CN116918700B - Method for rapidly obtaining snakegourd tissue culture seedlings by utilizing solid-liquid double-layer culture medium - Google Patents
Method for rapidly obtaining snakegourd tissue culture seedlings by utilizing solid-liquid double-layer culture medium Download PDFInfo
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- CN116918700B CN116918700B CN202310536174.9A CN202310536174A CN116918700B CN 116918700 B CN116918700 B CN 116918700B CN 202310536174 A CN202310536174 A CN 202310536174A CN 116918700 B CN116918700 B CN 116918700B
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- 239000001963 growth medium Substances 0.000 title claims abstract description 63
- 235000008326 Trichosanthes anguina Nutrition 0.000 title claims abstract description 25
- 239000007788 liquid Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 19
- 240000006567 Trichosanthes anguina Species 0.000 title 1
- 244000078912 Trichosanthes cucumerina Species 0.000 claims abstract description 27
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 10
- 239000007787 solid Substances 0.000 claims description 25
- 238000009630 liquid culture Methods 0.000 claims description 22
- 238000012258 culturing Methods 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 210000001099 axilla Anatomy 0.000 claims description 2
- 240000006023 Trichosanthes kirilowii Species 0.000 claims 2
- 235000009818 Trichosanthes kirilowii Nutrition 0.000 claims 2
- 239000002609 medium Substances 0.000 claims 2
- 238000004161 plant tissue culture Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 10
- 230000006698 induction Effects 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 4
- 229960003987 melatonin Drugs 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000012883 rooting culture medium Substances 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 239000006244 Medium Thermal Substances 0.000 description 1
- 241000243785 Meloidogyne javanica Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 238000012090 tissue culture technique Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
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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
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture and seedling culture, and discloses a method for rapidly obtaining a snakegourd tissue culture seedling by utilizing a solid-liquid double-layer culture medium. The invention has the advantages of simple procedure, short period, high propagation coefficient, strong operability and the like, and provides theoretical and practical basis for the quick acquisition of high-quality seedlings of snakegourd fruits.
Description
Technical Field
The invention relates to the technical field of plant tissue culture and seedling culture, in particular to a method for rapidly obtaining a snakegourd tissue culture seedling by utilizing a solid-liquid double-layer culture medium.
Background
The female and male plants of the snakegourd fruit are free from obvious morphological difference before flowering, are difficult to distinguish, the female and male plants are often caused to be more and less after sowing and planting, the yield and economic benefit are seriously influenced, the snakegourd fruit seedling cultivation becomes a technical problem, the female and male plant identification and seedling reproduction problems are solved by common root tuber reproduction in production, but diseases such as root knot nematode are easily caused to occur and accumulate by long-term root tuber reproduction, the diseases are frequently caused, and the yield and quality are seriously reduced. The tissue culture technique is helpful for directionally culturing a large number of high-quality snakegourd seedlings. The research on the tissue culture technology of snakegourd fruit mostly uses solid culture medium at present, and the production of tissue culture seedlings is completed gradually by adding different hormone ratios according to basic steps of sterile seedling obtaining, rooting-free seedling propagation, rooting, seedling hardening, transplanting and the like. The production process is troublesome, the seedling rate is low, the cost is high, and the period is long.
Melatonin (melatonin, MT) belongs to indole compounds, has the same synthesis precursor tryptophan as indoleacetic acid, has the effect of regulating plant growth, and can also improve the resistance of plants to adversity stress. The application of melatonin in tissue culture of snakegourd fruits is not seen at present.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides a method for rapidly obtaining the snakegourd tissue culture seedlings by utilizing solid-liquid double-layer culture media, which simplifies the tissue culture path by utilizing two different culture media combined with a solid-liquid double-layer culture technology, realizes the purpose of obtaining high-quality snakegourd tissue culture seedlings in short time and is easier to obtain strong seedlings by applying melatonin in the snakegourd tissue culture media. The invention has simple procedure, high seedling rate, low cost, short period and strong operability.
The technical scheme is as follows: the invention provides a method for rapidly obtaining snakegourd tissue culture seedlings by utilizing a solid-liquid double-layer culture medium, which comprises the following steps:
A. Preparing a solid-liquid culture medium: the formula of the solid culture medium is 1/2 MS+0.5-1.0 mg.L -1MT +0.1～0.5 mg·L-1 IAA; the formula of the liquid culture medium is MS+0.5-2 mg.L -16-BA + 0.05～0.1 mg·L-1 MT;
B. split charging of solid-liquid culture medium: firstly, subpackaging a solid culture medium into culture bottles, and injecting a sterilized liquid culture medium after the solid culture medium is cooled and solidified;
C. preparation of explants: selecting a strong snakegourd plant, collecting tender materials with terminal buds or axillary buds, sterilizing the surfaces of the tender materials, and cutting the tender materials into stem segments with buds to obtain an explant;
D. inoculating and culturing: and B, inoculating the explant into a culture bottle after the solid-liquid culture medium is subpackaged in the step B, and covering a bottle cap for culture to obtain the snakegourd tissue culture seedlings.
Further, in the step A, the formula of the solid culture medium also comprises 30 g-L -1 of sucrose and 6 g-L -1 of agar.
Further, in the step B, the split charging amount of the solid culture medium in the culture bottle is 2/3-3/4 of the conventional split charging amount, and the liquid culture medium is 1/4-1/3 of the conventional split charging amount; the liquid culture medium is covered on the solid culture medium, and the liquid culture medium is kept to be a thin layer of 1-2 mm.
Further, the conventional split charging amount is 20-25 mL.
Further, in the step C, the surface sterilization specifically includes: washing 15-30 min with running water, rinsing with 75% alcohol to sterilize 15-30 s, and sterilizing 15-30 min with 2% sodium hypochlorite.
Further, in step C, the length of the explant is 0.3-0.5 cm, the apical incision is 0.1-0.2. 0.2 cm on the bud, and the basal incision is near the underside of the node.
Further, the base incision is chamfered as much as possible to form a chamfer, and the chamfer faces upwards during inoculation.
Further, in the step D, the specific steps of inoculation and cultivation are as follows:
1) Shallow inserting the explants into a solid culture medium, and inoculating 2-3 explants to each culture bottle;
2) Culturing the culture flask in a light-proof environment at 20-25 ℃ for 3-5 d, and culturing under 12 h light after the incision at the lower end of the explant is provided with a white protrusion.
In the step D, when adventitious buds appear at the base of the explant and lateral buds are formed at axilla, the adventitious buds or the lateral buds grow to 0.3-0.5 cm, and the adventitious buds or the lateral buds can be cut off and inoculated into a new culture flask for bud proliferation.
The beneficial effects are that: the invention has the following specific beneficial effects:
the solid culture medium is mainly used for inducing rooting, so that the explant incision can differentiate the root in a short time, the liquid culture medium can induce lateral buds or adventitious buds to provide sterile materials for proliferation and subculture, therefore, the solid-liquid double-culture-medium culture can give consideration to the induction differentiation of the buds and the root, a set of tissue culture procedures for single culture, such as obtaining the buds from the primary culture, proliferating the buds to rooting the buds, and the like, in the traditional tissue culture are optimized, the tissue culture link is simplified, the seedling culture period is shortened, and the propagation coefficient is improved.
Therefore, the invention provides a method for rapidly obtaining the snakegourd tissue culture seedlings by utilizing the solid-liquid double-layer culture medium, which can simplify the production process of the snakegourd tissue culture seedlings, shorten the seedling culture period and improve the propagation coefficient.
Drawings
FIG. 1 shows the induction of roots and shoots by solid-liquid double-layer culture according to embodiment 1;
FIG. 2 shows the rooting induction of the solid media of comparative example 1 and comparative example 2.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
Embodiment 1:
a method for rapidly obtaining tissue culture seedlings of snakegourd fruit by utilizing solid-liquid double-layer culture medium comprises the following steps:
A. Preparing a solid-liquid culture medium: the formula of the solid culture medium is 1/2 MS+0. mg.L -1MT +0.2 mg·L- 1 IAA, 30 g.L -1 of sucrose and 6 g.L -1 of agar are added; the liquid culture medium is MS+1 mg.L -16-BA + 0.05 mg·L- 1 MT, and sucrose and agar are not added.
B. Split charging of solid-liquid culture medium: packaging solid culture medium, sterilizing at high temperature and high pressure at 15 mL per bottle, cooling the solid culture medium, packaging sterilized liquid culture medium into culture bottles on an ultra-clean workbench, wherein the packaging amount is 5 mL, and keeping the upper layer liquid as a thin layer of about 1-2 mm.
C. preparation of explants: selecting a strong snakegourd plant, collecting tender materials with terminal buds or axillary buds, washing for 30 min with running water, rinsing with 75% alcohol for sterilization for 20 s, and sterilizing with 2% sodium hypochlorite for 20 min; then cutting the sterilized fructus Trichosanthis material into stem segments with buds of about 0.5cm, cutting the top end of the stem segments on the buds about 0.2 cm, and cutting the base part of the stem segments near the lower part of the knots, wherein the base part of the stem segments is a bevel.
D. Inoculating and culturing: shallow inserting the explants into a solid culture medium, and inoculating 2 explants to each bottle; firstly, placing the culture flask in the dark at about 25 ℃ for culture, changing the white protrusion of the incision of the base part of the explant into 12 h illumination culture after 5-7 d, and obtaining a large number of secondary roots and tertiary roots after 30 d, wherein the adventitious buds and lateral buds are formed by differentiation, and the number of stem and vine nodes reaches 4-5.
E. Proliferation culture: 40 d, cutting off the lateral buds and the adventitious buds which grow to about 0.5 cm, inoculating the lateral buds and the adventitious buds into a new culture medium, and continuing to proliferate the buds, wherein the proliferation multiple of the buds can reach 6-10 times.
Comparative example 1:
The comparative example adopts a bud-first root culture way, adopts a solid culture medium in the bud induction stage, adopts a solid culture medium formula MS+1 mg.L -16-BA + 0.05 mg·L-1 MT, adds 30 g.L -1 of sucrose and 6 g.L -1 of agar, and adopts 12 h illumination to culture about 30 d of culture medium after each bottle of split charging 20mL of culture medium, so as to form adventitious buds and lateral buds, and can proliferate 3-5 times; cutting buds, inoculating the buds into a rooting culture medium, wherein the rooting culture medium has the formula same as that of embodiment 1,1/2 MS+0. mg.L -1MT +0.2 mg·L-1 IAA, adding sucrose 30 g.L -1 and agar 6 g.L -1, and culturing under the culture conditions same as that of embodiment 1 in the dark and then in the light, so as to form a complete plant after 30-40 d.
Comparative example 2:
The comparative example adopts a solid culture mode of directly inducing rooting, and the formula of a solid culture medium is the same as that of embodiment 1,1/2 MS+0.5 mg.L -1MT +0.2 mg·L-1 IAA, and sucrose 30 g.L -1 and agar 6 g.L -1 are added; explants were prepared according to embodiment 1 under the same conditions as in embodiment 1, with a dark and light culture followed by about 40 d to form whole plants.
Fig. 1 shows the induction conditions of the solid-liquid double-layer culture of embodiment 1 on roots and buds, and fig. 2 shows the induction rooting conditions of the solid culture medium of comparative examples 1 and 2, and it can be seen that the solid-liquid culture of embodiment 1 can simultaneously achieve root and bud induction and differentiation, the culture period is about 30 d, the comparative example 1 is a conventional culture path, and the culture period is longer from the bud induction to rooting and is about 70-80 d. In addition, the embodiment 1 can realize the proliferation of the bud seedling at the same time, the proliferation is 6-10 times, the comparative example 1 can only root after the bud, the proliferation is 3-5 times, and the main purpose of the comparative example 2 is to induce the rooting, so that the complete plant is formed.
The foregoing embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement the same, not to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (8)
1. The method for rapidly obtaining the snakegourd tissue culture seedlings by utilizing the solid-liquid double-layer culture medium is characterized by comprising the following steps of:
A. Preparing a solid-liquid culture medium: the formula of the solid culture medium is 1/2 MS+0.5-1.0 mg.L -1 MT +0.1～0.5 mg·L-1 IAA, 30 g.L -1 of sucrose and 6 g.L -1 of agar; the formula of the liquid culture medium is MS+0.5-2 mg.L -1 6-BA + 0.05～0.1 mg·L-1 MT;
B. split charging of solid-liquid culture medium: firstly, subpackaging a solid culture medium into culture bottles, and injecting a sterilized liquid culture medium after the solid culture medium is cooled and solidified;
C. preparation of explants: selecting a strong snakegourd plant, collecting tender materials with terminal buds or axillary buds, sterilizing the surfaces of the tender materials, and cutting the tender materials into stem segments with buds to obtain an explant;
D. inoculating and culturing: and B, inoculating the explant into a culture bottle after the solid-liquid culture medium is subpackaged in the step B, and covering a bottle cap for culture to obtain the snakegourd tissue culture seedlings.
2. The method for rapidly obtaining the tissue culture seedlings of the snakegourd fruit by utilizing the solid-liquid double-layer culture medium according to claim 1, wherein in the step B, the split charging amount of the solid culture medium in the culture bottle is 2/3-3/4 of the conventional split charging amount, and the liquid culture medium is 1/4-1/3 of the conventional split charging amount; the liquid culture medium is covered on the solid culture medium, and the liquid culture medium is kept to be a thin layer of 1-2 mm.
3. The method for rapidly obtaining tissue culture seedlings of snakegourd fruit by utilizing solid-liquid double-layer culture medium according to claim 2, wherein the conventional split charging amount is 20-25 mL.
4. The method for rapidly obtaining tissue culture seedlings of snakegourd fruit by utilizing a solid-liquid double-layer culture medium according to claim 1, wherein in the step C, the surface sterilization is specifically as follows: washing 15-30 min with running water, rinsing with 75% alcohol to sterilize 15-30 s, and sterilizing 15-30 min with 2% sodium hypochlorite.
5. The method for rapidly obtaining tissue culture seedlings of trichosanthes kirilowii using a solid-liquid double-layer medium according to claim 1, wherein in step C, the length of the explant is 0.3-0.5 cm, the top incision is 0.1-0.2. 0.2 cm on the bud, and the base incision is near the lower part of the node.
6. The method for rapidly obtaining tissue culture seedlings of snakegourd using solid-liquid double-layer culture medium according to claim 5, wherein the base incision is chamfered as much as possible to form a chamfer, and the chamfer faces upwards when inoculating.
7. The method for rapidly obtaining tissue culture seedlings of snakegourd fruit by utilizing a solid-liquid double-layer culture medium according to claim 1, wherein in the step D, the specific steps of inoculating and culturing are as follows:
1) Shallow inserting the explants into a solid culture medium, and inoculating 2-3 explants to each culture bottle;
2) Culturing the culture flask in a light-proof environment at 20-25 ℃ for 3-5 d, and culturing under 12 h light after the incision at the lower end of the explant is provided with a white protrusion.
8. The method for rapidly obtaining tissue culture seedlings of trichosanthes kirilowii using a solid-liquid double-layer medium according to any one of claims 1 to 7, wherein in step D, when adventitious buds appear at the base of the explant and lateral buds are formed at the axilla, the adventitious buds or lateral buds can be sheared off and inoculated into a new culture flask for bud propagation when the adventitious buds or lateral buds grow to 0.3-0.5 cm.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101699989A (en) * | 2009-11-20 | 2010-05-05 | 杨保成 | Method for tissue culture and rapid propagation of Trichosanthes kirilowii Maxim |
| CN109258459A (en) * | 2018-08-08 | 2019-01-25 | 芜湖职业技术学院 | Snakegourd Fruit vitro proliferation culture medium and preparation method thereof and Snakegourd Fruit subculture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE60006367T2 (en) * | 1999-03-25 | 2004-08-12 | University Of Guelph, Guelph | MICROPROPAGATION AND PRODUCTION OF PHYTOPHARMACEUTICAL PLANTS |
| KR102158657B1 (en) * | 2018-08-10 | 2020-09-22 | 한국 한의학 연구원 | Medium composition for producing in vitro culture and seminal root of herbal plant of Trichosanthes kirilowii and mass production method of herbal plant of Trichosanthes kirilowii using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101699989A (en) * | 2009-11-20 | 2010-05-05 | 杨保成 | Method for tissue culture and rapid propagation of Trichosanthes kirilowii Maxim |
| CN109258459A (en) * | 2018-08-08 | 2019-01-25 | 芜湖职业技术学院 | Snakegourd Fruit vitro proliferation culture medium and preparation method thereof and Snakegourd Fruit subculture method |
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