CN108782252A - A kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube - Google Patents
A kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube Download PDFInfo
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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
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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
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Abstract
The method that the present invention provides micrografting in a kind of fresh water Chinese pear tissue-cultured seedling test tube, including：Fiber differentiation step：Stem with bud is inoculated into culture on inducing culture for explant and forms in vitro cuttings；Shoot proliferation step：In vitro cuttings are transferred to the unrooted tissue-cultured seedling that proliferation acquisition is carried out on proliferated culture medium, using the unrooted tissue-cultured seedling of proliferation as the scion of micrografting；Stock incubation step：Birchleaf pear seed is inoculated into culture on seed germination medium and forms stock；Micrografting step：The base portion that scion is whittled into wedge shape is inserted into the notch of stock, obtains micrografting seedling；Acclimatization and transplants step：Transplanting after grafting hardening is cultivated after transplanting in plastic canopy in cultivation matrix.Present invention combination tissue cultures and graft technology carry out micrografting using the tissue-cultured seedling and the birchleaf pear seedling that grows directly from seeds of proliferation, eliminate the Induction Process of tissue culture seedling rooting, simplify the program of nursery, shorten seedling raise period, improve the anti-adversity ability and high yield characteristic of nursery stock.
Description
Technical field
The present invention relates to Chinese pear seedling propagating technology fields, and in particular to micrografting in a kind of fresh water Chinese pear tissue-cultured seedling test tube
Method.
Background technology
Fresh water Chinese pear is the famous Chinese pear being named from the places where the products are manufactured, and main product endures general in Guangdong Huizhou city Huiyang District fresh water, i.e. leaf
Native place, fruit storage tolerance, the bright profit of color and luster, pulp is sharp and clear, succulence, is favored by people Hongkong and Macro and south east asia are deep,
Once the south of the Five Ridges is enjoyed great prestige.Fresh water Chinese pear is with development and local lichee, longan of the Huiyang District manufacture with processing industry after reform and opening-up
The popularizing planting of equal fruit trees, fresh water sand pear cultivation area are greatly reduced, and fresh water Chinese pear industry gradually declines.Fresh water Chinese pear produces at present
Industry have " not depositing " saying (Huiyang Zhao Fei, Ni Genjin, Zhang Jiaen fresh water sand pear cultivation history examine states agriculturals archaeology, 2013,
(3):158-165), fresh water Chinese pear germplasm causes anxiety.It is high to adapt to south as the peculiar industrial crops in place for fresh water Chinese pear
Warm high humidity weather has unique and apparent germplasm advantage, to promote the development of fresh water Chinese pear industry and preserving to have place special
The germ plasm resource of color, inventor herein, which once enables reaching, has studied the side that fresh water sand pear is quickly bred by method for tissue culture
Method, it was found that be suitble to the Initial culture of fresh water Chinese pear, the method for squamous subculture and rooting induction and corresponding culture medium prescription.It is light
To improve the anti-adversity ability and high yield characteristic of fresh water Chinese pear in the production of water sand pears traditional cultivation, with the excellent maternal plant of fresh water Chinese pear
Branch does scion, does stock using local birchleaf pear seedling and is grafted and cultivates excellent grafting.Pass through method for tissue culture
Though the fresh water Chinese pear nursery stock quickly bred has the excellent characteristic of fresh water Chinese pear maternal plant, and has the whole series identical with excellent maternal plant
Hereditary information, but the tissue-cultured seedling directly formed by test tube rooting of vitro seedling do not have birchleaf pear grow directly from seeds root system namely pears seedling does not have
There is the root system for adapting to local soil environment, without anti-adversity ability possessed by tradition grafting and high yield basis.In addition, Chinese pear is
Xylophyta difficult to take root directly promotees to be proliferated rooting of vitro seedling using tissue cultures, because of the influence of the physiological status of seedling test tube,
Rooting rate is not high and has wild effect.
Invention content
The purpose of the present invention is to provide a kind of survival rate height, can quickly breed the strong fresh water Chinese pear tissue-cultured seedling of resistance
Micro-grafting method.To realize the above-mentioned technical purpose, the present invention uses technical solution below.
The method that the present invention provides micrografting in a kind of fresh water Chinese pear tissue-cultured seedling test tube, including：
Fiber differentiation step：The mercuric chloride solution that fresh water Chinese pear explant is placed in 0.1%-0.3% sterilizes 10-16 minutes,
Then aseptic water washing 5-6 times is used, after aseptic filter paper suck dry moisture, is cut into 1-3 terminal bud and/or lateral bud with vaccinating lancet
The stem with bud is that explant is inoculated into and has sterilized and on the inducing culture that solidifies by the stem with bud of a length of 0.5-2cm,
By inducing culture elder generation light culture 5-10 days after inoculation, then induce under the conditions of optical culture the terminal bud or lateral bud of explant stem section
It sprouts, forms in vitro cuttings；
Shoot proliferation step：Aseptically the in vitro cuttings of a height of 2-4cm of Fiber differentiation are cut with vaccinating lancet
Under be transferred to and sterilized and rised in value on the proliferated culture medium that solidifies, and carry out optical culture 30-40 days, promote in vitro cuttings
Lateral bud or adventitious bud sprout the unrooted tissue-cultured seedling for obtaining and growing thickly, and with vaccinating lancet cutting are aseptically single by unrooted tissue-cultured seedling
Strain tissue-cultured seedling is chosen the single plant tissue-cultured seedling higher than 1.5cm and is transferred on proliferated culture medium, and multiple squamous subculture obtains unrooted tissue culture
Seedling, scion of the unrooted tissue-cultured seedling as micrografting after squamous subculture 30-40 days；
Stock incubation step：The birchleaf pear seed that Aging storage is crossed is rinsed 30-60 minutes with slow flowing water, is used after drying
70% alcohol treatment 30s is drained after aseptic water washing 3 times, is sterilized 4-8 minutes with 0.1%-0.3% mercuric chloride solutions, disinfection terminates
With rinsed with sterile water 5-6 times, it is then seeded into and has sterilized and on the seed germination medium that solidifies, the seed after inoculation is sprouted
Then culture medium elder generation light culture 5-10 days induces seed to sprout under the conditions of optical culture, form sterile birchleaf pear seedling, birchleaf pear children
Stock when seedling grows into second true leaf formation as micrografting；
Micrografting step in test tube：Tinfoil is cut into the bar shaped of 0.5cm × 1.5cm, it is spare after high pressure sterilization；Sterile
Under the conditions of, stock is taken out by its truncation, stays the stem of 3-4cm long, bottom lateral bud and blade are extractd with tweezers, along stock stem section top
The longitudinal sectional formation length in portion is the notch of 0.4-0.6cm；Height is chosen as scion similar in 2-5cm, rugosity and stock stem section, is being connect
2-6 piece leaves are stayed at the top of fringe, and its base portion is whittled into the wedge structure that length is 0.4-0.6cm, by the wedge base of scion
It is inserted into the notch of rootstock seedling stem section, keeps notch closely connected, the edge alignment on stock and scion at least one side will be grafted with tinfoil
Mouth bondage, obtains micrografting seedling；And micrografting seedling is put into the test tube for filling fluid nutrient medium for having sterilized and being cooled to room temperature
In, the 1/4 of Liquid Culture fiduciary point test tube volume, Liquid Culture primary surface, which is put into tinfoil and so that liquid with fixing grafting seedling, does not transfer
Micrografting seedling is carried out optical culture and is promoted graft union healing and seedling by the rhizome position of seedlings picking with sterile ParafilmTM test tube mouth
Growth；
Acclimatization and transplants step：By grafting after optical culture 15 days, when stock root starts to grow, and scion leaf is in just
Chang Yese simultaneously has test tube is placed in hardening 10-15 days in natural light when young leaves growth, then after removing sealed membrane hardening 1 day, transplanting
In volume ratio be 2:In 1 peat and the cultivation matrix of perlite mixture, protected in closed plastic canopy inside holding after transplanting
Wet culture, gradually ventilation after a week, until removing plastic canopy.
Further, further include explant pre-treatment step before the Fiber differentiation step, outside the fresh water Chinese pear
Implant is formed by fresh water Chinese pear young sprout after explant pre-treatment step, and the explant pre-treatment step is：By the length of acquisition
It rinses surface irregularities well for the fresh water Chinese pear young sprout of 5-10cm and is placed in the washing powder solution of saturation and impregnate 5-10 minutes, change
Water-removing rinsing, which is placed on for 3-5 times in flowing tap water, to be rinsed 5-12 hours, finally is sealed to install with sealed bag and is placed in 4-10 DEG C of ice
It is spare in case.
Further, in Fiber differentiation step, the stem with bud is the stem of single bud of the band comprising terminal bud or lateral bud
Section, band comprising terminal bud and lateral bud or be lateral bud two buds three buds of stem section or band comprising terminal bud and lateral bud stem
Section.
Further, the Aging storage in stock incubation step is：Acquisition no disease and pests harm, development are good when birchleaf pear fruit maturation
Good fruit is put in outdoor stacking and macerates rotten, the height 20-30cm of stacking, makes the damage seed that do not generate heat at fruit accumulation center, etc.
Fruit it is soft it is ripe after, mash fruit, clean seed is set shady and cool ventilation by the floater that washing removes rotten pulp and swims
Loading plastic seal pocket is put into spare after 2-4 DEG C of refrigerator preserves 30 days after shade is dry.
Further, the light culture is the culture of cultivation temperature no light under conditions of 23-27 DEG C；The optical culture
Daily illumination 12-16 hours, the culture of intensity of illumination 1500-2100lx under conditions of 23-27 DEG C for cultivation temperature.
Further, the step of sterilizing is in 1.1-1.5kgf/cm2With under the conditions of 121 DEG C of high pressure-temperature in steam copper
Interior disinfection 18-20 minutes.
Further, the inducing culture described in Fiber differentiation step is：Using MS as minimal medium, 0.5- is added
1.0mg/L 6- benzyls aminoadenine/6-BA, 0.08-0.15mg/L heteroauxin/IBA, additional 2.5-3.0% sucrose and 0.4-
0.7% agar, pH value 5.60-5.80；
Proliferated culture medium described in shoot proliferation step is：Using MS as minimal medium, 2.0-4.0mg/L 6- are added
Benzyl aminoadenine/6-BA, 0.04-0.08mg/L heteroauxin/IBA adds 2.5-3.0% sucrose and 0.4-0.7% agar,
PH value is 5.60-5.80；
Seed germination medium described in stock incubation step is：Using MS as minimal medium, 0.1-0.5mg/L is added
6- benzyls aminoadenine/6-BA, 0.08-0.15mg/L heteroauxin/IBA adds 2.5-3.0% sucrose and 0.4-0.7% fine jades
Fat, pH value 5.60-5.80；
Fluid nutrient medium in test tube described in micrografting step is：Separately add 0.3- by minimal medium of 1/2MS
0.7mg/L heteroauxins/IBA and 2.0-2.5% sucrose, pH value 5.60-5.80.1/2MS is a large amount of in MS culture medium prescriptions
Element halves, other elements it is constant and prepare culture medium.
Further, the MS culture medium prescriptions are made of following each compound, are divided into a great number of elements, micro member
Element, four substance of molysite and organic matter：
A great number of elements contains potassium nitrate/KNO31900mg/L, ammonium nitrate/NH4NO31650mg/L, calcium chloride/CaCl2·
2H2O 440mg/L, magnesium sulfate/MgSO4·7H2O 370mg/L, potassium dihydrogen phosphate/KH2PO4170mg/L；
Trace element contains potassium iodide/KI 0.83mg/L, boric acid/H3BO36.2mg/L, manganese sulfate/Mn SO4·4H2O
22.3mg/L, zinc sulfate/Zn SO4·7H2O 8.6mg/L, sodium molybdate/Na2MoO4·2H2O 0.25mg/L, copper sulphate/Cu
SO4·5H2O 0.025mg/L, cobalt chloride/CoCl2·6H2O 0.025mg/L；
Molysite sulfur acid ferrous iron/Fe SO4·7H2O 27.8mg/L, disodium ethylene diamine tetraacetate/Na2-EDTA·2H2O
37.3mg/L；
Organic substance contains：Inositol 100mg/L, niacin 0.5mg/L, puridoxine hydrochloride/vitamin B60.5mg/L, hydrochloric acid sulphur
Amine element/vitamin B10.1mg/L, glycine 2.0mg/L.
The method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube provided by the invention has the advantages that very prominent, explanation
It is as follows：
1. the method that the present invention induces fresh water Chinese pear terminal bud, lateral bud etc. by tissue cultures, obtains in short period of time
A large amount of scion simultaneously carry out micrografting, can get a large amount of grafting, establish fresh water Chinese pear tissue culture micrografting reproduction technique system,
A new way is provided for the breeding of fresh water Chinese pear；
2. the stock of micrografting and the non-lignifying of scion or ability semi-lignified in fresh water Chinese pear test tube are easy to generate callus,
Graft union is easy to heal, and micrografting carries out under sterile environment, avoid bacterium and virus infection, be conducive to grafting at
Living, survival rate is up to 90% or more；
3. the method micrografting nursery using the present invention can be obtained intact plant in 30 days or so, really realize efficiently fast
Speed；The method not only eliminates the process of rooting induction, and also growth is rapid, and the grafting cultivated can preserve fresh water Chinese pear
Merit, also with birchleaf pear to the adaptability and resistance of soil；
4. due to the operations such as stock in the method for the present invention, the acquisition of scion and micrografting, culture and acclimatization and transplants be
It realizes, therefore is not limited by environmental factors such as season, weather and temperature under artificial control, it can whole year production；
Can also be the guarantor of germ plasm resource 5. the micrografting of fresh water Chinese pear cannot be only used for nursery stock quickly breeding and breed improvement
Deposit, the quick detection of plant virus, fruit tree quarantine, the identification of grafting affinity and affinity numerous technical courses such as early prediction
Topic lays the first stone.
The beneficial effects of the present invention are：In conjunction with tissue cultures and graft technology, using proliferation test tube seedling and grow directly from seeds
Wild birchleaf pear seedling carries out micrografting, eliminates the Induction Process of rooting of vitro seedling, the program of nursery is simplified, when shortening nursery
Between, it is also fully utilized by the characteristic that stock adapts to local soil environment, improves the anti-adversity ability and high yield characteristic of nursery stock.
Specific implementation mode
In the following detailed description, some exemplary embodiments of the present invention are described by way of explanation.It retouches
It states and is regarded as illustrative in nature, be not intended to limit the scope of the claims.
Embodiment 1
The method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube provided in this embodiment, including：
Explant pre-treatment step：The tender young sprout of fresh water Chinese pear children for acquiring a length of 5-10cm in the wild, removes spire at once
After be put into set in ice chest in sealed bag and take back, rinsing the surface irregularities such as dirt, dust, silt well with tap water is placed on saturation
Washing powder solution in impregnate 5 minutes, then washing powder water is rinsed with tap water and changes water 3 times, then releases dynamic tap water punching 5
Hour, finally with sealed bag sealing install be placed in it is spare in 4 DEG C of refrigerators.
Fiber differentiation step：Fresh water Chinese pear explant is placed in 0.1% mercuric chloride solution and is sterilized 16 minutes, nothing is then used
Bacterium water rinses 5 times, after aseptic filter paper suck dry moisture, be cut into terminal bud with vaccinating lancet and lateral bud be simple bud a length of 0.5-1cm
Stem section be inoculated on inducing culture rapidly, full light culture under the conditions of 23 DEG C, culture went under light and cultivates after 5 days, light intensity
For 1500lx, daily illumination induces the terminal bud of explant stem section or lateral bud to sprout to adventitious bud is grown in 12 hours, forms sterile examination
Guan Miao；
The inducing culture used for:MS+6-BA 0.5mg/L+IBA 0.08mg/L+ sucrose 2.5%+ agar 0.4%,
PH value is 5.60.
The formula of MS is made of following each compound, can be divided into four class such as a great number of elements, trace element, molysite and organic matter
Substance.The MS formulas of other steps are identical as the MS formulas of this step in embodiment.A great number of elements contains potassium nitrate/KNO3
1900mg/L, ammonium nitrate/NH4NO31650mg/L, calcium chloride/CaCl2·2H2O 440mg/L, magnesium sulfate MgSO4·7H2O
370mg/L, potassium dihydrogen phosphate/KH2PO4170mg/L.Trace element contains potassium iodide/KI 0.83mg/L, boric acid/H3BO3
6.2mg/L, manganese sulfate/Mn SO4·4H2O 22.3mg/L, zinc sulfate/Zn SO4·7H2O 8.6mg/L, sodium molybdate/
Na2MoO4·2H2O 0.25mg/L, copper sulphate/Cu SO4·5H2O 0.025mg/L, cobalt chloride/CoCl2·6H2O
0.025mg/L.Molysite sulfur acid ferrous iron/Fe SO4·7H2O 27.8mg/L, disodium ethylene diamine tetraacetate/Na2-EDTA·2H2O
37.3mg/L.Organic substance contains：Inositol 100mg/L, niacin 0.5mg/L, puridoxine hydrochloride/vitamin B60.5mg/L, hydrochloric acid
Thiamine/vitamin B10.1mg/L, glycine 2.0mg/L.
Shoot proliferation step：Aseptically the in vitro cuttings of the high 2-4cm of Fiber differentiation are solved with vaccinating lancet
Cut open cutter cuts to be transferred to and sterilize and on the proliferated culture medium that solidifies, then the proliferated culture medium after inoculation is carried out optical culture 30
It, the lateral bud or adventitious bud for promoting in vitro cuttings sprout the unrooted tissue-cultured seedling to be formed and be grown thickly, by unrooted tissue-cultured seedling in aseptic condition
Lower with vaccinating lancet cutting is single plant tissue-cultured seedling, chooses the single plant tissue-cultured seedling higher than 1.5cm and is transferred on proliferated culture medium, through subculture
Scion of the unrooted tissue-cultured seedling that culture obtains after 30 days as micrografting；
The proliferated culture medium used for:MS+6-BA 2.0mg/L+IBA 0.04mg/L+ sucrose 2.5%+ agar 0.4%,
PH value is 5.60.
Aging storage step：By the end of September or acquisition no disease and pests harm when birchleaf pear fruit maturation in October, well-developed fruit
It is put in outdoor stacking and macerates rotten, the height 20-30cm of stacking, make the damage seed that do not generate heat at the centers Guo Dui.Equal fruits it is soft it is ripe after, smash
Decayed fruit is real, the floater that washing removes rotten pulp and swims, and clean seed is set to be packed into after shady and cool ventilation shade is done and is moulded
Material sealed bag is put into spare after 2-4 DEG C of refrigerator preserves 30 days.
Stock incubation step：The birchleaf pear seed that Aging storage is crossed is rinsed 30 minutes before inoculated and cultured with slow flowing water,
With 70% alcohol treatment 30s after drying, drains, sterilized 8 minutes with 0.1% mercuric chloride solution, disinfection terminates after aseptic water washing 3 times
With rinsed with sterile water 5 times, it is then seeded into and has sterilized and on the seed germination medium that solidifies.Seed with seed sprouts culture
Then base elder generation light culture 5 days induces seed to sprout, forms sterile birchleaf pear test tube seedling, growth of seedling under the conditions of optical culture
It can be used as the stock of micrografting when second true leaf formation.
The seed germination medium used for：MS+6-BA 0.1mg/L+IBA 0.08mg/L+ sucrose 2.5%+ agar
0.4%, pH value 5.60.
Micrografting step in test tube：Tinfoil is cut into the bar shaped of 0.5cm × 1.5cm, it is spare after high pressure sterilization；Sterile
Under the conditions of, stock is taken out by its truncation, stays the stem of 3-4cm long, bottom lateral bud and blade are extractd with tweezers, along stock stem section top
The longitudinal sectional formation length in portion is the notch of 0.4-0.6cm；Height is chosen as scion similar in 2-5cm, rugosity and stock stem section, is being connect
2 leaves are stayed at the top of fringe, and its base portion is whittled into the wedge structure that length is 0.4-0.6cm, and the wedge base of scion is inserted
In the notch for entering rootstock seedling stem section, keep notch closely connected, the edge on stock and scion at least one side alignment, with tinfoil by graft union
Bondage obtains micrografting seedling；Grafting is put into and has been sterilized and in the test tube for filling fluid nutrient medium that is cooled to room temperature, liquid
The 1/4 of fiduciary point test tube volume is cultivated, Liquid Culture primary surface is put into tinfoil makes liquid not have grafting with fixing grafting seedling
Grafting is carried out optical culture and is promoted graft union healing and growth of seedling by rhizome position with sterile ParafilmTM test tube mouth；
The fluid nutrient medium used for：MS+IBA 0.3mg/L+ sucrose 2.5%+ agar 0.4%, pH value 5.60.
Acclimatization and transplants step：Grafting is after optical culture 15 days, and when stock root starts to grow, and scion leaf is in normal
Leaf color and after having test tube being placed in natural light hardening 10 days when young leaves growth, and sealed membrane hardening is removed after 1 day, it transplants in body
Product is than being 2:In 1 peat and the cultivation matrix of perlite mixture, cultivation matrix is mixed evenly with carbendazim solution.It moves
Carbendazim solution is sprayed after cultivation, pre- disease prevention occurs, gradually logical after a week in closed plastic canopy inside holding moisturizing culture
Wind, until removing plastic canopy.
In the present embodiment, the light culture is the culture of cultivation temperature no light under conditions of 23 DEG C；The light training
Support daily illumination 12 hours, the culture of intensity of illumination 1500lx under conditions of 23 DEG C for cultivation temperature.
In the present embodiment, the step of sterilizing is in 1.1-1.5kgf/cm2It is being steamed under the conditions of 121 DEG C of high pressure-temperature
Disinfection 18 minutes in steam-boiler.
Embodiment 2
The method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube provided in this embodiment, including：
Explant pre-treatment step：The tender young sprout of fresh water Chinese pear children for acquiring a length of 5-10cm in the wild, removes spire at once
After be put into set in ice chest in sealed bag and take back, rinsing the surface irregularities such as dirt, dust, silt well with tap water is placed on saturation
Washing powder solution in impregnate 7.5 minutes, then washing powder water is rinsed with tap water and changes water 4 times, then releases dynamic tap water
Punching 8.5 hours, finally with sealed bag sealing install be placed in it is spare in 7 DEG C of refrigerators.
Fiber differentiation step：Fresh water Chinese pear explant is placed in 0.2% mercuric chloride solution and is sterilized 13 minutes, nothing is then used
Bacterium water rinse 5 times, after aseptic filter paper suck dry moisture, be cut into terminal bud and lateral bud with vaccinating lancet or be all lateral bud containing 2 buds it is a length of
The stem section of 0.5-1.5cm is inoculated on inducing culture rapidly, full light culture under the conditions of 25 DEG C, and culture is gone to after 5 days under light
Culture, light intensity 1800lx, the terminal bud or lateral bud of the induction explant stem section of daily illumination 14 hours are sprouted to growing adventitious bud, shape
At in vitro cuttings；
The inducing culture used for:MS+6-BA 0.75mg/L+IBA 0.12mg/L+ sucrose 2.5%+ agar
0.55%, pH value 5.70.
The formula of MS is made of following each compound, can be divided into four class such as a great number of elements, trace element, molysite and organic matter
Substance.The MS formulas of other steps are identical as the MS formulas of this step in embodiment.A great number of elements contains potassium nitrate/KNO3
1900mg/L, ammonium nitrate/NH4NO31650mg/L, calcium chloride/CaCl2·2H2O 440mg/L, magnesium sulfate MgSO4·7H2O
370mg/L, potassium dihydrogen phosphate/KH2PO4170mg/L.Trace element contains potassium iodide/KI 0.83mg/L, boric acid/H3BO3
6.2mg/L, manganese sulfate/Mn SO4·4H2O 22.3mg/L, zinc sulfate/Zn SO4·7H2O 8.6mg/L, sodium molybdate/
Na2MoO4·2H2O 0.25mg/L, copper sulphate/Cu SO4·5H2O 0.025mg/L, cobalt chloride/CoCl2·6H2O
0.025mg/L.Molysite sulfur acid ferrous iron/Fe SO4·7H2O 27.8mg/L, disodium ethylene diamine tetraacetate/Na2-EDTA·2H2O
37.3mg/L.Organic substance contains：Inositol 100mg/L, niacin 0.5mg/L, puridoxine hydrochloride/vitamin B60.5mg/L, hydrochloric acid
Thiamine/vitamin B10.1mg/L, glycine 2.0mg/L.
Shoot proliferation step：Aseptically the in vitro cuttings of the high 2-4cm of Fiber differentiation are solved with vaccinating lancet
Cut open cutter cuts to be transferred to and sterilize and on the proliferated culture medium that solidifies, and carries out optical culture 35 days, promotees the lateral bud of in vitro cuttings
Or adventitious bud sprouts the unrooted tissue-cultured seedling to be formed and be grown thickly, and with vaccinating lancet cutting is aseptically single plant group by unrooted tissue-cultured seedling
Seedling is trained, chooses and is transferred on proliferated culture medium higher than the single plant tissue-cultured seedling of 1.5cm, unrooted tissue culture is obtained after 35 days through squamous subculture
Scion of the seedling as micrografting；
The proliferated culture medium used for:MS+6-BA 3.0mg/L+IBA 0.06mg/L+ sucrose 2.75%+ agar
0.55%, pH value 5.70.
Aging storage step：By the end of September or acquisition no disease and pests harm when birchleaf pear fruit maturation in October, well-developed fruit
It is put in outdoor stacking and macerates rotten, the height 20-30cm of stacking, make the damage seed that do not generate heat at the centers Guo Dui.Equal fruits it is soft it is ripe after, smash
Decayed fruit is real, the floater that washing removes rotten pulp and swims, and clean seed is set to be packed into after shady and cool ventilation shade is done and is moulded
Material sealed bag is put into spare after 2-4 DEG C of refrigerator preserves 30 days.
Stock incubation step：The birchleaf pear seed that Aging storage is crossed is rinsed 45 minutes before inoculated and cultured with slow flowing water,
With 70% alcohol treatment 30s after drying, drains, sterilized 6 minutes with 0.2% mercuric chloride solution, disinfection terminates after aseptic water washing 3 times
With rinsed with sterile water 5 times, it is then seeded into and has sterilized and on the seed germination medium that solidifies.Seed with seed sprouts culture
Then base elder generation light culture 8 days induces seed to sprout, forms sterile birchleaf pear test tube seedling, growth of seedling under the conditions of optical culture
It can be used as the stock of micrografting when second true leaf formation.
The seed germination medium used for：MS+6-BA 0.25mg/L+IBA 0.12mg/L+ sucrose 2.75%+ agar
0.55%, pH value 5.70.
Micrografting step in test tube：Tinfoil is cut into the bar shaped of 0.5cm × 1.5cm, it is spare after high pressure sterilization；Sterile
Under the conditions of, stock is taken out by its truncation, stays the stem of 3-4cm long, bottom lateral bud and blade are extractd with tweezers, along stock stem section top
The longitudinal sectional formation length in portion is the notch of 0.4-0.6cm；Height is chosen as scion similar in 2-5cm, rugosity and stock stem section, is being connect
4 leaves are stayed at the top of fringe, and its base portion is whittled into the wedge structure that length is 0.4-0.6cm, and the wedge base of scion is inserted
In the notch for entering rootstock seedling stem section, keep notch closely connected, the edge on stock and scion at least one side alignment, with tinfoil by graft union
Bondage obtains micrografting seedling；Grafting is put into and has been sterilized and in the test tube for filling fluid nutrient medium that is cooled to room temperature, liquid
The 1/4 of fiduciary point test tube volume is cultivated, Liquid Culture primary surface is put into tinfoil makes liquid not have grafting with fixing grafting seedling
Grafting is carried out optical culture and is promoted graft union healing and growth of seedling by rhizome position with sterile ParafilmTM test tube mouth；
The fluid nutrient medium used for：MS+IBA 0.5mg/L+ sucrose 2.2%, pH value 5.70.
Acclimatization and transplants step：Grafting is after optical culture 15 days, and when stock root starts to grow, and scion leaf is in normal
Leaf color and after having test tube being placed in natural light hardening 12 days when young leaves growth, and sealed membrane hardening is removed after 1 day, it transplants in body
Product is than being 2:In 1 peat and the cultivation matrix of perlite mixture, cultivation matrix is mixed evenly with carbendazim solution.It moves
Carbendazim solution is sprayed after cultivation, pre- disease prevention occurs, gradually logical after a week in closed plastic canopy inside holding moisturizing culture
Wind, until removing plastic canopy.
In the present embodiment, the light culture is the culture of cultivation temperature no light under conditions of 25 DEG C；The light training
Support daily illumination 14 hours, the culture of intensity of illumination 1800lx under conditions of 25 DEG C for cultivation temperature.
In the present embodiment, the step of sterilizing is in 1.1-1.5kgf/cm2It is being steamed under the conditions of 121 DEG C of high pressure-temperature
Disinfection 19 minutes in steam-boiler.
Embodiment 3
The method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube provided in this embodiment, including：
Explant pre-treatment step：The tender young sprout of fresh water Chinese pear children for acquiring a length of 5-10cm in the wild, removes spire at once
After be put into set in ice chest in sealed bag and take back, rinsing the surface irregularities such as dirt, dust, silt well with tap water is placed on saturation
Washing powder solution in impregnate 10 minutes, then washing powder water is rinsed with tap water and changes water 6 times, then releases the punching of dynamic tap water
12 hours, finally with sealed bag sealing install be placed in it is spare in 10 DEG C of refrigerators.
Fiber differentiation step：Fresh water Chinese pear explant is placed in 0.3% mercuric chloride solution and is sterilized 10 minutes, nothing is then used
Bacterium water rinses 6 times, after aseptic filter paper suck dry moisture, is cut into a length of 10-2cm's that terminal bud and lateral bud are 3 buds with vaccinating lancet
Stem section is inoculated on inducing culture rapidly, full light culture under the conditions of 27 DEG C, and culture goes under light after 10 days and cultivates, and light intensity is
2000lx, daily illumination induce the terminal bud of explant stem section or lateral bud to sprout to adventitious bud is grown for 16 hours, form sterile test tube
Seedling；
The inducing culture used for:MS+6-BA 1.0mg/L+IBA 0.15mg/L+ sucrose 3.0%+ agar 0.7%,
PH value is 5.80.
The formula of MS is made of following each compound, can be divided into four class such as a great number of elements, trace element, molysite and organic matter
Substance.The MS formulas of other steps are identical as the MS formulas of this step in embodiment.A great number of elements contains potassium nitrate/KNO3
1900mg/L, ammonium nitrate/NH4NO31650mg/L, calcium chloride/CaCl2·2H2O 440mg/L, magnesium sulfate MgSO4·7H2O
370mg/L, potassium dihydrogen phosphate/KH2PO4170mg/L.Trace element contains potassium iodide/KI 0.83mg/L, boric acid/H3BO3
6.2mg/L, manganese sulfate/Mn SO4·4H2O 22.3mg/L, zinc sulfate/Zn SO4·7H2O 8.6mg/L, sodium molybdate/
Na2MoO4·2H2O 0.25mg/L, copper sulphate/Cu SO4·5H2O 0.025mg/L, cobalt chloride/CoCl2·6H2O
0.025mg/L.Molysite sulfur acid ferrous iron/Fe SO4·7H2O 27.8mg/L, disodium ethylene diamine tetraacetate/Na2-EDTA·2H2O
37.3mg/L.Organic substance contains：Inositol 100mg/L, niacin 0.5mg/L, puridoxine hydrochloride/vitamin B60.5mg/L, hydrochloric acid
Thiamine/vitamin B10.1mg/L, glycine 2.0mg/L.
Shoot proliferation step：Aseptically the in vitro cuttings of the high 2-4cm of Fiber differentiation are solved with vaccinating lancet
Cut open cutter cuts to be transferred to and sterilize and on the proliferated culture medium that solidifies, and carries out optical culture 40 days, promotees the lateral bud of in vitro cuttings
Or adventitious bud sprouts the unrooted tissue-cultured seedling to be formed and be grown thickly, and with vaccinating lancet cutting is aseptically single plant group by unrooted tissue-cultured seedling
Seedling is trained, chooses and is transferred on proliferated culture medium higher than the single plant tissue-cultured seedling of 1.5cm, unrooted tissue culture is obtained after 40 days through squamous subculture
Seedling is as scion；
The proliferated culture medium used for:MS+6-BA 4.0mg/L+IBA 0.08mg/L+ sucrose 3.0%+ agar 0.7%,
PH value is 5.80.
Aging storage step：By the end of September or acquisition no disease and pests harm when birchleaf pear fruit maturation in October, well-developed fruit
It is put in outdoor stacking and macerates rotten, the height 20-30cm of stacking, make the damage seed that do not generate heat at the centers Guo Dui.Equal fruits it is soft it is ripe after, smash
Decayed fruit is real, the floater that washing removes rotten pulp and swims, and clean seed is set to be packed into after shady and cool ventilation shade is done and is moulded
Material sealed bag is put into spare after 2-4 DEG C of refrigerator preserves 30 days.
Stock incubation step：The birchleaf pear seed that Aging storage is crossed is rinsed 60 minutes before inoculated and cultured with slow flowing water,
With 70% alcohol treatment 30s after drying, drains, sterilized 4 minutes with 0.3% mercuric chloride solution, disinfection terminates after aseptic water washing 3 times
With rinsed with sterile water 6 times, it is then seeded into and has sterilized and on the seed germination medium that solidifies.Seed with seed sprouts culture
Then base elder generation light culture 10 days induces seed to sprout, forms sterile birchleaf pear test tube seedling, growth of seedling under the conditions of optical culture
It can be used as the stock of grafting when second true leaf formation.
The seed germination medium used for：MS+6-BA 0.5mg/L+IBA 0.15mg/L+ sucrose 3.0%+ agar
0.7%, pH value 5.80.
Micrografting step in test tube：Tinfoil is cut into the bar shaped of 0.5cm × 1.5cm, it is spare after high pressure sterilization；Sterile
Under the conditions of, stock is taken out by its truncation, stays the stem of 3-4cm long, bottom lateral bud and blade are extractd with tweezers, along stock stem section top
The longitudinal sectional formation length in portion is the notch of 0.4-0.6cm；Height is chosen as scion similar in 2-5cm, rugosity and stock stem section, is being connect
6 leaves are stayed at the top of fringe, and its base portion is whittled into the wedge structure that length is 0.4-0.6cm, and the wedge base of scion is inserted
In the notch for entering rootstock seedling stem section, keep notch closely connected, the edge on stock and scion at least one side alignment, with tinfoil by graft union
Bondage obtains micrografting seedling；Grafting is put into and has been sterilized and in the test tube for filling fluid nutrient medium that is cooled to room temperature, liquid
The 1/4 of fiduciary point test tube volume is cultivated, Liquid Culture primary surface is put into tinfoil makes liquid not have grafting with fixing grafting seedling
Grafting is carried out optical culture and is promoted graft union healing and growth of seedling by rhizome position with sterile ParafilmTM test tube mouth；
The fluid nutrient medium used for：MS+IBA0.7mg/L+ sucrose 2.5%, pH value 5.80.
Acclimatization and transplants step：Grafting is after optical culture 15 days, and when stock root starts to grow, and scion leaf is in normal
Leaf color and after having test tube being placed in natural light hardening 12 days when young leaves growth, and sealed membrane hardening is removed after 1 day, it transplants in body
Product is than being 2:In 1 peat and the cultivation matrix of perlite mixture, cultivation matrix is mixed evenly with carbendazim solution.It moves
Carbendazim solution is sprayed after cultivation, pre- disease prevention occurs, gradually logical after a week in closed plastic canopy inside holding moisturizing culture
Wind, until removing plastic canopy.
In the present embodiment, the light culture is the culture of cultivation temperature no light under conditions of 27 DEG C；The light training
Support daily illumination 16 hours, the culture of intensity of illumination 2100lx under conditions of 27 DEG C for cultivation temperature.
In the present embodiment, the step of sterilizing is in 1.1-1.5kgf/cm2It is being steamed under the conditions of 121 DEG C of high pressure-temperature
Disinfection 20 minutes in steam-boiler.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (8)
1. a kind of method of micrografting in fresh water Chinese pear tissue-cultured seedling test tube, which is characterized in that including：
Fiber differentiation step：By fresh water Chinese pear explant be placed in 0.1%-0.3% mercuric chloride solution sterilize 10-16 minutes, then
With aseptic water washing 5-6 times, after aseptic filter paper suck dry moisture, it is cut into vaccinating lancet a length of with 1-3 terminal bud and/or lateral bud
The stem with bud is that explant is inoculated into and has sterilized and on the inducing culture that solidifies, will connect by the stem with bud of 0.5-2cm
Inducing culture elder generation light culture after kind 5-10 days, then induce the terminal bud of explant stem section or lateral bud to sprout under the conditions of optical culture
Hair forms in vitro cuttings；
Shoot proliferation step：The in vitro cuttings of a height of 2-4cm of Fiber differentiation are cut with vaccinating lancet aseptically and are turned
It is connected to and has sterilized and be proliferated on the proliferated culture medium that solidifies, and carry out optical culture 30-40 days, promote the lateral bud of in vitro cuttings
Or adventitious bud is sprouted and obtains the unrooted tissue-cultured seedling grown thickly, with vaccinating lancet cutting is aseptically single plant group by unrooted tissue-cultured seedling
Seedling to be trained, the single plant tissue-cultured seedling higher than 1.5cm is chosen and is transferred on proliferated culture medium, multiple squamous subculture obtains unrooted tissue-cultured seedling,
Scion of the unrooted tissue-cultured seedling as micrografting after squamous subculture 30-40 days；
Stock incubation step：The birchleaf pear seed that Aging storage is crossed is rinsed 30-60 minutes with slow flowing water, with 70% second after drying
Alcohol handles 30s, is drained after aseptic water washing 3 times, is sterilized 4-8 minute with 0.1%-0.3% mercuric chloride solutions, and disinfection end is with sterile
Water rinses 5-6 times, is then seeded into and has sterilized and on the seed germination medium that solidifies, by the seed germination medium after inoculation
Then first light culture 5-10 days induces seed to sprout, forms sterile birchleaf pear seedling, birchleaf pear growth of seedling under the conditions of optical culture
To the stock as micrografting when second true leaf formation；
Micrografting step in test tube：Tinfoil is cut into the bar shaped of 0.5cm × 1.5cm, it is spare after high pressure sterilization；In aseptic condition
Under, stock is taken out by its truncation, stays the stem of 3-4cm long, bottom lateral bud and blade are extractd with tweezers, it is vertical at the top of stock stem section
Cut the notch that formation length is 0.4-0.6cm；Height is chosen for scion similar in 2-5cm, rugosity and stock stem section, in scion
2-6 piece leaves are stayed at top, and its base portion is whittled into the wedge structure that length is 0.4-0.6cm, and the wedge base of scion is inserted into
In the notch of rootstock seedling stem section, keep notch closely connected, the edge alignment on stock and scion at least one side is tied up graft union with tinfoil
It ties up, obtains micrografting seedling；And micrografting seedling is put into and has been sterilized and in the test tube for filling fluid nutrient medium that is cooled to room temperature, liquid
The 1/4 of body culture fiduciary point test tube volume, Liquid Culture primary surface is put into tinfoil makes liquid not have grafting with fixing grafting seedling
Rhizome position micrografting seedling is subjected to optical culture and is promoted graft union healing and growth of seedling with sterile ParafilmTM test tube mouth；
Acclimatization and transplants step：After grafting optical culture 15 days, when stock root starts to grow, and scion leaf is in normal leaf color
And have and test tube is placed in hardening 10-15 days in natural light when young leaves growth, then after removing sealed membrane hardening 1 day, transplant in volume
Than being 2:In 1 peat and the cultivation matrix of perlite mixture, in closed plastic canopy inside holding moisturizing culture after transplanting,
Gradually ventilation after a week, until removing plastic canopy.
2. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：It is lured described
It further includes before explant pre-treatment step to lead incubation step, and the fresh water Chinese pear explant is by fresh water Chinese pear young sprout through explant
It is formed after pre-treatment step, the explant pre-treatment step is：
The fresh water Chinese pear young sprout of a length of 5-10cm of acquisition is rinsed well surface irregularities to be placed in the washing powder solution of saturation
It impregnates 5-10 minute, changes water-removing rinsing and be placed on for 3-5 time in flowing tap water and rinse 5-12 hours, finally with sealed bag sealing dress
It is placed in well spare in 4-10 DEG C of refrigerator.
3. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：It is trained in induction
It supports in step, the stem with bud is the stem section of single bud of the band comprising terminal bud or lateral bud, band comprising terminal bud and lateral bud or is
The stem section of three buds of stem section or band comprising terminal bud and lateral bud of two buds of lateral bud.
4. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：It is trained in stock
It further includes before Aging storage step to support step：No disease and pests harm is acquired when birchleaf pear fruit maturation, well-developed fruit is put in room
Rotten, the height 20-30cm of stacking is macerated in outer stacking, and make not generate heat at fruit accumulation center damage seed, wait fruits it is soft it is ripe after, mash
Clean seed is set after shady and cool ventilation shade is done and is packed into plastics by fruit, the floater that washing removes rotten pulp and swims
Sealed bag is put into spare after 2-4 DEG C of refrigerator preserves 30 days.
5. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：The dark training
It supports as the culture of cultivation temperature no light under conditions of 23-27 DEG C；The optical culture is condition of the cultivation temperature at 23-27 DEG C
Daily illumination 12-16 hours down, the culture of intensity of illumination 1500-2100lx.
6. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：The step of sterilizing
Rapid is in 1.1-1.5kgf/cm2With sterilized 18-20 minutes in steam copper under the conditions of 121 DEG C of high pressure-temperature.
7. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 1, it is characterised in that：Fiber differentiation
Inducing culture described in step is：Using MS as minimal medium, 0.5-1.0mg/L 6- benzyls aminoadenine/6- is added
BA, 0.08-0.15mg/L heteroauxin/IBA, additional 2.5-3.0% sucrose and 0.4-0.7% agar, pH value 5.60-
5.80；
Proliferated culture medium described in shoot proliferation step is：Using MS as minimal medium, 2.0-4.0mg/L 6- benzyl ammonia is added
Base adenine/6-BA, 0.04-0.08mg/L heteroauxin/IBA adds 2.5-3.0% sucrose and 0.4-0.7% agar, pH value
For 5.60-5.80；
Seed germination medium described in stock incubation step is：Using MS as minimal medium, 0.1-0.5mg/L 6- are added
Benzyl aminoadenine/6-BA, 0.08-0.15mg/L heteroauxin/IBA adds 2.5-3.0% sucrose and 0.4-0.7% agar,
PH value is 5.60-5.80；
Fluid nutrient medium in test tube described in micrografting step is：Separately add 0.3-0.7mg/L Yin as minimal medium using 1/2MS
Indolylbutyric acid/IBA and 2.0-2.5% sucrose, pH value 5.60-5.80,1/2MS are that a great number of elements halves in MS culture medium prescriptions,
The culture medium that other elements are constant and prepare.
8. the method for micrografting in fresh water Chinese pear tissue-cultured seedling test tube according to claim 5, it is characterised in that：The MS
Culture medium prescription is made of following each compound, is divided into a great number of elements, four substance of trace element, molysite and organic matter：
A great number of elements contains potassium nitrate/KNO31900mg/L, ammonium nitrate/NH4NO31650mg/L, calcium chloride/CaCl2·2H2O
440mg/L, magnesium sulfate/MgSO4·7H2O 370mg/L, potassium dihydrogen phosphate/KH2PO4170mg/L；
Trace element contains potassium iodide/KI 0.83mg/L, boric acid/H3BO36.2mg/L, manganese sulfate/Mn SO4·4H2O 22.3mg/
L, zinc sulfate/Zn SO4·7H2O 8.6mg/L, sodium molybdate/Na2MoO4·2H2O 0.25mg/L, copper sulphate/Cu SO4·5H2O
0.025mg/L, cobalt chloride/CoCl2·6H2O 0.025mg/L；
Molysite sulfur acid ferrous iron/Fe SO4·7H2O 27.8mg/L, disodium ethylene diamine tetraacetate/Na2-EDTA·2H2O
37.3mg/L；
Organic substance contains：Inositol 100mg/L, niacin 0.5mg/L, puridoxine hydrochloride/vitamin B60.5mg/L, thiamine hydrochloride/
Vitamin B10.1mg/L, glycine 2.0mg/L.
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