CN103155864A - Rapid in vitro breeding method of zucchini - Google Patents
Rapid in vitro breeding method of zucchini Download PDFInfo
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- CN103155864A CN103155864A CN 201110419721 CN201110419721A CN103155864A CN 103155864 A CN103155864 A CN 103155864A CN 201110419721 CN201110419721 CN 201110419721 CN 201110419721 A CN201110419721 A CN 201110419721A CN 103155864 A CN103155864 A CN 103155864A
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Abstract
The invention discloses a rapid in vitro breeding method of zucchini, relates to a plant tissue culture technique, and belongs to the technical field of biological engineering. The invention has the beneficial effect that compared with the prior art, a tissue culture method can accelerate propagation speed, and under the culture conditions of the invention, the culture of aseptic seedlings, callus induction, bud differentiation and rooting culture greatly shorten a physiological cycle of zucchini; more importantly, the obtained callus tissue is suitable for further zucchini cell culture, and study on the secondary metabolites of zucchini cell; and the significance of the invention lays in that the method lays foundation for molecular breeding of zucchini, for the rapid propagation of zucchini variety and can obtain useful secondary metabolites of zucchini.
Description
Technical field
The present invention relates to a kind of plant tissue culture technique, belong to technical field of bioengineering, specifically a kind of method of custard squash Vitro Quick Reproduction.
Background technology
Custard squash (Cucurbita pepo L.), another name: hay melon, pumpkin etc., the annual climing property herbaceous plant of Curcurbitaceae Cucurbita.Originate in south, North America, the present extensively cultivates.The custard squash that China cultivates spring ahead of time is melon a kind of vegetables of listing the earliest in early spring, for crossing the light positive contribution of having made of vegetables spring, is also a kind of principal item that the vegetable grower obtains Efficient Cultivation.In recent years, found that custard squash had great value in health care, nutrition, aspect medical; It is rich in moisture and has the effects such as reducing fever and causing diuresis, relieving restlessness are quenched the thirst, moistened the lung and relieve the cough, dispersing swelling and dissipating binds, moist skin; Also be used for the diseases such as supplemental treatment oedema abdominal distension, polydipsia, sore and ephritis, cirrhotic ascites; Modern study shows, custard squash contains a kind of inducer of interferon, can stimulate body to produce interferon, improves immunity, bring into play antiviral and effect tumour.Therefore custard squash is subject to paying close attention to more and more widely, its cultivation obtains greater advance with conventional breeding, but conventional breeding can't satisfy need of production, and the fine genes of many custard squashs is not used, particularly aspect degeneration-resistant and the breeding of high-load secondary metabolites, research is comparatively weak.Along with the develop rapidly of biotechnology, for the secondary metabolites breeding research of custard squash provides important foundation.
Seminal propagation is often adopted in the breeding of custard squash at present, repoductive time is long, reproduction coefficient is low, and is subjected to the restriction of variety and genetype, and the differentiation rate of bud is relatively low, and pollution rate is higher, and the Brown problem occurs, utilizing during callus carries out cell cultivation process of later stage, cellular biomass is not too high, these are cultivated for utilizing the pumpkin tissue to carry out cell, and production secondary metabolites etc. all has limitation.
Summary of the invention
The present invention aims to provide a kind of method of custard squash Vitro Quick Reproduction, will greatly shorten the physiological period of custard squash in the differentiation of the inducing of custard squash Aseptic seedling culture callus, bud, culture of rootage.
In order to realize the method for a kind of custard squash Vitro Quick Reproduction of the present invention, the technical scheme steps of employing is as follows:
One, choose full, of the same size custard squash seed, the 7h～8h that soaks seed in the thermostatted water of 25 ℃, then on super-clean bench, the alcohol surface sterilization 30s with 70% sets to 0 the HgCl of .1%
24～8min sterilizes respectively in solution, during sterilization otherwise stop shaking, then use aseptic water washing 4～5 times, under aseptic condition, it is put into respectively on the aseptic seedling germination medium after seed disinfection and cultivate, described germination medium is by adding agar 8g, sucrose 20g prescription in every 1LMS, the pH value is transferred to 5.8,25 ± 1 ℃ of incubator temperature, dark culturing 1～2 day;
Two, the seed that will sprout carries out the 6h dark, 16～20h illumination, and illuminance is 80%, gets its cotyledon, hypocotyl, radicle and do sugarcane explants through callus induction when the aseptic seedling cotyledon has just turned green; Take cotyledon and be seeded on calli induction media, first remove the cotyledon margin; With remaining crosscut, get its nearly petiole end, every cotyledon gets two approximately explants of 2mm * 6mm; The explant surface that obtains is seeded in calli induction media up cultivates;
When three, 1. taking off plumular axis and be evoked callus, induce step to be seeded on calli induction media for taking hypocotyl, hypocotyl is got under cotyledon approximately 0.3cm position, the segment of being cut into 0.5cm～1.0cm; The explant minute horizontal positioned that is obtained by hypocotyl or lower end are inserted in calli induction media and are cultivated, and cultivate 20～40 days to get callus under 25 ± 1 ℃ of temperature condition; When 2. being taken as the radicle evoked callus, induce step to be: to take radicle and be seeded on calli induction media, radicle is got the section near plumular axis, be cut into the segment of 0.5cm～1.0cm, the explant minute horizontal positioned that is obtained by radicle and lower end are inserted in calli induction media and are cultivated, after inoculation, be placed on 25 ± 1 ℃ of cultivations in incubator;
Four, the choosing callus of growing preferably is cut into volume and is about 0.5～1.0cm
3Stripping and slicing and change in the bud differential medium 25 ± 1 ℃ over to and cultivated 20～40 days, every two weeks are changed subcultures, callus is differentiation and bud formation progressively;
Five, the bud with differentiation downcuts from base portion, and in the insertion root media, 25 ± 1 ℃ are cultured to the blastogenesis root;
Calli induction media in described step 3 is: MS+2.0mg/L 6BA+0.2mg/L NAA.
Bud in described step 4 is divided into: with the material of cultured callus as further differentiation cultivation, be cut into the stripping and slicing that volume is about the 0.5cm3 left and right, and change in the bud differential medium, cultivated 28～32 days at 25 ± 1 ℃ of temperature F, every two weeks are changed a subculture, Calli Differentiation becomes bud, and the bud differential medium consists of: add 1.0mg/L BA and 0.1mg/L NAA, i.e. MS+1.0mg/LBA+0.1mg/LNAA at MS.
Culture of rootage in described step 5 is: the bud of differentiation is cut F from base portion, insert in root media, cultivate at 25 ± 1 ℃ of temperature, root media is: add 0.1mg/L NAA mother liquor, that is: 1/2MS+0.1mg/L NAA at 1/2MS.
The method of a kind of custard squash Vitro Quick Reproduction of the present invention, its beneficial effect is: but to organize the method accelerates multiplication speed of cultivating, greatly shorten the physiological period of custard squash in the differentiation of the inducing of the cultivation of aseptic seedling, callus, bud, culture of rootage, the more important thing is that the callus that obtains is fit to further carry out the custard squash cell and cultivates, carry out custard squash cell secondary metabolite research, its significance also shows in addition: establish group training basis for custard squash carries out molecular breeding; For the Fast-propagation of some pumpkin varieties lays the foundation; Obtain the useful secondary metabolite of custard squash.
Embodiment
Below with specific embodiment, technical scheme of the present invention is described.
Embodiment 1
One, choose full, of the same size custard squash seed, the 7h that soaks seed in the thermostatted water of 25 ℃, then on super-clean bench, the alcohol surface sterilization 30s with 70% sets to 0 the HgCl of .1%
28min sterilizes respectively in solution, during sterilization otherwise stop shaking, then use aseptic water washing 4 times, under aseptic condition, it is put into respectively on the aseptic seedling germination medium after seed disinfection and cultivate, described germination medium is by adding agar 8g, sucrose 20g prescription in every 1LMS, the pH value is transferred to 5.8,25 ℃ of incubator temperature, dark culturing 1 day;
Two, the seed that will sprout carries out the 6h dark, 20h illumination, and illuminance is 80%, gets its cotyledon, hypocotyl, radicle and do sugarcane explants through callus induction when the aseptic seedling cotyledon has just turned green; Take cotyledon and be seeded on calli induction media, first remove the cotyledon margin; With remaining crosscut, get its nearly petiole end, every cotyledon gets two approximately explants of 2mm * 6mm; The explant surface that obtains is seeded in calli induction media up cultivates;
When three, 1. taking off plumular axis and be evoked callus, induce step to be seeded on calli induction media for taking hypocotyl, hypocotyl is got under cotyledon approximately 0.3cm position, the segment of being cut into 0.5cm; The explant minute horizontal positioned that is obtained by hypocotyl or lower end are inserted in calli induction media and are cultivated, and cultivate 20～40 days to get callus under 25 ℃ of temperature condition, described calli induction media: MS+1.0mg/L 6BA+0.1mg/LNAA; When 2. being taken as the radicle evoked callus, induce step to be: to take radicle and be seeded on calli induction media, radicle is got the section near plumular axis, be cut into the segment of 0.5cmcm, the explant minute horizontal positioned that is obtained by radicle and lower end are inserted in calli induction media and are cultivated, after inoculation, be placed on 25 ± 1 ℃ of cultivations in incubator, described calli induction media: MS+2.0mg/L 6BA+0.2mg/L NAA;
Four, the choosing callus of growing preferably is cut into volume and is about 0.5cm
3Stripping and slicing and change in the bud differential medium 25 ± 1 ℃ over to and cultivated 20 days, every two weeks are changed subcultures, callus is differentiation and bud formation progressively, described bud differential medium is: MS+0.1mg/L BA+0.05mg/L NAA;
Five, the bud with differentiation downcuts from base portion, and in the insertion root media, 25 ± 1 ℃ are cultured to the blastogenesis root, and described root media is: 1/2MS+0.05mg/L NAA.
Claims (2)
1. the method for a custard squash Vitro Quick Reproduction is characterized in that: the physiological period that greatly shortens custard squash in the differentiation of the inducing of the cultivation of aseptic seedling, callus, bud, culture of rootage; Calli induction media is: MS+2.0mg/L 6BA+0.2mg/L NAA.
2. a kind of method of custard squash Vitro Quick Reproduction as claimed in claim 1 is characterized in that: callus of induce cultivate for degree be 25 ± 1 ℃.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110419721 CN103155864A (en) | 2011-12-14 | 2011-12-14 | Rapid in vitro breeding method of zucchini |
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| CN 201110419721 CN103155864A (en) | 2011-12-14 | 2011-12-14 | Rapid in vitro breeding method of zucchini |
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| CN 201110419721 Pending CN103155864A (en) | 2011-12-14 | 2011-12-14 | Rapid in vitro breeding method of zucchini |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082140A (en) * | 2014-06-30 | 2014-10-08 | 苏州派腾生物医药科技有限公司 | Rapid propagation method of lobed actinostemma herb |
| CN106937599A (en) * | 2017-04-19 | 2017-07-11 | 贵州省园艺研究所 | Pumpkin do not inseminate Ovary culture in vitro induction gynogenesis culture medium and application |
| CN113826552A (en) * | 2021-09-24 | 2021-12-24 | 内蒙古农业大学 | In-vitro rapid propagation method for seed cucurbita pepo |
-
2011
- 2011-12-14 CN CN 201110419721 patent/CN103155864A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104082140A (en) * | 2014-06-30 | 2014-10-08 | 苏州派腾生物医药科技有限公司 | Rapid propagation method of lobed actinostemma herb |
| CN104082140B (en) * | 2014-06-30 | 2016-07-06 | 苏州派腾生物医药科技有限公司 | A kind of Lobed Actinostemma Herb rapid propagation method |
| CN106937599A (en) * | 2017-04-19 | 2017-07-11 | 贵州省园艺研究所 | Pumpkin do not inseminate Ovary culture in vitro induction gynogenesis culture medium and application |
| CN113826552A (en) * | 2021-09-24 | 2021-12-24 | 内蒙古农业大学 | In-vitro rapid propagation method for seed cucurbita pepo |
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Application publication date: 20130619 |