CN104885936A - Seed liquid culture-based method used for rapid propagation of rhizoma bletillae seedlings - Google Patents
Seed liquid culture-based method used for rapid propagation of rhizoma bletillae seedlings Download PDFInfo
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- CN104885936A CN104885936A CN201510241161.4A CN201510241161A CN104885936A CN 104885936 A CN104885936 A CN 104885936A CN 201510241161 A CN201510241161 A CN 201510241161A CN 104885936 A CN104885936 A CN 104885936A
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Abstract
The invention discloses a seed liquid culture-based method used for rapid propagation of rhizoma bletillae seedlings, and belongs to the technical field of plant seedlings rapid propagation. According to the seed liquid culture-based method, rhizoma bletillae seeds are cultured in a sterile liquid culture medium so as to obtain protocorms; and the protocorms are delivered into a growing media for continuous culturing. Beneficial effects are that: the seed liquid culture-based method is friendly to the environment; large amounts of pesticides and bactericidal agents are not needed; production technology is simple; only one time of sterile operation is needed in the whole production process; production cost is low; the liquid culture medium is used for culturing, so that problems of culturing using solid culture mediums are avoided, and less manual work is needed, wherein the problems are that seeding quantity is small, a plurality of times of subculturing are needed, root systems of obtained seedlings are intertwined, and much manual work is needed for soil planting; seedling period is short; seed germination rate and seedlings rate are both higher than 95%; and the seed liquid culture-based method possesses high promotional value on large scale seedling culturing and variety breeding using rhizoma bletillae seeds.
Description
Technical field
The present invention relates to a kind of method of breeding bletilla seedling, be specifically related to a kind of method of the Fast-propagation bletilla seedling based on seed liquid culture, namely first bletilla seed is cultivated in aseptic liquid nutrient medium and form protocorm, again protocorm is seeded in cultivation matrix and continues to cultivate, belong to plant seedling quick propagating technology field.
Background technology
Orchid Seeds is very little, is called as " dust " seed (dust seed), is a key character of orchid.The long 0.05-6.0mm of seed of orchid, diameter 0.01-0.9mm, single seed heavily about 0.31-24 μ g, embryo is also very little, as orchid (Acanthephippium sylhetense) 0.058 × 0.042mm spent by altar, in one piece of fruit, seed amount is up to 400 ten thousand; Roberts, Zhang Yan seed to bletilla is measured as: long 1.20mm, wide 0.23mm, the long 0.38mm of embryo, and the measurement result of wide 0.22mm, Kiyohara is: the long 1.80mm of seed, wide 0.23mm, the long 0.49mm of embryo, wide 0.21mm.
Because Orchid Seeds is very little, it has been generally acknowledged that the nutriment stored in Orchid Seeds is not enough to make it sprout and produces a strain from the seedling of nourishing one's nature, therefore be required with mycosymbiosis, but the degree of dependence of available data display orchid to fungi there are differences.It is very large that dissimilar orchid relies on difference to mycorrhizal fungi (mycorrhiza) trophism; May being associated with fungi in the seed germination stage of type of growing nonparasitically upon another plant, and adult nutritionally may not rely on mycorrhizal fungi; It is that continuation relies on that the kind of land form then shows mycorrhizal fungi, the tongue lip orchid (Plantanthera) of the U.S., the honeybee orchid (Ophrys) in Europe and the Hymenocallis americana (Caladenia) in Australia are the representatives of land form, and mycorrhizal fungi grows most important to it.
Bletilla (Bletilla Striata, also known as the bletilla striata) is that the orchid family bletilla belongs to perennial land form draft, this genus about 6 kinds, 4 kinds, China, Shan Nan distributes 3 kinds, and not only ornamental value is higher, also be one of conventional Chinese medicine, it act as astringing to arrest bleeding, detumescence and promoting granulation, for haematemesis of spitting blood, traumatism and bleeding, sore swollen toxin, chapped skin, hemoptysis of pulmonary tuberculosis, ulcer haemorrhage.Bletilla not only medical value is high, and has ornamental value, the important raw and processed materials of the modern medicine industry of Ye Shi China and cosmetics industry.
In recent years, along with the development of China's traditional Chinese medicine, bletilla range of application clinically constantly expands, and the medication demand of bletilla sharply increases.
Bletilla seed is very tiny and without endosperm, is difficult under field conditions (factors) sprout and growth.The main employing of current bletilla breeding is nourished and generated, and has two kinds of modes:
1, division propagation; for bletilla Sterile culture mode; be divided into two by bletilla stem tuber; division propagation spring or autumn all can carry out; but its cycle is long, and reproductive efficiency is low, and consumption kind of amount is large; in addition long-term vegetative propagation easily causes kind of matter to degenerate, and is difficult to adapt to the needs of large-scale planting and meet the needs of a large amount of cultivation.
2, Plant Tissue Breeding, before and after nineteen sixty, first Morel finds to utilize plant tissue culture technique can vegetative propagation orchid, and this technology is widely used in the breeding of multiple the orchid family subsequently, especially to the breeding of artificial culture Hybrid; Domestic and international researcher has also carried out cultured in vitro research widely to bletilla, mainly uses solid culture medium, and Yuan Ning utilizes bletilla seed to carry out axenic germination (being also called non-symbiosis germination), forms test-tube plantlet; Tian Yingcui etc. adopt bletilla striata young root to go out bletilla striata protocorm as explant induction, and Yang Junhui etc. cut the Stem tip induction Multiple Buds of bletilla striata aseptic seedlings, and Multiple Buds culture of rootage is strain; Zhu Yuqiu etc. then utilize bletilla striata seeds to sprout the protocorm of formation as explant, induced synthesis protocorm; Yu Chaoxiu etc. utilize stem apex and the lateral bud of cultivating plants, transplant, test-tube plantlet survival rate more than 90% after proliferative induction Buds formation root; The aseptic seeding technology to not homeomorphism bletilla striata seeds in age such as Fu Zhihui is explored; Arditti describes and utilizes the tip of a root (Root Tip), stipes (Stem Node) and pseudobulb (Pseudobulb) cultured in vitro, and evoking adventive bud carries out in-vitro propagate.
It is feasible for utilizing plant tissue culture technique to carry out bletilla Fast-propagation, at present application in production, but this technology has the following disadvantages:
1, comparatively complicated technical conditions are needed;
2, sterile working labour intensity is large, and personnel need technical training;
3, test-tube plantlet domestication survival rate is low.
Therefore, plant tissue culture technique is utilized to carry out the production cost of bletilla Fast-propagation higher.And liquid culture is carried out to form protocorm to Orchid Seeds have some to report, comprise Chu (2006), Gangaprasad (2013), Hsu (2012), Popov (2004), Pedroso (1992), Popova (2003), Hossain (2010), Nhut (2005), Minea (2004), Paul (2012), Nagananda (2012), Thorpe (2011), the people such as nearest Wang Kai (Wang Kai, Li Yue, Zhang Yun peak, Tian Baoqiang, Yang Xiumei, Pu Jia is peaceful, tight victory seven. the efficient sprouting of bletilla striata seeds and the structure of vegetative propagation system thereof. Yunnan Normal University's journal (natural science edition), 2014, 34 (4): 71-78) liquid culture is carried out to bletilla seed, the 1/2MS liquid nutrient medium of result display interpolation 1.0mg/L 6-BA is seed germination optimal medium, their work mainly carries out theoretical research, comprise solid, liquid nutrient medium contrast, seed vitality detection, the impact of different culture media, different developmental condition seed germination effects etc., the research carried out is all carry out in the liquid nutrient medium of smaller size smaller, after seed germination forms protocorm, all protocorm is not proceeded to soil matrix and cultivate seedling, thus form large-scale breeding technology.
In addition, patent document related to the present invention has:
1, bletilla seed stem production method (application number: 201310579744.9),
2, a kind of bletilla striata seeds seedling-cultivating method (application number: 201210350910.3),
3, the bletilla striata seed tissue culture propagation method (application number: 201310282939.7),
4, utilize bletilla striata seeds Fast-propagation seedling method (application number: 200910102668.6),
5, by static liquid cultivate produce orchid seedling method (U.S.: 7073289),
5 kinds of methods are all adopt bletilla mature seed above, carry out aseptic seeding, and namely Vitro Plant is cultivated.
6, the method (application number: 201310152721.X) of bletilla fast seedling raising,
The method need adopt sprinkling carbendazim and insecticide to occur to suppress miscellaneous bacteria and insect.
7, a kind of bletilla seed propagation method (application number: 201310085109.5),
The method need spray 1/2MS carbonless base paper liquid to culture matrix, after planting needs regularly to spray nutrient solution (component: 6-benzyl aminopurine 0 ~ 2.0mg/L, 1-methyl α-naphthyl acetate 0 ~ 1.0mg/L, solvent is 1/2MS or MS carbonless base paper liquid).
Visible, have not yet to see to utilize in bletilla seed liquid medium within and form protocorm, then protocorm is seeded into the technology report carrying out quick sapling multiplication in cultivation matrix.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of bletilla seed that utilizes in aseptic liquid nutrient medium, cultivate formation protocorm, again protocorm is seeded in cultivation matrix the method continuing to cultivate Fast-propagation bletilla seedling, the method is environmentally friendly, technique is simple, growing-seedling period is short, and seed germination rate and planting percent are all higher.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
Based on a method for the Fast-propagation bletilla seedling of seed liquid culture, it is characterized in that, comprise the following steps:
Step1: by aseptic inoculation after bletilla seed disinfection in liquid nutrient medium, is placed in quiescent culture under illumination, inoculates 20mg bletilla seed in every 100ml liquid nutrient medium;
Step2: after the protocorm of diameter 1-2mm to be obtained, protocorm is taken out medium, be seeded into after flushing sterile-processed, be rich in the cultivation matrix of humus;
Step3: protocorm is germination and growth in cultivation matrix, can be used for selling or field planting land for growing field crops when tubercle diameter reaches 0.5-2cm.
The method of the aforesaid Fast-propagation bletilla seedling based on seed liquid culture, is characterized in that, in Step1, the disinfectant used bletilla seed disinfection is 70% alcohol and 0.1%HgCl
2or the clorox of 20% carry out with the use of.
The method of the aforesaid Fast-propagation bletilla seedling based on seed liquid culture, is characterized in that, in Step1, aforementioned liquids medium is MS medium, B5 medium, N6 medium or Knudson medium.
The method of the aforesaid Fast-propagation bletilla seedling based on seed liquid culture, is characterized in that, in Step1, under illumination, the cultivation temperature of quiescent culture is 20-25 DEG C, and humidity is 50%-90%, and intensity of illumination is 3000-6000 lux.
The method of the aforesaid Fast-propagation bletilla seedling based on seed liquid culture, it is characterized in that, in Step2, aforementioned soil matrix is the mixture of peat and common cultivation soil or natural soils, the mass percentage of aforementioned peat is 20-50%.
The method of the aforesaid Fast-propagation bletilla seedling based on seed liquid culture, it is characterized in that, in Step3, the initial stage that protocorm grows in cultivation matrix shelters from heat or light with shade net, intensity of illumination is increased gradually, by building plastic greenhouse or keeping the water content of cultivation matrix in the scope of 75%-95% by spraying humidification in whole cultivation after taking root.
Usefulness of the present invention is:
(1) this method does not need a large amount of use of insecticide, bactericidal agent, environmentally friendly;
(2) production technology is simple, a sterile working is only needed in whole production process, liquid nutrient medium is used to cultivate, avoid use the seeding quantity brought of solid culture medium little, need repeatedly squamous subculture, the seedlings root of formation is wound around mutually, field planting soil time need the more problem such as manually, thus need less artificial on the whole, production cost is low;
(3) growing-seedling period is short, and seed germination rate, planting percent, all up to more than 95%, carry out extensive nursery for utilizing bletilla seed and breed breeding has good promotional value.
Embodiment
Below in conjunction with specific embodiment, concrete introduction is done to the present invention.
Embodiment 1
One, seed is prepared
Take 0.05g bletilla seed, be placed in 90 order gauzes, running water 1 day, then carries out disinfection to seed.
The disinfectant used bletilla seed disinfection can be 70% alcohol and 0.1%HgCl
2the two carry out with the use of, can also be 70% alcohol and 20% both clorox carry out with the use of.
Two, medium is prepared
250ml MS liquid nutrient medium is filled in 250ml triangular flask, moist heat sterilization 40 minutes at 121 DEG C.
Liquid nutrient medium can also select the medium cultivated for Vitro Plant of other kind, such as B5 medium, N6 medium, Knudson medium.
Three, seed inoculated and cultured
Under sterile working, seed is inoculated in triangular flask (other container also can), then triangular flask is placed in quiescent culture under illumination.
Cultivation temperature: 20-25 DEG C, humidity: 50%-90%, intensity of illumination: 3000-6000 lux.
In this step, by the illumination adopting liquid nutrient medium, static state (non-oscillating) to cultivate, give appropriate amount, cultured in vitro is carried out to a large amount of seeds, a large amount of, discrete, diameter can have been obtained and be about 2mm, green protocorm.
Present method avoids use solid culture medium, therefore it also avoid use the seeding quantity brought of solid culture medium little, need repeatedly squamous subculture, the seedlings root of formation is wound around mutually, field planting soil time need the more problem such as manually, thus needing less to artificial on the whole, production cost reduces.
Four, cultivation matrix and facility is prepared
Prepare the cultivation matrix being rich in humus, in the present embodiment, the mixture of what cultivation matrix was selected is peat and common cultivation soil, wherein, the mass percentage of peat is 20% (mass percentage of peat is in the scope of 20%-50%).
By 121 DEG C of damp and hot methods to cultivation matrix disinfection, then cultivation matrix is covered cultivation bed top layer.After cultivation matrix disinfection, the seedling that protocorm is quick, efficient formation has root, leaf can be contributed to.
Cultivating facility is round-the-clock greenhouse or plastics Small plastic shed.
Five, protocorm sowing
Seed illumination cultivation is after about 45 days, and the diameter of protocorm can reach 1-2mm, now gets final product bottle outlet.
Protocorm is placed in 90 order gauzes, cleaning, is then evenly disseminated on cultivation matrix.
Six, cultivation management
At the initial stage that protocorm grows in cultivation matrix, the shade net of different shading rate is selected to shelter from heat or light according to light conditions.
After protocorm is taken root in cultivation matrix, increase intensity of illumination gradually.
In whole cultivation, protect by building plastic greenhouse or keep the water content of cultivation matrix in the scope of 75%-95% by spraying humidification.
Protocorm sowing is about after 3 months, and tubercle diameter reaches 0.5-1cm, now can sell or field planting land for growing field crops.
Through observing, when adopting method of the present invention to breed seedling, the germination rate of seed, planting percent are all up to more than 95%.
Embodiment 2
One, seed is prepared
Get 1 piece of non-dehiscent capsule, be placed in flowing water and rinse 1 hour, then capsule is carried out disinfection, under sterile working, strip seed, and prepare to be inoculated in medium.
To capsule sterilize the disinfectant that uses can be both 70% alcohol and 0.1%HgCl2 carry out with the use of, can also be 70% alcohol and 20% both clorox carry out with the use of.
Two, medium is prepared
1000ml B5 liquid nutrient medium is filled in 1000ml reagent bottle, moist heat sterilization 40 minutes at 121 DEG C.
Three, seed inoculated and cultured
Under sterile working, seed is inoculated in reagent bottle, then reagent bottle is placed in quiescent culture under illumination.
Cultivation temperature: 20-25 DEG C, humidity: 50%-90%, intensity of illumination: 3000-6000 lux.
Four, cultivation matrix and facility is prepared
Prepare the cultivation matrix being rich in humus, in the present embodiment, the mixture of what cultivation matrix was selected is peat and natural soils, wherein, the mass percentage of peat is 50% (mass percentage of peat is in the scope of 20%-50%).
With 0.5% carbendazim to cultivation matrix disinfection, then cultivation matrix is covered cultivation bed top layer.
Cultivating facility is round-the-clock greenhouse or plastics Small plastic shed.
Five, protocorm sowing
Seed illumination cultivation is after about 45 days, and the diameter of protocorm can reach 1-2mm, now gets final product bottle outlet.
Protocorm is placed in 180 order gauzes, cleaning, is then evenly disseminated on cultivation matrix.
Six, cultivation management
At the initial stage that protocorm grows in cultivation matrix, the shade net of different shading rate is selected to shelter from heat or light according to light conditions.
After protocorm is taken root in cultivation matrix, increase intensity of illumination gradually.
In whole cultivation, protect by building plastic greenhouse or keep the water content of cultivation matrix in the scope of 75%-95% by spraying humidification.
Protocorm sowing is about after 7 months, and tubercle diameter reaches 1-2cm, now can sell or field planting land for growing field crops.
Through observing, when adopting method of the present invention to breed seedling, the germination rate of seed, planting percent are all up to more than 95%.
As can be seen here, method of the present invention has following advantage:
1, this method is environmentally friendly, does not need to use a large amount of insecticide, bactericidal agent;
2, production technology is simple, only needs a sterile working in whole production process;
3, production cost is low, uses liquid nutrient medium to cultivate, avoid use the seeding quantity brought of solid culture medium little, need repeatedly squamous subculture, the seedlings root of formation is wound around mutually, field planting soil time need the more problem such as manually;
4, seed germination rate, planting percent are all up to more than 95%;
5, growing-seedling period is short, average about 5 months;
6, for utilizing bletilla seed to carry out extensive nursery and breed breeding has good promotional value.
It should be noted that, above-described embodiment does not limit the present invention in any form, the technical scheme that the mode that all employings are equal to replacement or equivalent transformation obtains, and all drops in protection scope of the present invention.
Claims (6)
1., based on the method for the Fast-propagation bletilla seedling of seed liquid culture, it is characterized in that, comprise the following steps:
Step1: by aseptic inoculation after bletilla seed disinfection in liquid nutrient medium, is placed in quiescent culture under illumination, inoculates 20mg bletilla seed in every 100ml liquid nutrient medium;
Step2: after the protocorm of diameter 1-2mm to be obtained, protocorm is taken out medium, be seeded into after flushing sterile-processed, be rich in the cultivation matrix of humus;
Step3: protocorm is germination and growth in cultivation matrix, can be used for selling or field planting land for growing field crops when tubercle diameter reaches 0.5-2cm.
2. the method for the Fast-propagation bletilla seedling based on seed liquid culture according to claim 1, is characterized in that, in Step1, the disinfectant used bletilla seed disinfection is 70% alcohol and 0.1%HgCl
2or the clorox of 20% carry out with the use of.
3. the method for the Fast-propagation bletilla seedling based on seed liquid culture according to claim 1, is characterized in that, in Step1, described liquid nutrient medium is MS medium, B5 medium, N6 medium or Knudson medium.
4. the method for the Fast-propagation bletilla seedling based on seed liquid culture according to claim 1, it is characterized in that, in Step1, under illumination, the cultivation temperature of quiescent culture is 20-25 DEG C, humidity is 50%-90%, and intensity of illumination is 3000-6000 lux.
5. the method for the Fast-propagation bletilla seedling based on seed liquid culture according to claim 1, it is characterized in that, in Step2, described soil matrix is the mixture of peat and common cultivation soil or natural soils, and the mass percentage of described peat is 20-50%.
6. the method for the Fast-propagation bletilla seedling based on seed liquid culture according to claim 1, it is characterized in that, in Step3, the initial stage that protocorm grows in cultivation matrix shelters from heat or light with shade net, intensity of illumination is increased gradually, by building plastic greenhouse or keeping the water content of cultivation matrix in the scope of 75%-95% by spraying humidification in whole cultivation after taking root.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105165368A (en) * | 2015-10-15 | 2015-12-23 | 向君民 | Method for rapidly breeding seedlings by using bletilla striata tubers |
| CN105325287A (en) * | 2015-11-24 | 2016-02-17 | 长江大学 | Disinfection and inoculation method of microphyte seeds and pollen |
| CN105325302A (en) * | 2015-12-11 | 2016-02-17 | 电子科技大学 | Method for bletilla striata seedling production based on liquid medium |
| CN105474831A (en) * | 2015-12-16 | 2016-04-13 | 湖南省西瓜甜瓜研究所 | Bletilla striata bare seed disinfection method |
| CN105766459A (en) * | 2016-03-16 | 2016-07-20 | 陕西理工学院 | Simple and convenient method for rhizome-bletillae-protocorm seedling culturing with biodegradable membranous material |
| CN105815222A (en) * | 2016-04-15 | 2016-08-03 | 成都大学 | Bletillae plant rapid-propagation method taking bletillae rhizomes as explants |
| CN106258903A (en) * | 2016-08-29 | 2017-01-04 | 浙江金石生物科技有限公司 | A kind of method of large-scale production Pseudobulbus Bletillae (Rhizoma Bletillae) seedling |
| CN106922536A (en) * | 2017-04-13 | 2017-07-07 | 毕节市农业科学研究所 | A kind of method that fast energy-saving cultivates bletilla seedling |
| CN108668893A (en) * | 2018-04-23 | 2018-10-19 | 福建省农业科学院农业生物资源研究所 | A kind of method of silk ribbon attached to an official seal or a medal grass seed fast seedling growing |
| CN108719070A (en) * | 2018-06-12 | 2018-11-02 | 中国科学院武汉植物园 | A kind of tissue culture seed disinfection of bletilla and inoculation method |
| CN109601322A (en) * | 2019-01-16 | 2019-04-12 | 陕西理工大学 | A method of nursery is carried out using bletilla seed |
| CN110073979A (en) * | 2019-06-04 | 2019-08-02 | 红河学院 | A kind of method for culturing seedlings of orchid |
| CN114080962A (en) * | 2021-11-17 | 2022-02-25 | 杭州市农业科学研究院 | Bletilla striata seedling growth promoting method based on Piriformospora indica |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105165368A (en) * | 2015-10-15 | 2015-12-23 | 向君民 | Method for rapidly breeding seedlings by using bletilla striata tubers |
| CN105325287A (en) * | 2015-11-24 | 2016-02-17 | 长江大学 | Disinfection and inoculation method of microphyte seeds and pollen |
| CN105325302A (en) * | 2015-12-11 | 2016-02-17 | 电子科技大学 | Method for bletilla striata seedling production based on liquid medium |
| CN105474831B (en) * | 2015-12-16 | 2017-11-17 | 湖南省西瓜甜瓜研究所 | A kind of bletilla striata naked seed sterilization method |
| CN105474831A (en) * | 2015-12-16 | 2016-04-13 | 湖南省西瓜甜瓜研究所 | Bletilla striata bare seed disinfection method |
| CN105766459A (en) * | 2016-03-16 | 2016-07-20 | 陕西理工学院 | Simple and convenient method for rhizome-bletillae-protocorm seedling culturing with biodegradable membranous material |
| CN105815222A (en) * | 2016-04-15 | 2016-08-03 | 成都大学 | Bletillae plant rapid-propagation method taking bletillae rhizomes as explants |
| CN105815222B (en) * | 2016-04-15 | 2019-06-07 | 成都大学 | It is a kind of using bletilla rhizomes as the bletilla plant rapid propagation method of explant |
| CN106258903A (en) * | 2016-08-29 | 2017-01-04 | 浙江金石生物科技有限公司 | A kind of method of large-scale production Pseudobulbus Bletillae (Rhizoma Bletillae) seedling |
| CN106922536A (en) * | 2017-04-13 | 2017-07-07 | 毕节市农业科学研究所 | A kind of method that fast energy-saving cultivates bletilla seedling |
| CN108668893A (en) * | 2018-04-23 | 2018-10-19 | 福建省农业科学院农业生物资源研究所 | A kind of method of silk ribbon attached to an official seal or a medal grass seed fast seedling growing |
| CN108719070A (en) * | 2018-06-12 | 2018-11-02 | 中国科学院武汉植物园 | A kind of tissue culture seed disinfection of bletilla and inoculation method |
| CN109601322A (en) * | 2019-01-16 | 2019-04-12 | 陕西理工大学 | A method of nursery is carried out using bletilla seed |
| CN110073979A (en) * | 2019-06-04 | 2019-08-02 | 红河学院 | A kind of method for culturing seedlings of orchid |
| CN114080962A (en) * | 2021-11-17 | 2022-02-25 | 杭州市农业科学研究院 | Bletilla striata seedling growth promoting method based on Piriformospora indica |
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