CN107155888A - A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions - Google Patents
A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions Download PDFInfo
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- CN107155888A CN107155888A CN201710408320.4A CN201710408320A CN107155888A CN 107155888 A CN107155888 A CN 107155888A CN 201710408320 A CN201710408320 A CN 201710408320A CN 107155888 A CN107155888 A CN 107155888A
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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
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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
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Abstract
The present invention relates to the method that Camellia nitidissima embryo under a kind of sunlight conditions cultivates tissue-cultured seedling.The present invention includes explant sterilization and bud Initial culture, the culture of bud shoot proliferation, 4 steps of bud culture of rootage and nursery stock transplanting, the light source that the present invention is used comes from sunshine, by the regulation to intensity of illumination, growth-promoting effect of the sunshine to Tissue Culture Seedings of Chrysantha is given full play to.Golden flower tea shoots cultivate 40 d under sunlight conditions, growth coefficient 9~13, Tissue Culture Seedings of Chrysantha rooting rate is up to 90%~95%, more than 20% is improved than using incandescent lamp as the method rooting rate of light source, same root media, the time of the long root point of bud is 20~25 d under sunlight conditions, the bud long root point time is 30~35 d under incandescent lamp, the former shortens 5~10 d at longer than the latter bud point time, and transplanting survival rate is up to more than 95%, has reached the requirement of industrialization production Camellia nitidissima nursery stock.
Description
Technical field
The present invention relates to plant Fast Asexual Propagation Technique field, more particularly, to cultivation Camellia nitidissima kind under a kind of sunlight conditions
The method of embryo tissue-cultured seedling.
Background technology
Camellia nitidissima（Camellia nittidissima Chi）, belong to Theaceae Camellia, uniquely had in camellia family
There is the species of golden yellow petal, be the distinctive traditional famous flower of China.It is also one of 8 kinds of one-level national key protected plants of country, in state
It is inside and outside all famous.It is domestic that Camellia nitidissima is described as " tea race queen ", " giant panda of plant kingdom ", it is external then be referred to as " to have fantasies of
In yellow camellia ", " magical " Dongfang " magic tea ".Not only ornamental value is high for Camellia nitidissima, or rare medicinal herbs, its medical value
Obtain being widely recognized as society.Camellia nitidissima contains that the right organic germaniums of d, zinc, selenium, alum etc. are a variety of important health-care effect to human body
Amino acid, vitamin etc. necessary to trace element, and Tea Polyphenols and human body, in reduction blood glucose, blood fat, anti-cancer, Fighting cancer club
There is special efficacy in face.At present, the golden flower tea products of the in the market extremely masses favor, and Camellia nitidissima main producing region Guangxi can only meet it
The 1/10 of sales volume.Camellia nitidissima market prospects are very wide as can be seen here.
As Camellia nitidissima medical value is widely recognized as by society, product is in great demand, and expensive, resource is well sold and in short supply, Camellia nitidissima kind
Plant industry, which springs up like bamboo shoots after a spring rain, rapidly to be developed.In order to which golden flower tea resources are applied into society as early as possible, professional scholars carry out
The research of Camellia nitidissima tissue culture technique, the report of correlative theses has：Yan Muqin etc. had weight to seven kinds successively from 1980
Valve, great Hua, the Camellia nitidissima kind of the characteristic such as fragrance carries out Study on tissue culture, and intact plant and transplant survival were obtained in 1981；
Liao Han swords in 1987 etc. have carried out the tissue cultures of Shoot from Camellia chryntha, and rooting rate is 75%, and transplant survival, will be cut without offspring
On the sprout stock for being grafted onto C. olelfera or Camellia Vietnamensis, survival rate is 78 %；Huang Xiaona etc. is tired with regard to being taken root in Camellia nitidissima bottle
Difficulty has carried out the research of tissue-cultured seedling Rooting ex vitro, and rooting efficiency can reach 85%.The Patents that country authorizes have：1. it is a kind of golden
Jasmine tea tissue culture and rapid propagation method（ZL 20140755440.8）, using 7 to August part Camellia nitidissima blade as explant, inducer blade is produced
Callus, callus is inoculated in bud inducement cultivation base, is placed in 25~28 DEG C of temperature, and brightness condition of culture culture 35~
40 d, callus differentiation budding；Bud is inoculated in root media, 25~28 DEG C of temperature, the training of brightness condition of culture is placed in
20~30 d are supported, tissue culture seedling is turned out；Bud induction culture medium prescription be：The g/L sucrose of MS+6~8 g/L agar+25~29
The mg/L NAA of+0.3~0.8 mg/L 6-BA+0.8~1.5, the g/L sugarcanes of root media MS+6~8 g/L agar+25~29
The mg/L activated carbons of mg/L NAA+0.8 of+0.8~2.5 mg/L IAA+0.5 of sugar~1.5~1.5（WC）+ 0.8~1.5 mg/L
Ferrous sulfate.2. a kind of Camellia nitidissima tissue culture enrichment procedure（ZL201410581398.2）Using Camellia nitidissima Mature Embryos Among as explant
Body, 5%~10% NaCl220~30 min of solution disinfection sterilizing are inoculated in primary with sterile bud is obtained in culture medium, and sterile bud exists
Repeat to cultivate in proliferated culture medium and strong buds medium, cultivation cycle 5~6 weeks；The strong bud of shearing and strong bastem portion is dipped in 500~
10~20 min are transferred to culture, rooting rate 95%~98% in root media again in 1000 mg/L IBA, and nursery stock transplanting is survived
Rate more than 90%；Initial culture base is the mg/ of mg/L 6-BA+0.2 of+30 g/L sucrose of MS+6 g/L agar+0.2~0.5~0.5
L NAA, proliferated culture medium is the mg/L of mg/L 6-BA+1.5 of+40 g/L sucrose of MS+6 g/L agar+1.5~2.0~2.0
KT；Strong buds medium is the mg/L IAA of MS+6 g/L agar+40g% sucrose+0.5~1.0, and illumination training is carried out using incandescent lamp
Support, the h of illumination 16, the h of light culture 8, the Lx of intensity of illumination 2000~3000,25 ± 3 DEG C~28 DEG C of temperature.3. Camellia nitidissima tissue training
Support base（ZL85102805）Improve ER；NH4NO3 1200 mg/L, KNO3 1800 mg/L, CaCI2 200 mg/L, KH2PO4 400
Mg/L, H3BO3 1 mg/L, MnSO4·H2O 0.5 mg/L, ZnSO4 ·7H2O 1 mg/L, NaM0O4 ·H2The mg/L of O 1,
CuSO4·5H2O 0.1 mg/L, FeSO4+Na2EDTA【(27.8+37.3 complex compound 5ml)】, the mg/L of nicotinic acid 10, glycine
2.0 mg/L, the mg/L of inositol 5, the mg/L of puridoxine hydrochloride 1, the mg/L of Tyiamine Hd 4, the g/L of agar 6, the g/L of sucrose 30,
The mg/L of 6-BA 1~5 mg/L, IAA 1~3.Main use incandescent lamp offer illumination condition reported above, but incandescent light
Spectrum only feux rouges, green glow and blue spectrum is stronger, and the effect to plant growth is far weaker than sunshine.
Sunshine spectrum enriches, and has 7 kinds of visible rays（Red orange red malachite royal purple）With 2 kinds of black lights（Ultraviolet and infrared
Line）.Blue violet light in sunshine plays the role of very big with is formationed of the green light to plant growth and young shoot, and suppression plant internode is stretched
It is long, promote the differentiation of bud.Blue light is also to dominate the most important spectrum of cell differentiation, while influenceing the phototropism of plant.Blue violet light
Be conducive to the synthesis of protein and organic acid, contribute to the synthesis of anthocyanidin and vitamin.Feux rouges and infrared ray are conducive to carbon
The synthesis of hydrate, promotes the sprouting of seed and the elongation of stem, promotes the decomposition of carbon dioxide and the formation of chlorophyll.Cause
This, by adjusting the intensity of illumination of sunshine, is favorably improved Tissue Culture Seedings of Chrysantha photosynthesis and promotes the growth of tissue-cultured seedling.
But the document and patent on carrying out industrial seedling rearing to Tissue Culture Seedings of Chrysantha using sunshine are there are no at present.
The content of the invention
The present invention seeks to overcome the shortcomings of that there is provided one using the cultivation of incandescent lamp progress Tissue Culture Seedings of Chrysantha in the prior art
The method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under kind of sunlight conditions, makes the tissue-cultured seedling of Camellia nitidissima cultivate not by region and weather
Limitation, can keep the genetic stability of Camellia nitidissima merit, and the energy, cost-effective is saved again, is quickly trained beneficial to industrialization
Educate the excellent tissue-cultured seedling of Camellia nitidissima.
To achieve the above object, the present invention uses following technical scheme：
A kind of method that Camellia nitidissima embryo cultivates tissue-cultured seedling under sunlight conditions, comprises the following steps：
（1）Explant is sterilized and bud Initial culture：Gather 7~8 ripe, the Camellia nitidissima fruits that shell is in red green June~July,
Running water rinses fruit appearance dust, peels off exocarp, is placed in superclean bench, and 30 S, sterilized water are sterilized with 75% alcoholic solution
Cleaning 3~4 times, the HgCl2 solution of mass fraction 1 ‰ soaks 8 min, and sterile water wash 3~4 times is peelled off endocarp, is inoculated in
In Initial culture base, be placed under the conditions of 300~800 Lx solar irradiation and carry out the d of Initial culture 7~10, afterwards dislocation in
Continue to cultivate 20~23 d under the conditions of 1000~2500 Lx solar irradiation, to high 2~5 cm of plumule；
（2）Bud shoot proliferation culture：The shearing of 2~5 cm high plumules is transferred in subculture multiplication medium, it is placed in 1500~
The d of bud shoot proliferation culture 10~15 is carried out under the conditions of 4000 Lx solar irradiation, then is placed in 4000~8000 Lx sunshine
Continue to cultivate 20 ~ 25 d according under the conditions of, growth coefficient is 8~13；
（3）Bud culture of rootage：By high 3~5 cm in shoot proliferation bud, blade is emerald green, leaf surface brightening, band keratin, there is blade 3~5
The simple bud shearing opened, straight cutting is placed in 15~40 d of culture under the conditions of 2000~8000 Lx solar irradiations in root media,
As root system 0.5~1 cm of length, hardening in the hardening canopy of natural environment, rooting rate 90 ~ 95% are moved closer to；
（4）Nursery stock transplanting：Bottle seedling d of hardening 20~30 in hardening canopy, seedling root is moved when being changed into canescence from white
Plant, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up the root media adhered on root system, transplant in warp
In the nutrition cup for crossing matrix sterilization, 15~28 DEG C of temperature, humidity 70~85%, degree of shading 70~80%, shady and cool environment bar are placed in
To cultivate, managed and protected through fertilization strategy under part, nursery stock is high >=30 cm, garden or upper mug culture seedlings can be gone out during ground diameter >=0.3 cm.
It is preferred that, in the bud shoot proliferation culture, plumule is sheared, is transferred in subculture multiplication medium, is placed in
The d of bud shoot proliferation culture 10~15 is carried out under the conditions of 2500~4000 Lx solar irradiation, 4000~8000 Lx are placed in too
Sunlight continues the d of shoot proliferation culture 20 ~ 25, growth coefficient 8~13 under the conditions of shining.
It is preferred that, in the bud culture of rootage, by high 3~5 cm in shoot proliferation bud, blade is emerald green, leaf surface brightening, band
Keratin, the simple bud for having 3~5, blade is sheared, and straight cutting is placed in 4000~8000 Lx solar irradiation conditions in root media
The lower d of progress bud culture of rootage 15~40, as root system 0.5~1 cm of length, moves closer to hardening in the hardening canopy of natural environment,
Rooting rate 90 ~ 95%.
It is preferred that, the formula of the Initial culture base is：MS+50 mg/L Na2SO4+2 mg/L 6- benzyl aminoadenines
The g/L sucrose of+4.5 g/L agar powders of+0.5 mg/L methyl α-naphthyl acetates+300 mg/L caseinhydrolysates of+10 mg/L vitamin Cs+30,
pH=5.8。
It is preferred that, the formula of the subculture multiplication medium is：MS+50 mg/L Na2SO4+3 mg/L 6- benzyl amino glands
The mg/L caseinhydrolysates+4.5 of+15~20 mg/L glutaminases of the mg/L of purine+1+15 mg/L vitamin Cs of methyl α-naphthyl acetate+300
The g/L sucrose of g/L agar powders+30, pH=5.8.
It is preferred that, the formula of the root media is：2/3 (MS+50 mg/L Na2SO4)+3 mg/L indoles fourths
The g/L sucrose of+4.5 g/L agar powders of sour+0.3 mg/L methyl α-naphthyl acetates of+5 mg/L heteroauxins+30, or 2/3（MS+50 mg/L
Na2SO4- 1650 mg/L NH4NO3+100 mg/L Ca(NO3)2·4H2O）The mg/L indoles second of+3 mg/L indolebutyric acids+5
The g/L sucrose of+4.5 g/L agar powders of sour+0.3 mg/L methyl α-naphthyl acetates+30, pH=5.8.
Increase the Na of debita spissitudo in MS culture mediums+The osmotic potential of plant cell can be adjusted, promotes cell division, carries
The photosynthesis of high blade, promotes nursery stock robust growth, in culture of rootage, and MS culture mediums are removed into NH4NO3Increase by 100
mg/L Ca(NO3)2·4H2O, can solve in culture medium that N content is relatively low, N phenomenons be lacked in bud growth course of taking root, it is to avoid leaf
Piece yellow, influences the rooting rate of bud.
It is preferred that, the bud Initial culture, the culture of bud shoot proliferation and bud culture of rootage are double glazing in wall 80%
Carried out in the culturing room of window.
It is preferred that, the light application time of the culturing room is 11~14 h/d, and temperature is 22 ~ 28 DEG C.
The present invention possesses following advantage and good effect：
（1）The light source that the present invention is used comes from sunshine, by being adjusted to intensity of illumination and light application time, gives full play to sunshine
The facilitation grown to Tissue Culture Seedings of Chrysantha, Tissue Culture Seedings of Chrysantha rooting rate is up to 90%~95%, than using incandescent lamp conduct
The method rooting rate of light source improves more than 20%, same root media, under sunlight conditions the time of the long root point of bud for 20~
The bud long root point time is 30~35 d under 25d, incandescent lamp, and the former shortens 5~10d at longer than the latter bud point time.
（2）The Tissue Culture Seedings of Chrysantha growth coefficient that the present invention is cultivated is up to 8~13, and transplanting survival rate more than 95% reaches
The requirement of industrialization production Camellia nitidissima nursery stock.
（3）The explant of the present invention derives from the plant with growth vigor, riotous growth, Hua Duo（2500/a. plants with
On）, produce and spend biennial bearing unobvious, use the tissue culture method of " to plant numerous bud ", the tissue-cultured seedling genetic stability of cultivation is good, can keep
The excellent character of elite stand.
（4）Basal culture medium is MS culture mediums, and the Na of low concentration is added in MS culture mediums2SO4, Na+Plant can be adjusted thin
The osmotic potential of born of the same parents, promotes cell division, improves the photosynthesis of blade, promotes nursery stock robust growth, is trained in primary and propagation
The caseinhydrolysate that appropriate concentration is added in base is supported, enzymatic activity can be promoted, has obvious rush to the propagation and differentiation of cell and tissue
Enter effect.
（5）Present invention process is simple, workable, without using incandescent light source, reduces power cost institute of the present invention
Use base.
Brief description of the drawings
Fig. 1：Culturing room；
Fig. 2：Culturing room's sunshine spectrogram；
Fig. 3：Incandescent light spectrogram；
Fig. 4：Shoot proliferation bud form comparison diagram, the left shoot proliferation bud cultivated for embodiment 1 of figure, figure is right to be cultivated for comparative example 1
Shoot proliferation bud：
Fig. 5：Rooted seedling Root morphology comparison diagram, the left rooted seedling cultivated for embodiment 1 of figure, the right life cultivated for comparative example 1 of figure
Offspring；
Fig. 6：Rooted seedling production figure.
Embodiment
The method for describing to cultivate Camellia nitidissima embryo tissue-cultured seedling under a kind of sunlight conditions of the invention below in conjunction with example, these
Description is not the restriction to present invention.
Embodiment 1
Golden flower is cultivated under a kind of method that sunshine using seed as explant cultivates Tissue Culture Seedings of Chrysantha, a kind of sunlight conditions
The method of tea embryo tissue-cultured seedling comprises the following steps：
（1）Explant is sterilized and bud Initial culture：Gather 7~8 ripe, the Camellia nitidissima fruits that shell is in red green, running water June
Fruit appearance dust is rinsed, exocarp is peelled off, is placed in superclean bench, 30 S, sterile water wash 3 are sterilized with 75% alcoholic solution
It is secondary, the HgCl of mass fraction 1 ‰2Solution soaks 8 min, and sterile water wash 3 times peels off endocarp, is inoculated in Initial culture base
In, the formula of Initial culture base is MS+50 mg/L Na2SO4The mg/L methyl α-naphthyl acetates of+2 mg/L 6- benzyls aminoadenine+0.5+
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of 10 mg/L vitamin Cs+30, pH=5.8 are placed in wall
80% is in the culturing room of double pane（Fig. 1）, the culture h/d of indoor illumination time 11~14,22 ~ 28 DEG C of temperature, 300~
The d of Initial culture 8 is carried out under the conditions of 800 Lx solar irradiation, dislocation is under the conditions of 1000~2500 Lx solar irradiation afterwards
Continue to cultivate 22 d, embryo germination rate >=98% generally, cultivates 10~15d embryo rudiments, when rudiment high 2~5cm,
Bud is inoculated in proliferated culture medium induction Multiple Buds；
（2）Bud shoot proliferation culture：Plumule is sheared, is transferred in subculture multiplication medium, the formula of subculture multiplication medium
For：MS+50 mg/L Na2SO4The mg/ of+3+15 mg/L vitamin Cs of mg/L 6- benzyls+1 mg/L methyl α-naphthyl acetates of aminoadenine+20
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of L glutaminases+30, pH=5.8 are placed in 2500~4000
The d of bud shoot proliferation culture 10 is carried out under the conditions of Lx solar irradiation, then is placed under the conditions of 4000~8000 Lx solar irradiation
Continue to cultivate, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature,：The d of shoot proliferation culture the 35th, bud high 3~5 cm, it is emerald green,
Stem semi-lignified, blade 2/3 deploys, growth coefficient 13；The d of shoot proliferation culture the 40th, high 3~6 cm of bud, emerald green, stem
Lignifying, blade 2/3 deploys, band keratin, leaf surface brightening, growth coefficient 13；The d of shoot proliferation culture the 50th, bud is high by 3~6
Cm, emerald green, stem lignifying is stronger, and blade 2/3 deploys, larger, leaf surface brightening, keratin, growth coefficient 13；
（3）Bud culture of rootage：High 3~6 cm of bud in 35 d of culture, 40 d and 50 d shoot proliferation bud is taken respectively, and blade is emerald green
Green, leaf surface brightening, band keratin, the simple bud for having 3~5, blade is sheared, direct insertion to insert in root media, root media
It is formulated as 2/3 (MS+50 mg/L Na2SO4)+0.3 mg/L methyl α-naphthyl acetates of+5 mg/L heteroauxins of+3 mg/L indolebutyric acids+
The g/L sucrose of 4.5 g/L agar powders+30, pH=5.8 are placed under the conditions of 4000~8000 Lx solar irradiations and carry out bud and take root training
Support, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature, 35 d of culture shoot proliferation bud is grown in the 16th d of culture of rootage
Root point, the cm of the 25th d root systems length 0.5 ~ 1.0, average rooting rate 96.7%；40 d shoot proliferation bud is cultivated in culture of rootage
15th d sends out roots a little, the cm of the 24th d root systems length 0.5 ~ 1.0, average rooting rate 98.3%；The shoot proliferation bud for cultivating 50 d exists
30th d of culture of rootage sends out roots a little, the cm of the 45th d root systems length 0.5 ~ 1.0, average rooting rate 83.3%；
（4）Nursery stock transplanting：The cm of root system length 0.5~1 bottle seedling is moved into the d of hardening 20 in hardening canopy, seedling root is become by white
Transplanted during for canescence, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up the life adhered on root system
Root culture medium, transplants in the nutrition cup sterilized by matrix, is placed in 15~28 DEG C of temperature, humidity 70~85%, and degree of shading 70~
80%, to cultivate under shady and cool environmental condition, managed and protected through fertilization strategy, nursery stock is high >=30 cm, garden can be gone out during ground diameter >=0.3 cm
Or upper mug culture seedlings, average transplanting survival rate 99.4%.
Embodiment 2
A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions, comprises the following steps：
（1）Explant is sterilized and bud Initial culture：Gather 7~8 ripe, the Camellia nitidissima fruits that shell is in red green, running water June
Fruit appearance dust is rinsed, exocarp is peelled off, is placed in superclean bench, 30 S, sterile water wash 4 are sterilized with 75% alcoholic solution
It is secondary, the HgCl of mass fraction 1 ‰2Solution soaks 8 min, and sterile water wash 4 times peels off endocarp, is inoculated in Initial culture base
In, the formula of Initial culture base is MS+50 mg/L Na2SO4The mg/L methyl α-naphthyl acetates of+2 mg/L 6- benzyls aminoadenine+0.5+
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of 10 mg/L vitamin Cs+30, pH=5.8 are placed in wall
80% is in the culturing room of double pane（Fig. 1）, the culture h/d of indoor illumination time 11~14,22 ~ 28 DEG C of temperature, 300~
The d of Initial culture 7 is carried out under the conditions of 800 Lx solar irradiation, dislocation is under the conditions of 1000~2500 Lx solar irradiation afterwards
Continue to cultivate 23 d, embryo germination rate >=98% generally, cultivates 10~15 d embryo rudiments, treats high 2~5 cm of rudiment
When, bud is inoculated in proliferated culture medium induction Multiple Buds；
（2）Bud shoot proliferation culture：Plumule is sheared, is transferred in subculture multiplication medium, the formula of subculture multiplication medium
For：MS+50 mg/L Na2SO4Mg/L vitamin Cs+the 16mg/L of+3+1 mg/L methyl α-naphthyl acetates of mg/L 6- benzyls aminoadenines+15
The g/L sucrose of+4.5 g/L agar powders of the mg/L of glutaminase+300 caseinhydrolysates+30, pH=5.8 are placed in 1500~3000 Lx
Solar irradiation under the conditions of carry out the d of bud shoot proliferation culture 10, then be placed under the conditions of 4000~6000 Lx solar irradiation after
Continuous culture, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature：The d of shoot proliferation culture the 35th, high 2~4 cm of bud, emerald green, stem
Dry semi-lignified, blade 1/2 deploys, growth coefficient 10；The d of shoot proliferation culture the 40th, high 3~4 cm of bud, emerald green, stem is firm
Lignifying, blade 2/3 deploys, band keratin, leaf surface brightening, growth coefficient 11；The d of shoot proliferation culture the 50th, high 3~5 cm of bud,
Emerald green, stem lignifying, blade 2/3 deploys, leaf surface brightening, keratin, growth coefficient 11；
（3）Bud culture of rootage：High 3~5 cm of bud in 35 d of culture, 40 d and 50 d shoot proliferation bud is taken respectively, and blade is emerald green
Green, leaf surface brightening, band keratin, the simple bud for having 3~5, blade is sheared, direct insertion to insert in root media, root media
It is formulated as 2/3 (MS+50 mg/L Na2SO4)+0.3 mg/L methyl α-naphthyl acetates of+5 mg/L heteroauxins of+3 mg/L indolebutyric acids+
The g/L sucrose of 4.5 g/L agar powders+30, pH=5.8 are placed under the conditions of 3000~6000 Lx solar irradiations and carry out bud and take root training
Support, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature, 35 d of culture shoot proliferation bud is grown in the 20th d of culture of rootage
Root point, the cm of the 30th d root systems length 0.5 ~ 1.0, average rooting rate 93.3%；40 d shoot proliferation bud is cultivated in culture of rootage
17th d sends out roots a little, the cm of the 26th d root systems length 0.5 ~ 1.0, average rooting rate 94.8%；The shoot proliferation bud for cultivating 50 d exists
30th d of culture of rootage sends out roots a little, the cm of the 45th d root systems length 0.5 ~ 1.0, average rooting rate 85.3%；
（4）Nursery stock transplanting：The cm of root system length 0.5~1 bottle seedling is moved into the d of hardening 20 in hardening canopy, seedling root is become by white
Transplanted during for canescence, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up the life adhered on root system
Root culture medium, transplants in the nutrition cup sterilized by matrix, is placed in 15~28 DEG C of temperature, humidity 70~85%, and degree of shading 70~
80%, to cultivate under shady and cool environmental condition, managed and protected through fertilization strategy, nursery stock is high >=30 cm, garden can be gone out during ground diameter >=0.3 cm
Or upper mug culture seedlings, average transplanting survival rate 98.9%.
Embodiment 3
A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions, comprises the following steps：
（1）Explant is sterilized and bud Initial culture：Gather the Camellia nitidissima fruit that 7~8 ripe shells are in red green, running water July
Fruit appearance dust is rinsed, exocarp is peelled off, is placed in superclean bench, 30 S, sterile water wash 4 are sterilized with 75% alcoholic solution
It is secondary, the HgCl of mass fraction 1 ‰2Solution soaks 8 min, and sterile water wash 4 times peels off endocarp, is inoculated in Initial culture base
In, the formula of Initial culture base is MS+50 mg/L Na2SO4The mg/L methyl α-naphthyl acetates of+2 mg/L 6- benzyls aminoadenine+0.5+
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of 10 mg/L vitamin Cs+30, pH=5.8 are placed in wall
80% is in the culturing room of double pane（Fig. 1）, the culture h/d of indoor illumination time 11~14,22 ~ 28 DEG C of temperature, 300~
The d of Initial culture 8 is carried out under the conditions of 800 Lx solar irradiation, dislocation is under the conditions of 1000~2500 Lx solar irradiation afterwards
Continue to cultivate 22 d, embryo germination rate >=98% generally, cultivates 10~15 d embryo rudiments, treats high 2~5 cm of rudiment
When, bud is inoculated in proliferated culture medium induction Multiple Buds；
（2）Bud shoot proliferation culture：Plumule is sheared, is transferred in subculture multiplication medium, the formula of subculture multiplication medium
For：MS+50 mg/L Na2SO4The mg/ of+3+15 mg/L vitamin Cs of mg/L 6- benzyls+1 mg/L methyl α-naphthyl acetates of aminoadenine+18
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of L glutaminases+30, pH=5.8 are placed in 1500~2500
The d of bud shoot proliferation culture 10 is carried out under the conditions of Lx solar irradiation, then is placed under the conditions of 4000~5000 Lx solar irradiation
Continue to cultivate, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature；The d of shoot proliferation culture the 35th, bud high 2~4 cm, it is emerald green,
Stem semi-lignified, blade 1/2 deploys, growth coefficient 8.0；Shoot proliferation culture the 40th d, high 3~4 cm, emerald green, stem is firm
Lignifying, blade 2/3 deploys, band keratin, leaf surface brightening, growth coefficient 9；The d of shoot proliferation culture the 50th, high 3~5 cm of bud,
Emerald green, stem lignifying, blade 2/3 deploys, leaf surface brightening, keratin, growth coefficient 9.5；
（3）Bud culture of rootage：High 3~5 cm of bud in 35 d of culture, 40 d and 50 d shoot proliferation bud is taken respectively, and blade is emerald green
Green, leaf surface brightening, band keratin, the simple bud for having 3~5, blade is sheared, direct insertion to insert in root media, root media
It is formulated as 2/3 (MS+50 mg/L Na2SO4)+0.3 mg/L methyl α-naphthyl acetates of+5 mg/L heteroauxins of+3 mg/L indolebutyric acids+
The g/L sucrose of 4.5 g/L agar powders+30, pH=5.8 are placed under the conditions of 2000~5000 Lx solar irradiations and carry out bud and take root training
Support, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature.The shoot proliferation bud for cultivating 35 d is grown in the 25th d of culture of rootage
Root point, the cm of the 35th d root systems length 0.5 ~ 1.0, average rooting rate 90.1%；40 d shoot proliferation bud is cultivated in culture of rootage
20th d sends out roots a little, the cm of the 30th d root systems length 0.5 ~ 1.0, average rooting rate 91.3%；The shoot proliferation bud for cultivating 50 d exists
25th d of culture of rootage sends out roots a little, the cm of the 40th d root systems length 0.5 ~ 1.0, average rooting rate 86.3%；
（4）Nursery stock transplanting：The cm of root system length 0.5~1 bottle seedling is moved into the d of hardening 22 in hardening canopy, seedling root is become by white
Transplanted during for canescence, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up the life adhered on root system
Root culture medium, transplants in the nutrition cup sterilized by matrix, is placed in 15~28 DEG C of temperature, humidity 70~85%, and degree of shading 70~
80%, to cultivate under shady and cool environmental condition, managed and protected through fertilization strategy, nursery stock is high >=30 cm, garden can be gone out during ground diameter >=0.3 cm
Or upper mug culture seedlings, average transplanting survival rate 96.7%.
Embodiment 4
A kind of method that Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions, comprises the following steps：
（1）Explant is sterilized and bud Initial culture：The 6 Camellia nitidissima fruits that collection 7~8 is ripe month in and month out, shell is in red green, originally
Water rinses fruit appearance dust, peels off exocarp, is placed in superclean bench, and 30 S, sterile water wash are sterilized with 75% alcoholic solution
3 times, the HgCl of mass fraction 1 ‰2Solution soaks 8 min, and sterile water wash 3 times peels off endocarp, is inoculated in Initial culture base
In, the formula of Initial culture base is MS+50 mg/L Na2SO4The mg/L methyl α-naphthyl acetates of+2 mg/L 6- benzyls aminoadenine+0.5+
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of 10 mg/L vitamin Cs+30, pH=5.8 are placed in wall
80% is in the culturing room of double pane（Fig. 1）, the culture h/d of indoor illumination time 11~14,22 ~ 28 DEG C of temperature, 300~
The d of Initial culture 10 is carried out under the conditions of 800 Lx solar irradiation, dislocation is in 1000~2500 Lx solar irradiation condition afterwards
Lower to continue to cultivate 20 d, embryo germination rate >=98% generally, cultivates 10d~15d embryo rudiments, treats that rudiment is high by 2~5
During cm, bud is inoculated in proliferated culture medium induction Multiple Buds；
（2）Bud shoot proliferation culture：Plumule is sheared, is transferred in subculture multiplication medium, the formula of subculture multiplication medium
For：MS+50 mg/L Na2SO4The mg/ of+3+15 mg/L vitamin Cs of mg/L 6- benzyls+1 mg/L methyl α-naphthyl acetates of aminoadenine+15
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of L glutaminases+30, pH=5.8 are placed in 2500~4000
The d of bud shoot proliferation culture 10 is carried out under the conditions of Lx solar irradiation, 4000~8000 Lx solar irradiation condition is placed in afterwards
Under proceed the h/d of bud shoot proliferation culture light application time 11~14,22 ~ 28 DEG C of temperature：The d of shoot proliferation culture the 35th, bud
High 3~6 cm, red green, stem is more crisp, and blade 1/2 deploys, growth coefficient 9.0；The d of shoot proliferation culture the 40th, bud is high by 3.5
~6 cm, emerald green, stem semi-lignified, blade 2/3 deploys, band keratin, leaf surface brightening, growth coefficient 10；Shoot proliferation culture
50th d, high 4~6 cm of bud, emerald green, stem lignifying, blade 2/3 deploys, leaf surface brightening, keratin, growth coefficient 10；
（3）Bud culture of rootage：High 3~6 cm of bud in 35 d of culture, 40 d and 50 d shoot proliferation bud is taken respectively, and blade is emerald green
Green, leaf surface brightening, band keratin, the simple bud for having 3~5, blade is sheared, direct insertion to insert in root media, root media
It is formulated as 2/3（MS+50 mg/L Na2SO4- 1650 mg/L NH4NO3+100 mg/L Ca(NO3)2·4H2O）+3 mg/L
The g/L sucrose of+4.5 g/L agar powders of the mg/L of indolebutyric acid+5+0.3 mg/L methyl α-naphthyl acetates of heteroauxin+30, pH=5.8 are placed in
Bud culture of rootage, the h/d of light application time 11~14,22 ~ 28 DEG C of temperature, culture are carried out under the conditions of 4000~8000 Lx solar irradiations
35 d shoot proliferation bud sends out roots a little in the 21st d of culture of rootage, the cm of the 29th d root systems length 0.5 ~ 1.0, average rooting rate
91.3%；The shoot proliferation bud for cultivating 40 d sends out roots a little in the 18th d of culture of rootage, the cm of the 30th d root systems length 0.5 ~ 1.0,
Average rooting rate 92.6%；The shoot proliferation bud for cultivating 50 d sends out roots a little in the 25th d of culture of rootage, and the 43rd d root systems are long
0.5 ~ 1.0 cm, average rooting rate 85.3%；
（4）Nursery stock transplanting：The cm of root system length 0.5~1 bottle seedling is moved into hardening 28d in hardening canopy, seedling root is changed into from white
Transplanted during canescence, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up and adhere to taking root on root system
Culture medium, transplants in the nutrition cup sterilized by matrix, is placed in 15~28 DEG C of temperature, humidity 70~85%, and degree of shading 70~
80%, to cultivate under shady and cool environmental condition, managed and protected through fertilization strategy, nursery stock is high >=30 cm, garden can be gone out during ground diameter >=0.3 cm
Or upper mug culture seedlings, average transplanting survival rate 94.1%.
Comparative example 1
The difference of comparative example 1 and embodiment 1 is that the light source that comparative example 1 is used is incandescent lamp, and sunshine and incandescent lamp spectrum are such as
Shown in Fig. 2, Fig. 3, comparative example 1 is in bud Initial culture, shoot proliferation culture, each step of culture of rootage, all using after inoculation 8
D is placed in culture under the conditions of 300~800Lx solar irradiation, is placed in 2500 Lx incandescent light cultures afterwards, used in comparative example 1
The explant processing method, Initial culture base, subculture medium, root media and culturing room's temperature environment and example 1 are same；
In bud Initial culture, 10d~15d embryo rudiments are cultivated, bud is weaker, when rudiment high 1.5~2cm, is inoculated in proliferated culture medium
Induce Multiple Buds, embryo germination rate 97%；In bud shoot proliferation culture, the d of shoot proliferation culture the 35th, bud high 2~3 cm are red
Green, stem is more crisp, and blade 1/3 deploys, and blade face is dim, growth coefficient 2.5；The d of shoot proliferation culture the 40th, bud is high by 2.5~4
Cm, green brown, stem is more crisp, and blade 1/3 deploys, growth coefficient 3.0；The d of shoot proliferation culture the 50th, bud high 3~5 cm are green
Brown, stem semi-lignified, blade 1/2 deploys, and blade face is glossy, growth coefficient 3.5；In bud culture of rootage, 35 d are cultivated
Shoot proliferation bud sent out roots a little in the 35th d of culture of rootage, the cm of the 55th d root systems length 0.5 ~ 1.0, average rooting rate
33.1%；The shoot proliferation bud for cultivating 40 d sends out roots a little in the 30th d of culture of rootage, the cm of the 50th d root systems length 0.5 ~ 1.0,
Average rooting rate 38.5%；The shoot proliferation bud for cultivating 50 d sends out roots a little in the 25th d of culture of rootage, and the 45th d root systems are long
0.5 ~ 1.0 cm, average rooting rate 62.0%, the average survival after transplanting is 51.7%.
Comparative example 2
The difference of comparative example 2 and embodiment 2 is that the light source that comparative example 2 is used is incandescent lamp, and sunshine and incandescent lamp spectrum are such as
Shown in Fig. 2, Fig. 3, comparative example 2 is used in bud Initial culture, the culture of bud shoot proliferation, each step of bud culture of rootage, all and connect
7 d are placed in culture under the conditions of 300~800Lx solar irradiation after kind, and 2500 Lx incandescent light cultures, comparative example 2 are placed in afterwards
The explant processing method used, Initial culture base, subculture medium, root media and culturing room's temperature environment and example
2 is consistent；In bud Initial culture, 10d~15d embryo rudiments are cultivated, bud is weaker, when rudiment high 1.5~2cm, is inoculated in increasing
Grow culture medium induction Multiple Buds, embryo germination rate 97.1%；In bud shoot proliferation culture, the d of shoot proliferation culture the 35th, bud is high
2~4 cm, reddish violet, stem is more crisp, and blade 1/3 deploys, and blade face is dim, growth coefficient 2.6；The d of shoot proliferation culture the 40th,
High 2.5~4 cm of bud, green brown, stem is more crisp, and blade 1/3 deploys, growth coefficient 3.0；Shoot proliferation culture the 50th d is high by 3
~5 cm, green brown, stem semi-lignified, blade 1/2 deploys, and blade face is glossy, growth coefficient 3.3；In bud culture of rootage,
The shoot proliferation bud for cultivating 35 d sends out roots a little in the 35th d of culture of rootage, the cm of the 55th d root systems length 0.5 ~ 1.0, average raw
Root rate 32.2%；The shoot proliferation bud for cultivating 40 d sends out roots a little in the 30th d of culture of rootage, the 50th d root systems length 0.5 ~ 1.0
Cm, average rooting rate 33.6%；The shoot proliferation bud for cultivating 50 d sends out roots a little in the 25th d of culture of rootage, the 45th d root systems
Long 0.5 ~ 1.0 cm, average rooting rate 61.8%, the average survival after transplanting is 52.4%.
Comparative example 3
The difference of comparative example 3 and embodiment 3 is that the light source that comparative example 2 is used is incandescent lamp, and sunshine and incandescent lamp spectrum are such as
Shown in Fig. 2, Fig. 3, comparative example 2 is used in bud Initial culture, the culture of bud shoot proliferation, each step of bud culture of rootage, all and connect
8 d are placed in culture under the conditions of 300~800 Lx solar irradiation after kind, and 2500 Lx incandescent light cultures are placed in afterwards.Comparative example
The explant processing method, Initial culture base, subculture medium, root media and culturing room's temperature environment and reality used in 3
Example 3 is consistent；In bud Initial culture, 10d~15d embryo rudiments are cultivated, bud is weaker, when rudiment high 1.5~2cm, is inoculated in
Proliferated culture medium induces Multiple Buds, embryo germination rate 97.5%；In bud shoot proliferation culture, the d of shoot proliferation culture the 35th, bud
High 2~4 cm, reddish violet, stem is more crisp, and blade 1/3 deploys, and blade face is dim, growth coefficient 2.5；Shoot proliferation culture the 40th
D, high 2.5~4 cm of bud, green brown, stem is more crisp, and blade 1/3 deploys, growth coefficient 2.8；The d of shoot proliferation culture the 50th, bud
High 3~5 cm, green brown, stem semi-lignified, blade 1/2 deploys, and blade face is glossy, growth coefficient 3.2；In bud culture of rootage
In, 35 d of culture shoot proliferation bud sends out roots a little in the 35th d of culture of rootage, and the cm of the 55th d root systems length 0.5 ~ 1.0 is average
Rooting rate 30.6%；The shoot proliferation bud for cultivating 40 d sends out roots a little in the 30th d of culture of rootage, and the 50th d root systems length 0.5 ~
1.0 cm, average rooting rate 35.8%；The shoot proliferation bud for cultivating 50 d sends out roots a little in the 25th d of culture of rootage, the 45th d
The cm of root system length 0.5 ~ 1.0, average rooting rate 60.2%, the average survival after transplanting is 51.3%.
Comparative example 4
The difference of comparative example 4 and embodiment 4 is that the light source that comparative example 2 is used is incandescent lamp, and sunshine and incandescent lamp spectrum are such as
Shown in Fig. 2, Fig. 3, comparative example 2 is used in bud Initial culture, the culture of bud shoot proliferation, each step of bud culture of rootage, all and connect
10 d are placed in culture under the conditions of 300~800 Lx solar irradiation after kind, and 2500 Lx incandescent light cultures are placed in afterwards.Contrast
The explant processing method, Initial culture base, subculture medium, root media and culturing room's temperature environment used in example 4 with
Example 4 is consistent；In bud Initial culture, 10~15d embryo rudiments are cultivated, bud is weaker, when rudiment high 1.5~2cm, is inoculated in
Proliferated culture medium induces Multiple Buds, embryo germination rate 97.8%；In bud shoot proliferation culture, the d of shoot proliferation culture the 35th, bud
High 2~4 cm, reddish violet, stem is more crisp, and blade 1/3 deploys, and blade face is dim, growth coefficient 2.5；Shoot proliferation culture the 40th
D, high 2.5~4 cm of bud, green brown, stem is more crisp, and blade 1/3 deploys, growth coefficient 2.8；The d of shoot proliferation culture the 50th, bud
High 3~5 cm, green brown, stem semi-lignified, blade 1/2 deploys, and blade face is glossy, growth coefficient 3.0；In bud culture of rootage
In, 35 d of culture shoot proliferation bud sends out roots a little in the 38th d of culture of rootage, and the cm of the 60th d root systems length 0.5 ~ 1.0 is average
Rooting rate 29.5%；The shoot proliferation bud for cultivating 40 d sends out roots a little in the 35th d of culture of rootage, and the 65th d root systems length 0.5 ~
1.0 cm, average rooting rate 32.8%；The shoot proliferation bud for cultivating 50 d sends out roots a little in the 32nd d of culture of rootage, the 46th d
The cm of root system length 0.5 ~ 1.0, average rooting rate 55.2%, the average survival after transplanting is 46.8%.
Found out by above example and comparative example, the d of the bud shoot proliferation culture 40 every physical signs of tissue-cultured seedling is optimal,
Actual production also uses the d of bud shoot proliferation culture 40 technique, below with regard to the bud shoot proliferation culture 40 of embodiment and comparative example
The technique effect that d is obtained is compared, as shown in Fig. 4, Fig. 5 and table 1：
The embodiment of table 1 and the d of comparative example bud shoot proliferation culture 40 technique effect are contrasted
| The d of bud shoot proliferation culture 40 | Bud culture of rootage is to the long 0.5-1 cm of root system | Transplant into Motility rate | |
| Embodiment 1 | High 3~6 cm of shoot proliferation bud, emerald green, stem lignifying, blade 2/3 deploys, band keratin, Leaf surface brightening, growth coefficient 13 | 15d sends out roots a little, the cm of the 24th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 98.3% | 99.4% |
| Comparative example 1 | High 2.5~4 cm of shoot proliferation bud, green brown, stem is more crisp, and blade 1/3 deploys, propagation Coefficient 3.0 | 30d sends out roots a little, the cm of the 50th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 38.5% | 51.7% |
| Embodiment 2 | High 3~4 cm of shoot proliferation bud, emerald green, the firm lignifying of stem, blade 2/3 deploys, belt leather Matter, leaf surface brightening, growth coefficient 11 | 17d sends out roots a little, the cm of the 26th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 94.8% | 98.9% |
| Comparative example 2 | High 2.5~4 cm of shoot proliferation bud, green brown, stem is more crisp, and blade 1/3 deploys, propagation Coefficient 3.0 | 30d sends out roots a little, the cm of the 50th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 33.6% | 52.4% |
| Embodiment 3 | High 3~4 cm of shoot proliferation bud, emerald green, the firm lignifying of stem, blade 2/3 deploys, belt leather Matter, leaf surface brightening, growth coefficient 9 | 20d sends out roots a little, the cm of the 30th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 91.3% | 96.7% |
| Comparative example 3 | High 2.5~4 cm of shoot proliferation bud, green brown, stem is more crisp, and blade 1/3 deploys, propagation Coefficient 2.8 | 30d sends out roots a little, the cm of the 50th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 35.8% | 51.3% |
| Embodiment 4 | High 3.5~6 cm of shoot proliferation bud, emerald green, stem semi-lignified, blade 2/3 deploys, band Keratin, leaf surface brightening, growth coefficient 10 | 18d sends out roots a little, the cm of the 30th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 92.6% | 94.1% |
| Comparative example 4 | High 2.5~4 cm of shoot proliferation bud, green brown, stem is more crisp, and blade 1/3 deploys, propagation Coefficient 2.8 | 35d sends out roots a little, the cm of the 65th d root systems length 0.5 ~ 1.0, puts down Equal rooting rate 32.8% | 46.8% |
Shown in Fig. 4, Fig. 5 and table 1, Camellia nitidissima embryo cultivates the method for tissue-cultured seedling and compares incandescent lamp under a kind of sunlight conditions of the invention
Under the conditions of cultivate tissue-cultured seedling method it is excellent：Subculture bud cultivation cycle is short, and bud growth coefficient is high, robust growth；Root time bud beginning is short,
Rooting rate is high, and transplanting survival rate of nursery stocks is high.Therefore, method of the invention is more efficient than the method that tissue-cultured seedling is cultivated under incandescent lamp, more
It is suitable for large-scale production（Fig. 6）.
Claims (8)
1. a kind of method that Camellia nitidissima embryo cultivates tissue-cultured seedling under sunlight conditions, it is characterised in that comprise the following steps：
（1）Explant is sterilized and bud Initial culture：Gather 7~8 ripe, the Camellia nitidissima fruits that shell is in red green June~July,
Running water rinses fruit appearance dust, peels off exocarp, is placed in superclean bench, and 30 S, sterilized water are sterilized with 75% alcoholic solution
Cleaning 3~4 times, the HgCl of mass fraction 1 ‰2Solution soaks 8 min, and sterile water wash 3~4 times is peelled off endocarp, is inoculated in
In Initial culture base, be placed under the conditions of 300~800 Lx solar irradiation and carry out the d of Initial culture 7~10, afterwards dislocation in
Continue to cultivate 20~23 d under the conditions of 1000~2500 Lx solar irradiation, to high 2~5 cm of plumule；
（2）Bud shoot proliferation culture：The shearing of 2~5 cm high plumules is transferred in subculture multiplication medium, it is placed in 1500~
The d of bud shoot proliferation culture 10~15 is carried out under the conditions of 4000 Lx solar irradiation, then is placed in 4000~8000 Lx sunshine
Continue to cultivate 20 ~ 25 d according under the conditions of, growth coefficient is 8~13；
（3）Bud culture of rootage：By high 3~5 cm in shoot proliferation bud, blade is emerald green, leaf surface brightening, band keratin, there is blade 3~5
The simple bud shearing opened, straight cutting is placed in 15~40 d of culture under the conditions of 2000~8000 Lx solar irradiations in root media,
As root system 0.5~1 cm of length, hardening in the hardening canopy of natural environment, rooting rate 90 ~ 95% are moved closer to；
（4）Nursery stock transplanting：Bottle seedling d of hardening 20~30 in hardening canopy, seedling root is moved when being changed into canescence from white
Plant, during nursery stock transplanting, first open bottle cap, take out rooted seedling, clean up the root media adhered on root system, transplant in warp
In the nutrition cup for crossing matrix sterilization, 15~28 DEG C of temperature, humidity 70~85%, degree of shading 70~80%, shady and cool environment bar are placed in
To cultivate, managed and protected through fertilization strategy under part, nursery stock is high >=30 cm, garden or upper mug culture seedlings can be gone out during ground diameter >=0.3 cm.
2. the method for Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions according to claim 1, it is characterised in that institute
State in bud shoot proliferation culture, plumule is sheared, is transferred in subculture multiplication medium, be placed in 2500~4000 Lx sun
The d of bud shoot proliferation culture 10~15 is carried out under illumination condition, then is placed in continuation under the conditions of 4000~8000 Lx solar irradiation
The d of shoot proliferation culture 20 ~ 25, growth coefficient 8~13.
3. the method for Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions according to claim 1, it is characterised in that institute
State in bud culture of rootage, by high 3~5 cm in shoot proliferation bud, blade is emerald green, leaf surface brightening, band keratin has 3~5, blade
Simple bud shearing, straight cutting is placed under the conditions of 4000~8000 Lx solar irradiations in root media and carries out bud culture of rootage 15
~40 d, as root system 0.5~1 cm of length, move closer to hardening in the hardening canopy of natural environment, rooting rate 90 ~ 95%.
4. the method for Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions according to claim 1, it is characterised in that institute
The formula for stating Initial culture base is：MS+50 mg/L Na2SO4The mg/L methyl α-naphthyl acetates of+2 mg/L 6- benzyls aminoadenine+0.5+
The g/L sucrose of+4.5 g/L agar powders of+300 mg/L caseinhydrolysates of 10 mg/L vitamin Cs+30, pH=5.8.
5. cultivating the method for Camellia nitidissima embryo tissue-cultured seedling under sunlight conditions according to claim 1 or 2, its feature exists
In the formula of the subculture multiplication medium is：MS+50 mg/L Na2SO4The mg/L naphthalenes of+3 mg/L 6- benzyls aminoadenine+1
The g/L agar powders+30 of+300 mg/L caseinhydrolysates of the mg/L of acetic acid+15+15~20 mg/L glutaminases of vitamin C+4.5
G/L sucrose, pH=5.8.
6. cultivating the method for Camellia nitidissima embryo tissue-cultured seedling under the sunlight conditions according to claim 1 or 3, its feature exists
In the formula of the root media is：2/3 (MS+50 mg/L Na2SO4)+5 mg/L indoles of+3 mg/L indolebutyric acids
The g/L sucrose of+4.5 g/L agar powders of the mg/L of acetic acid+0.3 methyl α-naphthyl acetates+30, or 2/3（MS+50 mg/L Na2SO4- 1650 mg/
L NH4NO3+100 mg/L Ca(NO3)2·4H2O）The mg/L naphthalene second of+5 mg/L heteroauxins of+3 mg/L indolebutyric acids+0.3
The sour g/L sucrose of+4.5 g/L agar powders+30, pH=5.8.
7. cultivating the method for Camellia nitidissima embryo tissue-cultured seedling under the sunlight conditions according to any one of claim 1 ~ 6, it is special
Levy and be, the bud Initial culture, the culture of bud shoot proliferation and bud culture of rootage are in the culture that wall 80% is double pane
Indoor progress.
8. the method for Camellia nitidissima embryo tissue-cultured seedling is cultivated under sunlight conditions according to claim 7, it is characterised in that institute
The light application time for stating culturing room is 11~14 h/d, and temperature is 22 ~ 28 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107711247A (en) * | 2017-10-25 | 2018-02-23 | 黄海民 | A kind of breeding method of camellia seedling |
| CN108967023A (en) * | 2018-10-31 | 2018-12-11 | 安徽东旭大别山农业科技有限公司 | A kind of nursery implantation methods of oil tea |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972291A (en) * | 2012-11-23 | 2013-03-20 | 广西壮族自治区林业科学研究院 | Tissue culture and propagation method and inductive culture mediums for Chongzuo camellia nitidissima |
| CN104145824A (en) * | 2014-09-01 | 2014-11-19 | 广西壮族自治区林业科学研究院 | Method for cultivating subcultured bud of upright crown tissue culture seedling of fir wood |
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
-
2017
- 2017-06-02 CN CN201710408320.4A patent/CN107155888B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972291A (en) * | 2012-11-23 | 2013-03-20 | 广西壮族自治区林业科学研究院 | Tissue culture and propagation method and inductive culture mediums for Chongzuo camellia nitidissima |
| CN104145824A (en) * | 2014-09-01 | 2014-11-19 | 广西壮族自治区林业科学研究院 | Method for cultivating subcultured bud of upright crown tissue culture seedling of fir wood |
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
Non-Patent Citations (3)
| Title |
|---|
| 李春龙等: "《植物组织培养》", 31 July 2016, 西南交通大学出版社 * |
| 洪永辉等: ""防城金花茶优良个体组培快繁体系研究"", 《林业勘察设计》 * |
| 蔡玲等: ""几种因子对油茶组培芽增殖与生长的影响"", 《广西林业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107711247A (en) * | 2017-10-25 | 2018-02-23 | 黄海民 | A kind of breeding method of camellia seedling |
| CN108967023A (en) * | 2018-10-31 | 2018-12-11 | 安徽东旭大别山农业科技有限公司 | A kind of nursery implantation methods of oil tea |
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