CN112273231A - Method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration - Google Patents
Method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration Download PDFInfo
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Abstract
The invention discloses a method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration, which takes stem segments with axillary buds of aseptically germinated seeds as explants to perform axillary bud induction, cluster bud proliferation, rooting culture and hardening seedling transplantation to obtain the regeneration of asparagus cochinchinensis plants. By adopting the method, the axillary bud induction rate can reach 85.05 percent, the multiple of cluster bud multiplication can reach 3.65 percent, the rooting rate can reach 78.27 percent, and the transplanting survival rate can reach 87.10 percent, so that the production cost can be effectively reduced, the tissue culture rapid propagation period can be shortened, and the method can be applied to large-scale seedling breeding and resource protection of radix asparagi.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration.
Technical Field
Asparagus cochinchinensis (Lour.) Merr, which is perennial climbing herbaceous plant of Asparagus of Liliaceae, is named as Asparagus cochinchinensis, Angelica gigas root, etc.; asparagus cochinchinensis is recorded in Shen nong Ben Cao Jing of east Han Dynasty, and its dry root tuber is used as a medicine, which has the effects of nourishing yin, moistening dryness, clearing lung-heat and lowering fire, and is mainly used for treating cough due to dryness-heat, tuberculosis due to yin deficiency, body fluid injury due to fever, internal heat, diabetes, constipation due to intestinal dryness, sore throat, etc. The radix asparagi has a long medicinal history in China, integrates medicinal, edible and ornamental values into a whole, and has wide development and utilization prospects.
In recent years, wild asparagus resource is rapidly reduced due to the fact that people abundantly adopt and dig without restriction, and the wild asparagus resource is listed as a national third-level key point for protecting wild medicinal material species. Therefore, the development of artificial planting of the asparagus cochinchinensis has important significance for protecting wild asparagus cochinchinensis resources and realizing the sustainable utilization of the wild asparagus cochinchinensis resources. At present, the propagation of the seedlings of the radix asparagi is mainly based on seed propagation and plant division propagation, but the problems of long seedling period, low propagation multiple, high production cost and the like exist, and the large-scale production requirement is difficult to meet. The rapid propagation technology of the seedlings of the radix asparagi can be established by means of tissue culture, and particularly the cluster bud multiplication and plant regeneration technology established by the research of the subject group lays a good foundation for realizing the industrialization of the radix asparagi seedlings.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration, which overcomes the problems of difficult explant induction, low cluster bud proliferation efficiency, etc. in the prior art.
In order to realize the aim, the invention provides a method for inducing proliferation of asparagus cochinchinensis multiple buds and plant regeneration, which takes stem segments with axillary buds of sterile germinated seeds as explants to carry out axillary bud induction, multiple bud proliferation culture, rooting culture, seedling hardening and transplanting. The method comprises the following specific steps:
(1) seed treatment and germination into seedlings: selecting mature and plump cochinchnese asparagus seeds, soaking for 4-6min by using 98% concentrated sulfuric acid, and soaking for 4-6h by using warm water at 35 ℃; placing on a clean bench, soaking in 75% alcohol for 0.5-1min, soaking in 0.1% mercuric chloride for 10-12min, and washing with sterile water for 3-5 times; inoculating the seeds on a seedling culture medium, inducing the seeds to generate sterile germination seedlings under the culture conditions of 25 +/-2 ℃, 2500 + 3000lx illumination intensity and 12h/d illumination time, and germinating the seeds into sterile germination seedlingsThe seedling culture medium is as follows: MS + GA30.5mg/L +6-BA 1.0mg/L + KT 0.1mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0;
(2) axillary bud germination: selecting a strong bud seedling, cutting a stem segment with axillary buds, and inoculating the stem segment with the axillary buds into an axillary bud induction culture medium, wherein the axillary bud induction culture medium comprises: MS + NAA0.1 mg/L +6-BA 1.0mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0. The culture conditions were the same as for seed germination (the same applies below);
(3) and (3) cluster bud multiplication: cutting single buds or cluster buds of axillary buds, and inoculating to a proliferation culture medium, wherein the proliferation culture medium is as follows: MS +6-BA0.5 mg/L + KT 1.5mg/L + IAA 1.5mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0. If the browning phenomenon occurs in the proliferation culture process, adding 0.5g/L polyvinylpyrrolidone (PVP) into the proliferation culture medium for subculture;
(4) rooting culture: when the cluster buds grow to be more than 3cm, cutting off bud blocks of 2-3 buds, inoculating the bud blocks on a rooting culture medium for rooting culture, wherein the rooting culture medium comprises: 1/2MS + PUT 1.0mg/L + IAA 1.5mg/L + IBA 1.5mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0; when browning occurs in the rooting process, 0.5g/L of Activated Carbon (AC) is added into a rooting culture medium;
(5) hardening and transplanting seedlings: opening a bottle cap in a tissue culture room for hardening off the rooted seedlings with more than two roots and extending stem branches for 8-10 days, taking out the rooted seedlings, cleaning a root culture medium, and transplanting the rooted seedlings into a mixed matrix of coconut coir, perlite and garden soil in a volume ratio of 2:2: 1; during transplanting, the roots should be shallow to the position where the roots are completely covered by the matrix, so that the roots are fully contacted with the matrix, and the tissue culture seedlings are kept in a straight state; compacting, pouring water, and placing in an environment with light transmittance of 20% -30%; after one week of seedling recovery, 1000 times diluted flower-rich compound fertilizer (N: P: K: 1:1:1) is applied every 15 days, and watering is carried out by adopting a dry-wet method.
Preferably, the germinated seedlings of the seeds in the step (2) grow robustly and are more than 3cm high.
Preferably, axillary buds germinate after 15-20 days in the step (3), and single buds and cluster buds which have the axillary bud induction rate higher than 60% and are strong in growth are selected, cut and inoculated on the multiplication culture medium.
The invention has the beneficial effects that:
(1) overcomes the defects of long propagation and plant division propagation period, low propagation coefficient and the like of the seeds of the asparagus cochinchinensis.
(2) The invention obtains the seed germination seedling by inducing the seed germination seedling, takes the stem section with the bud as the explant, the material source of the explant is not limited by the age and time of the parent, the regeneration frequency of the plant is high, the axillary bud inductivity reaches 85.05 +/-4.35%, the multiple of the cluster bud proliferation reaches 3.65 +/-0.10, the rooting rate reaches 78.27 +/-2.56%, and the transplanting survival rate reaches 87.10 +/-1.08%; can be applied to the large-scale seedling breeding and resource protection of the radix asparagi.
(3) According to the tissue culture scheme, the callus induction culture stage is not needed, and the culture period of the asparagus cochinchinensis tissue is shortened by 20-50 d; through axillary bud induction and proliferation culture, the method can greatly improve the cluster bud induction efficiency and reduce the production cost.
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The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this application.
FIG. 1: a is cochinchnese asparagus root seeds; b is the growth condition of the seeds when the seeds are cultured on a germination and seedling culture medium for 36 d; c is selected strong bud seedlings; d is a stem segment with axillary buds;
FIG. 2: a is the growth condition of the bud when a stem section of 0.5-1.0cm is cultured on an induction culture medium for 21 days; B-C is the proliferation effect of the single bud and the cluster bud on a proliferation culture medium for 21 d;
FIG. 3: a is the growth condition on rooting culture medium for 27 days; b, rooting seedlings; c is the growth condition of the tissue culture seedlings when the seedlings are transplanted for 60 d.
Detailed Description
The following embodiments are provided to explain the embodiments of the present invention in detail, and how to apply technical means to solve the technical problems and achieve the technical effects of the implementation process, according to the embodiments, the implementation process can be fully understood and implemented.
Example 1
(1) Seed treatment and germination into seedlings
The plant material, Asparagus cochinchinensis (Lour.) Merr, seed in this example was from Sichuan county, Guizhou provinceAn asparagus cochinchinensis planting base. Selecting mature and plump cochinchnese asparagus seeds, soaking the seeds for 4min by using 98% concentrated sulfuric acid, soaking the seeds for 4h in warm water at 35 ℃, then placing the seeds on an ultra-clean workbench, sequentially soaking the seeds for 0.5 to 1min by using 75% of alcohol, soaking the seeds for 10 to 12min by using 0.1% of mercury bichloride, and washing the seeds for 3 to 5 times by using sterile water for later use; inoculating the seeds on a seedling culture medium, and inducing the seeds to germinate into seedlings under the culture conditions of the temperature of 25 +/-2 ℃, the illumination intensity of 2500-; the germination and seedling culture medium is MS + GA30.5mg/L +6-BA 1.0mg/L + KT 0.1mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0; when the seedlings are cultured for 15 days, the seeds begin to expose white, and the seedlings grow to be more than 3cm after about 36 days of culture, so that the seedlings grow well.
(2) Germination of axillary buds
Selecting sterile sprouting seedlings which are higher than 3cm and grow well, cutting the sprouting seedlings into 0.5-1.0cm stem sections with axillary buds by using a dissecting knife in a sterile environment, inoculating the stem sections with the axillary buds on an axillary bud induction culture medium, and culturing under the culture conditions of the embodiment 1, wherein the axillary bud induction culture medium is as follows: MS + NAA0.1 mg/L +6-BA 1.0mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0, its inductivity is 85.05 + -4.35%, and the bud is strong and strong.
(3) Multiplication of cluster buds
Cutting the germinated single bud or cluster bud, inoculating on a proliferation culture medium, and culturing under 2500-: MS +6-BA0.5 mg/L + KT 1.5mg/L + IAA 1.5mg/L + PVP 0.5g/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0, after culturing for 21d, the multiplication multiple reaches 3.65 +/-0.10.
(4) Rooting culture
When the cluster buds grow to be more than 3cm, cutting off bud blocks of 2-3 buds, and inoculating the bud blocks on a rooting culture medium for rooting culture. The rooting culture medium comprises: 1/2MS + PUT 1.0mg/L + IAA 1.5mg/L + IBA 1.5mg/L + AC 0.5g/L + sucrose 30g/L + agar 7g/L, pH 5.8-6.0. After the plant grows on the rooting culture medium for 27 days, the rooting rate reaches 78.27 +/-2.56 percent, the number of roots is 5.22 +/-0.42, and the growth state is good.
(5) Hardening and transplanting seedlings
Placing more than two rooted seedlings with spread stems and leaves in a tissue culture room, unscrewing a bottle cap for 3 days, and opening the bottle cap for 5-7 days. After hardening, taking out the rooted seedlings, cleaning a root culture medium, transplanting the rooted seedlings into a mixed matrix of coconut chaff, perlite and garden soil in a volume ratio of 2:2:1, planting the obtained tissue culture seedlings into a nutrition pot with the diameter of 12cm and the height of 10cm, wherein the roots are shallow until the matrix completely covers the root generating part, keeping the tissue culture seedlings in a straight state, fully contacting the root system with the matrix during transplanting, compacting, watering thoroughly, and placing in an environment with the light transmittance of 20-30%. After one week of seedling recovery, 1000 times diluted flower-rich compound fertilizer (N: P: K: 1:1:1) is applied every 15 days, and watering is carried out by adopting a dry-wet method. When the seedlings grow for 60 days, the survival rate reaches 86.11 +/-1.62%, the root-crown ratio is 1.04 +/-0.03, the seedling strengthening index is 0.87 +/-0.06, and the seedlings grow robustly.
Example 2
(1) Seed treatment and germination into seedlings
The plant material, cochinchinensis (Lour.) Merr, seed in this example was from the cochinchinensis planting base in the city, Guizhou province. Selecting mature and plump cochinchnese asparagus seeds, soaking the seeds for 4min by using 98% concentrated sulfuric acid, soaking the seeds for 4h in warm water at 35 ℃, then placing the seeds on an ultra-clean workbench, sequentially soaking the seeds for 0.5 to 1min by using 75% of alcohol, soaking the seeds for 10 to 12min by using 0.1% of mercury bichloride, and washing the seeds for 3 to 5 times by using sterile water for later use; inoculating the seeds on a seedling culture medium, and inducing the seeds to germinate into seedlings under the culture conditions of the temperature of 25 +/-2 ℃, the illumination intensity of 2500-; the germination and seedling culture medium is MS + GA31.0mg/L +6-BA 1.5mg/L + KT 0.1mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0; when the seeds are cultured for 15 days, the seeds begin to expose white, and the sprouts grow to be more than 3cm after about 36 days of culture.
(2) Germination of axillary buds
Selecting sterile sprouting seedlings which are higher than 3cm and grow well, cutting the sprouting seedlings into 0.5-1.0cm stem sections with axillary buds by using a dissecting knife in a sterile environment, inoculating the stem sections with the axillary buds on an axillary bud induction culture medium, and culturing under the culture conditions of the embodiment 1, wherein the axillary bud induction culture medium is as follows: MS + NAA0.1 mg/L +6-BA0.5 mg/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0. The bud inductivity is 77.15 +/-4.55%, and the buds grow well.
(3) Multiplication of cluster buds
Cutting the germinated single bud or cluster bud, inoculating on a proliferation culture medium, and culturing under 2500-: MS +6-BA0.5 mg/L + KT 1.0mg/L + IAA 1.0mg/L + PVP 0.5g/L + sucrose 30g/L + agar 7g/L, pH5.8-6.0, after culturing for 21d, the multiplication multiple is 2.74 +/-0.24.
(4) Rooting culture
When the cluster buds grow to be more than 3cm, cutting off bud blocks of 2-3 buds, and inoculating the bud blocks on a rooting culture medium for rooting culture. The rooting culture medium comprises: 1/2MS + PUT 1.0mg/L + IBA 0.5mg/L + AC 0.5g/L + sucrose 30g/L + agar 7g/L, pH 5.8-6.0. After the plant grows on the rooting culture medium for 27 days, the rooting rate reaches 71.36 +/-1.92 percent, the number of roots is 3.60 +/-1.20, and the growth state is good.
(5) Hardening and transplanting seedlings
Placing more than two rooted seedlings with spread stems and leaves in a tissue culture room, unscrewing a bottle cap for 3 days, and opening the bottle cap for 5-7 days. After hardening, taking out the rooted seedlings, cleaning a root culture medium, transplanting the rooted seedlings into a mixed matrix of coconut chaff, perlite and vermiculite in a volume ratio of 1:1:1, planting the obtained tissue culture seedlings into a nutrition pot with the diameter of 12cm and the height of 10cm, wherein the roots are shallow until the matrix completely covers the root generating part, keeping the tissue culture seedlings in a straight state, fully contacting the root system with the matrix during transplanting, compacting, watering thoroughly, and placing in an environment with the light transmittance of 20-30%. After one week of seedling recovery, 1000 times diluted flower-rich compound fertilizer (N: P: K: 1:1:1) is applied every 15 days, and watering is carried out by adopting a dry-wet method. When the seedlings grow for 60 days, the survival rate reaches 87.10 +/-1.08%, the root-crown ratio is 1.06 +/-0.07, the strong seedling index is 1.10 +/-0.02, the root system is developed, and the seedlings grow robustly.
The foregoing embodiments are merely illustrative of the principles and methods of the present invention and are not to be construed as limiting thereof. It will be appreciated by those skilled in the art that variations may be made in the methods and principles of the invention described herein without departing from the scope of the inventive concept as defined by the above teachings or the skill or knowledge of the relevant art, and yet still be within the scope of the appended claims.
Claims (8)
1. A method for inducing proliferation of asparagus cochinchinensis cluster buds and plant regeneration is characterized by comprising the following steps of taking stem segments with axillary buds of sterile germinated seeds as explants to perform axillary bud germination culture, cluster bud proliferation culture, rooting culture and hardening seedling transplantation, and the method comprises the following specific steps:
(1) seed treatment and germination into seedlings: selecting mature and plump cochinchnese asparagus seeds, soaking the seeds for 4 to 6min by concentrated sulfuric acid and soaking the seeds for 4 to 6h by warm water; soaking in 75% alcohol for 0.5-1min, soaking in mercury for 10-12min, and washing with sterile water for 3-5 times; inoculating the seeds on a seedling culture medium, controlling the pH to be 5.8-6.0, and inducing the seeds to germinate to generate aseptic seedlings under the culture conditions that the temperature is 25 +/-2 ℃, the illumination intensity is 2500-;
(2) axillary bud germination: selecting a strong aseptic bud seedling, cutting a stem segment with axillary buds, inoculating the stem segment on an axillary bud induction culture medium, and enabling the axillary buds to germinate to generate single buds or multiple buds at the pH of 5.8-6.0; the culture condition is the same as that of the seed germination;
(3) and (3) cluster bud multiplication: cutting single bud or multiple bud of axillary bud, inoculating to proliferation culture medium, and proliferating at pH of 5.8-6.0;
(4) rooting culture: when the cluster buds grow to be more than 3cm, cutting off bud blocks with 2-3 buds, inoculating the bud blocks on a rooting culture medium at the pH value of 5.8-6.0, and enabling the bud blocks to root to form a regeneration plant;
(5) hardening and transplanting seedlings: opening a bottle cap of a rooted seedling with more than two roots and spread stems and branches in a tissue culture room, hardening the seedling for 8-10 days, taking out the rooted seedling, cleaning a root culture medium, and transplanting the rooted seedling into a mixed matrix with the volume ratio of coconut chaff to perlite to garden soil being 2: 1.
2. The method of claim 1, wherein the seedling medium is MS + GA30.5mg/L +6-BA 1.0mg/L + KT 0.1mg/L + sucrose 30g/L + agar 7 g/L.
3. The method for inducing the proliferation of the clustered shoots of asparagus cochinchinensis and the regeneration of plants as claimed in claim 1, wherein the aseptic sprouts obtained in step (2) are robust and grow at a height of more than 3 cm.
4. The method for inducing the proliferation of clustered shoots and regeneration of plants as claimed in claim 1, wherein said axillary bud induction medium is MS + NAA0.1 mg/L +6-BA 1.0mg/L + sucrose 30g/L + agar 7 g/L.
5. The method for inducing the proliferation and plant regeneration of asparagus cochinchinensis clump buds as claimed in claim 1, wherein the proliferation medium MS +6-BA0.5 mg/L + KT 1.5mg/L + IAA 1.5mg/L + sucrose 30g/L + agar 7 g/L.
6. The method for inducing the proliferation and plant regeneration of asparagus cochinchinensis clump buds according to claim 1, wherein in the step (3), when axillary buds germinate after 15-20 days, single buds and clump buds with axillary bud induction rate higher than 60% and strong growth are selected, cut off and inoculated on a proliferation culture medium, and if the browning phenomenon occurs in the proliferation culture process, 0.5g/L of polyvinylpyrrolidone (PVP) is added into the proliferation culture medium for subculture.
7. The method for inducing the proliferation of clustered shoots of asparagus as claimed in claim 1, wherein the rooting medium 1/2MS + Putrescine (PUT)1.0mg/L + IAA 1.5mg/L + IBA 1.5mg/L + sucrose 30g/L + agar 7 g/L.
8. The method for inducing the proliferation of the clustered shoots of asparagus cochinchinensis and the regeneration of the plants as claimed in claim 1, wherein in the step (4), when the browning phenomenon occurs during the rooting process, 0.5g/L of activated carbon is added to the rooting medium.
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| CN111616048A (en) * | 2020-04-27 | 2020-09-04 | 云南省农业科学院药用植物研究所 | Novel tissue culture and rapid propagation method for asparagus cochinchinensis |
| CN113383654A (en) * | 2021-07-01 | 2021-09-14 | 云南省热带作物科学研究所 | Passion flower micro bud seedling grafting method |
| CN116458427A (en) * | 2023-04-14 | 2023-07-21 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
| CN116746486A (en) * | 2022-12-29 | 2023-09-15 | 福建省中科生物股份有限公司 | Induction method for improving induction rate of asparagus meat tuberous root |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111616048A (en) * | 2020-04-27 | 2020-09-04 | 云南省农业科学院药用植物研究所 | Novel tissue culture and rapid propagation method for asparagus cochinchinensis |
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| CN113383654A (en) * | 2021-07-01 | 2021-09-14 | 云南省热带作物科学研究所 | Passion flower micro bud seedling grafting method |
| CN116746486A (en) * | 2022-12-29 | 2023-09-15 | 福建省中科生物股份有限公司 | Induction method for improving induction rate of asparagus meat tuberous root |
| CN116458427A (en) * | 2023-04-14 | 2023-07-21 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
| CN116458427B (en) * | 2023-04-14 | 2024-01-26 | 广西壮族自治区农业科学院 | Method for producing root-knot potatoes in asparagus bottle |
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