CN113412788A - Method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems - Google Patents
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention discloses a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stem nodes, which takes polygonatum cyrtonema stem nodes as explants, buds with stems and leaves can be cut into stem sections with internodes to perform cluster bud induction in the stages of primary induction culture and subculture multiplication culture, so that the multiplication coefficient is further improved, the buds are kept in a seedling forming state with the stems and leaves through strong seedling culture, then rooting and stem-forming culture is performed, and finally polygonatum cyrtonema tissue culture seedlings with the stems and leaves are obtained, and can be transplanted to a field for planting after temporary planting for 1-3 months, thereby greatly shortening the seedling culture time, saving the time and the field for temporary planting and transplanting, and reducing the seedling culture cost. The method of the invention, according to the characteristics of keeping all properties of original crops by the plant tissue culture technology, extracts the stem as the mark of the polygonatum cyrtonema big seedling, and the stem section is easy to take materials, the sterilization is easy to pass, the tuber of the plant is not damaged, the plant can normally grow and harvest, and the method has great significance for the development of polygonatum cyrtonema industry.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems.
Background
Polygonatum cyrtonema Hua (rhizoma Polygonati)PolygonatumcyrtonemaHua) is a perennial herb of Polygonatum of Liliaceae, is a bulk medicinal and edible Chinese medicinal material in China, and has sexual activityThe Chinese medicine is sweet in taste, can enter spleen, kidney and lung channels, has the functions of tonifying kidney and replenishing vital essence, relieving epigastric distention and benefiting vital energy, nourishing Yin and moistening dryness, promoting the production of body fluid and invigorating spleen, can be used for treating diseases such as deficiency of the kidney, weakness of the spleen and the stomach, lung deficiency and dry cough, tiredness and hypodynamia and the like for a long time, can also be used for treating diseases such as cardiovascular diseases, tuberculosis, chronic hepatitis, palpitation and shortness of breath, body fluid deficiency and thirst due to long-term illness, diabetes, hypertension and the like, is an important component of 150 Chinese patent medicines, and is very widely applied.
Polygonatum cyrtonema is distributed in the large part of the south of the Yangtze river, and although the distribution range is wide, the medicinal resources are mainly wild resources. Along with the improvement of living standard of people, the health preservation is more emphasized, polygonatum cyrtonema has good treatment effect on a plurality of modern chronic diseases, has no toxic or side effect, can be taken for a long time, leads to the rapid increase of market demand, the phenomenon that wild resources are dug by disorder is serious, the wild storage amount is rapidly reduced, and the artificial cultivation is imperative.
In recent years, polygonatum cyrtonema starts to be cultivated artificially, and the seedlings mainly comprise seed seedlings and tuber seedlings. Although many polygonatum cyrtonema seeds exist, the seed seedling raising time is very long, the seedlings need to grow for 3 years, the stems with leaves are taken out to form large seedlings, and the survival rate of transplanting the polygonatum cyrtonema seeds into a field is guaranteed. The seed sources of the tuber seedlings are mixed, the problems of seed degeneration, susceptibility to diseases and the like caused by virus accumulation exist, the tubers are easy to cause mechanical damage and rot during transportation, and the yield and the quality of the harvested tubers are uneven.
The plant tissue culture technology has the advantages of purification and rejuvenation, capability of producing a large number of seedlings in a short time and the like, and is widely applied to crop seedling breeding. Many researchers have studied the tissue culture and rapid propagation technical research of polygonatum plants such as polygonatum sibiricum, polygonatum cyrtonema and polygonatum kingianum, and the like, mainly uses new buds or seeds on tubers as explants, and because the plant tissue culture technology has the characteristic of maintaining all properties of original crops, and the seeds or tuber buds are in the seedling stage of polygonatum cyrtonema growth, even after the tissue culture seedling with tubers is taken out of a bottle, the seedlings can still need to be temporarily planted for 1 year to become the big seedlings with the stems, although the time is 2 years shorter than that of the seed seedlings, the time is longer than that of the other crops, and therefore, the application of the polygonatum cyrtonema tissue culture seedlings is greatly limited. And the tuber sprout of polygonatum cyrtonema basically can not grow again after being cut off, the tuber can not be used as seed stem, but only as medicinal material, and the tuber sprout grows in soil, so that the variety of microorganisms is various, the sterilization is difficult to pass, endophyte is easy to grow, the pollution rate is high, and the cost of tissue culture seedlings is increased.
Disclosure of Invention
The invention provides a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems, aiming at the problem of long seedling cultivation time of polygonatum cyrtonema. The polygonatum multiflorum stem nodes are used as explants, buds with stems and leaves can be cut into stem sections with internodes for cluster bud induction in the stages of primary induction culture and secondary multiplication culture, the multiplication coefficient is further improved, the buds are kept in a seedling state with the stems and leaves through strong seedling culture, rooting agglomeration stem culture is carried out, polygonatum multiflorum tissue culture seedlings with the stems and leaves are finally obtained, the polygonatum multiflorum can be transplanted to a field for planting after temporary planting for 1-3 months, the seedling culture time is greatly shortened, the time and the field for temporary planting and transplanting are saved, and the seedling culture cost is reduced.
The technical scheme for realizing the purpose of the invention is as follows:
a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems comprises the following steps:
(1) taking explant materials and pretreating: taking young and tender stem buds of polygonatum cyrtonema, cutting off leaves, soaking in washing powder water, stirring at intervals, washing with flowing clear water, and filtering to remove water drops for later use;
(2) sterilization and primary induction culture: cutting Polygonatum sibiricum stems into 1 cm long stem segments with internodes on a superclean bench, and adding 0.1% mercuric chloride (HgCL)2) Sterilizing the solution, cleaning the solution with sterile water, putting the solution on sterilized filter paper, sucking water, cutting off old wounds at two ends, inoculating the solution on an initial induction culture medium, and culturing for 30 days, wherein the bud induction rate is 62.1-95.80%;
the primary induction culture medium comprises: MS +6-BA 0.5-2.0 mg/L + ZT 0.1-0.7 mg/L + TDZ 0.1-0.5 mg/L + IAA 0.01-0.1 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8;
(3) subculture multiplication culture: cutting the sterile buds induced in the step (2) into stem sections with internodes on an ultraclean workbench, inoculating the stem sections into a multiplication medium, culturing for 30 days, inducing multiple buds with single-leaf buds in the middle and buds with stems and leaves in the part, and having a multiplication coefficient of 3.2-5.9;
the proliferation culture medium is as follows: MS + ZT 0.1-0.5 mg/L +6-BA 0.5-2.0 mg/L + NAA 0.01-0.1 mg/L +2, 4-D0.1-0.5 mg/L + white sugar 30 g/L + agar 5 g/L, and pH value is 5.8;
(4) strong bud culture: taking out the cluster buds obtained in the step (3) on an ultraclean workbench, cutting the cluster buds into small cluster buds with 2 buds, inoculating the small cluster buds into a strong bud culture medium for culturing for 30 days, wherein the single-leaf buds grow into buds with stem leaves, the buds are stronger, and the bud height is 2-5 cm;
the strong bud culture medium is as follows: 0.5-2.0 mg/L of MS +6-BA, 0.1-1.0 mg/L of KT, 0.05-0.5 mg/L of NAA, 30 g/L of white sugar and 5 g/L of agar, wherein the pH value is 5.8;
(5) and (3) rooting and stem agglomeration culture: cutting the cluster buds with the stem and the leaf obtained in the step (4) into single buds, inoculating the single buds into a rooting and stem-forming culture medium, and culturing for 60 days to obtain tissue culture seedlings with tubers, wherein the tubers are 0.4-1.2 cm in size, and the average number of roots is 5.4-24.1;
the rooting and stem-forming culture medium comprises: 1/2MS + NAA 0.5-2.0 mg/L + IAA 0.5-2.0 mg/L + DA-60.1-0.5 mg/L + white sugar 35 g/L + agar 5 g/L, pH value is 5.8;
the culture conditions of steps (2) to (5) are: the culture temperature is 25 + -1 deg.C, the illumination time is 12 hr/day, and the illumination intensity is 2000 Lx.
The primary induction medium is preferably: MS +6-BA1.0mg/L + ZT0.4mg/L + TDZ0.3mg/L + IAA0.05 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8.
The proliferation medium is preferably: MS + ZT0.3mg/L +6-BA1.0mg/L + NAA0.05 mg/L +2,4-D0.3 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8.
The strong bud culture medium is preferably: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8.
The rooting and stem-forming culture medium is preferably: 1/2MS + NAA1.0 mg/L + IAA1.0 mg/L + DA-60.2 mg/L + white sugar 35 g/L + agar 5 g/L, pH 5.8.
In the culture medium, ZT is zeatin, 6-BA is benzylaminopurine, KT is kinetin, DA-6 is amine fresh fat, 2,4-D is 2, 4-dichlorophenoxyacetic acid, TDZ is thiadiazophenyl urea, NAA is alpha-naphthylacetic acid, IAA is indole-3-acetic acid, reagents are analytically pure, and water is ultrapure water.
The plant growth regulator in the culture medium is combined in different types and concentrations, and has different effects on different growth stages of polygonatum cyrtonema tissue culture seedlings. 6-BA has the functions of promoting the formation of buds and inducing the generation of callus; ZT can promote plant cell division, prevent chlorophyll and protein degradation, slow respiration, and maintain cell activity; 2,4-D has the function of promoting the germination of axillary buds of polygonatum cyrtonema; TDZ can promote the regeneration and reproduction of plant buds and break the dormancy of the buds; glutamine has the functions of promoting protein synthesis in cells, promoting the growth and differentiation of cells and the like; DA-6 is a high-efficiency cytokinin and mainly has the effects of promoting the growth of crops, promoting the rooting of the crops and the like; KT has the functions of delaying the aging of in vitro leaves, inducing bud differentiation and increasing the opening degree of stomata besides the function of promoting cell division; NAA promotes cell growth and induces adventitious root formation; IAA can regulate plant growth and organ establishment, and promote cell growth.
The method of the invention, according to the characteristics of keeping all properties of original crops by the plant tissue culture technology, extracts the stem as the mark of the polygonatum cyrtonema big seedling, and the stem section is easy to take materials, the sterilization is easy to pass, the tuber of the plant is not damaged, the plant can normally grow and harvest, and the method has great significance for the development of polygonatum cyrtonema industry.
The method has the advantages of simple operation, good stability, good repeatability, low variation rate, energy conservation, high efficiency and the like.
Detailed Description
The present invention will be described in further detail with reference to examples, but the present invention is not limited thereto.
Example 1:
a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems comprises the following steps:
(1) taking explant materials and pretreating: cutting young stem buds of Polygonatum multiflorum Thunb, cutting off leaves, soaking in washing powder water for 10 min, stirring at intervals, washing with flowing clear water for 20 min, and draining water droplets;
(2) sterilization and primary induction culture: cutting Polygonatum sibiricum stems into 1 cm long stem segments with internodes on a superclean bench, and adding 0.1% mercuric chloride (HgCL)2) Sterilizing the solution for 6 min, cleaning with sterile water for 5 times, placing on sterilized filter paper, sucking water, cutting off two ends of old wound, inoculating into primary induction culture medium, and culturing for 30 days with bud induction rate of 62.15%;
the primary induction culture medium comprises: MS +6-BA0.5 mg/L + ZT0.1mg/L + TDZ0.1 mg/L + IAA0.01 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(3) subculture multiplication culture: cutting the sterile buds induced in the step (2) into stem sections with internodes on an ultraclean workbench, inoculating the stem sections into a multiplication medium for culturing for 30 days, wherein the middle parts of the stem sections induced clustered buds are single-leaf buds, the parts of the stem sections induced clustered buds are buds with stems and leaves, and the multiplication coefficient is 3.2;
the proliferation culture medium is as follows: MS + ZT0.1mg/L +6-BA0.5 mg/L + NAA0.01mg/L +2,4-D0.1 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(4) strong bud culture: taking out the buds proliferated in the step (3) on an ultraclean workbench, cutting the buds into small cluster buds with 2 buds, inoculating the small cluster buds into a strong bud culture medium for culturing for 30 days, wherein the single-leaf buds grow into buds with stem leaves, the buds are strong, and the bud height is 2-5 cm;
the strong bud culture medium is as follows: MS +6-BA0.5 mg/L + KT0.1mg/L + NAA0.05 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(5) and (3) rooting and stem agglomeration culture: cutting the cluster buds with the stem and the leaf obtained in the step (4) into single buds, inoculating the single buds into a rooting and caking stem culture medium for culturing for 60 days to obtain a tissue culture seedling with a tuber, wherein the tuber is 0.4 cm in size, and the average number of the roots is 5.4;
the rooting and stem-forming culture medium comprises: 1/2MS + NAA0.5 mg/L + IAA0.5 mg/L + DA-60.1 mg/L + white sugar 35 g/L + agar 5 g/L, pH 5.8.
Example 2:
a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems comprises the following steps:
(1) taking explant materials and pretreating: cutting young stem buds, cutting off leaves, soaking in washing powder water for 10 min, stirring, washing with flowing clear water for 20 min, and draining water;
(2) sterilization and primary induction culture: cutting Polygonatum sibiricum stems into 1 cm long stem segments with internodes on a superclean bench, and adding 0.1% mercuric chloride (HgCL)2) Sterilizing the solution for 6 min, cleaning with sterile water for 5 times, placing on sterilized filter paper, sucking water, cutting off two ends of old wound, inoculating into primary induction culture medium, and culturing for 30 days with bud induction rate of 83.67%;
the primary induction culture medium comprises: MS +6-BA2.0mg/L + ZT0.7mg/L + TDZ0.5 mg/L + IAA0.1 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8;
(3) subculture multiplication culture: cutting the sterile buds induced in the step (2) into stem sections with internodes on an ultraclean workbench, inoculating the stem sections into a multiplication medium for culturing for 30 days, wherein the middle parts of the stem sections induced clustered buds are single-leaf buds, the parts of the stem sections induced clustered buds are buds with stems and leaves, and the multiplication coefficient is 4.9;
the proliferation culture medium is as follows: MS + ZT0.5 mg/L +2.0 mg/L + NAA0.1 mg/L +2,4-D0.5 mg/L + white sugar 30 g/L + agar 5 g/L, pH 5.8;
(4) strong bud culture: taking out the buds proliferated in the step (3) on an ultraclean workbench, cutting the buds into small cluster buds with 2 buds, inoculating the small cluster buds into a strong bud culture medium for culturing for 30 days, wherein the single-leaf buds grow into buds with stem leaves, the buds are generally strong, and the bud height is 2-5 cm;
the strong bud culture medium is as follows: MS +6-BA2.0mg/L + KT1.0mg/L + NAA0.5 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(5) and (3) rooting and stem agglomeration culture: cutting the cluster buds with the stem and the leaf obtained in the step (4) into single buds, inoculating the single buds into a rooting and caking stem culture medium for culturing for 60 days to obtain a tissue culture seedling with a tuber, wherein the tuber is 1.0 cm in size, and the average number of roots is 17.2;
the rooting and stem-forming culture medium comprises: 1/2MS + NAA2.0 mg/L + IAA2.0 mg/L + DA-60.5 mg/L + white sugar 35 g/L + agar 5 g/L, pH 5.8.
Example 3:
a method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems comprises the following steps:
(1) taking explant materials and pretreating: cutting young stem buds, cutting off leaves, soaking in washing powder water for 10 min, stirring, washing with flowing clear water for 20 min, and draining water;
(2) sterilization and primary induction culture: cutting Polygonatum sibiricum stems into 1 cm long stem segments with internodes on a superclean bench, and adding 0.1% mercuric chloride (HgCL)2) Sterilizing the solution for 6 min, cleaning with sterile water for 5 times, placing on sterilized filter paper, sucking water, cutting off two ends of old wound, inoculating into primary induction culture medium, and culturing for 30 days with bud induction rate of 95.80%;
the primary induction culture medium comprises: MS +6-BA1.0mg/L + ZT0.4mg/L + TDZ0.3mg/L + IAA0.05 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8;
(3) subculture multiplication culture: cutting the sterile buds induced in the step (2) into stem sections with internodes on an ultraclean workbench, inoculating the stem sections into a multiplication medium for culturing for 30 days, wherein the middle parts of the stem sections induced clustered buds are single-leaf buds, the parts of the stem sections induced clustered buds are buds with stems and leaves, and the multiplication coefficient is 5.9;
the proliferation culture medium is as follows: MS + ZT0.3mg/L +6-BA1.0mg/L + NAA0.05 mg/L +2,4-D0.3 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(4) strong bud culture: taking out the buds proliferated in the step (3) on an ultraclean workbench, cutting the buds into small cluster buds with 2 buds, inoculating the small cluster buds into a strong bud culture medium for culturing for 30 days, wherein the single-leaf buds grow into buds with stem leaves, the buds are strong, and the bud height is 2-5 cm;
the strong bud culture medium is as follows: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8;
(5) and (3) rooting and stem agglomeration culture: cutting the cluster buds with the stem and the leaf obtained in the step (4) into single buds, inoculating the single buds into a rooting and caking stem culture medium for culturing for 60 days to obtain a tissue culture seedling with a tuber, wherein the tuber is 1.2 cm in size, and the average number of the roots is 24.1;
the rooting and stem-forming culture medium comprises: 1/2MS + NAA1.0 mg/L + IAA1.0 mg/L + DA-60.2 mg/L + white sugar 35 g/L + agar 5 g/L, pH 5.8.
The culture conditions of the steps (2) to (5) described in examples 1 to 3 were: the culture temperature is 25 + -1 deg.C, the illumination time is 12 hr/day, and the illumination intensity is 2000 Lx.
Claims (5)
1. A method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems is characterized by comprising the following steps:
(1) taking explant materials and pretreating:
taking young and tender stem buds of polygonatum cyrtonema, cutting off leaves, soaking in washing powder water, stirring at intervals, washing with flowing clear water, and filtering to remove water drops for later use;
(2) sterilization and primary induction culture:
on an ultraclean workbench, cutting polygonatum cyrtonema stems into stem segments with 1 cm long internodes, sterilizing the stem segments by using 0.1 percent mercuric chloride solution, cleaning the stem segments by using sterile water, putting the stem segments on sterilized filter paper to absorb water, cutting old wounds at two ends, inoculating the stem segments on a primary induction culture medium, and culturing for 30 days, wherein the bud induction rate is 62.1-95.80 percent;
the primary induction culture medium comprises: MS +6-BA 0.5-2.0 mg/L + ZT 0.1-0.7 mg/L + TDZ 0.1-0.5 mg/L + IAA 0.01-0.1 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8;
(3) subculture multiplication culture:
cutting the sterile buds induced in the step (2) into stem sections with internodes on an ultraclean workbench, inoculating the stem sections into a multiplication medium, culturing for 30 days, inducing multiple buds with single-leaf buds in the middle and buds with stems and leaves in the part, and having a multiplication coefficient of 3.2-5.9;
the proliferation culture medium is as follows: MS + ZT 0.1-0.5 mg/L +6-BA 0.5-2.0 mg/L + NAA 0.01-0.1 mg/L +2, 4-D0.1-0.5 mg/L + white sugar 30 g/L + agar 5 g/L, and pH value is 5.8;
(4) strong bud culture:
taking out the cluster buds obtained in the step (3) on an ultraclean workbench, cutting the cluster buds into small cluster buds with 2 buds, inoculating the small cluster buds into a strong bud culture medium for culturing for 30 days, wherein the single-leaf buds grow into buds with stem leaves, the buds are stronger, and the bud height is 2-5 cm;
the strong bud culture medium is as follows: 0.5-2.0 mg/L of MS +6-BA, 0.1-1.0 mg/L of KT, 0.05-0.5 mg/L of NAA, 30 g/L of white sugar and 5 g/L of agar, wherein the pH value is 5.8;
(5) and (3) rooting and stem agglomeration culture:
cutting the cluster buds with the stem and the leaf obtained in the step (4) into single buds, inoculating the single buds into a rooting and stem-forming culture medium, and culturing for 60 days to obtain tissue culture seedlings with tubers, wherein the tubers are 0.4-1.2 cm in size, and the average number of roots is 5.4-24.1;
the rooting and stem-forming culture medium comprises: 1/2MS + NAA 0.5-2.0 mg/L + IAA 0.5-2.0 mg/L + DA-60.1-0.5 mg/L + white sugar 35 g/L + agar 5 g/L, pH value is 5.8;
the culture conditions of steps (2) to (5) are: the culture temperature is 25 + -1 deg.C, the illumination time is 12 hr/day, and the illumination intensity is 2000 Lx.
2. The method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems as claimed in claim 1, wherein:
the primary induction culture medium in the step (2) is as follows: MS +6-BA1.0mg/L + ZT0.4mg/L + TDZ0.3mg/L + IAA0.05 mg/L + glutamine 0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8.
3. The method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems as claimed in claim 1, wherein:
the proliferation culture medium in the step (3) is as follows: MS + ZT0.3mg/L +6-BA1.0mg/L + NAA0.05 mg/L +2,4-D0.3 mg/L + white sugar 30 g/L + agar 5 g/L, pH value is 5.8.
4. The method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems as claimed in claim 1, wherein:
the strong bud culture medium in the step (4) is as follows: MS +6-BA1.0mg/L + KT0.5mg/L + NAA0.2mg/L + white sugar 30 g/L + agar 5 g/L, and the pH value is 5.8.
5. The method for cultivating tissue culture seedlings by using polygonatum cyrtonema stems as claimed in claim 1, wherein:
the rooting and stem-forming culture medium in the step (5) comprises: 1/2MS + NAA1.0 mg/L + IAA1.0 mg/L + DA-60.2 mg/L + white sugar 35 g/L + agar 5 g/L, pH 5.8.
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| CN113678737A (en) * | 2021-10-11 | 2021-11-23 | 浙江省亚热带作物研究所 | Open tissue culture method for polygonatum cyrtonema |
| CN114403010A (en) * | 2022-02-22 | 2022-04-29 | 福建农林大学 | Polygonatum cyrtonema micro-tuber dark energy-saving in-vitro propagation method |
| CN116530415A (en) * | 2023-05-30 | 2023-08-04 | 贵州省植物园 | Circularly operated polygonatum cyrtonema tissue culture breeding method |
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| CN114403010B (en) * | 2022-02-22 | 2022-11-15 | 福建农林大学 | Polygonatum cyrtonema micro-tuber dark energy-saving in-vitro propagation method |
| CN116530415A (en) * | 2023-05-30 | 2023-08-04 | 贵州省植物园 | Circularly operated polygonatum cyrtonema tissue culture breeding method |
| CN116530415B (en) * | 2023-05-30 | 2024-04-02 | 贵州省植物园 | Circularly operated polygonatum cyrtonema tissue culture breeding method |
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