CN112385542A - Tissue culture and rapid propagation seedling method for leaves of polygala tenuifolia - Google Patents
Tissue culture and rapid propagation seedling method for leaves of polygala tenuifolia Download PDFInfo
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- CN112385542A CN112385542A CN202011326969.XA CN202011326969A CN112385542A CN 112385542 A CN112385542 A CN 112385542A CN 202011326969 A CN202011326969 A CN 202011326969A CN 112385542 A CN112385542 A CN 112385542A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention relates to a tissue culture and rapid propagation seedling method for polygala tenuifolia leaves, which comprises the following steps: selecting an explant, performing induction culture on the explant, performing proliferation culture on the explant, performing strong seedling culture on the explant, and performing rooting culture on the explant. The invention adopts the leaves which are easy to collect and have a large number as explants, and can quickly culture the rooted seedlings which have stable characters, high propagation efficiency, white root systems and strong stems.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine cultivation, in particular to a tissue culture and rapid propagation seedling method for polygala tenuifolia leaves in southwest.
Background
The polygala plants in polygalaceae in southwest are about 600 in number, are widely distributed in the world, and are 42 in China and 24 in Yunnan. Polygala tenuifolia and Polygala sibirica have been listed in the national pharmacopoeia in 2005 and 2010 and are well known Chinese herbal medicines from ancient times to present. Listed as the superior product in Shen nong Ben Cao Jing, it is regarded as a life-nourishing drug. The Va nationality is called 'Niang mu Liang' in China, also called 'pig large intestine', and the Va nationality people have been used for hundreds of years and play an important role in the survival, health and reproduction of the Va nationality people, so far, the Va nationality people are still main wild drug resources for ensuring the health of the vast masses in the locality and are called 'herb mothers', and the Va nationality people have the effects of strengthening the body, tonifying the kidney and strengthening yang, resisting fatigue, nourishing the heart and tranquilizing the mind, dispelling diseases and relieving spasm, relaxing muscles and stimulating the blood circulation and the like. Can be used for treating sexual hypofunction such as sexual impotence and premature ejaculation due to kidney deficiency, cough with asthma due to kidney deficiency, fatigue due to wind-cold-heat, senile dementia, scrofula, tuberculosis, rheumatic numbness, female with clear and thin vaginal discharge due to cold womb, and infertility due to cold womb. Through preliminary medical observation, the 'Polygala crotalarioides' has better effects on the reproductive system, the cardiovascular system and the nervous system of a human body. In the region near cang, due to the long-term utilization of natural resources of 'Polygala crotalarioides', wild resources are seriously lost and are difficult to find traces and almost extinct. In order to save the valuable resource in the national medical treasury, the Va nationality people's mother-of-medicine' enables the Va nationality people to be continuously utilized, benefits the people, strengthens the research of introduction, domestication and cultivation without losing time, changes the wild into the family life, and is not only needed but also more urgent.
The main propagation method of polygala tenuifolia at present tends to be traditionally propagated by seeds. A small amount of stem segments and seeds are used for tissue culture and propagation. The seed planting and tissue culture have the problems that the seed propagation is sexual propagation, the character is unstable, variation is easy to generate, the flowering phases are inconsistent, the quantity of the seeds which are difficult to harvest is small, large-scale planting and seed tissue culture are difficult to realize, the seeds are extremely small and difficult to operate, pollution is caused by carelessness, previous work is abandoned, the germination rate is low, the stem section of the polygala tenuifolia far away from the south is short, the lignification degree is higher, explants are difficult to collect, the tissue culture difficulty is high, and the pollution rate is high.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the difficulties and provide a method for tissue culture and rapid propagation of polygala tenuifolia leaves into seedlings, which can realize efficient, rapid and stable propagation by using a small amount of leaves and has great significance for protecting and promoting large-scale planting of polygalaceae plants in southwestern.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: the tissue culture and rapid propagation seedling method of polygala tenuifolia leaves is characterized in that: comprises the following steps of (a) carrying out,
(1) and selecting an explant: taking newly-emerged leaves on stem segments of polygala tenuifolia in the south west as explants, selecting 3-4 pm in sunny days, and disinfecting after the selection is finished;
(2) and (3) induction culture of explants: placing the sterilized explant subjected to the sterilization treatment in the step (1) in a sterilized stainless steel disc, cutting a necrotic part at the base part of the explant by using a sterile scalpel, then horizontally placing the leaf surface in an induction culture medium upwards, then placing the explant after the operation in a culture room, setting the temperature to be 22-25 ℃, setting the illumination time to be 14 h/day, setting the illumination intensity to be 3000LX, differentiating cluster buds within 30 days, and differentiating 15-25 cluster buds from each leaf;
(3) and proliferation culture of explants: cutting the cluster buds in the step (2) into 5-6 small blocks of cluster buds, transferring the small blocks into a proliferation culture medium, setting the temperature to be 22-25 ℃, setting the illumination time to be 16 h/day, setting the illumination intensity to be 3000LX, and culturing for 15-20 days to obtain cluster seedlings;
(4) and culturing strong seedlings of explants: dividing the clumped seedlings in the step (3) into small blocks, putting 8-10 clumped seedlings in a strong seedling culture medium, and culturing at the same temperature and light intensity as the propagation culture of explants to obtain robust and healthy seedlings after 20-25 days;
(5) and rooting culture of the explant: dividing the seedlings in the step (4) into single seedlings, inserting the single seedlings into a rooting culture medium, then putting the explants subjected to operation into a culture room, setting the temperature to be 22-25 ℃, performing dark culture for 48 hours, setting the illumination time to be 12 hours/day, setting the illumination intensity to be 2000LX, and obtaining the rooting seedlings of the polygala tenuifolia with white roots and strong stems after 15-20 days.
As a modification, the sterilization method in the step (1) is as follows:
s1: soaking the explant in detergent water for 5 minutes, slightly stirring, flushing with running water for three minutes, and finally placing in a dry sterile bottle for later use;
s2: the explants are put into sterile water for rinsing once, then put into 75% alcohol for soaking for 30 seconds, put into sterile water for rinsing once again, put into 5% sodium hydrosulfite for soaking for 5 minutes, and put into sterile water for rinsing once again.
As an improvement, the running water is tap water.
As an improvement, the induction culture medium in the step (2) is MS + NAA0.5mg/L +6-BA0.5mg/L, potato juice 100g/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
As an improvement, the enrichment medium in the step (3) is MS + KT3mg/L + NAA0.2mg/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
As an improvement, the strong seedling culture medium in the step (4) is MS +6-BA0.5mg/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
As an improvement, the rooting medium in the step (5) is 1/2MS + IBA0.2mg/L, 1g/L of activated carbon, 4.5g/L of agar, 0.1g/L of inositol and 20g/L of white sugar.
As a modification, in the steps (2) to (5), the set temperature is preferably 23 ℃.
The invention has the following advantages: the leaves are simple to collect and large in quantity, compared with the method that the influence of season limiting factors is avoided when seeds are collected and stem sections are collected, the culture effect is superior to that of other two explants, and the characteristics are stable; the multiplication efficiency is high, and the multiplication multiple of each generation is 4-6 times on the premise of ensuring the quality of the nursery stock; the proliferation and rooting period is short, the proliferation time of each generation only needs 15-20 days, and the rooting time is 20-25 days, which is far shorter than the proliferation and rooting culture time of the other two explants.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves is characterized in that: comprises the following steps of (a) carrying out,
(1) and selecting an explant: taking newly-emerged leaves on stem segments of polygala tenuifolia in the south west as explants, selecting 3-4 pm in sunny days, and disinfecting after the selection is finished;
the disinfection steps are as follows:
firstly, putting explants into detergent water to be soaked for 5 minutes and lightly stirred, then washing the explants for three minutes by tap water, and finally putting the explants into a dry sterile bottle for later use;
and secondly, putting the explant into sterile water for rinsing once, then putting the explant into 75% alcohol for soaking for 30 seconds, putting the explant into sterile water for rinsing once again, putting the explant into 5% sodium hypochlorite for soaking for 5 minutes, and putting the explant into sterile water for rinsing once again.
(2) And (3) induction culture of explants: placing the sterilized explant subjected to the sterilization treatment in the step (1) in a sterilized stainless steel disc, cutting a necrotic part at the base part of the explant by using a sterile scalpel, then horizontally placing the leaf surface upwards in an induction culture medium, then placing the explant after the operation in a culture room, setting the temperature to be 23 ℃, setting the illumination time to be 14 h/day, setting the illumination intensity to be 3000LX, differentiating cluster buds within 30 days, differentiating 15-25 cluster buds per leaf, wherein the induction culture medium is MS + NAA0.5mg/L +6-BA0.5mg/L, 100g/L potato juice, 4.5g/L agar, 0.1g/L inositol and 30g/L white granulated sugar.
(3) And proliferation culture of explants: and (3) cutting the cluster buds in the step (2) into 5-6 small blocks of cluster buds, transferring the small blocks into a multiplication culture medium, setting the temperature at 23 ℃, the illumination time at 16 h/day, the illumination intensity at 3000LX, and the culture period at 15-20 days to obtain cluster seedlings, wherein the multiplication culture medium comprises MS + KT3mg/L + NAA0.2mg/L, agar at 4.5g/L, inositol at 0.1g/L and white granulated sugar at 30 g/L.
(4) And culturing strong seedlings of explants: dividing the cluster seedlings in the step (3) into small blocks, placing 8-10 cluster seedlings into a strong seedling culture medium, wherein the temperature and light intensity are the same as those of the propagation culture of explants, and obtaining robust and healthy seedlings after 20-25 days, wherein the strong seedling culture medium is MS +6-BA0.5mg/L, agar is 4.5g/L, inositol is 0.1g/L, and white granulated sugar is 30 g/L.
(5) And rooting culture of the explant: dividing the nursery stock in the step (4) into single seedlings, inserting the single seedlings into a rooting culture medium, then placing the explant after operation into a culture room, setting the temperature to be 23 ℃, carrying out dark culture for 48 hours, setting the illumination time to be 12 h/day, setting the illumination intensity to be 2000LX, and obtaining the West south polygala root seedling with a white and strong stem after 15-20 days, wherein the rooting culture medium comprises 1/2MS + IBA0.2mg/L, 1g/L of activated carbon, 4.5g/L of agar, 0.1g/L of inositol and 20g/L of white sugar.
The invention adopts the leaves which are easy to collect and have a large number as the explants, has no influence of season limiting factors compared with the collection of seeds and stem sections, has excellent culture effect, stable character and high proliferation efficiency, has the proliferation multiple of 4-6 times for each generation on the premise of ensuring the quality of seedlings, has short proliferation and rooting period, only needs 15-20 days for each generation of proliferation time, takes root for 20-25 days, and is far shorter than the proliferation and rooting culture time of other two explants, which is a great breakthrough to the tissue culture of the polygala tenuifolia.
The present invention and the embodiments thereof have been described above, but the description is not limited to the embodiments, but only one of the embodiments of the present invention, and the actual embodiments are not limited thereto. In conclusion, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves is characterized in that: comprises the following steps of (a) carrying out,
(1) and selecting an explant: taking newly-emerged leaves on stem segments of polygala tenuifolia in the south west as explants, selecting 3-4 pm in sunny days, and disinfecting after the selection is finished;
(2) and (3) induction culture of explants: placing the sterilized explant subjected to the sterilization treatment in the step (1) in a sterilized stainless steel disc, cutting a necrotic part at the base part of the explant by using a sterile scalpel, then horizontally placing the leaf surface in an induction culture medium upwards, then placing the explant after the operation in a culture room, setting the temperature to be 22-25 ℃, setting the illumination time to be 14 h/day, setting the illumination intensity to be 3000LX, differentiating cluster buds within 30 days, and differentiating 15-25 cluster buds from each leaf;
(3) and proliferation culture of explants: cutting the cluster buds in the step (2) into 5-6 small blocks of cluster buds, transferring the small blocks into a proliferation culture medium, setting the temperature to be 22-25 ℃, setting the illumination time to be 16 h/day, setting the illumination intensity to be 3000LX, and culturing for 15-20 days to obtain cluster seedlings;
(4) and culturing strong seedlings of explants: dividing the clumped seedlings in the step (3) into small blocks, putting 8-10 clumped seedlings in a strong seedling culture medium, and culturing at the same temperature and light intensity as the propagation culture of explants to obtain robust and healthy seedlings after 20-25 days;
(5) and rooting culture of the explant: dividing the seedlings in the step (4) into single seedlings, inserting the single seedlings into a rooting culture medium, then putting the explants subjected to operation into a culture room, setting the temperature to be 22-25 ℃, performing dark culture for 48 hours, setting the illumination time to be 12 hours/day, setting the illumination intensity to be 2000LX, and obtaining the rooting seedlings of the polygala tenuifolia with white roots and strong stems after 15-20 days.
2. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: the disinfection method in the step (1) comprises the following steps:
s1: soaking the explant in detergent water for 5 minutes, slightly stirring, flushing with running water for three minutes, and finally placing in a dry sterile bottle for later use;
s2: the explants are put into sterile water for rinsing once, then put into 75% alcohol for soaking for 30 seconds, put into sterile water for rinsing once again, put into 5% sodium hydrosulfite for soaking for 5 minutes, and put into sterile water for rinsing once again.
3. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 2, which is characterized in that: the running water is tap water.
4. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: the induction culture medium in the step (2) is MS + NAA0.5mg/L +6-BA0.5mg/L, potato juice 100g/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
5. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: the proliferation culture medium in the step (3) is MS + KT3mg/L + NAA0.2mg/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
6. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: the strong seedling culture medium in the step (4) is MS +6-BA0.5mg/L, agar 4.5g/L, inositol 0.1g/L and white granulated sugar 30 g/L.
7. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: the rooting culture medium in the step (5) is 1/2MS + IBA0.2mg/L, 1g/L of activated carbon, 4.5g/L of agar, 0.1g/L of inositol and 20g/L of white sugar.
8. The tissue culture and rapid propagation seedling method of polygala tenuifolia leaves according to claim 1, which is characterized in that: in steps (2) to (5), the setting temperature is preferably 23 ℃.
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| CN202011326969.XA CN112385542A (en) | 2020-11-24 | 2020-11-24 | Tissue culture and rapid propagation seedling method for leaves of polygala tenuifolia |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113207698A (en) * | 2021-06-24 | 2021-08-06 | 云南省农业科学院药用植物研究所 | Novel tissue culture seedling raising method for one-step seedling raising by utilizing polygala tenuifolia |
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| CN105532458A (en) * | 2015-12-29 | 2016-05-04 | 李永华 | Tissue culture method of polygala crotalarioides |
| CN106577281A (en) * | 2016-12-13 | 2017-04-26 | 福建省农业科学院农业生物资源研究所 | High-seedling-rate culture method for tissue culture of Polygala fallax stems |
| CN110692521A (en) * | 2019-11-25 | 2020-01-17 | 云南善源生物科技发展有限公司 | High-seedling-rate cultivation method for stem tissue culture of polygala tenuifolia |
| CN113207698A (en) * | 2021-06-24 | 2021-08-06 | 云南省农业科学院药用植物研究所 | Novel tissue culture seedling raising method for one-step seedling raising by utilizing polygala tenuifolia |
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- 2020-11-24 CN CN202011326969.XA patent/CN112385542A/en active Pending
Patent Citations (4)
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| CN105532458A (en) * | 2015-12-29 | 2016-05-04 | 李永华 | Tissue culture method of polygala crotalarioides |
| CN106577281A (en) * | 2016-12-13 | 2017-04-26 | 福建省农业科学院农业生物资源研究所 | High-seedling-rate culture method for tissue culture of Polygala fallax stems |
| CN110692521A (en) * | 2019-11-25 | 2020-01-17 | 云南善源生物科技发展有限公司 | High-seedling-rate cultivation method for stem tissue culture of polygala tenuifolia |
| CN113207698A (en) * | 2021-06-24 | 2021-08-06 | 云南省农业科学院药用植物研究所 | Novel tissue culture seedling raising method for one-step seedling raising by utilizing polygala tenuifolia |
Non-Patent Citations (2)
| Title |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113207698A (en) * | 2021-06-24 | 2021-08-06 | 云南省农业科学院药用植物研究所 | Novel tissue culture seedling raising method for one-step seedling raising by utilizing polygala tenuifolia |
| CN113207698B (en) * | 2021-06-24 | 2021-11-23 | 云南省农业科学院药用植物研究所 | Tissue culture seedling raising method for one-step seedling raising by utilizing polygala tenuifolia |
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