CN105123515A - Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage - Google Patents
Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage Download PDFInfo
- Publication number
- CN105123515A CN105123515A CN201510449041.3A CN201510449041A CN105123515A CN 105123515 A CN105123515 A CN 105123515A CN 201510449041 A CN201510449041 A CN 201510449041A CN 105123515 A CN105123515 A CN 105123515A
- Authority
- CN
- China
- Prior art keywords
- eggplant
- sprouting
- medium
- sterilizing
- stem section
- 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
- 244000061458 Solanum melongena Species 0.000 title claims abstract description 50
- 235000002597 Solanum melongena Nutrition 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000009396 hybridization Methods 0.000 title description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 87
- 239000012879 subculture medium Substances 0.000 claims abstract description 15
- 238000005520 cutting process Methods 0.000 claims abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 10
- 201000010099 disease Diseases 0.000 claims abstract description 9
- 241000238631 Hexapoda Species 0.000 claims abstract description 6
- 230000004083 survival effect Effects 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 44
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- 238000004659 sterilization and disinfection Methods 0.000 claims description 29
- 238000005204 segregation Methods 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 16
- 229960002523 mercuric chloride Drugs 0.000 claims description 16
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 230000002073 mitogenetic effect Effects 0.000 claims description 13
- UUTKICFRNVKFRG-WDSKDSINSA-N (4R)-3-[oxo-[(2S)-5-oxo-2-pyrrolidinyl]methyl]-4-thiazolidinecarboxylic acid Chemical compound OC(=O)[C@@H]1CSCN1C(=O)[C@H]1NC(=O)CC1 UUTKICFRNVKFRG-WDSKDSINSA-N 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 5
- 239000011435 rock Substances 0.000 claims description 4
- 238000011160 research Methods 0.000 abstract description 9
- 241000196324 Embryophyta Species 0.000 abstract description 8
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 230000002068 genetic effect Effects 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 241000607479 Yersinia pestis Species 0.000 abstract 1
- 230000007613 environmental effect Effects 0.000 abstract 1
- 208000015181 infectious disease Diseases 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 239000012883 rooting culture medium Substances 0.000 abstract 1
- 230000000050 nutritive effect Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000036512 infertility Effects 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 5
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 3
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 3
- 239000006013 carbendazim Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000003020 moisturizing effect Effects 0.000 description 3
- 239000006870 ms-medium Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 238000005728 strengthening Methods 0.000 description 3
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 230000032459 dedifferentiation Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012214 genetic breeding Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000009399 inbreeding Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 241000207763 Solanum Species 0.000 description 1
- 229930014669 anthocyanidin Natural products 0.000 description 1
- 150000001452 anthocyanidin derivatives Chemical class 0.000 description 1
- 235000008758 anthocyanidins Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000006160 differential media Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
A method for preserving the separated group or distant filial generation of eggplant by micro-cuttage with stem segments includes such steps as choosing the first oneSterilizing a proper eggplant stem section, inserting the eggplant stem section on a sprout meristem culture medium, growing sprouts at leaf axillary positions in succession after 7-10 days, cutting off the sprouts from a near base part when the sprouts grow to 1-2cm, sterilizing, transferring the sprouts to a subculture medium for culture, transferring the sprouts to a rooting culture medium for culture when the sprouts grow to 3-4cm, growing robust roots after 10-15 days to form new eggplant seedlings, and planting the eggplant seedlings in a field after hardening the seedlings for a period of time, wherein the survival rate is 95% -100%. For long-term storage F2The population is transferred on the subculture medium all the time, and the transfer is carried out once every 30 days. Compared with a cuttage method and a common tissue culture method, the method has simple steps, integrates the advantages of the two methods, is short in using time, small in occupied area, free of infection of plant diseases and insect pests, easy to control environmental conditions, convenient to manage, strong in stability of new buds generated from stem segments, and not easy to generate variation. Therefore, the method has very important application value in genetic and breeding research.
Description
Technical field
The present invention relates to a kind of method of preserving eggplant new germ plasm and interim segregation population, be adapted to the preservation of the germ plasm resource cannot or hardly preserved by sexual propagation, especially study with interim segregation population or the preservation of distant hybrid progeny with distant hybrid sterility or sterility, i.e. stem section micro cuttage method, be a kind of vegetative store method, belong to plant biotechnology field.
Background technology
Micro cuttage method is preserved Eggplant Germplasm Resources and is had broad application prospects in Crop Genetic Breeding theory and practice research, fast and effeciently the germ plasm resource that eggplant cannot or hardly be preserved by sexual propagation can be regenerated by micro cuttage, by its stable preserving, not only can solve the difficult problem that the kind matter that causes due to distant hybrid sterility and sterility cannot be preserved, recurrence is carried out in the scientific research that also to make with interim segregation population be examination material becomes possibility.
In eggplant genetic breeding research, there is RIL (RIL) in the colony that people often use, doubled haploid (DH) colony, F
2colony three kinds.RIL is the types of populations for genetic analysis and mapping in biology, by F
2the individual inbreeding of more generation of colony produces, and in colony, each strain is isozygotied.In practical study, we often adopt " standard " RIL colony in selfing 6-7 generation, therefore build RIL colonial need cost considerable time, drop into a large amount of man power and materials.DH colony is the dliploid that monoploid is formed through chromosome doubling, and each DH plant genotype is isozygotied, and belongs to permanent population.But produce DH plant and depend on colored training technology, the anther culture of eggplant is very difficult, and differentiation rate is extremely low.In addition, flower training process can produce selection effect to the pollen of different genotype, destroys the genetic structure of DH colony, causes more serious inclined segregation phenomenon, thus the accuracy of influence research.F
2colony is temporary transient segregation population, build this colonial need time shorter, the genotype that parent is all is contained in colony, gene effect can be estimated and detect dissimilar mutual work etc., but this types of populations is once selfing or inbreeding, its genetic constitution will change, and cannot use for a long time, is therefore preserved significant by certain means.
Hybridization between eggplant cultivated species and other Solanum species is limited due to reproductive disorder, therefore wants to utilize the merit such as disease-resistant, degeneration-resistant of eggplant wild species very difficult.Due to distant hybrid F
1there is the feature of hybrid sterility, cause part distant hybridization F
1in generation, does not become plant that is complete or maturation; Often have sterility, namely Post flowering is difficult to obtain fertilized egg, or can not grow up to the plant of complete health after forming fertilized egg, so obtain distant hybridization F
2colony becomes distant hybrid F
1more difficult.Research distant hybrid F
1for efficient store method, for groping the effective ways overcoming distant hybridization obstacle, thus distant hybridization F can be obtained
2colony races against time, and creates chance.Therefore, in eggplant breeding work, obtaining distant hybrid progeny is a thing very not easily, and their preservation is one and significantly works.
Eggplant distant hybridization F
1and F
2the Techniques of preserving of segregation population rarely has report in the world, the method having reported out be at present (A.Frary, S.Doganlar, M.C.Daunay, etal., 2003) such as A.Frary by cuttage by a distant hybridization F
2colony has successfully transported to France from the U.S..LorenzoBarchi etc. (BarchiL, LanteriS, PortisE, etal., 2012), in order to obtain more reasonable anthocyanidin content accurately, utilize the method for cuttage that the F2 colony of ' 305E40 ' x ' 67/3 ' is replicated 3 times for investigation.But cuttage exists, and floor space greatly, easily infects damage by disease and insect, management process is loaded down with trivial details and be difficult to obtain the shortcomings such as constant condition of culture.Some are also had to utilize tissue culture method to set up the efficient genetic system of eggplant to preserve Eggplant Germplasm Resources, general procedure is first utilize tissues different on eggplant or organ to obtain a large amount of callus through dedifferentiation, then break up again, callus grows new tender shoots, received by tender shoots after root media carries out culture of rootage a period of time, field planting is to field.Although this method also can preserve Eggplant Germplasm Resources, but process is more loaded down with trivial details, time used is longer, and from callus, break up the tender shoots obtained again easily morph, the more important thing is that different genotype has stronger selectivity to medium component and hormone combination, thus have impact on applying of the method.Stem of eggplant section micro cuttage method can solve the problem, and it can persistence eggplant F
1and F
2segregation population, step is simple, and the used time is short and floor space is few, and do not have infecting of damage by disease and insect, condition of culture easily controls, and is convenient to management, and strong by the stability of stem section generation sprouting, and variation is less likely to occur.Therefore, in heredity and breeding research, there is very important using value.
Summary of the invention
The present invention aims to provide a kind of method of simple and quick preservation eggplant segregation population or distant hybrid progeny.
General principle of the present invention is: adopt suitable sterilizing methods to carry out sterilization treatment to the axis section containing leaf bud, under certain condition, after cultured in vitro a period of time, at axil, director goes out sprouting, by when being transferred on appropriate media after newborn tender shoots sterilizing, complete regeneration plant can be obtained.
The innovative point that the present invention gives prominence to is: by research segregation population or be not easy obtain distant hybridization Germ-plasma resources protection get off, can study for many years, step is simple, explant dedifferentiation is formed callus, and the process simplification being then differentiated to form tender shoots again, for directly to form tender shoots from explant, has saved the time, and floor space is few, do not have infecting of damage by disease and insect, condition of culture is easy to control, and is convenient to management.
Preserve the method (innovation of the present invention) of eggplant research segregation population or distant hybrid progeny:
1. the choosing and process of stem of eggplant section: in robust growth, without the newborn side shoot eggplant plant of damage by disease and insect gathering long about the 15cm not with inflorescence, remove nearly top and petiole, tiltedly be cut into little stem section containing 1-2 blade as little cuttings, cutting length 2-3cm, bud point indistinctly can be seen at axil place, upper cut is 1.5-2cm place above bud, and lower cut is 0.5-1.0cm place below bud, crack-free.
2. the selection of medium: the medium for micro cuttage (sprouting is mitogenetic) is MS+1.0mg/L6-BA+0.1mg/LNAA, medium for sprouting subculture is MS+0.02mg/LNAA+1.0mg/L6-BA, and the medium for taking root is the MS medium not adding any hormone.
3. the first sterilizing of stem section: the stem of eggplant Duan Xianyong sterilizing paper rinsed well is blotted surface moisture, then alcohol sterilization 30 second of 70% is used, blot with sterilizing filter paper again, go to sterilizing 7-8 minute in the conical flask of the mercuric chloride filling 0.1%, period frequently rocks, to reach the more sufficient effect of sterilizing.Then stem section is taken out, with aseptic water washing 4-5 time, to blot the cuttage of morphology lower end after surface moisture on the mitogenetic medium of sprouting with sterilizing filter paper.
4. stem section sterilizing again: once find the stem explants of long bacterium, uses 0.1% mercuric chloride sterilizing 2-3min again with after 70% alcohol sterilization 30s, blots surface moisture, be transferred on the mitogenetic medium of new sprouting with after aseptic water washing 3-4 time.After two-stage sterilization, the stem explants of long bacterium is given up and is no longer used again.
5. the first sterilizing of sprouting: after stem section culture 7-10 days, can go out sprouting the axil director of stem section successively, until sprouting grow to about 1.0-2.0cm long time it is cut from base portion, note not switching to stem section.0.1% mercuric chloride sterilizing 2-3min, blots surface moisture with after aseptic water washing 3-4 time, is transferred on sprouting subculture medium.
6. sprouting sterilizing again: sprouting was cultivated after 3-4 days, part there will be long bacterium situation, uses 0.1% mercuric chloride sterilizing 2-3min again with after 70% alcohol sterilization 30s, blots surface moisture, be transferred on new subculture medium, continuation cultivation with after aseptic water washing 3-4 time.After two-stage sterilization, the sprouting of long bacterium gives up and no longer uses again.
7. sprouting subculture is numerous with expansion: sprouting may have grown into the high plant of 3-4cm cultivate 25-30d on subculture medium after, now can carry out expanding numerous work, namely with aseptic operation cutter, new section is cut into several segment, each segment at least containing a leaf bud, is then transferred on subculture medium.
8. hardening: after newborn stem section cultivates 15-20 days on root media, can bear new root, at this moment open blake bottle bottleneck, other conditions are constant, carry out hardening, every day sprays some sterile water moisturizings in bottle, cultivates 4 days.Common seedling medium is watered to 60% water content, at 121 DEG C, is placed to room temperature under 0.1Mpa condition after sterilizing 40min, is then dispensed in aseptic nutritive cube.Slowly taken out by newborn seedling in medium, wash the medium of root with the clear water of flowing off, be then put in the taking root liquid containing the former powder G.A-6 of 0.1% carbendazim+0.1% root taking sprout strengthening agent and soak after 30 seconds, field planting is in the medium filling sterilization matrix.Temperature 26 DEG C, humidity 90%, cultivates under the condition of photoperiod 16h after 15-20 days and can descend ground field planting.
The present invention is completed by following steps:
1. stem of eggplant section is selected and process
The eggplant side shoot stem section of healthy growth is gathered in the morning of continuous sunny, stem section requires: the newborn side shoot of long about the 15cm not with inflorescence, remove nearly top and petiole, tiltedly be cut into little stem section containing 1-2 blade as little cuttings, cutting length 2-3cm, indistinctly can see bud point at axil place, upper cut is 1.5-2cm place above bud, lower cut is 0.5-1.0cm place below bud, crack-free (as Fig. 1).
2. the first sterilizing of stem section and cuttage
Wash away earth and impurity, rinse 20 minutes under flowing tap water, first blot surface moisture with sterilizing filter paper, then use alcohol sterilization 30 second of 70%, then blot with sterilizing filter paper, be transferred to sterilizing 7-8 minute in the mercuric chloride of 0.1%, period frequently rocks, to reach the more sufficient effect of sterilizing.Then stem section is taken out, with aseptic water washing 4-5 time, to blot the cuttage of morphology lower end after surface moisture on the mitogenetic medium of sprouting (as Fig. 2) with sterilizing paper.
3. stem section sterilizing again with switching cuttage
Cultivate under these conditions after 3-4 days, part stem section there will be the situation of long bacterium, at this moment taken out as early as possible, cut position of carrying disease germs, 0.1% mercuric chloride sterilizing 2-3min is used again with after 70% alcohol sterilization 30s, blot surface moisture with after aseptic water washing 3-4 time, be transferred on the mitogenetic medium of new sprouting, continue to cultivate.Remove stem section that is dead and long bacterium again in time.
4. the first sterilizing of sprouting and switching
After stem section culture 7-10 days, can go out sprouting (as Fig. 3) axil director successively, when sprouting grows to about 1.0-2.0cm length, it cuts from base portion by (as Fig. 4), notes not switching to stem section.With 0.1% mercuric chloride sterilizing 2-3min, then blot surface moisture after aseptic water washing 3-4 time, be transferred on sprouting subculture medium.
5. sprouting sterilizing and switching again
Sprouting was cultivated after 3-4 days, and part there will be long bacterium situation, uses 0.1% mercuric chloride sterilizing 2-3min again with after 70% alcohol sterilization 30s, blots surface moisture, be transferred on new sprouting subculture medium, continuation cultivation (as Fig. 5) with after aseptic water washing 3-4 time.Remove again the sprouting of long bacterium in time.
6. culture of rootage
Sprouting was cultivated after 25-30 days, can grow up to the newborn stem section (as Fig. 6) that 3-4cm is high, now can receive that sprouting subculture medium to carry out expansion numerous, also can be transferred on root media and cultivate, promote that it is taken root.
7. first hardening
After newborn stem section cultivates 15-20 days on root media, can bear new root (as Fig. 7, Fig. 8), at this moment open blake bottle bottleneck, other conditions are constant, carry out hardening, and every day sprays some sterile water moisturizings in bottle, cultivate 4 days.
8. the sterilizing of hardening matrix and packing again
Nutritive cube soaks 30min and carries out sterilization in 75% alcohol.Common seedling medium adds water to 60% water content, and at 121 DEG C, sterilizing 40min under 0.1Mpa condition, is dispensed in aseptic nutritive cube after being placed to room temperature.
9. hardening again
Newborn seedling after carrying out hardening for the first time is slowly taken out, the medium of root is washed off with the clear water of flowing, then be put in the taking root liquid containing the former powder G.A-6 of 0.1% carbendazim+0.1% root taking sprout strengthening agent and soak after 30 seconds, field planting is in the medium filling sterilization matrix.
10. field planting
Again can by newborn Miao Dingzhi to large Tanaka after hardening 15-20 days in nutritive cube, survival rate 95%-100%.
In above-mentioned steps 2,3,4,6 component of medium and proportioning as follows:
The mitogenetic medium of sprouting (cuttage medium): MS minimal medium, 0.1mg/LNAA, 1.0mg/L6-BA, 30g/L sucrose, 7g/L agar, adds water to 1L, the pH to 5.8-6.0 of adjustment medium before sterilizing.
Sprouting subculture and expansion breeding culture medium: MS minimal medium, 0.02mg/LNAA, 1.0mg/L6-BA, 30g/L sucrose, 7g/L agar, adds water to 1L, the pH to 5.8-6.0 of adjustment medium before sterilizing;
Root media: MS minimal medium, 30g/L sucrose, 7g/L agar, adds water to 1L, the pH to 5.8-6.0 of adjustment medium before sterilizing.
Above-mentioned medium all at 121 DEG C, sterilizing 20min under 0.1Mpa HTHP.
Above-mentioned stem section or sprouting carry out transferring in the medium, subculture, be all placed on room temperature 26 DEG C after operation of taking root, and the photoperiod is cultivate in the constant temperature illumination cultivation room of 16h.
In the present invention, described plant hormone defines and is called for short as follows: naa (NAA), 6-benzyl aminoadenine (6-BA).The minimal medium used sees the following form for MS medium, each component and concentration.
Unit: mg/L.
Accompanying drawing explanation
Fig. 1 is the eggplant cuttings figure of band axillalry bud; Fig. 2 is that the cuttage of eggplant cuttings is in mitogenetic medium figure; Fig. 3 is eggplant cuttings axillary bud sprouting figure; Fig. 4 is cuttage stem section and switchable sprouting figure; Fig. 5 is sprouting squamous subculture figure; Fig. 6 is regrowth (not taking root) figure; Fig. 7 is regrowth (taking root) figure; Fig. 8 is regrowth root figure
Embodiment
According to technical scheme of the present invention, concrete implementation step is:
1. try the determination of material
Choose eggplant distant hybrid 11-435(and cultivate eggplant) the wild eggplant of × 11-587() and self progeny F
2as test material.
2. the preparation of medium
Minimal medium is MS medium, and concrete composition refers to top list.
The mitogenetic medium of sprouting (cuttage medium): MS minimal medium+0.1mg/LNAA+1.0mg/L6-BA, 30g/L sucrose, 7g/L agar, adds water to 1L, and pH is adjusted to 5.8-6.0, at 121 DEG C, sterilizing 20 minutes under 0.1Mpa condition.
Sprouting subculture and expansion breeding culture medium: MS minimal medium+0.02mg/LNAA+1.0mg/L6-BA, 30g/L sucrose, 7g/L agar, adds water to 1L, and pH is adjusted to 5.8-6.0, at 121 DEG C, sterilizing 20 minutes under 0.1Mpa condition.
Root media: MS minimal medium, 30g/L sucrose, 7g/L agar, adds water to 1L, and pH is adjusted to 5.8-6.0, at 121 DEG C, sterilizing 20 minutes under 0.1Mpa condition.
3. stem of eggplant section is selected and process
The distant hybrid F of healthy growth is gathered in the morning of continuous sunny
111-435(cultivates eggplant) the wild eggplant of × 11-587() and self progeny F
2eggplant side shoot stem section, removes nearly top and petiole, is tiltedly cut into the little stem section containing 1-2 leaf bud, washes away earth and impurity, rinses 20 minutes under flowing tap water.
4. the first sterilizing of stem section and cuttage
The stem of eggplant Duan Xianyong sterilizing paper rinsed well is blotted surface moisture, and then use alcohol sterilization 30 second of 70%, then blot with sterilizing paper, be transferred to sterilizing 7-8 minute in the conical flask filling 0.1% mercuric chloride, period frequently rocks conical flask.Then stem section taken out, with aseptic water washing 4-5 time, be inoculated on sprouting differential medium morphology lower end after blotting surface moisture with sterilizing paper, seal with sealed membrane, be placed on 26 DEG C, the photoperiod is cultivate in the constant temperature illumination cultivation room of 16h.
5. stem section sterilizing and switching cuttage again
Cultivate under these conditions after 3-4 days, there is the situation of long bacterium in part stem section, at this moment taken out as early as possible, cut position of carrying disease germs, 0.1% mercuric chloride sterilizing 2-3min is used again with after 70% alcohol sterilization 30s, blot surface moisture with after aseptic water washing 3-4 time, cuttage, on the mitogenetic medium of new sprouting, continues to cultivate.Remove stem section that is dead or long bacterium again at any time.
6. the first sterilizing of sprouting and switching
After stem section culture 7-10 days, not long bacterium with process the stem section of transferring and go out sprouting the axil place president of stem section successively, when sprouting grows to about 1.0-2.0cm length, it is cut from base portion.0.1% mercuric chloride sterilizing 2-3min, blot surface moisture with after aseptic water washing 3-4 time, be transferred on sprouting subculture medium, seal with sealed membrane, be placed on 26 DEG C, the photoperiod is cultivate in the constant temperature illumination cultivation room of 16h.
7. sprouting sterilizing and subculture again
Sprouting was cultivated after 3-4 days, and long bacterium situation appears in part, uses 0.1% mercuric chloride sterilizing 2-3min again with after 70% alcohol sterilization 30s, blots surface moisture, be transferred to continuation cultivation on new subculture medium with after aseptic water washing 3-4 time.Remove again the sprouting of long bacterium at any time.
8. expand numerous
After sprouting cultivates 30 days, grow up to the bud section of the long left and right of 4cm, be cut into several segment with aseptic operation cutter on superclean bench, every section, at least containing a bud point, is then transferred on new sprouting subculture medium and cultivates.
9. culture of rootage
Expand numerous squamous subculture after one month, the tender tip of new life at axil place grows to about 3cm length, it is cut from base portion, is transferred on root media, seals with sealed membrane, be placed on 26 DEG C, and the photoperiod is cultivate in the constant temperature illumination cultivation room of 16h.
10. the sterilizing of hardening matrix and packing
Nutritive cube soaks 30min and carries out sterilization in 70% alcohol.Common seedling medium is watered to 60% water content, and at 121 DEG C, sterilizing 40min under 0.1Mpa condition, is dispensed in aseptic nutritive cube after being placed to room temperature.
10. hardening
Root media was cultivated after 15 days, and open blake bottle bottleneck, other conditions are constant, carry out hardening, and every day sprays some sterile water moisturizings in bottle.Continue cultivation after 4 days, slowly taken out by newborn seedling, wash the medium of root with the clear water of flowing off, be then put in the taking root liquid containing the former powder G.A-6 of 0.1% carbendazim+0.1% root taking sprout strengthening agent and soak after 30 seconds, field planting is in the nutritive cube filling sterilization matrix.
11. field planting
In nutritive cube hardening after 20 days by newborn Miao Dingzhi to large Tanaka, survival rate 100%.
Claims (7)
1. utilize stem section micro cuttage to preserve the method for eggplant segregation population or distant hybrid progeny, it is characterized in that: choose suitable stem of eggplant section and carry out first sterilization treatment, then cuttage is on the mitogenetic medium of sprouting, sterilization treatment is again carried out behind some skies, sprouting is gone out axil director successively after 7-10d, when sprouting grows to 1-2cm, it is cut from nearly base portion, carry out first sterilization treatment, then be transferred on sprouting subculture medium and cultivate, sterilization treatment is again carried out behind some skies, forward to when sprouting grows to 3-4cm on root media and cultivate, healthy and strong root is grown after 10-15d, form new eggplant seedling, after hardening a period of time, field planting is to field, survival rate 95%-100%.
2. the store method of eggplant segregation population according to claim 1 or distant hybrid progeny, it is characterized in that: described in choose stem section step in robust growth, without the newborn side shoot eggplant plant of damage by disease and insect gathering long about the 15cm not with inflorescence, remove nearly top and petiole, tiltedly be cut into little stem section containing 1-2 blade as little cuttings, cutting length 2-3cm, bud point indistinctly can be seen at axil place, upper cut is 1.5-2cm place above bud, lower cut is 0.5-1.0cm place below bud, crack-free.
3. the store method of eggplant segregation population according to claim 2 or distant hybrid progeny, it is characterized in that: the stem of eggplant Duan Xianyong sterilizing paper rinsed well is blotted surface moisture by the first sterilization steps of described stem section, then alcohol sterilization 30 second of 70% is used, blot with sterilizing filter paper again, be transferred to sterilizing 7-8 minute in the conical flask filling 0.1% mercuric chloride, period frequently rocks, to reach the more sufficient effect of sterilizing, then stem section is taken out, with aseptic water washing 4-5 time, to blot the cuttage of morphology lower end after surface moisture on the mitogenetic medium of sprouting with sterilizing filter paper.
4. the store method of eggplant segregation population according to claim 2 or distant hybrid progeny, it is characterized in that: described stem section again sterilization steps once find the stem explants of long bacterium, 0.1% mercuric chloride sterilizing 2-3min is used again with after 70% alcohol sterilization 30s, blot surface moisture with after aseptic water washing 3-4 time, be transferred on the mitogenetic medium of new sprouting.
5. the store method of eggplant segregation population according to claim 3 or distant hybrid progeny, is characterized in that: the mitogenetic medium of sprouting (cuttage medium) consists of MS minimal medium (0.7% agar, 3% sucrose), 1mg/L6-BA, 0.1mg/LNAA.
6. the store method of eggplant segregation population according to claim 4 or distant hybrid progeny, it is characterized in that: described sprouting first sterilization steps stem section culture is after 7-10 days, stem section can go out sprouting axil director successively, when sprouting grows to about 1.0-2.0cm length, it is cut from base portion, note not switching to stem section, with 0.1% mercuric chloride sterilizing 2-3min, blot surface moisture with after aseptic water washing 3-4 time, be transferred on sprouting subculture medium.
7. the store method of eggplant segregation population according to claim 5 or distant hybrid progeny, it is characterized in that: described sprouting again sterilization steps sprouting is cultivated after 3-4 days, part there will be long bacterium situation, 0.1% mercuric chloride sterilizing 2-3min is used again with after 70% alcohol sterilization 30s, surface moisture is blotted with after aseptic water washing 3-4 time, be transferred on new subculture medium, continue to cultivate, after two-stage sterilization, the sprouting of long bacterium gives up and no longer uses again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510449041.3A CN105123515B (en) | 2015-07-28 | 2015-07-28 | Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510449041.3A CN105123515B (en) | 2015-07-28 | 2015-07-28 | Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105123515A true CN105123515A (en) | 2015-12-09 |
| CN105123515B CN105123515B (en) | 2017-06-30 |
Family
ID=54709412
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510449041.3A Expired - Fee Related CN105123515B (en) | 2015-07-28 | 2015-07-28 | Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105123515B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113287513A (en) * | 2021-07-01 | 2021-08-24 | 金陵科技学院 | Eggplant haploid plant sexual propagation doubling method based on improvement of pollen activity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101379950A (en) * | 2008-10-24 | 2009-03-11 | 中国热带农业科学院热带作物品种资源研究所 | Method for quickly breeding millettia speciosa champ germchit using test tube micro cuttage |
| CN103805632A (en) * | 2014-01-28 | 2014-05-21 | 浙江省农业科学院 | Preparation method of transgenic eggplant |
| CN104145689A (en) * | 2014-08-26 | 2014-11-19 | 河北农业大学 | Cuttage and rapid propagation method for angelica keiskei |
| CN104335901A (en) * | 2014-11-14 | 2015-02-11 | 中国科学院西北高原生物研究所 | In vitro microcuttage rapid-propagation method for high-quality Qaidam wolfberry seedlings |
-
2015
- 2015-07-28 CN CN201510449041.3A patent/CN105123515B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101379950A (en) * | 2008-10-24 | 2009-03-11 | 中国热带农业科学院热带作物品种资源研究所 | Method for quickly breeding millettia speciosa champ germchit using test tube micro cuttage |
| CN103805632A (en) * | 2014-01-28 | 2014-05-21 | 浙江省农业科学院 | Preparation method of transgenic eggplant |
| CN104145689A (en) * | 2014-08-26 | 2014-11-19 | 河北农业大学 | Cuttage and rapid propagation method for angelica keiskei |
| CN104335901A (en) * | 2014-11-14 | 2015-02-11 | 中国科学院西北高原生物研究所 | In vitro microcuttage rapid-propagation method for high-quality Qaidam wolfberry seedlings |
Non-Patent Citations (1)
| Title |
|---|
| 黄志银等: "茄子组织培养高效再生植株体系的优化", 《长江蔬菜》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113287513A (en) * | 2021-07-01 | 2021-08-24 | 金陵科技学院 | Eggplant haploid plant sexual propagation doubling method based on improvement of pollen activity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105123515B (en) | 2017-06-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102227976B (en) | Litsea cubeba pers asexual quick propagation and seedling breeding method | |
| Sadhu | Plant propagation | |
| CN100553445C (en) | New lilium longiflorum production of hybrid seeds parent's tissue-culturing rapid propagation and cross-breeding method | |
| Vijayan et al. | Application of tissue culture techniques for propagation and crop improvement in mulberry (Morus spp.) | |
| Faisal et al. | In vitro plant regeneration from alginate-encapsulated microcuttings of Rauvolfia tetraphylla L | |
| CN107155898A (en) | A kind of dendrobium candidum carries out expanding numerous method using stem section thin slice | |
| CN104255489A (en) | Rapid vegetative propagation technical method for psammosilene tunicoides with axillary buds and immature stems | |
| CN104145814B (en) | Method for obtaining regeneration plants by stem tissue culture of cerasus cerasoides (var. cerasoides) | |
| CN101731137B (en) | Method for improving salt-tolerant properties of Brassicaceae crops | |
| CN104782482B (en) | Stable high-efficient method for ex-vivo preservation and growth recovery of strawberry germplasm resource | |
| CN105941162B (en) | The method for building up of tillered-onion high-efficiency regeneration system | |
| CN105230473A (en) | Creation method of salt-resistant corn | |
| CN109156338B (en) | Method for obtaining stevia rebaudiana selfing progeny through embryo rescue | |
| CN105123515B (en) | Method for preserving segregating population or distant hybridization progeny of eggplant by utilizing stem micro-cuttage | |
| CN105145358A (en) | Tissue culture and rapid propagation method for common fibraurea stem | |
| Singh et al. | Recent advances in production of healthy planting material for disease management in potato | |
| Panda et al. | Improving macropropagation and seed germination techniques for conservation of threatened species | |
| CN108849500A (en) | The culture medium and method of a kind of rescue of lotus embryo and the development of offspring's fast-growth | |
| Abd Alhady | In Vitro Plant Regeneration from Alginate Encapsulated Shoot Tips of Casimairoa Edulis L. | |
| Vongvijitra | Traditional vegetative propagation and tissue culture of some Thai bamboos | |
| CN105850748A (en) | Conifer seed germination method | |
| Karachi | Sesbania species as potential hosts to root-knot nematode (Meloidogyne javanica) in Tanzania | |
| CN101015272B (en) | Method for breeding novel varieties of mellow-type ground-cover chrysanthemum utilizing D.indicum var.aromaticum | |
| Balestri et al. | Isolation of leaf protoplasts from Pancratium maritimum L. and two other dune plants: possible applications | |
| Li et al. | Regeneration of plants from protoplasts of Arachis species (peanut) |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170630 Termination date: 20190728 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |