CN101002540A - High effective tissue culture regeneration of cucumber, and method for transplanting of said seedlings with high viability - Google Patents
High effective tissue culture regeneration of cucumber, and method for transplanting of said seedlings with high viability Download PDFInfo
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- CN101002540A CN101002540A CN 200710013242 CN200710013242A CN101002540A CN 101002540 A CN101002540 A CN 101002540A CN 200710013242 CN200710013242 CN 200710013242 CN 200710013242 A CN200710013242 A CN 200710013242A CN 101002540 A CN101002540 A CN 101002540A
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Abstract
A method for the tissue culture and transplanting of cucumber includes such steps as taking the aseptic seedling of cucumber, choosing and inoculating explant, inducing regeneration bud, differentiating, growing, rooting culture, pratisizing seedlings and transplanting.
Description
Technical field:
The invention belongs to biological technical field, be specifically related to the transplanting method of hormone combination, temperature control and the regrowth of different medium in cucumber regenerative process, the regenerative process.
Background technology:
The main at present conventional method that adopts of cucumber (cucumis sativus.L) breeding.Because the germ plasm resource scarcity, genetic background is narrow, leans on existing resource and conventional breeding method to be difficult to select the good kind of strong stress resistance.Utilize biotechnology to assist traditional breeding method, improve cucumber quality and resistance, caused extensive concern by engineered method.Setting up efficient cucumber regenerating system fast is the prerequisite of utilizing technique for gene engineering.The existing many reports of the plant regeneration of relevant cucumber, for example:
1) " cucumber cotyledons tissue culture regeneration plant " [J] of Zhao Junliang, Ma Rongli and Li Changhua. Shanxi agricultural science, 1996,24 (1): 23;
2) " cucumber Study on tissue culture " (bulletin) [J] of Dong Lingdi. Agricultural University Of Southwest's journal, 2000,22 (1): 23;
3) " cucumber Study on tissue culture " [J] of Mei Qian and Zhang Xingguo. Agricultural University Of Southwest's journal, 2002,24 (3): 266-267;
4) " research of cucumber cotyledons joint Regeneration in Vitro system " [J] of Zhao Juan, Wang Hua and Pan Junsong etc. Shanghai Communications University's journal, 2004,22 (1): 43-47;
5) Wang Yanrong, Chen Limei, " foundation of cucumber cotyledons high-efficiency regeneration system and genetic transformation " [J] of Pan Junsong etc.Shanghai Communications University's journal (agricultural science version) 2006, vol 24, No.2,153-164.
In these reports in the past, people have carried out the research of plant regeneration to explants such as cucumber cotyledons, true leaf, hypocotyl, radicles, but find no matter to adopt which kind of explant, all there are many problems in the plant regeneration of cucumber, mainly be: regeneration frequency is not high, plant is downgraded, bloom in the regeneration plant bottle, regrowth is difficult to take root, transplant survival difficulty etc., and these technical problems restrict the extensive use of biotechnology in breed cucumber always.
Summary of the invention:
The purpose of this invention is to provide a kind of tissue culture regeneration of cucumber efficiently and regrowth transplant survival method thereof, this method can reduce the influence of genotype to the cucumber regeneration capacity, improve the regeneration frequency of cucumber, shorten the regeneration period, solving cucumber regeneration plant internode shortens, bloom in the bottle, aging and regrowth is difficult to take root, and is difficult to the problem of transplant survival, transforms the regenerating system of setting up stability and high efficiency for cucumber gene.
Technical scheme of the present invention is: tissue culture regeneration of cucumber and regrowth transplant survival method thereof efficiently, its feature comprises the steps:
1) acquisition of aseptic seedling: select full seed, seed of the same size, soaked 30～40 minutes with running water, treat after kind of the skin deliquescing earlier with 75% Ethanol Treatment 30 seconds, used 2% NaClO surface sterilization again 10 minutes, sterile water washing 3～4 times is scattered on waterlogged absorbent cotton/filter paper, and the dark place reason is after 24 hours under 30 ℃ of environment, place 25 ℃ of temperature and provide in the incubator of illumination and cultivate, obtain aseptic seedling;
2) explant selection and inoculation:
Get 2～4 days the cucumber aseptic seedling cotyledon in " showing money or valuables one carries unintentionally " back, cut the cotyledon of nearly petiole end 1/2～2/3, the surface flush up in bud induce, on the differential medium; Described bud is induced, differential medium is: MS+6-BA 1mg/L+ABA (0.5～1) mg/L+AgNO
3The solid culture of (0～1) mg/L+Ades (1～40) mg/L+3% sucrose+0.7% agar;
3) induce, differentiation culture:
To place 25 ℃ of temperature, photoperiod be that 12 hours/day, intensity of illumination are that the culturing room of 1500lux cultivated 10～15 days with inoculating the inducing of cotyledon, differential medium;
4) the regeneration bud elongation is cultivated:
Cotyledon is induced, differentiation culture is after 10～15 days, at the visible a large amount of bud of growing thickly in the edge of otch; Transfer to bud elongation medium with explant this moment, and elongation medium is: MS+GA
3(0.1～0.5) mg/L+3% sucrose+0.7% agar;
5) culture of rootage:
The regeneration bud that will be higher than 1cm downcuts, and is transferred to root media, and root media is: 1/2MS+IBA0.5mg/L+1.5% sucrose+0.7% agar; Regeneration plant has many adventive root to produce after one week;
6) hardening and transplanting:
With the regrowth of having taken root more than the height of seedling 5cm, open bottle cap in culturing room, after carrying out 2 days adaptability hardening, take out, clean the residual agar of root, place the above-mentioned liquid root media that does not add agar to cultivate 5～10 days, temperature is controlled at 25～32 ℃, as seen there are a large amount of new roots to produce, will refine the good direct field planting of regrowth this moment to the vegetable garden.
Remarkable result of the present invention is:
The inducing, break up and extend of the selection of the acquisition of the present invention by comprising the cucumber aseptic seedling, explant material and inoculation, regeneration bud, the control of the environmental conditions such as temperature of step such as culture of rootage, acclimatization and transplants and this process, can reduce the influence of genotype to the cucumber regeneration capacity, improve the regeneration frequency of cucumber, shorten the regeneration period, it is short to solve cucumber regeneration plant internode, bloom in the bottle, aging and regrowth is difficult to take root, the problem that is difficult to transplant survival transforms the regenerating system of having set up stability and high efficiency for cucumber gene.
Experiment showed, according to method of the present invention, can back about 45 days of inoculation promptly obtain to grow normal, growth is vigorous, the regrowth that can transplant, behind the foster root of hardening transplanting survival rate more than 90% near 100%.
Embodiment:
Embodiment 1
Select full seed, South China type kind of the same size " green clothing angel " parental seed Tm, after 30 minutes,, use 2% NaClO surface sterilization 10min again with the running water immersion earlier with 75% Ethanol Treatment 30s, sterile water washing 3 times is scattered on waterlogged absorbent cotton/filter paper.30 ℃ of dark places reason is after one day, and placing 25 ℃, photoperiod is that 12hr/ days, intensity of illumination are the cultivation indoor cultivation of 1500lux, obtains aseptic seedling.
Get " showing money or valuables one carries unintentionally " back 2 days cucumber aseptic seedling cotyledon, cut the cotyledon of nearly petiole end 1/2, the surface flushes the 1mg/L+AgNO in MS+6-BA 1mg/L+ABA up
30.5mg/L+Ades the bud of 20mg/L+3% sucrose+0.7% agar is induced, on the differential medium; Then explant material is placed the culturing room of similarity condition to cultivate, after 12 days at the visible a large amount of bud of growing thickly in the edge of otch.Transfer to MS+GA with explant this moment
3The bud elongation medium of 0.5mg/L+3% sucrose+0.7% agar.Downcut when indefinite bud is higher than 1cm, be transferred to the root media of 1/2MS+IBA 0.5mg/L+1.5% sucrose+0.7% agar, regeneration plant has many adventive root to produce after the week.With the regrowth of having taken root more than the height of seedling 5cm, open bottle cap in culturing room, carry out 2 days adaptability hardening after, take out, clean the residual agar of root, place the liquid root media that does not add agar to cultivate a week, temperature is controlled at 30 ℃, as seen has the new root of a large amount of whites to produce.Directly field planting is to the vegetable garden for the good regrowth of refining this moment, and survival rate is more than 90%.
The statistics of embodiment 1:
| Inoculation explant number | The state of explant (12dpi) | Go out bud explant percentage (14dpi) | Number of seedling (42dpi) | Each explant number of seedling (42dpi) | Transplanting survival rate (50dpi) | ||
| The explant size | The explant color | The callus state | |||||
| 20 | It is original 4～5 times that explant increases | The leaf look dark green | The callus consolidation, yellow green | 90％ | 38 | 1.90 | 94.7％ |
Embodiment 2
Select full seed, North China of the same size type strain " the close thorn in Xintai City " to separate progeny seed 007-71321, after 30 minutes,, use 2% NaClO surface sterilization 10min again with the running water immersion earlier with 75% Ethanol Treatment 30s, sterile water washing 3 times is scattered on waterlogged absorbent cotton/filter paper.30 ℃ of dark places reason is after one day, and placing 25 ℃, photoperiod is that 12hr/ days, intensity of illumination are the cultivation indoor cultivation of 1500lux, obtains aseptic seedling.
Get " showing money or valuables one carries unintentionally " back 2 days cucumber aseptic seedling cotyledon, cut the cotyledon of nearly petiole end 2/3, the surface flushes the 1mg/L+AgNO in MS+6-BA 1mg/L+ABA up
3The bud of 1mg/L+Ades 20mg/L+3% sucrose+0.7% agar is induced, on the differential medium; Then explant material is placed the culturing room of similarity condition to cultivate, after 15 days at the visible a large amount of bud of growing thickly in the edge of otch.Transfer to MS+GA with explant this moment
3The bud elongation medium of 0.5mg/L+3% sucrose+0.7% agar.Downcut when indefinite bud is higher than 1cm, be transferred to the root media of 1/2MS+IBA 0.5mg/L+1.5% sucrose+0.7% agar, regeneration plant has many adventive root to produce after the week.With the regrowth of having taken root more than the height of seedling 5cm, open bottle cap in culturing room, carry out 2 days adaptability hardening after, take out, clean the residual agar of root, place the liquid root media that does not add agar to cultivate a week, temperature is controlled at 30 ℃, as seen has the new root of a large amount of whites to produce.Directly field planting is to the vegetable garden for the good regrowth of refining this moment, and survival rate is more than 90%.
The statistics of embodiment 2:
| Inoculation explant number | The state of explant (14dpi) | Go out bud explant percentage (16dpi) | Number of seedling (47dpi) | Each explant number of seedling (47dpi) | Transplanting survival rate (56dpi) | ||
| The explant size | The explant color | The callus state | |||||
| 21 | It is original 4 ~ 5 times that explant increases | The leaf look dark green | The callus consolidation, yellow green | 85.7％ | 39 | 1.86 | 94.9％ |
Embodiment 3
Select full seed, American-European type strain of the same size " cherub No. 2 " parental seed HL7, after 30 minutes,, use 2% NaClO surface sterilization 10min again with the running water immersion earlier with 75% Ethanol Treatment 30s, sterile water washing 3 times is scattered on waterlogged absorbent cotton/filter paper.30 ℃ of dark places reason is after one day, and placing 25 ℃, photoperiod is that 12hr/ days, intensity of illumination are the cultivation indoor cultivation of 1500lux, obtains aseptic seedling.
Get " showing money or valuables one carries unintentionally " back 2 days cucumber aseptic seedling cotyledon, cut the cotyledon of nearly petiole end 1/2, the surface flush up in the bud of MS+6-BA 1mg/L+ABA1mg/L+Ades 20mg/L+3% sucrose+0.7% agar induce, on the differential medium; Then explant material is placed the culturing room of similarity condition to cultivate, after 12 days at the visible a large amount of bud of growing thickly in the edge of otch.Transfer to MS+GA with explant this moment
3The bud elongation medium of 0.5mg/L+3% sucrose+0.7% agar.Downcut when indefinite bud is higher than 1cm, be transferred to the root media of 1/2MS+IBA 0.5mg/L+1.5% sucrose+0.7% agar, regeneration plant has many adventive root to produce after the week.With the regrowth of having taken root more than the height of seedling 5cm, open bottle cap in culturing room, carry out 2 days adaptability hardening after, take out, clean the residual agar of root, place the liquid root media that does not add agar to cultivate a week, temperature is controlled at 30 ℃, as seen has the new root of a large amount of whites to produce.Directly field planting is to the vegetable garden for the good regrowth of refining this moment, and survival rate is more than 90%.
The statistics of embodiment 3:
| Inoculation explant number | The state of explant (12dpi) | Go out bud explant percentage (14dpi) | Number of seedling (40dpi) | Each explant number of seedling (40dpi) | Transplanting survival rate (50dpi) | ||
| The explant size | The explant color | The callus state | |||||
| 20 | It is original 4 ~ 5 times that explant increases | The leaf look dark green | The callus consolidation, yellow green | 85％ | 37 | 1.85 | 100％ |
Claims (1)
1, tissue culture regeneration of cucumber and regrowth transplant survival method thereof efficiently is characterized in that, comprise the steps:
1) acquisition of aseptic seedling: select full seed, seed of the same size, soaked 30～40 minutes with running water, treat after kind of the skin deliquescing earlier with 75% Ethanol Treatment 30 seconds, used 2% NaClO surface sterilization again 10 minutes, sterile water washing 3～4 times is scattered on waterlogged absorbent cotton/filter paper, and the dark place reason is after 24 hours under 30 ℃ of environment, place 25 ℃ of temperature and provide in the incubator of illumination and cultivate, obtain aseptic seedling;
2) explant selection and inoculation:
Get 2～4 days the cucumber aseptic seedling cotyledon in " showing money or valuables one carries unintentionally " back, cut the cotyledon of nearly petiole end 1/2～2/3, the surface flush up in bud induce, on the differential medium; Described bud is induced, differential medium is: MS+6-BA 1mg/L+ABA (0.5～1) mg/L+AgNO
3The solid culture of (0～1) mg/L+Ades (1～40) mg/L+3% sucrose+0.7% agar;
3) induce, differentiation culture:
To place 25 ℃ of temperature, photoperiod be that 12 hours/day, intensity of illumination are that the culturing room of 1500lux cultivated 10～15 days with inoculating the inducing of cotyledon, differential medium;
4) the regeneration bud elongation is cultivated:
Cotyledon is induced, differentiation culture is after 10～15 days, at the visible a large amount of bud of growing thickly in the edge of otch; Transfer to bud elongation medium with explant this moment, and elongation medium is: MS+GA
3(0.1～0.5) mg/L+3% sucrose+0.7% agar;
5) culture of rootage:
The regeneration bud that will be higher than 1cm downcuts, and is transferred to root media, and root media is: 1/2MS+IBA 0.5mg/L+1.5% sucrose+0.7% agar; Regeneration plant has many adventive root to produce after one week;
6) hardening and transplanting:
With the regrowth of having taken root more than the height of seedling 5cm, open bottle cap in culturing room, after carrying out 2 days adaptability hardening, take out, clean the residual agar of root, place the above-mentioned liquid root media that does not add agar to cultivate 5～10 days, temperature is controlled at 25～32 ℃, as seen there are a large amount of new roots to produce, will refine the good direct field planting of regrowth this moment to the vegetable garden.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773060B (en) * | 2009-01-08 | 2011-10-19 | 中国科学院植物研究所 | Method for accelerating cucumber growth and special culture medium thereof |
| CN103098714A (en) * | 2013-02-28 | 2013-05-15 | 上海交通大学 | Method for improving regeneration in vitro and conversion rate of cucumber cotyledonary node |
| CN106069094A (en) * | 2016-06-29 | 2016-11-09 | 固镇县华原家庭农场 | A kind of hardening off method improving cucumber seedling vigor |
| CN109258466A (en) * | 2018-10-12 | 2019-01-25 | 深圳大学 | A method of building pakchoi vitro Regeneration System |
-
2007
- 2007-01-18 CN CN 200710013242 patent/CN101002540A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773060B (en) * | 2009-01-08 | 2011-10-19 | 中国科学院植物研究所 | Method for accelerating cucumber growth and special culture medium thereof |
| CN103098714A (en) * | 2013-02-28 | 2013-05-15 | 上海交通大学 | Method for improving regeneration in vitro and conversion rate of cucumber cotyledonary node |
| CN106069094A (en) * | 2016-06-29 | 2016-11-09 | 固镇县华原家庭农场 | A kind of hardening off method improving cucumber seedling vigor |
| CN109258466A (en) * | 2018-10-12 | 2019-01-25 | 深圳大学 | A method of building pakchoi vitro Regeneration System |
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Open date: 20070725 |