CN105165622A - Tissue culture rapid propagation method for paph appletonianum - Google Patents
Tissue culture rapid propagation method for paph appletonianum Download PDFInfo
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Abstract
The invention discloses a tissue culture rapid propagation method for paph appletonianum and belongs to the field of plant tissue culture and research. The method comprises the first step of explant sterilization, the second step of seed germination culture, the third step of enrichment culture, the fourth step of differentiation culture, the fifth step of rooting culture and the sixth step of acclimatization and transplanting. By means of the method, tissue culture and rapid and stable propagation of paph appletonianum are achieved, obtained regeneration plants are stable in character, uniform and strong, the rooting rate reaches higher than 88%, the transplanting survival rate is higher than 95%, large-scale commercialization seedling culture production can be carried out, and great significance is achieved on the protection, development and utilization of precious wild germplasm resources.
Description
Technical field
The invention belongs to Plant Tissue Breeding research field, be specifically related to the blue tissue culture and rapid propagation method of a kind of volume calyx pocket.
Background technology
Volume calyx pocket orchid (Paphiopedilumappletonianum(Gower) Rolfe) belong to the orchid family Paphiopedilum, it is herbaceos perennial, mainly be distributed in Hainan Province of China, south, Guangxi province, also there are distribution in Vietnam, Laos, Cambodia and Thailand, be born on the leaf mould in dark and damp, the acid sandstone face of sylvan life of height above sea level 300-1200 rice, all can grow in 12 ~ 30 DEG C of temperature ranges.Volume calyx pocket blue blade square round shape is oval, there is the green alternate grid spot of the depth above, flower shape is peculiar, and pattern is beautiful, and the florescence is long, maintenance management is simple, quite liking having high ornamental value and higher economic worth by consumer, is the emerging kind of development pot flowers industry, also be excellent breeding parent, the market demand potential is huge.
At present, all wild species of Paphiopedilum are all listed in first-grade state protection plant, and the wild resource of volume calyx pocket orchid is excavated and environmental disruption due to year after year excessive, and population quantity reduces year by year, urgently takes safeguard measure, to protect its germ plasm resource and wild stocks.The blue general method adopting division propagation and seminal propagation of volume calyx pocket; but there is the problems such as reproduction coefficient is low, survival rate is not high; utilize tissue culture method can available protecting wild resource, but the group culturation rapid propagating technology rolling up calyx pocket orchid so far succeed, thus constrain its scale commodity production.
Summary of the invention
The object of the invention is to solve prior art Problems existing, utilize plant tissue culture method numerous soon to obtain the blue seedling of a large amount of high-quality volume calyx pocket, the technical scheme of employing is as follows:
1.the blue tissue culture and rapid propagation method of a kind of volume calyx pocket, is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 130d, gather do not ftracture, full capsule as explant, with 75% alcohol sterilizing 20s, 0.1% mercuric chloride sterilizing 10min, aseptic water washing 6 times, then use 0.1% mercuric chloride second time sterilizing 3min, aseptic water washing 6 times, blots surface moisture with aseptic filter paper;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, illumination cultivation 40 ~ 45d, seed development becomes protocorm, described seed germination medium is VW+6-BA0.1 ~ 0.3mg/L+NAA0.1 ~ 0.3mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, illumination cultivation 55 ~ 60d obtains the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0 ~ 2.5mg/L+NAA0.5 ~ 0.8mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, illumination cultivation 45 ~ 55d obtains the seedling with 2 ~ 3 leaves, described differential medium is: 1/2MS+6-BA0.2 ~ 0.5mg/L+NAA0.6 ~ 0.9mg/L+KT0.4 ~ 0.7mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(5) culture of rootage: seedling is proceeded to root media, illumination cultivation 55 ~ 60d obtains the test-tube plantlet of high 4 ~ 5cm, described root media is: 1/2MS+IBA0.2 ~ 0.5mg/L+NAA0.8 ~ 1.0mg/L+ peptone 2.0g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(6) acclimatization and transplants: select robust growth, test-tube plantlet that root system grows to 2 ~ 3cm, band bottle moves to normal temperature, without under direct solar environment, hardening 10 ~ 15d, takes out test-tube plantlet, cleans medium with clear water, be transplanted in medium of seedling bed, environmental requirement well-ventilated, shading 70% ~ 75%, relative air humidity 75% ~ 80%, temperature 15 ~ 28 DEG C, 15 ~ 20d test-tube plantlet can survive;
2.the blue tissue culture and rapid propagation method of aforesaid a kind of leaf roll pocket, it is characterized in that, the medium of seedling bed laying method described in step (6) is: the coarse sand that seedbed lower floor paving 5 ~ 7cm is thick, the leaf mould that upper strata paving 8 ~ 10cm is thick.
Beneficial effect of the present invention is embodied in: the tissue cultures and the fast and stable breeding that achieve volume calyx pocket orchid, and the Regenerated Plant Characters obtained is stable, Miao Qi, Miao Zhuan, rich cheek; Differentiation rate reaches more than 80%, and rooting rate reaches more than 88%, and transplanting survival rate reaches more than 95%, can carry out large-scale commercial nursery production, for this precious Wild ornamental resources protection and develop significant.
Embodiment
Embodiment 1
The blue tissue culture and rapid propagation method of a kind of volume calyx pocket, comprises the following steps:
(1) explant sterilization: when the corolla rolling up calyx pocket orchid just launches, remove lip, the pollen grain on stamen is taken off, be coated onto on pistil stigma and complete artificial pollination, pollination 130d after, gather do not ftracture, full capsule as explant, with 75% alcohol sterilizing 20s, 0.1% mercuric chloride sterilizing 10min, aseptic water washing 6 times, then use 0.1% mercuric chloride second time sterilizing 3min, aseptic water washing 6 times, blot surface moisture, for subsequent use;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, illumination cultivation 40 ~ 45d, seed development becomes protocorm, described seed germination medium is VW+6-BA0.1mg/L+NAA0.1mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, illumination cultivation 55 ~ 60d obtains the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.5mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, illumination cultivation 45 ~ 55d obtains the seedling with 2 ~ 3 leaves, differentiation rate is 85%, described differential medium is: 1/2MS+6-BA0.2mg/L+NAA0.6mg/L+KT0.4mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(5) culture of rootage: seedling is proceeded to root media, illumination cultivation 55 ~ 60d obtains the test-tube plantlet of high 4 ~ 5cm, rooting rate is 92%, described root media is: 1/2MS+IBA0.2mg/L+NAA0.8mg/L+ peptone 2.0g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(6) acclimatization and transplants: select robust growth, the test-tube plantlet band bottle that root system grows to 2 ~ 3cm moves to normal temperature, without under direct light condition, hardening 10 ~ 15d, take out test-tube plantlet, medium is cleaned with clear water, transplant in the leaf mould in seedbed, seedbed lower floor used is covered with the thick coarse sand of 5 ~ 7cm, be conducive to draining and breathe freely, upper strata is covered with the thick leaf mould of 8 ~ 10cm, volume calyx pocket orchid is shallow root plant, the depth bounds of root system vertical distribution is 0 ~ 8cm, planting environment requires well-ventilated, shading 70% ~ 75%, relative air humidity 75% ~ 80%, temperature 15 ~ 28 DEG C, 15 ~ 20d test-tube plantlet can survive, transplanting survival rate reaches 98%,
(7) culturing room's illumination cultivation condition is: temperature 25 ± 2 DEG C, illumination 10 ~ 12h/d, intensity of illumination 1100 ~ 1400Lx.
Embodiment 2
The blue tissue culture and rapid propagation method of a kind of volume calyx pocket, comprises the following steps:
(1) explant sterilization: after artificial pollination 130d, gather do not ftracture, full capsule as explant, with 75% alcohol sterilizing 20s, 0.1% mercuric chloride sterilizing 10min, aseptic water washing 6 times, then use 0.1% mercuric chloride second time sterilizing 3min, aseptic water washing 6 times, blots surface moisture with aseptic filter paper;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, illumination cultivation 40 ~ 45d, seed development becomes protocorm, described seed germination medium is VW+6-BA0.3mg/L+NAA0.3mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, illumination cultivation 55 ~ 60d obtains the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA2.5mg/L+NAA0.8mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, illumination cultivation 45 ~ 55d obtains the seedling with 2 ~ 3 leaves, differentiation rate is 80%, described differential medium is: 1/2MS+6-BA0.5mg/L+NAA0.9mg/L+KT0.7mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(5) culture of rootage: seedling is proceeded to root media, illumination cultivation 55 ~ 60d obtains the test-tube plantlet of high 4 ~ 5cm, rooting rate for the root media described in 90% is: 1/2MS+IBA0.5mg/L+NAA1.0mg/L+ peptone 2.0g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(6) acclimatization and transplants: select robust growth, test-tube plantlet band bottle that root system grows to 2 ~ 3cm moves to normal temperature, without under direct light condition, hardening 10 ~ 15d, take out test-tube plantlet, medium is cleaned with clear water, transplant in the leaf mould in seedbed, the coarse sand that seedbed lower floor paving 5 ~ 7cm used is thick, the leaf mould that upper strata paving 8 ~ 10cm is thick, planting environment requires well-ventilated, shading 70% ~ 75%, relative air humidity 75% ~ 80%, temperature 15 ~ 28 DEG C, 15 ~ 20d test-tube plantlet can survive, and transplanting survival rate reaches 95%;
(7) culturing room's illumination cultivation condition is: temperature 25 ± 2 DEG C, illumination 10 ~ 12h/d, intensity of illumination 1100 ~ 1400Lx.
Embodiment 3
The blue tissue culture and rapid propagation method of a kind of volume calyx pocket, comprises the following steps:
(1) explant sterilization: after artificial pollination 130d, gather do not ftracture, full capsule as explant, with 75% alcohol sterilizing 20s, 0.1% mercuric chloride sterilizing 10min, aseptic water washing 6 times, then use 0.1% mercuric chloride second time sterilizing 3min, aseptic water washing 6 times, blots surface moisture with aseptic filter paper;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, illumination cultivation 40 ~ 45d, seed development becomes protocorm, described seed germination medium is VW+6-BA0.2mg/L+NAA0.2mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, illumination cultivation 55 ~ 60d obtains the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA2.0mg/L+NAA0.7mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, illumination cultivation 45 ~ 55d obtains the seedling with 2 ~ 3 leaves, differentiation rate is 82%, described differential medium is: 1/2MS+6-BA0.3mg/L+NAA0.7mg/L+KT0.5mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(5) culture of rootage: seedling is proceeded to root media, illumination cultivation 55 ~ 60d obtains the test-tube plantlet of high 4 ~ 5cm, rooting rate is 88%, described root media is: 1/2MS+IBA0.3mg/L+NAA0.9mg/L+ peptone 2.0g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(6) acclimatization and transplants: select robust growth, test-tube plantlet band bottle that root system grows to 2 ~ 3cm moves to normal temperature, without direct light condition lower refining seedling 10 ~ 15d, take out test-tube plantlet, medium is cleaned with clear water, transplant in the leaf mould in seedbed, the coarse sand that seedbed lower floor paving 5 ~ 7cm used is thick, the leaf mould that upper strata paving 8 ~ 10cm is thick, planting environment requires well-ventilated, shading 70% ~ 75%, relative air humidity 75% ~ 80%, temperature 15 ~ 28 DEG C, 15 ~ 20d test-tube plantlet can survive, and transplanting survival rate reaches 97%;
(7) culturing room's illumination cultivation condition is: temperature 25 ± 2 DEG C, illumination 10 ~ 12h/d, intensity of illumination 1100 ~ 1400Lx.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in previous embodiment, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of embodiment of the present invention technical scheme.
Claims (2)
1. roll up the blue tissue culture and rapid propagation method of calyx pocket, it is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 130d, gather do not ftracture, full capsule as explant, with 75% alcohol sterilizing 20s, 0.1% mercuric chloride sterilizing 10min, aseptic water washing 6 times, then use 0.1% mercuric chloride second time sterilizing 3min, aseptic water washing 6 times, blots surface moisture with aseptic filter paper;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, illumination cultivation 40 ~ 45d, seed development becomes protocorm, described seed germination medium is VW+6-BA0.1 ~ 0.3mg/L+NAA0.1 ~ 0.3mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, illumination cultivation 55 ~ 60d obtains the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0 ~ 2.5mg/L+NAA0.5 ~ 0.8mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, illumination cultivation 45 ~ 55d obtains the seedling with 2 ~ 3 leaves, described differential medium is: 1/2MS+6-BA0.2 ~ 0.5mg/L+NAA0.6 ~ 0.9mg/L+KT0.4 ~ 0.7mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(5) culture of rootage: seedling is proceeded to root media, illumination cultivation 55 ~ 60d obtains the test-tube plantlet of high 4 ~ 5cm, described root media is: 1/2MS+IBA0.2 ~ 0.5mg/L+NAA0.8 ~ 1.0mg/L+ peptone 2.0g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.5 ~ 7.2;
(6) acclimatization and transplants: select robust growth, test-tube plantlet that root system grows to 2 ~ 3cm, band bottle moves to normal temperature, without under direct solar environment, hardening 10 ~ 15d, takes out test-tube plantlet, cleans medium with clear water, be transplanted in medium of seedling bed, environmental requirement well-ventilated, shading 70% ~ 75%, relative air humidity 75% ~ 80%, temperature 15 ~ 28 DEG C, 15 ~ 20d test-tube plantlet can survive.
2. the blue tissue culture and rapid propagation method of a kind of leaf roll pocket according to claim 1, it is characterized in that, the medium of seedling bed laying method described in step (6) is: the coarse sand that seedbed lower floor paving 5 ~ 7cm is thick, the leaf mould that upper strata paving 8 ~ 10cm is thick.
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Cited By (5)
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| CN106561456A (en) * | 2016-10-28 | 2017-04-19 | 中国科学院华南植物园 | Method for aseptically sowing and rapidly propagating paphiopedilum helenae |
| CN106718876A (en) * | 2016-11-23 | 2017-05-31 | 中国科学院华南植物园 | The Cord blood culture medium and Cord blood propagation method of a kind of Helen's pocket orchid protocorm |
| CN107079817A (en) * | 2017-06-21 | 2017-08-22 | 贵州省林业科学研究院 | Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method |
| JP2018505648A (en) * | 2016-01-13 | 2018-03-01 | 中国科学院華南植物園 | Tissue culture and rapid propagation of high quality seedlings of Paphiopedilum mordiae |
| CN108633376A (en) * | 2018-04-21 | 2018-10-12 | 云南省林业科学院 | A kind of cultural method of the sub- non-symbiosis germination of Gree pocket orchid species |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018505648A (en) * | 2016-01-13 | 2018-03-01 | 中国科学院華南植物園 | Tissue culture and rapid propagation of high quality seedlings of Paphiopedilum mordiae |
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| CN106718876A (en) * | 2016-11-23 | 2017-05-31 | 中国科学院华南植物园 | The Cord blood culture medium and Cord blood propagation method of a kind of Helen's pocket orchid protocorm |
| CN106718876B (en) * | 2016-11-23 | 2018-12-07 | 中国科学院华南植物园 | A kind of the cryo-conservation culture medium and cryo-conservation propagation method of Helen's pocket orchid protocorm |
| CN107079817A (en) * | 2017-06-21 | 2017-08-22 | 贵州省林业科学研究院 | Pocket Lanzhou and Xinjiang kind " excellent pocket is blue " tissue culture and rapid propagation method |
| CN108633376A (en) * | 2018-04-21 | 2018-10-12 | 云南省林业科学院 | A kind of cultural method of the sub- non-symbiosis germination of Gree pocket orchid species |
| CN108633376B (en) * | 2018-04-21 | 2021-01-12 | 云南省林业和草原科学院 | Culture method for non-symbiotic germination of paphiopedilum glaucescens seeds |
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Application publication date: 20151223 |