CN104663433B - A kind of Camellia nitidissima Chi quickly tissue culture method - Google Patents
A kind of Camellia nitidissima Chi quickly tissue culture method Download PDFInfo
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- CN104663433B CN104663433B CN201410755414.5A CN201410755414A CN104663433B CN 104663433 B CN104663433 B CN 104663433B CN 201410755414 A CN201410755414 A CN 201410755414A CN 104663433 B CN104663433 B CN 104663433B
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Abstract
The present invention discloses a kind of Camellia nitidissima Chi quickly tissue culture method, it is characterised in that comprise the steps：（1）The sterilization of explant；（2）Callus culture；（3）Calluss Bud Differentiation；（4）Bud is taken root.The present invention provides a kind of Camellia nitidissima Chi quickly tissue culture method, can reduce the cost bred, and growth cycle is short, breeding potential is high, conveniently manage, beneficial to industrialized production and the automatically-monitored soil for greatly having saved human and material resources and field crops.
Description
Technical field
The invention belongs to tissue culture's production technical field, the present invention relates to a kind of Camellia nitidissima Chi quickly tissue culture method.
Background technology
Camellia nitidissima Chi belongs to Theaceae, Camellia, is twin sisters with tea, Flos Camelliae Japonicae, South Mountain tea, oil tea, Camellia sasanqua Thunb. etc..Camellia nitidissima Chi
Spend golden yellow, it is dazzling brilliant, seemingly apply coating of wax, it is sparkling and crystal-clear and glossy, seemingly have translucent sense.Camellia nitidissima Chi is singly born in leaf
Axil, when the flowers are in blossom, has cup-shaped, gyalectiform or bowl-shape, delicate and charming colourful, beautiful grace.In the past, people did not see pattern gold
Yellow species.Nineteen sixty, Chinese science worker are found that a kind of flavous Flos Camelliae Japonicae in one band of Nanning first, are ordered
Entitled Camellia nitidissima Chi.It is external to be referred to as magical " Dongfang " magic tea, it is described as " plant kingdom giant panda ", " tea race queen ".
Camellia nitidissima Chi is a kind of ancient plant, extremely rare, and distribution is extremely narrow, and the wild Camellia nitidissima Chi in the whole world 90% is only
Be distributed in blue one band of mountain offshoot of Guangxi China Fangchenggang City Shiwan Dashan, be grown on below 700 meters of height above sea level, with height above sea level 200～
More typically, the lower limit of vertical distribution is 20 meters or so of height above sea level to scope between 500 meters.If Camellia nitidissima Chi is near Fangcheng County king river
Strand hills tableland be still distributed.The upper limit of vertical distribution up to 890 meters of height above sea level, such as that Tao great Shan of Ningming County still visible
Other Camellia parvipetala, quantity are few, are rare species rare in the world.Camellia nitidissima Chi likes warm and moist weather, likes draining good
Good acid ground, seedling stage happiness are covered, and into after the florescence, quite happiness transmits sunlight.Require that to soil not sternly, subacidity to neutrality is
Can grow in soil.It is barren-resistant, fertilizer is also liked, waterlogging power is strong.
According in Nutrition and Food Safety Office of China Disease Prevention and control Centre, Guangxi Zhuang Autonomous Region prevention and control of diseases
It is the heart, Guangxi Zhuang Autonomous Region sanitary monitoring test center, the center nutrition of Beijing disease prevention and control center and food safety institute, wide
Authoritative institution's check tables such as western Zhuang autonomous region analysis testing research center, Colleges Of Traditional Chinese Medicine Of Guangxi, Guangxi Agriculture College experimental center
It is bright：The nontoxic level of JINHUA Camellia, containing more than 400 kinds of nutrient substance, have no toxic side effect.Rich in tea polysaccharide, tea polyphenols, Total saponin, total
Flavone, tea pigment, caffeine, protein, vitamin B1, B2, vitamin C, Vitamin E, Folic Acid, fatty acid, B- carotene etc.
Various natural nutrition compositions；Camellia nitidissima Chi contains tens kinds of aminoacid such as theanine, threonine, and various to human body tool rich in having
There are a trace element such as the natural organic germanium (Ge) of important health-care effect, selenium (Se), molybdenum (Mo), zinc (Zn), vanadium (V), and potassium (K),
The macroelements such as calcium (Ca), magnesium (Mg).Since first Camellia nitidissima Chi section was successfully held from 2009, Camellia nitidissima Chi section becomes Port of Fangcheng
One of four leading macroculture festival celebration brands of city.JINHUA tea industry is also developed rapidly, emerge osmanthus Ren Tang Camellia nitidissima Chis, in
The a collection of leading enterprises such as port high-tech national treasure Camellia nitidissima Chi, hundred happiness Camellia nitidissima Chis, Camellia nitidissima Chi series of products separately win Guangxi best brand of product, Guangxi
Famous mark, product enjoy a good market both at home and abroad market, and annual value of production is more than 1,200,000,000 yuan.
Camellia nitidissima Chi is deeply loved by the public as can be seen here, and improving the yield of Camellia nitidissima Chi, will to carry out offspring to Camellia nitidissima Chi numerous
Grow, in order to meet this demand, the present invention provides a kind of Camellia nitidissima Chi quickly tissue culture method, quickly can carry out group to Camellia nitidissima Chi
Culture breeding is knitted, and emergence rate is higher.
The content of the invention
The present invention is directed to above-mentioned problem, there is provided a kind of Camellia nitidissima Chi quickly tissue culture method, can reduce the cost bred,
Growth cycle is short, breeding potential is high, convenient management, automatically-monitored saved beneficial to industrialized production and greatly human and material resources
With the soil of field crops.
The solution of the present invention is by being achieved in that：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
The sterilization of explant：The leaf of Camellia nitidissima Chi in 6～July is plucked, and 400～600 is diluted using 20～40% dimethoate emulsions
Times immersion steeps 5～10min, be put into after taking-up superclean bench use after 4% vitamin C aqueous solution soaking 30min again 3% time
Chloric acid carries out disinfection 5～10min, then with aseptic water washing 3～5 times, finally blots leaf of Camellia nitidissima Chi surface using sterilizing filter paper
Sterilized water；The firm leaf of Camellia nitidissima Chi plucked is carried out disinfection using dimethoate emulsion, after can creep the insect on leaf of Camellia nitidissima Chi
Harmful liquid under residual is removed, and is then soaked using vitamin C and can not only effectively be delayed explant brown with hypochlorous acid sterilization again
Change time and holding explant vitality of subject, and the antibacterial on leaf of Camellia nitidissima Chi can be killed, reuse aseptic water washing, it is ensured that JINHUA
On Folium Camelliae sinensis, other liquid noresidues, finally blot the sterilized water on leaf of Camellia nitidissima Chi surface using sterilizing filter paper, in case by leaf of Camellia nitidissima Chi
There is diluting effect to culture medium in being put into callus culture base.
(2) callus culture：Callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out length
The leaf silver of 15～18mm, wide 10～15mm, leaf silver is put into and is had been loaded in the culture dish of callus culture base, light to press
Leaf silver, light culture 8～15 days at 29～31 DEG C then proceed to the light culture for carrying out at 26～28 DEG C 8～15 days, obtain gold
Scented tea calluss；By the way of light light culture, the time of light culture is former more than optical culture, can avoid leaf of Camellia nitidissima Chi reparation
Period excessive illumination makes which carry out photosynthesis and generates saccharide, loses a part of matter and energy in leaf of Camellia nitidissima Chi body, makes
The total amount that the matter and energy of repair process must be completed is reduced, and causes the time to form calluss slack-off.
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium be MS+6～8g/L agar+25～29g/L sucrose+0.3～0.8mg/L 6- benzyl purines+0.8～
1.5mg/L naphthalene acetic acids, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0-0.4mg/L, 3- indolebutyric acid 0.4-2.5mg/；Adjust
Section culture medium pH value be 6.0～6.5, under the conditions of 25～28 DEG C, light light culture after 30～45 days calluss differentiation sprout；
Addition 6- benzyl purines have suppression leaves of plants inner chlorophyll, nucleic acid, the decomposition of protein in the medium, protect green anti-old；Will
Aminoacid, auxin, inorganic salt etc. are to various efficiency such as treatment site allocation and transportation.
(4) bud is taken root：When in step (3), bud length is to 5～6cm, by bud dislocation root induction culture medium, wherein, root is lured
The formula for leading culture medium is MS+6～8g/L agar+25～29g/L sucrose+0.8～2.5mg/L+0.5～1.5mg/ of heteroauxing
The ferrous sulfate of L naphthalene acetic acid+0.8～1.5mg/L+0.8～1.5mg/L of activated carbon, under the conditions of 25～28 DEG C, light light culture 20
～30 days, seedling is turned out, tissue culture terminates.Adopt naphthalene acetic acid and can promote cell point for the substance of one of culture medium
Split and expand, it is inducible to take root；And the ferrous sulfate of anti-browning agent+0.8～1.5mg/L of activated carbon can reduce tissue culture process China and foreign countries
Implant, protocorm and Multiple Buds produce browning, have reached induction at the beginning of explant, the control of protocorm subculture multiplication melting brown rate at present and have existed
Less than 15%, Multiple Buds root induction melting brown rate is less than less than 5%.
In the present invention, used as further illustrating, described each culture dish is put into the leaf silver of 3～5.Leaf bar is put into suitably
Piece can guarantee that every leaf silver can sufficiently absorb enough nutrients, prevents nutrient deficiency from causing culture failure, that is, improves breeding
Rate.
In the present invention, as further illustrating, the formula of the callus culture base be MS+6～8g/L agar+25～
29g/L sucrose+0.3～0.8mg/L 6- benzyl purine+0.8～1.5mg/L naphthalene acetic acids, adjust culture medium pH value be 6.5～
7.0；Culture medium is put into into high temperature sterilize pot, in 121～130 DEG C, 1.1～1.5kg/cm2Under the conditions of carry out sterilizing 30～
50min, to then take out after being cooled to 45 DEG C and passes through the sterilizing of high temperature sterilize pot
Culture dish in.
In the present invention, used as further illustrating, the specification of the culture dish is 90 × 15mm；In the loading culture dish
Culture medium for culture dish volume 1/3～1/2.Method using directly toppling over, compares and measures quantitative culture medium using graduated cylinder,
With quick, easy advantage.
In the present invention, used as further illustrating, the brightness cultivation cycle in the step (3) and step (4) is optical culture
20～22h, 2～4h of light culture.Germinate and during taking root in calluss, need to absorb enough light sources formed chlorophyll after
And photosynthesis can be completed by absorbing light source, photosynthesis can synthesize the energy, supplement the energy needed for growth.
In the present invention, used as further illustrating, in the step (3), the intensity of illumination of optical culture is 1500～1700 Le gram
This, in step (4), the intensity of illumination of optical culture is 2000～3000 luxs.Light during callus culture using the low light level is trained
Support, improve the proliferation times of Camellia nitidissima Chi tissue culture, shorten the time of cycle culture, and can be reduced using the optical culture compared with the low light level
The power consumption produced during daylight lamp light filling and the heat for producing, reduce the expense needed for cooling, therefore can improve life using the method
Efficiency is produced, production cost, the development of favourable industry is reduced.Bud root culture strengthens the intensity of illumination, complies with the life of Camellia nitidissima Chi
Habit living, promotes its Rapid Rooting to germinate.
In the present invention, as further illustrating, the leaf of Camellia nitidissima Chi is that color is light green, complete, no disease and pests harm；Leaf of Camellia nitidissima Chi
Plucking time be at 1～2 point in evening.At dead of night leaf of Camellia nitidissima Chi is plucked, the leaf of Camellia nitidissima Chi in the late into the night has stopped carrying out light
Cooperation is used, but still carries out Repiration, is consumed internal starch, is carried out callus culture for leaf of Camellia nitidissima Chi and provide good
Good condition, reduction form the time of calluss.After first plucking in prior art
The operation for carrying out callus culture again is simpler.
The substantive distinguishing features of the protrusion of the present invention and marked improvement are：
1. the present invention carries out tissue culture using the leaf of Camellia nitidissima Chi in the late into the night, carries out callus culture for leaf of Camellia nitidissima Chi and provides well
Condition, can quickly make leaf of Camellia nitidissima Chi form leaf of Camellia nitidissima Chi calluss, and then reduce the time of tissue culture.
2. the present invention formed leaf of Camellia nitidissima Chi calluss after germinate, taking root light light culture of all carry out, not only by light train
Support and promote leaf of Camellia nitidissima Chi Callus formation chlorophyll, carry out the photosynthesis synthesis energy and energy needed for growth, Er Qietong are provided
Crossing light culture promotes which to carry out Repiration, and material conversion promotes growth.
3. optical culture of the present invention uses weaker intensity of illumination, not only increases the proliferation times of Camellia nitidissima Chi tissue culture,
The time of shortening cycle culture, and the power consumption that produces when can reduce daylight lamp light filling using the optical culture of the low light level and the heat for producing
Amount, reduce cooling needed for expense, therefore using the method can improve production efficiency, reduce production cost, favourable industry send out
Exhibition, reaches more than 75% by the germination percentage of method of the present invention Camellia nitidissima Chi.
4. the method for the present invention is conducive to industrialized production and automatically-monitored, has greatly saved human and material resources and field
The soil of interrow crop；There is in JINHUA tea shoot cultivation and production field great promotional value.
Specific embodiment
The present invention is further illustrated below by specific embodiment, so that advantages and features of the invention are easier to be managed
Solution, it should be understood that embodiments of the invention are only used for the present invention, rather than limitation of the present invention.
Embodiment 1：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
(1) sterilization of explant：Pluck at 1 point in evening June that color is light green, complete, no disease and pests harm leaf of Camellia nitidissima Chi, so
400 times of immersions are diluted using 20% dimethoate emulsion afterwards and steep 5min, be put into superclean bench and use 4% vitamin C water-soluble after taking-up
After immersion bubble 30min, 3% hypochlorous acid carries out disinfection 5min again, then with aseptic water washing 3 times, is finally inhaled using sterilizing filter paper
The sterilized water on dry leaf of Camellia nitidissima Chi surface；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 15mm, wide 10mm's
Leaf silver is during the leaf silver of 3 to be put into the specification for having been loaded into callus culture base for the culture dish of 90 × 15mm, light to press
Leaf silver, light culture 8 days at 29 DEG C then proceed to the light culture for carrying out at 26 DEG C 8 days, obtain JINHUA tea callus, its
In, the formula of callus culture base is MS+6g/L agar+25g/L sucrose+0.3mg/L 6- benzyl purine+0.8mg/L naphthalene second
Acid, the pH value for adjusting culture medium are 6.5；Culture medium is put into into high temperature sterilize pot, in 121 DEG C, 1.1kg/cm2Under the conditions of gone out
Bacterium 30min, to then take out after being cooled to 45 DEG C and passes through high temperature sterilize pot and go out
In the culture dish of bacterium, load the culture medium in culture dish for the 1/3 of culture dish volume；
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium is MS+6g/L agar+25g/L sucrose+0.3mg/L 6- benzyl purine+0.8mg/L naphthalene acetic acids, and is added auxiliary
Expect, adjuvant is：Indole-3-acetic acid 1.4mg/L, 3- indolebutyric acid 0.4mg/；The pH value for adjusting culture medium is 6.0, in 25 DEG C of bars
Under part, carry out optical culture 20h, the light light culture of light culture 4h after 30 days calluss differentiation sprout, wherein, the illumination of optical culture
Intensity is 1500 luxs；
(4) bud is taken root：When in step (3), bud length is to 5cm, by bud dislocation root induction culture medium, wherein, root induction training
The formula of foster base is MS+6g/L agar+25g/L sucrose+0.8mg/L heteroauxing+0.5mg/L naphthalene acetic acid+0.8mg/L activated carbons
The ferrous sulfate of+0.8mg/L, under the conditions of 25 DEG C, carries out optical culture 20h, the light light culture of light culture 4h 20 days, wherein, light
The intensity of illumination of culture is 3000 luxs, and to seedling is turned out, tissue culture terminates.
Embodiment 2：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
(1) sterilization of explant：Pluck at 5 points in evening July that colors are light green, complete, no disease and pests harm leaf of Camellia nitidissima Chi, so
600 times of immersions are diluted using 40% dimethoate emulsion afterwards and steep 10min, be put into superclean bench and use 4% vitamin C water-soluble after taking-up
After immersion bubble 30min, 3% hypochlorous acid carries out disinfection 10min again, then with aseptic water washing 5 times, finally using sterilizing filter paper
Blot the sterilized water on leaf of Camellia nitidissima Chi surface；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 18mm, wide 15mm's
Leaf silver is during the leaf silver of 5 to be put into the specification for having been loaded into callus culture base for the culture dish of 90 × 15mm, light to press
Leaf silver, light culture 15 days at 31 DEG C then proceed to the light culture for carrying out at 28 DEG C 15 days, obtain JINHUA tea callus,
Wherein, the formula of callus culture base is MS+8g/L agar+29g/L sucrose+0.8mg/L 6- benzyl purine+1.5mg/L naphthalenes
Acetic acid, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0.15mg/L, 3- indolebutyric acid 1.5mg/；Adjust the pH value of culture medium
For 6.0；Culture medium is put into into high temperature sterilize pot, in 130 DEG C, 1.5kg/cm2Under the conditions of carry out sterilizing 50min, then take out cold
But toppled in superclean bench to after 45 DEG C rapidly and be dispensed in the culture dish for passing through the sterilizing of high temperature sterilize pot, load training
Culture medium in foster ware for culture dish volume 1/2；
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium is MS+8g/L agar+29g/L sucrose+0.8mg/L 6- benzyl purine+1.5mg/L naphthalene acetic acids, adjusts culture
The pH value of base is 7.5, under the conditions of 28 DEG C, carries out optical culture 22h, light light culture calluss differentiation after 45 days of light culture 2h
Sprout, wherein, the intensity of illumination of optical culture is 1700 luxs；
(4) bud is taken root：When in step (3), bud length is to 6cm, by bud dislocation root induction culture medium, wherein, root induction training
The formula of foster base is MS+8g/L agar+29g/L sucrose+2.5mg/L heteroauxing+1.5mg/L naphthalene acetic acid+1.5mg/L activated carbons
The ferrous sulfate of+1.5mg/L, under the conditions of 28 DEG C, carries out optical culture 22h, the light light culture of light culture 2h 30 days, wherein, light
The intensity of illumination of culture is 2000 luxs, and to seedling is turned out, tissue culture terminates.
Embodiment 3：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
(1) sterilization of explant：In June evening 1: 40 timesharing pluck that color is light green, complete, no disease and pests harm JINHUA
Folium Camelliae sinensis, then dilute 450 times of immersions using 25% dimethoate emulsion and steep 6min, be put into superclean bench and use 4% dimension life after taking-up
After plain C aqueous solution soakings 30min, 3% hypochlorous acid carries out disinfection 6min again, then with aseptic water washing 4 times, finally using going out
Bacterium filter paper blots the sterilized water on leaf of Camellia nitidissima Chi surface；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 16mm, wide 11mm's
Leaf silver is during the leaf silver of 4 to be put into the specification for having been loaded into callus culture base for the culture dish of 90 × 15mm, light to press
Leaf silver, light culture 9 days at 30 DEG C then proceed to the light culture for carrying out at 27 DEG C 9 days, obtain JINHUA tea callus, its
In, the formula of callus culture base is MS+7g/L agar+26g/L sucrose+0.4mg/L 6- benzyl purine+0.9mg/L naphthalene second
Acid, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0.11mg/L 3- indolebutyric acid 0.22mg/；Adjust culture medium pH value be
6.2；Culture medium is put into into high temperature sterilize pot, in 122 DEG C, 1.2kg/cm2Under the conditions of carry out sterilizing 35min, then take out cooling
Toppled in superclean bench to after 45 DEG C rapidly and be dispensed in the culture dish for passing through the sterilizing of high temperature sterilize pot, load culture
Culture medium in ware for culture dish volume 1/3；
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium is MS+7g/L agar+26g/L sucrose+0.4mg/L 6- benzyl purine+0.9mg/L naphthalene acetic acids, adjusts culture
The pH value of base is 6.6, under the conditions of 26 DEG C, carries out optical culture 21h, light light culture calluss differentiation after 33 days of light culture 3h
Sprout, wherein, the intensity of illumination of optical culture is 1550 luxs；
(4) bud is taken root：When in step (3), bud length is to 5.5cm, by bud dislocation root induction culture medium, wherein, root induction
The formula of culture medium is that MS+7g/L agar+26g/L sucrose+0.9mg/L heteroauxing+0.8mg/L naphthalene acetic acids+0.9mg/L are active
The ferrous sulfate of charcoal+1.0mg/L, under the conditions of 26 DEG C, carries out optical culture 21h, the light light culture of light culture 3h 25 days, wherein,
The intensity of illumination of optical culture is 2300 luxs, and to seedling is turned out, tissue culture terminates.
Embodiment 4：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
(1) sterilization of explant：In July evening 1: 55 timesharing pluck that color is light green, complete, no disease and pests harm JINHUA
Folium Camelliae sinensis, then dilute 500 times of immersions using 35% dimethoate emulsion and steep 7min, be put into superclean bench and use 4% dimension life after taking-up
After plain C aqueous solution soakings 30min, 3% hypochlorous acid carries out disinfection 7min again, then with aseptic water washing 3 times, finally using going out
Bacterium filter paper blots the sterilized water on leaf of Camellia nitidissima Chi surface；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 17mm, wide 12mm's
Leaf silver is during the leaf silver of 3 to be put into the specification for having been loaded into callus culture base for the culture dish of 90 × 15mm, light to press
Leaf silver, light culture 14 days at 29 DEG C then proceed to the light culture for carrying out at 27 DEG C 13 days, obtain JINHUA tea callus,
Wherein, the formula of callus culture base is MS+7g/L agar+28g/L sucrose+0.7mg/L 6- benzyl purine+1.0mg/L naphthalenes
Acetic acid, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0.3mg/L, 3- indolebutyric acid 2.5mg/；
The pH value for adjusting culture medium is 6.1；Culture medium is put into into high temperature sterilize pot, in 128 DEG C, 1.4kg/cm2Under the conditions of
Sterilizing 40min is carried out, to be then taken out after being cooled to 45 DEG C and is passed through high temperature and go out
In the culture dish of bacterium pot sterilizing, load the culture medium in culture dish for the 1/2 of culture dish volume；
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium is MS+7g/L agar+26g/L sucrose+0.7mg/L 6- benzyl purine+1.3mg/L naphthalene acetic acids, adjusts culture
The pH value of base is 7.0, under the conditions of 27 DEG C, carries out optical culture 20h, light light culture calluss differentiation after 32 days of light culture 4h
Sprout, wherein, the intensity of illumination of optical culture is 1600 luxs；
(4) bud is taken root：When in step (3), bud length is to 5.3cm, by bud dislocation root induction culture medium, wherein, root induction
The formula of culture medium is that MS+7g/L agar+28g/L sucrose+2.0mg/L heteroauxing+1.0mg/L naphthalene acetic acids+1.0mg/L are active
The ferrous sulfate of charcoal+1.1mg/L, under the conditions of 26 DEG C, carries out optical culture 20h, the light light culture of light culture 4h 27 days, wherein,
The intensity of illumination of optical culture is 2100 luxs, and to seedling is turned out, tissue culture terminates.
Embodiment 5：
A kind of Camellia nitidissima Chi quickly tissue culture method, comprises the steps：
(1) sterilization of explant：In May evening 1: 40 timesharing pluck that color is light green, complete, no disease and pests harm JINHUA
Folium Camelliae sinensis, then dilute 550 times of immersions using 22% dimethoate emulsion and steep 9min, be put into superclean bench and use 4% dimension life after taking-up
After plain C aqueous solution soakings 30min, 3% hypochlorous acid carries out disinfection 9min again, then with aseptic water washing 4 times, finally using going out
Bacterium filter paper blots the sterilized water on leaf of Camellia nitidissima Chi surface；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 17mm, wide 14mm's
Leaf silver is during the leaf silver of 3 to be put into the specification for having been loaded into callus culture base for the culture dish of 90 × 15mm, light to press
Leaf silver, light culture 11 days at 29 DEG C then proceed to the light culture for carrying out at 26 DEG C 11 days, obtain JINHUA tea callus,
Wherein, the formula of callus culture base is MS+6g/L agar+25g/L sucrose+0.7mg/L 6- benzyl purine+1.2mg/L naphthalenes
Acetic acid, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0.25mg/L, 3- indolebutyric acid 0.35mg/；Adjust the pH value of culture medium
For 6.4；Culture medium is put into into high temperature sterilize pot, in 129 DEG C, 1.2kg/cm2Under the conditions of carry out sterilizing 45min, then take out cold
But toppled in superclean bench to after 45 DEG C rapidly and be dispensed in the culture dish for passing through the sterilizing of high temperature sterilize pot, load training
Culture medium in foster ware for culture dish volume 1/2；
(3) calluss Bud Differentiation：By in calluss dislocation bud inducement cultivation base obtained above, wherein, bud induction
The formula of culture medium is MS+6g/L agar+26g/L sucrose+0.7mg/L 6- benzyl purine+1.3mg/L naphthalene acetic acids, adjusts culture
The pH value of base is 7.3, under the conditions of 25 DEG C, carries out optical culture 21h, light light culture calluss differentiation after 42 days of light culture 3h
Sprout, wherein, the intensity of illumination of optical culture is 1650 luxs；
(4) bud is taken root：When in step (3), bud length is to 5cm, by bud dislocation root induction culture medium, wherein, root induction training
The formula of foster base is MS+6g/L agar+25g/L sucrose+2.3mg/L heteroauxing+0.9mg/L naphthalene acetic acid+1.3mg/L activated carbons
The ferrous sulfate of+1.3mg/L, under the conditions of 25 DEG C, carries out optical culture 22h, the light light culture of light culture 2h 29 days, wherein, light
The intensity of illumination of culture is 2500 luxs, and to seedling is turned out, tissue culture terminates.
As embodiment 1～5 it is each under the conditions of carry out tissue culture under conditions of, the sum, wound healing to the tissue culture of each embodiment
Primary vane was carried out every 3 days during the situation change in tissue culture's stage and emergence rate statistical table such as table 1, callus culture
The record of color change, summarizes variation tendency.
Claims (6)
1. a kind of Camellia nitidissima Chi quickly tissue culture method, it is characterised in that comprise the steps：
(1) sterilization of explant：The leaf of Camellia nitidissima Chi in 5～July is plucked, and 400～600 times is diluted using 20～40% dimethoate emulsions
Immersion steeps 5～10min, be put into after taking-up superclean bench use after 4% vitamin C aqueous solution soaking 30min again with 3% it is secondary
Chloric acid carries out disinfection 5～10min, then with aseptic water washing 3～5 times, finally blots leaf of Camellia nitidissima Chi surface using sterilizing filter paper
Sterilized water；
(2) callus culture：Using dissecting knife along leaf of Camellia nitidissima Chi stem position it is crosscutting go out long 15～18mm, it is wide by 10～
The leaf silver of 15mm, leaf silver is put into and is had been loaded in the culture dish of callus culture base, light pressure leaf silver, 29～31
Light culture 8～15 days at DEG C, then proceed to the light culture for carrying out at 26～28 DEG C 8～15 days, obtain JINHUA tea callus；
(3) calluss Bud Differentiation：Calluss obtained above are moved in bud inducement cultivation base, wherein, bud inducement cultivation
The formula of base is MS+6～8g/L agar+25～29g/L sucrose+0.3～0.8mg/L 6- benzyl purine+0.8～1.5mg/L naphthalenes
Acetic acid, and adjuvant is added, adjuvant is：Indole-3-acetic acid 0-0.4mg/L, 3- indolebutyric acid 0.4-2.5mg/；
The pH value for adjusting culture medium is 6.0～6.5, under the conditions of 25～28 DEG C, light light culture calluss point after 30～45 days
Dissolve bud；
(4) bud is taken root：When in step (3), bud length is to 5～6cm, bud is moved in root induction culture medium, wherein, root induction training
The formula of foster base is MS+6～8g/L agar+25～29g/L sucrose+0.8～2.5mg/L heteroauxing+0.5～1.5mg/L naphthalenes
The ferrous sulfate of acetic acid+0.8～1.5mg/L+0.8～1.5mg/L of activated carbon, under the conditions of 25～28 DEG C, light light culture 20～
30 days, seedling is turned out, tissue culture terminates,
The formula of the callus culture base is MS+6～8g/L agar+25～29g/L sucrose+0.3～0.8mg/L 6- benzyls
Base purine+0.8～1.5mg/L naphthalene acetic acids, the pH value for adjusting culture medium are 6.5～7.0；Culture medium is put into into high temperature sterilize pot,
In 121～130 DEG C, 1.1～1.5kg/cm2Under the conditions of carry out 30～50min of sterilizing, then take out after being cooled to 45 DEG C and to exist rapidly
Topple in superclean bench and be dispensed in the culture dish for passing through the sterilizing of high temperature sterilize pot.
2. Camellia nitidissima Chi quickly tissue culture method according to claim 1, it is characterised in that each culture dish is put into 3～5
Leaf silver.
3. Camellia nitidissima Chi quickly tissue culture method according to claim 1, it is characterised in that the specification of the culture dish is 90 ×
15mm；Load the culture medium in culture dish for the 1/3～1/2 of culture dish volume.
4. Camellia nitidissima Chi quickly tissue culture method according to claim 1, it is characterised in that in the step (3) and step (4)
Brightness cultivation cycle be 20～22h of optical culture, 2～4h of light culture.
5. Camellia nitidissima Chi quickly tissue culture method according to claim 1, it is characterised in that optical culture in the step (3)
Intensity of illumination is 1500～1700 luxs, and in step (4), the intensity of illumination of optical culture is 2000～3000 luxs.
6. Camellia nitidissima Chi quickly tissue culture method according to claim 1, it is characterised in that the leaf of Camellia nitidissima Chi is that color is tender
The blade of green, complete, no disease and pests harm；The plucking time of leaf of Camellia nitidissima Chi is at 1～2 point in evening.
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| CN105684913A (en) * | 2016-04-27 | 2016-06-22 | 覃伟 | Tissue culture fast propagation method for garcinia paucinervis |
| CN107125136A (en) * | 2017-06-08 | 2017-09-05 | 合肥市风达农业有限责任公司 | A kind of Camellia nitidissima tissue culture mating system |
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| JPH0292222A (en) * | 1988-09-30 | 1990-04-03 | Kyowa Hakko Kogyo Co Ltd | Production of indefinite embryo |
| CN1460713A (en) * | 2003-04-29 | 2003-12-10 | 广西富新科技股份有限公司 | Camellia chrysantha culture medium for tissue culture and quick breeding |
| 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 |
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