CN104488712A - Camellia nitidissima sprout tissue culture and rapid propagation method - Google Patents
Camellia nitidissima sprout tissue culture and rapid propagation method Download PDFInfo
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Abstract
The invention discloses a camellia nitidissima sprout tissue culture and rapid propagation method. The camellia nitidissima sprout tissue culture and rapid propagation method is characterized by comprising the following steps: (1) disinfecting explants; (2) culturing calluses; (3) differentiating shoots from the calluses; and (4) rooting the shoots. The camellia nitidissima sprout tissue culture and rapid propagation method disclosed by the invention has the advantages of propagation cost reduction, short growth cycle, high propagation rate and convenience of management, is suitable for industrial production and automatic monitoring and is beneficial to saving manpower, material resources and crop field greatly.
Description
Technical field
The invention belongs to tissue cultures production technical field, the present invention relates to the tissue culture and rapid propagation method of a kind of tender shoots Camellia nitidissima.
Background technology
Camellia nitidissima belongs to Theaceae, Camellia, is twin sisters with tea, camellia, South Mountain tea, oil tea, tea plum etc.Camellia nitidissima spend golden yellow, dazzling brilliant, be coated with coating of wax seemingly, sparkling and crystal-clear and glossy, seemingly have translucent 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.In the past, people did not see the golden yellow kind of pattern.Nineteen sixty, Chinese science worker has found a kind of flavous camellia at Nanning one band first, is named as Camellia nitidissima.Be referred to as magical " Dongfang " magic tea abroad, be described as " vegetative kingdom giant panda ", " tea race queen ".
Camellia nitidissima is a kind of ancient plant, very rare, distribute extremely narrow, the wild Camellia nitidissima in the whole world 90% is only distributed in blue mountain offshoot one band of Guangxi China Fangchenggang City Shiwan Dashan, be grown on height above sea level less than 700 meters, more common with the scope between height above sea level 200 ~ 500 meters, the lower limit of vertical distribution is height above sea level about 20 meters.As the strand hills tableland of Camellia nitidissima near Fangcheng County king river still has distribution.The upper limit of vertical distribution can reach height above sea level 890 meters, and as that Tao great Shan of Ningming County still can see indivedual Camellia parvipetala, quantity is few, is rare species rare in the world.Camellia nitidissima happiness warm and moist weather, like well-drained acid ground, seedling stage, happiness covered, and after entering the florescence, quite liked transmission sunlight.Require not tight to soil, subacidity can grow to neutral all soil.Barren-resistant, also like fertilizer, waterlogging power is strong.
Check according to authoritative institutions such as Nutrition and Food Safety Office of China Disease Prevention and control Centre, Guangxi Center for Disease Control and Prevention, sanitary monitoring test center, Guangxi Zhuang Autonomous Region, the center nutrition of Beijing disease prevention and control center and food security institute, Analysis-Test Research Center, Guangxi Zhuang Autonomous Region, Colleges Of Traditional Chinese Medicine Of Guangxi, Guangxi Agriculture College experimental centers and show: the nontoxic level of golden flower Camellia, containing 400 multiple nutrients materials, to have no side effect.Be rich in the multiple natural nutrition compositions such as tea polysaccharide, Tea Polyphenols, Total saponin, general flavone, Tea Pigment, caffeine, protein, vitamin B1, B2, vitamin C, vitamin E, folic acid, fatty acid, B-carotin; Camellia nitidissima contains tens seed amino acid such as theanine, threonine, and be rich in the multiple trace element such as natural organic germanium (Ge), selenium (Se), molybdenum (Mo), zinc (Zn), vanadium (V) human body to important health-care effect, and the macroelement such as potassium (K), calcium (Ca), magnesium (Mg).Since successfully holding first Camellia nitidissima joint from 2009, Camellia nitidissima joint becomes one of four leading macroculture festival celebration brands of Fangchenggang City.Camellia nitidissima industry have also been obtained and develops rapidly, emerge a collection of leading enterprises such as osmanthus Ren Tang Camellia nitidissima, middle port high-tech national treasure Camellia nitidissima, hundred happiness Camellia nitidissimas, Camellia nitidissima series of products separately win Guangxi best brand of product, Guangxi famous mark, and product enjoys a good market both at home and abroad market, and annual value of production is more than 1,200,000,000 yuan.
Camellia nitidissima is deeply loved by the public as can be seen here, the output improving Camellia nitidissima will carry out offspring's breeding to Camellia nitidissima, in order to meet this demand, the invention provides the tissue culture and rapid propagation method of a kind of tender shoots Camellia nitidissima, can carry out tissue culture propagating to Camellia nitidissima fast, and emergence rate is higher.
Summary of the invention
The present invention is directed to above-mentioned Problems existing, the tissue culture and rapid propagation method of a kind of tender shoots Camellia nitidissima is provided, the cost of breeding can be reduced, growth cycle is short, reproduction rate is high, conveniently manage, be beneficial to suitability for industrialized production and automatically-monitoredly saved the soil of human and material resources and field crops greatly.
The solution of the present invention is by realizing like this:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
The sterilization of explant: the golden camellia tea plucking for 6 ~ July, use 20 ~ 40% dimethoate emulsion dilution, 400 ~ 600 times of immersion bubble 5 ~ 10min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 5 ~ 10min, then use aseptic water washing 3 ~ 5 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface; Dimethoate emulsion is used to carry out disinfection to the golden camellia tea just plucked, harmful liquid under residual after insect on golden camellia tea being creeped is removed, then use vitamin C to soak effectively to delay the Brown time with hypochlorous acid sterilization again and keep explant vigor, and the bacterium on golden camellia tea can be killed, re-use aseptic water washing, guarantee other liquid noresidues on golden camellia tea, sterilizing filter paper is finally used to blot the sterile water on golden camellia tea surface, in case golden camellia tea is put into callus tissue culture base have diluting effect to medium.
(2) callus tissue culture: callus tissue culture: use scalpel to go out long 15 ~ 18cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 10 ~ 15cm, leaf silver is put into the culture dish loading callus tissue culture base, light pressure leaf silver, light culture 8 ~ 15 days at 29 ~ 31 DEG C, then proceed to the light culture carried out at 26 ~ 28 DEG C 8 ~ 15 days, obtain Camellia nitidissima callus; Adopt the mode of light light culture, the time of light culture, the former light that is greater than was cultivated, illumination too much during can avoiding golden camellia tea reparation makes it carry out photosynthesis and generates carbohydrate, lose a part of matter and energy in golden camellia tea body, the total amount of the matter and energy of repair process is reduced, has caused the time forming callus slack-off.
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+6 ~ 8g/L agar+25 ~ 29 g/L sucrose+0.3 ~ 0.8mg/L 6 ~ benzyl purine+0.8 ~ 1.5mg/L methyl α-naphthyl acetate, the pH value regulating medium is 6.0 ~ 6.5, under 25 ~ 28 DEG C of conditions, light light culture after 30 ~ 45 days Calli Differentiation sprout; Add 6 ~ benzyl purine and there is decomposition in the medium that suppress leaves of plants inner chlorophyll, nucleic acid, protein, protect green anti-old; By amino acid, growth hormone, mineral salt etc. to multiple usefulness such as treatment sites allocation and transportation.
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.4 mg/L to be that 22 ~ 25 DEG C of clear water were added in culture dish in follow-up 5 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 5 ~ 6cm in step (3), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+6 ~ 8g/L agar+25 ~ 29g/L sucrose+0.8 ~ 2.5mg/L heteroauxin+0.5 ~ 1.5mg/L methyl α-naphthyl acetate+0.8 ~ 1.5mg/L active carbon+0.8 ~ 1.5mg/L, under 25 ~ 28 DEG C of conditions, light light culture 20 ~ 30 days, turns out seedling, and group training terminates.Methyl α-naphthyl acetate is adopted to be that one of them additive mass-energy of medium enough promotes cell division and expansion, can root induction; And the ferrous sulfate of anti-brown agent active carbon+0.8 ~ 1.5mg/L can reduce explant, protocorm and Multiple Buds in group training process and produces brownization, reached at present that explant is just induced, protocorm shoot proliferation melting brown rate controls below 15%, Multiple Buds root induction melting brown rate is lower than less than 5%.
In the present invention, as further illustrating, the leaf silver of 3 ~ 5 put into by described each culture dish.Suitably put into leaf silver and can ensure that every bar leaf silver can absorb enough nutrients fully, prevent nutrient deficiency from causing and cultivate unsuccessfully, namely improve reproduction rate.
In the present invention, as further illustrating, the formula of described callus tissue culture base is MS+6 ~ 8g/L agar+25 ~ 29 g/L sucrose+0.3 ~ 0.8mg/L 6 ~ benzyl purine+0.8 ~ 1.5mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.5 ~ 7.0; Medium is put into high-temperature sterilization pot, at 121 ~ 130 DEG C, 1.1 ~ 1.5 kg/cm
2carry out sterilizing 30 ~ 50min under condition, in superclean bench, after then taking-up is cooled to 45 DEG C, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing.
In the present invention, as further illustrating, the specification of described culture dish is 90 × 15mm; Medium in described loading culture dish is 1/3 ~ 1/2 of culture dish volume.Adopt the method for directly toppling over, compare and use graduated cylinder to measure quantitative medium, have fast, easy advantage.
In the present invention, as further illustrating, the light light culture cycle in described step (3) and step (5) is that light cultivates 20 ~ 22h, light culture 2 ~ 4h.Germinate at callus and take root period, need to absorb enough light sources to form chlorophyll then by absorbing light source to complete photosynthesis, photosynthesis can synthesize the energy, supplements the energy needed for growth.
In the present invention, as further illustrating, the intensity of illumination that in described step (3), light is cultivated is 1500 ~ 1700 luxs, and the intensity of illumination that in step (5), light is cultivated is 2000 ~ 3000 luxs.The light of the low light level is adopted to cultivate during callus tissue culture, improve the proliferation times of Sectio Chrysantha training, the time that the shortening cycle cultivates, and adopt the heat of power consumption and the generation produced when cultivating can reduce fluorescent lamp light filling compared with the light of the low light level, reduce the expense needed for cooling, therefore adopt the method to enhance productivity, reduce production cost, the development of favourable industry.Bud culture of rootage strengthens the intensity of illumination, complies with the life habit of Camellia nitidissima, promotes that its Rapid Rooting germinates.
In the present invention, as further illustrating, described golden camellia tea be color light green, complete, without damage by disease and insect; The plucking time of golden camellia tea is point in evening 1 ~ 2.At dead of night golden camellia tea is plucked, the golden camellia tea in the late into the night has stopped carrying out photosynthesis, but still carries out respiration, consumes the starch in body, for golden camellia tea carries out the condition that callus tissue culture provides good, reduce the time forming callus.With first pluck blade in prior art after carry out light culture a period of time after to carry out the operation of callus tissue culture simpler.
Outstanding substantive distinguishing features of the present invention and marked improvement are:
1. the present invention utilizes the golden camellia tea in the late into the night to carry out group training, for golden camellia tea carries out the condition that callus tissue culture provides good, golden camellia tea can be made fast to form golden camellia tea callus, and then decrease the time of group training.
2. germinate after the present invention forms golden camellia tea callus, take root and all carry out light light culture, and cultivate before sprouting, tender shoots growth result can be increased.Not only cultivated by light and promote golden camellia tea Callus formation chlorophyll, carrying out the photosynthesis synthesis energy provides growth institute energy requirement, and promotes that it carries out respiration by light culture, and material is changed, growth promoting effects.
3. light of the present invention is cultivated and is used more weak intensity of illumination, not only increase the proliferation times of Sectio Chrysantha training, the time that the shortening cycle cultivates, and adopt the light of the low light level to cultivate the heat of power consumption and the generation produced when can reduce fluorescent lamp light filling, reduce the expense needed for cooling, therefore adopt the method to enhance productivity, reduce production cost, the development of favourable industry, reaches more than 75% by the germination rate of method Camellia nitidissima of the present invention.
4. method of the present invention is conducive to suitability for industrialized production and automatically-monitored, has saved the soil of human and material resources and field crops greatly; In golden flower tea shoot cultivation production field, there is great promotional value.
Embodiment
Further illustrate the present invention below by specific embodiment, be easier to make advantages and features of the invention be understood, it should be understood that embodiments of the invention are only used for the present invention, instead of limitation of the present invention.
embodiment 1:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
(1) sterilization of explant: at 1 in evening June pluck color light green, complete, without the golden camellia tea of damage by disease and insect, then 20% dimethoate emulsion is used to dilute 400 times of immersion bubble 5min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 5min, then use aseptic water washing 3 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 15cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 10cm, it is the culture dish of 90 × 15mm that the leaf silver of 3 is put into the specification loading callus tissue culture base, light pressure leaf silver, light culture 8 days at 29 DEG C, then proceed to the light culture carried out at 26 DEG C 8 days, obtain Camellia nitidissima callus, wherein, the formula of callus tissue culture base is MS+6g/L agar+25 g/L sucrose+0.3mg/L 6 ~ benzyl purine+0.8mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.5; Medium is put into high-temperature sterilization pot, at 121 DEG C, 1.1kg/cm
2carry out sterilizing 30min under condition, in superclean bench, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing after then taking-up is cooled to 45 DEG C, the medium loaded in culture dish is 1/3 of culture dish volume;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+6g/L agar+25g/L sucrose+0.3mg/L 6 ~ benzyl purine+0.8mg/L methyl α-naphthyl acetate, the pH value regulating medium is 6.0, under 25 DEG C of conditions, carry out light cultivate the light light culture of 20h, light culture 4h after 30 days Calli Differentiation sprout, wherein, the intensity of illumination that light is cultivated is 1500 luxs;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.4 mg/L to be that 22 ~ 25 DEG C of clear water were added in culture dish in follow-up 5 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 5cm in step (4), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+6g/L agar+25g/L sucrose+0.8mg/L heteroauxin+0.5mg/L methyl α-naphthyl acetate+0.8mg/L active carbon+0.8mg/L, under 25 DEG C of conditions, carry out light and cultivate 20h, the light light culture of light culture 4h 20 days, wherein, the intensity of illumination that light is cultivated is 3000 luxs, to turning out seedling, group training terminates.
embodiment 2:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
(1) sterilization of explant: at 2 in evening July pluck colors light green, complete, without the golden camellia tea of damage by disease and insect, then 40% dimethoate emulsion is used to dilute 600 times of immersion bubble 10min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 10min, then use aseptic water washing 5 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 18cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 15cm, it is the culture dish of 90 × 15mm that the leaf silver of 5 is put into the specification loading callus tissue culture base, light pressure leaf silver, light culture 15 days at 31 DEG C, then proceed to the light culture carried out at 28 DEG C 15 days, obtain Camellia nitidissima callus, wherein, the formula of callus tissue culture base is MS+8g/L agar+29 g/L sucrose+0.8mg/L 6 ~ benzyl purine+1.5mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.0; Medium is put into high-temperature sterilization pot, at 130 DEG C, 1.5 kg/cm
2carry out sterilizing 50min under condition, in superclean bench, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing after then taking-up is cooled to 45 DEG C, the medium loaded in culture dish is 1/2 of culture dish volume;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+8g/L agar+29 g/L sucrose+0.8mg/L 6 ~ benzyl purine+1.5mg/L methyl α-naphthyl acetate, the pH value regulating medium is 7.5, under 28 DEG C of conditions, carry out light cultivate the light light culture of 22h, light culture 2h after 45 days Calli Differentiation sprout, wherein, the intensity of illumination that light is cultivated is 1700 luxs;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.2 ~ 0.3 mg/L to be that 22 ~ 25 DEG C of clear water were added in culture dish in follow-up 5 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 6cm in step (4), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+8g/L agar+29g/L sucrose+2.5mg/L heteroauxin+1.5mg/L methyl α-naphthyl acetate+1.5mg/L active carbon+1.5mg/L, under 28 DEG C of conditions, carry out light and cultivate 22h, the light light culture of light culture 2h 30 days, wherein, the intensity of illumination that light is cultivated is 2000 luxs, to turning out seedling, group training terminates.
embodiment 3:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
(1) sterilization of explant: in evening 1: 20 in June timesharing pluck color light green, complete, without the golden camellia tea of damage by disease and insect, then 25% dimethoate emulsion is used to dilute 450 times of immersion bubble 6min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 6min, then use aseptic water washing 4 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 16cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 11cm, it is the culture dish of 90 × 15mm that the leaf silver of 4 is put into the specification loading callus tissue culture base, light pressure leaf silver, light culture 9 days at 30 DEG C, then proceed to the light culture carried out at 27 DEG C 9 days, obtain Camellia nitidissima callus, wherein, the formula of callus tissue culture base is MS+7g/L agar+26 g/L sucrose+0.4mg/L 6 ~ benzyl purine+0.9mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.2; Medium is put into high-temperature sterilization pot, at 122 DEG C, 1.2 kg/cm
2carry out sterilizing 35min under condition, in superclean bench, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing after then taking-up is cooled to 45 DEG C, the medium loaded in culture dish is 1/3 of culture dish volume;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+7g/L agar+26 g/L sucrose+0.4mg/L 6 ~ benzyl purine+0.9mg/L methyl α-naphthyl acetate, the pH value regulating medium is 6.6, under 26 DEG C of conditions, carry out light cultivate the light light culture of 21h, light culture 3h after 33 days Calli Differentiation sprout, wherein, the intensity of illumination that light is cultivated is 1550 luxs;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.6 mg/L to be that 23 ~ 27 DEG C of clear water were added in culture dish in follow-up 6 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 5.5cm in step (4), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+7g/L agar+26g/L sucrose+0.9mg/L heteroauxin+0.8mg/L methyl α-naphthyl acetate+0.9mg/L active carbon+1.0mg/L, under 26 DEG C of conditions, carry out light and cultivate 21h, the light light culture of light culture 3h 25 days, wherein, the intensity of illumination that light is cultivated is 2300 luxs, to turning out seedling, group training terminates.
embodiment 4:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
(1) sterilization of explant: in evening 1: 30 in July timesharing pluck color light green, complete, without the golden camellia tea of damage by disease and insect, then 35% dimethoate emulsion is used to dilute 500 times of immersion bubble 7min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 7min, then use aseptic water washing 3 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 17cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 12cm, it is the culture dish of 90 × 15mm that the leaf silver of 3 is put into the specification loading callus tissue culture base, light pressure leaf silver, light culture 14 days at 29 DEG C, then proceed to the light culture carried out at 27 DEG C 13 days, obtain Camellia nitidissima callus, wherein, the formula of callus tissue culture base is MS+7g/L agar+28 g/L sucrose+0.7mg/L 6 ~ benzyl purine+1.0mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.1; Medium is put into high-temperature sterilization pot, at 128 DEG C, 1.4 kg/cm
2carry out sterilizing 40min under condition, in superclean bench, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing after then taking-up is cooled to 45 DEG C, the medium loaded in culture dish is 1/2 of culture dish volume;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+7g/L agar+26 g/L sucrose+0.7mg/L 6 ~ benzyl purine+1.3mg/L methyl α-naphthyl acetate, the pH value regulating medium is 7.0, under 27 DEG C of conditions, carry out light cultivate the light light culture of 20h, light culture 4h after 32 days Calli Differentiation sprout, wherein, the intensity of illumination that light is cultivated is 1600 luxs;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.6 mg/L to be that 23 ~ 27 DEG C of clear water were added in culture dish in follow-up 6 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 5.3cm in step (4), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+7g/L agar+28g/L sucrose+2.0mg/L heteroauxin+1.0mg/L methyl α-naphthyl acetate+1.0mg/L active carbon+1.1mg/L, under 26 DEG C of conditions, carry out light and cultivate 20h, the light light culture of light culture 4h 27 days, wherein, the intensity of illumination that light is cultivated is 2100 luxs, to turning out seedling, group training terminates.
embodiment 5:
A tissue culture and rapid propagation method for tender shoots Camellia nitidissima, comprises the steps:
(1) sterilization of explant: in evening 1: 50 in May timesharing pluck color light green, complete, without the golden camellia tea of damage by disease and insect, then 22% dimethoate emulsion is used to dilute 550 times of immersion bubble 9min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 9min, then use aseptic water washing 4 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 17cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 14cm, it is the culture dish of 90 × 15mm that the leaf silver of 3 is put into the specification loading callus tissue culture base, light pressure leaf silver, light culture 11 days at 29 DEG C, then proceed to the light culture carried out at 26 DEG C 11 days, obtain Camellia nitidissima callus, wherein, the formula of callus tissue culture base is MS+6g/L agar+25 g/L sucrose+0.7mg/L 6 ~ benzyl purine+1.2mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.4; Medium is put into high-temperature sterilization pot, at 129 DEG C, 1.2 kg/cm
2carry out sterilizing 45min under condition, in superclean bench, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing after then taking-up is cooled to 45 DEG C, the medium loaded in culture dish is 1/2 of culture dish volume;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+6g/L agar+26 g/L sucrose+0.7mg/L 6 ~ benzyl purine+1.3mg/L methyl α-naphthyl acetate, the pH value regulating medium is 7.3, under 25 DEG C of conditions, carry out light cultivate the light light culture of 21h, light culture 3h after 42 days Calli Differentiation sprout, wherein, the intensity of illumination that light is cultivated is 1650 luxs;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.6 mg/L to be that 23 ~ 27 DEG C of clear water were added in culture dish in follow-up 6 ~ 10 days; The growth of tender shoots effectively can be accelerated by clear water.
(5) blastogenesis root: when bud grows to 5cm in step (4), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+6g/L agar+25g/L sucrose+2.3mg/L heteroauxin+0.9mg/L methyl α-naphthyl acetate+1.3mg/L active carbon+1.3mg/L, under 25 DEG C of conditions, carry out light and cultivate 22h, the light light culture of light culture 2h 29 days, wherein, the intensity of illumination that light is cultivated is 2500 luxs, to turning out seedling, group training terminates.
Under carrying out the condition of group training under condition as each in embodiment 1 ~ 5, to the sum of the tissue cultures of each embodiment, the change of the situation in callus tissue culture stage and emergence rate statistical form as table 1, carry out the record of primary vane color change during callus tissue culture every 3 days, sum up variation tendency.
Table 1 emergence rate statistical form
| Statistical item | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Tissue culture flasks number | 20 | 20 | 20 | 20 | 20 |
| The sheet number of tissue cultures | 61 | 102 | 83 | 64 | 60 |
| The change of leaf color during callus tissue culture | Light green ~ 1/3 green 2/3 is yellow ~ pale yellow | Light green ~ 1/2 green 1/2 is yellow ~ pale yellow | Green 1/2 Huang ~ 1/3 green 2/3, light green ~ 1/2 is yellow ~ pale yellow | Green 2/3 Huang ~ 1/4 green 3/4, light green ~ 1/3 is yellow ~ pale yellow | Green 2/3 Huang ~ 1/4 green 3/4, light green ~ 1/3 is yellow ~ pale yellow |
| Sprout sheet number | 53 | 89 | 73 | 64 | 56 |
| A seedling number | 55 | 74 | 62 | 52 | 53 |
| Planting percent | 83% | 74% | 77.5% | 86.6% | 88.3% |
As table 1 is reached a conclusion, when other conditions are constant, each culture dish is adopted to be applicable to cultivation 4 blades, enough energy can be provided, make callus energy fast germination, along with the number of blade of cultivating in culture dish is more, planting percent reduces gradually, so suitably control to cultivate blade sheet number, can improve by tissue cultures planting percent.
Claims (7)
1. a tissue culture and rapid propagation method for tender shoots Camellia nitidissima, is characterized in that, comprises the steps:
(1) sterilization of explant: the golden camellia tea plucking for 5 ~ July, use 20 ~ 40% dimethoate emulsion dilution, 400 ~ 600 times of immersion bubble 5 ~ 10min, put into after taking-up superclean bench use 4% vitamin C aqueous solution soaking 30min after again 3% hypochlorous acid to carry out disinfection 5 ~ 10min, then use aseptic water washing 3 ~ 5 times, finally use sterilizing filter paper to blot the sterile water on golden camellia tea surface;
(2) callus tissue culture: use scalpel to go out long 15 ~ 18cm along the stem position crosscut of golden camellia tea, the leaf silver of wide 10 ~ 15cm, leaf silver is put into the culture dish loading callus tissue culture base, light pressure leaf silver, light culture 8 ~ 15 days at 29 ~ 31 DEG C, then proceed to the light culture carried out at 26 ~ 28 DEG C 8 ~ 15 days, obtain Camellia nitidissima callus;
(3) Calli Differentiation bud: by callus dislocation bud inducement medium obtained above, wherein, the formula of bud inducement medium is MS+6 ~ 8g/L agar+25 ~ 29 g/L sucrose+0.3 ~ 0.8mg/L 6 ~ benzyl purine+0.8 ~ 1.5mg/L methyl α-naphthyl acetate, the pH value regulating medium is 6.0 ~ 6.5, under 25 ~ 28 DEG C of conditions, light light culture after 30 ~ 45 days Calli Differentiation sprout;
(4) blastogenesis root nursing in early stage: use the temperature containing sucrose 0.3 ~ 0.4 mg/L to be that 22 ~ 25 DEG C of clear water were added in culture dish in follow-up 5 ~ 10 days;
(5) blastogenesis root: when bud grows to 5 ~ 6cm in step (3), by in bud dislocation root induction medium, wherein, the formula of root induction medium is the ferrous sulfate of MS+6 ~ 8g/L agar+25 ~ 29g/L sucrose+0.8 ~ 2.5mg/L heteroauxin+0.5 ~ 1.5mg/L methyl α-naphthyl acetate+0.8 ~ 1.5mg/L active carbon+0.8 ~ 1.5mg/L, under 25 ~ 28 DEG C of conditions, light light culture 20 ~ 30 days, turns out seedling, and group training terminates.
2. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, is characterized in that, the leaf silver of 3 ~ 5 put into by described each culture dish.
3. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, it is characterized in that, the formula of described callus tissue culture base is MS+6 ~ 8g/L agar+25 ~ 29 g/L sucrose+0.3 ~ 0.8mg/L 6 ~ benzyl purine+0.8 ~ 1.5mg/L methyl α-naphthyl acetate, and the pH value regulating medium is 6.5 ~ 7.0; Medium is put into high-temperature sterilization pot, at 121 ~ 130 DEG C, 1.1 ~ 1.5 kg/cm
2carry out sterilizing 30 ~ 50min under condition, in superclean bench, after then taking-up is cooled to 45 DEG C, topple over rapidly being dispensed in the culture dish of high-temperature sterilization pot sterilizing.
4. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, is characterized in that, the specification of described culture dish is 90 × 15mm; Medium in described loading culture dish is 1/3 ~ 1/2 of culture dish volume.
5. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, is characterized in that, the light light culture cycle in described step (3) and step (5) is that light cultivates 20 ~ 22h, light culture 2 ~ 4h.
6. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, is characterized in that, the intensity of illumination that in described step (3), light is cultivated is 1500 ~ 1700 luxs, and the intensity of illumination that in step (5), light is cultivated is 2000 ~ 3000 luxs.
7. the tissue culture and rapid propagation method of tender shoots Camellia nitidissima according to claim 1, is characterized in that, described golden camellia tea be color light green, complete, without damage by disease and insect; The plucking time of golden camellia tea is point in evening 1 ~ 2.
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| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
| CN107135948A (en) * | 2017-06-02 | 2017-09-08 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima spray cultivates tissue-cultured seedling under sunlight conditions |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105494107A (en) * | 2016-02-25 | 2016-04-20 | 湖南农业大学 | Tea tree tissue culture method |
| CN107135948A (en) * | 2017-06-02 | 2017-09-08 | 广西壮族自治区南宁良凤江国家森林公园 | A kind of method that Camellia nitidissima spray cultivates tissue-cultured seedling under sunlight conditions |
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