

Preferably, the time for culturing the seeds in the liquid culture medium is 20-30 d.

Preferably, the obtaining manner of the seeds comprises: cleaning and disinfecting the dendrobe capsules, and splitting to obtain seeds.

Preferably, the hardening off comprises closing the cover in a greenhouse for hardening off for 14-21 d, and then opening the cover for hardening off for 5-7 d; when the cover is opened for hardening seedlings, the temperature in the greenhouse is 10-15 ℃, the humidity is 60% -70%, and the shading degree is 65% -75%.

Preferably, the transplanted culture medium comprises one or more of pine bark, coconut coir and wood chips.

Has the advantages that:

the invention provides a dendrobium culture medium which comprises a liquid culture medium, wherein the liquid culture medium takes an N6 culture medium as a basic culture medium and further comprises NAA 0-0.5 mg/L and sucrose 28-32 g/L, and the pH of the culture medium is 5.8-6.0. According to the liquid culture medium disclosed by the invention, the germination rate of the dendrobium seeds is improved, the dendrobium seeds can turn green, and protocorms can be seen by naked eyes after the dendrobium seeds turn green, so that the problem that the dendrobium seeds are unevenly distributed in a tissue culture bottle due to low germination rate, and further the dendrobium grows unevenly is solved.

Furthermore, the dendrobium seedlings can be uniformly distributed in the culture medium through the dibble seeding technology, the number of the dendrobium seedlings in each tissue culture bottle can be controlled by controlling the number of the dibble seeding protocorms, the problem that continuous transfer is needed due to insufficient space of the culture bottles is solved, and further seedling formation is realized.

Furthermore, the invention simplifies the industrialized seedling raising steps of the dendrobium seedlings by utilizing a liquid culture medium and a spot sowing one-step seedling raising technology, shortens the seedling raising period, saves the mass production cost, greatly reduces the labor cost and avoids unnecessary pollution loss.

Furthermore, the culture medium prepared by screening the seedling culture medium and utilizing the mutual matching of the pine bark, the coconut husk and the sawdust is breathable, water-retaining and loose, and is convenient for the attachment of the roots of the dendrobium, so that the survival rate of the test-tube plantlets reaches 95 percent.

Drawings

FIG. 1 is a diagram of a protocorm diluent according to an embodiment of the present invention;

FIG. 2 is a diagram of a seed suspension aspirated by a pipette according to application example 1 of the present invention;

FIG. 3 is a diagram of a tissue culture bottle after dibbling in application example 1 of the present invention;

FIG. 4 is a diagram showing the height of test-tube plantlets in example 1 of the present invention;

FIG. 5 is a height diagram of a test-tube plantlet in application example 2 of the present invention;

FIG. 6 is a height diagram of test-tube plantlets in application example 3 of the present invention;

FIG. 7 is a bottom view of a tissue culture bottle with 4-5 tube seedlings according to the application example 1;

FIG. 8 is a graph of a dilution of a comparative example 3 protocorm of the present invention;

FIG. 9 is a diagram of a test-tube plantlet of comparative example 3 according to the present invention;

FIG. 10 is a diagram of a germination medium containing dendrobe seeds according to comparative example 4 of the present invention;

FIG. 11 is a diagram of the seedling strengthening of Dendrobium nobile in comparative example 4 of the present invention;

FIG. 12 is a diagram showing the rooting of test-tube plantlets in comparative example 4 of the present invention;

FIG. 13 is a test tube plantlet diagram before seedling hardening in comparative example 4 of the present invention;

FIG. 14 is a drawing of a liquid medium containing dendrobe seeds according to comparative example 5 of the present invention;

FIG. 15 is a drawing showing a liquid medium containing dendrobe seeds according to comparative example 6 of the present invention;

FIG. 16 is a diagram of seedlings after washing in example 1;

FIG. 17 is a diagram of seedlings after washing in example 11 of the present invention;

FIG. 18 is a diagram of seedlings after washing in example 12 of the present invention;

FIG. 19 is a morphological diagram of comparative example 7 seed culture 7d according to the present invention;

FIG. 20 is a morphological diagram of comparative example 7 seed culture 14d according to the present invention;

FIG. 21 is a schematic diagram showing a seed culture 21d according to comparative example 7 of the present invention;

FIG. 22 is a morphological diagram of comparative example 7 seed culture 28d according to the present invention;

FIG. 23 is a schematic diagram of a seed culture 35d in comparative example 7 of the present invention;

FIG. 24 is a schematic diagram showing a seed culture 49d according to comparative example 7 of the present invention;

FIG. 25 is a diagram of protocorms observed with a microscope in application example 1 of the present invention;

FIG. 26 is a diagram of protocorms observed with a microscope in application example 4 of the present invention;

FIG. 27 is a diagram of protocorms observed with a microscope in application example 5 of the present invention;

FIG. 28 is a diagram of a transplanted Dendrobium seedling of the present invention application example 1.

Detailed Description

The invention provides a dendrobium culture medium which comprises a liquid culture medium, wherein the liquid culture medium takes an N6 culture medium as a basic culture medium and also comprises 0-0.4 mg/L of NAA and 28-32 g/L of cane sugar, further preferably comprises 0.3-0.4 mg/L of NAA and 30g/L of cane sugar, most preferably comprises 0.4mg/L of NAA and 30g/L of cane sugar, and the pH value of the culture medium is 5.8-6.0.

In the invention, the culture medium preferably further comprises a solid culture medium, wherein the solid culture medium comprises 80-120 g/L of potatoes, 40-60 g/L of bananas, 0.3-0.5 mg/L of NAA, 28-32 g/L of sucrose and 6-7 g/L of agar, the pH value of the culture medium is 5.8-6.0, and the content of Huabao No. 1 is 0.5-1.5 g/L; further preferably comprises 100g/L of potatoes, 50g/L of bananas, 0.4mg/L of NAA0, MS culture medium and Huabao No. 1, 30g/L of cane sugar and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0; the content of the Huabao No. 1 is 0.8-1.2 g/L; most preferably comprises 100g/L of potatoes, 50g/L of bananas, 0.4mg/L of NAA0, 6.5g/L of MS culture medium, 6.5g/L of agar and 30g/L of cane sugar, wherein the pH value of the culture medium is 5.8-6.0; the content of the Huabao No. 1 is 1 g/L. The invention has no special requirements on the sources of all components in the dendrobium culture medium, and can adopt commercially available commodities well known by technical personnel in the field.

The present invention preferably uses MS medium, N6 medium, sucrose and agar produced by Nanjing shouder Biotech Co.

In the present invention, the limited form of the potato or banana includes potato juice or banana juice; the preparation method of the potato juice or the banana juice preferably comprises the following steps: cleaning potato, peeling or directly peeling banana, cutting into pieces, and respectively crushing with a juicer. The invention has no special requirements for the source of the juicer, and can adopt commercial products which are well known to those skilled in the art.

The liquid culture medium solves the problems that the dendrobium seeds lack endosperm and lack fungi symbiosis in plant tissue culture, improves the germination rate of the dendrobium seeds by soaking the dendrobium seeds in the liquid culture medium, enables the dendrobium seeds to turn green, enables protocorms to be visible by naked eyes after the dendrobium seeds turn green, and solves the problems that the dendrobium seeds are low in germination rate, so that seedlings are unevenly distributed in a tissue culture bottle, and the dendrobium grows unevenly.

The invention also provides application of the dendrobium culture medium in the technical scheme in improving germination rate of dendrobium seeds and high-efficiency seedling culture.

In the present invention, the application preferably comprises the steps of:

(2) diluting the protocorm liquid, dibbling the diluted protocorm liquid into a tissue culture bottle containing a solid culture medium in the dendrobium culture medium, and carrying out illumination culture to obtain a test-tube plantlet;

In the present invention, said dendrobium capsule preferably comprises a mature uncracked dendrobium capsule. The invention selects the mature and uncracked dendrobe capsule, which not only can avoid seed contamination, but also is beneficial to the cleaning and disinfection treatment of the capsule.

In the present invention, the obtaining manner of the seeds preferably includes: cleaning and disinfecting the dendrobe capsules, and splitting to obtain seeds.

In the present invention, the washing preferably includes: firstly, cutting off part of a pedicel and a flower base at the front end of a capsule by using a scalpel, cleaning, then cleaning the surface by using a detergent, and washing for 10-30 min by using running water, and further preferably for 20 min; the detergent preferably comprises one or more of washing powder, liquid detergent and hand sanitizer. The dendrobium stem capsule cleaning agent is cleaned by the detergent and washed by running water, so that stains on the surface of the dendrobium stem capsule can be washed off, and the follow-up disinfection can be carried out more easily and successfully.

In the present invention, the sterilization preferably includes: disinfecting with 70% alcohol for 30-60 s, rinsing with sterile water for 3-5 times, disinfecting with mercuric chloride for 8-10 min, and rinsing with sterile water for 5-7 times; further preferred includes sterilizing with 70% ethanol for 30s, rinsing with sterile water for 3 times, sterilizing with mercuric chloride for 10min, and rinsing with sterile water for 5 times.

In the present invention, the conditions for the dark culture preferably include: the temperature is 23-25 ℃, the humidity is 75-85%, and the time is 5-7 d; the conditions for the light culture preferably include: the temperature is 23-25 ℃, the humidity is 75-85%, the illumination intensity is 2000-2500 Lx, and the illumination time is 12-14 h/d.

In the invention, the time for culturing the seeds in the liquid culture medium is preferably 20-30 d. The time for culturing the seeds in the liquid culture medium is preferably determined according to the types of dendrobium, and the preferable dendrobium nobile ranges from 26 days to 30 days, the preferable dendrobium candidum ranges from 28 days to 32 days, the preferable dendrobium chrysotoxum ranges from 18 days to 22 days, the preferable dendrobium nobile ranges from 23 days to 27 days, and the preferable dendrobium nobile ranges from 23 days to 27 days. The method determines different culture times according to different dendrobium varieties, can avoid the problems that the culture time is too long, the protocorm of the dendrobium is too large and is not beneficial to subsequent dibbling operation, and solves the problems that the quality of the protocorm is reduced and the survival rate is reduced because the gas in the liquid is seriously consumed along with the prolonging of the culture time and the respiratory action and the photosynthesis of the protocorm cells are influenced.

After obtaining the protocorm liquid, the protocorm liquid is diluted and dibbled into a tissue culture bottle containing a solid culture medium in the dendrobium culture medium for illumination culture, and then the test-tube plantlet is obtained.

In the present invention, the concentration of the stock solution after dilution is preferably 25 to 35 grains/300. mu.L, more preferably 28 to 32 grains/300. mu.L, and most preferably 30 grains/300. mu.L. The invention can control the quantity of the original corms dibbled by each bottle by controlling the volume of the dibbling original corm diluent, not only has convenient operation, but also can relatively accurately control the quantity of the original corms dibbled by each bottle.

In the invention, the dibble seeding quantity is preferably determined according to the area of the solid culture medium, and is specifically 0.9-1.25 granules/cm²More preferably 1.0 to 1.14 particles/cm²Most preferably 1.07 grains/cm²(ii) a The dibbling mode is preferably dibbled into a tissue culture bottle containing a solid culture medium in the dendrobium culture medium; the tissue culture bottle preferably comprises 250ml of specification, and the bottle bottom area is 28cm²(ii) a Preferably dibbling 25-35 grains per bottle, further preferably 28-32 grains, and most preferably 30 grains per bottle; the dibbling preferably comprises sucking 300 mu L of protocorm diluent by using a pipette gun, and uniformly dibbling on a solid culture medium in the dendrobium culture medium. According to the invention, the number of dendrobium seedlings in each tissue culture bottle can be controlled by controlling the number of the dibbled dendrobium protocorms, and the problem of continuous transfer due to insufficient space of the culture bottle is further solved by combining the dilution concentration setting of protocorm liquid, so that one-step seedling formation is realized.

The invention simplifies the industrialized seedling raising steps of the dendrobium seedlings by utilizing the liquid culture medium and the spot sowing one-step seedling raising technology, shortens the seedling raising period, saves the mass production cost, greatly reduces the labor cost and avoids unnecessary pollution loss at the same time.

After the test-tube plantlet is obtained, the test-tube plantlet is subjected to seedling hardening, seedling washing and transplanting to obtain the dendrobium seedling. In the invention, the seedling exercising preferably comprises closing the cover in a greenhouse for exercising for 14-21 d, and then opening the cover for exercising for 5-7 d; when the cover is opened for hardening seedlings, the temperature in the greenhouse is 10-15 ℃, the humidity is 60% -70%, and the shading degree is 65% -75%; further preferably, the method comprises the steps of closing a cover in a greenhouse for hardening seedlings for 15d, and then opening the cover for hardening the seedlings for 7 d; when the cover is opened for hardening seedlings, the temperature in the greenhouse is 10-15 ℃, the humidity is 60-70%, and the shading degree is 70%.

In the invention, the seedling washing preferably comprises the steps of cleaning the culture medium remained on the root by using clear water, soaking in 800 times of diluted carbendazim solution for 10-15 min, and finally spreading the washed dendrobium seedlings to a shade place until the root is whitened.

In the present invention, the specification of the transplanted seedling raising bag preferably includes 10 × 10 cm; the transplanted culture medium preferably comprises one or more of pine bark, coconut coir and wood chips, and the pine bark preferably comprises small-size pine bark; when the culture medium is pine bark and coconut coir, the volume ratio of the pine bark to the coconut coir is preferably 5 (1-2); when the cultivation medium is pine bark and wood chips, the volume ratio of the pine bark to the wood chips is 5 (1-2). According to the invention, the survival rate of the test-tube plantlets reaches 95% by controlling the seedling hardening time, temperature and humidity and selecting a seedling culture substrate suitable for the growth of dendrobium.

In order to further illustrate the present invention, the culture medium of dendrobium and the application thereof provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparing a dendrobium liquid culture medium, taking an N6 culture medium as a basic culture medium, and only containing NAA0.4mg/L and sucrose 30g/L, wherein the pH value of the culture medium is 5.8-6.0.

Example 2

Preparing a dendrobium liquid culture medium, taking an N6 culture medium as a basic culture medium, and only containing NAA0.3mg/L and 30g/L of cane sugar, wherein the pH value of the culture medium is 5.8-6.0.

Example 3

Preparing a dendrobe liquid culture medium, taking an N6 culture medium as a basic culture medium, and only containing 30g/L of sucrose, wherein the pH value of the culture medium is 5.8-6.0.

Example 4

Preparing a dendrobium solid culture medium, taking an MS culture medium as a basic culture medium, and only containing 100g/L of potato juice, 50g/L of banana juice, NAA0.4mg/L, 1g/L of Huabao No. 1, 30g/L of cane sugar and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0.

Example 5

Preparing a dendrobium solid culture medium, taking an MS culture medium as a basic culture medium, and only containing 100g/L of potato juice, 50g/L of banana juice, NAA0.3mg/L, 1g/L of Huabao No. 1, 30g/L of sucrose and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0.

Example 6

Preparing a dendrobium solid culture medium, taking an MS culture medium as a basic culture medium, and only containing 100g/L of potato juice, 50g/L of banana juice, NAA0.5mg/L, 1g/L of Huabao No. 1, 30g/L of sucrose and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0.

Example 7

Preparing a solid culture medium, taking an MS culture medium as a basic culture medium, and only containing 100g/L of potato juice, 50g/L of banana juice, 0.4mg/L of NAA0, 30g/L of cane sugar and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0.

Example 8

Preparing a solid culture medium which consists of 100g/L of potato juice, 50g/L of banana juice, NAA0.4mg/L, 1g/L of Huabao No. 1, 30g/L of cane sugar and 6.5g/L of agar, wherein the pH value of the culture medium is 5.8-6.0.

Comparative example 1

Preparing a liquid culture medium, taking an MS culture medium as a basic culture medium, and only containing NAA0.4mg/L and cane sugar 30g/L, wherein the pH value of the culture medium is 5.8-6.0.

Comparative example 2

Preparing a liquid culture medium, taking 1/2MS culture medium as a basic culture medium, and only containing NAA0.4mg/L and sucrose 30g/L, wherein the pH value of the culture medium is 5.8-6.0.

Application example 1

The application of the culture medium prepared in the embodiments 1 and 4 in improving the germination rate of dendrobium seeds and raising seedlings with high efficiency comprises the following steps:

(1) taking mature and uncracked dendrobium stem capsules as explants, firstly cutting off partial pedicles and flower bases at the front end of the capsules by using a scalpel, cleaning up, then cleaning the surface by using washing powder, washing for 10min by using running water, then transferring into a super clean workbench, disinfecting for 30s by using 70% alcohol, rinsing for 3 times by using sterile water, disinfecting for 10min by using mercuric chloride, and rinsing for 5 times by using sterile water. Splitting a capsule, shaking seeds into the liquid culture medium prepared in the example 1, uniformly shaking, performing dark culture for a period of time, and performing illumination culture;

the dark culture conditions are as follows: the temperature is 24 +/-1 ℃, the humidity is 80 +/-5 percent, and the time is 5 d;

the illumination culture conditions are as follows: the temperature is 24 +/-1 ℃, the humidity is 80 +/-5%, the illumination intensity is 2000Lx, the illumination is 12h/d, and the time is 23 d.

(2) Diluting the protocorm to 40 grains/300 mu L (see figure 1), shaking to prepare a uniform seed suspension, observing the protocorm by a 40-fold microscope (see figure 25), sucking 300 mu L of the seed suspension by a pipette (see figure 2), uniformly dibbling the seed suspension onto a one-step seedling solid culture medium (see figure 3), and carrying out illumination culture to obtain a test-tube seedling;

the illumination culture conditions are as follows: the temperature is 24 +/-1 ℃, the humidity is 80 +/-5%, the illumination intensity is 2500Lx, the illumination is 12h/d, and the time is 240 d.

(3) When the test-tube seedlings cultured in the step (2) grow to 6-7 cm high (shown in figure 4) and have 4-5 roots (shown in figure 7), closing the cover in the greenhouse to harden the seedlings for 15 days, opening the cover to harden the seedlings for 7 days, spraying water mist once in the morning and evening during the opening cover hardening period, controlling the temperature in the greenhouse to be 10-15 ℃, controlling the humidity to be 60-70% and controlling the shading temperature to be 70%; washing the seedlings, firstly, cleaning the culture medium remained on the roots with clear water, then, soaking the seedlings in 800 times of diluted carbendazim solution for 10-15 min, and finally, spreading the washed dendrobium seedlings to a shade place until the roots turn white (see figure 16); the seedling raising bag for transplanting is 10 multiplied by 10cm (see figure 28), and the culture medium is: bark of small-size pine: and 5:1 of coconut coir.

Application example 2

An application method similar to that of application example 1 is only different in that the solid medium in step (2) is the solid medium prepared in example 5, and test-tube plantlets grow to 6-7 cm high (see fig. 5).

Application example 3

An application method similar to that of application example 1 is only different in that the solid medium in step (2) is the solid medium prepared in example 6, and test-tube plantlets grow to 6-7 cm high (see fig. 6).

Application example 4

An application method similar to application example 1 except that the liquid medium in step (1) was the liquid medium prepared in example 2, and the protocorm was observed with a 40-fold microscope (see FIG. 26).

Application example 5

An application method similar to application example 1 except that the liquid medium in step (1) was the liquid medium prepared in example 3, and the protocorm was observed with a 40-fold microscope (see FIG. 27).

Application example 6

An application method similar to application example 1 is characterized in that dendrobium officinale is adopted as dendrobium officinale, dark culture time in step (1) is 5 days, and illumination culture time is 25 days.

Application example 7

An application method similar to application example 1, which is characterized in that dendrobium chrysotoxum is adopted, the dark culture time in the step (1) is 5 days, and the illumination culture time is 15 days.

Application example 8

An application method similar to application example 1, which is characterized in that the dendrobium nobile is dendrobium bigelovii, the dark culture time in the step (1) is 5 days, and the illumination culture time is 20 days.

Application example 9

An application method similar to application example 1, which is characterized in that the dendrobium nobile is dendrobium bicolor, the dark culture time in the step (1) is 5 days, and the illumination culture time is 20 days.

Application example 10

An application method similar to application example 1, which is characterized in that the dendrobium nobile is short-rod dendrobium nobile, the dark culture time in the step (1) is 5 days, and the illumination culture time is 20 days.

Comparative example 3

A method of application similar to that of application example 1, except that the liquid medium in step (1) was sterilized water (see FIG. 8), and the cultured test-tube plantlets were as shown in FIG. 9.

As can be seen from the graphs 1-9 and 25-27, the liquid culture medium prepared by the method provided by the invention obviously improves the germination rate of the dendrobium seeds, so that the dendrobium seeds can turn green, the protocorms can be seen by naked eyes after turning green, and the embryo growth in the seeds can be observed under a microscope to break through the seed coats, so that the seeds grow into the protocorms capable of germinating, and the problems that the germination rate of the dendrobium seeds is low, seedlings are unevenly distributed in a tissue culture bottle, and the dendrobium grows unevenly are solved.

Comparative example 4

The traditional three-step seedling application.

(1) Seed germination: cut a osculum with stem of noble dendrobium capsule front end, clip the carpopodium of capsule with tweezers, gently all fall the stem of noble dendrobium seed on sprouting culture medium (see figure 10), sprout the culture medium: MS + 13% banana;

(2) and (3) proliferation and seedling strengthening stage: when the dendrobium seedlings in the step (1) are cultured for 80 days and grow to 1-2 cm, transferring the dendrobium seedlings into a strong seedling culture medium (see figure 11), and proliferating the strong seedling culture medium: MS + 13% potatoes;

(3) and (3) rooting period: when the dendrobium proliferated seedlings in the step (2) are cultured for 100 days and grow to 3-4 cm, transferring the test-tube seedlings into a rooting culture medium (see figure 12), wherein the rooting culture medium comprises: 1/2MS + 15% banana + NAA0.5mg/L;

(4) and (4) hardening, washing and transplanting the test-tube plantlets when the dendrobium nobile is rooted and cultured for 120 days in the step (3).

The culture conditions of seedling hardening, seedling washing and transplanting are the same as the application example 1, and the test-tube seedlings before seedling hardening are shown in figure 13.

As can be seen from the drawings 1-7 and 10-13, the dendrobium seedlings can be uniformly distributed in the culture medium by the dibbling technology, the number of the dendrobium seedlings in each tissue culture bottle can be controlled by controlling the number of the dibbling protocorms, the problem that continuous transfer is needed due to insufficient space of the culture bottles is solved, and further one-step seedling establishment is realized.

In addition, the method simplifies the traditional industrialized seedling raising steps of the dendrobium seedlings by utilizing a liquid culture medium and a spot sowing one-step seedling raising technology, shortens the seedling raising time by 32 days compared with the traditional three-step seedling raising method, obviously shortens the seedling raising period, saves the mass production cost, greatly reduces the labor cost, and simultaneously avoids unnecessary pollution loss.

Comparative example 5

A method of application similar to that of application example 1, except that the liquid medium in step (1) was the liquid medium prepared in comparative example 1 (see FIG. 14).

Comparative example 6

A method of application similar to that of application example 1, except that the liquid medium in step (1) was the liquid medium prepared in comparative example 2 (see FIG. 15).

As can be seen from fig. 1, 14 and 15, the liquid culture medium prepared in comparative example 1 cannot make the seeds turn green, while the culture medium prepared in comparative example 2 can make part of the seeds turn green but cannot further develop into protocorms, but the liquid culture medium prepared in the invention significantly improves the germination rate of the dendrobium seeds, so that the dendrobium seeds can turn green, and the protocorms developed after the green change are visible to the naked eye, thereby solving the problem that the dendrobium seeds are not uniformly distributed in the tissue culture bottle due to low germination rate, and further the dendrobium grows non-uniformly.

Application example 11

An application method similar to that of application example 1, except that the solid medium in step (2) was the solid medium prepared in example 7, and the seedling was washed as shown in FIG. 17.

Application example 12

An application method similar to that of application example 1, except that the solid medium in step (2) was the solid medium prepared in example 8, and the seedling was washed as shown in FIG. 18.

As can be seen from FIGS. 16 to 18, different solid culture media prepared by the method can realize one-step seedling formation, and the dendrobium seedlings are basically consistent and good in growth vigor; the seedling growth vigor of the seedling in the culture medium prepared in the example 4 or 5 is poorer than that of the seedling in the culture medium prepared in the example 4, mainly because the culture medium prepared in the example 5 or 6 is added with the Huabao 1 or MS culture medium, and the culture medium added with the Huabao 1 or MS culture medium can obviously reduce the culture cost on the premise of meeting the requirement of one-step seedling formation.

In conclusion, the solid culture medium prepared by the invention is more suitable for the growth of protocorms, and the quantity of dendrobium seedlings in each tissue culture bottle can be controlled by controlling the quantity of the dendrobium protocorms in the dibbling mode, so that the problem that continuous transfer is required due to insufficient space of the culture bottles is solved, and further one-step seedling formation is realized.

Comparative example 7

An application method similar to that of application example 1 is characterized in that the light culture time in (1) is 44d, and the growth conditions of seeds at 7d, 14d, 21d, 28d, 35d, 42d and 49d, which are measured from the shaking of the seeds into the liquid culture medium prepared in example 1, are recorded, and the pictures corresponding to tables 1 and 7-49 d are shown in FIGS. 19-25.

TABLE 1 growth of protocorms at different cultivation times

It can be known from table 1 and fig. 19-25 that the culture time of the seeds in the liquid culture medium is not as long as possible, the culture time is too long, the protocorm of dendrobium nobile lindl is too large to facilitate the subsequent dibble seeding operation, and along with the extension of the culture time, the gas in the liquid is seriously consumed, the protocorm cell is affected to carry out respiration and photosynthesis, the quality of the protocorm is reduced, and the survival rate is reduced.

The method determines different culture times according to different dendrobium varieties, can avoid the problems that the culture time is too long, the protocorm of the dendrobium is too large and is not beneficial to subsequent dibbling operation, and solves the problems that the quality of the protocorm is reduced and the survival rate is reduced because the gas in the liquid is seriously consumed along with the prolonging of the culture time and the respiratory action and the photosynthesis of the protocorm cells are influenced.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. The dendrobe culture medium is characterized by comprising a liquid culture medium, wherein the liquid culture medium takes an N6 culture medium as a basic culture medium and further comprises 0-0.5 mg/L of NAA and 28-32 g/L of sucrose, and the pH value of the culture medium is 5.8-6.0.

2. The culture medium according to claim 1, further comprising a solid culture medium, wherein the solid culture medium comprises 80-120 g/L of potatoes, 40-60 g/L of bananas, 0.3-0.5 mg/L of NAA, MS culture medium and/or Huabao No. 1, 28-32 g/L of sucrose and 6-7 g/L of agar, and the pH value of the culture medium is 5.8-6.0;

the content of the Huabao No. 1 is 0.5-1.5 g/L.

3. The application of the dendrobium culture medium of claim 1 or 2 in improving germination rate of dendrobium seeds and raising seedlings with high efficiency.

4. Use according to claim 3, characterized in that it comprises the following steps:

5. The use of claim 4, wherein the amount of dibble seeding is determined by the area of the solid medium, specifically 0.9-1.25 particles/cm²。

6. The use according to claim 4, wherein the conditions of the dark culture comprise: the temperature is 23-25 ℃, the humidity is 75-85%, and the time is 5-7 d;

7. The use according to claim 4, wherein the seeds are cultured in the liquid medium for 20-30 days.

8. The use according to claim 4 or 7, wherein the seeds are obtained by: cleaning and disinfecting the dendrobe capsules, and splitting to obtain seeds.

9. The application of claim 4, wherein the acclimatization comprises closing a cover in a greenhouse for acclimatization for 14-21 d, and then opening the cover for acclimatization for 5-7 d; when the cover is opened for hardening seedlings, the temperature in the greenhouse is 10-15 ℃, the humidity is 60% -70%, and the shading degree is 65% -75%.

10. The use according to claim 4, wherein the transplanted culture substrate comprises one or more of pine bark, coconut coir and wood chips.