CN105325298A - Primary culture method of lotus root stem tip tissue - Google Patents
Primary culture method of lotus root stem tip tissue Download PDFInfo
- Publication number
- CN105325298A CN105325298A CN201510800063.XA CN201510800063A CN105325298A CN 105325298 A CN105325298 A CN 105325298A CN 201510800063 A CN201510800063 A CN 201510800063A CN 105325298 A CN105325298 A CN 105325298A
- Authority
- CN
- China
- Prior art keywords
- lotus rhizome
- tip tissue
- medium
- stem
- culture method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a primary culture method of lotus root stem tip tissue. The method comprises steps as follows: 1) collection of lotus root buds; 2) pretreatment of the buds; 3) disinfection and inoculation; 4) expansion culture of stem tip tissue; 5) leaf expansion culture. According to the method, explant tissue browning is prevented with a proper culture medium and a proper culture method; teratological variation of leaves is prevented by the aid of a leaf expansion culture medium, and stems and leaves develop normally. With the adoption of the method, the seedling rate of lotus root stem tip apical meristem can exceed 90%.
Description
Technical field
The present invention relates to a kind of lotus rhizome stem-tip tissue Initial culture method, belong to agricultural, biological technical field.
Background technology
Plant Tissue Breeding refers to from plant corpus isolates the tissue, organ or the cell that suit the requirements, protoplast etc., by sterile working, under manual control condition, carry out cultivating to obtain the technology that the whole plant of regeneration or production have other products of economic worth.This technology has wide practical use in the preservation etc. of the plant of Fast-propagation rare plant or high economic worth, plant corpus virus-free and plant germplasm resource.
Lotus rhizome is asexually propagated plant, and maternal plant easily produces subterranean stem, rhizomatous joint can produce new plant, breed offspring.The disease of parent and physiological disorder etc. are easily passed to offspring by this propagation method, and can, by generation accumulation, cause the yield and quality of Progeny plants to decline.Utilize lotus rhizome stem-tip tissue to carry out artificial culture and propagation offspring, effectively can prevent the propagation of pathogeny, guarantee that offspring can keep good strains of seeds.Lotus rhizome stem-tip tissue cultivates point Initial culture, a Multiplying culture, 4 steps such as culture of rootage and domestication, wherein stem-tip tissue Initial culture refers to the process being inoculated into by lotus rhizome stem-tip tissue and specific medium obtaining without pathogeny plant, being obtain the important step without pathogeny plant, is also the key technology that lotus rhizome stem-tip tissue is cultivated.
In current lotus rhizome stem apex Initial culture, Problems existing is: it is high that browning rate easily occurs inoculation rear stem-tip tissue, and the blade of Newborn Leaves can not launch, and petiole can not extend, and growth deformity, inoculation survival rate is low, and aberration rate is high, and planting percent is low.
Summary of the invention
The object of this invention is to provide a kind of lotus rhizome stem-tip tissue Initial culture method.The method can make the planting percent of lotus rhizome stem apex apical meristem reach more than 90%.
Applicant finds after deliberation: lotus rhizome stem-tip tissue Initial culture process can be divided into explant to expand and cauline leaf grows two stages.
Existing lotus rhizome stem-tip tissue cultural method, employing be solid culture medium cultivate, inoculation after explant easily there is Necrosis.Through applicant's research and experiment, find that dissimilar medium and training method are to the brown stain of explant with expand and there is notable difference, have developed lotus rhizome explant and expand medium, adopt this medium to carry out liquid concussion and cultivate the generation that can prevent brown stain, the browning rate of explant is reduced within 5%.
Existing lotus rhizome stem-tip tissue cultural method, dysplasia after stem-tip tissue inoculation, the blade of Newborn Leaves can not launch, and petiole can not extend, growth deformity.Applicant is through large quantifier elimination and experiment, find that medium launches there is significant impact to lotus rhizome cauline leaf, have developed lotus rhizome stem-tip tissue exhibition leaf medium, after explant inoculation after expanding and cultivating 30-40d in medium, be transformed in lotus rhizome exhibition leaf medium and cultivate, can prevent deformity from occurring, the explantation tissue of expanding can be grown by normal growth.
Based on above-mentioned research, the present invention prevents explantation tissue's brown stain by adopting the method for suitable medium and cultivation; By the generation adopting exhibition leaf medium to prevent leaf malformation from making a variation, its cauline leaf is grown; Its concrete technical scheme is as follows:
A kind of lotus rhizome stem-tip tissue Initial culture method, comprises the steps:
1. the collection of lotus rhizome sprout: acquisition time is the 5-8 month, chooses rhizomatous terminal bud;
2. the pre-treatment of sprout: terminal bud is left and taken top 3-5cm, with 84 thimerosal process and flowing water cleaning;
3. sterilization and inoculation: sterilize and inoculate and all carry out in superclean bench, specific as follows:
3.1 sterilizations:
3.1.1 the alcohol disinfecting 30 seconds of 75% is used;
3.1.2 effective chlorine density is the liquor natrii hypochloritis of 1%, sterilizes 30 minutes;
3.1.3 after aqua sterilisa cleaning, for subsequent use;
3.2 inoculations:
3.2.1 the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under multiplication factor is 10-40x anatomical lens, until growing point exposes.
3.2.2 with dissecting needle needle point picking growing point top tissue, being inoculated into lotus rhizome expands in medium: the composition of medium is: 1/2MS+NAA (0.1-0.3mg/L)+BA (0.2-0.5mg/L)+sucrose (3%), and pH is 5.8-6.0; The test tube of culture vessel to be diameter be 25mm, medium consumption is 15-20ml.
4. stem-tip tissue expands cultivation: the test tube inoculated is placed on to rotate on concussion shaking table and carries out concussion cultivation, and vibration velocity is 2rmp, condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, and light application time is 16hr; Cultivation cycle 30-40d forms the sprout of expanding;
5. open up leaf to cultivate:
Stem-tip tissue is expanding after medium culture 30-40d, and medium is replaced with exhibition leaf medium, the composition of medium is MS+BA (0.1-0.3mg/L)+GA (0.2-0.5mg/L)+sucrose (5%), and pH is 5.8-6.0; Culture vessel is 300ml triangular flask, medium consumption 100ml, condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, and light application time is 16hr, cultivation cycle 40-50 days.
Step 1) in, in order to ensure the purity of kind, sprout gathers and will carry out in kind garden, and acquisition length is 5-10cm, to guarantee the quality of stolon.
Step 2) in pre-treatment be with 400 times of 84 medicining liquid dipping sprout 10-15 minute, then use distilled water flushing 10-15 minute, aforementioned 400 times refer to and dilute 84 thimerosals with water by the volume ratio of 400:1.
Step 2) in from the reason of head clip stolon 3-5cm be convenient to sterilization inoculation time easy to operate;
Step 3) in aqua sterilisa cleaning 3-5 time.
Step 3) described in the multiplication factor of anatomical lens be 10-40x so that clear observation growing point, convenient sampling.
Step 3) in, be ensure explant size ﹤ 0.5mm, described dissecting needle pin rugosity is Φ=0.2-0.4mm, as contrast during operation, needle point get the rugosity that growing point tissue is no more than pin.
Step 3) in, during described Initial culture, culture vessel is test tube Φ=2.5cm, and often pipe medium addition is 15-20ml.
Tool of the present invention has the following advantages:
1, adopt liquid to expand medium and can accelerate the speed of expanding, carry out shaking the generation of cultivating and can prevent brown stain, ensure that explant survives and reach more than 90%.
2, the sprout that the exhibition leaf medium Neng Shi explantation tissue adopted is formed can open up leaf normally, prevents the generation made a variation, ensure that the purity of offspring's kind property.
3, adopt the dissecting needle of rugosity Φ=0.2-0.4mm, as contrast during sampling, the diameter that effectively can control explant is less than 0.5mm, ensure that explant is without pathogeny, offspring's totally nontoxic that it breeds.
Accompanying drawing explanation
Fig. 1 is lotus rhizome stem apex pictorial diagram of the present invention.
Fig. 2 is that stem-tip tissue of the present invention expands cultivation pictorial diagram.
Fig. 3 is that sprout of the present invention exhibition leaf cultivates pictorial diagram.
Fig. 4 is lotus rhizome Initial culture plant pictorial diagram of the present invention.
Embodiment
Embodiment
1, the collection of sprout: choose respectively lotus root varieties for ' No. 4, E Lian ', ' beauty is red ', ' waft flower, and ' three kind cultivate.Carry out to ensure that the purity sprout of kind is captured in kind garden, acquisition time is the 5-8 month, chooses loose rhizomatous terminal bud, and field sampling sprout length is 5-8cm.
2, sprout process: the sprout gathered flushing from the beginning, after 15-20 minute, with 400 times of 84 medicining liquid dipping 10-15 minute, then is used distilled water flushing 10-15 minute.
3, sterilization and inoculation: sterilize and inoculate and all carry out in superclean bench
3.1 sterilizations:
3.1.1 by the sprout of above-mentioned process, clip 3-5cm from top.
3.1.2 the alcohol disinfecting 15-20 second of 75% is used;
3.1.3 effective chlorine density is the liquor natrii hypochloritis of 1%, sterilizes 30 minutes
3.1.4 aqua sterilisa is for subsequent use after cleaning 3-5 time.
3.2 inoculations:
3.2.1 the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under 10-40x anatomical lens, until growing point exposes.
3.2.2 be the dissecting needle needle point picking growing point top tissue of 0.2-0.4mm with pin rugosity Φ, be inoculated in medium, be contrast with dissecting needle during operation, needle point get the rugosity that growing point tissue is no more than pin, the composition of medium is: 1/2MS+NAA (0.1-0.3mg/L)+BA (0.2-0.5mg/L)+sucrose (3%), and pH is 5.8-6.0; The test tube of culture vessel to be diameter be 25mm, medium consumption is 15-20ml.
4, stem-tip tissue expands cultivation: the test tube inoculated exists, and is placed on to rotate on concussion shaking table to carry out concussion cultivation, and vibration velocity is 2rmp, condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, and light application time is 16hr; Cultivation cycle 30-40d forms the sprout of expanding;
5, Zhan Ye cultivates:
Stem-tip tissue is expanding after medium culture 30-40d, and medium is replaced with exhibition leaf medium, the composition of medium is MS+BA (0.1-0.3mg/L)+GA (0.2-0.5mg/L)+sucrose (5%), and pH is 5.8-6.0; Culture vessel is 300ml blake bottle, medium consumption 100ml, condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, and light application time is 16hr, cultivates and within 40-50 days, just can obtain just for plant.Seedling rate through above-mentioned incubation ' No. 4, E Lian ', ' beauty is red ' and ' waft flower ' Initial culture is respectively 93.5%, 92.6% and 97.8%.
Claims (10)
1. a lotus rhizome stem-tip tissue Initial culture method, is characterized in that, comprise the steps:
1) collection of lotus rhizome sprout: acquisition time is the 5-8 month, chooses rhizomatous terminal bud;
2) pre-treatment of sprout: terminal bud is left and taken top 3-5cm, with thimerosal process and flowing water cleaning;
3) sterilization and inoculation:
Described inoculation method is:
A. the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under anatomical lens, until growing point exposes;
B. with dissecting needle needle point picking growing point top tissue, be inoculated into lotus rhizome and expand in medium; The composition of described medium is: 1/2MS+NAA (0.1-0.3mg/L)+BA (0.2-0.5mg/L)+sucrose (3%), and pH is 5.8-6.0;
4) stem-tip tissue expands cultivation: carry out concussion after inoculation and cultivate, vibration velocity is 2rmp, condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, and light application time is 16hr; Cultivation cycle 30-40d forms the sprout of expanding;
5) Zhan Ye cultivates: stem-tip tissue is expanding after medium culture 30-40d, medium is replaced with exhibition leaf medium, the composition of medium is MS+BA (0.1-0.3mg/L)+GA (0.2-0.5mg/L)+sucrose (5%), and pH is 5.8-6.0; Condition of culture temperature 23-25 DEG C, light intensity 2000-3000lx, light application time is 16hr, cultivation cycle 40-50 days.
2., according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterized in that, step 1) in, in order to ensure the purity of kind, sprout gathers and will carry out in kind garden, and acquisition length is 5-10cm, to guarantee the quality of stolon.
3., according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterized in that, step 2) in from the reason of head clip stolon 3-5cm be convenient to sterilization inoculation time easy to operate.
4. according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterized in that, step 2) in thimerosal process and flowing water cleaning be with 400 times of 84 medicining liquid dipping sprout 10-15 minute, use distilled water flushing 10-15 minute again, described 400 times refer to and dilute 84 thimerosals with water by the volume ratio of 400:1.
5. lotus rhizome stem-tip tissue Initial culture method according to claim 1, is characterized in that, step 3) in sterilization method be:
A. the alcohol disinfecting 30 seconds of 75% is used;
B. effective chlorine density is the liquor natrii hypochloritis of 1%, sterilizes 30 minutes;
C. after aqua sterilisa cleaning, for subsequent use.
6. lotus rhizome stem-tip tissue Initial culture method according to claim 1, is characterized in that, step 3) described in the multiplication factor of anatomical lens be 10-40x so that clear observation growing point, convenient sampling.
7. lotus rhizome stem-tip tissue Initial culture method according to claim 1, is characterized in that, step 3) in, for ensureing explant size ﹤ 0.5mm, described dissecting needle pin rugosity is Φ=0.2-0.4mm, as contrast during operation, needle point get the rugosity that growing point tissue is no more than pin.
8., according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterized in that, step 3) in sterilization and inoculation all carry out in superclean bench.
9. lotus rhizome stem-tip tissue Initial culture method according to claim 1, is characterized in that, step 4) in the test tube of culture vessel to be diameter be 25mm, medium consumption is 15-20ml.
10. lotus rhizome stem-tip tissue Initial culture method according to claim 1, is characterized in that, step 5) in culture vessel be 300ml triangular flask, medium consumption 100ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510800063.XA CN105325298B (en) | 2015-11-18 | 2015-11-18 | A kind of lotus rhizome stem-tip tissue Initial culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510800063.XA CN105325298B (en) | 2015-11-18 | 2015-11-18 | A kind of lotus rhizome stem-tip tissue Initial culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105325298A true CN105325298A (en) | 2016-02-17 |
| CN105325298B CN105325298B (en) | 2017-12-22 |
Family
ID=55276289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510800063.XA Active CN105325298B (en) | 2015-11-18 | 2015-11-18 | A kind of lotus rhizome stem-tip tissue Initial culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105325298B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0466032A (en) * | 1990-07-04 | 1992-03-02 | Mamoru Yamabe | Acclimation and mass-production of virus-freed seedling of nelumbo nucifera gaertn. |
| CN1258435A (en) * | 1999-12-22 | 2000-07-05 | 扬州大学 | Fast lotus root propagating method in detoxicated seed-breeding field |
| CN1631075A (en) * | 2004-11-26 | 2005-06-29 | 扬州大学 | Domestication method for lotus root group cultivating de-poisoning breeding |
| CN102067818A (en) * | 2010-11-19 | 2011-05-25 | 武汉市蔬菜科学研究所 | Inducing technology of test tube lotus root |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
-
2015
- 2015-11-18 CN CN201510800063.XA patent/CN105325298B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0466032A (en) * | 1990-07-04 | 1992-03-02 | Mamoru Yamabe | Acclimation and mass-production of virus-freed seedling of nelumbo nucifera gaertn. |
| CN1258435A (en) * | 1999-12-22 | 2000-07-05 | 扬州大学 | Fast lotus root propagating method in detoxicated seed-breeding field |
| CN1631075A (en) * | 2004-11-26 | 2005-06-29 | 扬州大学 | Domestication method for lotus root group cultivating de-poisoning breeding |
| CN102067818A (en) * | 2010-11-19 | 2011-05-25 | 武汉市蔬菜科学研究所 | Inducing technology of test tube lotus root |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
Non-Patent Citations (3)
| Title |
|---|
| 徐达勋等: "莲藕茎尖培养技术的研究", 《中国果菜》 * |
| 李良俊等: "莲藕茎尖培养苗的快繁技术", 《南京农业大学学报》 * |
| 王振龙: "《植物组织培养》", 30 June 2007, 中国农业大学出版社 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105325298B (en) | 2017-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103283595B (en) | Primary culture method of stem tip tissue of strawberry | |
| CN103931492B (en) | The tissue culture fast seedling-cultivating method of apple rootstock M9 | |
| CN101926287B (en) | Method for culturing tissue of 'Zhongzhen No.1' | |
| JP2016140318A (en) | Recovering method of rubber tree, proliferation method of rubber tree, induction method of shoot, growth method of shoot, root method of shoot and conditioning method for infant plant | |
| CN103704130A (en) | Chinese orchid and cymbidium hybridum hybrid seedling raising method | |
| CN101347098B (en) | Method of propagation of Tilia miqueliana by tissue culture | |
| CN102202495B (en) | Somatic embryogenesis of jatropha curcas from ovules | |
| CN104604689B (en) | The method of quick acquisition witloof explant and its healing rate of raising | |
| CN102613087B (en) | Method for culturing and breeding Correa carmen by using biological tissue | |
| CN109042330A (en) | A kind of method for tissue culture of spindle tree | |
| CN106106147A (en) | A kind of renovation process of Flos Nelumbinis unmature subleaf somatic embryo development ways | |
| CN103477988A (en) | Culture in vitro and rapid propagation method for syzygium grijsii | |
| CN104012406B (en) | The in-vitro regeneration method of sweet cherry variety red pearl in evening | |
| CN108782252A (en) | A kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube | |
| CN107568069B (en) | A kind of smoothbark birch tissue-cultured seedling high efficiently multiplying method | |
| CN105145358A (en) | Tissue culture and rapid propagation method for common fibraurea stem | |
| CN104429940A (en) | Method for acquiring virus-free strawberry seedlings | |
| CN108849500A (en) | The culture medium and method of a kind of rescue of lotus embryo and the development of offspring's fast-growth | |
| CN104137773A (en) | Creation method of switchgrass mutants | |
| CN104429958A (en) | Method for in-vitro rejuvenation of ulmus pumila tissue culture seedlings | |
| CN108450332A (en) | A kind of Chinese toon stem section method for tissue culture | |
| CN107182783A (en) | A kind of method of Hainan rhizomes of Panax notoginseng bastem portion regeneration induction plant | |
| CN103202226A (en) | Quick and efficient rooting method for Chinese cabbage tissue culture seedlings | |
| CN105325298A (en) | Primary culture method of lotus root stem tip tissue | |
| CN105794650A (en) | Method for preserving minimum population Guangxi bilberry offspring by means of immature seeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |