CN105766640A - Method for establishing efficient regeneration system for lagenaria siceraria by taking cotyledonary nodes as explants - Google Patents
Method for establishing efficient regeneration system for lagenaria siceraria by taking cotyledonary nodes as explants Download PDFInfo
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- CN105766640A CN105766640A CN201610168309.0A CN201610168309A CN105766640A CN 105766640 A CN105766640 A CN 105766640A CN 201610168309 A CN201610168309 A CN 201610168309A CN 105766640 A CN105766640 A CN 105766640A
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- cucurbit
- regeneration system
- cotyledonary node
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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
- A01H4/001—Culture apparatus for tissue culture
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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
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- Developmental Biology & Embryology (AREA)
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Abstract
The invention particularly relates to a method for establishing an efficient regeneration system for lagenaria siceraria by taking cotyledonary nodes as explants. The method includes steps: (1) selecting ripe and plump seeds, dehulling to obtain kernels, sterilizing, transferring to an MS basal culture medium, and culturing to obtain sterile seedlings; (2) taking the sterile seedlings to obtain cotyledonary node explants; (3) inoculating the cotyledonary node explants into an adventitious bud induction medium MS1, and culturing to obtain adventitious bud clusters; (4) stripping the adventitious bud clusters off the explants, transferring to a subculture medium MS2, and separating the adventitious bud clusters to obtain single adventitious buds when the adventitious buds grow to 3-4cm in height; (5) transferring the single adventitious buds to a rooting medium MS3, and culturing until rooting is realized; (6) subjecting the rooted regenerated plants to hardening treatment; (7) after hardening is finished, transplanting seedlings into a seedling culture substrate, and performing greenhouse culture. The method is high in bud induction rate, simple in operation, easy to implement and applicable to gene manipulation including germplasm innovation, genetic improvement and the like of lagenaria siceraria.
Description
Technical field
The invention belongs to field of plant genetic, being specifically related to a kind of is that outer implant is set up with cotyledonary node
The method of cucurbit high-efficiency regeneration system.
Background technology
Cucurbit (Lagenaria siceraria Standl.) is Curcurbitaceae (Cucurbitaceae) hois spp, 1 year
Raw herbaceous plant.Originate from Africa.Its tender fruit edible, mellow fruit can make container.Additionally, cucurbit also has
There is a special purposes, i.e. can be used as the graft stock of watermelon and muskmelon.The purpose of original adoption cucurbit grafting
It is to control watermelon blight (Fusarium oxysporum f.sp.niveum).Droop is that watermelon produces upper one
Destructive soil-borne disease, belongs to Fusarium oxysporum f. sp. niveum, all can occur whole breeding time at watermelon,
Germ is mainly main source of infection with mycelia, chlamydospore or sclerotium, and second crop soil is typically fallen ill seriously, even
Total crop failure.By grafting, utilize the feature that cucurbit stock height is anti-or immune, be effectively improved watermelon and soil is passed
The resistance of disease, simultaneously because its can effectively overcome continuous cropping obstacle, reduce environmental pollution, add easy to operate,
It is suitable for scale seedling raising and the comprehensive advantages such as watermelon resistance can be effectively improved, being able to the most aborning extensively
Application.
Experimental results demonstrate that cucurbit stock not only can improve the western muskmelon resistance to soil-borne disease both at home and abroad, moreover it is possible to
Strengthen western muskmelon to other sick (epidemic disease, powdery mildew, fruit rot, verticillium wilt etc.), worm (root-knot nematode)
Evil and the resistivity of virosis.Meanwhile, cucurbit stock grafting also improves western muskmelon to abiotic stress such as
The resistance of low temperature stress.
The seed selection of excellent cucurbit stock new varieties and supporting grafting cultivation technology are the successes of western muskmelon grafting cultivation
Key.Traditional cucurbit genetic improvement mainly uses conventional breeding methods, is hybridized by selection cross F1 generation
Kind.But, cucurbit conventional breeding cycle length, difficulty is big and inhereditary feature is unstable, greatly constrains excellent
The seed selection process of good cucurbit stock variety.By contrast, transgenic technology have simple to operate, low cost,
The feature that breeding cycle is short, is widely used in crop improvement.Technique for gene engineering means are used to carry out kind of a matter wound
Newly, first have to set up efficient plant regeneration system.At present in ground family crop existing cucumber, muskmelon,
Watermelon, winter squash etc. report plant regeneration system, and the research report that the rarely seen tissue cultures about cucurbit is relevant
Road.The foundation of regenerating system by genotype, outer implant type, conditions of tissue culture (as type of culture medium,
Hormonal readiness etc.) etc. the impact of factor.Although other crops of Curcurbitaceae have established regenerating system, but due to
Ground family crop hereditary basis is narrow, and existing regenerating system can not be successfully applied to cucurbit, in practical operation
During poor repeatability, and regeneration rate is extremely low.Therefore, research and development it are badly in need of a kind of efficiently, stable and simple and easy to do
Cucurbit regenerating system, for being carried out germplasm innovation and the genetic improvement of cucurbit further by genetic engineering means,
And then cultivate excellent cucurbit stock new varieties and lay the foundation.
Summary of the invention
Invention broadly provides a kind of is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
The method not only bud induction rate provided is high, simple to operate, easy to implement, it is adaptable to cucurbit germplasm innovation, something lost
Pass the genetic manipulations such as improvement.Its technical scheme is as follows:
A kind of is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node, comprises the following steps:
(1) seed of picking mature and plump, shell to obtain seed, is disinfected by seed, then is transferred to nothing
On the MS basal medium of hormone incubated to seed germination under dark surrounds, it is thus achieved that aseptic seedling；
(2) taking aseptic seedling, cut plumule and hypocotyl, separately cotyledon, every half cotyledon is crosscutting, it is thus achieved that son
The outer implant of leaf segment；
(3) outer for cotyledonary node implant being inoculated in adventitious bud induction culture base MS1 cultivation, period is the most more
Change culture medium, it is thus achieved that adventitious bud bunch；
(4) adventitious bud bunch is peeled off in outer implant, is forwarded in subculture medium MS2, continue to cultivate,
Period periodic replacement culture medium, when adventitious bud grows to 3-4cm height, separately adventitious bud bunch, it is thus achieved that single not
Normal bud seedling；
(5) single adventitious shoot is forwarded in root media MS3, cultivates to taking root；
(6) regeneration plant taken root is carried out hardening process；
(7), after hardening completes, take out seedling, wash away root culture medium, transplant in seedling medium, be put in
Hot-house culture.
Preferably, the method in step (1) disinfected seed is, is first the nothing of 70% with volume content
Water-ethanol is to seed surface sterilization 35-45S, with aseptic water washing 2-3 time, then is 10% by mass concentration
NaClO sterilizes 8-12min, then with aseptic water washing 2-3 time, is finally placed on aseptic filter paper and drains moisture.
Preferably, step (2) is chosen the aseptic seedling of sprouting 3d.
Preferably, in step (3), adventitious bud induction culture base MS1 includes: MS culture medium, 3.0mg L-1
6-BA, 0.01mg L-12,4-D, 0.3mg L-1AgNO3、30g·L-1Sucrose and 8g L-1
Agar, the pH of adventitious bud induction culture base MS1 is 5.6-6.0.
Preferably, cotyledonary node outer planting body incubation time in adventitious bud induction culture base MS1 in step (3)
All for 3-4, condition of culture is: cultivation temperature is 25 DEG C, 16h illumination and 8h dark alternate treatment, 3000Lux
Illumination, and often 10d changes a subculture.
Preferably, in step (4), incubation time is 2-3 week, and the frequency changing culture medium is that every 10d changes
One subculture.
Preferably, in step (4), subculture medium MS2 includes: MS culture medium, 3.0mg L-16-BA,
0.01mg·L-12,4-D, 0.3mg L-1AgNO3、30g·L-1Sucrose and 8g L-1Agar,
The pH of subculture medium MS2 is 5.6-6.0.
Preferably, in step (5), root media MS3 includes: MS culture medium, 0.05mg L-1's
NAA、30g·L-1Sucrose and 8g L-1Agar, the pH of root media MS3 is 5.6-6.0, training
Support to the time taken root be 2 weeks.
Preferably, in step (6) plant root length to be regenerated to 2cm length, regrowth grow to 5-6cm height time,
Open tissue culture bottle lid, carry out practicing seedling and process.
Using above-mentioned is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node, the present invention have with
Lower advantage:
The method that the present invention provides is simple to operate, easy to implement, and adventitious bud induction frequency is high simultaneously, the most favorably
In the genetic stability of holding cucurbit cultivars, may additionally facilitate the preservation of cucurbit germ plasm resource, it is adaptable to cucurbit kind
The genetic manipulations such as matter innovation, genetic improvement.Meanwhile, the indefinite of different medium culture different times is used
Bud, makes culture efficiency high.
Accompanying drawing explanation
Fig. 1 is the cucurbit seed sterilized；
Fig. 2 is the aseptic seedling after cucurbit seed sprouts 3d；
Fig. 3 is the outer implant of cotyledonary node；
Fig. 4 is the adventitious bud bunch induced；
Fig. 5 is the adventitious shoot after adventitious bud bunch elongation；
Fig. 6 is the adventitious shoot taken root；
Fig. 7 is that regeneration plant practices seedling；
Fig. 8 is the regrowth of transplant survival；
Fig. 9 is ratoon growth performance.
Specific embodiment
1, cucurbit stock variety surpasses the cultivation of Feng Kangsheng king's aseptic seedling: the cucurbit stock of picking mature and plump is super rich
The seed of antibiosis king, manually shell acquisition seed, by seed first with 70% absolute ethyl alcohol surface sterilization 40S,
Aseptic water washing 2-3 time, then sterilize 8-12min with NaClO that mass concentration is 10%, aseptic water washing
2-3 time, it is placed on aseptic filter paper and drains moisture, then be transferred to the adventitious bud induction culture base MS1 without hormone
Upper (as shown in Figure 1), it is placed under 25 DEG C of dark conditions and cultivates to seed germination, it is thus achieved that aseptic seedling is (such as figure
Shown in 2), wherein adventitious bud induction culture base MS1 includes: MS culture medium, 3.0mg L-16-BA,
0.01mg·L-12,4-D, 0.3mg L-1AgNO3、30g·L-1Sucrose and 8g L-1Agar,
Its pH is 5.6-6.0, preferably 5.8.
2, take the aseptic seedling of 3d after seed is sprouted, cut plumule and hypocotyl, the most separately cotyledon, every half
Sheet cotyledon is crosscutting, it is thus achieved that the outer implant (as shown in Figure 3) of cotyledonary node.
3, outer for cotyledonary node implant is inoculated in the adventitious bud induction culture base MS1 containing hormon, tool
Body different hormone combinations and proportioning are shown in Table 1, be placed in 25 DEG C, 16h/8h illumination/dark, 3000Lux illumination bar
Cultivate under part.Every 10d changes a subculture.After cultivating 3-4 week, the inductivity of statistics adventitious bud is (such as figure
Shown in 4).As it can be seen from table 1 different plant growth regulator can the generation of evoking adventive bud,
But, adventitious bud induction frequency is differed greatly by different ratio.Wherein, when 2,4-D (2,4-dichlorphenoxyacetic acid)
Concentration is 0.01mg L-1, 6-BA (6 benzyl purine) concentration be 3.0mg L-1、AgNO3Content is
0.3mg·L-1Time, the inductivity of adventitious bud is the highest, is 56.2%.Therefore, with MS+3.0mg L-1
6-BA+0.01mg·L-12,4-D+0.3mg·L-1AgNO3+30g·L-1Sucrose+8g L-1Agar, pH
Be 5.8 be adventitious bud bunch induction optimal medium.
Table 1 different hormone combinations and the proportioning induction to adventitious bud
4, adventitious bud bunch is peeled off in outer implant, be forwarded on subculture medium MS2 continue to cultivate, often
10d changes a subculture.Wherein, the consisting of of MS2 culture medium: MS+3.0mg L-16-BA+0.01
mg·L-12,4-D+0.3mg·L-1AgNO3+30g·L-1Sucrose+8g L-1Agar, pH is 5.8.No
Normal bud on MS2 culture medium can normal growth extending, cultivate 2-3 week, treat that adventitious bud grows to 3-4cm
Gao Shi, careful separately adventitious bud bunch, it is thus achieved that single adventitious shoot (as shown in Figure 5).
5, the single adventitious shoot of 3-4cm is forwarded in root media MS3, cultivates 2 weeks and can give birth to
Root (as shown in Figure 6).Wherein, adventitious bud rooting culture medium MS3 consists of: 1/2MS+0.05mg L-1
NAA (methyl α-naphthyl acetate)+30g L-1Sucrose+8g L-1Agar, pH is 5.8.
6, the domestication of regeneration plant, transplanting: it is high that plant root length to be regenerated to 2cm length, plant grow to 5-6cm
Time, open tissue culture bottle lid, carry out practicing seedling (as shown in Figure 7).White silk seedling, after 1 week, takes out seedling, washes away
Root culture medium, transplants in seedling medium, is put in hot-house culture, waters a water every 2 days, after transplanting
Survival rate can reach for 98% (as shown in Figure 8).Regrowth is planted in test booth, it was found that
Cucurbit regrowth identical with seedling growing way (as shown in Figure 9).
It will be apparent to those skilled in the art that can technical scheme as described above and design, make it
Its various corresponding changes and deformation, and all these changes and deformation all should belong to present invention power
Within the protection domain that profit requires.
Claims (9)
1. one kind is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node, it is characterised in that: bag
Include following steps:
(1) seed of picking mature and plump, shell to obtain seed, is disinfected by seed, then is transferred to nothing
On the MS basal medium of hormone incubated to seed germination under dark surrounds, it is thus achieved that aseptic seedling；
(2) taking aseptic seedling, cut plumule and hypocotyl, separately cotyledon, every half cotyledon is crosscutting, it is thus achieved that son
The outer implant of leaf segment；
(3) outer for cotyledonary node implant being inoculated in adventitious bud induction culture base MS1 cultivation, period is the most more
Change culture medium, it is thus achieved that adventitious bud bunch；
(4) adventitious bud bunch is peeled off in outer implant, is forwarded in subculture medium MS2, continue to cultivate,
Period periodic replacement culture medium, when adventitious bud grows to 3-4cm height, separately adventitious bud bunch, it is thus achieved that single not
Normal bud seedling；
(5) single adventitious shoot is forwarded in root media MS3, cultivates to taking root；
(6) regeneration plant taken root is carried out hardening process；
(7), after hardening completes, take out regrowth, wash away root culture medium, transplant in seedling medium, put
In hot-house culture.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: the method in step (1) disinfected seed is, be first 70% with volume content
Absolute ethyl alcohol is to seed surface sterilization 35-45S, with aseptic water washing 2-3 time, then is 10% by mass concentration
NaClO sterilize 8-12min, then with aseptic water washing 2-3 time, be finally placed on aseptic filter paper and drain moisture.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: step (2) is chosen the aseptic seedling sprouting 3d.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: in step (3), adventitious bud induction culture base MS1 includes: MS culture medium, 3.0mg L-1
6-BA, 0.01mg L-12,4-D, 0.3mg L-1AgNO3、30g·L-1Sucrose and 8g L-1
Agar, the pH of adventitious bud induction culture base MS1 is 5.6-6.0.
The most according to claim 4 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: cotyledonary node outer planting body incubation time in adventitious bud induction culture base MS1 in step (3)
All for 3-4, condition of culture is: cultivation temperature is 25 DEG C, 16h illumination and 8h dark alternate treatment, 3000Lux
Illumination, and often 10d changes a subculture.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: in step (4), incubation time is 2-3 week, the frequency changing culture medium is that every 10d changes
One subculture.
The most according to claim 6 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: in step (4), subculture medium MS2 includes: MS culture medium, 3.0mg L-16-BA,
0.01mg·L-12,4-D, 0.3mg L-1AgNO3、30g·L-1Sucrose and 8g L-1Agar,
The pH of subculture medium MS2 is 5.6-6.0.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: in step (5), root media MS3 includes: MS culture medium, 0.05mg L-1's
NAA、30g·L-1Sucrose and 8g L-1 agar, the pH of root media MS3 is 5.6-6.0,
Cultivation to the time taken root is 2 weeks.
The most according to claim 1 is the method that outer implant sets up cucurbit high-efficiency regeneration system with cotyledonary node,
It is characterized in that: in step (6) plant root length to be regenerated to 2cm length, regrowth grow to 5-6cm height time,
Open tissue culture bottle lid, carry out practicing seedling and process.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106234047A (en) * | 2016-07-22 | 2016-12-21 | 中国农业科学院作物科学研究所 | A kind of outer implant system of selection converted for agriculture bacillus mediated soybean cotyledon node and application |
| CN109566420A (en) * | 2019-01-31 | 2019-04-05 | 江苏省农业科学院 | A kind of tissue culture and rapid propagation method of cucurbit |
| CN109997690A (en) * | 2018-11-15 | 2019-07-12 | 齐齐哈尔大学 | A method of improving watermelon tissue-cultured seedling transplanting survival rate |
| CN113412786A (en) * | 2021-06-17 | 2021-09-21 | 宁波市农业科学研究院 | Culture medium for promoting regeneration of cucurbit cotyledon and application thereof |
| CN114431150A (en) * | 2022-03-07 | 2022-05-06 | 福建农林大学 | Jute tissue culture method taking cotyledonary node as explant |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106234047A (en) * | 2016-07-22 | 2016-12-21 | 中国农业科学院作物科学研究所 | A kind of outer implant system of selection converted for agriculture bacillus mediated soybean cotyledon node and application |
| CN106234047B (en) * | 2016-07-22 | 2019-07-26 | 中国农业科学院作物科学研究所 | A kind of explant selection method and application for the conversion of mediated by agriculture bacillus soybean cotyledon node |
| CN109997690A (en) * | 2018-11-15 | 2019-07-12 | 齐齐哈尔大学 | A method of improving watermelon tissue-cultured seedling transplanting survival rate |
| CN109566420A (en) * | 2019-01-31 | 2019-04-05 | 江苏省农业科学院 | A kind of tissue culture and rapid propagation method of cucurbit |
| CN113412786A (en) * | 2021-06-17 | 2021-09-21 | 宁波市农业科学研究院 | Culture medium for promoting regeneration of cucurbit cotyledon and application thereof |
| CN114431150A (en) * | 2022-03-07 | 2022-05-06 | 福建农林大学 | Jute tissue culture method taking cotyledonary node as explant |
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