阅读说明：本技术 基于器官直接发生途径的半夏快速繁殖方法 (Pinellia ternata rapid propagation method based on organ direct generation approach ) 是由 罗雯 刘艳梅 陈王涛 张同林 匡全 郭志鸿 许继国 卢雪兰 黄琼英 王梦丽 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种基于器官直接发生途径的半夏快速繁殖方法,属于无性繁殖技术领域,该方法选取健康无病害的半夏块茎作为母体,除去泥土杂质,清洗,自然晾干；采用酒精、双氧水和次氯酸钠溶液对半夏块茎进行浸泡灭菌；半夏块茎切块；将半夏块茎切块在植物生长调节剂溶液中浸泡后,再采用防腐剂溶液浸泡后,直接作为人工种子进行种植；本发明证明了通过切块繁殖直接诱导半夏再生的可行性,既克服了传统繁殖方法存在的繁殖周期长、繁殖系数低的缺点,也避免了组培方法的复杂程序和高成本,该方法获得的半夏切块可以直接作为人工种子,且方法安全,简便易行,并可以规模化生产,是半夏种子生产的一种有效途径。(The invention discloses a pinellia ternate rapid propagation method based on an organ direct generation way, which belongs to the technical field of vegetative propagation, and the method selects healthy disease-free pinellia ternate tubers as a matrix, removes soil impurities, cleans the pinellia ternate tubers, and naturally dries the pinellia ternate tubers; soaking and sterilizing tuber of pinellia with alcohol, hydrogen peroxide and sodium hypochlorite solution; cutting pinellia tuber into pieces; cutting pinellia tuber into blocks, soaking in plant growth regulator solution, soaking in antiseptic solution, and planting as artificial seed; the invention proves the feasibility of directly inducing the regeneration of the pinellia ternata through the block-cutting propagation, overcomes the defects of long propagation period and low propagation coefficient of the traditional propagation method, and avoids the complex procedure and high cost of the tissue culture method.)

1. A pinellia ternata rapid propagation method based on an organ direct generation approach is characterized by comprising the following steps:

(1) selecting healthy disease-free pinellia tuber as a matrix, removing soil impurities, cleaning, and naturally drying;

(2) soaking and sterilizing tuber of pinellia with alcohol, hydrogen peroxide and sodium hypochlorite solution;

(3) cutting pinellia tuber into pieces;

(4) and (3) cutting the tuber of the pinellia ternata into blocks, soaking the cut tuber of the pinellia ternata in a plant growth regulator solution, soaking the cut tuber of the pinellia ternata in a preservative solution, covering soil in soil for cultivation, and performing normal field management.

2. The rapid propagation method of pinellia ternata according to claim 1, wherein the diameter of the tuber of pinellia ternata is 1.5-2 cm.

3. The rapid propagation method of pinellia ternata as claimed in claim 1, wherein in step (1), the washing comprises: washing the tuber of pinellia with washing powder until the water is clear.

4. The rapid propagation method of pinellia ternata as claimed in claim 1, wherein in the step (2), the volume concentration of the alcohol is 75%, the volume concentration of the hydrogen peroxide is 10%, and the volume concentration of the sodium hypochlorite solution is 4%.

5. The rapid propagation method of pinellia ternata as claimed in claim 4, wherein the specific operations of soaking and sterilizing comprise: soaking in ethanol for 2min, hydrogen peroxide for 15min, and sodium hypochlorite solution for 20 min.

6. The rapid propagation method of pinellia ternata as claimed in claim 1, wherein in step (3), the cutting comprises: longitudinally cutting the tuber of pinellia into 4-8 pieces.

7. The rapid propagation method of pinellia ternata as claimed in claim 1, wherein in step (4), the plant growth regulator comprises IAA or 6-BA, and the concentration of the plant growth regulator solution is 0.1-1 mg/L.

8. The rapid propagation method of pinellia ternata as claimed in claim 1, wherein in step (4), the preservative is potassium carbonate, and the preservative solution has a mass concentration of 0.1-0.2%.

Technical Field

The invention relates to the technical field of asexual propagation, in particular to a pinellia ternate rapid propagation method based on an organ direct generation way.

Background

Pinellia ternate (Pinellia ternata (Thunb.) Breit.) is a perennial medicinal plant of the family of the Araceae, preferring a mild humid climate and a concealed environment. Pinellia ternate recorded in Chinese pharmacopoeia is an important Chinese medicinal material taking dried tubers as medicine, and the tubers of the pinellia ternate contain chemical components such as volatile oil, nicotine, glutamic acid, sitosterol and the like. Has effects in lowering adverse qi, stopping vomiting, eliminating dampness, eliminating phlegm, relieving distension and fullness, and resolving hard mass, and can be used for treating phlegm, cough, asthma, nausea, emesis, giddiness, etc. Because the good application value of the pinellia ternate is widely concerned by people in various countries, the pinellia ternate is always in a state of short supply and short demand for a long time, and the market price is continuously increased.

Pinellia ternate producing areas are all available in various places of China, mainly produced in Sichuan, Hubei, Jiangsu and other places, and although the pinellia ternate resources are widely distributed, wild resources are nearly exhausted due to environmental damage and excessive mining of people; the traditional artificial cultivation and breeding has long period and low propagation coefficient, and the traditional artificial cultivation and breeding is damaged by viruses, so that the conditions of insufficient yield, low quality, weak pesticide effect and the like of medicinal materials are caused. Although the use of tissue culture technology provides a feasible scheme in the aspect of the production of pinellia ternata artificial seeds, the method for quickly propagating the pinellia ternata seedlings by the tissue culture method mainly focuses on the aspects of explants, culture conditions, culture media and regeneration approaches. The common pinellia tuber material used in tissue culture of pinellia tuber includes leaf, petiole, tuber and bulbil. The early-stage material-taking induction effect is found to be better by culturing the leaves, the petioles and the tubers of the pinellia ternata in different growth periods of the Xia Haiwu (1994). The researches of Wanmeiliang et al (1995) show that the induction effect of the petiole is best, the leaf is second, both the petioles and the leaf are started quickly, and the induction rate can reach 90%; the tubers and the bulbils are poor. There are two ways of regenerating pinellia tuber plants: the organ pathway and the embryoid body pathway. Zhuzhongrong (1991) and the like are cultured by cutting petioles and tubers, and a plurality of spheroids are directly generated from the surface of an explant without obvious callus in the process, and then green buds grow from the upper part of the spheroids and roots grow from the lower part of the spheroids. The stem of Heiyukun (1994) is connected to differentiation culture medium to directly produce small tuber or bulb, and then the complete plant is differentiated. In some cases, regenerated shoots can also be formed through the callus phase. The research lays a foundation for the tissue culture and rapid propagation of the pinellia ternata, and complete plants can be obtained finally by culturing the pinellia ternata leafstalks, leaves, bulblets and tubers which are used as explants. However, these methods all require culturing buds or plantlets from pinellia ternata explants, and then transplanting the explants into the land, so that most of the researches are in the laboratory stage, and the problems of expensive equipment, high cost, complex operation, high technical requirements and the like exist, and various plant diseases and insect pests are encountered in the cultivation process, and the problems limit the application of the methods in the production practice of pinellia ternata.

Disclosure of Invention

The invention aims to provide a pinellia ternate rapid propagation method based on an organ direct generation way, so as to solve the problems in the prior art, the method optimizes the pinellia ternate tuber block-cutting propagation method, can directly plant the processed pinellia ternate tuber in a field, is simple and easy to implement, is easy for large-scale production, and has no obvious difference between the content of medicinal active ingredients of the harvested tuber and a wild group.

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

the invention provides a pinellia ternate rapid propagation method based on an organ direct generation way, which comprises the following steps:

(1) selecting healthy disease-free pinellia tuber as a matrix, removing soil impurities, cleaning, and naturally drying;

(2) soaking and sterilizing tuber of pinellia with alcohol, hydrogen peroxide and sodium hypochlorite solution;

(3) cutting pinellia tuber into pieces;

(4) and (3) cutting the tuber of the pinellia ternata into blocks, soaking the cut tuber of the pinellia ternata in a plant growth regulator solution, soaking the cut tuber of the pinellia ternata in a preservative solution, covering soil in soil for cultivation, and performing normal field management.

Further, the diameter of the tuber of pinellia ternata is 1.5-2 cm.

Further, in the step (1), the washing includes: washing the tuber of pinellia with washing powder until the water is clear.

Further, in the step (2), the volume concentration of the alcohol is 75%, the volume concentration of the hydrogen peroxide is 10%, and the volume concentration of the sodium hypochlorite solution is 4%.

Further, the specific operation of the soaking sterilization comprises the following steps: soaking in ethanol for 2min, hydrogen peroxide for 15min, and sodium hypochlorite solution for 20 min.

Further, in the step (3), the dicing includes: longitudinally cutting the tuber of pinellia into 4-8 pieces.

Further, in the step (4), the plant growth regulator comprises IAA or 6-BA, and the concentration of the plant growth regulator solution is 0.1-1 mg/L.

Further, in the step (4), the preservative is potassium carbonate, and the preservative solution has a mass concentration of 0.1-0.2%.

The invention discloses the following technical effects:

the invention proves the feasibility of directly inducing the regeneration of the pinellia ternata through the block-cutting propagation, overcomes the defects of long propagation period and low propagation coefficient of the traditional propagation method, and avoids the complex procedure and high cost of the tissue culture method.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a diagram of a non-diced pinellia ternate pot culture;

FIG. 2 shows 0.1% K of cut pinellia tuber₂CO₃Soaking (no hormone induction) the potted plant map;

FIG. 3 shows that cut pinellia tuber is induced by 1 mg/L6-BA and 0.1% K₂CO₃Potted plant pictures after soaking;

FIG. 4 shows the harvested seed stems per pot after 6 months of potting without cutting the tuber pinellia;

FIG. 5 shows that cut pinellia tuber was induced by 1 mg/L6-BA and 0.1% K₂CO₃The seed stems were harvested per pot 6 months after the soaking.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

In the embodiment of the invention, the tuber of pinellia ternata in the west of the Yangtze river is used as an experimental material.

Example 1

A pinellia ternata rapid propagation method based on an organ direct generation approach comprises the following steps:

(1) selecting healthy disease-free tuber of pinellia ternata with diameter of 1.5-2cm as parent, washing with washing powder, washing with tap water until water is clear, and naturally drying in the ventilated place;

(2) soaking and sterilizing tuber pinellia by using 75% alcohol, 10% hydrogen peroxide and 4% sodium hypochlorite solution in sequence, wherein the soaking process is as follows: soaking in ethanol for 2min, soaking in hydrogen peroxide for 15min, soaking in sodium hypochlorite solution for 20min, washing with sterile water for 3 times, and air drying the surface;

(3) longitudinally cutting pinellia tuber into 4 pieces;

(4) and (3) cutting pinellia tubers into blocks, soaking the blocks in 1 mg/L6-BA solution for 30min, soaking the blocks in 0.1 wt% potassium carbonate solution for 30min, covering soil in soil for cultivation, and performing normal field management.

Example 2

1. Screening of optimum sterilization conditions for tuber pinellia block-cutting propagation

Washing the tuber of pinellia with washing powder, washing with tap water until the water is clear, and air drying in a ventilated place. Each seed stem (diameter 1.5-2cm) was cut longitudinally into 4 pieces and the best sterilization conditions for pinellia tuber cut propagation were screened using an orthogonal experiment, the orthogonal design is as shown in table 1:

TABLE 1

Experiment number	75% alcohol	10% hydrogen peroxide	4% sodium hypochlorite
				1	1min	5min	15min
2	1min	10min	20min
				3	1min	15min	10min
4	30s	5min	15min
				5	30s	10min	20min
6	30s	15min	10min
				7	2min	5min	15min
8	2min	10min	20min
				9	2min	15min	10min

Repeating 5 times for each experiment group, sterilizing, soaking in 1 mg/L6-BA solution for 30min, culturing at 26 deg.C on soaked sterile absorbent cotton, and lighting for 8 h/d. Culturing for 20 days, and counting the number of new buds. The results are shown in Table 2:

TABLE 2

Test No	Repetition of 1	Repetition 2	Repetition of 3	Repetition of 4	Repetition 5
						1	30	30	39	30	42
2	33	29	36	42	24
						3	44	57	45	30	44
4	45	36	44	51	42
						5	50	42	47	44	59
6	51	39	55	30	33
						7	35	57	36	49	39
8	33	45	51	41	41
						9	33	41	36	62	60

According to statistics, the optimal sterilization conditions are 75% ethanol for 2min, 10% hydrogen peroxide for 15min and 4% sodium hypochlorite solution for 20 min.

2. Screening of pinellia tuber cut block regenerated plant growth regulator

Washing the tuber of pinellia with washing powder, washing with tap water until the water is clear, and air drying in a ventilated place. Soaking in 75% ethanol for 2min, 10% hydrogen peroxide for 15min, 4% sodium hypochlorite solution for 20min, washing with sterile water for 3 times, and air drying. After the tuber of pinellia ternata is disinfected, the tuber of pinellia ternata is longitudinally cut into 3 sections, and the 3 sections are respectively placed in absorbent cotton tissue culture bottles containing a plant growth regulator, wherein the plant growth regulator is IAA, NAA, 6-BA or KT, and the concentration is 0.1mg/L or 1 mg/L. Each concentration of each plant growth regulator is set to 3 times, and the plant growth regulators are cultured at 26 ℃ with the illumination period of 8 h/d. The observed data were subjected to one-way anova, and the results are shown in table 3.

TABLE 3 Effect of plant growth regulator concentration on the number of regenerated shoots of Semiazeri tubers

The results show that: the number of regenerated buds is subjected to one-way anova, the addition of 0.5mg/L IAA or 1 mg/L6-BA shows significant difference (P is less than 0.05), and the mean value of the number of regenerated buds is significantly higher than that of other treatment groups.

3. Slicing mode screening for influencing pinellia tuber slicing regeneration

Sterilizing tuber of rhizoma Pinelliae (the same manner as above), air drying on surface, cutting seed stem with diameter of 1.5-2cm into 4, 8, 16 pieces, placing experimental group in 1 mg/L6-BA, placing control group in sterile water, soaking at 4 deg.C for 24 hr, and air drying on surface. Each treatment was repeated 3 times. Placing on soaked sterile absorbent cotton, culturing at 26 deg.C, and lighting period 8 h/d. After 20 days of culture, the number of new seed buds was counted, and the results are shown in Table 4.

TABLE 4 Effect of cut size on the number of regenerated shoots in semi-summer

The results show that the longitudinal cutting of 4 or 8 tubers with the addition of 1 mg/L6-BA promotes the development of new buds on the cut tuber of pinellia.

4. Screening of pinellia tuber cutting antiseptic treatment conditions

The cut pinellia ternata is extremely easy to rot after being covered with soil and cultivated, and can hardly regenerate and survive. With K₂CO₃Treating under the above optimal sterilization conditions as antiseptic, and soaking in 1 mg/L6-BA for 24 hr to obtain pieces of rhizoma Pinelliae with different concentrations of K₂CO₃After the solution soaking treatment (repeating for 3 times per treatment), soil-covered pot culture (flowerpot with diameter of 15 cm) is carried out, regular observation is carried out, and the number of regenerated plants is counted. Tubers were harvested after 6 months of culture and weighed, and the results are given in Table 5.

The results show that: longitudinally cutting seed stem of pinellia tuber with diameter of 1.5-2cm into 4 pieces with similar size, and placing cut pinellia tuber at 0.1% K₂CO₃The solution is soaked for 30min, so that the regeneration efficiency of the plants can be effectively improved, and the higher concentration can not improve the regeneration efficiency of the plants; as shown in Table 6, when the pinellia tuber stems 1.5-2cm in diameter were longitudinally cut into 4 pieces, the number of the harvested progeny tubers was the largest and significantly higher than the yield of the non-cut seed stems. The cut pieces are too small (8 pieces), and although the number of regenerated plants is large, the yield of seed stems harvested in the same year is remarkably reduced, so that the cut pieces are not suitable to be smaller than 0.5 cm.

TABLE 5K₂CO₃Influence of concentration on half-summer cutting and earthing cultivation effect

TABLE 6 influence of rhizoma Pinelliae cutting size on earthing cultivation yield

Photographing at 40 days after the above treatment, it can be seen that the plants (figure 1) obtained by directly cultivating the uncut tuber seeds have good growth, but the number of the plants is the same as that of the cultivated tuber seeds, and the number of the plants is not increased(ii) a The plants obtained by longitudinally cutting the seed stems of pinellia ternata into 4 pieces without hormone induction (figure 2) were slower in growth, but the number was slightly increased compared with that of the plants without cutting; and the number of plants obtained by longitudinally cutting 4 pinellia ternata seed stems and inducing with 1 mg/L6-BA (figure 3) is obviously increased. The cut rhizoma Pinelliae is 0.1% K₂CO₃And (5) potting after soaking.

After 6 months of cultivation, seed stems were harvested separately for each pot. It can be seen that the seed stems obtained by planting the non-diced pinellia tuber are relatively large in single individuals (fig. 4); cut pinellia tuber induced by 1 mg/L6-BA and 0.1% K₂CO₃The individual stems obtained by the cultivation after soaking were relatively small but in large amounts (FIG. 5).

Example 3

Analysis of pharmaceutical ingredients of pinellia tuber

The tuber of pinellia which grows naturally and the tuber which grows in a cutting way by adopting the method of the embodiment 1 are harvested, non-targeted metabolome detection experiments are carried out on the tuber of pinellia which grows by a wild group and a cutting regeneration group, the average number of relative quantitative information of tuber metabolites of quercetin, kaempferol and coniferol is shown in the table 7, the relative quantitative information of the tuber metabolites of quercetin, kaempferol and coniferol is carried out single-factor variance analysis, and the result shows that the difference between the quercetin, kaempferol and coniferol of the wild group and the cutting regeneration group is not obvious (p is more than 0.05). In conclusion, the content of the medicinal active ingredients of the tubers harvested by the cutting regeneration is not obviously different from that of the wild group. This approach proved to be applicable to production.

TABLE 7 relative quantitative information of the metabolites Quercetin, Kaempferol, coniferol of the wild group and the chum stem of the cut regeneration group

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.