CN113207697A - Water chestnut stem tip tissue culture method - Google Patents

Water chestnut stem tip tissue culture method Download PDF

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Publication number
CN113207697A
CN113207697A CN202110681478.5A CN202110681478A CN113207697A CN 113207697 A CN113207697 A CN 113207697A CN 202110681478 A CN202110681478 A CN 202110681478A CN 113207697 A CN113207697 A CN 113207697A
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water
culture medium
buds
culture
water chestnut
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江文
高美萍
黄诚梅
蒋慧萍
方彦蓉
黄诗宇
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Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
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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
    • 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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  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

A water chestnut stem tip tissue culture method comprises the following steps: s1 preparing a culture medium mother solution, wherein the culture medium mother solution is prepared from macroelements, microelements, chelated iron, organic matters, cane sugar, condensate and water; s2, preparing a plant growth regulator, wherein the plant growth regulator is prepared from cytokinin, auxin and plant retardant; s3 preparing a culture medium, wherein the culture medium is prepared from 50-60 ℃ water, carrageenan, mother liquor and sugar; s4 selecting and sterilizing the water chestnut culture buds; s5 cultivating, selecting and hardening the water chestnut sprout to obtain the water chestnut stem tip. The culture method can well keep the variety characteristics of the water chestnuts and exert the excellent advantages of new varieties.

Description

Water chestnut stem tip tissue culture method
[ technical field ] A method for producing a semiconductor device
The invention relates to a stem tip tissue culture method, in particular to a water chestnut stem tip tissue culture method.
[ background of the invention ]
Water chestnut (the name of school: helleocharis dulcis (burm. f.) Trin.), water chestnut, combined spicebush root, linden and the like, belongs to the family of monocotyledonous cyperaceae and is a perennial herbaceous plant. The water chestnut is one of famous and excellent agricultural products in Guangxi province and is one of the traditional important export-earning products in China. The water chestnut industry in China develops rapidly in the last decade, but for a long time, farmers remain the planting mode of bulb propagation by themselves, so that the variety degradation is serious, the pest and disease damage is serious, the prevention and treatment cost is greatly increased, the yield is remarkably reduced, the big fruit rate is reduced, the commodity is poor, and the unit benefit is remarkably reduced. In order to maintain the excellent properties of new species, researchers develop water chestnut tissue culture technology, maintain the species characteristics of the water chestnuts and exert the excellent advantages of the new species.
[ summary of the invention ]
In order to solve the problems, the invention provides the water chestnut stem tip tissue culture method, which can well keep the variety characteristics of water chestnuts and exert the excellent advantages of new varieties.
The invention is realized by the following technical scheme, and provides a water chestnut stem tip tissue culture method, which comprises the following steps:
s1 preparing a culture medium mother solution, wherein the culture medium mother solution is prepared from macroelements, microelements, chelated iron, organic matters, cane sugar, condensate and water;
s2, preparing a plant growth regulator, wherein the plant growth regulator is prepared from cytokinin, auxin and plant retardant;
s3, preparing a culture medium, wherein the culture medium is prepared from 50-60 ℃ water, carrageenan, culture medium mother liquor and sugar according to a ratio of 167:1:28: 4;
s4 selecting and sterilizing the water chestnut culture buds;
s5 cultivating, selecting and hardening the water chestnut sprout to obtain the water chestnut stem tip.
Particularly, the pH value of the culture medium mother liquor in the S1 is 5.8-6.2, and the macroelements are NH4NO3、KNO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O is prepared according to the concentrations of 1650mg/L, 1900mg/L, 370mg/L, 170mg/L and 440mg/L respectively; the microelement uses MnSO4·4H2O、 ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·H2O、CoCl2·6H2O is prepared according to the concentration of 22.3mg/L, 8.6mg/L, 6.2mg/L, 0.83mg/L, 0.25mg/L, 0.025mg/L and 0.025mg/L respectively; the chelated iron is Na2-EDTA、FeSO4·7H2O is prepared according to the concentration of 37.3 mg/and 27.8 mg/respectively; the organic matter is prepared from glycine, VB1, nicotinic acid, VB6 and inositol according to the concentration of 2.0mg/L, 0.1mg/L, 0.5mg/L and 100 mg/L; the concentration of the sucrose is 20000 mg/L-30000 mg/L, if the concentration of the sucrose is lower than 20000mg/L, the later-stage water chestnuts grow slowly and weakly, the subculture period is correspondingly prolonged, and if the concentration of the sucrose is higher than 30000mg/L, the growth potential is weak, and the material is easy to age; agar or carrageenan with the concentration of 3500 mg/L-5000 mg/L is selected as the condensate, if the condensate concentration is lower than 3500mg/L, the culture medium is liquefied and not solidified, the growth of the subculture material is irregular, and the hardness of the culture medium with the concentration higher than 5000mg/L is too high, so that the material access and nutrient absorption are not facilitated; distilled water or redistilled water is selected as the water, so that the influence of the concentration of the culture medium with trace elements in tap water can be avoided, the culture medium mother liquor is prepared and then stored in an environment at 4 ℃ for standby, and thus, the deterioration, oxidation and the like of nutritional ingredients such as amino acid, chelated iron and the like in the mother liquor culture medium can be prevented.
Particularly, the cytokinin in the S2 is formed by mixing 1-3 mol of 6-benzyl adenine and 0.5-2.5 mol of 6-furfuryl aminopurine, the auxin is formed by mixing 0.01-1.0 mol of indole acetic acid and 0.01-1.0 mol of naphthalene acetic acid, and the plant retardant is 0.1-0.35 mol of paclobutrazol.
Specifically, the S4 is specifically implemented by the following method:
selecting bulbs without damages of pests, rinsing the bulbs with clear water, airing surface water, wiping the surface with 70% alcohol, cutting complete terminal buds and side buds, placing the cut terminal buds and side buds in a sterile bottle, then soaking the terminal buds and side buds in 70% alcohol for 2min on an ultra-clean bench, washing the terminal buds and side buds with sterile water for 2 times, then soaking the terminal buds and side buds with 0.1% mercuric chloride for 10-15 min, and then washing the terminal buds and side buds with sterile water for 3-5 times.
Specifically, the S5 is specifically implemented by the following method:
s51, placing the water chestnut culture buds obtained in S4 on sterile filter paper or an aluminum sheet, cutting bud tips with the length of 0.1-0.2 cm and containing 1-2 knots, placing the bud tips into a mixed solution, mixing the mixed solution after the culture medium mother solution, the plant growth regulator and the culture medium are configured, dissolving the mixture by using 0.1M hydrochloric acid or 0.1M sodium hydroxide, regulating the pH value to be 5.8-6.2, and then adding distilled water for fixation to obtain the water chestnut culture buds;
s52, cultivating the water chestnut culture buds obtained in S51 at 26 +/-2 ℃ for 15 days, selecting dark green and strong materials, transferring the materials to a culture medium of MS +6-BA 1 mg-2.5 mg/L + NAA 0.01 mg-0.1 mg/L for primary culture, inducing cluster buds, simultaneously taking materials, sending virus for detection, and eliminating toxic strains to obtain the cluster buds;
s53, cutting the clustered shoots with more than 5 roots of non-pollution leafy stems into small blocks with 2-3 bud points, and placing the small blocks on a prepared MS subculture medium for culturing for 25-30 d in a subculture period, wherein in the culturing process, the proliferation coefficient is controlled within the range of 3.0-5.0, and the subculture frequency is controlled within 10 periods;
s54 selecting non-pollution leafy stem more than 5 from the subculture material, adding PP333 with different concentrations into the culture medium, processing 50 bottles each time, inoculating on the rooting culture medium of MS + PP3330.1mg/L-0.35 mg/L + NAA 0.5 mg/L-1 mg/L, placing in the illumination culture room with temperature (28 ℃ +/-2 ℃) for culture, counting the height, number and length of the seedling after 25 days, and counting the average number of each cluster.
The invention provides a water chestnut stem tip tissue culture method, which comprises the steps of preparing a culture medium, selecting and sterilizing water chestnuts, inoculating, carrying out primary culture, carrying out secondary culture, selecting and culturing rooting materials, and hardening seedlings to obtain water chestnut stem tip tissues.
[ detailed description ] embodiments
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent.
The invention provides a water chestnut stem tip tissue culture method, which comprises the following steps:
s1 preparing mother liquid of culture medium from macroelements (NH), trace elements (trace elements), chelated iron, organic matter, cane sugar, condensate and water4NO3、KNO3、 MgSO4·7H2O、KH2PO4、CaCl2·2H2O is prepared according to the concentrations of 1650mg/L, 1900mg/L, 370mg/L, 170mg/L and 440mg/L respectively; the microelement uses MnSO4·4H2O、ZnSO4·7 H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·H2O、CoCl2·6H2O is prepared according to the concentration of 22.3mg/L, 8.6mg/L, 6.2mg/L, 0.83mg/L, 0.25mg/L, 0.025mg/L and 0.025mg/L respectively; the chelated iron is Na2-EDTA、FeSO4·7H2O is prepared according to the concentration of 37.3 mg/and 27.8 mg/respectively; the organic matter is prepared from glycine, VB1, nicotinic acid, VB6 and inositol according to the concentration of 2.0mg/L, 0.1mg/L, 0.5mg/L and 100 mg/L; the concentration of the sucrose is 20000 mg/L-30000 mg/L; the condensate is agar or carrageenan with the concentration of 3500 mg/L-5000 mg/L; distilled water or redistilled water is selected as the water, the pH value of the prepared culture medium mother liquor is 5.8-6.2, the prepared culture medium mother liquor is prepared and then stored in an environment at 4 ℃ for standby application, and therefore the deterioration, oxidation and the like of nutritional ingredients such as amino acid, chelated iron and the like in the mother liquor culture medium can be prevented.
S2, preparing a plant growth regulator, wherein the plant growth regulator is prepared from cytokinin, auxin and plant retardant, the cytokinin in S2 is formed by mixing 6-benzyl adenine with the concentration of 1-3 mol and 6-furfuryl aminopurine with the concentration of 0.5-2.5 mol, the auxin is formed by mixing indoleacetic acid with the concentration of 0.01-1.0 mol and naphthylacetic acid with the concentration of 0.01-1.0 mol, and the plant retardant is formed by mixing paclobutrazol with the concentration of 0.1-0.35 mol.
S3, preparing a culture medium, wherein the culture medium is prepared from 50-60 ℃ water, carrageenan, culture medium mother liquor and sugar according to a ratio of 167:1:28: 4.
S4 selection and disinfection of water chestnut culture buds are realized by the following method:
selecting bulbs without damages of pests, rinsing the bulbs with clear water, airing surface water, wiping the surface with 70% alcohol, cutting complete terminal buds and side buds, placing the cut terminal buds and side buds in a sterile bottle, then soaking the terminal buds and side buds in 70% alcohol for 2min on an ultra-clean bench, washing the terminal buds and side buds with sterile water for 2 times, then soaking the terminal buds and side buds with 0.1% mercuric chloride for 10-15 min, and then washing the terminal buds and side buds with sterile water for 3-5 times.
S5 cultivation, selection and seedling hardening of the water chestnut cultivation bud to obtain the water chestnut stem tip, which is realized by the following method:
s51, placing the water chestnut culture buds obtained in S4 on sterile filter paper or an aluminum sheet, cutting bud tips with the length of 0.1-0.2 cm and containing 1-2 knots, placing the bud tips into a mixed solution, mixing the mixed solution after the culture medium mother solution, the plant growth regulator and the culture medium are configured, dissolving the mixture by using 0.1M hydrochloric acid or 0.1M sodium hydroxide, adjusting the pH value to 5.8-6.2, and adding distilled water to fix the volume to obtain the water chestnut culture buds;
s52, cultivating the water chestnut culture buds obtained in S51 at 26 +/-2 ℃ for 15 days, selecting dark green and strong materials, transferring the materials to a culture medium of MS +6-BA 1 mg-2.5 mg/L + NAA 0.01 mg-0.1 mg/L for primary culture, inducing cluster buds, simultaneously taking materials, sending virus for detection, and eliminating toxic strains to obtain the cluster buds;
s53, cutting the clustered shoots with more than 5 roots of non-pollution leafy stems into small blocks with 2-3 bud points, and placing the small blocks on a prepared MS subculture medium for culturing for 25-30 d in a subculture period, wherein in the culturing process, the proliferation coefficient is controlled within the range of 3.0-5.0, and the subculture frequency is controlled within 10 periods;
s54 selecting non-pollution leafy stem more than 5 from the subculture material, adding PP333 with different concentrations into the culture medium, processing 50 bottles each time, inoculating on the rooting culture medium of MS + PP3330.1mg/L-0.35 mg/L + NAA 0.5 mg/L-1 mg/L, placing in the illumination culture room with temperature (28 ℃ +/-2 ℃) for culture, counting the height, number and length of the seedling after 25 days, and counting the average number of each cluster.
In order to verify the feasibility of the method provided by the invention, the following experiments are carried out to compare and obtain table 2, wherein table 1 shows the results of different concentrations of 15% agricultural paclobutrazol on the water chestnut tissue culture rooting, and table 2 shows the statistical results of different concentrations of 15% agricultural paclobutrazol on the transplantation survival of the water chestnut tissue culture rooting seedlings.
TABLE 1
Figure BDA0003122784530000051
Figure BDA0003122784530000061
As can be seen from Table 1, the formulation of MS + NAA0.6mg/l + PP3330.3 mg/l is the best, and the obtained seedlings are normal, have thick and short root systems and thick leaf-shaped stems.
TABLE 2
Figure BDA0003122784530000062

Claims (6)

1. A water chestnut stem tip tissue culture method is characterized by comprising the following steps:
s1 preparing a culture medium mother solution, wherein the culture medium mother solution is prepared from macroelements, microelements, chelated iron, organic matters, cane sugar, condensate and water;
s2, preparing a plant growth regulator, wherein the plant growth regulator is prepared from cytokinin, auxin and plant retardant;
s3, preparing a culture medium, wherein the culture medium is prepared from 50-60 ℃ water, carrageenan, culture medium mother liquor and sugar according to a ratio of 167:1:28: 4;
s4 selecting and sterilizing the water chestnut culture buds;
s5 cultivating, selecting and hardening the water chestnut sprout to obtain the water chestnut stem tip.
2. The method for tissue culture of water chestnut stem tips according to claim 1, wherein the pH value of the culture medium mother liquor in S1 is 5.8-6.2, and the pH value isMacroelement selected from NH4NO3、KNO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O is prepared according to the concentrations of 1650mg/L, 1900mg/L, 370mg/L, 170mg/L and 440mg/L respectively, and the microelements are MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·H2O、CoCl2·6H2O is prepared according to the concentration of 22.3mg/L, 8.6mg/L, 6.2mg/L, 0.83mg/L, 0.25mg/L, 0.025mg/L and 0.025mg/L respectively, and the chelated iron is Na2-EDTA、FeSO4·7H2The culture medium is characterized in that the culture medium is prepared from O according to the concentration of 37.3 mg/and 27.8 mg/respectively, the organic matter is prepared from glycine, VB1, nicotinic acid, VB6 and inositol according to the concentration of 2.0mg/L, 0.1mg/L, 0.5mg/L and 100mg/L, the concentration of sucrose is 20000 mg/L-30000 mg/L, the condensate is agar or carrageenan with the concentration of 3500 mg/L-5000 mg/L, the water is distilled water or redistilled water, and the culture medium mother liquor is stored in an environment at 4 ℃ for later use after being prepared.
3. The method for tissue culture of water chestnut stem tips as claimed in claim 2, wherein the cytokinin in S2 is formed by mixing 6 benzyl adenine with the concentration of 1-3 mol and 6-furfuryl aminopurine with the concentration of 0.5-2.5 mol, the auxin is formed by mixing indoleacetic acid with the concentration of 0.01-1.0 mol and naphthylacetic acid with the concentration of 0.01-1.0 mol, and the plant retardant is paclobutrazol with the concentration of 0.1-0.35 mol.
4. The method for cultivating the corm Eleocharitis stem tip tissue as claimed in claim 3, wherein the culture medium mother liquor, the plant growth regulator and the culture medium are mixed after configuration is completed, then dissolved by 0.1M hydrochloric acid or 0.1M sodium hydroxide, the pH value is adjusted to 5.8-6.2, and then distilled water is added to the mixture to obtain a mixed solution.
5. The method for tissue culture of water chestnut stem tips according to claim 1, wherein the step S4 is realized by adopting the following steps:
selecting bulbs without damages of pests, rinsing the bulbs with clear water, airing surface water, wiping the surface with 70% alcohol, cutting complete terminal buds and side buds, placing the cut terminal buds and side buds in a sterile bottle, then soaking the terminal buds and side buds in 70% alcohol for 2min on an ultra-clean bench, washing the terminal buds and side buds with sterile water for 2 times, then soaking the terminal buds and side buds with 0.1% mercuric chloride for 10-15 min, and then washing the terminal buds and side buds with sterile water for 3-5 times.
6. The method for tissue culture of water chestnut stem tips according to claim 4, wherein the step S5 is realized by adopting the following steps:
s51, placing the water chestnut culture buds obtained in the step S4 on sterile filter paper or an aluminum sheet, cutting bud tips with the length of 0.1-0.2 cm and containing 1-2 knots, and placing the bud tips into the mixed solution;
s52, cultivating the water chestnut culture buds obtained in S51 at 26 +/-2 ℃ for 15 days, selecting dark green and strong materials, transferring the materials to a culture medium of MS +6-BA 1 mg-2.5 mg/L + NAA 0.01 mg-0.1 mg/L for primary culture, inducing cluster buds, simultaneously taking materials, sending virus for detection, and eliminating toxic strains to obtain the cluster buds;
s53, cutting the clustered shoots with more than 5 roots of non-pollution leafy stems into small blocks with 2-3 bud points, and placing the small blocks on a prepared MS subculture medium for culturing for 25-30 d in a subculture period, wherein in the culturing process, the proliferation coefficient is controlled within the range of 3.0-5.0, and the subculture frequency is controlled within 10 periods;
s54 selecting non-pollution leafy stem more than 5 from the subculture material, adding PP333 with different concentrations into the culture medium, processing 50 bottles each time, inoculating on the rooting culture medium of MS + PP3330.1mg/L-0.35 mg/L + NAA 0.5 mg/L-1 mg/L, placing in the illumination culture room with temperature (28 ℃ +/-2 ℃) for culture, counting the height, number and length of the seedling after 25 days, and counting the average number of each cluster.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102487829A (en) * 2011-12-19 2012-06-13 广州甘蔗糖业研究所湛江甘蔗研究中心 Method of comprehensive detoxification and rapid propagation for starch-type water chestnut
CN103070072A (en) * 2013-01-22 2013-05-01 武汉市蔬菜科学研究所 Method for fast propagation of test tube water chestnut by tissue culture
CN106857253A (en) * 2017-02-10 2017-06-20 台州市农业科学研究院 A kind of effective water chestnut virus poison-removing method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102487829A (en) * 2011-12-19 2012-06-13 广州甘蔗糖业研究所湛江甘蔗研究中心 Method of comprehensive detoxification and rapid propagation for starch-type water chestnut
CN103070072A (en) * 2013-01-22 2013-05-01 武汉市蔬菜科学研究所 Method for fast propagation of test tube water chestnut by tissue culture
CN106857253A (en) * 2017-02-10 2017-06-20 台州市农业科学研究院 A kind of effective water chestnut virus poison-removing method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
王碧琴: ""PP333对荸荠试管苗增殖调控的研究"", 《江西科学》 *
蔡佳燕: ""荸荠组织培养及其休眠的研究"", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 *

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