CN107006367B - 'sunshine' cherry tissue culture rapid propagation method - Google Patents
'sunshine' cherry tissue culture rapid propagation method Download PDFInfo
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- CN107006367B CN107006367B CN201710259822.5A CN201710259822A CN107006367B CN 107006367 B CN107006367 B CN 107006367B CN 201710259822 A CN201710259822 A CN 201710259822A CN 107006367 B CN107006367 B CN 107006367B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses a tissue culture and rapid propagation method of 'sunshine' cherry. The invention uses the annual semi-lignified branch of the adult excellent plant as an explant through the setting of a basic culture medium and the optimization of different hormone components and concentration level proportion, forms a complete plant through axillary bud induction, axillary bud proliferation and rooting culture, and obtains the healthy and strong seedling with regular growth and consistent phenotype after being transplanted to the matrix. The invention has the advantages of high propagation coefficient, short cultivation period, no limitation of seasons and the like, and the inductivity reaches 100 percent; the multiplication coefficient reaches 5.1, the multiplication buds grow quickly, the number of cultured 18d buds is more than 2.5cm, and the number of effective buds reaches 35 per bottle on average; rooting culture for 10 days, wherein the rooting rate is more than 98 percent, and the number of roots reaches more than 5 per plant; the tissue culture rooted seedlings are robust, and the transplanting survival rate is up to more than 92 percent; in practical production application, annual large-scale rapid seedling culture can be carried out, healthy, tidy and consistent 'sunshine' cherry container seedlings are produced, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture rapid propagation method of 'sunshine' cherry.
Background
'sunshine' cherry (Cerasus youkou) is a horticultural cultivar bred by hybridizing Tiancheng Jiye (C.yedonensis cv. Amagi-yoshin) and Hanseng cherry (C.caululanta), is a deciduous arbor, and is in a shape of a tree cup; leaves are opened first, an umbrella-shaped inflorescence is formed, 3 flowers are bundled, and the flowers are horizontally slightly drooping; the calyx is long and bell-shaped, hairless, dark purple red or red, the long oval sepals are in the shape of needles, and the sepals are full of edges and hairless; the flower diameter is 3.8-4.6 cm, the flower has 5 petals, is light red and purple, has obvious veins, and the tip is deep and has 2 cracks; late 3 months, early 4 months (middle and lower Yangtze river), and middle 4 months (Beijing and Shandong). The cherry blossom is large and bright in color, has a florescence close to that of the Ji-Ye dyed well, can be matched with the Ji-Ye dyed well to form a red-white-phase cherry blossom tunnel, is called as cherry-Miao in Japan, and is widely popular with people. The cherry blossom is high in ornamental value, strong in tree vigor, strong in disease resistance and stress resistance and good in heat resistance, is a few ornamental cherry blossom varieties which can adapt to the climate of the south and north of China at present, and has a wide development prospect.
At present, the 'sunshine' cherry is propagated by grafting in a conventional way, and the propagation quantity is limited due to the limitation of factors such as the number of scions, the variety of stocks, the grafting time and the like, so that the market demand can not be met far away. The conventional propagation speed is low, the rapid popularization and application of the rapid propagation method are severely restricted, the rapid propagation method needs to be used for propagation in a tissue culture rapid propagation technology, the large-scale seedling culture is carried out by taking the adult excellent plants of the cerasus serrulata 'as stock plants, the industrial development of the cerasus serrulata' is promoted, and the method is practical and important.
Through literature search, no report about 'sunshine' cherry tissue culture rapid propagation is found at present. If the 'sunshine' cherry is subjected to tissue culture and rapid propagation by referring to the related tissue culture medium of the existing cerasus plants, the conditions of yellowing of tissue culture buds, slow extension and growth, small and weak buds, small number of effective buds, vitrification phenomena and the like can occur, so that the propagation coefficient is small, the seedling quality is poor, the propagation speed is slow, and the production cost is high.
disclosure of Invention
the invention aims to: the tissue culture rapid propagation method of the 'sunshine' cherry is provided to overcome the problems that the tissue culture rapid propagation method of the 'sunshine' cherry is deficient and the tissue culture rapid propagation effect of the related tissue culture medium to the 'sunshine' cherry is poor in the prior art.
in order to realize the aim, the invention provides a 'sunshine' cherry tissue culture rapid propagation method, which comprises the following steps:
(1) Pretreatment and collection of explants: before cutting the explant, alternately spraying a sunshine cherry tree body for 2-3 times by using carbendazim or chlorothalonil solution every 3-4 days, cutting the upper half lignified branches of the sunshine cherry tree body as the explant (preferably cutting the explant at 10 am in sunny weather), placing the explant under an ultraviolet lamp for irradiation for 20-30 min, removing leaves, soaking the explant for 5min by using a detergent solution, carefully and lightly scrubbing the stem (cutting off the leaves at the base of a petiole without brushing) by using a cotton ball or a soft brush pen dipped with the detergent solution, then cleaning the stem by using purified water, and preserving moisture at low temperature for later use;
(2) explant sterilization and axillary bud induction: sterilizing explants by using benzalkonium bromide and mercuric chloride, inoculating the sterilized explants to an induction culture medium to obtain axillary buds, wherein the induction culture medium takes YG as a basic culture medium, 0.5-1.5 mg of 6-BA (6-benzylamino adenine), 0-0.2 mg of IBA (indolebutyric acid), 0.05-0.2 mg of NAA (naphthalene acetic acid), 20-40 g of sucrose and 6g of agar are added per liter, and the pH value is 5.8-6.0;
(3) and (3) proliferation culture: cutting axillary buds, inoculating the axillary buds to a proliferation culture medium to culture to obtain proliferation seedlings, wherein the proliferation culture medium takes YG as a basic culture medium, and 0.4-1.0 mg of 6-BA, 0.05-0.2 mg of NAA, 0.05-0.2 mg of IBA, and GA are added to each liter3(gibberellin) 0.5-1.5 mg, cane sugar 20-40 g, agar 6g, and pH 5.8-6.0;
(4) rooting culture: cutting tender shoots of the proliferated seedlings, inoculating the tender shoots into a rooting culture medium, and culturing to obtain rooted seedlings, wherein the rooting culture medium takes 1/2YG culture medium as a basic culture medium, and each liter of the rooting culture medium is added with 0.2-0.5 mg of NAA, 0.2-0.5 mg of IBA, 15-20 g of sucrose, 6g of agar and 5.8-6.0 of pH;
(5) Hardening and transplanting seedlings: moving the rooted seedlings to a greenhouse for hardening and transplanting to obtain 'sunshine' cherry container seedlings.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, in the steps (2) and (3), the culture conditions are 24 +/-1 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/day.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, in the step (4), the culture condition is 25 +/-1 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h/day.
as an improvement of the 'sunshine' cherry tissue culture rapid propagation method, the YG culture medium contains the following components: 1000mg/L NH4NO3、950mg/L KNO3、220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、370mg/L MgSO4·7H2O、300mg/L NaH2PO4、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、6.2mg/L H3BO3、0.83mg/L KI、0.25mg/L Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L CoCl2、33.4mg/L FeSO4·7H2O、44.8mg/L Na2·EDTA·H2O, 100mg/L inositol, 2.0mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, pH 5.8, and water as solvent; the 1/2YG medium is NH in the YG medium4NO3、KNO3、Ca(NO3)2·4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4the dosage is reduced by half, and the other components are unchanged.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, the disinfection in the step (2) is to soak 1 wt% of benzalkonium bromide solution for 5-6 min, pour out the benzalkonium bromide solution, wash with sterile water for 3-4 times, then add 0.1 wt% of mercuric chloride solution to soak for 1-5 min, continuously shake during the process, pour out the mercuric chloride solution, and wash with sterile water for 5-6 times.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, the step (2) of inoculating on the induction culture medium is to cut off the injured parts at the two ends of the leaf supporting, the leaf stalk and the stem section of the explant by using a sterile blade, then inoculating on the induction culture medium, and inoculating one explant in one bottle.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, in the step (3), after the axillary buds are cut off and inoculated into a propagation culture medium, the axillary buds are differentiated to form a new bud cluster, the buds with the height of more than or equal to 3cm in the new bud cluster are cut into stem sections with the length of 1.0-1.5 cm, and then the stem sections are transferred into a new propagation culture medium for culture, so that propagation seedlings are obtained.
As an improvement of the 'sunshine' cherry tissue culture rapid propagation method, in the step (5), a rooting seedling which is subjected to rooting culture for 10-15 days is moved to a greenhouse to be adaptive to the environment for 5-7 days, then the seedling is hardened for 1 day (the tissue culture bottle cover is fully opened from half), the tissue culture seedling is taken out and cleaned to be a culture medium, and the tissue culture seedling is transplanted to a culture medium with a volume ratio of peat: perlite: and (3) on a mixed matrix of rice chaff ash which is 3:1:1, moisturizing by using a covering film 4-5 days before transplanting, and then unfastening the covering film according to conventional seedling management.
The invention sets a basic culture medium (YG culture medium, the basic culture medium specially set for the physiological state of 'sunshine' cherry), optimizes the proportion of different hormone components and concentration levels, takes the annual semi-lignified branch of an adult excellent plant as an explant, forms a complete plant through axillary bud induction, axillary bud proliferation and rooting culture, and obtains the healthy and strong seedling with regular growth and consistent phenotype after being transplanted to a substrate. The invention has the advantages of high speed of inducing the axillary buds of the explant and high induction rate reaching 100 percent; the propagation coefficient is high and reaches 5.1, the culture period is short, the method is not limited by seasons, the propagation bud grows fast, and the cultured 18d buds reach more than 3 cm; rooting culture for 10 days, wherein the rooting rate is more than 98 percent, and the number of roots reaches more than 5 per plant; the tissue culture rooted seedlings are robust, and the transplanting survival rate is up to more than 90 percent; in practical production application, annual large-scale rapid seedling culture can be carried out, and healthy, tidy and consistent 'sunshine' cherry tissue culture seedlings are produced.
Compared with the prior art, the invention has the following advantages:
The method has the advantages of high induction speed, high induction rate, large multiplication coefficient, strong cluster buds, high elongation and growth speed, high rooting rate, developed root system, strong and strong rooted seedlings, high transplanting survival rate, strong and tidy seedling growth and low production cost. The method provides an effective way for the large-scale seedling production of the 'sunshine' cherry seedlings, can obtain the 'sunshine' cherry seedlings on a large scale by the large-scale seedling culture, provides technical support for asexual popularization of the 'sunshine' cherry, provides a new excellent cherry variety and high-quality seedlings for landscaping and ecological civilization construction in Guangdong and even south China, and has great significance and wide application prospect.
Drawings
The method for tissue culture and rapid propagation of 'sunshine' cherry and the beneficial effects thereof are explained in detail in the following by combining the attached drawings and the specific implementation mode.
FIG. 1 is a diagram showing axillary bud induction according to the present invention.
FIG. 2 shows propagation flasks in the propagation medium of the present invention.
FIG. 3 is the root system of the rooted seedling in the rooting medium of the present invention.
FIG. 4 shows the tissue-cultured rooted seedlings of the present invention.
FIG. 5 shows the growth of the tissue-cultured rooted seedlings after transplantation.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results.
The YG media of the following examples all contained the following ingredients: 1000mg/L NH4NO3、950mg/L KNO3、220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、370mg/L MgSO4·7H2O、300mg/L NaH2PO4、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、6.2mg/L H3BO3、0.83mg/L KI、0.25mg/L Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L CoCl2、33.4mg/L FeSO4·7H2O、44.8mg/L Na2·EDTA·H2O, 100mg/L inositol, 2.0mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, and water as solvent. The 1/2YG medium contains macroelements (NH) contained in YG culture4NO3、KNO3、Ca(NO3)2·4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4) The dosage is reduced by half, and the other components and the preparation method are unchanged.
The components and the contents of the formula are uniformly mixed, the pH value is adjusted to be 5.8, and the sterilization is carried out for 18-20 min at the temperature of 121 ℃, so as to obtain the YG culture medium for later use.
Example 1
a tissue culture and rapid propagation method of 'sunshine' cherry comprises the following steps:
(1) explant collection: and (3) cutting strong semi-lignified branches without diseases and insect pests from a sunshine cherry stock plant in the sunny morning of 4-5 months, carrying the branches back to a laboratory after moisturizing treatment, removing leaves from the collected twigs, cleaning the twigs with purified water, and placing the twigs serving as explants in a refrigerator at 4 ℃ for storage. And before the explants are collected, spraying 800 times of carbendazim and chlorothalonil alternately for 2-3 times every 3-4 days.
(2) And (3) explant surface disinfection: cutting branches (explants) into axillary stems with leaves and about 4cm long, cleaning the axillary stems with leaves on a super-clean workbench by using sterile water , then using a neogill solution with the mass fraction of 1%, soaking for 5-6 min, pouring the neogill solution, washing for 3 times by using sterile water, then adding a mercuric chloride solution with the mass fraction of 0.1%, soaking for 1-5 min according to the tender degree of the explants, continuously shaking the process, pouring the mercuric chloride solution, and then washing for 5-6 times by using sterile water. And (3) absorbing water drops on the surface of the sterile material by sterile filter paper, cutting injured parts at two ends of a leaf supporting part, a petiole and a stem section by using a sterile blade, and then cutting the stem section into 2cm axillary stem sections with leaves for later use to obtain sterilized axillary sections with leaves.
(3) axillary bud induction: inoculating the sterilized axillary stem segment (explant) with leaves to an induction culture medium for culture, wherein the culture conditions are as follows: the axillary buds are obtained by inoculating one explant per bottle at 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the 3 rd axillary buds begin to germinate, the axillary buds stretch quickly, the 18 th buds can grow to 2.5-3 cm (figure 1), the induction rate is 100%, YG is used as a basic culture medium for an induction culture medium, 0.8mg of 6-BA, 0.1mg of IBA0.1mg, 0.1mg of NAA0.1mg, 30g of sucrose and 6g of agar (hormones purchased from Sigma-Aldrich company) are added into each liter of the induction culture medium, and the pH is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use).
(4) and (3) proliferation culture: the induced axillary buds are cut into 1.0-1.5 cm stem sections, and the stem sections are transferred into a proliferation culture medium for culture, wherein the culture conditions are as follows: 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the YG is taken as a basic culture medium, and 6-BA 0.6mg, IBA0.1mg, NAA0.1mg and GA are added into each liter30.5mg, 30g of cane sugar and 6g of agar, wherein the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use), after axillary buds are inoculated into a multiplication culture medium, the buds grow fast, single buds form bud clusters, the multiplication coefficient is increased and then decreased along with the extension of the subculture time (table 1), the 18d multiplication coefficient is the highest and reaches 5.1, and the height of seedlings is high>2.5cm, and the number of effective buds reaches 35 pieces/bottle (FIG. 2), thereby obtaining a proliferated seedling.
TABLE 1 Effect of different subculture cycles on proliferation of 'sunshine' cherry
(5) rooting culture: selecting strong effective buds (bud height is more than 1.5cm) from the proliferated seedlings, cutting and transferring the strong effective buds into a rooting culture medium for culture, wherein the culture conditions are as follows: 25 +/-1 ℃, the illumination intensity is 2500lx, the illumination time is 12h/d, the rooting culture medium takes 1/2YG as a basic culture medium, 0.2mg of NAA, 0.5mg of IBA, 15g of cane sugar and 6g of agar are added into each liter, the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use), the rooting rate is more than 98% in 10d, and the average rooting number of each plant is more than 5 (figure 3 and figure 4).
(6) hardening and transplanting seedlings: moving a rooting bottle seedling into a greenhouse to adapt to the external environment for 5-7 days, then hardening the seedling for 1d from half-open to full-open, carefully taking out the seedling in the bottle after hardening, cleaning a culture medium adhered to the seedling, transplanting the seedling into seedling culture cups filled with a peat, perlite and rice chaff ash mixed matrix of 3:1:1 (volume ratio), planting one seedling in each seedling cup, just covering the root system by the depth of transplanting covering soil, compacting the matrix to enable the seedling root to be in close contact with the matrix, and thoroughly pouring root fixing water after the transplanting is finished.
(7) Seedling cultivation: after transplanting, managing the moisture, fertilizer and germ resistance of the seedlings:
Moisture management: after the bottle seedlings are transplanted, the matrix is kept moist, the relative humidity of air is preferably 80% -90%, the moisture can be preserved by covering a membrane for 4-5 days, then water is supplemented by spraying, the culture temperature is controlled to be 25 +/-1 ℃, and a shading shed is shaded by a shading net with 70%;
Management of fertilizer and germ prevention: spraying a chlorothalonil or carbendazim solution with the mass fraction of 0.1% on the third day after transplantation for disinfection and sterilization, and then spraying the solution once every 10 days, wherein the chlorothalonil and the carbendazim are alternately used; when new buds germinate and new roots grow, 1500 times of nutrient solution is sprayed, and the nutrient solution is sprayed once every 15 days according to the growth condition of seedlings;
And thirdly, after the seedlings grow stably and grow new buds and new roots, the illumination intensity is gradually increased until full-illumination conventional management is carried out, the survival rate is higher than 96 percent (figure 5) after transplanting in one month, and when the seedlings grow to be 15cm high ('sunshine' cherry seedlings), the seedlings can be moved to a field to be cultivated into large seedlings.
example 2
A tissue culture and rapid propagation method of 'sunshine' cherry comprises the following steps:
(1) Explant collection: and (3) cutting strong semi-lignified branches without diseases and insect pests from a sunshine cherry stock plant in the sunny morning of 4-5 months, carrying the branches back to a laboratory after moisturizing treatment, removing leaves from the collected twigs, cleaning the twigs with purified water, and placing the twigs serving as explants in a refrigerator at 4 ℃ for storage. And before the explants are collected, spraying 800 times of carbendazim and chlorothalonil alternately for 2-3 times every 3-4 days.
(2) and (3) explant surface disinfection: cutting branches (explants) into axillary stems with leaves and about 4cm long, cleaning the axillary stems with leaves on a super-clean workbench by using sterile water , then using a neogill solution with the mass fraction of 1%, soaking for 5-6 min, pouring the neogill solution, washing for 3 times by using sterile water, then adding a mercuric chloride solution with the mass fraction of 0.1%, soaking for 1-5 min according to the tender degree of the explants, continuously shaking the process, pouring the mercuric chloride solution, and then washing for 5-6 times by using sterile water. And (3) absorbing water drops on the surface of the sterile material by sterile filter paper, cutting injured parts at two ends of a leaf supporting part, a petiole and a stem section by using a sterile blade, and then cutting the stem section into 2cm axillary stem sections with leaves for later use to obtain sterilized axillary sections with leaves.
(3) Axillary bud induction: inoculating the sterilized axillary stem segment (explant) with leaves to an induction culture medium for culture, wherein the culture conditions are as follows: 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, one explant is inoculated in each bottle, the 3 rd axillary bud begins to sprout, the bud grows well, the induction rate is 90.7%, the YG is used as a basic culture medium (shown in table 2, the YG is the best basic culture medium for inducing the axillary bud of 'sunshine' cherry), 6-BA1.0mg, NAA0.1mg, 30g of sucrose and 6g of agar (hormones are purchased from Sigma-Aldrich company) are added into each liter of the induction culture medium, and the pH is 5.8 (the preparation method is that the components are uniformly mixed, the pH value is adjusted, and the mixture is sterilized for later use), so that the axillary bud is obtained.
TABLE 2 Induction Effect of 'sunshine' cherry on different minimal media
(4) and (3) proliferation culture: the induced axillary buds are cut into 1.0-1.5 cm stem sections, and the stem sections are transferred into a proliferation culture medium for culture, wherein the culture conditions are as follows: 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the proliferation culture medium takes YG as a basic culture medium, and 6-BA 0.4mg, IBA0.1mg, NAA 0.05mg and GA are added into each liter31.0mg, 30g of cane sugar and 6g of agar, wherein the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use), after axillary buds are inoculated into a multiplication culture medium, the 18d multiplication coefficient reaches 3.2, the leaves are green and unfolded, the stems are strong, and the internodes are long, so that the multiplied seedlings are obtained.
(5) Rooting culture: selecting strong effective buds (bud height is more than 1.5cm) from the proliferated seedlings, cutting and transferring the strong effective buds into a rooting culture medium for culture, wherein the culture conditions are as follows: 25 +/-1 ℃, illumination intensity of 2500lx and illumination time of 12h/d, wherein the rooting medium takes 1/2YG as a basic medium, 0.2mg of NAA, 0.2mg of IBA, 15g of sucrose and 6g of agar are added into each liter, the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use), and the rooting rate reaches 83.7% after 10 d.
(6) Hardening and transplanting seedlings: moving a rooting bottle seedling into a greenhouse to adapt to the external environment for 5-7 days, then hardening the seedling for 1d from half-open to full-open, carefully taking out the seedling in the bottle after hardening, cleaning a culture medium adhered to the seedling, transplanting the seedling into seedling culture cups filled with a peat, perlite and rice chaff ash mixed matrix of 3:1:1 (volume ratio), planting one seedling in each seedling cup, just covering the root system by the depth of transplanting covering soil, compacting the matrix to enable the seedling root to be in close contact with the matrix, and thoroughly pouring root fixing water after the transplanting is finished.
(7) Seedling cultivation: after transplanting, managing the moisture, fertilizer and germ resistance of the seedlings:
Moisture management: after the bottle seedlings are transplanted, the matrix is kept moist, the relative humidity of air is preferably 80% -90%, the moisture can be preserved by covering a membrane for 4-5 days, then water is supplemented by spraying, the culture temperature is controlled to be 25 +/-1 ℃, and a shading shed is shaded by a shading net with 70%;
Management of fertilizer and germ prevention: spraying a chlorothalonil or carbendazim solution with the mass fraction of 0.1% on the third day after transplantation for disinfection and sterilization, and then spraying the solution once every 10 days, wherein the chlorothalonil and the carbendazim are alternately used; when new buds germinate and new roots grow, 1500 times of nutrient solution is sprayed, and the nutrient solution is sprayed once every 15 days according to the growth condition of seedlings;
And thirdly, after the seedlings grow stably and grow new buds and new roots, the illumination intensity is gradually increased until full-illumination conventional management is carried out, the survival rate is higher than 93% after transplanting in one month, and when the seedlings grow to be 15cm high ('sunshine' cherry seedlings), the seedlings can be moved to a field to be cultivated.
Example 3
A tissue culture and rapid propagation method of 'sunshine' cherry comprises the following steps:
(1) Explant collection: and (3) cutting strong semi-lignified branches without diseases and insect pests from a sunshine cherry stock plant in the sunny morning of 4-5 months, carrying the branches back to a laboratory after moisturizing treatment, removing leaves from the collected twigs, cleaning the twigs with purified water, and placing the twigs serving as explants in a refrigerator at 4 ℃ for storage. And before the explants are collected, spraying 800 times of carbendazim and chlorothalonil alternately for 2-3 times every 3-4 days.
(2) And (3) explant surface disinfection: cutting branches (explants) into axillary stems with leaves and about 4cm long, cleaning the axillary stems with leaves on a super-clean workbench by using sterile water , then using a neogill solution with the mass fraction of 1%, soaking for 5-6 min, pouring the neogill solution, washing for 3 times by using sterile water, then adding a mercuric chloride solution with the mass fraction of 0.1%, soaking for 1-5 min according to the tender degree of the explants, continuously shaking the process, pouring the mercuric chloride solution, and then washing for 5-6 times by using sterile water. And (3) absorbing water drops on the surface of the sterile material by sterile filter paper, cutting injured parts at two ends of a leaf supporting part, a petiole and a stem section by using a sterile blade, and then cutting the stem section into 2cm axillary stem sections with leaves for later use to obtain sterilized axillary sections with leaves.
(3) Axillary bud induction: inoculating the sterilized axillary stem segment (explant) with leaves to an induction culture medium for culture, wherein the culture conditions are as follows: the axillary buds are obtained by inoculating one explant into each bottle at 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the 3 rd of the axillary buds start to sprout, the buds grow well, the induction rate is 89%, the induction culture medium takes YG as a basic culture medium, and 6-BA1.0mg, IBA0.1mg, NAA0.1mg, sucrose 30g and agar 6g (hormones purchased from Sigma-Aldrich company) are added into each liter of the induction culture medium, and the pH value is 5.8 (the preparation method is that the components are uniformly mixed, the pH value is adjusted, and the mixture is sterilized for later use).
(4) And (3) proliferation culture: the induced axillary buds are cut into 1.0-1.5 cm stem sections, and the stem sections are transferred into a proliferation culture medium for culture, wherein the culture conditions are as follows: 24 +/-1 ℃, the illumination intensity is 2000lx, the illumination time is 12h/d, the YG is taken as a basic culture medium, and 6-BA 0.8mg, IBA0.2mg, NAA 0.05mg and GA are added into each liter31.0mg, 35g of cane sugar and 6g of agar, the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the above-mentioned components, regulating pH value and sterilizing for standby), after the axillary bud is inoculated into proliferation culture medium, the 18d proliferation coefficient can be up to 4.3, the leaf is green, spread and its stem is thick and thickThe internodes were generally long, and thus, the proliferated seedlings were obtained.
(5) Rooting culture: selecting strong effective buds (bud height is more than 1.5cm) from the proliferated seedlings, cutting and transferring the strong effective buds into a rooting culture medium for culture, wherein the culture conditions are as follows: 25 +/-1 ℃, illumination intensity of 2500lx and illumination time of 12h/d, wherein the rooting medium takes 1/2YG as a basic medium, 0.5mg of NAA, 0.2mg of IBA0.2mg, 20g of sucrose and 6g of agar are added into each liter, the pH value is 5.8 (the preparation method comprises the steps of uniformly mixing the components, adjusting the pH value and sterilizing for later use), and the rooting rate reaches 91% after 10 d.
(6) Hardening and transplanting seedlings: moving a rooting bottle seedling into a greenhouse to adapt to the external environment for 5-7 days, then hardening the seedling for 1d from half-open to full-open, carefully taking out the seedling in the bottle after hardening, cleaning a culture medium adhered to the seedling, transplanting the seedling into seedling culture cups filled with a peat, perlite and rice chaff ash mixed matrix of 3:1:1 (volume ratio), planting one seedling in each seedling cup, just covering the root system by the depth of transplanting covering soil, compacting the matrix to enable the seedling root to be in close contact with the matrix, and thoroughly pouring root fixing water after the transplanting is finished.
(7) Seedling cultivation: after transplanting, managing the moisture, fertilizer and germ resistance of the seedlings:
moisture management: after the bottle seedlings are transplanted, the matrix is kept moist, the relative humidity of air is preferably 80% -90%, the moisture can be preserved by covering a membrane for 4-5 days, then water is supplemented by spraying, the culture temperature is controlled to be 25 +/-1 ℃, and a shading shed is shaded by a shading net with 70%;
Management of fertilizer and germ prevention: spraying a chlorothalonil or carbendazim solution with the mass fraction of 0.1% on the third day after transplantation for disinfection and sterilization, and then spraying the solution once every 10 days, wherein the chlorothalonil and the carbendazim are alternately used; when new buds germinate and new roots grow, 1500 times of nutrient solution is sprayed, and the nutrient solution is sprayed once every 15 days according to the growth condition of seedlings;
And thirdly, after the seedlings grow stably and grow new buds and new roots, the illumination intensity is gradually increased until full-illumination conventional management is carried out, the survival rate is higher than 92% after transplanting in one month, and when the seedlings grow to be 15cm high ('sunshine' cherry seedlings), the seedlings can be moved to a field to be cultivated.
Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Claims (3)
1. a 'sunshine' cherry tissue culture rapid propagation method is characterized by comprising the following steps:
(1) pretreatment and collection of explants: before cutting the explant, alternately spraying sunshine cherry tree bodies for 2-3 times by using carbendazim or chlorothalonil solution every 3-4 days, cutting upper half lignified branches of the sunshine cherry tree bodies as the explant, placing the explant under an ultraviolet lamp for irradiation for 20-30 min, removing leaves, soaking for 5min by using detergent solution, cleaning, and preserving moisture at low temperature for later use;
(2) Explant sterilization and axillary bud induction: disinfecting an explant by using benzalkonium bromide and mercuric chloride, inoculating the explant to an induction culture medium to obtain axillary buds, wherein the induction culture medium takes YG as a basic culture medium, and each liter of the induction culture medium is added with 0.5-1.5 mg of 6-BA, 0.05-0.2 mg of IBA 0-0.2 mg of NAA, 20-40 g of cane sugar and 6g of agar, and the pH value is 5.8-6.0;
(3) and (3) proliferation culture: cutting axillary buds, inoculating the axillary buds to a proliferation culture medium to culture to obtain proliferation seedlings, wherein the proliferation culture medium takes YG as a basic culture medium, and 0.4-1.0 mg of 6-BA, 0.05-0.2 mg of NAA, 0.05-0.2 mg of IBA, and GA are added to each liter30.5-1.5 mg, 20-40 g of cane sugar and 6g of agar, wherein the pH value is 5.8-6.0;
(4) rooting culture: cutting tender shoots of the proliferated seedlings, inoculating the tender shoots into a rooting culture medium, and culturing to obtain rooted seedlings, wherein the rooting culture medium takes 1/2YG culture medium as a basic culture medium, and each liter of the rooting culture medium is added with 0.2-0.5 mg of NAA, 0.2-0.5 mg of IBA, 15-20 g of sucrose, 6g of agar and 5.8-6.0 of pH;
(5) hardening and transplanting seedlings: moving the rooted seedlings to a greenhouse for hardening and transplanting to obtain 'sunshine' cherry container seedlings;
The YG medium contained the following ingredients: 1000mg/L NH4NO3、950mg/L KNO3、220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、370mg/L MgSO4·7H2O、300mg/L NaH2PO4、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、6.2mg/L H3BO3、0.83mg/L KI、0.25mg/L Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L CoCl2、33.4mg/L FeSO4·7H2O、44.8mg/L Na2·EDTA·H2o, 100mg/L inositol, 2.0mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, pH 5.8, and water as solvent; the 1/2YG medium is NH in the YG medium4NO3、KNO3、Ca(NO3)2·4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4the dosage is reduced by half, and the other components are unchanged;
In the step (2), the culture condition is 24 +/-1 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/day;
In the step (3), the culture condition is 24 +/-1 ℃, the illumination intensity is 2000lx, and the illumination time is 12 h/day;
In the step (4), the culture condition is 25 +/-1 ℃, the illumination intensity is 2500lx, and the illumination time is 12 h/day;
The disinfection in the step (2) is carried out by soaking 1 wt% of benzalkonium bromide solution for 5-6 min, then washing with sterile water for 3-4 times, then adding 0.1 wt% of mercuric chloride solution, soaking for 1-5 min, and then washing with sterile water for 5-6 times;
The step (2) of inoculating the explant on the induction culture medium is to cut the injured parts at the two ends of the leaf, the petiole and the stem section of the explant by using a sterile blade and then inoculate the explant on the induction culture medium.
2. The 'sunshine' cherry tissue culture and rapid propagation method according to claim 1, wherein in the step (3), after the axillary buds are cut and inoculated to the enrichment culture medium, the axillary buds are differentiated to form a new bud cluster, buds in the new bud cluster with the height of more than or equal to 3cm are cut into stem sections with the length of 1.0-1.5 cm, and then the stem sections are transferred to the new enrichment culture medium for culture, so that the proliferated seedlings are obtained.
3. The 'sunshine' cherry tissue culture and rapid propagation method according to claim 1, wherein in the step (5), the rooted seedlings are moved to a greenhouse to be adaptive to the environment for 5-7 days, then the seedlings are hardened for 1 day, the tissue culture seedlings are removed from the clean culture medium and are transplanted to peat: perlite: and (3) on a mixed matrix of rice chaff ash which is 3:1:1, moisturizing by using a covering film 4-5 days before transplanting, and then unfastening the covering film according to conventional seedling management.
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