阅读说明：本技术 一种粉葛茎尖培养快速繁殖方法 (Rapid propagation method for culturing stem tip of pueraria thomsonii ) 是由 王继华 梅瑜 李向荣 蔡时可 顾艳 周芳 徐世强 孙铭阳 李静宇 于 2020-11-23 设计创作，主要内容包括：本发明提供了一种粉葛茎尖培养快速繁殖方法,包括以下步骤：(1)外植体选取与消毒；(2)培养基的制备；(3)茎尖分离与不定芽诱导培养：将消毒外植体的茎尖接种于不定芽诱导培养基中进行诱导培养,得到粉葛不定芽；(4)继代增殖：将粉葛不定芽接入继代增殖培养基中进行继代增殖培养,得到粉葛无菌苗；(5)无菌生根：将粉葛无菌苗进行切繁,接入无菌生根培养基中进行生根培养,得到粉葛幼苗,然后炼苗、移栽；(6)脱毒种苗鉴定：取植株叶片进行RNA鉴定,选择无毒核酸相应序列的株系作为健康种苗进行扩繁。本发明粉葛茎尖培养快速繁殖方法可以快速培养出没有病原菌聚集的粉葛种苗,提高繁殖系数,且培养周期短。(The invention provides a rapid propagation method for culturing a stem tip of pueraria thomsonii, which comprises the following steps: (1) selecting and disinfecting explants; (2) preparing a culture medium; (3) separating stem tips and carrying out adventitious bud induction culture: inoculating the stem tip of the sterilized explant into an adventitious bud induction culture medium for induction culture to obtain an adventitious bud of the pueraria thomsonii; (4) subculture proliferation: inoculating adventitious buds of radix Puerariae into a subculture multiplication medium for subculture multiplication to obtain aseptic seedlings of radix Puerariae; (5) sterile rooting: cutting and propagating the sterile pueraria thomsonii seedlings, inoculating the sterile pueraria thomsonii seedlings into a sterile rooting culture medium for rooting culture to obtain pueraria thomsonii seedlings, and then hardening and transplanting the seedlings; (6) identification of detoxified seedlings: and (3) taking plant leaves for RNA identification, and selecting a strain with a corresponding sequence of non-toxic nucleic acid as a healthy seedling for propagation. The rapid propagation method for culturing the stem tip of the pueraria thomsonii can rapidly culture the seedlings of the pueraria thomsonii without gathering pathogenic bacteria, improve the propagation coefficient and have short culture period.)

1. A rapid propagation method for culturing the stem tip of the pueraria thomsonii is characterized by comprising the following steps:

(1) selecting and disinfecting explants: selecting tender branches of pueraria thomsonii as explants to be disinfected to obtain disinfected explants;

(2) preparation of a culture medium: preparing an improved MS culture medium, and then respectively preparing an adventitious bud induction culture medium, a subculture multiplication culture medium and a sterile rooting culture medium on the basis of the improved MS culture medium; the formula of the improved MS culture medium is that polyvinylpyrrolidone with the concentration of 10-20mg/L is added into the MS culture medium;

(3) separating stem tips and carrying out adventitious bud induction culture: carrying out aseptic separation on the buds of the sterilized explants under a dissecting mirror, inoculating the buds into an adventitious bud induction culture medium for induction culture to obtain the adventitious buds of the pueraria thomsonii;

(4) subculture proliferation: inoculating adventitious buds of radix Puerariae into a subculture multiplication medium for subculture multiplication to obtain aseptic seedlings of radix Puerariae;

(5) sterile rooting: cutting and propagating the sterile pueraria thomsonii seedlings, inoculating the sterile pueraria thomsonii seedlings into a sterile rooting culture medium for rooting culture to obtain pueraria thomsonii seedlings, and then hardening and transplanting the seedlings;

(6) identification of detoxified seedlings: and (3) taking plant leaves for RNA identification, and selecting a strain with a corresponding sequence of non-toxic nucleic acid as a healthy seedling for propagation.

2. The method for rapid propagation of stem tip of Pueraria thomsonii according to claim 1, wherein the adventitious bud induction medium is prepared by adding 6-BA and IBA on the modified MS medium; the concentration of 6-BA in the adventitious bud induction culture medium is 0.05-0.2mg/L, and the concentration of IBA is 0.05-0.5 mg/L.

3. The method for rapid propagation of stem tip culture of Pueraria thomsonii according to claim 2, wherein the concentration of 6-BA in the medium for adventitious bud induction is 1mg/L and the concentration of IBA is 0.2 mg/L.

4. The method for rapid propagation of stem tip of Pueraria thomsonii according to claim 2, wherein the inducing culture conditions are a temperature of 28 ± 2 ℃, a humidity of 60-80%, an illumination time of 14 h/day, and an illumination intensity of 800-; the culture time is 18-22 days.

5. The method for rapid propagation of stem tip culture of pueraria thomsonii according to claim 1 or 2, wherein the secondary propagation culture medium is formulated by adding 6-BA and IBA on the modified MS culture medium; the concentration of 6-BA in the subculture multiplication medium is 0.5-1mg/L, and the concentration of IBA is 0.05-0.5 mg/L.

6. The method for rapid propagation of stem tip culture of Pueraria thomsonii according to claim 5, wherein the concentration of 6-BA in the medium for subculture propagation is 1mg/L and the concentration of IBA is 0.5 mg/L.

7. The method for rapid propagation of stem tip of Pueraria thomsonii according to claim 5, wherein the subculture conditions include a temperature of 28 ± 2 ℃, a humidity of 60-80%, an illumination time of 14 h/day, and an illumination intensity of 800-; the culture time is 20-25 days.

8. The method for rapid propagation of stem tip of Pueraria thomsonii according to claim 1 or 7, wherein the formulation of the sterile rooting medium is to add IBA and NAA on the basis of the modified MS medium, the concentration of IBA in the rooting medium is 0.1-1mg/L, and the concentration of NAA in the rooting medium is 1-2.5 mg/L.

9. The method for rapid propagation of stem tip culture of Pueraria thomsonii according to claim 8, wherein the concentration of IBA in the medium for sterile rooting is 0.5mg/L and the concentration of NAA is 1 mg/L.

10. The method for rapid propagation of stem tip of Pueraria thomsonii according to claim 8, wherein the rooting culture conditions are temperature of 26 ± 3 ℃, humidity of 50-65%, illumination time of 14 h/day, illumination intensity of 800-; the culture time is 15-20 days.

Technical Field

The invention belongs to the technical field of plant rapid propagation, and particularly relates to a rapid propagation method for culturing a stem tip of pueraria thomsonii.

Background

Pueraria thomsoni Benth is a perennial vine plant of Pueraria of Leguminosae, is a traditional Chinese herbal medicine for relieving exterior syndrome with pungent and cool natures, and is named as 'southern ginseng'. The pachyrhizua angulatus is widely distributed, has a long planting history in most areas of China, and part of pachyrhizua angulatus also obtains national geographical signs, thereby being an important plant with homology of medicine and food. Radix Puerariae has effects in expelling pathogenic factors from muscles and skin, clearing and activating the channels and collaterals, and relieving alcoholism, and is prepared from radix Puerariae as main material, and can be made into SANGGEJIANGZHI pill, GEGETANG tablet, GEGENGTANG granule, GEGEQINLIAN pill, and GEQINLIAN tablet. The pueraria thomsonii has rich germplasm resources, contains components such as starch, flavone, pueraria thomsonii and the like, mostly has the health-care function, can be processed into functional food, and has extremely wide market prospect.

The propagation of the pueraria thomsonii mainly depends on the branching of adult stems, the old stems are rich in medicinal components such as puerarin and the like, are excellent raw materials for preparing the pueraria tea and have lower propagation coefficient as seedlings; meanwhile, the cost is higher; in addition, the seed stems are different in size and growth, so that the later-stage standard production is not facilitated, pathogenic bacteria are gathered in vivo to influence the growth in long-term asexual propagation, the economic value of the radix puerariae is reduced, and healthy seedlings which grow vigorously and are consistent in growth are lacked in the market. The stem tip detoxification technology is a common technology for cultivating healthy seedlings in production, such as sugarcane, citrus, taro and the like, and generates good economic benefit. The healthy seedlings of the radix puerariae cultivated by combining stem tip detoxification with tissue culture industrialized seedling technology have good market prospect.

Disclosure of Invention

The invention aims to provide a rapid propagation method for culturing the stem tip of the pueraria thomsonii, which solves the defects that the conventional propagation of the pueraria thomsonii adopts adult stems with high medicinal value to propagate, the cost is high, seedlings are irregular, seed stems are provided with germs, and the propagation coefficient is low.

The purpose of the invention is realized by the following technical scheme:

a method for culturing and rapidly propagating stem tips of pueraria thomsonii comprises the following steps:

(1) selecting and disinfecting explants: selecting tender branches of pueraria thomsonii as explants to be disinfected to obtain disinfected explants;

(2) preparation of a culture medium: preparing an improved MS culture medium, and then respectively preparing an adventitious bud induction culture medium, a subculture multiplication culture medium and a sterile rooting culture medium on the basis of the improved MS culture medium; the formula of the improved MS culture medium is that polyvinylpyrrolidone (PVP) is added into the MS culture medium, and the concentration is 10-20 mg/L;

(3) separating stem tips and carrying out adventitious bud induction culture: carrying out aseptic separation on the buds of the sterilized explants under a dissecting mirror, inoculating the buds into an adventitious bud induction culture medium for induction culture to obtain the adventitious buds of the pueraria thomsonii;

(4) subculture proliferation: inoculating adventitious buds of radix Puerariae into a subculture multiplication medium for subculture multiplication to obtain aseptic seedlings of radix Puerariae;

(5) sterile rooting: cutting and propagating the sterile pueraria thomsonii seedlings, inoculating the sterile pueraria thomsonii seedlings into a sterile rooting culture medium for rooting culture to obtain pueraria thomsonii seedlings, and then hardening and transplanting the seedlings;

(6) identification of detoxified seedlings: and (3) taking plant leaves for RNA identification, and selecting a strain with a corresponding sequence of non-toxic nucleic acid as a healthy seedling for propagation.

In the invention, the disinfection treatment is to soak in alcohol and then put mercuric chloride solution for disinfection.

Further, the alcohol soaking is to soak for 10 to 30 seconds by adopting 70 percent alcohol, and then to wash by using sterile water; the mercuric chloride solution is sterilized by 0.1-0.3% mercuric chloride solution for 8-12min, and then washed by sterile water.

The invention can be improved in the following way, the formula of the adventitious bud induction culture medium is that 6-BA and IBA are added on the improved MS culture medium; the concentration of 6-BA in the adventitious bud induction culture medium is 0.05-0.2mg/L, and the concentration of IBA is 0.05-0.5 mg/L.

Preferably, the concentration of 6-BA in the medium for adventitious bud induction is 1mg/L and the concentration of IBA is 0.2 mg/L.

In the present invention, the bud of the sterilized explant is an axillary bud of a stem segment of Pueraria thomsonii, and the size is 0.5 x 0.5 mm.

In the invention, the induction culture conditions are that the temperature is 28 +/-2 ℃, the humidity is 60-80%, the illumination time is 14 hours/day, and the illumination intensity is 800-12001 x; the culture time is 18-22 days.

The invention can be further improved in the following way, the formulation of the subculture multiplication medium is that 6-BA and IBA are added on the improved MS medium; the concentration of 6-BA in the subculture multiplication medium is 0.5-1mg/L, and the concentration of IBA is 0.05-0.5 mg/L.

Preferably, the concentration of 6-BA in the medium for subculture proliferation is 1mg/L and the concentration of IBA is 0.5 mg/L.

In the invention, the subculture proliferation conditions comprise a temperature of 28 +/-2 ℃, a humidity of 60-80%, an illumination time of 14 hours/day and an illumination intensity of 800-; the culture time is 20-25 days.

The invention can be further improved, the formula of the sterile rooting medium is that IBA and NAA are added on the basis of the improved MS medium, the concentration of IBA in the rooting medium is 0.1-1mg/L, and the concentration of NAA in the rooting medium is 1-2.5 mg/L.

Preferably, the concentration of IBA in the medium for sterile rooting is 0.5mg/L and the concentration of NAA is 1 mg/L.

In the invention, the rooting culture conditions comprise that the temperature is 26 +/-3 ℃, the humidity is 50-65%, the illumination time is 14 hours/day, and the illumination intensity is 800-; the culture time is 15-20 days.

Compared with the prior art, the invention has the following beneficial effects:

(1) the rapid propagation method for culturing the stem tip of the pueraria thomsonii can rapidly culture the seedlings of the pueraria thomsonii plants without gathering pathogenic bacteria, the stem tip culture has small variation without a callus approach, and the excellent characters of a female parent can be better maintained; can effectively improve the propagation coefficient, has short culture period and greatly improves the planting benefit, and can effectively solve the problem of impure pueraria thomsonii seedlings.

(2) According to the breeding and growth characteristics of the pueraria thomsonii seedlings, a stem tip separation method is established, and a culture medium for adventitious bud induction, a culture medium for subculture multiplication and a culture medium for sterile rooting which are suitable for tissue culture of the pueraria thomsonii are prepared on the basis of an MS culture medium.

(3) The rapid propagation method for culturing the stem tip of the radix puerariae provided by the invention has the advantages that the produced seedlings grow neatly and consistently, later-stage management is facilitated, the production cost is reduced, the consistency of the seedlings is ensured, the income of planting and sellers is increased, and the development of the radix puerariae industry is promoted.

Drawings

FIG. 1 shows the separation of the stem tip of Pueraria thomsonii of the present invention;

FIG. 2 shows the induction culture of the adventitious bud of Pueraria thomsonii according to the present invention;

FIG. 3 shows the case of the proliferation culture of adventitious buds of Pueraria thomsonii according to the present invention;

FIG. 4 shows the rooting culture of Pueraria thomsonii of the present invention;

FIG. 5 shows the hardening-off of tissue culture seedlings of Pueraria thomsonii of the present invention;

FIG. 6 shows the case of healthy tissue culture of Pueraria thomsonii of the present invention;

FIG. 7 shows the transplanting of the seedlings of Pueraria thomsonii of the present invention.

Detailed Description

The present invention is further described below in conjunction with specific examples to better understand and implement the technical solutions of the present invention for those skilled in the art.

Example 1

A method for culturing and rapidly propagating Pueraria volcanic stem tips comprises the following steps:

(1) selecting and disinfecting explants:

selecting young and tender branches of healthy radix puerariae as explants,

secondly, washing the branches with a neutral detergent to remove dirt on the surfaces of the branches, then removing redundant leaves, cleaning paper towels to suck up surface moisture,

③ soaking the mixture on a clean bench for 20s by 70 percent alcohol, washing the mixture for 3 times by sterile water, 4min each time,

fourthly, immediately transferring the explant into 0.2% mercuric chloride solution for disinfection for 10min, and finally washing the explant with sterile water for 4 times, 5min each time, so as to obtain a disinfected explant;

(2) preparation of improved MS culture medium:

the formula of the improved MS culture medium is as follows: the preparation of the improved MS culture medium comprises the following components: polyvinylpyrrolidone (PVP) was added to the MS medium at a concentration of 15 mg/L.

(3) Stem tip stripping and adventitious bud induction:

cutting the sterilized explant obtained in the step (1) into single-bud stem sections with the length of 0.4-0.5cm, taking stem tip growing points with the size of about 0.5mm under a stereomicroscope, inoculating the stem tip growing points to a culture medium for adventitious bud induction, and performing induction culture for 20 days to obtain the pueraria thomsonii adventitious buds; the induction culture conditions were as follows: the temperature is 28 +/-2 ℃, the humidity is 60-80%, the illumination time is 14 hours/day, and the illumination intensity is 10001 x; culturing for about 1 month to form bud-shaped bulge, and allowing part of adventitious bud to appear; the formula of the culture medium for adventitious bud induction is as follows: adding 6-BA and IBA on the modified MS medium; wherein the concentration of 6-BA is 1mg/L, and the concentration of IBA is 0.2 mg/L;

(4) subculture proliferation:

inoculating the adventitious buds of the pueraria thomsonii obtained in the step (3) into a culture medium for subculture proliferation, and carrying out subculture for 23 days to obtain aseptic seedlings of the pueraria thomsonii; the subculture conditions were as follows: the temperature is 28 +/-2 ℃, the humidity is 60-80%, the illumination time is 14 hours/day, and the illumination intensity is 10001 x; the formula of the culture medium for subculture proliferation is as follows: adding 6-BA and IBA on the basis of the improved MS culture medium; wherein the concentration of 6-BA is 1mg/L, and the concentration of IBA is 0.5 mg/L;

(5) sterile rooting:

cutting and propagating the sterile seedlings of the radix puerariae obtained in the step (4), keeping each plant with an adventitious bud, and inoculating the seedlings of the radix puerariae into a culture medium for sterile rooting to perform rooting culture for 18 days to obtain the seedlings of the radix puerariae; the rooting culture conditions were as follows: the culture temperature is 26 + -3 deg.C, humidity is 50-65%, illumination time is 14 hr/day, and illumination intensity is 10001 x; the formula of the culture medium for sterile rooting is as follows: adding IBA and NAA on the basis of the improved MS culture medium, wherein the concentration of IBA is 0.5mg/L, and the concentration of NAA is 1 mg/L;

after the Pueraria thomsonii seedlings grow to 0.5cm in root length and 3cm in seedling height in the step (5), the bottle caps of culture bottles are opened, indoor hardening culture is carried out for 5 days, then root culture media are cleaned, 0.1% carbendazim solution is soaked for 10 minutes, tissue culture planting is carried out in nutrient cups filled with peat soil, the humidity of leaf surfaces is kept, and the survival rate is more than 95%.

(6) Identification of detoxified seedlings:

taking plant leaves to carry out extraction and quality inspection of total RNA, library construction, high-throughput sequencing, quality control and the like, and selecting sRNA with the length of 18-36nt from clearreads to carry out sequence splicing to obtain contigs (contigs). And comparing the contigs obtained by splicing with a nucleic acid database (NCBI) to obtain a highly homologous sequence, comparing with a virus nucleic acid database and a virus protein database, and identifying whether the sequence contains small RNA (ribonucleic acid) so as to judge whether the sequence is a healthy seedling, and selecting a strain without a corresponding sequence as the healthy seedling for propagation.

Example 2

A method for culturing and rapidly propagating stem tips of Pueraria thomsonii comprises the following steps:

(1) selecting and disinfecting explants:

selecting young and tender branches of healthy radix puerariae as explants,

secondly, washing the branches with a neutral detergent to remove dirt on the surfaces of the branches, then removing redundant leaves, cleaning paper towels to suck up surface moisture,

thirdly, on a clean bench, soaking the workpiece for 25s with 70 percent alcohol, then washing the workpiece for 3 times with sterile water, 3min each time,

fourthly, immediately transferring the explant into 0.25% mercuric chloride solution for disinfection for 10min, and finally washing the explant with sterile water for 4 times, 5min each time, so as to obtain a disinfected explant;

(2) preparation of improved MS culture medium:

the formula of the improved MS culture medium is as follows: the preparation of the improved MS culture medium comprises the following components: polyvinylpyrrolidone (PVP) was added to the MS medium at a concentration of 15 mg/L.

(3) Stem tip stripping and adventitious bud induction:

cutting the sterilized explant obtained in the step (1) into single-bud stem sections with the length of 0.4-0.5cm, taking stem tip growing points with the size of about 0.5mm under a stereomicroscope, inoculating the stem tip growing points to a culture medium for adventitious bud induction, and performing induction culture for 22 days to obtain the pueraria thomsonii adventitious bud; the induction culture conditions were as follows: the temperature is 28 +/-2 ℃, the humidity is 60-80%, the illumination time is 14 hours/day, and the illumination intensity is 9001 x; culturing for about 1 month to form bud-shaped bulge, and allowing part of adventitious bud to appear; the formula of the culture medium for adventitious bud induction is as follows: adding 6-BA and IBA on the modified MS medium; wherein the concentration of 6-BA is 1mg/L, and the concentration of IBA is 0.2 mg/L;

(4) subculture proliferation:

inoculating the adventitious buds of the pueraria thomsonii obtained in the step (3) into a culture medium for subculture proliferation, and carrying out subculture for 24 days to obtain aseptic seedlings of the pueraria thomsonii; the subculture conditions were as follows: the temperature is 28 +/-2 ℃, the humidity is 60-80%, the illumination time is 14 hours/day, and the illumination intensity is 9001 x; the formula of the culture medium for subculture proliferation is as follows: adding 6-BA and IBA on the basis of the improved MS culture medium; wherein the concentration of 6-BA is 1mg/L, and the concentration of IBA is 0.5 mg/L;

(5) sterile rooting:

cutting and propagating the sterile seedlings of the radix puerariae obtained in the step (4), keeping each plant with an adventitious bud, and inoculating the seedlings of the radix puerariae into a culture medium for sterile rooting to perform rooting culture for 19 days to obtain the seedlings of the radix puerariae; the rooting culture conditions were as follows: the culture temperature is 26 +/-3 ℃, the humidity is 50-65%, the illumination time is 14 hours/day, and the illumination intensity is 9001 x; the formula of the culture medium for sterile rooting is as follows: adding IBA and NAA on the basis of the improved MS culture medium, wherein the concentration of IBA is 0.5mg/L, and the concentration of NAA is 1 mg/L;

after the Pueraria thomsonii seedlings grow to 0.5cm in root length and 3cm in seedling height in the step (5), the bottle caps of culture bottles are opened, indoor hardening culture is carried out for 5 days, then root culture media are cleaned, 0.1% carbendazim solution is soaked for 10 minutes, tissue culture planting is carried out in nutrient cups filled with peat soil, the humidity of leaf surfaces is kept, and the survival rate is more than 95%.

(6) Identification of detoxified seedlings:

taking plant leaves to carry out extraction and quality inspection of total RNA, library construction, high-throughput sequencing, quality control and the like, and selecting sRNA with the length of 18-36nt from clean reads to carry out sequence splicing to obtain contigs (contigs). And comparing the contigs obtained by splicing with a nucleic acid database (NCBI) to obtain a highly homologous sequence, comparing with a virus nucleic acid database and a virus protein database, and identifying whether the homologous sequence contains small RNA (ribonucleic acid) so as to judge whether the seedlings are healthy seedlings, and selecting a strain without the corresponding sequence as the healthy seedlings for propagation.

The above embodiments illustrate various embodiments of the present invention in detail, but the embodiments of the present invention are not limited thereto, and those skilled in the art can achieve the objectives of the present invention based on the disclosure of the present invention, and any modifications and variations based on the concept of the present invention fall within the scope of the present invention, which is defined by the claims.