CN108419672B

CN108419672B - Method for calluses of dracaena cochinchinensis to form seedlings

Info

Publication number: CN108419672B
Application number: CN201810077131.8A
Authority: CN
Inventors: 程玲
Original assignee: Yangtze University
Current assignee: Guangxi Hongge Biotechnology Co ltd
Priority date: 2018-01-26
Filing date: 2018-01-26
Publication date: 2021-09-28
Anticipated expiration: 2038-01-26
Also published as: CN108419672A

Abstract

The invention provides a method for calluses and seedlings of dracaena cochinchinensis, which specifically comprises the following steps: (1) selecting an explant; (2) inducing to obtain callus; (3) performing proliferation culture on the callus; (4) performing differentiation culture on the callus, and adding 1-3mg/L of thidiazuron TDZ, 0.1-0.5mg/L of kinetin KT, 0.1-0.5mg/L of brassinolide and 2mg/L of polyvinylpyrrolidone PVP into an MS differentiation culture medium; (5) rooting culture, wherein 1/2MS rooting culture medium is added with 0.5-1.0mg/L of NAA and 2g/L of active carbon; (6) hardening and transplanting the seedlings. The multiplication coefficient of the clumped buds of the dracaena cochinchinensis reaches 5-12 times, the clumped buds are strong and strong, the dracaena cochinchinensis seedlings can easily take root after being inoculated into a rooting culture medium, the rooting rate reaches more than 90%, the transplanting survival rate is more than 95%, and a large number of dracaena cochinchinensis seedlings suitable for cultivation can be cultivated in a short time.

Description

Method for calluses of dracaena cochinchinensis to form seedlings

Technical Field

The invention relates to the technical field of vegetative propagation of seedlings, in particular to a method for growing seedlings from calluses of dracaena cochinchinensis.

Background

Dracaena cochinchinensis (Lour.) harms s.c. chen) belongs to Dracaena of liliaceae, also called millennia wood, dragon's blood tree and Dracaena cochinchinensis, has sweet, warm and salty taste, has the effects of promoting blood circulation to remove blood stasis, relieving swelling and pain and astringing to stop bleeding, is mainly used for traumatic injury, incised wound bleeding, angina pectoris and the like, and belongs to the important protection of wild plants and endangered rare species in China. Dracaena cochinchinensis has high medicinal value and is a traditional and rare Chinese medicinal material in China. The dragon blood tree with flag leaves in China has less resources and the use amount of traditional Chinese medicine resources is increased rapidly, and in recent years, the market has increased the dragon blood, so that people have devastating felling and endangering of wild resources of the dragon blood tree with flag leaves, and the dragon blood is listed as rare or endangered plants by many countries.

The tissue culture mode is adopted for propagation, the propagation speed is high, the seedling quality is good, and the purpose of large-scale production can be achieved. The tissue culture conditions of dracaena cochinchinensis are harsh, and the dracaena cochinchinensis cultured under the general tissue culture conditions or the browning rate of the dracaena cochinchinensis cultured under the general tissue culture conditions is extremely high. For this reason, the difficulty of tissue culture of dracaena cochinchinensis lies in: the method comprises the following steps of firstly, exploring the optimal hormone factors and conditions for callus induction of dracaena cochinchinensis so as to increase the induction rate of the callus; a better browning inhibiting method is found by adding a browning preventing substance, changing the culture environment and the like so as to effectively reduce the browning rate and establish a high-quality cochliobolus glauca callus culture system; and thirdly, exploring the optimal hormone factors and conditions for differentiation and rooting culture of the calluses of the dracaena cochinchinensis so as to improve the proliferation coefficient and rooting rate of the clumps of the dracaena cochinchinensis. At present, only two comparison documents are available for the research of propagating dracaena cochinchinensis by using a tissue culture technology, wherein the comparison document 1: the patent application number is 'CN 201510755473.7', the patent name is 'a method for inducing dracaena cochinchinensis loose callus', the technical problem is one; comparison document 2: the patent application number is 'CN 201410735185.0', the patent name is 'a method for inducing calluses of dracaena cochinchinensis and preventing browning', and the technical problem II is solved; the two comparison documents do not explore and solve the optimal hormone factors and conditions for the subsequent differentiation and rooting culture of the calluses of the dracaena cochinchinensis in the third problem.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for growing seedlings of dracaena cochinchinensis calluses, the multiplication coefficient of clump buds of dracaena cochinchinensis obtained by the culture method reaches 5-12 times, the clump buds are strong and can easily root after being inoculated into a rooting culture medium, the rooting rate can reach more than 90%, the transplanting survival rate is more than 95%, a large amount of dracaena cochinchinensis seedlings suitable for cultivation can be cultivated in a short time, the multiplication coefficient and the seedling quality of the dracaena cochinchinensis seedlings are guaranteed, large-scale production is realized, and the production requirement is met.

The invention is realized by the following steps:

the invention provides a method for calluses of dracaena cochinchinensis to form seedlings, which specifically comprises the following steps:

step 1, selection of explants: taking a flag leaf dragon tree sterile test-tube plantlet as an explant;

step 2, inducing the culture medium to obtain callus: placing the explant in an induction culture medium for induction to obtain callus;

step 3, callus proliferation culture: placing the callus obtained in the step 2 in an MS subculture medium for propagation culture to obtain a large amount of callus;

step 4, callus differentiation culture: placing the callus obtained in the step 3 in an MS differentiation culture medium for differentiation culture to obtain a large number of cluster buds, wherein 2.0mg/L of thidiazuron TDZ, 0.2mg/L of kinetin KT, 0.5mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar are added into the MS differentiation culture medium, and the pH value of the culture medium is 5.8;

step 5, rooting culture: dividing the cluster buds obtained in the step (4) into single buds, and culturing the single buds in 1/2MS rooting culture medium to obtain complete rooted seedlings, wherein 1.0mg/L NAA, 2g/L active carbon, 10g/L sucrose and 5g/L agar are added into 1/2MS rooting culture medium, and the pH value of the culture medium is 5.8;

step 6, hardening and transplanting seedlings: opening the bottle cap of the complete seedling with roots in a room with the room temperature of 25 ℃, adding 30ml of tap water into the bottle, hardening the seedling for 4-7 days, taking out the seedling after surface cutin is formed, cleaning the root culture medium, and immediately transplanting the root culture medium into a sterilized culture medium.

Specifically, the method for taking the dracaena cochinchinensis aseptic test-tube plantlet as the explant in the step 1 comprises the steps of disinfecting plump seeds or buds of the dracaena cochinchinensis aseptic test-tube plantlet germinated in river sand, cleaning surface dirt with a detergent water solution, placing the washed surface dirt in a beaker for washing for 5-8 min with running water, then moving the beaker to a super clean workbench, soaking the seeds and the buds for 30s with 75% v/v alcohol, washing the seeds and the buds for 1 time with sterile water, soaking the seeds and the buds with 0.1% v/v HgC12 for 3 times with sterile water, sucking the surface moisture with sterile filter paper, inoculating the soaked seeds and the buds to an MS induction culture medium, and growing the dracaena cochinchinensis aseptic test-tube plantlet.

The invention has the following beneficial effects:

1. according to the method for calluses and seedling formation of dracaena cochinchinensis, the multiplication coefficient of the calluses and buds of the dracaena cochinchinensis obtained by the culture method reaches 5-12 times, the rooting rate is above 90%, the transplanting survival rate is above 95%, high-quality seedlings of dracaena cochinchinensis with high survival rate can be provided in a short time, and the problem of large-scale seedling culture of dracaena cochinchinensis is effectively solved.

2. The callus differentiation culture of the invention is that 1-3mg/L thidiazuron TDZ, 0.1-0.5mg/L kinetin KT, 0.1-0.5mg/L brassinolide and 2mg/L polyvinylpyrrolidone PVP are added into an MS differentiation culture medium used by the callus differentiation culture, wherein the thidiazuron TDZ, the kinetin KT and the brassinolide with proper concentration and proportion can promote the mass multiplication of the buds of the dracaena cochinchinensis clumps; polyvinylpyrrolidone PVP is effective in preventing browning.

3. 0.5-1.0mg/L of NAA naphthalene acetic acid and 2g/L of active carbon are added into an 1/2MS rooting culture medium used in the invention, the NAA naphthalene acetic acid with proper concentration can improve the rooting rate of dracaena cochinchinensis, the active carbon is used for adsorbing some harmful secondary metabolites generated by plants, and the active carbon has the function of reducing browning.

Detailed Description

(I) exploration of auxin proportion in MS differentiation culture medium for dracaena cochinchinensis tooth proliferation culture

Example 1

(1) Selection of explants: taking a flag leaf dragon blood tree sterile test-tube plantlet as an explant.

(2) Inducing a culture medium to obtain callus: the obtained explant is placed in a clean bench, a 0.5cm section is cut by a dissecting knife, inoculated into an MS culture medium, and cultured for 40 days under the condition of dark culture at the culture temperature of 23-27 ℃ to obtain a large amount of callus, wherein 1.0mg/L of 6-benzyladenine 6-BA, 0.2mg/L of naphthylacetic acid NAA, 1.0mg/L of dichlorophenoxyacetic acid 2.4-D, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar are added into the MS culture medium, and the pH value of the culture medium is 5.8.

(3) And (3) callus proliferation culture: and (3) placing the callus obtained in the step (2) into an MS culture medium, and culturing for 40 days under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 1500lux and the illumination time is 8-10 hours/day to obtain a differentiable callus part, wherein 1.0mg/L of thidiazuron TDZ, 0.5mg/L of dichlorophenoxyacetic acid 2.4-D, 1.0mg/L of 6-benzyladenine 6-BA, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar are added into the MS subculture medium, and the pH value of the culture medium is 5.8. Here, 1.0mg/L of thiadiazine TDZ means that 1.0mg of thiadiazine TDZ is added to 1L of MS medium, and the same applies to the addition of other substances.

(4) Differentiation culture of callus: and (3) placing the callus obtained in the step (3) into an MS differentiation culture medium, and culturing for 30 days under the conditions that the culture temperature is 23-27 ℃, the illumination intensity is 1500lux and the illumination time is 8-10 hours/day to obtain a large number of cluster buds, wherein 3.0mg/L of thidiazuron TDZ, 0.5mg/L of kinetin KT, 0.3mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar are added into the MS differentiation culture medium, and the pH value of the culture medium is 5.8.

(5) Rooting culture: dividing the cluster buds obtained in the step (4) into single buds, and putting the single buds into 1/2MS rooting culture medium to be cultured for 30 days under the conditions of culture temperature of 23-27 ℃, illumination intensity of 1500lux and illumination time of 8-10 hours/day to obtain complete rooted seedlings, wherein 1.0mg/L of NAA, 2g/L of activated carbon, 10g/L of sucrose and 5g/L of agar are added into 1/2MS rooting culture medium, and the pH value of the culture medium is 5.8.

(6) Hardening and transplanting seedlings: opening the bottle cap of the complete seedling with roots in a room with the room temperature of 25 ℃, adding 30ml of tap water into the bottle, hardening the seedling for 4-7 days, taking out the seedling after surface cutin is formed, cleaning the root culture medium, and immediately transplanting the root culture medium into a sterilized culture medium.

Example 2

And (4) adding 2.0mg/L of thidiazuron TDZ, 0.2mg/L of kinetin KT, 0.5mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar into the MS differentiation culture medium used in the step 4, wherein the pH value of the culture medium is 5.8. The other operation steps are the same as in example 1.

Example 3

And (4) adding 1.5mg/L of thidiazuron TDZ, 0.2mg/L of kinetin KT, 0.3mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar into the MS differentiation culture medium used in the step 4, wherein the pH value of the culture medium is 5.8. The other operation steps are the same as in example 1.

Example 4

In the step 4, 1.5mg/L of thidiazuron TDZ, 0.5mg/L of kinetin KT, 0.5mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar are added into an MS differentiation culture medium, and the pH value of the culture medium is 5.8. The other operation steps are the same as in example 1.

Example 5

In the step 4, 1mg/L of thidiazuron TDZ, 0.1mg/L of kinetin KT, 0.1mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar are added into an MS differentiation culture medium, and the pH value of the culture medium is 5.8. The other operation steps are the same as in example 1.

Example 6

In the step 4, 3mg/L of thidiazuron TDZ, 0.5mg/L of kinetin KT, 0.5mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of cane sugar and 5g/L of agar are added into an MS differentiation culture medium, and the pH value of the culture medium is 5.8. The other operation steps are the same as in example 1.

Comparative example 1

Different from the example 1, in the step 4, when the test-tube plantlet cluster buds are rapidly propagated and cultured, the MS differentiation culture medium is the basic component of the MS culture medium, and no other components are added. The other steps were the same as in example 1.

The above examples 1-6 and comparative example 1 were repeated at the same time at least 3 times, and the multiplication times of the shoots of the flag leaf dragon tree in the above examples 1-6 (i.e. how many shoots a shoot grows after being inoculated into the culture medium) were observed and calculated respectively and counted as follows:

TABLE 1

As can be seen from Table 1 above, the germination rate was the highest in example 1, which is the most preferable example when 2.0mg/L of thidiazuron TDZ, 0.2mg/L of kinetin KT, 0.5mg/L of brassinolide, and 2mg/L of polyvinylpyrrolidone PVP were added to the MS differentiation medium. Thidiazuron TDZ, kinetin KT and brassinolide with proper concentration and proportion can promote the massive proliferation of the clumped buds of dracaena cochinchinensis; polyvinylpyrrolidone PVP is effective in preventing browning.

(II) exploring auxin proportion in rooting culture medium

As is clear from the above, the germination rate was the highest in example 1, and therefore, example 7 was set up, in which the MS differentiation medium was the same as in example 1, except that 0.5mg/L NAA, 2g/L activated carbon, 10g/L sucrose and 5g/L agar were added to the 1/2MS rooting medium in step 5, and the pH of the medium was 5.8. The other steps were the same as in example 1. The rooting rate of the dracaena cochinchinensis is improved by exploring a proper proportion.

Comparative example 2: 1/2MS rooting medium contains no other components.

The above examples 1, 7 and 2 were repeated at the same time at least 3 times, and the rooting rate of the dracaena cochinchinensis in the above examples 1 and 7 was observed and calculated respectively and counted as follows:

TABLE 2

As can be seen from the above Table 2, the rooting rate of example 1 is the highest, namely 1.0mg/L NAA naphthalene acetic acid, 2g/L active carbon, and NAA naphthalene acetic acid with a proper concentration can improve the rooting rate of dracaena cochinchinensis, the active carbon is used for adsorbing some harmful secondary metabolites produced by plants, and the active carbon has the effect of reducing browning.

(III) comparison of transplanting conditions

The following are comparisons of the transplanting of the tissue culture seedlings obtained in example 1 and comparative example 1.

Culturing seedlings of the two rooting culture media for 20 days, simultaneously hardening the seedlings of the two tissue culture media to obtain complete plants with roots, opening a bottle cap in a room with the room temperature of 25 degrees, adding a small amount of tap water into the bottle, hardening the seedlings for 2-4 days, taking out the seedlings after surface cutin is formed, cleaning the root culture media, immediately transplanting the seedlings into ventilated and low-light sandy soil, and transplanting the seedlings to a field after the seedlings grow in the sandy soil for one month. Opening a bottle cap of the complete seedling with the roots indoors at the room temperature of 25 ℃, adding 30ml of tap water into the bottle, hardening the seedling for 4-7 days, taking out the seedling after surface cutin is formed, cleaning the root culture medium, immediately transplanting the seedling into the well-ventilated and weak-light sandy soil, and transplanting the seedling to a field after the seedling grows for one month in the sandy soil. Spraying 3-5 times every day from 8 am to 6 am within one week after transplanting, each time for 10min, and spraying 1 time each time from 8 am and 6 am, each time for 10 min; the temperature condition during transplanting is 20-28 ℃, the relative humidity is 75-80%, and the sun-shading rate is 70%. After transplanting, watering the seedlings thoroughly, spraying water once in the morning and evening each day, transplanting the dracaena cochinchinensis seedlings for 20 days, spraying 0.1-0.3% monopotassium phosphate foliar fertilizer every seven days, and watering the nutrient solution regularly.

And observing and recording every day, and after 12 days of transplanting, surveying and counting the transplanting survival rate of the two tissue culture seedlings by adopting the following formula.

The transplanting survival rate (%) (the number of survival plants (clumps) of the tissue culture seedlings of dracaena cochinchinensis transplanted per treatment/the total number of the survival plants (clumps) of the tissue culture seedlings of dracaena cochinchinensis transplanted per treatment) x 100%.

The transplanting survival rate is compared by two statistical methods according to the plant height of the dracaena cochinchinensis tissue culture seedlings, namely the survival rate of the total seedlings before transplanting and the survival rate of the dracaena cochinchinensis seedlings of more than 5cm, and specific data are shown in the table 3 of the transplanting conditions of the tissue culture seedlings of the embodiment 1 and the comparative embodiment 1.

TABLE 3

The data comparison in table 3 shows that: the survival rate of the tissue culture seedling transplanting assembly of dracaena cochinchinensis in example 1 is 95.0%, which is far higher than the survival rate of transplanting by traditional MS culture by 16.0%; in addition, the number of days required for reversion after transplantation is earlier than that of comparative example 1 in example 1, which fully indicates that when the technical scheme of the invention is adopted for carrying out dracaena cochinchinensis tissue culture, the dracaena cochinchinensis seedling has good quality and stronger root absorption capacity, the environment adaptation capacity after transplantation is stronger than that of the traditional MS culture, and the reversion period after transplantation is early and easy to survive.

In conclusion, the dracaena cochinchinensis clump bud multiplication coefficient obtained by the culture method reaches 5-12 times, the rooting rate is above 90%, the transplanting survival rate is above 95%, high-quality dracaena cochinchinensis seedlings with high survival rate can be provided in a short time, and the problem of large-scale seedling raising of dracaena cochinchinensis is effectively solved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for calluses of dracaena cochinchinensis to form seedlings is characterized by comprising the following steps:

step 2, inducing the culture medium to obtain callus: putting the explant into an induction culture medium for induction to obtain callus, wherein the induction culture medium is prepared by adding 1.0mg/L of 6-benzyladenine 6-BA, 0.2mg/L of naphthylacetic acid NAA, 1.0mg/L of dichlorophenoxyacetic acid 2.4-D, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar into an MS culture medium, the pH value of the culture medium is 5.8, and the culture conditions are as follows: culturing at 23-27 deg.C for 40 days in dark;

step 3, callus proliferation culture: placing the callus obtained in the step 2 in an MS subculture medium for propagation culture to obtain a large amount of callus; in the step 3, 1.0mg/L of thidiazuron TDZ, 0.5mg/L of dichlorophenoxyacetic acid 2.4-D, 1.0mg/L of 6-benzyladenine 6-BA, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar are added into the MS subculture medium, the pH value of the culture medium is 5.8, and the culture conditions are as follows: culturing at 23-27 deg.C under illumination intensity of 1500lux for 40 days under illumination time of 8-10 hr/day;

step 4, callus differentiation culture: placing the callus obtained in the step 3 in an MS differentiation culture medium for differentiation culture to obtain a large number of cluster buds, wherein 1-3mg/L of thidiazuron TDZ, 0.1-0.5mg/L of kinetin KT, 0.1-0.5mg/L of brassinolide, 2mg/L of polyvinylpyrrolidone PVP, 30g/L of sucrose and 5g/L of agar are added into the MS differentiation culture medium, and the pH value of the culture medium is 5.8;

step 5, rooting culture: dividing the cluster buds obtained in the step 4 into single buds, and culturing the single buds in 1/2MS rooting culture medium to obtain complete rooted seedlings, wherein 0.5-1.0mg/L of NAA, 2g/L of active carbon, 10g/L of sucrose and 5g/L of agar are added into 1/2MS rooting culture medium, and the pH value of the culture medium is 5.8;

step 6, hardening and transplanting seedlings: after the seedlings with the complete roots are hardened, the root culture medium is cleaned and immediately transplanted into a sterilized culture medium.

2. The method for callus seedling of dracaena cochinchinensis as claimed in claim 1, wherein the specific method for taking the aseptic test-tube seedling of dracaena cochinchinensis as the explant in step 1 is to firstly sterilize the plump seeds or the buds of dracaena cochinchinensis which are sowed in river sand, wash the surface dirt with the aqueous solution of detergent, wash the surface dirt in a beaker for 5-8 min with running water, then move the beaker to an ultra-clean bench, soak the seeds and the buds with 75% v/v alcohol for 30s, wash the surface dirt with the aseptic water for 1 time, and then use 0.1% v/v HgCl₂Soaking in sterile water for 3 times, drying surface water with sterile filter paper, inoculating to MS induction culture medium, and growing sterile test-tube plantlet of dracaena cochinchinensis.

3. The method for callus seedling formation of dracaena cochinchinensis as claimed in claim 1, wherein in the step 4, the culture conditions are as follows: culturing at 23-27 deg.C under illumination intensity of 1500lux for 30 days at 8-10 hr/day.

4. The method for callus seedling formation of dracaena cochinchinensis as claimed in claim 1, wherein in the step 5, the culture conditions are: culturing at 23-27 deg.C under illumination intensity of 1500lux for 30 days under illumination time of 8-10 hr/day.

5. The method for callus seedling of dracaena cochinchinensis as claimed in claim 1, wherein in step 6, the cap of the whole rooted seedling is opened in a room at room temperature of 25 ℃, 30ml of tap water is added into the bottle, the seedling is hardened for 4-7 days, the seedling is taken out after surface cutin is formed, and the root medium is washed and immediately transplanted into the sterilized culture medium.