CN109496864B - Tissue culture method for rapidly propagating cymbidium hybridum - Google Patents
Tissue culture method for rapidly propagating cymbidium hybridum Download PDFInfo
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- CN109496864B CN109496864B CN201811509884.8A CN201811509884A CN109496864B CN 109496864 B CN109496864 B CN 109496864B CN 201811509884 A CN201811509884 A CN 201811509884A CN 109496864 B CN109496864 B CN 109496864B
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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 discloses a tissue culture method for rapidly propagating cymbidium hybridum, which takes cymbidium hybridum petals as explants, soaks the cymbidium hybridum petals with phosphate buffer solution during disinfection, induces protocorm-like bulbs with WPN culture medium containing grape juice and fish oil, proliferates in specific DKW culture medium containing lecithin, and then proliferates in specific B5The method has the advantages of high induction rate, high multiplication factor, short cultivation time, strong growth of the planted seedlings and emerald green color.
Description
Technical Field
The invention belongs to the field of flower cultivation and propagation, and particularly relates to a tissue culture method for rapidly propagating cymbidium hybridum.
Background
Cymbidium hybridum (Cymbidium hybridum) is an evergreen perennial epiphytic herb of Cymbidium in the family of orchidaceae, and is a product population cultivated by artificially hybridizing large epiphytic species, small epiphytic species and some Cymbidium hybridum for more than one hundred years. The cymbidium hybridum leaves are verdurous, wild and wild in flower posture, luxurious and beautiful, and is a famous new orchid star in the world. It has the delicate fragrance and elegance of Chinese orchid and the rich and colorful property of Chinese orchid, is very popular in the international flower market and is deeply favored by flower lovers.
The cymbidium hybridum has incomplete seed development and almost no endosperm, and under natural conditions, the germination of seeds needs to be symbiotic with fungi, so that the germination rate of the seeds is extremely low, the traditional plant division propagation is extremely slow, and in order to rapidly propagate the cymbidium hybridum in large quantities and realize the orchid industrialization road, the production applies the plant tissue culture rapid propagation technology, but the prior art has the problems of unsuccessful induction of protocorm, low inductivity, low increment rate, long cultivation time, poor quality of regenerated seedlings and the like.
Disclosure of Invention
The present invention is to solve the above problems and to provide a tissue culture method for cymbidium hybridum, which has high induction rate of protocorm, high proliferation rate, less browning, short cultivation time, and better quality of regenerated seedlings.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a tissue culture method for rapidly propagating cymbidium hybridum, which is characterized by comprising the following steps of:
(1) explant disinfection: taking cymbidium hybridum petals which bloom at 1-2 days, washing with sterile water for 15min, soaking with phosphate buffer solution with pH of 7.0 for 30min, wiping with 75% alcohol for 30s, and then wiping with 0.1% HgCl2Soaking for 4-5 min, washing for 20min with sterile water, sucking water, and cutting into sections of 0.6-0.8 cm;
(2) pseudoprotocorm induction: inoculating the explant obtained in step (1) into a first medium consisting of: WPM basic culture medium, 6-BA0.5mg/L, NAA1.0mg/L, grape juice 5g/L and fish oil 5g/L, and culturing for 30-40 days to obtain protocorm;
(3) and (3) proliferation culture: cutting the protocorm obtained in the step (2), and inoculating the protocorm to a second culture medium, wherein the culture medium comprises the following components: culturing DKW basic culture medium, TDZ3.0mg/L, active carbon 1.0g/L and lecithin 3g/L for 50-60 days to obtain a proliferated protocorm;
(4) rooting culture: transferring the protocorm proliferated in the step (3) into a third culture medium, wherein the third culture medium comprises the following components: b is5Culturing a basic culture medium, NAA2.5mg/L, agar 8g/L and 6-BA2mg/L for 15-20 days, transferring to a third culture medium again, and culturing for 25-35 days to obtain a rooted seedling;
(5) hardening and transplanting seedlings: transplanting the rooted seedlings obtained in the step (4) into a culture medium, domesticating for 2 weeks, and normally culturing after hardening and reviving the seedlings.
The cymbidium hybridum petals with high difficulty in inducing the protocorm are used as explants, the cymbidium hybridum petals are soaked in a phosphate buffer solution during disinfection, the induction rate is improved, the specific WPM culture medium containing grape juice and fish oil is used for performing pseudoprotocorm induction, the induction rate is successfully over 75 percent to obtain the protocorm, the protocorm is further proliferated in the specific DKW culture medium containing lecithin, the proliferation multiple is 8-10 times, and then specific B is used for proliferating the cymbidium hybridum petals in the specific DKW culture medium5Performing secondary rooting culture in a culture medium, hardening and transplanting seedlings, and finally performing rapid propagation for about 6 months to obtain planted seedlings which are robust in growth and emerald green in color.
In a preferred embodiment of the present invention, the phosphate buffer solution prepared in step (1) is prepared by: taking 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, and shaking up to obtain the potassium dihydrogen phosphate. The buffer solution prepared by potassium dihydrogen phosphate-sodium hydroxide can effectively stimulate the explant, activate the growing point cells and further improve the induction rate of the protocorm.
In a preferred embodiment of the present invention, the culture environment of step (2) is: the culture temperature is 23 ℃, the daily illumination is 9h, and the illumination intensity is 1500lx, which is beneficial to improving the induction speed and shortening the induction time.
In a preferred embodiment of the present invention, the culture environment of step (3) is: the culture temperature is 25 deg.C, the daily illumination is 11h, and the illumination intensity is 2500lx, which is helpful for accelerating the proliferation speed and shortening the proliferation time.
In a preferred embodiment of the present invention, the culture environment of step (4) is: the culture temperature is 25 ℃, the illumination is 8 hours per day, and the illumination intensity is 2000lx, which is beneficial to accelerating the rooting speed and shortening the culture time.
In a preferred embodiment of the present invention, the protocorm in step (3) is cut into 0.5cm pieces, which facilitates the proliferation of protocorms and accelerates subculture.
In a preferred embodiment of the invention, the first medium has a pH of 5.4, which helps to increase the induction rate and prevent browning.
In a preferred embodiment of the invention, the pH value of the second culture medium is 6.8, which is helpful for increasing the multiplication times and increasing the multiplication speed.
In a preferred embodiment of the invention, the third medium has a pH of 6.0 and is conducive to the formation of strong shoots, strong seedlings, dark green leaves, and more hairy roots.
In summary, compared with the prior art, the invention has the following beneficial effects: the cymbidium hybridum petals are used as explants, the cymbidium hybridum tissue culture rapid propagation is realized, the induction rate is high, the multiplication times are high, the cultivation time is short, the cultivation quality is high, the planted seedlings grow strongly, the color is emerald green, and the commercial value is high.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
Example 1
Tissue culture of cymbidium hybridum was performed as follows.
(1) Explant disinfection: taking cymbidium hybridum petals blooming on day 2, washing with sterile water for 15min, soaking in phosphate buffer solution with pH of 7.0 for 30min (the phosphate buffer solution is prepared by adding 0.2mol/L potassium dihydrogen phosphate solution 250ml, and 0.2mol/L sodium hydroxide solution 118ml, diluted to 1000ml with water, shaken well) and then wiped with 75% alcohol for 30s, followed by 0.1% HgCl2Soaking for 5min, washing with sterile water for 20min, draining, and cutting into 0.8cm sections;
(2) pseudoprotocorm induction: inoculating the explant obtained in step (1) into a first medium consisting of: WPM basic culture medium, 6-BA0.5mg/L, NAA1.0mg/L, grape juice 5g/L and fish oil 5g/L, wherein the pH is adjusted to 5.0, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12h per day with illumination intensity of 2000lx for 40 days to obtain protocorm;
(3) and (3) proliferation culture: cutting the protocorm obtained in the step (2) into small pieces of 0.8cm, and inoculating the small pieces to a second culture medium, wherein the culture medium comprises the following components: DKW basic culture medium, TDZ3.0mg/L, active carbon 1.0g/L, lecithin 3g/L, pH is adjusted to 6.0, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12h per day with illumination intensity of 2000lx for 60 days to obtain proliferated protocorm;
(4) rooting culture: transferring the protocorm proliferated in the step (3) into a third culture medium, wherein the third culture medium comprises the following components: b is5The culture medium comprises a basic culture medium, NAA2.5mg/L, agar 8g/L and 6-BA2mg/L, wherein the pH is adjusted to 7.0, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12h per day with illumination intensity of 2000lx for 20 days, transferring to the third culture medium, and culturing for 35 days to obtain rooted seedling;
(5) hardening and transplanting seedlings: transplanting the rooted seedlings obtained in the step (4) into a culture medium, domesticating for 2 weeks, and normally culturing after hardening and reviving the seedlings.
Example 2
Tissue culture of cymbidium hybridum was performed as follows.
(1) Explant disinfection: washing cymbidium hybridum petals on day 1 with sterile water for 15min, soaking in phosphate buffer solution with pH of 7.0 for 30min (the phosphate buffer solution is prepared by collecting 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, shaking, wiping with 75% alcohol for 30s, and wiping with 0.1% HgCl2Soaking for 5min, washing with sterile water for 20min, draining, and cutting into 0.6cm sections;
(2) pseudoprotocorm induction: inoculating the explant obtained in step (1) into a first medium consisting of: WPM basic culture medium, 6-BA0.5mg/L, NAA1.0mg/L, grape juice 5g/L and fish oil 5g/L, wherein the pH is adjusted to 5.4, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12 hr per day with illumination intensity of 2000lx for 36 days to obtain protocorm;
(3) and (3) proliferation culture: cutting the protocorm obtained in the step (2) into small pieces of 0.5cm, and inoculating the small pieces to a second culture medium, wherein the culture medium comprises the following components: DKW basic culture medium, TDZ3.0mg/L, active carbon 1.0g/L, lecithin 3g/L, pH is adjusted to 6.8, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12h per day with illumination intensity of 2000lx for 55 days to obtain proliferated protocorm;
(4) rooting culture: transferring the protocorm proliferated in the step (3) into a third culture medium, wherein the third culture medium comprises the following components: b5 minimal medium, NAA2.5mg/L, agar 8g/L, 6-BA2mg/L, pH is adjusted to 6.0, and the culture environment is as follows: culturing at 25 deg.C under illumination for 12h per day with illumination intensity of 2000lx for 18 days, transferring to the third culture medium, and culturing for 30 days to obtain rooted seedling;
(5) hardening and transplanting seedlings: transplanting the rooted seedlings obtained in the step (4) into a culture medium, domesticating for 2 weeks, and normally culturing after hardening and reviving the seedlings.
Example 3
Tissue culture of cymbidium hybridum was performed as follows.
(1) Explant disinfection: washing cymbidium hybridum petals on day 1 with sterile water for 15min, soaking in phosphate buffer solution with pH of 7.0 for 30min (the phosphate buffer solution is prepared by collecting 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, shaking, wiping with 75% alcohol for 30s, and wiping with 0.1% HgCl2Soaking for 5min, washing with sterile water for 20min, draining, and cutting into 0.7cm sections;
(2) pseudoprotocorm induction: inoculating the explant obtained in step (1) into a first medium consisting of: WPM basic culture medium, 6-BA0.5mg/L, NAA1.0mg/L, grape juice 5g/L and fish oil 5g/L, wherein the pH is adjusted to 5.4, and the culture environment is as follows: culturing at 23 deg.C under illumination for 9h per day with illumination intensity of 1500lx for 30 days to obtain protocorm;
(3) and (3) proliferation culture: cutting the protocorm obtained in the step (2) into small pieces of 0.5cm, and inoculating the small pieces to a second culture medium, wherein the culture medium comprises the following components: DKW basic culture medium, TDZ3.0mg/L, active carbon 1.0g/L, lecithin 3g/L, pH is adjusted to 6.8, and the culture environment is as follows: culturing at 25 deg.C under illumination for 11h per day with illumination intensity of 2500lx for 50 days to obtain proliferated protocorm;
(4) rooting culture: transferring the protocorm proliferated in the step (3) into a third culture medium, wherein the third culture medium comprises the following components: b5 minimal medium, NAA2.5mg/L, agar 8g/L, 6-BA2mg/L, pH is adjusted to 6.0, and the culture environment is as follows: culturing at 25 deg.C under illumination for 8h per day with illumination intensity of 2000lx for 15 days, transferring to the third culture medium, and culturing for 25 days to obtain rooted seedling;
(5) hardening and transplanting seedlings: transplanting the rooted seedlings obtained in the step (4) into a culture medium, domesticating for 2 weeks, and normally culturing after hardening and reviving the seedlings.
The tissue culture process of cymbidium hybridum of the example was observed, and the protocorm induction rate and proliferation fold were recorded and calculated, and the results are as follows.
| Examples | Rate of induction | Fold of proliferation |
| Example 1 | 78.2％ | 8.0 times of |
| Example 2 | 80.6％ | 9.2 times of |
| Example 3 | 85.8％ | 10.5 times of |
Claims (8)
1. A tissue culture method for rapidly propagating cymbidium hybridum is characterized by comprising the following steps:
(1) explant disinfection: taking cymbidium hybridum petals which bloom at 1-2 days, washing with sterile water for 15min, soaking with phosphate buffer solution with pH of 7.0 for 30min, wiping with 75% alcohol for 30s, and then wiping with 0.1% HgCl2Soaking for 4-5 min, washing for 20min with sterile water, sucking water, and cutting into sections of 0.6-0.8 cm;
(2) pseudoprotocorm induction: inoculating the explant obtained in step (1) into a first medium consisting of: WPM basic culture medium, 6-BA0.5mg/L, NAA1.0mg/L, grape juice 5g/L and fish oil 5g/L, and culturing for 30-40 days to obtain protocorm;
(3) and (3) proliferation culture: cutting the protocorm obtained in the step (2), and inoculating the protocorm to a second culture medium, wherein the culture medium comprises the following components: culturing DKW basic culture medium, TDZ3.0mg/L, active carbon 1.0g/L and lecithin 3g/L for 50-60 days to obtain a proliferated protocorm;
(4) rooting culture: transferring the protocorm proliferated in the step (3) into a third culture medium, wherein the third culture medium comprises the following components: b is5Culturing a basic culture medium, NAA2.5mg/L, agar 8g/L and 6-BA2mg/L for 15-20 days, transferring to a third culture medium again, and culturing for 25-35 days to obtain a rooted seedling;
(5) hardening and transplanting seedlings: transplanting the rooted seedlings obtained in the step (4) into a culture medium, domesticating for 2 weeks, and normally culturing after hardening and reviving the seedlings;
the preparation method of the phosphate buffer solution in the step (1) comprises the following steps: taking 250ml of 0.2mol/L potassium dihydrogen phosphate solution, adding 118ml of 0.2mol/L sodium hydroxide solution, diluting with water to 1000ml, and shaking up to obtain the potassium dihydrogen phosphate.
2. The method for culturing tissue of rapidly propagating cymbidium hybridum according to claim 1, wherein the culture environment in step (2) is: the culture temperature is 23 ℃, the illumination is 9 hours per day, and the illumination intensity is 1500 lx.
3. The method for culturing tissue of rapidly propagating cymbidium hybridum according to claim 1, wherein the culture environment in step (3) is: the culture temperature is 25 ℃, the illumination is 11 hours per day, and the illumination intensity is 2500 lx.
4. The method for culturing tissue of rapidly propagating cymbidium hybridum according to claim 1, wherein the culture environment in step (4) is: the culture temperature is 25 ℃, the daily illumination is 8h, and the illumination intensity is 2000 lx.
5. The tissue culture method for rapidly propagating cymbidium hybridum according to claim 1, wherein the protocorm of step (3) is cut into 0.5cm pieces.
6. The method for culturing the tissue of cymbidium hybridum according to any one of claims 1 to 5, wherein the pH value of the first culture medium is 5.4.
7. The method for culturing the tissue of cymbidium hybridum according to any one of claims 1 to 5, wherein the pH value of the second culture medium is 6.8.
8. The method for tissue culture of cymbidium hybridum for rapid propagation according to any one of claims 1 to 5, wherein the pH value of the third culture medium is 6.0.
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| CN1032814A (en) * | 1987-10-23 | 1989-05-10 | 三井石油化学工业株式会社 | The cultural method of plant tissue |
| CN103814815A (en) * | 2013-12-11 | 2014-05-28 | 柳州赛特生物科技研发中心 | Culture medium special for tissue culture of phalaenopsis amabilis |
| CN108308036A (en) * | 2018-05-08 | 2018-07-24 | 佛山市元诺科技有限公司 | A kind of ford nervilia leaf bulb quick breeding method for tissue culture |
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