CN103718969A - Regenerated plant in-vitro culture method for Sirindhorn michelia figo - Google Patents
Regenerated plant in-vitro culture method for Sirindhorn michelia figo Download PDFInfo
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- CN103718969A CN103718969A CN201410041803.1A CN201410041803A CN103718969A CN 103718969 A CN103718969 A CN 103718969A CN 201410041803 A CN201410041803 A CN 201410041803A CN 103718969 A CN103718969 A CN 103718969A
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- 241000196324 Embryophyta Species 0.000 title claims abstract description 45
- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 238000012136 culture method Methods 0.000 title abstract 6
- 240000008993 Magnolia figo Species 0.000 title abstract 5
- 235000015864 Michelia figo Nutrition 0.000 title abstract 4
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000009395 breeding Methods 0.000 claims abstract description 9
- 230000001488 breeding effect Effects 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 9
- 238000005286 illumination Methods 0.000 claims description 35
- 239000010977 jade Substances 0.000 claims description 33
- 239000011159 matrix material Substances 0.000 claims description 25
- 239000001963 growth medium Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 230000008929 regeneration Effects 0.000 claims description 19
- 238000011069 regeneration method Methods 0.000 claims description 19
- 230000012010 growth Effects 0.000 claims description 16
- 230000003203 everyday effect Effects 0.000 claims description 15
- 239000007921 spray Substances 0.000 claims description 12
- UUTKICFRNVKFRG-WDSKDSINSA-N (4R)-3-[oxo-[(2S)-5-oxo-2-pyrrolidinyl]methyl]-4-thiazolidinecarboxylic acid Chemical compound OC(=O)[C@@H]1CSCN1C(=O)[C@H]1NC(=O)CC1 UUTKICFRNVKFRG-WDSKDSINSA-N 0.000 claims description 10
- 238000005507 spraying Methods 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 9
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 5
- 241000238631 Hexapoda Species 0.000 claims description 5
- 230000028446 budding cell bud growth Effects 0.000 claims description 5
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 235000015097 nutrients Nutrition 0.000 claims description 5
- 239000003415 peat Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 239000002689 soil Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 230000018044 dehydration Effects 0.000 claims description 2
- 238000006297 dehydration reaction Methods 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 230000002786 root growth Effects 0.000 claims description 2
- 244000060011 Cocos nucifera Species 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000004083 survival effect Effects 0.000 abstract description 3
- 241000422846 Sequoiadendron giganteum Species 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 241000218377 Magnoliaceae Species 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 6
- 239000003337 fertilizer Substances 0.000 description 6
- 241000737241 Cocos Species 0.000 description 4
- 241000218394 Magnolia liliiflora Species 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000000050 nutritive effect Effects 0.000 description 3
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- 241000204619 Magnolia sirindhorniae Species 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 description 2
- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000567232 Ochnaceae Species 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
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Abstract
The invention discloses a regenerated plant in-vitro culture method for Sirindhorn michelia figo. The regenerated plant in-vitro culture method comprises the following six steps: (1) adopting explants and treating materials; (2) establishing a sterile propagule; (3) carrying out multiplication culture; (4) carrying out rooting culture; (5) transplanting; and (6) culturing seedlings. According to the regenerated plant in-vitro culture method for the Sirindhorn michelia figo, the inductivity and the survival rate are high; the steps are simple, convenient and feasible; the regenerated plant in-vitro culture method has very urgent and important meanings on preserving and rescuing world valuable and rare endangered michelia figo varieties and breeding extremely-endangered trees; the regenerated plant in-vitro culture method is used for carrying out tissue culture on big tree twigs of michelia figo plants; the problems of a tissue culture propagation technology that the pollution rate is low, the browning is serious, the inductivity is low, the transplanting survival rate is low and the like are overcome and the method has the very valuable academic research value.
Description
Technical field
The present invention relates to a kind of cultured in vitro regeneration plant method of plant, be specifically related to a kind of logical cultured in vitro regeneration plant method with a smile of poem beautiful jade, belong to biological in vitro tissue and cultivate seedling growing process field.
Background technology
Logical with a smile (the formal name used at school: Michelia sirindhorniae (Noot. et Chal.) N. H. Xia et X. H. Zhangj) current of poem beautiful jade, also generally using in the world poem beautiful jade wooden supporter blue (Magnolia sirindhorniae Noot. & Chalermglin) is one of world's Precious, Rare, Endangered Magnoliaceae kind, critically endangered seeds, original producton location Thailand, belong to aiphyllium, leaf, Huadu are very fragrant, can refine famous and precious plant essence.
Poem beautiful jade is logical is the evergreen megaphanerophyte of Magnoliaceae with a smile, and trunk is straight and upright, Gao Keda 30-35 m, and it is ivory white that flower is, and Dan Duohua can open 2-3 days, and the florescence is in the 6-7 month.The logical happiness with a smile of poem beautiful jade is born in the glam woods of high humidity, be unique seeds that can grow in water in Magnoliaceae extended familys, and growth fast, and it is wooden good famous and precious, and the imperial palace noble of Thailand locality be take and had the logical top-grade furniture of making of poem beautiful jade or article are flourish.Poem beautiful jade is logical is Thailand king because relatively make a pet of the logical princess of own daughter's poem beautiful jade with a smile, with its naming this Thailand's preciousness Ochnaceae in imminent danger treasure seeds.Therefore poem beautiful jade is logical is also called as " treasured of Thailand " with a smile, and by World Conservation Union, is classified as protected trees because its preciousness is rare.Within 2012, by China (middle mountain) south greening fair, be chosen as the best arbor gold medal of form.Poem beautiful jade is logical is good use material and flower garden ornamental tree species with a smile.
At present, there is the difficult problem that pollution rate is high, brownization is serious, inductivity is low, transplanting success is low in the tissue culture propagation of Magnoliacea plant, and these hinder the bottleneck of its propagation technique development especially for the breeding of logical this class endangered species with a smile of poem beautiful jade.
Summary of the invention
The object of the invention is to provide a kind of poem beautiful jade logical cultured in vitro regeneration plant method with a smile, to preserve and to rescue world's Precious, Rare, Endangered Magnoliaceae kind, realize the breeding of critically endangered Magnoliaceae seeds, for the large tree spray of research Magnoliacea plant, organize cultivation, overcoming the tissue culture propagation technology such as pollution rate is high, brownization is serious, inductivity is low, transplanting success is low provides valuable academic research experience.
Object of the present invention is achieved by the following technical programs: a kind of logical cultured in vitro regeneration plant method with a smile of poem beautiful jade, comprises the following steps:
(1) take explant and material processed thereof: take robust growth, sprout the shoot on the large tree of the logical life in 7 years with a smile of the poem beautiful jade mobile jib without damage by disease and insect, after Preservation Treatment, low temperature is taken back laboratory; Described material processed is the blade cutting off on shoot, after cleaning, with flowing water, rinses with bromogeramine, is cut into 2cm with the stem section of 1-2 axillalry bud;
(2) set up sterile propagation body: the stem section of having pruned, through cultivating, inducing, is set up to sterile propagation body;
(3) propagation is cultivated: the sterile propagation body of acquisition is inoculated in proliferated culture medium and is cultivated;
(4) culture of rootage: the Multiple Buds obtaining after propagation is cultivated is inoculated in root media to be cultivated;
(5) transplant;
(6) seedling culture.
The described concrete grammar of setting up sterile propagation body of step (2) is: the stem section of having pruned is under lab used to 0.1% mercury chloride sterilization 3-10 minute, again with aseptic washing 3-4 time, one bottle one section is inoculated on first culture base MS1, in room temperature, be 20-30 ℃, illumination every day 8-12 hour, under the condition of intensity of illumination 800-2000LX, cultivate 15-30 days, from axil, sprout green bud, the green bud of clip is inoculated on proliferated culture medium MS2, induce axillalry bud and indefinite bud growth and breeding continuously, complete the foundation of sterile propagation body.
The described propagation of step (3) is cultivated and is: sterile propagation body is inoculated in proliferated culture medium MS2, being placed on manual control room temperature is 20-30 ℃, illumination every day 8-12 hour, cultivates 15-30 days under the condition of intensity of illumination 800-2000LX, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 15-30 days, and bud clump propagation multiple 2-4 doubly, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation.
The described culture of rootage of step (4) is: when Multiple Buds high when 15mm is above, bud is cut from base portion, be divided into individual plant and be inoculated in root media MS3, be placed on room temperature 20-30 ℃, illumination every day 8-12 hour, intensity of illumination is 15-20 days under the condition of 1000-10000LX, seedling starts root, then is placed under 75% shading condition short root hardening 20-30 days in greenhouse, and bottle seedling leaf of taking root launches, elongation is healthy and strong, forms whole plant.
The described transplanting of step (5) refers to the tissue-culture container seedling of taking root is transplanted in seedling-raising cup, complete the process of bottle seedling from Artificial Control condition of culture provisions to seedling autotrophy, concrete grammar is: the seedling in bottle is carefully taken out, clean and stick the medium on seedling, be transplanted in the seedling-raising cup of filling up matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, be generally no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted.
Described matrix is to be fully mixed to get in the ratio of 2:1 by peat soil and coconut palm chaff.
Described transplanting is to carry out after the good bottle seedling of root growth is moved into greenhouse domestication again, a bottle seedling that starts root is directly moved into greenhouse domestication, bottle seedling tame in greenhouse, continue to grow root in the process of hardening, can shift to an earlier date 8-12 days a bottle seedling of having tamed is transplanted.
The described seedling culture of step (6) refers to that bottle seedling keeps matrix moistening after transplanting, and relative air humidity, more than 95%, prevents blade dehydration, progressively control afterwards water supply in media, relative moisture is controlled at 80%-60%, controls cultivation temperature 25-28 ℃, 70% shading net shading for cool canopy.
The described seedling culture of step (6) is to adopt 0.1% tpn spraying disinfection sterilizing in the 3rd day after transplanting, can effectively prevent that germ from growing, afterwards every spraying in 10-15 days once; When having sprouting sprouting, new root to grow, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once.
The described seedling culture of step (6) is that after transplanting, growth is stable, grows after sprouting and Xin Gen, progressively increases intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, moves to bigger flowerpot cultivating large seedling.
MS1 culture medium prescription of the present invention is first culture base, can contain the drug variety of following nutritive element and the ratio of quality in every 1 liter, is dissolved in the water and is mixed with just culture base, and PH is adjusted to 5.8
Add water to 1000ml, additional in addition: 6-benzyladenine 0.5-5.0mg/l, methyl α-naphthyl acetate 0.01-0.5mg/l.
MS2 culture medium prescription of the present invention is proliferated culture medium, can contain the drug variety of following nutritive element and the ratio of quality in every 1 liter, is dissolved in the water and is mixed with proliferated culture medium, and PH is adjusted to 5.8
Add water to 1000ml, additional in addition: 6-benzyladenine 0.2-2.0mg/l, 3-indolebutyric acid 0.01-0.5mg/l.
MS3 culture medium prescription of the present invention is root induction medium, can contain the drug variety of following nutritive element and the ratio of quality in every 1 liter, is dissolved in the water and is mixed with root media, and PH is adjusted to 5.8
Add water to 1000ml, additional in addition: 3-indolebutyric acid 0.2-2.0mg/l, methyl α-naphthyl acetate 0.05-0.8mg/l.
The logical cultured in vitro regeneration plant method inductivity with a smile of poem beautiful jade of the present invention and survival rate are high, step is simple and feasible, to preserving and rescue world's Precious, Rare, Endangered Magnoliaceae kind and breeding critically endangered seeds, there are very urgent and significance, for the large tree spray of research Magnoliacea plant, organize cultivation, overcome the tissue culture propagation technology such as pollution rate is high, brownization is serious, inductivity is low, transplanting success is low and there is extremely valuable research value.
Embodiment
Below by embodiment, the present invention is described in further details, these embodiment are only used for illustrating the present invention, do not limit the scope of the invention.
The following steps that adopt embodiment 1 realize the logical cultured in vitro regeneration plant with a smile of poem beautiful jade:
(1) take explant and material processed thereof: in lily magnolia garden, Xuwen County Divine Land, Guangdong Province, take robust growth, sprout the shoot on the large tree of the logical life in 7 years with a smile of the poem beautiful jade mobile jib without damage by disease and insect, after on-the-spot Preservation Treatment, low temperature is taken back laboratory; In laboratory, cut off the blade on shoot, after cleaning with bromogeramine, with flowing water, rinse, be cut into the stem section with 1-2 axillalry bud of 2cm;
(2) set up sterile propagation body: the stem section of having pruned is under lab used to 0.1% mercury chloride sterilization 3 minutes, use again aseptic washing 4 times, one bottle one section is inoculated on first culture base MS1, in room temperature, be 30 ℃, illumination every day 8 hours, cultivates under the condition of intensity of illumination 2000LX 15 days, from axil, sprout green bud, it is upper that the green bud of clip is inoculated in proliferated culture medium MS2, induces axillalry bud and indefinite bud growth and breeding continuously, completes the foundation of sterile propagation body;
(3) propagation is cultivated: sterile propagation body is inoculated in proliferated culture medium MS2, being placed on manual control room temperature is 30 ℃, illumination every day 8 hours, cultivates under the condition of intensity of illumination 1000LX 28 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 4 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation;
(4) culture of rootage: when Multiple Buds high when 15mm is above, bud is cut from base portion, be divided into individual plant and be inoculated in root media MS3, be placed on 28 ℃ of room temperatures, illumination every day 10 hours, intensity of illumination under the condition of 1000LX 15 days, seedling starts root, then is placed under 75% shading condition in greenhouse short root hardening 30 days, and bottle seedling leaf of taking root launches, elongation is healthy and strong, forms whole plant;
(5) transplant:
A prepares seedling medium: peat soil is fully mixed in the ratio of 2:1 with coconut palm chaff, mixed-matrix is filled in the seedling-raising cup in cool canopy standby;
B directly moves into greenhouse domestication by a bottle seedling that starts root, after domestication, the tissue-culture container seedling of taking root is carefully taken out, clean and stick the medium on seedling, be transplanted in the seedling-raising cup of filling up matrix, each cup plantation one young plant, transplants thickness of earth-fill cover and just covers root system, generally be no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted;
(6) seedling culture: after transplanting, carry out the management of moisture, fertilizer and anti-bacteria:
A water management: a bottle seedling keeps matrix moistening after transplanting, and relative air humidity is 95%, and relative moisture is controlled at 60%, controls 28 ℃ of cultivation temperature, 70% shading net shading for cool canopy;
The management of b fertilizer and anti-bacteria: adopt 0.1% tpn spraying disinfection sterilizing for the 3rd day after transplanting, afterwards every spraying in 15 days once; When having sprouting sprouting, new root to grow, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once;
C treats that growth is stable, grows after sprouting and Xin Gen, progressively increases intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, moves to bigger flowerpot cultivating large seedling.
The following steps that adopt embodiment 2 realize the logical cultured in vitro regeneration plant with a smile of poem beautiful jade:
(1) take explant and material processed thereof: in lily magnolia garden, Xuwen County Divine Land, Guangdong Province, take robust growth, sprout the shoot on the large tree of the logical life in 7 years with a smile of the poem beautiful jade mobile jib without damage by disease and insect, after on-the-spot Preservation Treatment, low temperature is taken back laboratory; In laboratory, cut off the blade on shoot, after cleaning with bromogeramine, with flowing water, rinse, be cut into the stem section with 1-2 axillalry bud of 2cm;
(2) set up sterile propagation body: the stem section of having pruned is under lab used to 0.1% mercury chloride sterilization 10 minutes, use again aseptic washing 3 times, one bottle one section is inoculated on first culture base MS1, in room temperature, be 20 ℃, illumination every day 12 hours, cultivates under the condition of intensity of illumination 1000LX 20 days, from axil, sprout green bud, it is upper that the green bud of clip is inoculated in proliferated culture medium MS2, induces axillalry bud and indefinite bud growth and breeding continuously, completes the foundation of sterile propagation body;
(3) propagation is cultivated: sterile propagation body is inoculated in proliferated culture medium MS2, being placed on manual control room temperature is 24 ℃, illumination every day 10 hours, cultivates under the condition of intensity of illumination 800LX 30 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 30 days, 3 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation;
(4) culture of rootage: when Multiple Buds high when 15mm is above, bud is cut from base portion, be divided into individual plant and be inoculated in root media MS3, be placed on 26 ℃ of room temperatures, illumination every day 8 hours, intensity of illumination under the condition of 20000LX 17 days, seedling starts root, then is placed under 75% shading condition in greenhouse short root hardening 28 days, and bottle seedling leaf of taking root launches, elongation is healthy and strong, forms whole plant;
(5) transplant:
A prepares seedling medium: peat soil is fully mixed in the ratio of 2:1 with coconut palm chaff, mixed-matrix is filled in the seedling-raising cup in cool canopy standby;
B directly moves into greenhouse domestication by a bottle seedling that starts root, after domestication, the tissue-culture container seedling of taking root is carefully taken out, clean and stick the medium on seedling, be transplanted in the seedling-raising cup of filling up matrix, each cup plantation one young plant, transplants thickness of earth-fill cover and just covers root system, generally be no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted;
(6) seedling culture: after transplanting, carry out the management of moisture, fertilizer and anti-bacteria:
A water management: a bottle seedling keeps matrix moistening after transplanting, and relative air humidity is 95%, and relative moisture is controlled at 88%, controls 25 ℃ of cultivation temperature, 70% shading net shading for cool canopy;
The management of b fertilizer and anti-bacteria: adopt 0.1% tpn spraying disinfection sterilizing for the 3rd day after transplanting, afterwards every spraying in 15 days once; When having sprouting sprouting, new root to grow, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once;
C treats that growth is stable, grows after sprouting and Xin Gen, progressively increases intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, moves to bigger flowerpot cultivating large seedling.
The following steps that adopt embodiment 3 realize the logical cultured in vitro regeneration plant with a smile of poem beautiful jade:
(1) take explant and material processed thereof: in lily magnolia garden, Xuwen County Divine Land, Guangdong Province, take robust growth, sprout the shoot on the large tree of the logical life in 7 years with a smile of the poem beautiful jade mobile jib without damage by disease and insect, after on-the-spot Preservation Treatment, low temperature is taken back laboratory; In laboratory, cut off the blade on shoot, after cleaning with bromogeramine, with flowing water, rinse, be cut into the stem section with 1-2 axillalry bud of 2cm;
(2) set up sterile propagation body: the stem section of having pruned is under lab used to 0.1% mercury chloride sterilization 5 minutes, use again aseptic washing 3 times, one bottle one section is inoculated on first culture base MS1, in room temperature, be 26 ℃, illumination every day 10 hours, cultivates under the condition of intensity of illumination 800LX 30 days, from axil, sprout green bud, it is upper that the green bud of clip is inoculated in proliferated culture medium MS2, induces axillalry bud and indefinite bud growth and breeding continuously, completes the foundation of sterile propagation body;
(3) propagation is cultivated: sterile propagation body is inoculated in proliferated culture medium MS2, being placed on manual control room temperature is 30 ℃, illumination every day 12 hours, cultivates under the condition of intensity of illumination 1200LX 15 days, from axil, sprouts aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivated after 22 days, 4 times of bud clump propagation multiples, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivated expanding propagation;
(4) culture of rootage: when Multiple Buds high when 15mm is above, bud is cut from base portion, be divided into individual plant and be inoculated in root media MS3, be placed on 26 ℃ of room temperatures, illumination every day 8 hours, intensity of illumination under the condition of 4000LX 20 days, seedling starts root, then is placed under 75% shading condition in greenhouse short root hardening 20 days, and bottle seedling leaf of taking root launches, elongation is healthy and strong, forms whole plant;
(5) transplant:
A prepares seedling medium: peat soil is fully mixed in the ratio of 2:1 with coconut palm chaff, mixed-matrix is filled in the seedling-raising cup in cool canopy standby;
B directly moves into greenhouse domestication by a bottle seedling that starts root, after domestication, the tissue-culture container seedling of taking root is carefully taken out, clean and stick the medium on seedling, be transplanted in the seedling-raising cup of filling up matrix, each cup plantation one young plant, transplants thickness of earth-fill cover and just covers root system, generally be no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after having transplanted;
(6) seedling culture: after transplanting, carry out the management of moisture, fertilizer and anti-bacteria:
A water management: a bottle seedling keeps matrix moistening after transplanting, and relative air humidity is 98%, and relative moisture is controlled at 80%, controls 28 ℃ of cultivation temperature, 70% shading net shading for cool canopy;
The management of b fertilizer and anti-bacteria: adopt 0.1% tpn spraying disinfection sterilizing for the 3rd day after transplanting, afterwards every spraying in 15 days once; When having sprouting sprouting, new root to grow, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once;
C treats that growth is stable, grows after sprouting and Xin Gen, progressively increases intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, moves to bigger flowerpot cultivating large seedling.
Claims (10)
1. poem beautiful jade leads to a cultured in vitro regeneration plant method with a smile, it is characterized in that: comprise the following steps:
(1) take explant and material processed thereof: take robust growth, sprout the shoot on the large tree of the logical life in 7 years with a smile of the poem beautiful jade mobile jib without damage by disease and insect, after Preservation Treatment, low temperature is taken back laboratory; Described material processed is the blade cutting off on shoot, after cleaning, with flowing water, rinses with bromogeramine, is cut into 2cm with the stem section of 1-2 axillalry bud;
(2) set up sterile propagation body: the stem section of having pruned, through cultivating, inducing, is set up to sterile propagation body;
(3) propagation is cultivated: the sterile propagation body of acquisition is inoculated in proliferated culture medium and is cultivated;
(4) culture of rootage: the Multiple Buds obtaining after propagation is cultivated is inoculated in root media to be cultivated;
(5) transplant;
(6) seedling culture.
2. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described concrete grammar of setting up sterile propagation body of step (2) is: the stem section of having pruned is under lab used to 0.1% mercury chloride sterilization 3-10 minute, again with aseptic washing 3-4 time, one bottle one section is inoculated on first culture base MS1, in room temperature, be 20-30 ℃, illumination every day 8-12 hour, under the condition of intensity of illumination 800-2000LX, cultivate 15-30 days, from axil, sprout green bud, the green bud of clip is inoculated on proliferated culture medium MS2, induce axillalry bud and indefinite bud growth and breeding continuously, complete the foundation of sterile propagation body.
3. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described propagation of step (3) is cultivated and is: sterile propagation body is inoculated in proliferated culture medium MS2, being placed on manual control room temperature is 20-30 ℃, illumination every day 8-12 hour, under the condition of intensity of illumination 800-2000LX, cultivate 15-30 days, from axil, sprout aseptic green bud, the green bud of clip is inoculated on proliferated culture medium MS2, bud clump propagation, cultivate after 15-30 days, bud clump propagation multiple 2-4 doubly, under aseptic condition, cut apart again switching, through subculture repeatedly, cultivate expanding propagation.
4. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described culture of rootage of step (4) is: when Multiple Buds high when 15mm is above, bud is cut from base portion, being divided into individual plant is inoculated in root media MS3, be placed on room temperature 20-30 ℃, illumination every day 8-12 hour, intensity of illumination is 15-20 days under the condition of 1000-10000LX, seedling starts root, be placed into again under 75% shading condition short root hardening 20-30 days in greenhouse, bottle seedling leaf of taking root launches, and elongation is healthy and strong, forms whole plant.
5. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described transplanting of step (5) refers to the tissue-culture container seedling of taking root is transplanted in seedling-raising cup, complete the process of bottle seedling from Artificial Control condition of culture provisions to seedling autotrophy, concrete grammar is: the seedling in bottle is carefully taken out, clean and stick the medium on seedling, be transplanted in the seedling-raising cup of filling up matrix, each cup plantation one young plant, transplant thickness of earth-fill cover and just cover root system, generally be no more than 1 centimetre, compress matrix, make shoot root and matrix close contact, the final singling water of drenching after transplanting completes.
6. the logical cultured in vitro regeneration plant method with a smile of poem beautiful jade according to claim 5, is characterized in that: described matrix is to be fully mixed to get in the ratio of 2:1 by peat soil and coconut palm chaff.
7. poem beautiful jade leads to cultured in vitro regeneration plant method with a smile according to claim 1 or 5, it is characterized in that: described transplanting is to carry out after the good bottle seedling of root growth is moved into greenhouse domestication again, a bottle seedling that starts root is directly moved into greenhouse domestication, bottle seedling tame in greenhouse, continue to grow root in the process of hardening, can shift to an earlier date 8-12 days a bottle seedling of having tamed is transplanted.
8. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described seedling culture of step (6) refers to that bottle seedling keeps matrix moistening after transplanting, relative air humidity is more than 95%, prevent blade dehydration, progressively control afterwards water supply in media, relative moisture is controlled at 80%-60%, controls cultivation temperature 25-28 ℃, 70% shading net shading for cool canopy.
9. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described seedling culture of step (6) is within the 3rd day after transplanting, to adopt 0.1% tpn spraying disinfection sterilizing, can effectively prevent that germ from growing, afterwards every spraying in 10-15 days once; When having sprouting sprouting, new root to grow, spray 1500 times of nutrient solutions, according to seedling growth, every two weeks, spray once.
10. poem beautiful jade according to claim 1 leads to cultured in vitro regeneration plant method with a smile, it is characterized in that: the described seedling culture of step (6) is that after transplanting, growth is stable, grow after sprouting and Xin Gen, progressively increase intensity of illumination, until carry out full exposure Routine Management, when seedling culture to 15 is centimetre high, move to bigger flowerpot cultivating large seedling.
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| CN117598204B (en) * | 2024-01-24 | 2024-03-22 | 西南林业大学 | Tissue culture and rapid propagation method and application of michelia yunnanensis |
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