CN105532467A - Method for endangered Chinese azalea in vitro tissue culture propagation and storage - Google Patents

Method for endangered Chinese azalea in vitro tissue culture propagation and storage Download PDF

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CN105532467A
CN105532467A CN201610012027.1A CN201610012027A CN105532467A CN 105532467 A CN105532467 A CN 105532467A CN 201610012027 A CN201610012027 A CN 201610012027A CN 105532467 A CN105532467 A CN 105532467A
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bud
chinese azalea
culture
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vitro
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CN105532467B (en
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罗向东
刘梦露
戴亮芳
刘李珠
杨雪琴
余尚耘
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Jiangxi Normal University
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques

Abstract

The invention discloses a method for endangered Chinese azalea in vitro tissue culture propagation and storage. The best rapid propagation system and storage system are built in the Chinese azalea tissue culture process. According to the method, an optimum explant selection method, an efficient multiple sterilization method, optimum culture medium components, adventive bud rooting, acclimatizating and transplanting condition control for Chinese azalea in vitro growth propagation and storage and other key technological problems are included, and finally large-quantity, rapid and high-quality propagation of Chinese azalea in a short time is achieved. The Chinese azalea test tube plantlet differentiation rate can reach 100%, the adventive bud growth coefficient month proliferation rate can reach 8-10.5, and the rooting rate and the acclimatizating survival rate are 85% or above after 40 d, so that the increasing requirement of people for Chinese azalea can be effectively met, then, damage by people to wild Chinese azalea is reduced, protection of the endangered wild Chinese azalea is facilitated, and huge scientific research value, economic value and ecologic value are achieved.

Description

A kind of Chinese azalea excised cotyledon breeding in imminent danger and the method for preserving
Technical field
The invention belongs to Plant Biotechnology and cell engineering field, relate to a kind of Chinese azalea excised cotyledon breeding in imminent danger and the method for preserving.
Background technology
Chinese azalea (RhododendronmolleG.Don), has another name called Rhododendron molle and Chinese azalea etc., and be that China sheep is walked to and for an only initial species in subgenus, spring blooms, and flower foresythia, can be used for viewing and admiring, and is the valuable source of cuckoo designs and varieties improvement.In addition, Chinese azalea complete stool organ is all containing various active composition, flower, root, fruit etc. all can hyoscines, its extract can treat rheumatoid disease, a kind of malaria, CGN and hypertension etc., as anaesthetic, anodyne on medical industry, being agriculturally used as insecticide, to blood fluke and various pests, all there is extraordinary insect killing effect.
Before the eighties in last century, Chinese azalea throughout Central China, south China, also there is a small amount of distribution southwest.Wild Chinese azalea is mainly by the mode such as seminal propagation, cottage propagation.But because Chinese azalea requires harsh to habitat, tiny seed germinates difficulty under field conditions (factors), and be also difficult to survive to its artificial cuttage or division propagation, add that digging excessively of people is disorderly adopted and the destruction in habitat, the reproduction link of Chinese azalea is had a strong impact on, area and the population quantity of the distribution of Chinese azalea field sharply reduce, habitat is in " islanding ", its trace is seen in some field, province difficulty, wild Chinese azalea cannot meet the needs of people to it already, the risk that Chinese azalea is on the brink of to become extinct is increasingly sharpened, effective method is urgently taked to be protected and breed, enable the production of scale, meet the demand that people are growing.
Plant Biotechnology and tissue culture technique breed endangered plants not by the impact of the factor such as season and environmental condition, are the important channels solving an above-mentioned difficult problem.The matter of utmost importance that the present invention will solve is exactly find the key technical problem such as control of the optimum media components of the effective sterilizing methods of explant and growth and breeding thereof in Chinese azalea tissue culture procedures and adventitious bud rooting, rooting culture condition, and then set up the rapid propagation system of Chinese azalea, finally realize Chinese azalea a large amount of, quick, high-quality breeding at short notice.This can meet people's demand growing to Chinese azalea effectively, and then reduces people to the destruction of wild Chinese azalea, is conducive to the protection of wild Chinese azalea in imminent danger, has huge scientific research value, economic worth and the ecological value.
Summary of the invention
The object of the present invention is to provide a kind of Chinese azalea excised cotyledon breeding in imminent danger and the method for preserving, the area distributed for Chinese azalea field and population quantity sharply reduce, tiny seed germinates difficulty under field conditions (factors), and be also difficult to survive to its artificial cuttage or division propagation, explant sterilization difficulty is there is in tissue culture procedures, the pick-up rate of aseptic explant is low, the deficiency of the problems such as explant Differentiation and proliferation difficulty, the invention provides the method for the in vitro biological control of a kind of Chinese azalea in imminent danger and preservation, a large amount of at short notice for realizing Chinese azalea, fast, high-quality breeding provides technical support, for the demand meeting people growing to Chinese azalea provides plantlet in vitro, technical support and reference is provided for protecting wild Chinese azalea in imminent danger.
The present invention is achieved through the following technical solutions:
(1) live body of explant is bred and is selected: in order to collect the good axillalry bud of more meristematic capacities, the terminal bud of field live body Chinese azalea is cut off, after 10-20 days, each branch newly can sprout the individual new tender axillalry bud of 3-5, when growing to 1-2cm Deng axillalry bud, from axillalry bud base portion clip axillalry bud, clean through tap water, it is for subsequent use that filter paper loads sterile chamber after blotting water droplet;
(2) multiple surface sterilizing flow process: explant banister bruss scrubs branches and leaves, wash away the dust on branches and leaves with running water after, aseptic bottle put into by super-clean bench, with the ethanol surface sterilizing 0.5-1 minute of 70% volume ratio, then multiple sterilizations method sterilizing, namely first uses the oxydol H of 10% 2o 2surface sterilizing 10min, described hydrogen peroxide contains the polysorbas20 of 0.5%, and aseptic water washing 2-3 time, then uses the mercuric chloride HgC1 of 0.1% again 2, described mercuric chloride contains the polysorbas20 of 0.5%, and sterilizing 5-8min, finally uses aseptic water washing 3-5 time, and the axillalry bud explant after sterilizing is for subsequent use;
(3) Initial culture is inoculated: remove explant and be seeded on the medium of axillalry bud startup differentiation, medium consists of 6 of improvement, 7-V+ZT zeatin 1.0mg/L, the dosage of sucrose 30g/L of medium, agar is 0.8%, pH value is 5.6, culture parameters is 25 scholar 2 DEG C, and relative moisture is 60-70%, and intensity of illumination is 2000-2500Lx, light application time 14h/d, technical indicator is that explant pollution rate is lower than 10%-20%;
(4) differentiation is cultivated: when the axillalry bud in step 3 is grown to 4-6cm, remove Lao Ye, get the explant terminal bud that newly grows and stem section about 1-2cm to be transferred to differentiation and to expand on breeding culture medium 6,7-V+ZT1.5mg/L+NAA0.05mg/L+2,4-D0.05mg/L carries out dedifferentiation and Multiplying culture 20-40d, and dedifferentiation frequency reaches 100%; Condition of culture is with step 3;
(5) Vitro Quick Reproduction is cultivated: go on propagating culture medium by the indefinite bud through dedifferentiation in step 4, cultivation composition and condition of culture are with step 3, the indefinite bud of every bottle of about 40-50ml inoculation of medium 3-4 1-2cm, tufted seedling is produced from the differentiation of plantlet in vitro basal part of stem, technical parameter is that adventitious bud proliferation coefficient reaches 8-10.5, the Plantlet in vitro medium of indefinite bud can adopt 1/2 6, 7-V+ zeatin ZT1.0mg/L, the dosage of sucrose 15/L of medium, cultivation temperature 15-20 DEG C, indefinite bud can be transferred once for 3-4 month, switching number of times can be greatly reduced.
(6) culture of rootage and rooting culture: cut in step 5 and get stalwartness, the bud of neat and consistent is used for taking root, root media is 1/26, 7-V minimal medium adds on the medium on 0.05mg/LNAA takes root, condition of culture is with step 3, the bottle cap throwing off seedling of having taken root after 30-40d makes plantlet in vitro reform of nature environment 2-5d, be transplanted into subsequently in nutritive cube, matrix is perlite, Nutrition Soil=1:2, carry out rooting culture, nutritive cube Small plastic shed moisturizing, relative moisture position 70-80%, temperature is 20-25 DEG C, canopy film is opened gradually after 10-15d, Small plastic shed film is opened completely after 15d, technical parameter is that rooting rate and domestication survival rate all reach more than 85%.
Technique effect of the present invention is: the matter of utmost importance that the present invention will solve is exactly find optimum media components and the adventitious bud rooting of the effective sterilizing methods of explant and growth and breeding thereof in Chinese azalea tissue culture procedures, the key technical problems such as the control of rooting culture condition, and then set up the rapid propagation system of Chinese azalea, finally realize Chinese azalea a large amount of at short notice, fast, high-quality breeding, this can meet people's demand growing to Chinese azalea effectively, and then reduce people to the destruction of wild Chinese azalea, be conducive to the protection of wild Chinese azalea in imminent danger, there is huge scientific research value, economic worth and the ecological value.
Accompanying drawing explanation
Fig. 1 is the Chinese azalea branch removing terminal bud, and live body branch sprouts axillalry bud; The axillalry bud that 1-2cm newly sprouts is used as explant.
Fig. 2 is that Chinese azalea carries out dedifferentiation on differential medium, regenerates the indefinite bud of some.
Fig. 3 is Chinese azalea indefinite bud Multiplying culture on proliferated culture medium, and 1 indefinite bud can breed 8-15 bud.
Fig. 4 is that the bud of Chinese azalea in-vitro propagate is taken root in culture of rootage.
Fig. 5 tames the Chinese azalea plantlet in vitro survived.
Embodiment
Describe below in conjunction with accompanying drawing embodiment the beneficial effect that the present invention has in detail, be intended to help reader to understand essence of the present invention better, but any restriction can not be formed to enforcement of the present invention and protection domain.
The present invention is achieved in that and said method comprising the steps of:
(1) select explant: cut off by the terminal bud of field live body Chinese azalea (RhododendronmolleG.Don) branch, after 10-20 days, each branch newly can sprout the individual new tender axillalry bud (Fig. 1) of 3-5.When growing to 1-2cm Deng axillalry bud, from axillalry bud base portion clip axillalry bud, clean through tap water, it is for subsequent use that filter paper loads sterile chamber after blotting water droplet; Experimental result shows, adopts new sprouting axillalry bud to be the regeneration rate high (more than 90%) of explant, pollution rate low (lower than 10%), cycle short (2-4 week gets final product differentiation and proliferation).If adopt terminal bud and branch to be explant, endophyte is many, seriously polluted (pollution rate is up to more than 80%), and regeneration rate is low;
(2) surperficial bacterium is killed: in experimentation of the present invention, successively have employed different explant sterilization methods, finally determine following best sterilizing flow process: by the explant rinsed well on super-clean bench with 70% (V/V) ethanol 0.5-1 minute, then use the hydrogen peroxide (H of 10% 2o 2) (polysorbas20 containing 0.5%) surface sterilizing 10min, aseptic water washing 2-3 time, uses the mercuric chloride (HgC1 of 0.1% subsequently again 2) (polysorbas20 containing 0.5%) sterilizing 5-8min, finally use aseptic water washing 3-5 time, the axillalry bud explant after sterilizing is for subsequent use.The pollution rate explant pollution rate of this multiple sterilizations method is lower than 10%-20%.And traditional be used alone 0.1% mercuric chloride (HgC1 2) the method pollution rate of sterilizing 5-8min is up to more than 80%;
(3) Initial culture: the explant (newborn axillalry bud) of different size (0.5-3cm) is seeded on different Initial culture bases (table 1), 20-40d adds up discovery, the newborn axillalry bud of 1.0-2.cm is in 6 of improvement, the upper growth of 7-V+ZT (zeatin) 1.0mg/L is fast, and growing way is good.The dosage of sucrose 30g/L of medium, agar is 0.8%, and pH value is 5.6.Culture parameters is 25 scholar 2 DEG C, and relative moisture is 60-70%, and intensity of illumination is 2000-2500Lx, light application time 14h/d.Technical indicator be explant pollution rate lower than 10%-20%, start differentiation rate reach more than 90%.The easy callus of explant on the medium of high hormone concentration, is not easy propagation.The axillary bud growth being less than 1.0cm is slow, easy callus.The axillalry bud being greater than 2.0cm not easily regenerates;
(4) differentiation is cultivated: when the axillalry bud in step (3) is grown to 4-6cm, remove Lao Ye, get the explant terminal bud that newly grows and stem section about 1-2cm is transferred to (table 1) on different culture media, statistics after 20-40d shows, 6,7-V+ZT1.5mg/L+NAA0.05mg/L+2,4-D0.05mg/L dedifferentiation is effective, dedifferentiation frequency reaches 100% (Fig. 2), indefinite bud through this medium dedifferentiation grows soon on proliferated culture medium subsequently, and growth coefficient is up to more than 8;
(5) Multiplying culture: the indefinite bud through dedifferentiation in step (4) is gone to (the 1-4 medium in table 1) on propagating culture medium, the indefinite bud of every bottle of about 40-50ml inoculation of medium 3-4 1-2cm, result shows No. 2 medium (6, adventitious bud proliferation effect 7-V+ZT1.0mg/L) is best, and each indefinite bud base portion propagation of about 40d produces 8-10.5 tufted seedling (Fig. 3).The indefinite bud moon, proliferation times reached 8-10.5, and the indefinite bud of acquisition is for Plantlet in vitro and take root domestication and further Multiplying culture.
(6) Plantlet in vitro of Chinese azalea indefinite bud is cultivated: in order to reduce switching number of times, the indefinite bud of cultured in vitro can in vitro retarding of growing, the medium adopted is 1/2 6,7-V+ zeatin ZT1.0mg/L, the dosage of sucrose 15/L of medium, cultivation temperature 15-20 DEG C, other same steps of environmental condition (3) of cultivating, the indefinite bud cultivated under this condition can be transferred once for 3-4 month, can significantly reduce switching number of times.
(7) culture of rootage and rooting culture: cut in step (5) and get stalwartness, the bud of neat and consistent is inoculated on the different root medias in table 2, statistics after 30-40d show R2 medium (1/2 6, adventitious bud rooting effect 7-V+0.05mg/LNAA) is best, rooting rate more than 85% (Fig. 4).The bottle cap of seedling of having taken root makes plantlet in vitro reform of nature environment 2-5d, be transplanted into (matrix is perlite: Nutrition Soil=1:2) in nutritive cube subsequently and carry out rooting culture, nutritive cube is with Small plastic shed moisturizing (relative moisture position 70-80%), temperature is 20-25 DEG C, canopy film is opened gradually after 10-15d, open Small plastic shed film completely after 15d, domestication survival rate all reaches more than 85% (Fig. 5).
The various Chinese azalea of table 1 organizes the medium combination of cultivating breeding in vitro
The medium combination of the various Chinese azalea off-body kidney of table 2
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.

Claims (1)

1. a Chinese azalea excised cotyledon breeding in imminent danger and the method for preserving, it is characterized in that, (1) live body of explant is bred and is selected: in order to collect the good axillalry bud of more meristematic capacities, the terminal bud of field live body Chinese azalea is cut off, after 10-20 days, each branch newly can sprout the individual new tender axillalry bud of 3-5, when waiting axillalry bud to grow to 1-2cm, from axillalry bud base portion clip axillalry bud, clean through tap water, it is for subsequent use that filter paper loads sterile chamber after blotting water droplet;
(2) multiple surface sterilizing flow process: explant banister bruss scrubs branches and leaves, wash away the dust on branches and leaves with running water after, aseptic bottle put into by super-clean bench, with the ethanol surface sterilizing 0.5-1 minute of 70% volume ratio, then multiple sterilizations method sterilizing, namely first uses the oxydol H of 10% 2o 2surface sterilizing 10min, described hydrogen peroxide contains the polysorbas20 of 0.5%, and aseptic water washing 2-3 time, then uses the mercuric chloride HgC1 of 0.1% again 2, described mercuric chloride contains the polysorbas20 of 0.5%, and sterilizing 5-8min, finally uses aseptic water washing 3-5 time, and the axillalry bud explant after sterilizing is for subsequent use;
(3) Initial culture is inoculated: remove explant and be seeded on the medium of axillalry bud startup differentiation, medium consists of 6 of improvement, 7-V+ZT zeatin 1.0mg/L, the dosage of sucrose 30g/L of medium, agar is 0.8%, pH value is 5.6, culture parameters is 25 scholar 2 DEG C, and relative moisture is 60-70%, and intensity of illumination is 2000-2500Lx, light application time 14h/d, technical indicator is that explant pollution rate is lower than 10%-20%;
(4) differentiation is cultivated: when the axillalry bud in step 3 is grown to 4-6cm, remove Lao Ye, get the explant terminal bud that newly grows and stem section about 1-2cm to be transferred to differentiation and to expand on breeding culture medium 6,7-V+ZT1.5mg/L+NAA0.05mg/L+2,4-D0.05mg/L carries out dedifferentiation and Multiplying culture 20-40d, and dedifferentiation frequency reaches 100%; Condition of culture is with step 3;
(5) Vitro Quick Reproduction is cultivated: go on propagating culture medium by the indefinite bud through dedifferentiation in step 4, cultivation composition and condition of culture are with step 3, the indefinite bud of every bottle of about 40-50ml inoculation of medium 3-4 1-2cm, produce tufted seedling from the differentiation of plantlet in vitro basal part of stem, technical parameter is that adventitious bud proliferation coefficient reaches 8-10.5;
(6) Plantlet in vitro of Chinese azalea indefinite bud is cultivated: the indefinite bud of cultured in vitro can in vitro retarding of growing, the medium adopted is 1/2 6,7-V+ zeatin ZT1.0mg/L, the dosage of sucrose 15/L of medium, cultivation temperature 15-20 DEG C, other same steps of environmental condition (3) of cultivating.Technical parameter is transfer once for indefinite bud 3-4 month;
(7) culture of rootage and rooting culture: cut in step 5 and get stalwartness, the bud of neat and consistent is used for taking root, root media is 1/26, 7-V minimal medium adds on the medium on 0.05mg/LNAA takes root, condition of culture is with step 3, the bottle cap throwing off seedling of having taken root after 30-40d makes plantlet in vitro reform of nature environment 2-5d, be transplanted into subsequently in nutritive cube, matrix is perlite, Nutrition Soil=1:2, carry out rooting culture, nutritive cube Small plastic shed moisturizing, relative moisture position 70-80%, temperature is 20-25 DEG C, canopy film is opened gradually after 10-15d, Small plastic shed film is opened completely after 15d, technical parameter is that rooting rate and domestication survival rate all reach more than 85%.
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CN106613973A (en) * 2016-12-14 2017-05-10 江苏省农业科学院 Method for quickly breeding rhododendron molle by approach of regenerating adventitious buds by utilizing tissue culture seedling leaves
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CN116649214A (en) * 2023-06-16 2023-08-29 华中农业大学 Method for establishing rhododendron molle tissue culture technology system
CN117204297A (en) * 2023-10-25 2023-12-12 湖南省植物园 Cultivation and management method for rhododendron molle and rhododendron molle
CN117204297B (en) * 2023-10-25 2024-04-16 湖南省植物园 Cultivation and management method for rhododendron molle and rhododendron molle

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