CN112806264A - Tissue culture and rapid propagation method for Acer catalpa - Google Patents
Tissue culture and rapid propagation method for Acer catalpa Download PDFInfo
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- CN112806264A CN112806264A CN202110105028.1A CN202110105028A CN112806264A CN 112806264 A CN112806264 A CN 112806264A CN 202110105028 A CN202110105028 A CN 202110105028A CN 112806264 A CN112806264 A CN 112806264A
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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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- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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Abstract
The invention discloses a rapid propagation method for tissue culture of Acer catalpa bunge. The invention takes the seeds of the acer catalpa as the explants, and carries out the large-scale production of the high-quality acer catalpa seedlings by utilizing the totipotency of plant tissue cells, the plant tissue culture and other biological technologies through the disinfection, the adventitious bud induction, the adventitious bud proliferation, the rooting culture, the test-tube seedling transplantation and other stages of the explants, thereby being beneficial to the protection and the sustainable utilization of the acer catalpa. The method has unique operation procedure and culture medium, high disinfection success rate, high propagation efficiency, high growth speed, good seedling quality and other characteristics, and has good market prospect. The invention can be implemented only by simple plant tissue culture equipment.
Description
The technical field is as follows:
the invention belongs to the technical field of plant biology, and particularly relates to a rapid propagation method for tissue culture of Acer catalpi Maxim.
Background art:
the catalpa bungei (Acer catalpifolium Rehder) is a deciduous tree of Aceraceae and a special seed in Sichuan province, belongs to a national secondary important protection plant and is listed as a national minimum population wild plant in 2012. The method has the advantages of narrow distribution area, small field population quantity, low fruiting rate, and often-underdeveloped embryo, especially because the seeds are forced to be dormant by low temperature and fruit wings, the seeds are easy to lose water and lose germination capacity after being mature, and the sowing germination rate is high.
At present, although the patent report of the tissue culture of the Acer catalpa bungei exists in China, the stem with buds is taken as the explant, and the pollution rate is high.
The invention content is as follows:
the invention aims to overcome the defects in the prior art, perform large-scale propagation of the acer catalpa seedlings and provide a rapid propagation method for tissue culture of the acer catalpa seedlings.
The invention is realized by the following technical scheme:
a tissue culture and rapid propagation method for Acer catalpa bunge comprises the following steps:
a. the explant disinfection method comprises the following steps: taking seeds of the Acer catalpifolium Rehder, removing fruit wings, then sterilizing, then inoculating the seeds into a seed germination culture medium for culture, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-; the seed germination culture medium contains per liter: 0.5 to 1.5 g of gibberellin, 15 to 25 g of cane sugar, a proper amount of agar (6 g of agar) and 1/2MS culture medium in balance, wherein the pH value is 5.8 to 6.0;
b. adventitious bud induction: cutting off hypocotyl and inoculating the hypocotyl to an adventitious bud induction culture medium for inducing adventitious buds 20-30 days after germination of seeds and when the height of the seedlings is 2-3 cm, and inducing the adventitious buds under the conditions that the culture temperature is 24-28 ℃, the illumination intensity is 1500-2000lx and the illumination is 12-16 hours/day; the adventitious bud induction culture medium contains per liter: 1-3 mg of kinetin, 5-15 ml of coconut milk, 20-30 g of cane sugar, a proper amount of agar (6 g of agar), and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
c. adventitious bud proliferation: cutting the induced adventitious bud into single buds, subculturing the single buds in an adventitious bud multiplication culture medium for adventitious bud multiplication, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-2000lx, and the illumination is 12-16 hours/day; the adventitious bud multiplication culture medium contains per liter: 0.1-0.3 mg of kinetin, 5-15 ml of coconut juice, 20-30 g of cane sugar, a proper amount of agar (6 g of agar), and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
d. rooting culture: cutting adventitious buds with the height of 3-4 cm into single buds, culturing the single buds on a rooting culture medium for rooting, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-; the rooting culture medium contains per liter: 0-0.1 mg of indapacetic acid, 10-20 g of cane sugar, a proper amount of agar (6 g of agar), and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
e. transplanting test-tube seedlings: after rooting culture for 30-40 days and test-tube plantlet growing to 4-5 cm high, hardening seedling under natural illumination for 5-7 days, then washing off root culture medium, and planting into culture medium for culture, thereby obtaining the Acer catalpa seedling;
the improved MS minimal medium is characterized in that potassium nitrate in the MS minimal medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the rest components are unchanged.
The disinfection treatment specifically comprises the following steps: soaking in 75% alcohol by volume for 10-30 s, soaking in hypochlorous acid solution of 1.0% available chlorine for 3-5 min, washing with sterile water for 4-5 times, sterilizing with 0.1% mercuric chloride solution for 5-8 min, and washing with sterile water for 4-5 times.
The culture medium comprises peat soil and perlite, and the volume ratio of the peat soil to the perlite is (2-4): 1.
The MS basic culture medium is an international universal culture medium, and the components and the preparation method thereof are referred to documents (Tantangheng, Daizhegang, ornamental plant tissue culture technology, Beijing, China forestry publishing house, 1991.); the 1/2MS culture medium is formed by reducing the concentration of macroelements in MS minimal medium by half and keeping the concentration of other components unchanged.
The invention takes the seeds of the acer catalpa as the explants, and carries out the large-scale production of the high-quality acer catalpa seedlings by utilizing the totipotency of plant tissue cells, the plant tissue culture and other biological technologies through the disinfection, the adventitious bud induction, the adventitious bud proliferation, the rooting culture, the test-tube seedling transplantation and other stages of the explants, thereby being beneficial to the protection and the sustainable utilization of the acer catalpa. The method has unique operation procedure and culture medium, high disinfection success rate, high propagation efficiency, high growth speed, good seedling quality and other characteristics, and has good market prospect. The invention can be implemented only by simple plant tissue culture equipment.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. The explant disinfection method comprises the following steps: taking well-developed seeds on a plant with vigorous growth of the Acer catalpifolium Rehder, removing fruit wings, firstly soaking in 75 percent by volume of alcohol for 10 seconds, soaking in 1.0 percent of available chlorine hypochlorous acid solution for 3 minutes, washing with sterile water for 4 times, then sterilizing with 0.1 percent by mass of mercuric chloride solution for 8 minutes, washing with sterile water for 5 times, inoculating into a seed germination culture medium, and culturing at 24 ℃, the illumination is 1500lx, and the illumination is 16 hours/day. Can break the dormancy of seeds, the disinfection success rate is 95 percent, and the seeds germinate after 30 days. The seed germination culture medium contains per liter: gibberellins (GA)3)0.5 g, 15 g of cane sugar, 6 g of agar and the balance of 1/2MS culture medium (the concentration of macroelements in the MS minimal medium is halved, and the concentrations of other components are unchanged), and the pH is 5.8.
2. Adventitious bud induction: after 20 days of seed germination and when the height of the seedling is 2 cm, cutting hypocotyl, inoculating the hypocotyl into an adventitious bud induction culture medium for adventitious bud induction, wherein the culture temperature is 24 ℃, the illumination intensity is 1500lx, and the illumination is 16 hours/day. Adventitious buds can be induced after 40 days of culture. The adventitious bud induction medium contains per liter: 1 mg of Kinetin (KT), 5 ml of coconut milk, 20 g of cane sugar and 6 g of agar, and the balance is an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the other components are unchanged), and the pH value is 5.8.
3. Adventitious bud proliferation: acer catalpa maxim is very sensitive to plant growth regulators, and a slightly higher plant growth regulator can generate a large amount of callus and resist the growth and proliferation of buds. Therefore, the induced adventitious bud is cut into single buds, and then subcultured in an adventitious bud proliferation medium for adventitious bud proliferation, wherein the culture temperature is 24 ℃, the illumination intensity is 1500lx, and the illumination is 16 hours/day. Generally 30 days are 1 subculture proliferation cycle. The fold of proliferation after each 1 subculture proliferation cycle was 3 fold. The adventitious bud propagation culture medium contains per liter: 0.1 mg of Kinetin (KT), 5 ml of coconut juice, 20 g of cane sugar, 6 g of agar and the balance of improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the balance of components are unchanged), and the pH value is 5.8.
4. Rooting culture: cutting adventitious buds with the height of 3 cm into single buds, culturing the single buds on a rooting culture medium for rooting, wherein the culture temperature is 24 ℃, the illumination intensity is 1500lx, the illumination is 16 hours/day, and test-tube plantlets are obtained, and the rooting rate can reach 95%. The rooting medium contains per liter: 0 mg of indacetic acid (IAA), 10 g of cane sugar and 6 g of agar, and the rest is an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the potassium sulfate content is 800 mg/L, and the rest components are unchanged), and the pH value is 5.8.
5. Transplanting test-tube seedlings: transplanting after rooting culture for 30 days and test-tube plantlet of 4 cm height. Hardening the seedlings in a transplanting greenhouse under natural illumination for 5 days before transplanting, then opening a bottle stopper, taking the test-tube seedlings out of a culture bottle by using forceps, washing off a root culture medium, planting the test-tube seedlings into a mixed medium with a culture medium of peat soil and perlite in a volume ratio of 2:1, and watering, shading and moisturizing at the early stage to obtain the acer catalpa seedlings, wherein the survival rate can reach 95%.
Example 2
1. The explant disinfection method comprises the following steps: taking well-developed seeds on a plant with vigorous growth of the Acer catalpifolium Rehder, removing fruit wings, firstly soaking in 75 percent by volume of alcohol for 20 seconds, soaking in 1.0 percent of available chlorine hypochlorous acid solution for 4 minutes, washing with sterile water for 4 times, then sterilizing with 0.1 percent by mass of mercuric chloride solution for 7 minutes, washing with sterile water for 5 times, inoculating into a seed germination culture medium, and culturing at the culture temperature of 26 ℃, the illumination intensity of 1800lx and the illumination intensity of 14 hours/day. Can break the dormancy of seeds, the disinfection success rate is 90 percent, and the seeds germinate after 28 days. The seed germination culture medium contains per liter: gibberellins (GA)3)1.0 g, 20 g of cane sugar, 6 g of agar and the balance of 1/2MS culture medium (the concentration of macroelements in the MS minimal medium is halved, and the concentration of other components is unchanged), pH 5.9。
2. Adventitious bud induction: after the seeds germinate for 25 days and the seedlings are 2.5 cm high, cutting hypocotyls, inoculating the hypocotyls into an adventitious bud induction culture medium, and inducing adventitious buds, wherein the culture temperature is 26 ℃, the illumination intensity is 1800lx, and the illumination time is 14 hours/day. Adventitious buds can be induced when the culture is carried out for 35 days. The adventitious bud induction medium contains per liter: 2 mg of Kinetin (KT), 10 ml of coconut milk, 25 g of cane sugar and 6 g of agar, and the balance is an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the other components are unchanged), and the pH value is 5.9.
3. Adventitious bud proliferation: acer catalpa maxim is very sensitive to plant growth regulators, and a slightly higher plant growth regulator can generate a large amount of callus and resist the growth and proliferation of buds. Therefore, the induced adventitious bud is cut into single buds, and then subcultured in an adventitious bud proliferation medium for adventitious bud proliferation, wherein the culture temperature is 26 ℃, the illumination intensity is 1800lx, and the illumination is 14 hours/day. Generally 30 days are 1 subculture proliferation cycle. The fold of proliferation after each 1 subculture proliferation cycle was 3.5 fold. The adventitious bud propagation culture medium contains per liter: 0.2 mg of Kinetin (KT), 10 ml of coconut juice, 25 g of cane sugar and 6 g of agar, and the balance of the improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the other components are unchanged), and the pH value is 5.9.
4. Rooting culture: cutting adventitious buds with the height of 3.5 cm into single buds, culturing the single buds on a rooting culture medium for rooting, wherein the culture temperature is 26 ℃, the illumination intensity is 1800lx, the illumination is 14 hours/day, and test-tube plantlets are obtained, and the rooting rate can reach 98%. The rooting medium contains per liter: 0.05 mg of indapacetic acid (IAA), 15 g of cane sugar and 6 g of agar, and the balance of an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the balance of the components are unchanged), and the pH value is 5.9.
5. Transplanting test-tube seedlings: transplanting after rooting culture for 35 days and test-tube plantlet of 4.5 cm height. Hardening the seedlings in a transplanting greenhouse under natural illumination for 6 days before transplanting, then opening a bottle stopper, taking the test-tube seedlings out of a culture bottle by using forceps, washing off a root culture medium, loading the test-tube seedlings into a mixed medium with a culture medium of peat soil and perlite in a volume ratio of 3:1, and watering, shading and moisturizing at the early stage to obtain the acer catalpa seedlings, wherein the survival rate can reach 98%.
Example 3
1. The explant disinfection method comprises the following steps: taking well-developed seeds on a plant with vigorous growth of the Acer catalpifolium Rehder, removing fruit wings, firstly soaking in 75 percent by volume of alcohol for 30 seconds, soaking in 1.0 percent of hypochlorous acid solution of effective chlorine for 5 minutes, washing with sterile water for 5 times, then sterilizing with 0.1 percent by mass of mercuric chloride solution for 5 minutes, washing with sterile water for 4 times, inoculating into a seed germination culture medium, and culturing at the culture temperature of 28 ℃, the illumination intensity of 2000lx and the illumination intensity of 12 hours/day. Can break the dormancy of seeds, the disinfection success rate is 95 percent, and the seeds germinate after 25 days. The seed germination culture medium contains per liter: gibberellins (GA)3)1.5 g, 25 g of cane sugar and 6 g of agar, and the balance is 1/2MS culture medium (the concentration of macroelements in the MS minimal medium is halved, and the concentrations of other components are unchanged), and the pH is 6.0.
2. Adventitious bud induction: 30 days after the seeds germinate and 3 cm high, cutting hypocotyls, inoculating the hypocotyls into an adventitious bud induction culture medium, and inducing adventitious buds under the conditions of the culture temperature of 28 ℃, the illumination intensity of 2000lx and the illumination intensity of 12 hours/day. Adventitious buds can be induced after 30 days of culture. The adventitious bud induction medium contains per liter: 3 mg of Kinetin (KT), 15 ml of coconut milk, 30 g of cane sugar and 6 g of agar, and the balance is an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the other components are unchanged), and the pH value is 6.0.
3. Adventitious bud proliferation: acer catalpa maxim is very sensitive to plant growth regulators, and a slightly higher plant growth regulator can generate a large amount of callus and resist the growth and proliferation of buds. Therefore, the induced adventitious bud is cut into single buds, and then subcultured in an adventitious bud proliferation medium for adventitious bud proliferation, wherein the culture temperature is 28 ℃, the illumination intensity is 2000lx, and the illumination is 12 hours/day. Generally 30 days are 1 subculture proliferation cycle. The fold of proliferation after each 1 subculture proliferation cycle was 4 fold. The adventitious bud propagation culture medium contains per liter: 0.3 mg of Kinetin (KT), 15 ml of coconut juice, 30 g of cane sugar and 6 g of agar, and the balance of the improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the other components are unchanged), and the pH value is 6.0.
4. Rooting culture: cutting adventitious buds with the height of 4 cm into single buds, culturing the single buds on a rooting culture medium for rooting, wherein the culture temperature is 28 ℃, the illumination intensity is 2000lx, the illumination is 12 hours/day, and test-tube plantlets are obtained, and the rooting rate can reach 100%. The rooting medium contains per liter: 0.1 mg of indapacetic acid (IAA), 20 g of cane sugar and 6 g of agar, and the rest is an improved MS basic culture medium (potassium nitrate in the MS basic culture medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the rest components are unchanged), and the pH value is 6.0.
5. Transplanting test-tube seedlings: transplanting after rooting culture for 40 days and test-tube plantlet growing to 5 cm high. Hardening the seedlings in a transplanting greenhouse under natural illumination for 7 days before transplanting, then opening a bottle stopper, taking the test-tube seedlings out of a culture bottle by using tweezers, washing off a root culture medium, planting the test-tube seedlings into a mixed medium with a culture medium of peat soil and perlite in a volume ratio of 4:1, and watering, shading and moisturizing at the early stage to obtain the acer catalpa seedlings, wherein the survival rate can reach 100%.
Claims (3)
1. A tissue culture and rapid propagation method for Acer catalpa bungei is characterized by comprising the following steps:
a. the explant disinfection method comprises the following steps: taking seeds of the Acer catalpifolium Rehder, removing fruit wings, then sterilizing, then inoculating the seeds into a seed germination culture medium for culture, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-; the seed germination culture medium contains per liter: 0.5 to 1.5 g of gibberellin, 15 to 25 g of cane sugar, a proper amount of agar and 1/2MS culture medium in balance, wherein the pH value is 5.8 to 6.0;
b. adventitious bud induction: cutting off hypocotyl and inoculating the hypocotyl to an adventitious bud induction culture medium for inducing adventitious buds 20-30 days after germination of seeds and when the height of the seedlings is 2-3 cm, and inducing the adventitious buds under the conditions that the culture temperature is 24-28 ℃, the illumination intensity is 1500-2000lx and the illumination is 12-16 hours/day; the adventitious bud induction culture medium contains per liter: 1-3 mg of kinetin, 5-15 ml of coconut juice, 20-30 g of cane sugar, a proper amount of agar and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
c. adventitious bud proliferation: cutting the induced adventitious bud into single buds, subculturing the single buds in an adventitious bud multiplication culture medium for adventitious bud multiplication, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-2000lx, and the illumination is 12-16 hours/day; the adventitious bud multiplication culture medium contains per liter: 0.1-0.3 mg of kinetin, 5-15 ml of coconut juice, 20-30 g of cane sugar, a proper amount of agar and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
d. rooting culture: cutting adventitious buds with the height of 3-4 cm into single buds, culturing the single buds on a rooting culture medium for rooting, wherein the culture temperature is 24-28 ℃, the illumination intensity is 1500-; the rooting culture medium contains per liter: 0-0.1 mg of indapacetic acid, 10-20 g of cane sugar, a proper amount of agar and the balance of an improved MS basic culture medium, wherein the pH value is 5.8-6.0;
e. transplanting test-tube seedlings: after rooting culture for 30-40 days and test-tube plantlet growing to 4-5 cm high, hardening seedling under natural illumination for 5-7 days, then washing off root culture medium, and planting into culture medium for culture, thereby obtaining the Acer catalpa seedling;
the improved MS minimal medium is characterized in that potassium nitrate in the MS minimal medium is replaced by potassium sulfate, the content of the potassium sulfate is 800 mg/L, and the rest components are unchanged.
2. The tissue culture rapid propagation method of Acer catalpa bungei according to claim 1, wherein the disinfection treatment specifically comprises: soaking in 75% alcohol by volume for 10-30 s, soaking in hypochlorous acid solution of 1.0% available chlorine for 3-5 min, washing with sterile water for 4-5 times, sterilizing with 0.1% mercuric chloride solution for 5-8 min, and washing with sterile water for 4-5 times.
3. The tissue culture rapid propagation method of Acer catalpa bungei according to claim 1 or 2, characterized in that the culture medium comprises peat soil and perlite, and the volume ratio of the peat soil to the perlite is (2-4): 1.
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| CN114600891A (en) * | 2022-03-09 | 2022-06-10 | 北京市园林绿化科学研究院 | Composition and method for seed germination |
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| CN114600891A (en) * | 2022-03-09 | 2022-06-10 | 北京市园林绿化科学研究院 | Composition and method for seed germination |
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