CN110050690A - Southern area sweet cherry and cherry interspecific hybridization rescue culture seedling establishment method - Google Patents
Southern area sweet cherry and cherry interspecific hybridization rescue culture seedling establishment method Download PDFInfo
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- CN110050690A CN110050690A CN201910387761.XA CN201910387761A CN110050690A CN 110050690 A CN110050690 A CN 110050690A CN 201910387761 A CN201910387761 A CN 201910387761A CN 110050690 A CN110050690 A CN 110050690A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/027—Apparatus for pollination
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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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Abstract
The present invention relates to the suitable cultivation sweet cherry in plant hybridization breeding field more particularly to a kind of southern area and cherry interspecific hybridization rescue culture seedling establishment methods.The advantages of this method combination gean and cherry, be female parent with ' gean ', and ' cherry ' is that male parent has carried out interspecific hybridization.Aiming at the problem that distant hybridization aborted embryo, rescue culture has been carried out to bybrid embryo.It has been determined that each cross combination rescue culture is suitable for the period of drawing materials, the concentration and ratio of exogeneous growth regulator needed for hybrid rataria is sprouted, subculture expands numerous, culture of rootage respectively and suitable acclimatization and transplants method.The southern area sweet cherry and cherry inter-species rescue culture system established using the technology, embryo germination rate reaches 80.00%, squamous subculture hybrid young sprout growth coefficient has reached 5.0, culture of rootage rooting rate 90.91%, it is good that embryo trains seedling growing way, robust growth, leaf color is green, and acclimatization and transplants survival rate reaches 77.27%.
Description
Technical field
The present invention relates to the suitable cultivation sweet cherry in plant hybridization breeding field more particularly to a kind of southern area and cherry kinds
Intermolecular hybrid rescue culture seedling establishment method.
Background technique
Cherry be rosaceae (Rosaceae) Prunus (Prunus)Cherry subgenus (CeraraS) arbor, fruit is sweet and delicious,
Nutritive value is high, and wherein iron content occupies fruit first place, is known as the good reputation of " all kinds of fruits first ".By geographical and weather conditions
It restricts, the main producing region of China's sweet cherry concentrates on the northern areas such as Dalian, Yantai for a long time, and southern area is because of summer high temperature height
The factors such as wet and winter chilling requirement deficiency cause sweet cherry only to spend fruitless no economic flow rate.In recent years, In Middle And Lower Reaches of Changjiang River kind
Sweet cherry just effect is planted, is reformed by breed breeding and cultivation technique, solves all the time puzzled ' colored and not firm '
Critical issue without economic flow rate, achieves breakthrough.However existing kind and stock are mostly using northern main producing region as breeding
Object, is not appropriate for that winter is warm and the development of the southern area of summer high temperature high humidity, to further develop southern sweet cherry, no
Only accomplish there is production, more to accomplish of fine quality, then must carry out autonomous breeding work, be as early as possible from 2 Breeding directions of kind and stock
Southern sweet cherry development provides germ plasm resource.
The main cultivation cherry in China is cherry and gean.Cherry (Prunus. pseudocerasus
L. China) is originated in, fructescence morning, self-flowering fruiting, disease resistance are strong.But fruit is small, pericarp is softer, not storage tolerance.Europe
Sweet cherry (Prunus. aviumL. Europe and western part of Asia) are originated in, fruit is big, and pulp is thick and solid, compared with storage tolerance.By European sweet tea
Cherry and cherry is excellent combines, carries out gean and cherry interspecific hybridization, miscellaneous to tone fruit trees
Breeding work is handed over to have important meaning, it is especially of far-reaching significance to the proper stock breeding of southern sweet cherry.But distant hybridization is not close
With and embryo early stage abortion to sweet cherry crossbreeding bring obstacle, the appearance and development of embryo rescue techniques mention in order to overcome the problems referred above
An effective solution approach is supplied.20 world's embryo rescue techniques sixties are applied in the fruit breeding work in China, it is
To early stage abortion, the embryo degenerated or seedling can not be sowed, the technological means of early stage in vitro culture is carried out, for overcoming distant hybridization
Not affine raising interspecific hybrid germination rate etc. has important role.
It is miscellaneous that Chinese invention patent (publication number CN105284620A, publication date 2016.02.03) discloses a kind of Superearly peach
The method for handing over rescue culture seedling wins seven to eight points of Superearly peach ripe fruits, and core is broken after fruit disinfection and takes out ovule and is inoculated with
Ovule development is carried out in growth medium, then rataria is seeded in differential medium and carries out rataria differentiation culture, will be broken
The embryo of suspend mode carries out embryo germination culture, then is transplanted.Patent progress Superearly peach hybridization rescue culture opportunity is suitable for, in ovule
Suitable culture medium is had developed during culture and embryo germination, the developmental rate of bybrid embryo is improved, improves the ratio of normal chick embryo
Example, embryo rescue seedlings are neatly healthy and strong, and embryonic development power can achieve 83% or more, and differentiating force reaches 60%, and Culture abilityi reaches 50%, greatly
The radix of Superearly peach filial generation rescue culture seedling is improved, greatly so as to obtain more Superearly peach filial generation plant
Group improves Superearly peach bybrid embryo and saves planting percent, and this method strong operability is easily mastered.
But during rescue culture, embryo age, embryo skin, low-temperature stratification mode, embryo disinfection way, type of culture medium, carbon source, plant
The factors such as the type and proportion and seedling replanting method of object growth regulator can all impact rescue culture success rate.South
Area plantation is short sweet cherry fruit growth cycle, and the maturity period shifts to an earlier date, and rataria is drawn materials the time, aborted embryo time and embryo culture method with
There is some difference for northern area, therefore the hybrid embryo rescue culture system that foundation is suitable for southern sweet cherry can be miscellaneous for the remote edge of sweet cherry
It hands over breeding and expands Germplasm Resources Diversity work and lay the foundation.
Summary of the invention
The object of the present invention is to provide a kind of suitable cultivation sweet cherry in southern area and cherry interspecific hybridization rescue culture seedlings
Method.It is preliminary to establish southern sweet cherry hybrid embryo rescue culture system, overcome the problems, such as sweet cherry interspecific hybridization aborted embryo, promotes hybrid
Embryonic development development, improves its germination rate and planting percent, accelerates sweet cherry crossbreeding job schedule.
In order to achieve the above purpose, southern area sweet cherry of the invention and cherry interspecific hybridization rescue culture seedling
Method, method includes the following steps:
1) pollen and pollination are collected: with cherry (Prunus. pseudocerasusL.) it is male parent, acquires the small bell florescence
It is natural flower；Anther is peeled indoors, is dried in the shade under conditions of 20 ~ 30 DEG C of dryings；After anther dehiscence loose powder, it is collected in small
In vial and sealing is placed in 1 ~ 6 DEG C of refrigerator and saves backup；With gean (Prunus. aviumIt L. is) mother
This, female parent selfing is not affine, and small bell phase flower carries out artificial not producing Crossbred；Sulfuric acid paper bag and 5 ~ 10 d are covered after pollination immediately
Draping bag afterwards；
2) fruit picking and cold stratification: the maternal hybridization young fruit for being in stone phase embryo length/kind of long PF > 0.5 of picking respectively is fresh
Fruit is put into transparent plastic crisper, sets and stores 30 ~ 40 d in 1 ~ 6 DEG C of low temperature refrigerator；
3) it rataria inoculation and sprouting culture: after low-temperature treatment, rinses fruit well, peels off pulp and take embryo；In ultra-clean work
It carries out disinfection in platform；Endotesta is peelled off to be inoculated on the 1/2 MS solid medium for promoting additive containing the first exogeneous growth,
It is placed in culturing room's culture；
4) subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 1.5 ~ 2.5 cm long of interception is inoculated in after being commissioned to train
It supports on base MS, subculture medium MS is added the second exogeneous growth and promotes additive；It is placed in culturing room and cultivates 35 ~ 45 d；
5) culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in additional
Third exogeneous growth promotes in additive root media 1/2MS；It is placed in culturing room and cultivates 35 ~ 45 d, obtain hybrid seedling；
6) acclimatization and transplants: by 10 ~ 20 d of hybrid seedling hardening, it is transplanted into nutritive cube；Transplanting time is mid-February.
As a further improvement, in the application step 1) male parent varieties be cherry ' short handle ' ' amethyst ' ' depressed place Ge Jia ' or
' black pearl ', parental varieties are sweet cherry ' Jiangnan is red ' ' southern exposure No.1 ' ' Changfeng No.1 ' or ' pioneer '.
As a further improvement, in the application step 2 hybrid rescue culture best embryo age are as follows: with ' Jiangnan is red ' be female parent
Cross combination be 31 d after pollination, be maternal cross combination with ' southern exposure No.1 ' be 37 ~ 48 d after pollinating, with ' Changfeng
No.1 ' it be maternal cross combination is 41 d after pollination, it be maternal cross combination with ' pioneer ' is 32 ~ 46 d after pollinating.
As a further improvement, it is 1.5 ~ 2.5 mg/L 6-BA, 1.5 that the first exogeneous growth of the application, which promotes additive,
~ 2.0mg/L IAA, 20 ~ 40 g/L sucrose and 2 ~ 8 g/L agar.
As a further improvement, it is 0.3 ~ 0.7 mg/L 6-BA, 0.1 that the second exogeneous growth of the application, which promotes additive,
~ 0.4 mg/L IAA or 0.1 ~ 0.5 mg/L IBA, 20 ~ 40 g/L sucrose and 2 ~ 8 g/L agar.
As a further improvement, it is 0.2 ~ 0.5 mg/L IBA, 1.0 that the application third exogeneous growth, which promotes additive,
~ 1.5mg/L IAA, 20 ~ 40g/L sucrose and 2 ~ 8 g/L agar.
As a further improvement, sterilizing in the application step 3) with 70% alcohol rinse 1 time, aseptic water washing 3 times, then use
0.1% HgCl2Sterilize 3min, and aseptic water washing 4 ~ 5 times.
As a further improvement, the application step 3), 4) and 5) in culturing room's condition of culture be 25 ~ 27 DEG C of temperature, light
According to 16 h/d, intensity of illumination is 2 000 lx.
As a further improvement, the hardening off method in the application step 6) are as follows: after 60 d of culture of rootage, selection main root 6 ~
9,6 or more effective true leaves, 2.5 cm or more of height of seedling, base portion train seedling without the embryo of callus, robust growth, in Sterile culture room
It is a little to open hybrid seedling bottle cap, is gradually increased the degree of ventilation of culture bottle every 5 d；Nutritive cube before transplanting 3 d with bacterium more than 500 times
Clever solution disinfection dries spare；Matrix is laid in pallet, carries out 12 h of solar exposure；Seedling is small from culture bottle when transplanting
The heart takes out, and the culture medium on root system is washed away under flowing water, and transplanting irrigates root water, overlay film equipped in the nutritive cube of matrix, places
In the small plastic canopy built；Periodically water spray keeps humidity and temperature appropriate in canopy, pays attention to ventilated；It is gone after 2 weeks
Film, and guarantee ventilation and penetrating light, obtain sweet cherry and cherry hybrid plant.
The present invention due to the adoption of the above technical solution, has the characteristics that following:
1, the present invention judges embryo age using the length relation (i.e. PF value) of hybrid seed and embryo, can clearly understand hybrid embryo
Development condition specifies embryo development best period, to carry out rescue culture.
2, after the present invention can abolish stone, then HgCl is used in superclean bench2Sterilize 3 min, saves sterilization time,
Simplify tissue culture operating procedure, it is easier to grasp.
3, according to determining materials period, by young fruit, 30 ~ 40 d of storage are the present invention directly in 1 ~ 6 DEG C of low temperature refrigerator
Can breaking dormancy, it is simple and convenient, shorten breeding time, improve embryo crash breeding efficiency.
4, the present invention increases the radix of hybrid seedling, can reduce dirty in tissue culture by expanding numerous culture to hybrid young sprout subculture
Germplasm risk of missing caused by dye or transplanting hybrid seedling mortality, is conducive to follow-up study.
Using technical method of the invention, Hybrid embryo is carried out before sweet cherry distant hybrid aborted embryo, is improved
The germination rate of hybrid embryo accelerates sweet cherry interspecific hybridization breeding process.The suitable sweet cherry variety planted in southern area is less at present
And based on introducing a fine variety, more lacks the suitable stock variety of southern kind, rescue culture is carried out to sweet cherry hybrid embryo and enriches sweet tea cherry
The germ plasm resource of peach establishes certain basis for further screening southern area sweet cherry hybrid new breed.The method of the present invention is established
Southern area sweet cherry and cherry inter-species rescue culture system, embryo germination rate reaches 80.00%, and squamous subculture hybrid is new
Tip growth coefficient has reached 5.0, and culture of rootage rooting rate 90.91%, embryo training seedling growing way is good, robust growth, and leaf color is green.Hardening is moved
It plants survival rate and reaches 77.27%, improve the planting percent of sweet cherry interspecific hybridization, shorten the fruit breeding period.
Detailed description of the invention
Fig. 1: different female parent materials period A: the red B in Jiangnan: Changfeng No.1 C: southern exposure No.1 D: pioneer.
Fig. 2: it ' southern exposure No.1 ' × ' depressed place Ge Jia ' hybrid embryo rescue culture different stages of growth: A: sprouts and grown cultures B: after
In generation, expands numerous C: culture of rootage D: transplanting.
Specific embodiment
Embodiment 1
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: with cherry ' amethyst ' ' short handle ' for male parent, the natural flower at acquisition small bell florescence.Indoors
Anther is peeled, is dried in the shade under conditions of 25 DEG C of dryings.After anther dehiscence loose powder, it is collected in vial and seals and be placed in 4
DEG C refrigerator in save backup.
It is female parent with sweet cherry ' southern exposure No.1 ', artificial not producing Crossbred is carried out to small bell phase flower.It is covered immediately after pollination
Draping bag after sulfuric acid paper bag and 7d.
B. fruit picking and cold stratification: as shown in Figure 1 C, most preferably draw materials period in hybrid rescue culture (stone phase PF >
0.5) hybridization young fruit is adopted, fresh fruit is put into transparent plastic crisper, sets and stores 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile
Water rinses 4 ~ 5 times, peels off endotesta and is inoculated on 1/2 MS solid medium of additional first exogeneous growth promotor.It sets
It is cultivated in culturing room.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 2 cm long of interception is inoculated in subculture medium
On MS, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in
In additional third exogeneous growth promotor root media 1/2MS.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.Transplanting time is mid-February.
Respectively hybridize the best materials period of rescue culture in step B are as follows: 48 d after ' southern exposure No.1 ' × ' amethyst ' pollination；' court
38 d after positive No.1 ' × ' short handle ' pollination.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.3 mg/L 6-BA, 0.3 mg/L IBA, 30 g/L sucrose, 5 g/ in step D
L agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step E
L agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, and intensity of illumination is 2 000
lx。
Embodiment 2
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: the natural flower at acquisition cherry ' depressed place Ge Jia ' small bell florescence.Peel anther indoors, 25
It dries in the shade under conditions of DEG C dry.After anther dehiscence loose powder, it is collected in vial and seals and protected in the refrigerator for be placed in 4 DEG C
It deposits spare.
It is female parent with sweet cherry ' southern exposure No.1 ', artificial not producing Crossbred is carried out to small bell phase flower.It is covered immediately after pollination
Draping bag after sulfuric acid paper bag and 7d.
B. fruit picking and cold stratification: ' southern exposure No.1 ' × ' depressed place Ge Jia ' rescue culture period of most preferably drawing materials is after pollination 37
D, fresh fruit are put into transparent plastic crisper, set and store 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile water
It rinses 4 ~ 5 times, peels off endotesta and be inoculated on 1/2 MS solid medium of additional first exogeneous growth promotor.It is placed in
Culturing room's culture.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 2 cm long of interception is inoculated in subculture medium
On MS, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in
In additional 1/2 MS of third exogeneous growth promotor root media.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.Transplanting time is mid-February.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.3 mg/L 6-BA, 0.4 mg/L IAA, 30g/L sucrose, 5 g/L in step D
Agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step E
L agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, and intensity of illumination is 2 000
lx。
Hardening off method in step F are as follows: 60 d of culture of rootage or so, the effective true leaves of selection 6 ~ 9,6, main root or more,
2.5 cm or more of height of seedling, base portion train seedling without the embryo of callus, robust growth, a little in sterile culture chamber opening hybrid seedling bottle cap,
It is gradually increased the degree of ventilation (uncap 1/5,1/3,1/2) of culture bottle every 5 d, (about corkage tempers 15 until completely removing bottle cap
D).Nutritive cube 3 d before transplanting are sterilized with 500 times of carbendazim solutions, are dried spare.Matrix is laid in pallet, carries out sunlight
Be exposed to the sun 12 h.Seedling is carefully taken out from culture bottle when transplanting, and the culture medium on root system is washed away under flowing water, and transplanting is equipped with matrix
Nutritive cube in, irrigate root water, overlay film, be placed in the small plastic canopy built；Periodically water spray keeps appropriate wet in canopy
Degree and temperature, pay attention to ventilated；Striping after 2 weeks, and guarantee ventilation and penetrating light, obtain sweet cherry and cherry hybrid plant.
Embodiment 3
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: the natural flower at acquisition cherry ' black pearl ' small bell florescence.Peel anther indoors, 25
It dries in the shade under conditions of DEG C dry.After anther dehiscence loose powder, it is collected in vial and seals and protected in the refrigerator for be placed in 4 DEG C
It deposits spare.
It is female parent with sweet cherry ' southern exposure No.1 ', small bell phase flower carries out artificial not producing Crossbred.Sulphur is covered after pollination immediately
Draping bag after sour paper bag and 7 d.
B. fruit picking and cold stratification: ' southern exposure No.1 ' × ' black pearl ' rescue culture period of most preferably drawing materials is after pollination 37
D, fresh fruit is put into transparent plastic crisper after fruit picking, sets and stores 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile
Water rinses 4 ~ 5 times, peels off endotesta and is inoculated on 1/2 MS solid medium of additional first exogeneous growth promotor.It is placed in
Culturing room's culture.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 2 cm long of interception is inoculated in subculture medium
On MS, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in
In additional 1/2 MS of third exogeneous growth promotor root media.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.Transplanting time is mid-February.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.7 mg/L 6-BA, 0.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step D
L agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/L in step E
Agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, and intensity of illumination is 2 000
lx。
Embodiment 4
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: the natural flower at acquisition cherry ' amethyst ' small bell florescence.Peel anther indoors, 25 DEG C
It dries in the shade under conditions of drying.After anther dehiscence loose powder, it is collected in vial and seals and saved in the refrigerator for be placed in 4 DEG C
It is spare.
It is female parent with sweet cherry ' Jiangnan is red ', small bell phase flower carries out artificial not producing Crossbred.Sulfuric acid is covered after pollination immediately
Draping bag after paper bag and 7 d.
B. fruit picking and cold stratification: as shown in Figure 1A, ' Jiangnan is red ' × ' amethyst ' the rescue culture most preferably period of materials is
31 d after pollination, fresh fruit is put into transparent plastic crisper after fruit picking, sets and stores 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile
Water rinses 4 ~ 5 times, peels off endotesta and is inoculated on 1/2 MS solid medium of additional first exogeneous growth promotor.It sets
It is cultivated in culturing room.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 2 cm long of interception is inoculated in subculture medium
On MS, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in
In additional 1/2 MS of third exogeneous growth promotor root media.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.3 mg/L 6-BA, 0.3 mg/L IBA, 30 g/L sucrose, 5 g/ in step D
L agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step E
L agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, and intensity of illumination is 2 000
lx。
Embodiment 5
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: the natural flower at acquisition cherry ' amethyst ' small bell florescence.Peel anther indoors, 25 DEG C
It dries in the shade under conditions of drying.After anther dehiscence loose powder, it is collected in vial and seals and saved in the refrigerator for be placed in 4 DEG C
It is spare.
It is female parent with sweet cherry ' Changfeng No.1 ', small bell phase flower carries out artificial not producing Crossbred.Sulphur is covered after pollination immediately
Draping bag after sour paper bag and 7 d.
B. fruit picking and cold stratification: as shown in Figure 1B, ' Changfeng No.1 ' × ' amethyst ' rescue culture is most preferably drawn materials period
For 41 d after pollination, fresh fruit is put into transparent plastic crisper after fruit picking, sets and stores 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile
Water rinses 4 ~ 5 times, peels off endotesta and is inoculated on the 1/2MS solid medium of additional first exogeneous growth promotor.It is placed in
Culturing room's culture.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, 2 cm long stem sections of interception are inoculated in subculture medium MS
On, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in
In additional 1/2 MS of third exogeneous growth promotor root media.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.3 mg/L 6-BA, 0.3 mg/L IBA, 30 g/L sucrose, 5 g/ in step D
L agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step E
L agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, intensity of illumination 2000
lx。
Embodiment 6
Southern area sweet cherry and cherry interspecific hybridization embryo rescue techniques, comprising the following steps:
A. pollen and pollination are collected: with cherry ' amethyst ' ' black pearl ' for male parent, the natural flower at acquisition small bell florescence.In room
Anther is inside peeled, is dried in the shade under conditions of 25 DEG C of dryings.After anther dehiscence loose powder, it is collected in vial and seals and be placed in 4
DEG C refrigerator in save backup.
It is female parent with sweet cherry ' pioneer ', small bell phase flower carries out artificial not producing Crossbred.Template is covered after pollination immediately
Bag and 7 d after draping bag.
B. fruit picking and cold stratification: as shown in figure iD, most preferably draw materials period in hybrid rescue culture (stone phase PF >
0.5) hybridization young fruit is adopted, fresh fruit is put into transparent plastic crisper after fruit picking, sets and stores 35 d in 4 DEG C of low temperature refrigerators.
C. rataria inoculation and sprouting culture: after low-temperature treatment, tap water rinses fruit well, peels off pulp and takes embryo.
In superclean bench with 70% alcohol rinse 1 time, aseptic water washing 3 times, then with 0.1% HgCl2Sterilize 3 min, sterile
Water rinses 4 ~ 5 times, peels off endotesta and is inoculated on 1/2 MS solid medium of additional first exogeneous growth promotor.It sets
It is cultivated in culturing room.
D. subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 2 cm long of interception is inoculated in subculture medium
On MS, the second exogeneous growth promotor is added in subculture medium MS.It is placed in culturing room and cultivates 40 d.
E. culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of the robust growth of 2 cm or more of plant height is inoculated with
In additional 1/2 MS of third exogeneous growth promotor root media.It is placed in culturing room and cultivates 40 d, obtain hybrid seedling.
F. acclimatization and transplants: by 15 d of hybrid seedling hardening or so, it is transplanted into nutritive cube.Transplanting time is mid-February.
Respectively hybridize the best materials period of rescue culture in step B are as follows: 46 d after ' pioneer ' × ' amethyst ' pollination；' pioneer ' ×
32 d after ' black pearl ' pollination.
The first exogeneous growth promotor is 2.0 mg/L 6-BA, 1.8 mg/L IAA, 30 g/L sucrose, 5 g/ in step C
L agar.
The second exogeneous growth promotor is 0.3 mg/L 6-BA, 0.3 mg/L IBA, 30 g/L sucrose, 5 g/ in step D
L agar.
Third exogeneous growth promotor is 0.3 mg/L IBA, 1.1 mg/L IAA, 30 g/L sucrose, 5 g/ in step E
L agar.
Step C, culturing room's condition of culture is 25 ~ 27 DEG C of temperature, 16 h/d of illumination in D, E, and intensity of illumination is 2 000
lx。
The above are the descriptions to the embodiment of the present invention to keep this field special by the foregoing description of the disclosed embodiments
Industry technical staff can be realized or using the present invention.Various modifications to these embodiments carry out those skilled in the art
Saying will be apparent.The general principles defined herein can be the case where not departing from the spirit or scope of the present invention
Under, it realizes in other embodiments.Therefore, the present invention is not intended to be limited to these implementation columns shown in this article, but to accord with
Close the widest scope consistent with principles disclosed herein and novel point.
Claims (9)
1. southern area sweet cherry and cherry interspecific hybridization rescue culture seedling establishment method, which is characterized in that this method include with
Lower step:
1) pollen and pollination are collected: with cherry (Prunus. pseudocerasusL.) it is male parent, acquires the small bell florescence
It is natural flower；Anther is peeled indoors, is dried in the shade under conditions of 20 ~ 30 DEG C of dryings；After anther dehiscence loose powder, it is collected in small
In vial and sealing is placed in 1 ~ 6 DEG C of refrigerator and saves backup；With gean (Prunus. aviumIt L. is) mother
This, small bell phase flower carries out artificial not producing Crossbred；Draping bag after sulfuric acid paper bag and 5 ~ 10 d is covered after pollination immediately；
2) fruit picking and cold stratification: picking is maternal long/kind long in stone phase PF > 0.5(embryo respectively) hybridization young fruit,
Fresh fruit is put into transparent plastic crisper after fruit picking, sets and stores 30 ~ 40 d in 1 ~ 6 DEG C of low temperature refrigerator；
3) it rataria inoculation and sprouting culture: after low-temperature treatment, rinses fruit well, peels off pulp and take embryo；In ultra-clean work
It carries out disinfection in platform；It peels off endotesta to be inoculated on the 1/2MS solid medium for promoting additive containing the first exogeneous growth, set
It is cultivated in culturing room；
4) subculture expands numerous culture: taking the sterile young sprout of the rataria of sprouting, the stem section of 1.5 ~ 2.5 cm long of interception is inoculated in after being commissioned to train
It supports on base MS, subculture medium MS is added the second exogeneous growth and promotes additive；It is placed in culturing room and cultivates 35 ~ 45 d；
5) culture of rootage: after hybrid seedling squamous subculture, the Multiple Buds young sprout of 2 cm or more robust growth of plant height is inoculated in additional
Third exogeneous growth promotes in additive root media 1/2MS；It is placed in culturing room and cultivates 35 ~ 45 d, obtain hybrid seedling；
6) acclimatization and transplants: by 10 ~ 20 d of hybrid seedling hardening, it is transplanted into nutritive cube；Transplanting time is mid-February.
2. the method according to claim 1, wherein male parent varieties are that cherry ' short handle ' ' is purple in step 1)
It is brilliant ' ' depressed place Ge Jia ' or ' black pearl ', parental varieties are sweet cherry ' Jiangnan is red ' ' southern exposure No.1 ' ' Changfeng No.1 ' or ' pioneer ', mother
This is selfed not affine.
3. the method according to claim 1, wherein in step 2 pollinate after cenospecies rescue culture best embryo age
Be 31 d after pollination to be maternal cross combination with ' Jiangnan is red ', using the cross combination that ' southern exposure No.1 ' be female parent as pollination after
It is 41 d after pollinating that 37 ~ 48 d, which are maternal cross combination with ' Changfeng No.1 ', is that maternal cross combination is with ' pioneer '
32 ~ 46 d after pollination.
4. the method according to claim 1, wherein it is 1.5 ~ 2.5 that the first exogeneous growth, which promotes additive,
Mg/L 6-BA, 1.5 ~ 2.0 mg/L IAA, 20 ~ 40 g/L sucrose and 2 ~ 8 g/L agar.
5. the method according to claim 1, wherein it is 0.3 ~ 0.7 that the second exogeneous growth, which promotes additive,
Mg/L 6-BA, 0.1 ~ 0.4 mg/L IAA or 0.1 ~ 0.5 mg/L IBA, 20 ~ 40g/L sucrose and 2 ~ 8 g/L fine jades
Rouge.
6. the method according to claim 1, wherein it is 0.2 ~ 0.5 that third exogeneous growth, which promotes additive,
Mg/L IBA, 1.0 ~ 1.5 mg/L IAA, 20 ~ 40 g/L sucrose and 2 ~ 8 g/L agar.
7. the method according to claim 1, wherein disinfection is with 70% alcohol rinse 1 time in step 3), sterile water
It rinses 3 times, then with 0.1% HgCl2Sterilize 3 min, and aseptic water washing 4 ~ 5 times.
8. the method according to claim 1, wherein step 3), 4) and 5) in culturing room's condition of culture be temperature
25 ~ 27 DEG C, 16 h/d of illumination, intensity of illumination is 2 000 lx.
9. the method according to claim 1, wherein the hardening off method in step 6) are as follows: after 60 d of culture of rootage,
Selection 6 ~ 9,6, main root or more effectively true leaf, 2.5 cm or more of height of seedling, base portion train seedling without the embryo of callus, robust growth,
It is a little in sterile culture chamber opening medium bottle lid, the degree of ventilation of culture bottle is gradually increased every 5 d；Nutritive cube is 3 before transplanting
D is sterilized with 500 times of carbendazim solutions, is dried spare；Matrix is laid in pallet, carries out 12 h of solar exposure；Seedling when transplanting
It is carefully taken out from culture bottle, the culture medium on root system is washed away under flowing water, transplanting irrigates equipped in the nutritive cube of matrix and determines root
Water, overlay film are placed in the small plastic canopy built；Periodically water spray keeps humidity and temperature appropriate in canopy, notices that ventilation is saturating
Gas；Striping after 2 weeks guarantees ventilation and penetrating light, obtains sweet cherry and cherry hybrid plant.
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| CN111480574A (en) * | 2020-04-25 | 2020-08-04 | 杭州市农业科学研究院 | Tissue culture method for rapid seedling formation of sweet cherry intraspecific hybridization F1 generation |
| CN111642399A (en) * | 2020-06-28 | 2020-09-11 | 禄劝普林生物科技有限责任公司 | Culture medium suitable for micro-somatic propagation of Chinese cherry and plum hybrid variety |
| CN111972074A (en) * | 2020-08-13 | 2020-11-24 | 河北省农林科学院昌黎果树研究所 | Seed treatment method for early-maturing sweet cherry variety and method for sowing seedlings in current year |
| CN111972074B (en) * | 2020-08-13 | 2022-04-22 | 河北省农林科学院昌黎果树研究所 | Seed treatment method for early-maturing sweet cherry variety and method for sowing seedlings in current year |
| CN113317202A (en) * | 2021-06-28 | 2021-08-31 | 大连大学 | Method for culturing crystal sugar crisp cherry embryos |
| CN115462312A (en) * | 2022-10-21 | 2022-12-13 | 湖南省植物园 | Method for obtaining distant hybridization progeny of oriental cherry through embryo rescue technology |
| CN115462312B (en) * | 2022-10-21 | 2023-09-26 | 湖南省植物园 | Method for obtaining oriental cherry distant hybridization offspring through embryo rescue technology |
| CN115623984A (en) * | 2022-11-02 | 2023-01-20 | 中国林业科学研究院经济林研究所 | Genome heterozygosity-based apricot plant distant hybridization high-affinity backbone parent selection method and cotyledon abortion hybrid embryo rescue method |
| CN115968785A (en) * | 2023-02-15 | 2023-04-18 | 四川农业大学 | Method for establishing Chinese cherry regeneration system |
| CN115968785B (en) * | 2023-02-15 | 2024-03-12 | 四川农业大学 | Method for establishing Chinese cherry regeneration system |
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