CN106035087B - A kind of method of Changshan grapefruit fast asexual propagation - Google Patents
A kind of method of Changshan grapefruit fast asexual propagation Download PDFInfo
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- CN106035087B CN106035087B CN201610430004.2A CN201610430004A CN106035087B CN 106035087 B CN106035087 B CN 106035087B CN 201610430004 A CN201610430004 A CN 201610430004A CN 106035087 B CN106035087 B CN 106035087B
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Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- 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 present invention discloses a kind of method of Changshan grapefruit fast asexual propagation, and the explant after sterilization is seeded in inducing clumping bud culture medium by methods described, at 20~24 DEG C, cultivated under the conditions of 1000~1200lux of illumination, every switching in 25~45 days once, switching culture 23 times, obtain Multiple Buds；After Multiple Buds to be cut into the individual with terminal bud, root media is seeded to, is cultivated 30 60 days under the conditions of 24~26 DEG C, 1000~1200lux of illumination, forms intact plant；Using this method, a rataria in the plant that 3 months or so can obtain at least more than 6 plants, has very high efficiency, can be bred in the short time and obtain substantial amounts of Changshan grapefruit seedling, applied in production practices by culture.
Description
(1) technical field
The present invention relates to a kind of method of Changshan grapefruit fast asexual propagation.
(2) background technology
Changshan grapefruit is a novel species of mandarine category, to originate in the peculiar mixed citrus breeding of Zhejiang Changshan County.Its
Completely filled fruit, outward appearance are in foresythia；Fruit is full in golden yellow, juice；The sour-sweet moderate, micro-sweet of aromatic flavour, taste；Deeply by group
Crowd welcomes, and embodies good economic benefit.Plantation garden increases Changshan grapefruit Orchard Construction dynamics, seed area in recent years
Constantly expand；Moreover, more pay attention to the yield and fruit quality of Hu shaddock.Fruit tree of the Changshan grapefruit after more generation breedings, is not easy to protect
Its some maternal merit is deposited, situations such as taste is bad occurs in fruit；Some orchard workers are frequently with cutting propagation, but skewer
Slotting reproduction speed is slower, and survival rate is not high, it is difficult to adapts to the needs of Changshan grapefruit production development.
(3) content of the invention
It is an object of the present invention to provide a kind of method of quick breeding Changshan grapefruit, this method is efficient and convenient, can be shorter
Time in obtain substantial amounts of good seed, so as to set up quick, efficient, high-quality Industrialization of seeds and seedlings production system.This
It is both the active demand of orchard worker, and the demand of market today.
The technical solution adopted by the present invention is：
The present invention provides a kind of method of quick breeding Changshan grapefruit, and methods described is：(1) acquisition and induction of explant
Culture：Selection is grown fine, the sterilization of the Changshan grapefruit explant of no disease and pests harm, the explant after being sterilized；After sterilization
Explant is seeded in inducing clumping bud culture medium, is placed in culturing room, at 22~24 DEG C, illumination 1000~1200lux conditions
Lower culture, every switching in 25~45 days once (preferably 25 days), switching culture 2-3 times, obtain Multiple Buds；The inducing clumping bud
Culture medium is (6-BA) 0.25~2.5mg/L+ brassinosteroids containing 6-benzyl aminopurine (BRs) 0.1mg/L+AgNO3 1mg/L
MS improved culture mediums, the MS improved culture mediums for addition polyvinylpyrrolidone (PVP) 0.5g/L+ arginine (Arg)
0.1g/L+ glutamine (Gln) 0.5g/L MS minimal mediums；(2) culture of rootage：The Multiple Buds that step (1) obtains are cut
After being segmented into the individual with terminal bud, it is seeded on root media, is cultivated under the conditions of 22~24 DEG C, 1000~1200lux of illumination
30-60 days, form intact plant；The root media is trained for the MS improvement of indole-3-acetic acid containing 0.1-0.2mg/L (IAA)
Support base；The MS improved culture mediums are addition PVP 0.5g/L+Arg 0.1g/L+Gln 0.5g/L MS minimal mediums.
Further, step (1) the Changshan grapefruit explant is young stem, spire or young fruit with young shoot, more preferably described
Changshan grapefruit explant is the Post flowering young fruit of 3 months, the young fruit after 4 DEG C are placed 3 days, taking-up wrapped up with preservative film and
37 DEG C are placed 60 minutes, clean with distilled water flushing after cleaning immersion 5 minutes with liquid detergent, the young fruit after being cleaned, are stripped
Rataria, as explant.
Further, step (1) the explant sterilization method is：10min is soaked with 20-28g/L aqueous sodium hypochlorite solutions,
Aseptic water washing 3-5 times, surface moisture is blotted with filter paper, the explant after being sterilized.
Further, step (1) the inducing clumping bud culture medium is containing 0.25~0.75mg/L+BRs of 6-BA 0.1mg/L
+AgNO31mg/L MS improved culture mediums.
Further, step (2) described root media is for the MS improved culture mediums of the IAA containing 0.1-0.2mg/L or containing 0.1-
0.2mg/L IAA+0.5mg/L 6-BA MS improved culture mediums.
Further, the MS minimal mediums final concentration composition is：NH4NO3 1.65g/L、KNO31.9g/L、CaCl2·
2H2O 0.44g/L、MgSO4·7H2O 0.37g/L、KH2PO4 0.17g/L、KI 0.83mg/L、H3BO3 5.2mg/L、
MgSO4·7H2O 22.3mg/L、ZnSO4·7H2O3.6mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O
0.025mg/L、CoCl2·6H2O0.025mg/L、Na2EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, inositol 100mg/
L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, 20~40g/L of sucrose,
3~12g/L of agar powder, solvent are water, and pH is 5.6~6.0.
Compared with prior art, beneficial effect of the present invention is mainly reflected in：So far, there is not yet Changshan grapefruit utilization is outer
Any research report of implant regeneration plant, the present invention are to report first；Moreover, using this method, a rataria passes through culture,
In the plant that 3 months or so can obtain at least more than 6 plants, there is very high efficiency, it is big that acquisition can be bred in the short time
The Changshan grapefruit seedling of amount, applied in production practices.This method is efficient and convenient, can be obtained within the relatively short time
Substantial amounts of good seed, so as to set up quick, efficient, high-quality Industrialization of seeds and seedlings production system.This is both compeling for orchard worker
It is essential and asks, and the demand of market today.
(4) illustrate
Crispaturaed after Fig. 1 spire cultures and callus photo, A and B are the spire of callus.
Fig. 2 Changshan grapefruit young stem cultures expand photo.
The callus and Multiple Buds that Fig. 3 IMMATURE EMBRYOS CULTUREs are formed；A：Callus；B：The Multiple Buds of callus regeneration；
C：The Multiple Buds of rataria Direct Regeneration.
The intact plant of Fig. 4 IMMATURE EMBRYOS CULTUREs regeneration；A：The intact plant of regeneration；B：The plant of transplant survival.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1：
(1) selection and processing of explant：
Young stem, spire and the young fruit with young shoot of Changshan grapefruit are selected as explant.Specifically, Changshan grapefruit plant in
Test in greenhouse, watering in good time keeps its upgrowth situation good.During the germination of Hu shaddock, clip obtains children of the Hu shaddock with bud
Stem, spire.If without sprouting, topping treatment is carried out to Hu shaddock, sprouting is grown through 2 weekly assemblies, the children for being adapted to experiment can be obtained through 3-4 weeks
Stem and spire.The Post flowering young fruit of 3 months or so, diameter 2-3cm young fruit, by young fruit 4 DEG C place 3 days after, take
Go out and wrapped up with preservative film and placed 60 minutes at 37 DEG C, it is clean with distilled water flushing after cleaning immersion 5 minutes with liquid detergent, obtain
Pretreated young fruit is obtained, on aseptic operating platform, strips out rataria, size is in 1-3mm.Using unpretreated young fruit as control.
(2) disinfecting process of explant：
After with liquid detergent Hu shaddock spire, young stem and rataria with bud are cleaned into immersion 5 minutes respectively, done with distilled water flushing
Only, then with 20-28g/L aqueous sodium hypochlorite solutions soak 10min.Then material clean 3-5 times after processing is used with sterilized water
Filter paper blots the moisture of excess surface, the explant after being sterilized.
(3) Fiber differentiation of explant：
On superclean bench, the explant after above-mentioned sterilization is inoculated into inducing clumping bud culture medium (inducing clumping bud
Culture medium is 6-BA0.25~2.5mg/L+BRs 0.1mg/L+AgNO31mg/L+ MS improved culture mediums, wherein 6-BA tool
Body content is shown in Table shown in 1, table 2 and table 3), every kind of 20 explants of inducing clumping bud culture medium inoculated.In 22 ± 2 DEG C of temperature, light
According to being cultivated under the conditions of 1000-1200lux.Explant is transferred on new inducing clumping bud culture medium within every 25 days, in time observation
Growth change situation simultaneously photographs to record, and after transferring 1 time, obtains Multiple Buds.Under similarity condition, with 6-BA0.25~2.5mg/L+
BRs 0.1mg/L+AgNO31mg/L MS minimal mediums (6-BA contents are shown in Table 1- tables 3) are control.
(4) culture of rootage of Multiple Buds：
After Multiple Buds are cut into the individual with terminal bud, root media is seeded to, at 24~26 DEG C, illumination 1000~
Cultivated 30 days or so under the conditions of 1200lux, form intact plant.
(5) experimental result
When the hormon that spire is inoculated into table 1, young stem is inoculated into table 2, rataria is inoculated into listed by table 3 matches 6-
BA0.25~2.5mg/L+BRs 0.1mg/L+AgNO3(PVP 0.5g/L+Arg are not added with 1mg/L MS minimal mediums
0.1g/L+Gln 0.5g/L MS culture mediums), there is browning in 5 days or so in culture, and is finally stopped growth and withered.
When the hormon that spire is inoculated into table 1, young stem is inoculated into table 2, rataria is inoculated into listed by table 3 matches inducing clumping bud culture
Base (0.25~2.5mg/L+BRs of 6-BA 0.1mg/L+AgNO3In 1mg/L MS improved culture mediums, MS improved culture mediums are
Add PVP 0.5g/L+Arg 0.1g/L+Gln 0.5g/L MS culture mediums), cultivate 5 days or so, still keep bud green state.
The young fruit of harvesting after the Disinfection Methods of same step (2), strips rataria without 4 DEG C of low temperature and 37 DEG C of Heat thermostabilities
Inducing clumping bud culture medium is inoculated into, even if culture was not sprouted still by more than 60 days, is sprouted extremely slow.And young fruit is put into
4 DEG C after 3 days, taking-up preservative film parcel is put into 37 DEG C of Heat thermostabilities 60 minutes, then after the Disinfection Methods of step (2), strips
Rataria is inoculated into inducing clumping bud culture medium, and 2 weeks or so i.e. visible sprouting.
Therefore, 0.25~2.5mg/L+BRs of 6-BA 0.1mg/L+AgNO are selected3In 1mg/L MS improved culture mediums
(addition PVP 0.5g/L+Arg 0.1g/L+Gln 0.5g/L MS culture mediums) is used as spire, young stem and IMMATURE EMBRYOS CULTURE induced bundle
The culture medium sprouted；Young fruit was put into 4 DEG C after 3 days, and taking-up is put into 37 DEG C of Heat thermostabilities by the use of preservative film parcel and is used as rataria in 60 minutes
Pretreatment condition before culture.
After inoculation spire is cultivated on inducing clumping bud culture medium, at 15 days or so, there is chrysanthemum death, portion in a small number of blades
Leaflet piece is crispaturaed, and further forms callus, and cultivation results are shown in Table 1 and Fig. 1.
The Changshan grapefruit spire cultivation results of table 1
After young stem is inoculated with inducing clumping bud culture medium, after inoculation 20-30 days, there is the situation of expanding, but non-shape in stem
Into obvious callus, concrete outcome is shown in Table 2 and Fig. 2.
The Changshan grapefruit young stem culture result of table 2
Young fruit is by being put into 4 DEG C after 3 days, after taking-up preservative film parcel is put into the processing in 60 minutes of 37 DEG C of Heat thermostabilities, stripping
It is seeded on inducing clumping bud culture medium and is carried out in incubation after the rataria sterilization taken, rataria started to sprout at 2 weeks or so.Children
During embryo culture, Multiple Buds can be directly differentiated under the conditions of low concentration 6-BA (0.25-0.75mg/L)；And higher
Under the conditions of concentration 6-BA (1.0-2.5mg/L), then only dedifferentiation is callus.Moreover, callus is transferred to containing relatively low dense
Spend 6-BA (0.25-0.75mg/L) culture medium on, also can shoot regeneration, the results are shown in Table 3 and Fig. 3.
The Changshan grapefruit IMMATURE EMBRYOS CULTURE result of table 3
In addition, it is checking BRs 0.1mg/L+AgNO3Whether 1mg/L promotes rataria to regenerate Multiple Buds, and inoculation rataria is to adding
Add or do not add BRs 0.1mg/L and AgNO3Tested on 1mg/L culture medium, experiment condition and result such as table 4.As a result
Show BRs 0.1mg/L+AgNO31mg/L, which has, remarkably promotes rataria regeneration Multiple Buds.
The BRs of table 4 and AgNO3Influence to IMMATURE EMBRYOS CULTURE
Embodiment 2
The rataria of pretreated young fruit separation in embodiment 1 is seeded to inducing clumping bud culture medium (6-BA 0.5mg/
L+BRs 0.1mg/L+AgNO31mg/L MS improved culture mediums), cultivated under the conditions of 22 ± 2 DEG C, illumination 1000-1200lux
30 days or so, Multiple Buds are obtained, after being cut into the individual with terminal bud, are transferred to root media (0.1-0.2mg/L IAA
MS improved culture mediums or 0.1-0.2mg/L IAA+0.5mg/L 6-BA MS improved culture mediums, be shown in Table on 5), in temperature 22
± 2 DEG C, cultivate 30 days or so under the conditions of illumination 1000-1200lux, eventually form intact plant.Average each Multiple Buds pass through
More than 6 plant can be formed after cutting.In MS improved culture medium+0.1-0.2mg/L IAA, and MS improved culture mediums+0.1-
On 0.2mg/LIAA+0.5mg/L 6-BA culture medium, it can take root.At 30 days or so, root long can reach 2-3 centimetres, enter
One step forms intact plant.Therefore, a rataria passes through above-mentioned culture, and Multiple Buds can be obtained in 60 days, then by 30 days
Left and right can obtain at least more than 6 plants of intact plant, i.e. 1 rataria can obtain at least more than 6 plants complete at 3 months or so
Plant, there is the characteristics of short time, efficiency high.Concrete outcome is shown in Table 5 and Fig. 4.
Multiple Buds take root for forming the result of intact plant under 5 different condition of culture of table
| MS improved culture mediums+different phytohormone concentrations (mg/L) | It can take root |
| 0.1mg/L IAA+0.5mg/L 6-BA | Energy |
| 0.1mg/L IAA | Energy |
| 0.2mg/L IAA+0.5mg/L 6-BA | Energy |
| 0.2mg/L IAA | Energy |
| 0.1mg/L NAA | No, base portion forms callus |
| 0.1mg/L NAA+0.5mg/L 6-BA | No, base portion forms callus |
| 0.2mg/L NAA | No, base portion forms callus |
| 0.2mg/L NAA+0.5mg/L 6-BA | No, base portion forms callus |
Claims (5)
- A kind of 1. method of Changshan grapefruit fast asexual propagation, it is characterised in that methods described is：(1) acquisition of explant and lure Lead culture：Selection is grown fine, the sterilization of the Changshan grapefruit explant of no disease and pests harm, the explant after being sterilized；After sterilizing Explant be seeded in inducing clumping bud culture medium, cultivated under the conditions of 20~24 DEG C, 1000~1200lux of illumination, every Switching in 25~45 days once, switching culture 2-3 times, obtains Multiple Buds；The inducing clumping bud culture medium is that the amino of benzyl containing 6- is fast Purine 0.25~2.5mg/L+ brassinosteroids 0.1mg/L+AgNO31mg/L MS improved culture mediums, the MS improved culture mediums To add polyvinylpyrrolidone 0.5g/L+ arginine 0.1g/L+ glutamine 0.5g/L MS minimal mediums；The Changshan Hu shaddock explant is spire, rataria or the young stem with young shoot, and the rataria is to place the Post flowering young fruit of 3 months 3 days at 4 DEG C Afterwards, taking-up is wrapped up with preservative film and placed 60 minutes at 37 DEG C, after cleaning immersion 5 minutes with liquid detergent, is done with distilled water flushing Only, the young fruit after being cleaned, strips rataria；(2) culture of rootage：The Multiple Buds that step (1) obtains are cut into terminal bud After individual, root media is seeded to, cultivates 30-60 days, has been formed under the conditions of 24~26 DEG C, 1000~1200lux of illumination Whole plant；The root media is the MS improved culture mediums of the indole-3-acetic acid containing 0.1-0.2mg/L, the MS improvement culture Base is addition polyvinylpyrrolidone 0.5g/L+ arginine 0.1g/L+ glutamine 0.5g/L MS minimal mediums.
- 2. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (1) described explant disappears Malicious method is：10min is soaked with 20-28g/L aqueous sodium hypochlorite solutions, aseptic water washing 3-5 times, surface water is blotted with filter paper Point, the explant after being sterilized.
- 3. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (1) described Multiple Buds lure It is containing 6-benzyl aminopurine 0.25~0.75mg/L+ brassinosteroids 0.1mg/L+AgNO to lead culture medium31mg/L MS improvement Culture medium.
- 4. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that step (2) described culture of rootage Base is the MS improved culture mediums of+0.5mg/L 6-benzyl aminopurines of indole-3-acetic acid containing 0.1-0.2mg/L.
- 5. the method for Changshan grapefruit fast asexual propagation as claimed in claim 1, it is characterised in that the MS minimal mediums are whole Concentration forms：NH4NO3 1.65g/L、KNO3 1.9g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O 0.37g/L、 KH2PO4 0.17g/L、KI 0.83mg/L、H3BO3 5.2mg/L、MgSO4·7H2O 22.3mg/L、ZnSO4·7H2O 3.6mg/ L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole Tremble alcohol 0.5mg/L, nicotinic acid 0.5mg/L, 20~40g/L of sucrose, 3~12g/L of agar powder, and solvent is water, and pH is 5.6~6.0.
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