CN100559934C - A kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method - Google Patents
A kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method Download PDFInfo
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- CN100559934C CN100559934C CNB2007100302660A CN200710030266A CN100559934C CN 100559934 C CN100559934 C CN 100559934C CN B2007100302660 A CNB2007100302660 A CN B2007100302660A CN 200710030266 A CN200710030266 A CN 200710030266A CN 100559934 C CN100559934 C CN 100559934C
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Abstract
A kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method, the present invention with the primulina tabacum plant leaf of field acquisition as explant, by cultivation stages such as inducing culture step, clump bud propagation steps, culture of rootage step, transplant step and open-air transplant steps, allotment and preferably induce, breed, take root, the culture medium prescription of each cultivation stage such as transplanting, make primulina tabacum can realize its effectively purpose of breeding.In conjunction with the ecological recovery principle test-tube plantlet is being found the locality plantation of primulina tabacum to realize that primulina tabacum survives and ecological recovery in the plantation of Proterozoic.
Description
Technical field
The present invention relates to a kind of to the {in vitro} conservation of rare or endangered species primulina tabacum (Primulina tabacum Hance) and the biotechnology of breeding, specifically, relate to a kind of tissue culture propagating and field planting method to primulina tabacum (Primulina tabacum Hance).
Background technology
Primulina tabacum (Primulina tabacum Hance) is the perennial small-sized herbaceous plant of Gesneriaceae, from American Henry in 1881 after having found this rare plant of primulina tabacum on the cliff in basin, Liaanjiang county, Lianzhou City, North Guangdong, it again mystery disappeared 120 years.Up to several years ago, domestic relevant media report found this first one-level of country of primulina tabacum protective plant in imminent danger again in Lianzhou City.Its discovery develops research south of the Five Ridges geologic climate, soil and animals and plants has great learning value undoubtedly.The utilization plant tissue culture technique is carried out protection and the breeding of plant and is succeedd on a lot of plant speciess.There is very big difference at aspects such as cultured in vitro desired culture conditions and methods according to dissimilar plants; and primulina tabacum is as the protective plant rare in imminent danger of first emphasis first class of protection of China because that this species living environment requires is special, geographical distribution is narrow and small, population quantity seldom.This rare or endangered species are protected and utilized to utilization Plant Biotechnology breeding at present and cultivation primulina tabacum, there is no report both at home and abroad.
Summary of the invention
Be this rare or endangered species of protection primulina tabacum (Primulina tabacum Hance); the applicant has carried out the research work of this Plant Tissue Breeding; successfully induced the formation of somatic embryo generation and indefinite bud; effective breeding system and plant regeneration system have been set up with this; and realized the plantation survival of primulina tabacum, thereby the present invention proposed at Proterozoic in conjunction with the ecology recovering principle.
The objective of the invention is to propose a kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method.
Primulina tabacum proposed by the invention (Primulina tabacum Hance) tissue culture propagating and field planting method, its technical scheme is as follows: as explant, cultivate breeding and cultivation with the primulina tabacum plant leaf of field acquisition according to the following steps:
(1) inducing culture step: primulina tabacum plant leaf explant is patched inducing culture cultivates, induce primulina tabacum leaf explant somatic embryo to take place or/and the formation of indefinite bud, under 20～30 ℃, in dark culturing or every day in 1～20 μ mol m
-2s
-1Low light level irradiation 12～14 hours was cultivated 50～70 days, after the outer planting body can be induced organizator blast or indefinite bud, continued to cultivate 20-30 days, and described inducing culture adds calcium 220～440mg L with the MS minimal medium
-1, sucrose 20～30g L
-1, pH value 6.5～7.5 is the basis, in addition with addition of NAA 0.5～1.0mg L is arranged
-1+ TDZ 0.5～1.0mgL
-1, or BAP 0.5～1.0mg L
-1+ TDZ 0.2～1.0mg L
-1, or BAP 0.5～1.0mg L
-1+ NAA0.5～1.0mg L
-1NAA 0.5～1.0mg L wherein
-1+ TDZ 0.5～1.0mg L
-1But the inductor cell stage takes place; BAP0.5～1.0mg L
-1+ NAA 0.5～1.0mg L
-1But the formation of evoking adventive bud; BAP 0.5～1.0mg L
-1+ TDZ0.2～1.0mg L
-1The then formation of inductor blast and indefinite bud simultaneously.Average each explant can be induced somatic embryo or the indefinite bud about 80～90.
(2) clump bud propagation steps: somatic embryo or indefinite bud be transferred on the propagating culture medium cultivate, condition is under 20～30 ℃, illumination every day 20～50 μ mol m
-2s
-114 hours, one month to the one and a half months subculture once, the breeding frequency can reach 5～8, described propagating culture medium is with the MS minimal medium, additional calcium 220～440mg L
-1, sucrose 20～30g L
-1, pH value 6.5～7.5 is the basis, in addition with addition of BAP 0.5～1.0mg L is arranged
-1+ NAA 0.1～0.5mg L
-1
(3) culture of rootage step: the clump bud cut be transferred on the root media illumination and cultivate root induction, become test-tube plantlet, condition is 20～30 ℃, illumination every day 20～50 μ mol m
-2s
-114 hours, root media added calcium 220～440mg with the MS minimal medium
-1, sucrose 20～30g L
-1, pH value 6.5～7.5 is the basis, in addition with addition of IBA 0.2～1.0mgL is arranged
-1Or/and 0.2% activated carbon, test-tube plantlet is transplanted after cultivating 1 month on this medium;
(4) transplant step: test-tube seedling transplanting that will long root is positioned over environment moistening, that shade and cultivates down in the matrix of being transplanted to husky basin, and the matrix in the husky basin is pressed lime stone: vermiculite: sandstone=(1～2): (2～4): the proportional arrangement of (1～2);
(5) open-air transplant step: will in above-mentioned husky basin, plant in karst cave choma border by the test-tube plantlet after strong sprout, and together plant with the companion species liver moss.
In above-mentioned inducing culture step, the optimization formula of described inducing culture is: with the MS minimal medium, and additional calcium 440mg L
-1, sucrose 20～30g L
-1, pH value 6.5～7.0 is the basis, in addition with addition of NAA 0.5mg L is arranged
-1+ TDZ 1.0mg L
-1, or BAP 0.5mg L
-1+ TDZ 0.2mg L
-1, or BAP 1.0mg L
-1+ NAA 0.5mg L
-1
In above-mentioned clump bud propagation steps, the optimization formula of described propagating culture medium is: with the MS minimal medium, and additional calcium 440mg L
-1, sucrose 20～30g L
-1, pH value 6.5～7.0 is the basis, in addition with addition of BAP 1.0mg L is arranged
-1+ NAA 0.1mg L
-1
In above-mentioned culture of rootage step, the preferential prescription of described root media is: with the MS minimal medium, and additional calcium 440mg L
-1, sucrose 20～30g L
-1, pH value 6.5～7.0 is the basis, in addition with addition of IBA 1.0mg L is arranged
-1Or/and 0.2% activated carbon.
In above-mentioned transplant step, the matrix in the husky basin that test-tube plantlet is transplanted is by lime stone: vermiculite: the proportional arrangement of sandstone=1: 2: 1 is good.
The present invention is by providing a series of suitable prescriptions and condition of culture, comprise from induce, breed, take root, the prescription of serial basic process such as transplanting, make primulina tabacum can realize its effectively purpose of breeding.In conjunction with the ecological recovery principle test-tube plantlet is being found the locality plantation of primulina tabacum to realize that primulina tabacum survives and ecological recovery in the plantation of Proterozoic.
A) the inducing culture stage:, can on purpose induce the formation of generation of primulina tabacum leaf explant somatic embryo or indefinite bud by regulating the growth regulating agent prescription;
B) the clump bud breeding stage: somatic embryo or indefinite bud be transferred to can set up clump bud breeding system on the propagating culture medium, the screening optimum medium is to obtain higher propagation frequency;
C) take root the stage: the clump bud is cut be transferred to illumination cultivation root induction on the suitableeest root media that is screened, test-tube plantlet is cultivated in 1 month on this medium and all can be taken root;
D) the transplanting stage: test-tube seedling transplanting that will long root is to outdoor, utilizing the matrix of lime stone, vermiculite and sandstone and adding places environment moistening, that shade to cultivate down, produce and adapt to the growing environment that primulina tabacum is liked high calcium and high pH value, guaranteed that test-tube plantlet has higher transplanting survival rate, its transplanting survival rate reaches 85～90%;
E) the open-air transplanting tested: utilize the RESTORATION ECOLOGY species to return principle, reproduce and recover the habitat, improve survival rate; By the utilization of this project, total survival rate is up to more than 80%, these country-level endangered plants are preserved and develops, and is beneficial to the revegetation at hole, limestone area, lays a good foundation for further utilizing these species from now on.
Embodiment
Embodiment one
As explant, carry out primulina tabacum (Primulina tabacumHance) tissue culture propagating and field cultivating with the primulina tabacum blade gathered from subterranean stream scenic spot, Lianzhou City, concrete steps are as follows:
(1) inducing culture step: primulina tabacum plant leaf explant is patched inducing culture cultivate, induce the formation of generation of primulina tabacum leaf explant somatic embryo and indefinite bud, inducing culture adds calcium 440mgL with the MS minimal medium
-1, sucrose 30g L
-1, pH value 7.0 is the basis, in addition with addition of BAP 0.5mg L is arranged
-1+ TDZ 0.2mg L
-1, condition of culture: 20～30 ℃, every day is in 10 μ mol m
-2s
-1Low light level irradiation 14 hours was cultivated 60 days, after the outer planting body can be induced organizator blast and indefinite bud, continued to cultivate 30 days.Average each explant can be induced somatic embryo or the indefinite bud about 80～90.
(2) clump bud propagation steps: somatic embryo or indefinite bud be transferred on the propagating culture medium cultivate, condition is under 20～30 ℃, illumination every day 30 μ mol m
-2s
-114 hours, a month subculture once, described propagating culture medium is with the MS minimal medium, additional calcium 440mg L
-1, sucrose 30g L
-1, pH value 6.5 is the basis, in addition with addition of BAP 1.0mg L is arranged
-1+ NAA 0.1mgL
-1
(3) culture of rootage step: the clump bud cut be transferred on the root media illumination and cultivate root induction, become test-tube plantlet, condition is 20～30 ℃, illumination every day 50 μ mol m
-2s
-114 hours, root media added calcium 440mg L with the MS minimal medium
-1, sucrose 20g L
-1, pH value 6.5 is the basis, in addition with addition of IBA 1.0mg L is arranged
-1With 0.2% activated carbon.Test-tube plantlet is transplanted after cultivating 1 month on this medium;
(4) transplant step: test-tube seedling transplanting that will long root is positioned over environment moistening, that shade and cultivates down in the matrix of being transplanted to husky basin, and the matrix in the husky basin is pressed lime stone: vermiculite: the proportional arrangement of sandstone=1: 2: 1;
(5) open-air transplant step: will in above-mentioned husky basin, plant in karst cave choma border by the test-tube plantlet after strong sprout, and together plant with the companion species liver moss.
Embodiment two
As explant, carry out primulina tabacum (Primulina tabacumHance) tissue culture propagating and field cultivating with the primulina tabacum blade gathered from subterranean stream scenic spot, Lianzhou City, concrete steps are as follows:
(1) inducing culture step: primulina tabacum plant leaf explant is patched inducing culture cultivate, induce primulina tabacum leaf explant somatic embryo to take place, inducing culture adds calcium 400mg L with the MS minimal medium
-1, sucrose 30g L
-1, pH value 6.5 is the basis, in addition with addition of NAA 0.5mg L is arranged
-1+ TDZ 1.0mg L
-1, condition of culture: 20～30 ℃, every day is in 5 μ mol m
-2s
-1Low light level irradiation 14 hours was cultivated 60 days, after the outer planting body can be induced the organizator blast, continued to cultivate 30 days.
(2) clump bud propagation steps: somatic embryo is transferred on the propagating culture medium cultivates, condition is under 20～30 ℃, illumination every day 20 μ mol m
-2s
-114 hours, a month subculture once, described propagating culture medium is with the MS minimal medium, additional calcium 400mg L
-1, sucrose 30g L
-1, pH value 7.0 is the basis, in addition with addition of BAP 1.0mg L is arranged
-1+ NAA 0.1mg L
-1
(3) culture of rootage step: the clump bud cut be transferred on the root media illumination and cultivate root induction, become test-tube plantlet, condition is 20～30 ℃, illumination every day 50 μ mol m
-2s
-114 hours, root media added calcium 440mg L with the MS minimal medium
-1, sucrose 20g L
-1, pH value 7.0 is the basis, in addition with addition of IBA 1.0mg L is arranged
-1Test-tube plantlet is transplanted after cultivating 1 month on this medium;
(4) transplant step: test-tube seedling transplanting that will long root is positioned over environment moistening, that shade and cultivates down in the matrix of being transplanted to husky basin, and the matrix in the husky basin is pressed lime stone: vermiculite: the proportional arrangement of sandstone=1: 2: 1;
(5) open-air transplant step: will in above-mentioned husky basin, plant in karst cave choma border by the test-tube plantlet after strong sprout, and together plant with the companion species liver moss.
Embodiment three
As explant, carry out primulina tabacum (Primulina tabacumHance) tissue culture propagating and field cultivating with the primulina tabacum blade gathered from subterranean stream scenic spot, Lianzhou City, concrete steps are as follows:
(1) inducing culture step: primulina tabacum plant leaf explant is patched inducing culture cultivate, inducing culture adds calcium 300mg L with the MS minimal medium
-1, sucrose 30g L
-1, pH value 7.0 is the basis, in addition with addition of BAP 1.0mgL is arranged
-1+ NAA 0.5mg L
-1, condition of culture: 20～30 ℃, every day is in 10 μ mol m
-2s
-1The low light level shone 14 hours, induced the formation of primulina tabacum leaf explant indefinite bud, cultivated 60 days, after the outer planting body can be induced the formation indefinite bud, continued to cultivate 30 days.
(2) clump bud propagation steps: indefinite bud is transferred on the propagating culture medium cultivates, condition is under 20～30 ℃, illumination every day 30 μ mol m
-2s
-114 hours, a month subculture once, described propagating culture medium is with the MS minimal medium, additional calcium 440mg L
-1, sucrose 30g L
-1, pH value 7.0 is the basis, in addition with addition of BAP 1.0mg L is arranged
-1+ NAA 0.1mg L
-1
(3) culture of rootage step: the clump bud cut be transferred on the root media illumination and cultivate root induction, become test-tube plantlet, condition is 20～30 ℃, illumination every day 50 μ mol m
-2s
-114 hours, root media added calcium 440mg L with the MS minimal medium
-1, sucrose 30g L
-1, pH value 7.0 is the basis, in addition with addition of 0.2% activated carbon is arranged.Test-tube plantlet is transplanted after cultivating 1 month on this medium;
(4) transplant step: test-tube seedling transplanting that will long root is positioned over environment moistening, that shade and cultivates down in the matrix of being transplanted to husky basin, and the matrix in the husky basin is pressed lime stone: vermiculite: the proportional arrangement of sandstone=1: 3: 1;
(5) open-air transplant step: will in above-mentioned husky basin, plant in karst cave choma border by the test-tube plantlet after strong sprout, and together plant with the companion species liver moss.
Claims (2)
1. a primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method is characterized in that primulina tabacum plant leaf with field acquisition as explant, cultivates breeding and cultivation according to the following steps:
(1) inducing culture step: primulina tabacum plant leaf explant is patched inducing culture cultivate, induce primulina tabacum leaf explant somatic embryo to take place or/and the formation of indefinite bud, under 20～30 ℃, every day is in 1～20 μ mol m
-2s
-1Low light level irradiation 12～14 hours was cultivated 50～70 days, after the outer planting body can be induced organizator blast or indefinite bud, continued to cultivate 20-30 days, and described inducing culture adds calcium 220～440mg L with the MS minimal medium
-1, sucrose 20～30g L
-1, pH value 6.5～7.5 is the basis, in addition with addition of NAA 0.5mg L is arranged
-1+ TDZ 1.0mg L
-1, or BAP 0.5mg L
-1+ TDZ 0.2mg L
-1, or BAP 1.0mg L
-1+ NAA 0.5mg L
-1
(2) clump bud propagation steps: somatic embryo or indefinite bud be transferred on the propagating culture medium cultivate, condition is under 20～30 ℃, illumination every day 20～50 μ mol m
-2s
-114 hours, one month to the one and a half months subculture once, described propagating culture medium is with the MS minimal medium, additional calcium 220～440mg L
-1, sucrose 20～30g L
-1, pH value 6.5～7.5 is the basis, in addition with addition of BAP 1.0mg L is arranged
-1+ NAA 0.1mg L
-1
(3) take root the stage: the clump bud is cut be transferred to illumination cultivation root induction on the root media, become test-tube plantlet, condition is 20～30 ℃, illumination every day 20～50 μ mol m
-2s
-114 hours, root media added calcium 440mg L with the MS minimal medium
-1, sucrose 20～30g L
-1, pH value 6.5～7.0 is the basis, in addition with addition of IBA 1.0mg L is arranged
-1And/or 0.2% activated carbon, test-tube plantlet is transplanted after cultivating 1 month on this medium;
(4) the transplanting stage: test-tube seedling transplanting that will long root is positioned over environment moistening, that shade and cultivates down in the matrix of being transplanted to husky basin, and the matrix in the husky basin is pressed lime stone: vermiculite: sandstone=(1～2): (2～4): the proportional arrangement of (1～2);
(5) the open-air transplanting stage: will in above-mentioned husky basin, plant in karst cave choma border by the test-tube plantlet after strong sprout, and together plant with the companion species liver moss.
2. primulina tabacum according to claim 1 (Primulina tabacum Hance) tissue culture propagating and field planting method, it is characterized in that in the transplanting stage matrix in the husky basin that test-tube plantlet is transplanted is by lime stone: vermiculite: the proportional arrangement of sandstone=1: 2: 1.
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| CNB2007100302660A CN100559934C (en) | 2007-09-17 | 2007-09-17 | A kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method |
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| CNB2007100302660A CN100559934C (en) | 2007-09-17 | 2007-09-17 | A kind of primulina tabacum (Primulina tabacum Hance) tissue culture propagating and field planting method |
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| CN101965800B (en) * | 2010-11-19 | 2012-07-04 | 中国科学院华南植物园 | Tissue culture breeding method for metabriggsia ovalifolia |
| CN102405755B (en) * | 2011-09-13 | 2012-10-10 | 中国科学院华南植物园 | Method for breeding adventitious buds and adventitious roots by cuttage induction of primulina tabacum hance petioles |
| CN103081681B (en) * | 2013-01-17 | 2014-06-25 | 北京林业大学 | Leaf cutting propagating method of primulina hance plant |
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| CN106417010A (en) * | 2016-08-29 | 2017-02-22 | 中国科学院昆明植物研究所 | Tissue culture method of gesneriaceae plant |
| CN106417015B (en) * | 2016-09-13 | 2018-09-28 | 中国科学院华南植物园 | A kind of Huaiji primulina tabacum tissue cultures and rapid propagation method |
| CN106718888B (en) * | 2016-12-01 | 2019-04-19 | 广西壮族自治区林业科学研究院 | A kind of in-vitro culture method of brown line primulina tabacum |
| CN106900559B (en) * | 2017-03-24 | 2019-01-08 | 广西壮族自治区农业科学院花卉研究所 | A kind of aseptic seeding and method for tissue culture of micro- spot primulina tabacum |
| CN107047302B (en) * | 2017-03-24 | 2019-04-05 | 广西壮族自治区农业科学院花卉研究所 | A kind of method for tissue culture of Guigang primulina tabacum |
| CN109089853A (en) * | 2018-07-10 | 2018-12-28 | 深圳市仙湖植物园管理处（深圳市园林研究中心） | A kind of cultural method of Gesneriaceae |
| CN111492979B (en) * | 2020-05-25 | 2022-03-22 | 四川天艺生态园林集团股份有限公司 | Primula forbesii somatic embryo induction method |
| CN112806265B (en) * | 2021-01-27 | 2022-08-16 | 中国科学院华南植物园 | Yaozhao tissue culture method taking leaves as explants |
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