CN107125136A - A kind of Camellia nitidissima tissue culture mating system - Google Patents
A kind of Camellia nitidissima tissue culture mating system Download PDFInfo
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- CN107125136A CN107125136A CN201710426483.5A CN201710426483A CN107125136A CN 107125136 A CN107125136 A CN 107125136A CN 201710426483 A CN201710426483 A CN 201710426483A CN 107125136 A CN107125136 A CN 107125136A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of Camellia nitidissima tissue culture mating system, comprise the following steps：1) selection and sterilization of explant：Choose standby after Camellia nitidissima seed is sterilized as explant；2) preparation of golden flower tea extracts：Take Camellia nitidissima blade to add water and boil to obtain golden flower tea extracts；3) primary Fiber differentiation：By seed, it is inoculated into primary inducing culture and cultivates 8 12 days, until the seed grows terminal bud；4) shoot proliferation culture：Terminal bud is inoculated into subculture multiplication medium and cultivated 16 20 days, induction terminal bud propagation obtains Multiple Buds；5) culture of rootage：When the simple bud length of Multiple Buds is to 3.0~5.0cm, Tissue Culture Seedings of Chrysantha is inoculated into root media and carries out root induction culture 22~26 days；6) domestication and transplanting of test tube seedling.This method is not by seasonal effect, and the cultivation time is short, and proliferative speed is fast, solves current Camellia nitidissima germ plasm resource rareness, and the low problem of fruiting rate realizes Camellia nitidissima breeding low cost and bred on a large scale.
Description
Technical field
Field is bred the invention belongs to plant tissue culture, and in particular to a kind of Camellia nitidissima tissue culture mating system.
Background technology
Camellia nitidissima belongs to Theaceae, Camellia, is twin sisters with tea, camellia, South Mountain tea, oil tea, tea plum etc., is country
One of first class of protection plant.Camellia nitidissima spends golden yellow, dazzling brilliant, seemingly applies coating of wax, sparkling and crystal-clear and glossy, seemingly has semi-transparent
Bright sense.Camellia nitidissima is singly born in axil, when the flowers are in blossom, have cup-shaped, gyalectiform or bowl-shape, delicate and charming colourful, beautiful grace.With
Before, people did not see the golden yellow species of pattern.Nineteen sixty, Chinese science worker is found that in the band of Guangxi region one first
A kind of golden yellow camellia, is named as Camellia nitidissima.Foreign countries are referred to as magical " Dongfang " magic tea, are described as " plant kingdom great Xiong
Cat ", " tea race queen ".
However, plant and seed material of traditional camellia propagation method (cuttage, grafting, press strip, sowing) to Camellia nitidissima
Demand is big, the protection of serious restriction Precious, Rare, Endangered Camellia nitidissima species and amount reproduction；Meanwhile, this is bred bottleneck and also causes one
The low camellia hybrid new breed of a little rare setting percentages is difficult to scale up promoting.Therefore, the quick breeding for realizing Camellia nitidissima is current
The task of top priority of Camellia nitidissima breeder.
Plant Tissue Breeding is aseptically, by the isolated organ of plant, tissue, cell or protoplast, culture
On the culture medium manually prepared, manual control culture medium condition makes its growth, differentiation, propagation, develops into the mistake of intact plant
Journey.Because tissue cultures are carried out under conditions of plant parent is departed from, so also referred to as cultured in vitro.Plant Tissue Breeding
It is few with required plant raw material, do not limited, cultivation cycle is short, made a variation low by the season of growth, maternal excellent heredity can be kept
The advantages of characteristic；Therefore, plant tissue culture technique can be used for rescue endangered plants, solve plant production seed it is few or without and can not
Prolific problem.Plant tissue culture technique is undoubtedly the Gospel of camellia plant breeder, and this is that breeding for Camellia nitidissima carries
Supply more feasible scheme, it is to avoid the purebred breeding work of first-filial generation, substantially reduce breeding time limit.Existing camellia is educated
Kind of worker has carried out the research of related fields, and Li Jianan etc. (2003) is carried out to the flower pesticide of camellia meiocarpa and C. semisererata
Cultured in vitro, and induced synthesis callus；Chen Lihua (2007) etc. using the tender shoots of Yunnan Red camellia oleosa and with bud stem segments as
Explant material, the explant processing method and clump bud inducement cultivation base of its stem tip tissue culture of research and probe, has filtered out cloud
The convenient clump bud inducement cultivation base of south carthamus tinctorius oil tea；Gao Yuqiong and Lai Zhongxiong (2010) are with camellia nitidissima cotyledonary embryos stripping and slicing and adult
Plant leaf is material, carries out callus tissue culture research, successfully induces callus, and can preferably subculture.
The content of the invention
For problems of the prior art, the invention provides a kind of Camellia nitidissima tissue culture mating system, this kind of tissue culture
Mating system is not by seasonal effect, and the cultivation time is short, and proliferative speed is fast, solves current Camellia nitidissima germ plasm resource rareness, as a result
The low problem of rate, realizes Camellia nitidissima breeding low cost and breeds on a large scale.
In order to achieve the above object, the present invention is achieved through the following technical solutions：
A kind of Camellia nitidissima tissue culture mating system, is followed the steps below：
(1) selection and sterilization of explant：The disease-free Camellia nitidissima seed of health is chosen as explant, first by seed through stream
Water is rinsed after 2-4min, on superclean bench with percentage by volume for 75% ethanol solution surface sterilizing 1-3min, it is sterile
Water is rinsed 3 times, and mass percent is 0.1% HgCl2Solution sterilization 4-6min, aseptic water washing 4 times；
(2) preparation of golden flower tea extracts：The Camellia nitidissima blade for taking proper amount of fresh light green is added in open extractor,
Add water and be heated to boil condition and decoct 40-60min, the material liquid volume ratio of blade and water is 1:2-4, pours out extract solution extremely afterwards
In fluid storage tanks；Water is added into extractor again and is heated to boil condition decoction 1-2h, and the material liquid volume ratio of blade and water
For 1:3-5, pours out extract solution into the fluid storage tanks, the extract solution in last concentrated liquid storage tank to the 1/ of original volume
5-1/3, obtains golden flower tea extracts；
(3) primary Fiber differentiation：The sterilized seed of step (1), it is inoculated into primary inducing culture, in sterile bar
Under part, cultivate 8-12 days, until the seed grows terminal bud；
Primary inducing culture be 0.8~1.2g/L+6-BA of 1/2MS minimal medium+golden flower tea extracts 0.4~
4.0~8.0g/L of 0.8mg/L+NAA 0.2~0.6mg/L+ sucrose 20~30g/L+ nutrient solutions；
(4) shoot proliferation culture：The terminal bud is inoculated into subculture multiplication medium and cultivated 16-20 days, terminal bud is induced
Propagation, obtains Multiple Buds；
Proliferated culture medium is 0.4~0.6mg/L of 1/2B5 minimal medium+golden flower 0.8~1.2g/L+6-BA of tea extract
4.0~6.0g/L of+NAA 0.3~0.5mg/L+ sucrose 20~30g/L+ agar；
(5) culture of rootage：When the simple bud length of the Multiple Buds is to 3.0~5.0cm, Tissue Culture Seedings of Chrysantha is inoculated into life
Root induction culture 22~26 days is carried out in root culture medium；
Root media is 0.6~0.8mg/L+NAA0.4 of DCR culture medium+golden flower 1.0~1.6g/L+IBA of tea extract
6.0~8.0g/L of~0.6mg/L+ sucrose 15~25g/L+ agar；
(6) domestication and transplanting of test tube seedling：Camellia nitidissima rooting tube plantlet is moved into vinyl house from culturing room and places 4~6
After it, bottle cap hardening is gone 2~4 days, take out Camellia nitidissima seedling, its root is handled after 1~2min with 0.1% carbendazim, transplant
In cultivation matrix into hole tray, overlay film moisturizing 3~5 days, therebetween the morning and afternoon it is each it is ventilated once, every time 20~
30min, shade 65-75%, takes off the thiophanate methyl that 600-800 times of liquid is sprayed after film, and according to 10g/m2Consumption sprayed
Apply, hereafter carry out normal cultivation management, you can.
Preferably, the nutrient solution includes following component and its corresponding concentration：
0.02~0.04g/L of ammonium sulfate, 0.01~0.03g/L of calcium chloride dihydrate, 0.01~0.03g/L of epsom salt,
0.04~0.06g/L of anhydrous potassium dihydrogenphosphate, 0.01~0.03g/L of zinc sulfate, 0.02~0.04g/L of KI.
Further, the condition of culture of the primary Fiber differentiation, shoot proliferation culture and culture of rootage：Environment temperature
24 ± 2 DEG C, 12~16h/d of light application time, intensity of illumination is 2000~3000lx.
Preferably, the composition and its content of the DCR culture mediums are：NH4NO30.03-0.05g/L、KH2PO40.06-
0.08g/L、CaCl2·2H2O 0.02-0.04g/L、Ca(NO3)2·4H2O 0.06-0.1g/L、MgSO4·7H2O0.06-
0.08g/L、FeSO4·7H2O 0.04-0.06g/L、ZnSO4·7H2O 0.05-0.07g/L, KI 0.04-0.08g/L, sweet dew
Alcohol 0.3-0.5g/L, VB10.01-0.03g/L, VC 0.02-0.04g/L.
Further, the primary inducing culture is 1/2MS minimal medium+golden flower tea extracts 1.0g/L+6-
BA0.6mg/L+NAA 0.4mg/L+ sucrose 25g/L+ nutrient solutions 6.0g/L.
Further, the proliferated culture medium is 1/2B5 minimal medium+golden flower tea extracts 1.0g/L+6-
BA0.5mg/L+NAA 0.4mg/L+ sucrose 25g/L+ agar 5.0g/L.
Further, the root media is DCR culture medium+golden flower tea extract 1.3g/L+IBA 0.7mg/L+
NAA0.5mg/L+ sucrose 20g/L+ agar 7.0g/L.
Preferably, described cultivation matrix includes following raw material by weight：30-50 parts of diatomite, charcoal 15-
25 parts, 10-20 parts of haydite, 8-12 parts of rice chaff ash, 5-15 parts of pond sludge；
The charcoal, which is prepared by the following method, to be obtained：Cotton stem, vegetables bar weedtree branch or/and melon seedling are dried
To water content below 20%, then cut or smash, in input carbide furnace, carbonization 2.5~3.5 is small at 420 DEG C~480 DEG C
When after go out charcoal, be cooled to room temperature with water filtration when going out charcoal, then dry charcoal, crush, produce charcoal.
The following beneficial effect of present invention tool：
(1) Camellia nitidissima tissue culture mating system of the invention substantially reduces the growing-seedling period of Camellia nitidissima, saves nursery material
Material, overcomes the problems such as nursery material requested is more, the cycle is long present in traditional Camellia nitidissima method for culturing seedlings, greatly reduces production
Cost, improves production efficiency, the problems such as solving low current Camellia nitidissima cuttage survival rate, breeding cycle length, quality deterioration；
(2) Camellia nitidissima tissue culture mating system of the invention has sapling multiplication fast, and sapling multiplication is not influenceed by season, and one
Year four seasons can all cultivate nursery stock, while the problem of breeding for solving scientific research seed selection is applied, conventional breeding production it is simple according to
Rely the method for building breeding garden, soil, funds saved using this method and the problem of breeding garden production cycle is long is solved,
Popularizing application prospect is good；
(3) using the Camellia nitidissima tissue culture mating system of the present invention, using golden flower tea extracts as nourishing additive agent, can have
Effect ground makes breeding by tissue culture vast propagation, with good economic benefit, social benefit and ecological benefits.
Embodiment
The embodiment of the present invention is further described with reference to embodiment, following examples are only used for more
Technical scheme is clearly demonstrated, and can not be limited the scope of the invention with this.
Embodiment 1
The condition of culture of following primary Fiber differentiation, shoot proliferation culture and culture of rootage：22 DEG C of environment temperature, illumination
Time 12h/d, intensity of illumination is 2000lx.
A kind of Camellia nitidissima tissue culture mating system, is followed the steps below：
(1) selection and sterilization of explant：The disease-free Camellia nitidissima seed of health is chosen as explant, first by seed through stream
Water is rinsed after 2min, with the ethanol solution surface sterilizing 1min that percentage by volume is 75% on superclean bench, sterilized water punching
Wash 3 times, mass percent is 0.1% HgCl2Solution sterilization 4min, aseptic water washing 4 times；
(2) preparation of golden flower tea extracts：The fresh light green Camellia nitidissima blades of 400g are taken to be added in open extractor,
Add water and be heated to boil condition and decoct 40min, the material liquid volume ratio of blade and water is 1:2, extract solution is poured out afterwards to liquid
In storage tank；Water is added into extractor again and is heated to boil condition decoction 1h, and the material liquid volume ratio of blade and water is 1:3,
Extract solution is poured out into the fluid storage tanks, the extract solution in last concentrated liquid storage tank obtains golden flower to the 1/5 of original volume
Tea extract；
(3) primary Fiber differentiation：The sterilized seed of step (1), it is inoculated into primary inducing culture, in sterile bar
Under part, cultivate 8 days, until the seed grows terminal bud；
Primary inducing culture is 1/2MS minimal medium+golden flower tea extract 0.8g/L+6-BA 0.4mg/L+
NAA0.2mg/L+ sucrose 20g/L+ nutrient solutions 4.0g/L；
(4) shoot proliferation culture：The terminal bud is inoculated into subculture multiplication medium and cultivated 16 days, induction terminal bud increases
Grow, obtain Multiple Buds；
Proliferated culture medium is 1/2B5 minimal medium+golden flower tea extract 0.8g/L+6-BA 0.4mg/L+NAA0.3mg/
L+ sucrose 20g/L+ agar 4.0g/L；
(5) culture of rootage：When the simple bud length of the Multiple Buds is to 3.0cm, Tissue Culture Seedings of Chrysantha is inoculated into training of taking root
Support and root induction culture 22 days is carried out in base；
Root media is DCR culture medium+golden flower tea extract 1.0g/L+IBA 0.6mg/L+NAA 0.4mg/L+ sucrose
15g/L+ agar 6.0g/L；
(6) domestication and transplanting of test tube seedling：Camellia nitidissima rooting tube plantlet is moved into vinyl house from culturing room to place 4 days
Afterwards, go bottle cap hardening 2 days, take out Camellia nitidissima seedling, its root is handled after 1min with 0.1% carbendazim, transplant into hole tray
Cultivation matrix in, overlay film moisturizing 3 days, therebetween the morning and afternoon it is each it is ventilated once, each 20min, shade 65%, take off film
The thiophanate methyl of 600 times of liquid is sprayed afterwards, and according to 10g/m2Consumption sprayed, hereafter carry out normal cultivation management,
.
Wherein, the nutrient solution includes following component and its corresponding concentration：Ammonium sulfate 0.02g/L, calcium chloride dihydrate
0.01g/L, epsom salt 0.01g/L, anhydrous potassium dihydrogenphosphate 0.04g/L, zinc sulfate 0.01g/L and KI 0.02g/L；
The composition and its content of the DCR culture mediums be：NH4NO30.03g/L、KH2PO40.06g/L、CaCl2·
2H2O0.02g/L、Ca(NO3)2·4H2O 0.06g/L、MgSO4·7H2O 0.06g/L、FeSO4·7H2O 0.04g/L、
ZnSO4·7H2O 0.05g/L, KI 0.04g/L, mannitol 0.3g/L, VB10.01g/L and VC 0.02g/L.
Described cultivation matrix includes following raw material：Diatomite 30kg, charcoal 15kg, haydite 10kg, rice chaff ash 8kg,
Pond sludge 5kg；Above-mentioned charcoal, which is prepared by the following method, to be obtained：Cotton stem and vegetables bar weedtree branch are dried to aqueous
Then amount is cut or is smashed below 20%, in input carbide furnace, goes out charcoal after being carbonized 2.5 hours at 420 DEG C DEG C, when going out charcoal
Room temperature is cooled to water filtration, then charcoal is dried, crushes, produces charcoal.
Embodiment 2
The condition of culture of following primary Fiber differentiation, shoot proliferation culture and culture of rootage：24 DEG C of environment temperature, illumination
Time 14h/d, intensity of illumination is 2500lx.
A kind of Camellia nitidissima tissue culture mating system, is followed the steps below：
(1) selection and sterilization of explant：The disease-free Camellia nitidissima seed of health is chosen as explant, first by seed through stream
Water is rinsed after 3min, with the ethanol solution surface sterilizing 2min that percentage by volume is 75% on superclean bench, sterilized water punching
Wash 3 times, mass percent is 0.1% HgCl2Solution sterilization 5min, aseptic water washing 4 times；
(2) preparation of golden flower tea extracts：The fresh light green Camellia nitidissima blades of 500g are taken to be added in open extractor,
Add water and be heated to boil condition and decoct 50min, the material liquid volume ratio of blade and water is 1:3, extract solution is poured out afterwards to liquid
In storage tank；Water is added into extractor again and is heated to boil condition decoction 1.5h, and the material liquid volume ratio of blade and water is 1:
4, extract solution is poured out into the fluid storage tanks, and the extract solution in last concentrated liquid storage tank obtains golden to the 1/4 of original volume
Jasmine tea extract solution；
(3) primary Fiber differentiation：The sterilized seed of step (1), it is inoculated into primary inducing culture, in sterile bar
Under part, cultivate 10 days, until the seed grows terminal bud；
Primary inducing culture is 1/2MS minimal medium+golden flower tea extract 1.0g/L+6-BA 0.6mg/L+
NAA0.4mg/L+ sucrose 25g/L+ nutrient solutions 6.0g/L；
(4) shoot proliferation culture：The terminal bud is inoculated into subculture multiplication medium and cultivated 18 days, induction terminal bud increases
Grow, obtain Multiple Buds；
Proliferated culture medium is 1/2B5 minimal medium+golden flower tea extract 1.0g/L+6-BA 0.5mg/L+NAA0.4mg/
L+ sucrose 25g/L+ agar 5.0g/L；
(5) culture of rootage：When the simple bud length of the Multiple Buds is to 4.0cm, Tissue Culture Seedings of Chrysantha is inoculated into training of taking root
Support and root induction culture 24 days is carried out in base；
Root media is DCR culture medium+golden flower tea extract 1.4g/L+IBA 0.7mg/L+NAA 0.5mg/L+ sucrose
20g/L+ agar 7.0g/L；
(6) domestication and transplanting of test tube seedling：Camellia nitidissima rooting tube plantlet is moved into vinyl house from culturing room to place 5 days
Afterwards, go bottle cap hardening 3 days, take out Camellia nitidissima seedling, its root is handled after 1.5min with 0.1% carbendazim, transplant to hole tray
In cultivation matrix in, overlay film moisturizing 4 days, therebetween the morning and afternoon it is each it is ventilated once, each 25min, shade 70%, take off
The thiophanate methyl of 700 times of liquid is sprayed after film, and according to 10g/m2Consumption sprayed, hereafter carry out normal cultivation pipe
Reason, you can.
Wherein, the nutrient solution includes following component and its corresponding concentration：Ammonium sulfate 0.03g/L, calcium chloride dihydrate
0.02g/L, epsom salt 0.02g/L, anhydrous potassium dihydrogenphosphate 0.05g/L, zinc sulfate 0.02g/L and KI 0.03g/L；
The composition and its content of the DCR culture mediums be：NH4NO30.04g/L、KH2PO40.07g/L、CaCl2·
2H2O0.03g/L、Ca(NO3)2·4H2O 0.08g/L、MgSO4·7H2O 0.07g/L、FeSO4·7H2O 0.05g/L、
ZnSO4·7H2O 0.06g/L, KI 0.06g/L, mannitol 0.4g/L, VB10.02g/L and VC 0.03g/L.
Described cultivation matrix includes following raw material：Diatomite 40kg, charcoal 20kg, haydite 15kg, rice chaff ash 10kg,
Pond sludge 10kg；Above-mentioned charcoal, which is prepared by the following method, to be obtained：Cotton stem, vegetables bar weedtree branch and melon seedling are dried
To water content below 20%, then cut or smash, in input carbide furnace, go out charcoal after being carbonized 3 hours at 450 DEG C, go out charcoal
When room temperature is cooled to water filtration, then charcoal is dried, crush, produce charcoal.
Embodiment 3
The condition of culture of following primary Fiber differentiation, shoot proliferation culture and culture of rootage：26 DEG C of environment temperature, illumination
Time 16h/d, intensity of illumination is 3000lx.
A kind of Camellia nitidissima tissue culture mating system, is followed the steps below：
(1) selection and sterilization of explant：The disease-free Camellia nitidissima seed of health is chosen as explant, first by seed through stream
Water is rinsed after 4min, with the ethanol solution surface sterilizing 3min that percentage by volume is 75% on superclean bench, sterilized water punching
Wash 3 times, mass percent is 0.1% HgCl2Solution sterilization 6min, aseptic water washing 4 times；
(2) preparation of golden flower tea extracts：The fresh light green Camellia nitidissima blades of 600g are taken to be added in open extractor,
Add water and be heated to boil condition and decoct 60min, the material liquid volume ratio of blade and water is 1:4, extract solution is poured out afterwards to liquid
In storage tank；Water is added into extractor again and is heated to boil condition decoction 2h, and the material liquid volume ratio of blade and water is 1:5,
Extract solution is poured out into the fluid storage tanks, the extract solution in last concentrated liquid storage tank obtains golden flower to the 1/3 of original volume
Tea extract；
(3) primary Fiber differentiation：The sterilized seed of step (1), it is inoculated into primary inducing culture, in sterile bar
Under part, cultivate 12 days, until the seed grows terminal bud；
Primary inducing culture is 1/2MS minimal medium+golden flower tea extract 1.2g/L+6-BA 0.8mg/L+
NAA0.6mg/L+ sucrose 30g/L+ nutrient solutions 8.0g/L；
(4) shoot proliferation culture：The terminal bud is inoculated into subculture multiplication medium and cultivated 20 days, induction terminal bud increases
Grow, obtain Multiple Buds；
Proliferated culture medium is 1/2B5 minimal medium+golden flower tea extract 1.2g/L+6-BA 0.6mg/L+NAA0.5mg/
L+ sucrose 30g/L+ agar 6.0g/L；
(5) culture of rootage：When the simple bud length of the Multiple Buds is to 5.0cm, Tissue Culture Seedings of Chrysantha is inoculated into training of taking root
Support and root induction culture 26 days is carried out in base；
Root media is DCR culture medium+golden flower tea extract 1.6g/L+IBA 0.8mg/L+NAA 0.6mg/L+ sucrose
25g/L+ agar 8.0g/L；
(6) domestication and transplanting of test tube seedling：Camellia nitidissima rooting tube plantlet is moved into vinyl house from culturing room to place 6 days
Afterwards, go bottle cap hardening 4 days, take out Camellia nitidissima seedling, its root is handled after 2min with 0.1% carbendazim, transplant into hole tray
Cultivation matrix in, overlay film moisturizing 5 days, therebetween the morning and afternoon it is each it is ventilated once, each 30min, shade 75%, take off film
The thiophanate methyl of 800 times of liquid is sprayed afterwards, and according to 10g/m2Consumption sprayed, hereafter carry out normal cultivation management,
.
Wherein, the nutrient solution includes following component and its corresponding concentration：Ammonium sulfate 0.04g/L, calcium chloride dihydrate
0.03g/L, epsom salt 0.03g/L, anhydrous potassium dihydrogenphosphate 0.06g/L, zinc sulfate 0.03g/L and KI 0.04g/L；
The composition and its content of the DCR culture mediums be：NH4NO30.05g/L、KH2PO40.08g/L、CaCl2·
2H2O0.04g/L、Ca(NO3)2·4H2O 0.1g/L、MgSO4·7H2O 0.08g/L、FeSO4·7H2O 0.06g/L、
ZnSO4·7H2O0.07g/L, KI 0.08g/L, mannitol 0.5g/L, VB10.03g/L and VC 0.04g/L.
Described cultivation matrix includes following raw material：Diatomite 50kg, charcoal 25kg, haydite 20kg, rice chaff ash 12kg
With pond sludge 15kg；Above-mentioned charcoal, which is prepared by the following method, to be obtained：By vegetables bar weedtree branch and melon seedling dry to containing
Then water is cut or is smashed below 20%, in input carbide furnace, goes out charcoal after being carbonized 3.5 hours at 480 DEG C, when going out charcoal
Room temperature is cooled to water filtration, then charcoal is dried, crushes, produces charcoal.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although with reference to foregoing reality
Apply example the present invention is described in detail, for those skilled in the art, it still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic.All essences in the present invention
God is with principle, and any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (8)
1. a kind of Camellia nitidissima tissue culture mating system, it is characterised in that comprise the following steps：
(1) selection and sterilization of explant：The disease-free Camellia nitidissima seed of health is chosen as explant, first rushes seed through flowing water
Wash after 2-4min, with the ethanol solution surface sterilizing 1-3min that percentage by volume is 75% on superclean bench, sterilized water punching
Wash 3 times, mass percent is 0.1% HgCl2Solution sterilization 4-6min, aseptic water washing 4 times；
(2) preparation of golden flower tea extracts：The Camellia nitidissima blade for taking proper amount of fresh light green is added in open extractor, is added
Water is heated to boil condition and decocts 40-60min, and the material liquid volume ratio of blade and water is 1:2-4, pours out extract solution to liquid afterwards
In storage tank；Water is added into extractor again and is heated to boil condition decoction 1-2h, and the material liquid volume ratio of blade and water is 1:
3-5, pours out extract solution into the fluid storage tanks, the 1/5-1/ of the extract solution in last concentrated liquid storage tank to original volume
3, obtain golden flower tea extracts；
(3) primary Fiber differentiation：The sterilized seed of step (1), it is inoculated into primary inducing culture, in aseptic condition
Under, cultivate 8-12 days, until the seed grows terminal bud；
Primary inducing culture is 0.4~0.8mg/L of 1/2MS minimal medium+golden flower 0.8~1.2g/L+6-BA of tea extract
4.0~8.0g/L of+NAA 0.2~0.6mg/L+ sucrose 20~30g/L+ nutrient solutions；
(4) shoot proliferation culture：The terminal bud is inoculated into subculture multiplication medium and cultivated 16-20 days, induction terminal bud propagation,
Obtain Multiple Buds；
Proliferated culture medium is 0.4~0.6mg/L+NAA of 1/2B5 minimal medium+golden flower 0.8~1.2g/L+6-BA of tea extract
0.3~0.5mg/L+ sucrose 20~30g/L+ agar, 4.0~6.0g/L；
(5) culture of rootage：When the simple bud length of the Multiple Buds is to 3.0~5.0cm, Tissue Culture Seedings of Chrysantha is inoculated into training of taking root
Support and root induction culture 22~26 days is carried out in base；
Root media be 0.6~0.8mg/L+NAA of DCR culture medium+golden flower 1.0~1.6g/L+IBA of tea extract 0.4~
6.0~8.0g/L of 0.6mg/L+ sucrose 15~25g/L+ agar；
(6) domestication and transplanting of test tube seedling：By Camellia nitidissima rooting tube plantlet after culturing room moves to vinyl house placement 4~6 days,
Go bottle cap hardening 2~4 days, take out Camellia nitidissima seedling, its root is handled after 1~2min with 0.1% carbendazim, transplant to hole tray
In cultivation matrix in, overlay film moisturizing 3~5 days, therebetween the morning and afternoon it is each it is ventilated once, 20~30min every time, shade
65-75%, takes off the thiophanate methyl that 600-800 times of liquid is sprayed after film, and according to 10g/m2Consumption sprayed, hereafter carry out
Normal cultivation management, you can.
2. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the nutrient solution includes following
Component and its corresponding concentration：
It is 0.02~0.04g/L of ammonium sulfate, 0.01~0.03g/L of calcium chloride dihydrate, 0.01~0.03g/L of epsom salt, anhydrous
0.04~0.06g/L of potassium dihydrogen phosphate, 0.02~0.04g/L of 0.01~0.03g/L of zinc sulfate and KI.
3. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the primary Fiber differentiation,
Shoot proliferation culture and the condition of culture of culture of rootage：24 ± 2 DEG C of environment temperature, 12~16h/d of light application time, intensity of illumination
For 2000~3000lx.
4. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the group of the DCR culture mediums
Into and its content be：NH4NO3 0.03-0.05g/L、KH2PO4 0.06-0.08g/L、CaCl2·2H2O 0.02-0.04g/L、
Ca(NO3)2·4H2O 0.06-0.1g/L、MgSO4·7H2O 0.06-0.08g/L、FeSO4·7H2O 0.04-0.06g/L、
ZnSO4·7H2O 0.05-0.07g/L, KI 0.04-0.08g/L, mannitol 0.3-0.5g/L, VB1 0.01-0.03g/L and VC
0.02-0.04g/L。
5. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the primary inducing culture
For 1/2MS minimal medium+golden flower tea extract 1.0g/L+6-BA 0.6mg/L+NAA 0.4mg/L+ sucrose 25g/L+ nutrition
Liquid 6.0g/L.
6. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the proliferated culture medium is 1/
2B5 minimal medium+golden flower tea extract 1.0g/L+6-BA 0.5mg/L+NAA 0.4mg/L+ sucrose 25g/L+ agar 5.0g/
L。
7. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that the root media is
DCR culture medium+golden flower tea extract 1.3g/L+IBA 0.7mg/L+NAA 0.5mg/L+ sucrose 20g/L+ agar 7.0g/L.
8. a kind of Camellia nitidissima tissue culture mating system according to claim 1, it is characterised in that described cultivation matrix includes
Raw material by weight below：30-50 parts of diatomite, 15-25 parts of charcoal, 10-20 parts of haydite, 8-12 parts of rice chaff ash and the pool
5-15 parts of mud；
The charcoal, which is prepared by the following method, to be obtained：By cotton stem, vegetables bar weedtree branch or/and melon seedling dry to containing
Then water is cut or is smashed below 20%, in input carbide furnace, after being carbonized 2.5~3.5 hours at 420 DEG C~480 DEG C
Go out charcoal, be cooled to room temperature with water filtration when going out charcoal, then dry charcoal, crush, produce charcoal.
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