CN105165629A - Tissue culture rapid propagation method of paphiopedilum markianum - Google Patents
Tissue culture rapid propagation method of paphiopedilum markianum Download PDFInfo
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- CN105165629A CN105165629A CN201510695739.3A CN201510695739A CN105165629A CN 105165629 A CN105165629 A CN 105165629A CN 201510695739 A CN201510695739 A CN 201510695739A CN 105165629 A CN105165629 A CN 105165629A
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Abstract
The invention discloses a tissue culture rapid propagation method of paphiopedilum markianum, belonging to the field of researches of plant tissue culture. The method comprises the following steps: (1) sterilizing explants; (2) carrying out germination culture on seeds; (3) carrying out multiplication culture; (4) carrying out differentiation culture; (5) carrying out rooting culture; and (6) carrying out acclimatization and transplanting. Paphiopedilum markianum capsules are collected at proper time; after sterilization, seeds are sowed into a sterile germination culture medium to germinate; different culture medium formulas are used for culturing at different culture phases, and then high-quality seedlings which are healthy and have stable inheritable characters are obtained by acclimatization and transplanting. According to the method, the propagation coefficient is high, the propagation period is short, the survival rate of transplanting is 92% and above, and large-scale commercialized seedling culture production can be carried out, so that the protection and development of paphiopedilum markianum wild resources are facilitated, and the application prospect is wide.
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 tiger spot pocket.
Background technology
Tiger spot pocket orchid (PaphiopedilummarkianumFowlie) is the raw or semiepiphyte in the orchid family pocket blue subgenus ground, special product of China kind, originate in From Western Yunnan, many growths are at height above sea level 1500 ~ 2000m, on the sparse woods tree of In Limestone Area, cover the rock Shang Huoduoshi district of liver moss, Wild ornamental resources is very rare, the narrow square of blade is circular, Hua great, pistac, 6 ~ August of florescence, can be used for botanical garden, potted plant, cut-flower or layout flower border, there is unique ornamental value and Breeding Potential, but due to the destruction in habitat, artificial destructiveness is excavated, sell, tiger spot pocket becomes one of species in imminent danger in the world, be put into " CITS ", urgent need takes measures to be protected.
Pocket orchid is that group trains one of the most difficult orchid, and about the research report of tiger spot pocket orchid is few, the tissue cultures of crossbreed is relatively easy, the tissue cultures of initial species is more difficult, and the stem of tiger spot pocket orchid is extremely short, again near soil-grown, cut explant very difficult, very easily by Browning.Tiger spot pocket is blue owing to having more seed in single fruit, thus utilizing seed asepsis sprouting to cultivate is conventional breeding practice, but be subject to provenance restriction, because the blue population quantity of tiger spot pocket is less gradually, almost be difficult to the population of seeking natural health growth at present in the wild, if bred with the seed that minority individuality produces for a long time, will the threat of dieback be faced after some generations, utilize seed as explant at present, the method for carrying out tissue cultures not yet makes a breakthrough.
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 tiger spot pocket, the technical scheme of employing is as follows:
(1) explant sterilization: after artificial pollination 140d, gathers uncracked capsule as explant, with 75% alcohol sterilizing 30 ~ 50s, then uses 10% clorox sterilizing 10 ~ 15min, aseptic water washing 5 ~ 7 times;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, seed development becomes protocorm, and described seed germination medium is 1/2RE+6-BA0.6mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 ~ 9 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.2mg/L+ tryptone 3g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the seedling with 1 ~ 2 leaf, described differential medium is: 1/2MS+6-BA0.5mg/L+KT0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(5) culture of rootage: seedling is proceeded to root media, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 2000 ~ 2500Lx condition, obtain the test-tube plantlet of the long 3 ~ 4cm of root, described root media is: 1/2MS+IBA0.3mg/L+NAA0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(6) acclimatization and transplants: test-tube plantlet band bottle is moved to normal temperature, without under direct solar environment, hardening 1 week, take out test-tube plantlet, clean medium with clear water, plant in nutrition cup with the sphagna parcel root soaked, environmental requirement well-ventilated, shading 65% ~ 70%, relative air humidity 70% ~ 75%, temperature 15 ~ 28 DEG C, after 2 weeks, test-tube plantlet can survive.
Beneficial effect of the present invention is embodied in: the present invention with the blue seed of tiger spot pocket for explant; establish tissue culture propagation system; achieve the breakthrough of the blue tissue culture and rapid propagation method of tiger spot pocket; adopt the plantlet in vitro stabilization characteristics of genetics that this method obtains; reproduction coefficient is high, and the breeding cycle is short, and transplanting survival rate reaches more than 92%; be conducive to the Con-servation and development of the blue wild resource of tiger spot pocket, be with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that seed germination of the present invention is cultivated.
Fig. 2 is the schematic diagram of Multiplying culture of the present invention.
Fig. 3 is the schematic diagram that differentiation of the present invention is cultivated.
Fig. 4 is the schematic diagram of culture of rootage of the present invention.
Fig. 5 is the schematic diagram that test-tube seedling transplanting of the present invention survives.
Embodiment
Embodiment 1
The blue tissue culture and rapid propagation method of a kind of tiger spot pocket, is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 140d, gathers uncracked capsule as explant, with 75% alcohol sterilizing 40s, then uses 10% clorox sterilizing 12min, aseptic water washing 6 times;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 52d under intensity of illumination 1600 ~ 2000Lx condition, seed development becomes protocorm, and described seed germination medium is 1/2RE+6-BA0.6mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, PH is 6.0 ~ 6.5, as shown in Figure 1;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, temperature 25 ± 2 DEG C, illumination 14h/d, 60d is cultivated under intensity of illumination 1600 ~ 2000Lx condition, obtain the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.2mg/L+ tryptone 3g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, temperature 25 ± 2 DEG C, illumination 14h/d, 52d is cultivated under intensity of illumination 1600 ~ 2000Lx condition, obtain the seedling with 1 ~ 2 leaf, described differential medium is: 1/2MS+6-BA0.5mg/L+KT0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 3;
(5) culture of rootage: seedling is proceeded to root media, temperature 25 ± 2 DEG C, illumination 14h/d, 50d week is cultivated under intensity of illumination 2000 ~ 2500Lx condition, obtain the test-tube plantlet of the long 3 ~ 4cm of root, described root media is: 1/2MS+IBA0.3mg/L+NAA0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 4;
(6) acclimatization and transplants: test-tube plantlet band bottle is moved to normal temperature, without under direct solar environment, hardening 1 week, takes out test-tube plantlet, cleans medium with clear water, plant in nutrition cup with the sphagna parcel root soaked, environmental requirement well-ventilated, shading 65% ~ 70%, relative air humidity 70% ~ 75%, temperature 15 ~ 28 DEG C, after 2 weeks, test-tube plantlet can survive, and transplanting survival rate reaches more than 93%, as shown in Figure 5.
Embodiment 2
The blue tissue culture and rapid propagation method of a kind of tiger spot pocket, is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 140d, gathers uncracked capsule as explant, with 75% alcohol sterilizing 30s, then uses 10% clorox sterilizing 10min, aseptic water washing 5 times;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 weeks under intensity of illumination 1600 ~ 2000Lx condition, seed development becomes protocorm, and described seed germination medium is 1/2RE+6-BA0.6mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, PH is 6.0 ~ 6.5, as shown in Figure 1;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.2mg/L+ tryptone 3g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the seedling with 1 ~ 2 leaf, described differential medium is: 1/2MS+6-BA0.5mg/L+KT0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 3;
(5) culture of rootage: seedling is proceeded to root media, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 weeks under intensity of illumination 2000 ~ 2500Lx condition, obtain the test-tube plantlet of the long 3 ~ 4cm of root, described root media is: 1/2MS+IBA0.3mg/L+NAA0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 4;
(6) acclimatization and transplants: test-tube plantlet band bottle is moved to normal temperature, without under direct solar environment, hardening 1 week, takes out test-tube plantlet, cleans medium with clear water, plant in nutrition cup with the sphagna parcel root soaked, environmental requirement well-ventilated, shading 65% ~ 70%, relative air humidity 70% ~ 75%, temperature 15 ~ 28 DEG C, after 2 weeks, test-tube plantlet can survive, and transplanting survival rate reaches more than 92%, as shown in Figure 5.
Embodiment 3
The blue tissue culture and rapid propagation method of a kind of tiger spot pocket, is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 140d, gathers uncracked capsule as explant, with 75% alcohol sterilizing 50s, then uses 10% clorox sterilizing 15min, aseptic water washing 7 times;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, seed development becomes protocorm, and described seed germination medium is 1/2RE+6-BA0.6mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, PH is 6.0 ~ 6.5, as shown in Figure 1;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 9 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.2mg/L+ tryptone 3g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 2;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the seedling with 1 ~ 2 leaf, described differential medium is: 1/2MS+6-BA0.5mg/L+KT0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 3;
(5) culture of rootage: seedling is proceeded to root media, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 weeks under intensity of illumination 2000 ~ 2500Lx condition, obtain the test-tube plantlet of the long 3 ~ 4cm of root, described root media is: 1/2MS+IBA0.3mg/L+NAA0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5, as shown in Figure 4;
(6) acclimatization and transplants: test-tube plantlet band bottle is moved to normal temperature, without under direct solar environment, hardening 1 week, takes out test-tube plantlet, cleans medium with clear water, plant in nutrition cup with the sphagna parcel root soaked, environmental requirement well-ventilated, shading 65% ~ 70%, relative air humidity 70% ~ 75%, temperature 15 ~ 28 DEG C, after 2 weeks, test-tube plantlet can survive, and transplanting survival rate reaches more than 96%, as shown in Figure 5.
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 (1)
1. the blue tissue culture and rapid propagation method of tiger spot pocket, is characterized in that comprising the following steps:
(1) explant sterilization: after artificial pollination 140d, gathers uncracked capsule as explant, with 75% alcohol sterilizing 30 ~ 50s, then uses 10% clorox sterilizing 10 ~ 15min, aseptic water washing 5 ~ 7 times;
(2) seed germination is cultivated: aseptically cut by the capsule after sterilizing, by seed uniform broadcasting on seed germination medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, seed development becomes protocorm, and described seed germination medium is 1/2RE+6-BA0.6mg/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(3) Multiplying culture: protocorm is transferred to proliferated culture medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 8 ~ 9 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the mixture of protocorms and bud, described proliferated culture medium is: 1/2MS+6-BA1.0mg/L+NAA0.2mg/L+ tryptone 3g/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(4) differentiation is cultivated: the mixture obtained by Multiplying culture is transferred to differential medium, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 1600 ~ 2000Lx condition, obtain the seedling with 1 ~ 2 leaf, described differential medium is: 1/2MS+6-BA0.5mg/L+KT0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(5) culture of rootage: seedling is proceeded to root media, temperature 25 ± 2 DEG C, illumination 14h/d, cultivate 7 ~ 8 weeks under intensity of illumination 2000 ~ 2500Lx condition, obtain the test-tube plantlet of the long 3 ~ 4cm of root, described root media is: 1/2MS+IBA0.3mg/L+NAA0.5mg/L+ fresh jujube juice 80ml/L+AC1.0g/L+ sucrose 20g/L+ agar 6g/L, and PH is 6.0 ~ 6.5;
(6) acclimatization and transplants: test-tube plantlet band bottle is moved to normal temperature, without under direct solar environment, hardening 1 week, take out test-tube plantlet, clean medium with clear water, plant in nutrition cup with the sphagna parcel root soaked, environmental requirement well-ventilated, shading 65% ~ 70%, relative air humidity 70% ~ 75%, temperature 15 ~ 28 DEG C, after 2 weeks, test-tube plantlet can survive.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106613960A (en) * | 2016-10-28 | 2017-05-10 | 中国科学院华南植物园 | Rapid propagation method of paphiopedilum helenae callus regeneration system |
| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106613960A (en) * | 2016-10-28 | 2017-05-10 | 中国科学院华南植物园 | Rapid propagation method of paphiopedilum helenae callus regeneration system |
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| CN113951144A (en) * | 2021-12-03 | 2022-01-21 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
| CN113951144B (en) * | 2021-12-03 | 2022-11-04 | 中国科学院昆明植物研究所 | Method for promoting sterile germination and seedling formation of paphiopedilum sansevieri seeds |
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