CN111869566A - Ginseng free microspore induction culture method - Google Patents
Ginseng free microspore induction culture method Download PDFInfo
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Abstract
The invention provides a method for inducing and culturing isolated microspores of ginseng, which comprises the following steps: (1) selection, pretreatment and sterilization of flower buds: collecting buds in a mononuclear border period at the initial flowering stage of the ginseng, pretreating the buds at a low temperature of 4 ℃ for 1-2 days, and sterilizing; (2) separating and purifying microspores; (3) heat shock pretreatment: resuspending microspores in an induction culture medium, subpackaging, and culturing in dark at 32 deg.C for 3 d; (4) and (3) induction culture: the culture dish is transferred to a dark condition at 25 ℃ for static culture, and when embryoid bodies can be seen by naked eyes, the culture dish is transferred to a shaking table for continuous culture. The invention also provides a ginseng isolated microspore induction culture medium, which has a remarkable promoting effect on embryogenesis of ginseng microspores by adding certain specific components. The invention discusses the induction method of the ginseng isolated microspore for the first time, obtains the embryoid of the ginseng microspore through induction culture, and lays a foundation for establishing a culture system of the ginseng isolated microspore.
Description
Technical Field
The invention relates to a method for inducing and culturing isolated microspores of ginseng, belonging to the field of medicinal plant biotechnology.
Background
Ginseng (A, B)Panax ginsengC.A. Mey) also called yellow ginseng, herba schizophragmatis integrifolii, shencao, etc., which are plants of Panax genus of Araliaceae family, and their roots and overground plants can be used as medicine. The ginseng is produced in China, Korea, Japan and the like, has sweet taste and warm nature, is fond of cool and shady, has the efficacies of greatly reinforcing primordial qi, regulating and reinforcing five internal organs, removing pathogenic qi, improving eyesight and intelligence, recovering pulse and relieving depletion and the like, is one of the Baicao king and is also called northeast three treasures. The ginseng contains various ginsenoside, amino acid, polypeptide, polysaccharide and ginseng enzyme, and has the effects of invigorating primordial qi, invigorating spleen, benefiting lung, nourishing, tranquilizing, and improving memory. Modern researches find that ginseng has the effects of reducing oxidative stress reaction, relieving depression, preventing and treating senile dementia, improving atherosclerosis, improving osteoblast survival rate,Inhibiting the growth of tumor cells, etc. With the improvement of living standard of people, ginseng is not only used as a medicinal material, but also widely applied to a plurality of fields such as medicated diet, beverage, health care, cosmetics and the like.
Ginseng is a rare Chinese medicinal material and has a long cultivation history in China. In long-term production practice, people accumulate abundant cultivation experience, but in the breeding work, due to the lack of effective technical means, the barrier of traditional breeding cannot be broken up so far, and the progress of the ginseng breeding work is greatly hindered. Although the types of the plants in the genus of ginseng are more, the variation is unstable, and in addition, the plants grow slowly and have long growth period, and a new breakthrough of breeding in a short time is difficult to realize without an effective breeding means. Therefore, combining the species characteristics, selecting a proper breeding method to obtain rich and stable genetic types and accelerating the breeding process are the urgent priority for developing the ginseng breeding work.
Haploid breeding refers to a breeding method in which a plant tissue culture technology (such as anther in vitro culture) is used for inducing to generate a haploid plant, and a chromosome set is doubled by some means (such as colchicine treatment), so that a new variety is bred. As only one gene of each haploid material is needed, a doubled haploid homozygous line with homozygous genotype can be obtained after doubling, the breeding period can be greatly shortened, and the breeding efficiency is improved. Anther culture and free microspore culture are the main approaches to obtain haploid plants, wherein free microspore culture is a tissue culture technology developed on the basis of anther culture in recent years and has more advantages compared with anther culture. Pure microspores are separated in the culture of the free microspores, and the influence of anther wall and tapetum tissues on the division, differentiation and development of microspore cells in the culture is eliminated. The microspore culture solves the problem of plant interference formed by anther somatic cells frequently occurring in anther culture, and obviously improves the regeneration rate of haploids. During the culture period, various external factors can directly act on microspores, and the generated regeneration plants are all from the microspores, so that the double haploid plants obtained by the method are high homozygous genetically. The pure line obtained by microspore culture can be directly used for cultivating new varieties or used as an excellent parent to be indirectly applied to variety improvement. Meanwhile, the microspore culture technology is combined with the transgenic technology and the conventional breeding technology, so that the breeding level of crops can be greatly improved.
The free microspore culture is to change the normal development direction of plant pollen under the condition of in vitro culture to form a haploid plant. In this process, microspores are susceptible to and controlled by various factors. Wherein, the internal factors mainly comprise the genotype of the donor plant, the growth environment of the donor plant, the microspore development period and the like, and the external factors mainly comprise pretreatment measures, culture medium types, culture medium additives, culture density, culture conditions and the like. In recent years, techniques for culturing free microspores have been greatly improved. However, because the difficulty of culturing the free microspores is high and the involved factors are many, although China has more researches, most of the methods stay in the establishment of a culture system and preliminary application research, the extent of randomly operating the microspores is not reached, and the application range of the methods is also limited by plant species, varieties and varieties. However, no report is found on the current research on culture of isolated microspores of ginseng.
Disclosure of Invention
Aiming at the problems, the invention takes ginseng of different varieties as test materials, systematically studies factors influencing the microspore induction of the ginseng, establishes a universally applicable ginseng isolated microspore induction system and provides a theoretical basis for further perfecting the ginseng microspore culture technology.
The technical scheme provided by the invention is as follows:
a method for inducing and culturing isolated microspores of ginseng is characterized in that: comprises the following steps:
(1) selection, pretreatment and sterilization of flower buds: in the initial flowering period of the ginseng, collecting buds in the mononuclear border period, and further determining the microspore development period under a microscope; moisturizing the flower buds, standing in a refrigerator at 4 ℃ for 1-2 days, and sterilizing;
(2) microspore separation and purification: draining water from sterilized flower bud with absorbent paper, placing into mortar, adding small amount of B5-13 extracting solution, gently squeezing with grinding rodDissociating microspore into extractive solution, filtering with 400 mesh nylon net to 10ml centrifuge tube, centrifuging at 800r/min for 3min, discarding supernatant, adding small amount of B into precipitate513, shaking the extracting solution evenly, centrifuging at 800r/min for 3min, and repeating twice; discarding the supernatant to obtain precipitate as pure microspore;
(3) heat shock pretreatment: resuspending the purified microspores by using an induction culture medium, adjusting the density, subpackaging the microspores into sterile culture dishes with the diameter of 60mm, wherein each dish is 2.5-3.5 ml, and sealing by using a Parafilm membrane; placing the culture dish in a dark condition at 32 ℃ for culturing for 3 d;
(4) and (3) induction culture: and (4) transferring the heat-shocked culture dish to a dark condition at 25 ℃ for standing culture, and transferring to a shaking table for continuous culture when embryoid can be seen by naked eyes.
The length of the flower buds in the edge-approaching period of the mononuclear in the step (1) is 1.1-1.5 mm, the diameter is 2.1-2.8 mm, and the color of the anther is green or yellow-green.
The sterilization method in the step (1) comprises the following steps: transferring the flower bud of Ginseng radix into a sterile beaker of a clean bench, adding 70% alcohol for surface sterilization, soaking for 1min, pouring off alcohol, adding 0.1% mercuric chloride solution, soaking for sterilization for 10min, and washing with sterile water for 3 times.
In the step (3), the density of the microspores is adjusted to 1.0 x 105~2.0×105One per ml.
The formula of the induction culture medium in the step (3) is as follows: ca (NO)3）2·4H2O 430-530mg/L，KNO3150-200mg/L，KH2PO4100-150mg/L，MgSO4·7H2O 52-76mg/L，NaCl 37-45mg/L，FeSO4·7H2O26.4-30.2mg/L，Na2-EDTA 35.7-39.3mg/L，H3BO310.6-14.4mg/L，MnSO4·4H2O 20.5-24.0mg/L，ZnSO4·7H2O 7.7-9.5mg/L，KI 0.5-0.6mg/L，Na2MoO4·2H2O 0.2-0.3mg/L，CuSO4·5H2O 0.05-0.06mg/L，CoCl2·6H20.02-0.03mg/L of O, 1.2-1.6mg/L of choline, 10.4-0.6mg/L of vitamin B, 60.4-0.6mg/L, 0.4-0.6mg/L nicotinamide, 0.4-0.6mg/L folic acid, 0.08-0.10mg/L biotin, 70-110 mg/L-serine, 1.0-3.0mg/L glycine, 15-25mg/L sodium pyruvate, 35-45mg/L cyclic adenosine monophosphate, 20-50 mg/L-glutathione, 60-90mg/L inositol, H2O280-120 mu mol/L, 0.8-1.0mg/L of triacontanol, 1.4-1.6mg/L of 2,4-D, 0.4-0.6mg/L of KT, 1.8-2.2mg/L of melatonin, 500mg/L of yeast extract 400-.
The temperature of shaking table culture in the step (4) is 25 ℃, and the rotating speed is 40-50 r/min.
And adjusting the pH value of the induction culture medium to 5.6-6.0, and filtering and sterilizing by adopting a 0.22 mu m microporous filter membrane.
The invention has the following beneficial effects:
(1) the invention initially explores the induction culture of the ginseng isolated microspore, researches factors influencing the embryogenic rate of the ginseng microspore such as pretreatment, culture conditions, culture medium and the like, determines a method suitable for culturing the induced embryoid by the ginseng isolated microspore and lays a foundation for establishing a complete and efficient ginseng isolated microspore culture system.
(2) The induction medium is the key influencing the formation of embryoid, and the optimal formula of the induction medium is determined by a large number of single-factor experiments. The culture medium is added with certain concentrations of choline, nicotinamide, sodium pyruvate, cyclic adenosine monophosphate (cAMP) and H2O2The plant growth regulator is composed of triacontanol and 2,4-D, KT 0.5.5 mg/L yeast extract, and has significant promoting effect on embryogenesis of ginseng microspore.
Detailed Description
In order that the invention may be better understood, it is further illustrated by the following specific examples, which are not to be construed as limiting the invention.
Example 1
Test materials: 2 varieties of ginseng, namely 'big Maya' and 'bamboo reed', are selected, tested plants are all grown for five years and come from a ginseng planting base of Liaoning Hua Shen source.
The ginseng isolated microspore induction culture is carried out according to the following steps:
(1) selection, pretreatment and sterilization of flower buds: collecting buds of the ginseng at the edge stage of the single core at the initial flowering stage, wherein the corresponding length is 1.1mm-1.5mm, the diameter is 2.1mm-2.8mm, the color of the anther is green or yellow-green, and further determining the development period of the microspore under a microscope; and (4) moisturizing the flower buds, standing in a refrigerator at 4 ℃ for 1-2 days, and sterilizing. The sterilization method comprises the following steps: transferring the flower bud of Ginseng radix into a sterile beaker of a clean bench, adding 70% alcohol for surface sterilization, soaking for 1min, pouring off alcohol, adding 0.1% mercuric chloride solution, soaking for sterilization for 10min, and washing with sterile water for 3 times.
(2) Microspore separation and purification: draining water from sterilized flower bud with absorbent paper, placing into mortar, adding small amount of B513 extracting solution, slightly squeezing with grinding rod to make microspore free in the extracting solution, filtering with 400 mesh nylon net to 10ml centrifuge tube, centrifuging at 800r/min for 3min, discarding supernatant, adding small amount of B into precipitate513, shaking the extracting solution evenly, centrifuging at 800r/min for 3min, and repeating twice; and discarding the supernatant to obtain a precipitate, namely the pure microspore.
(3) Heat shock pretreatment: the purified microspores were resuspended in induction medium and adjusted to a density of 1.0X 105~2.0×105Subpackaging the seeds/ml into sterile culture dishes with the diameter of 60mm, wherein each dish is 2.5-3.5 ml, and sealing by using a Parafilm film; the dishes were incubated at 32 ℃ in the dark for 3 d.
(4) And (3) induction culture: and (3) transferring the heat-shocked culture dish to a dark condition of 25 ℃ for static culture, transferring to a shaking table (the temperature is 25 ℃ and the rotating speed is 45 r/min) for continuous culture for 4 weeks when embryoids are visible by naked eyes, counting the number of microspore embryoids, and calculating the embryo yield.
The formulation of the induction medium is as follows: ca (NO)3）2·4H2O 480mg/L，KNO3175mg/L，KH2PO4125mg/L，MgSO4·7H2O 64mg/L，NaCl 41mg/L，FeSO4·7H2O 28.3mg/L，Na2-EDTA 37.5mg/L，H3BO312.5mg/L，MnSO4·4H2O 22.3mg/L，ZnSO4·7H2O 8.3mg/L，KI 0.55mg/L，Na2MoO4·2H2O0.25mg/L，CuSO4·5H2O 0.055mg/L，CoCl2·6H2O0.025 mg/L, choline 1.4mg/L, vitamin B10.5mg/L, vitamin B60.5mg/L, nicotinamide 0.5mg/L, folic acid 0.5mg/L, biotin 0.09mg/L, L-serine 90mg/L, glycine 2.0mg/L, sodium pyruvate 20mg/L, cyclic adenosine monophosphate 40mg/L, L-glutathione 35mg/L, inositol 75mg/L, H2O2100 mu mol/L, triacontanol 0.9mg/L, 2, 4-D1.5 mg/L, KT 0.5mg/L, melatonin 2.0mg/L, yeast extract 450mg/L, 2-morpholine ethanesulfonic acid 70mg/L, sucrose 130g/L and active carbon 100 mg/L.
Example 2: effect of Heat shock pretreatment on Induction of Ginseng microspore embryoid
A method consistent with example 1 was used, except that: during heat shock pretreatment, the culture dish is placed in a dark condition of 30 ℃ or 32 ℃ for 3d or 5d, then the culture dish is transferred to a dark condition of 25 ℃ for static culture, when embryoid can be seen by naked eyes, the culture dish is transferred to a shaking table (the temperature is 25 ℃, the rotating speed is 45 r/min) for continuous culture for 4 weeks, and then the embryo rate is counted, and the test result is shown in the following table 1.
Heat shock has been shown to be effective in initiating microspore transition from the gametophytic pathway to the sporozoite pathway, and has become a routine step in most plant microspore culture procedures, with the intensity of stress varying depending on the stage of microspore development and plant species. The results in Table 1 show that the ginseng microspores have high embryo extraction rate after heat shock treatment at 30 ℃ for 5 days and heat shock treatment at 32 ℃ for 3 days, wherein the heat shock treatment at 32 ℃ for 3 days has the best effect, the embryo extraction rate is the highest, and the treatment time is short. Heat shock treatment at 30 ℃ for 3d times is less effective, while heat shock treatment at 32 ℃ for 5d is less effective.
Example 3: effect of triacontanol on Induction of microspore embryoid of Ginseng radix
A method consistent with example 1 was used, except that: the concentration of triacontanol in the induction culture medium is set to be 0.3mg/L, 0.6mg/L, 0.9mg/L and 1.2mg/L, no triacontanol is added as a control group, heat shock pretreatment is firstly carried out after microspores are suspended in the induction culture medium, then induction culture is carried out, the embryo rate is counted, and the test result is shown in the following table 2.
The results in Table 2 show that the germ-shedding rate of the ginseng microspores can be improved by adding triacontanol with the concentration of 0.3-1.2 mg/L into the induction culture medium. When the triacontanol concentration is 0.9mg/L and 1.2mg/L, the embryo yield of 2 test materials is obviously different from that of a control group, but the embryo yield difference between 2 concentrations is not obvious. Therefore, the suitable concentration of triacontanol is 0.9-1.2 mg/L, and 0.9mg/L is selected from the viewpoint of cost saving.
Example 4: h2O2Effect on Induction of microspore embryoid of Ginseng radix
A method consistent with example 1 was used, except that: will induce H in the medium2O2Concentration of (2) was set to 50. mu.100 mol/L, 200. mu. mol/L, 400. mu. mol/L so as not to add H2O2As a control group, microspores were suspended in the above induction medium, heat shock pretreatment was performed, induction culture was performed, and the embryo yield was counted, and the test results are shown in Table 3 below.
The results in Table 3 show that H is added to the induction medium at a concentration of 50-200. mu. mol/L2O2Can increase the embryo yield of ginseng microspore, namely H2O2When the concentration is 100 mu mol/L, the germ production rate of the big horse teeth is 4.30, the germ production rate of the rhizoma panacis japonici is 8.22, and the difference with the control group reaches an extremely significant level; when H is present2O2At a concentration of 400. mu. mol/L, the embryo-out rates of the 2 test materials were all lower than those of the control group. Description of the present experiment H2O2Can induce the formation of embryoid of ginseng microsporeHowever, the concentration is not preferably too high, and is preferably 100. mu. mol/L.
Example 5: effect of melatonin on Induction of microspore embryoid of Ginseng radix
A method consistent with example 1 was used, except that: the concentration of melatonin in the induction culture medium is set to be 0.5mg/L, 1.0mg/L, 2.0mg/L and 5.0mg/L, melatonin is not added as a control group, heat shock pretreatment is firstly carried out after microspores are suspended in the induction culture medium, then induction culture is carried out, the embryo rate is counted, and the test result is shown in the following table 4.
As can be seen from Table 4, the melatonin concentration is within the range of 0.5-5.0 mg/L, the embryo-out rate of the ginseng microspores is improved to different degrees, and when the melatonin concentration is 2.0mg/L, the induction effect on embryoid bodies is the best, the embryo-out rate is the highest, namely 4.24 of the large horse teeth and 2.8 times of that of the control group (1.49), and the rate of the rhizoma panacis japonici 8.17 is 1.9 times of that of the control group (1.49), so that the proper concentration of the melatonin is 2.0 mg/L.
The above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby, and any modification made on the basis of the technical scheme according to the technical idea proposed by the present invention falls within the protection scope of the present invention; the technology not related to the invention can be realized by the prior art.
Claims (7)
1. A method for inducing and culturing isolated microspores of ginseng is characterized in that: comprises the following steps:
(1) selection, pretreatment and sterilization of flower buds: in the initial flowering period of the ginseng, collecting buds in the mononuclear border period, and further determining the microspore development period under a microscope; moisturizing the flower buds, standing in a refrigerator at 4 ℃ for 1-2 days, and sterilizing;
(2) microspore separation and purification: draining water from sterilized flower bud with absorbent paper, placing into mortar, adding small amount of B5-13 extracting the liquid, gently squeezing with a grinding rod to make microsporesDissociating semen into extractive solution, filtering with 400 mesh nylon net to 10ml centrifuge tube, centrifuging at 800r/min for 3min, discarding supernatant, adding small amount of B into precipitate513, shaking the extracting solution evenly, centrifuging at 800r/min for 3min, and repeating twice; discarding the supernatant to obtain precipitate as pure microspore;
(3) heat shock pretreatment: resuspending the purified microspores by using an induction culture medium, adjusting the density, subpackaging the microspores into sterile culture dishes with the diameter of 60mm, wherein each dish is 2.5-3.5 ml, and sealing by using a Parafilm membrane; placing the culture dish in a dark condition at 32 ℃ for culturing for 3 d;
(4) and (3) induction culture: and (4) transferring the heat-shocked culture dish to a dark condition at 25 ℃ for standing culture, and transferring to a shaking table for continuous culture when embryoid can be seen by naked eyes.
2. The method for inducing and culturing isolated microspores of ginseng according to claim 1, wherein the culture medium comprises: the length of the flower buds in the edge-approaching period of the mononuclear in the step (1) is 1.1-1.5 mm, the diameter is 2.1-2.8 mm, and the color of the anther is green or yellow-green.
3. The method for inducing and culturing isolated microspores of ginseng according to claim 1, wherein the culture medium comprises: the sterilization method in the step (1) comprises the following steps: transferring the flower bud of Ginseng radix into a sterile beaker of a clean bench, adding 70% alcohol for surface sterilization, soaking for 1min, pouring off alcohol, adding 0.1% mercuric chloride solution, soaking for sterilization for 10min, and washing with sterile water for 3 times.
4. The method for inducing and culturing isolated microspores of ginseng according to claim 1, wherein the culture medium comprises: in the step (3), the density of the microspores is adjusted to 1.0 x 105~2.0×105One per ml.
5. The method for inducing and culturing isolated microspores of ginseng according to claim 1, wherein the culture medium comprises: the formula of the induction culture medium in the step (3) is as follows: ca (NO)3）2·4H2O 430-530mg/L，KNO3150-200mg/L，KH2PO4100-150mg/L，MgSO4·7H2O 52-76mg/L，NaCl 37-45mg/L，FeSO4·7H2O 26.4-30.2mg/L，Na2-EDTA 35.7-39.3mg/L，H3BO310.6-14.4mg/L，MnSO4·4H2O 20.5-24.0mg/L，ZnSO4·7H2O7.7-9.5mg/L，KI 0.5-0.6mg/L，Na2MoO4·2H2O 0.2-0.3mg/L，CuSO4·5H2O 0.05-0.06mg/L，CoCl2·6H20.02-0.03mg/L of O, 1.2-1.6mg/L of choline, 10.4-0.6mg/L of vitamin B, 60.4-0.6 mg/L of vitamin B, 0.4-0.6mg/L of nicotinamide, 0.4-0.6mg/L of folic acid, 0.08-0.10mg/L of biotin, 70-110mg/L of L-serine, 1.0-3.0mg/L of glycine, 15-25mg/L of sodium pyruvate, 35-45mg/L of cyclic adenosine monophosphate, 20-50mg/L of L-glutathione, 60-90mg/L of inositol, H2O280-120 mu mol/L, 0.8-1.0mg/L of triacontanol, 1.4-1.6mg/L of 2,4-D, 0.4-0.6mg/L of KT, 1.8-2.2mg/L of melatonin, 500mg/L of yeast extract 400-.
6. The method for inducing and culturing isolated microspores of ginseng according to claim 1, wherein the culture medium comprises: the temperature of shaking table culture in the step (4) is 25 ℃, and the rotating speed is 40-50 r/min.
7. The method for inducing and culturing isolated microspores of ginseng according to claim 5, wherein the culture medium comprises: and adjusting the pH value of the induction culture medium to 5.6-6.0, and filtering and sterilizing by adopting a 0.22 mu m microporous filter membrane.
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