阅读说明：本技术 一种手参组织培养无菌体系建立的方法 (Method for establishing gymnadenia conopsea tissue culture sterile system ) 是由 王旭东 陈凯 于 2021-08-02 设计创作，主要内容包括：本发明属于中药材植物组织培养技术领域,尤其涉及一种手参组织培养无菌体系建立的方法,包括如下步骤：1)采集：采集有1-2cm长健壮芽的手参,经初步清洗后将芽从手参上完整的分离下来,轻剥去掉最外层1-2层叶片,用洗衣粉饱和溶液浸泡15min,并用软毛刷轻轻刷表面,再用流水冲洗2h后,置于超净工作台上；2)消毒：用体积分数为75％的酒精消毒30s,无菌水冲洗3次,再用质量分数为0.1％氯化汞消毒5-10min,无菌水冲洗5次,每次冲洗2min以上；3)诱导培养：将步骤2)消毒后的手参芽接种在诱导培养基上培养；本发明方法可获得手参无菌体系,为手参组织培养快繁体系和手参再生技术奠定基础,同时对于手参资源开发和保护效果显著。(The invention belongs to the technical field of tissue culture of Chinese herbal medicine plants, and particularly relates to a method for establishing a gymnadenia conopsea tissue culture sterile system, which comprises the following steps: 1) collecting: collecting 1-2cm length of gymnadenia conopsea, cleaning, completely separating bud from gymnadenia conopsea, slightly peeling off 1-2 layers of outermost leaves, soaking in washing powder saturated solution for 15min, slightly brushing surface with soft brush, washing with running water for 2 hr, and placing on a clean bench; 2) and (3) disinfection: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5-10min, washing with sterile water for 5 times, and washing for more than 2min each time; 3) and (3) induction culture: inoculating the sterilized conic gymnadenia rhizome buds obtained in the step 2) on an induction culture medium for culture; the method can obtain a gymnadenia conopsea sterile system, lays a foundation for a gymnadenia conopsea tissue culture rapid propagation system and a gymnadenia conopsea regeneration technology, and has obvious effects on development and protection of gymnadenia conopsea resources.)

1. A method for establishing a gymnadenia conopsea tissue culture sterile system is characterized by comprising the following steps:

1) collecting: collecting 1-2cm length of gymnadenia conopsea, cleaning, completely separating bud from gymnadenia conopsea, slightly peeling off 1-2 layers of outermost leaves, soaking in washing powder saturated solution for 15min, slightly brushing surface with soft brush, washing with running water for 2 hr, and placing on a clean bench;

2) and (3) disinfection: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5-10min, washing with sterile water for 5 times, and washing for more than 2min each time;

3) and (3) induction culture: inoculating the sterilized conic gymnadenia rhizome buds obtained in the step 2) on an induction culture medium for culturing, wherein the induction culture medium is MS culture medium +6-KT 0.3-0.9mg/L +6-BA 0.6-1.2mg/L + NAA 0.6-1.2mg/L + PVP 20-50mg/L + banana mud 10-40g/L + agar 5.8g/L + sucrose 30g/L, and the pH value is 5.8-6.2.

2. The method for establishing the tissue culture sterile system of gymnadenia conopsea according to claim 1, wherein in step 2), the disinfection time with 0.1% by mass of mercuric chloride is 8 min.

3. The method for establishing the tissue culture sterile system of gymnadenia conopsea according to claim 1, wherein in step 3), the induction medium is: MS culture medium +6-KT0.3mg/L +6-BA 1.0mg/L + NAA1.0mg/L + PVP 40mg/L + banana paste 30g/L + agar 5.8g/L + sucrose 30g/L, pH 6.2.

4. The method for establishing the tissue culture sterile system of the gymnadenia conopsea as claimed in claim 1, wherein in step 3), the culture conditions are: culturing at 25 deg.C in dark for 7d, and culturing under 2000lx illumination for 25 d.

Technical Field

The invention belongs to the technical field of tissue culture of Chinese herbal medicine plants, and particularly relates to a method for establishing a gymnadenia conopsea tissue culture sterile system.

Background

The rhizoma Gymnadeniae is root tuber of Gymnadeniae (Gymnadenia conopsea) of Gymnadeniae of Orchidaceae, and is prepared from Mongolian and Tibetan medicines, such as rhizoma Gymnadeniae, and radix Codonopsis. It is a perennial herb and is famous for its shape. According to the report of related data, the gymnadenia conopsea is distributed in northeast and northwest of China, Henan, Shanxi, Shaanxi, Gansu, Sichuan, Yunnan, Tibet and the like, but the quantity is very small, and the natural propagation is very difficult. Rhizoma Gymnadeniae has effects of replenishing blood, invigorating qi, promoting fluid production, and quenching thirst, and can be used for treating cough and asthma due to lung deficiency, asthenia, marasmus, and neurasthenia.

In the prior art, the propagation of the gymnadenia conopsea is mainly based on the division of plants, tubers can be cut into small pieces for planting, but the mode is easy to cause virus infection, the propagation coefficient is low, the requirement of large-scale production is difficult to meet, wild resources are gradually exhausted along with the excavation in successive years, and the market requirement cannot be met, so that the tissue culture research of the gymnadenia conopsea is increasingly concerned by people.

Disclosure of Invention

The invention provides a method for establishing a gymnadenia conopsea tissue culture sterile system to solve the technical problems.

The technical scheme for solving the technical problems is as follows: a method for establishing a gymnadenia conopsea tissue culture sterile system comprises the following steps:

1) collecting: collecting 1-2cm length of gymnadenia conopsea, cleaning, completely separating bud from gymnadenia conopsea, slightly peeling off 1-2 layers of outermost leaves, soaking in washing powder saturated solution for 15min, slightly brushing surface with soft brush, washing with running water for 2 hr, and placing on a clean bench;

2) and (3) disinfection: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5-10min, washing with sterile water for 5 times, and washing for more than 2min each time;

3) and (3) induction culture: inoculating the sterilized conic gymnadenia rhizome buds obtained in the step 2) on an induction culture medium for culturing, wherein the induction culture medium is MS culture medium +6-KT 0.3-0.9mg/L +6-BA 0.6-1.2mg/L + NAA 0.6-1.2mg/L + PVP 20-50mg/L + banana mud 10-40g/L + agar 5.8g/L + sucrose 30g/L, and the pH value is 5.8-6.2.

Further, in the step 2), the disinfection time is 8min by using mercuric chloride with the mass fraction of 0.1%.

Further, in step 3), the induction medium is: MS culture medium +6-KT0.3mg/L +6-BA 1.0mg/L + NAA1.0mg/L + PVP 40mg/L + banana paste 30g/L + agar 5.8g/L + sucrose 30g/L, pH 6.2.

Further, in step 3), the culture conditions are that the culture is carried out for 25d under the condition of 25 ℃ and the dark culture is carried out for 7d, and then the culture is switched to 2000lx illumination intensity.

The invention has the beneficial effects that: the method of the invention takes the bud as the explant, can obtain a gymnadenia conopsea tissue culture sterile system, effectively avoids virus infection, improves the propagation coefficient, is beneficial to meeting the requirement of large-scale production, provides raw materials for tissue culture and rapid propagation of gymnadenia conopsea, and lays a foundation for large-scale seedling culture of gymnadenia conopsea.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

A method for establishing a gymnadenia conopsea tissue culture sterile system comprises the following steps:

1) collecting: collecting 1-2cm length of gymnadenia conopsea, cleaning, completely separating bud from gymnadenia conopsea, slightly peeling off 1-2 layers of outermost leaves, soaking in washing powder saturated solution for 15min, slightly brushing surface with soft brush, washing with running water for 2 hr, and placing on a clean bench;

2) and (3) disinfection: sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5-10min, washing with sterile water for 5 times, and washing for more than 2min each time;

3) and (3) induction culture: inoculating the sterilized conic gymnadenia rhizome buds obtained in the step 2) on an induction culture medium for culturing, wherein the induction culture medium is MS culture medium +6-KT 0.3-0.9mg/L +6-BA 0.6-1.2mg/L + NAA 0.6-1.2mg/L + PVP 20-50mg/L + banana mud 10-40g/L + agar 5.8g/L + sucrose 30g/L, and the pH value is 5.8-6.2.

The MS culture medium is a common basic culture medium formula in plant tissue culture technology, the content composition is constant, and the formula is shown in table 1:

TABLE 1MS media composition Table

NAA (naphthylacetic acid) is one of auxin, can promote plant cell differentiation, and can promote seedling bud regeneration and proliferation by combining with cytokinin under proper concentration;

6-BA (6-benzylamino adenine) is one of plant cytokinins, can promote plant cell division and elongation in stem tip or bud culture, can break apical dominance, and can promote the generation and proliferation of lateral bud and cluster bud;

6-KT (6-furfurylaminopurine), one of the plant cytokinins;

PVP (polyvinylpyrrolidone K30) has an inhibiting effect on explant browning, the action mechanism is that PVP is a specific adsorbent of phenolic substances, CO-N in PVP has strong ability of complexing polyphenol compounds, and the PVP can enable the polyphenol compounds not to be substrates of polyphenol oxidase, so that browning is inhibited;

banana puree: mashing common bananas into paste, and adding the paste into a culture medium for decocting;

6-BA, 6-KT, PVP, NAA and banana mud in the induction culture medium cooperate to improve the bud induction rate of the gymnadenia conopsea explant, strengthen the growth of axillary buds and effectively reduce the browning.

Further, in the step 2), the disinfection time is 8min by using mercuric chloride with the mass fraction of 0.1%.

Further, in step 3), the induction medium is: MS culture medium +6-KT0.3mg/L +6-BA 1.0mg/L + NAA1.0mg/L + PVP 40mg/L + banana paste 30g/L + agar 5.8g/L + sucrose 30g/L, pH 6.2.

Further, in step 3), the culture conditions are that the culture is carried out for 25d under the condition of 25 ℃ and the dark culture is carried out for 7d, and then the culture is switched to 2000lx illumination intensity.

The method for establishing the gymnadenia conopsea tissue culture sterile system will be described in detail below with reference to examples and experimental data.

Example 1

1) Collecting Gymnadenia conopsea with strong bud (1-2cm long), cleaning, separating bud from Gymnadenia conopsea completely, peeling off the outermost 1-2 layers of leaves, soaking in saturated detergent solution for 15min, brushing the surface with soft brush, washing with running water for 2 hr, and placing on clean bench;

2) sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 5, 6, 7, 8, 9, and 10min, and washing with sterile water for 5 times (each for more than 2 min);

3) inoculating the disinfected gymnadenia conopsea buds on an induction culture medium (MS +6-KT0.3mg/L +6-BA 1.0mg/L + NAA1.0mg/L + PVP 40mg/L), inoculating 5 bottles in each group, inoculating 1 explant in each bottle, and setting 2 groups for repeated experiments; and (4) culturing each bottle at 25 ℃ in the dark for 7d, then culturing for 25d under 2000lx illumination intensity, and counting the pollution condition and the bud survival rate.

The results are shown in Table 2:

TABLE 2 Disinfection time gradient test results Table

As can be seen from table 2, the contamination rate gradually decreases with the increase of the disinfection time, and when the disinfection time is 8, 9, 10 minutes, the contamination rate decreases to 0, the disinfection effect is good, but the survival rate decreases, which may be the death of the explant caused by the stimulation of the disinfectant, therefore, the contamination rate is the lowest when the disinfection time is 8 minutes, the survival rate of the explant is the highest 80%, which is the optimal disinfection time for the gymnadenia conopsea explant.

Example 2

1) Collecting Gymnadenia conopsea with strong bud (1-2cm long), cleaning, separating bud from Gymnadenia conopsea completely, peeling off the outermost 1-2 layers of leaves, soaking in saturated detergent solution for 15min, brushing the surface with soft brush, washing with running water for 2 hr, and placing on clean bench;

2) sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 5 times, and washing for more than 2 min;

3) designing 4-factor 4 horizontal orthogonal experiments according to different concentrations of 6-KT, 6-BA, NAA and culture media with different pH values, inoculating disinfected gymnadenia conopsea buds into induction culture media consisting of different hormones and different pH values according to the table 3, inoculating 5 bottles in each group, inoculating 1 explant in each bottle, and setting 2 groups of repeated experiments; culturing each bottle at 25 deg.C in dark for 7d, culturing under 2000lx illumination for 25d, counting axillary bud induction and browning conditions, respectively using "+, + +" to indicate axillary bud growth, with more "+" indicating better axillary bud growth.

The results are shown in Table 3:

TABLE 3 hormone level orthogonal experimental design and results table

As shown in Table 3, the axillary bud induction rate of group 3 was 70% at the highest, the browning rate was 40% at the same time, and the axillary bud was the best, so the optimal hormone combination was 0.3 mg/L6-KT +1.0 mg/L6-BA +1.0mg/L NAA, and the optimal pH of the medium was 6.2.

Example 3

1) Collecting Gymnadenia conopsea with strong bud (1-2cm long), cleaning, separating bud from Gymnadenia conopsea completely, peeling off the outermost 1-2 layers of leaves, soaking in saturated detergent solution for 15min, brushing the surface with soft brush, washing with running water for 2 hr, and placing on clean bench;

2) sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 5 times, and washing for more than 2 min;

3) inoculating the disinfected conic gymnadenia rhizome buds to an induction culture medium (MS +6-KT0.3mg/L +6-BA 1.0mg/L + NAA1.0mg/L + agar 5.8g/L + sucrose 30g/L, and the pH value is 6.2), simultaneously adding PVP with different concentrations and banana puree with different concentrations shown in the table 4 into the culture medium, inoculating 5 bottles into each group, inoculating 1 explant into each bottle, setting 2 groups of repeated experiments, and taking the addition of 0mg/LPVP and 0g/L of banana puree as a blank group (CK); culturing each bottle at 25 deg.C in dark for 7d, culturing under 2000lx illumination for 25d, counting explant browning condition and axillary bud growth variation, respectively using "+, + + + +" to represent axillary bud growth, more "+" represents better axillary bud growth.

TABLE 4 Table for the additive concentration of PVP and banana puree

Group of	PVP(mg/L)	Banana mud (g/L)
			1	20	10
2	30	20
			3	40	30
4	50	40
			CK	0	0

The results are shown in Table 5:

TABLE 5 additional concentration test results of PVP and banana puree

Group of	PVP(mg/L)	Browning rate	Banana mud (g/L)	Growth of axillary bud
					1	20	35％	10	++
2	30	20％	20	+++
					3	40	10％	30	++++
4	50	10％	40	+++
					CK	0	40％	0	++

As can be seen from Table 5, when the PVP concentration is 40 and 50mg/L, the browning rate is 10% at the lowest, which is 30% lower than that of the blank group (CK), the browning inhibition effect is significant, and the addition amount of 50mg/L is more than 40 mg/L; when the concentration of the banana puree is 30g/L, the axillary buds grow best, and the differentiation effect of the banana puree on the axillary buds is obvious; therefore, 40mg/LPVP and 30g/L banana puree is selectively added, the optimal browning inhibition effect is realized, the axillary bud differentiation effect is obvious, and the economic benefit is met.

In conclusion, the optimal disinfection method of the invention is to disinfect for 30s by using alcohol with the volume fraction of 75 percent, wash the alcohol with sterile water for 3 times, then disinfect for 8min by using mercuric chloride with the mass fraction of 0.1 percent respectively, and wash the alcohol with sterile water for 5 times, wherein each time is more than 2 min; the most suitable induction culture medium is MS culture medium, 6-KT0.3mg/L, 6-BA 1.0mg/L, NAA1.0mg/L, PVP 40mg/L, banana puree 30g/L, agar 5.8g/L, sucrose 30g/L, and the pH is 6.2; the method can obtain a tissue culture sterile system of the gymnadenia conopsea, effectively avoids virus infection, improves the propagation coefficient, is beneficial to meeting the requirement of large-scale production, provides raw materials for tissue culture and rapid propagation of the gymnadenia conopsea, and lays a foundation for large-scale seedling culture of the gymnadenia conopsea.

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.