CN109863995A - A kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods - Google Patents
A kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods Download PDFInfo
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- CN109863995A CN109863995A CN201810773644.2A CN201810773644A CN109863995A CN 109863995 A CN109863995 A CN 109863995A CN 201810773644 A CN201810773644 A CN 201810773644A CN 109863995 A CN109863995 A CN 109863995A
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Abstract
The present invention provides a kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods, comprising steps of step (1) induces in vitro cuttings, choosing the tender pretreated stem with bud of young sprout of fresh water Chinese pear children is explant, obtains in vitro cuttings;The in vitro cuttings are cut into a height of 1-3cm, the in vitro cuttings stem section access Storaged media with 1-4 piece leaflet and save culture test tube seedling 6-7 months by step (2) succeeding preservation test tube seedling;Step (3) renewal cultivation test tube seedling, the stem-segment with node of the test tube seedling of preservation is inoculated in recovery media, renewal cultivation is carried out under room temperature and illumination condition, a kind of fresh water Chinese pear germplasm Plantlet in vitro provided by the present invention is that a kind of consumption electric energy is few with restoration methods, minimum living is saved as this preserving seed method, and this method is simple to operate, and process is few, this method saves the high survival rate of fresh water Chinese pear germ plasm resource, and renewal cultivation survival rate is up to 90% or more.
Description
Technical field
The present invention relates to Preservation of plant germplasin technical fields, and in particular to a kind of fresh water Chinese pear germplasm Plantlet in vitro
And restoration methods.
Background technique
The store method of existing fresh water Chinese pear germ plasm resource, which is all made of, is grafted by branch and is carried out in orchard cultivation
Save, this method is time-consuming and laborious, influenced by soil and orchard space enrironment it is very big, and by various natural calamities and pest and disease damage
It influences, germplasm is be easy to cause to lose, save fresh water Chinese pear germ plasm resource using general tissue culture technique, test tube seedling needs
Regular squamous subculture need to just transfer primary every 1 month or so, continuous repetition transit working causes a large amount of manpower and material resources
Waste, while transferring often, also increase opportunities for contamination, and use low temperature or cryopreservation technology, operation sequence ratio
Cumbersome, equipment cost is greatly, consume energy height, for this purpose, exploring fresh water Chinese pear germplasm under normal temperature conditions can slowly grow, can extend
Subculture cycle and the preserving seed technology for reducing preservation cost have a very important significance current germ plasm resource Plantlet in vitro.
Summary of the invention
The purpose of the present invention is to provide a kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods, to solve existing skill
Take up an area more, easily affected by environment and germplasm problem easy to be lost in art.
The present invention in order to solve the above problem used by technical solution are as follows:
The present invention provides a kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods, includes the following steps:
Step (1) induces in vitro cuttings:
Choosing the tender pretreated stem with bud of young sprout of fresh water Chinese pear children is explant, is utilized to the explant
The mercuric chloride solution of 0.1%-0.3% sterilizes 10-16 minutes, then washes 5-6 with sterile pond under superclean bench gnotobasis
It is secondary, then after aseptic filter paper suck dry moisture, the explant is cut into a length of 0.5-2.0cm with 1-3 bud with scalpel
Stem with bud, and be inoculated into induced medium, daily illumination 13-15 hours, intensity of illumination 1500-2100lx, temperature
Degree is cultivated under the conditions of being 23-27 DEG C, and the bud on the stem with bud is sprouted and growth obtains in vitro cuttings;
Step (2) succeeding preservation test tube seedling:
The in vitro cuttings that will be obtained in step (1), wipe out lower end with surgical scissors under superclean bench gnotobasis
Blade, and the in vitro cuttings are cut into the stem-segment with node of a height of 1-3cm, in vitro cuttings with 1-4 piece leaflet, take institute
In vitro cuttings stem section morphology lower end access Storaged media is stated, daily illumination 8-12 hours, intensity of illumination 1000-
1500lx, temperature save culture 6-7 months under conditions of being 23-27 DEG C;
Step (3) renewal cultivation test tube seedling:
The stem-segment with node of fresh water Chinese pear test tube seedling obtained in step (2) is cut, a length of 0.5-1.0cm is inoculated in recovery
Culture medium, daily illumination 13-15 hours, intensity of illumination 1500-2100lx, temperature be 23-27 DEG C under the conditions of restored
Culture, renewal cultivation 30-50 days test tube seedlings, which can be inoculated with, to be transferred to Storaged media and carries out continuing to save or being transferred to proliferated culture medium
It is proliferated or is transferred to rooting induction culture medium and carry out rooting induction.
Further, the tender young sprout pretreatment of the children of fresh water Chinese pear described in step (1), the method for processing is: will be from field
The tender young sprout of fresh water Chinese pear children of a length of 5-10cm of acquisition newly sprouted be rinsed with water clean surface dirt, dust, silt and its
His sundries, then impregnated 5-10 minute with saturation washing powder solution, it changes water-removing rinsing 3-5 time and is placed on and flow water purification flushing 5-12
Hour, it is saved backup until rinsing well and being placed in the environment that temperature is 4-10 DEG C.
Further, the component of induced medium described in step (1) include MS (Murashige and Skoog),
0.5-1.0mg/L 6-BA/6- benzyl aminoadenine, 0.10-0.20mg/LIBA/ heteroauxin, 25-30 g/L sucrose, 4-7g/
The combination of one or more of L agar, the induced medium pH 5.6-5.8.
Further, Storaged media component described in step (2) include 1/2-1/3MS, 1.0-1.5mg/L 6-BA,
0.1-0.30mg/L NAA/ methyl α-naphthyl acetate, 4-12mg/L PP333/ paclobutrazol, 25-30g/L sucrose, one in 4-7g/L agar
Kind or several combinations, the Storaged media pH 5.6-5.8.
Further, recovery media component described in step (3) include for MS (Murashige and Skoog),
1.0-1.5mg/L 6-BA, 0.05-0.10mg/L IBA, 0.05-0.10mg/L GA3/ gibberellin, 25-30g/L sucrose, 4-
The combination of one or more of 7g/L agar, the recovery media pH 5.6-5.8.
Further, the component of proliferated culture medium described in step (3) include MS (Murashige and Skoog),
The group of one or more of 2.0-4.0mg/L 6-BA, 0.1-0.30mg/L NAA, 25-30g/L sucrose, 4-7g/L agar
It closes, the proliferated culture medium pH 5.6-5.8.
Further, rooting induction nutrient media components described in step (3) include 1/2-1/4MS (Murashige
And Skoog), 0.4-0.6mg/L 6-BA, 0.1-0.2mg/L NAA, 25-30g/L sucrose, one of 4-7g/L agar
Or several combinations, the rooting induction medium pH 5.6-5.8.
Further, the condition of culture of Multiplying culture described in step (3) and rooting induction is in daily illumination 13-
15 hours, intensity of illumination 1500-2100lx, temperature cultivated under conditions of being 23-27 DEG C.
The beneficial effects of the present invention are:
A kind of fresh water Chinese pear germplasm Plantlet in vitro provided by the present invention has the advantages that with restoration methods
(1) it grows fresh water Chinese pear germplasm slowly, extend subculture cycle and does not have to
Special ultralow temperature is standby, is that a kind of consumption electric energy is few, and minimum living is saved as this preserving seed method.
(2) test tube seedling does not need to carry out switching squamous subculture every month in the method for the present invention, saves a large amount of manpower and material resources,
Reduce because transferring often due to bring opportunities for contamination.
(3) simple to operate in the method for the present invention, process is few, and the inoculation technique for grasping tissue cultures can be carried out
The operation of germplasm Plantlet in vitro.
(4) for culture medium prescription using MS culture medium as minimal medium, MS is the training of most plants tissue in the method for the present invention
Culture medium is used in supporting, prepares and easy to use, is conducive to production application and Technique Popularizing.
(5) the method for the present invention save fresh water Chinese pear germ plasm resource high survival rate, renewal cultivation survival rate up to 90% with
On.
Specific embodiment
Embodiments of the present invention are specifically illustrated below with reference to embodiment, these embodiments are only given to say
Bright purpose, can not be interpreted as limitation of the invention, only for reference and illustrate use, do not constitute and protect to the invention patent
The limitation of range is protected, because without departing from the spirit and scope of the present invention, many can be carried out to the present invention and changed
Become.
Embodiment 1
A kind of fresh water Chinese pear germplasm Plantlet in vitro provided in this embodiment and restoration methods, include the following steps:
(1) in vitro cuttings are induced: being used from the tender young sprout of fresh water Chinese pear children of a length of 5-10cm of field acquisition newly sprouted
Tap water rinses the sundries such as surface smut, dust, silt well, then is impregnated 5 minutes with saturation washing powder solution, changes water purification drift
Wash and be placed on flowing running water for 3 times and rinse 5 hours, until rinse well be placed in the environment that temperature is 4-10 DEG C save it is standby
With.The fresh water Chinese pear stem section for taking refrigerator to save backup is sterilized 16 minutes with 0.1% mercuric chloride solution, then in superclean bench
It is washed 5 times under gnotobasis with sterile pond, after aseptic filter paper suck dry moisture, is cut into the band bud with 1-3 bud with scalpel
Stem section (a length of 0.5-2.0cm) is inoculated into induced medium (MS, 0.5mg/L 6-BA/6- benzyl aminoadenine+0.10mg/
LIBA/ heteroauxin, 25g/L sucrose, 4g/L agar, pH 5.6) on, it is small that daily illumination 13 is placed at a temperature of 23-27 DEG C
When, intensity of illumination is cultivated under conditions of being 1500lx, and the bud in stem section is sprouted and growth obtains in vitro cuttings.
(2) succeeding preservation test tube seedling: the in vitro cuttings for taking step (1) to cultivate are used under superclean bench gnotobasis
After surgical scissors wipe out lower end blade, test tube seedling is cut into a height of 1-3cm, the stem section with 1-4 piece leaflet takes stem section morphology
Lower end access Storaged media (1/2MS, 1.0mg/L 6-BA, 0.1mg/L NAA/ methyl α-naphthyl acetate, 4mg/L PP333/ paclobutrazol,
25g/L sucrose, 4g/L agar, pH 5.6), daily illumination 8 hours is placed at a temperature of 23 DEG C, intensity of illumination is 1000lx's
Under the conditions of carry out preservation culture.
(3) it renewal cultivation test tube seedling: takes step (2) to save the fresh water Chinese pear test tube seedling that culture has 6 months, cuts test tube
Seedling stem-segment with node, a length of 0.5-1.0cm, be inoculated in recovery media (MS, 1.0mg/L 6-BA, 0.05mg/L IBA,
0.05mg/L GA3/ gibberellin, 25g/L sucrose, 4g/L agar, pH 5.6), daily illumination 13 is placed at a temperature of 25-26 DEG C
Hour, intensity of illumination carries out renewal cultivation under the conditions of being 1500lx.Renewal cultivation 30-40 days renewal cultivation test tube seedlings can connect
Kind is transferred to Storaged media and carries out continuing to save.The test tube seedling of renewal cultivation is transferred to proliferated culture medium if being proliferated
(MS, 2.0mg/L 6-BA, 0.1mg/L NAA, 25g/L sucrose, 4g/L agar, pH 5.6) is proliferated, if wanting growth-promoting root
It forms tool root tissue-cultured seedling and is then transferred to rooting induction culture medium (1/2MS, 0.4mg/L 6-BA, 0.1mg/L NAA, 25g/L sugarcane
Sugar, 4g/L agar, pH 5.6) carry out rooting induction.The condition of culture of Multiplying culture and rooting induction is underlying at 23-27 DEG C
In daily illumination 13 hours, intensity of illumination was cultivated under the conditions of being 1500lx.
Embodiment 2
A kind of fresh water Chinese pear germplasm Plantlet in vitro provided in this embodiment and restoration methods, include the following steps:
(1) in vitro cuttings are induced: being rushed from the tender young sprout of fresh water Chinese pear children of a length of 5-10cm of field acquisition with tap water
The sundries such as wash clean surface smut, dust, silt, then impregnated 7.5 minutes with saturation washing powder solution, after changing water-removing rinsing 4 times
It is placed in flowing running water to rinse 8.5 hours, be saved backup until rinsing well and being placed in the environment that temperature is 4-10 DEG C.Choosing
The stem with bud for taking fresh water Chinese pear refrigerator to save backup sterilizes 13 minutes through 0.2% mercuric chloride solution, then in ultra-clean work
It is washed 5 times under platform gnotobasis with sterile pond, after aseptic filter paper suck dry moisture, is cut into the band with 1-3 bud with scalpel
Leaf stem section (a length of 0.5-2.0cm) is inoculated into induced medium (MS, 0.8mg/L 6-BA, 0.15mg/L IBA, 28g/L sugarcane
Sugar, 6g/L agar, pH 5.7) on, daily illumination 14 hours is placed at a temperature of 23-27 DEG C, intensity of illumination is 1800lx's
Under the conditions of cultivated, bud in stem section is sprouted and growth obtains in vitro cuttings.
(2) succeeding preservation test tube seedling: the in vitro cuttings for taking step (1) to cultivate are used under superclean bench gnotobasis
After surgical scissors wipe out lower end blade, test tube seedling is cut into a height of 1-3cm, the stem section with 1-4 piece leaflet takes stem section morphology
Storaged media (1/2MS, 1.2mg/L 6-BA, 0.2mg/L NAA, 8mg/L PP333,28g/L sucrose, 6g/L are accessed in lower end
Agar, pH 5.7), daily illumination 10 hours is placed at a temperature of 25 DEG C, intensity of illumination carries out preservation training under the conditions of being 1250lx
It supports.
(3) it renewal cultivation test tube seedling: takes step (2) to save the fresh water Chinese pear test tube seedling cultivated 6 months, cuts test tube seedling
Stem-segment with node, a length of 0.5-1.0cm, is inoculated in recovery media, and daily illumination 14 hours, light are placed at a temperature of 23-27 DEG C
Renewal cultivation is carried out under the conditions of being 1800lx according to intensity.Renewal cultivation 30-50 days renewal cultivation test tube seedlings, which can be inoculated with, is transferred to guarantor
Culture medium is deposited to carry out continuing to save, if being proliferated inoculation be transferred to proliferated culture medium (MS, 3.0mg/L 6-BA,
0.20mg/L NAA, 28g/L sucrose, 6g/L agar, pH 5.7) it is proliferated, if root induction is wanted to be trained tool root test tube
Miao Ze inoculation is transferred to rooting induction culture medium (1/3MS, 0.5mg/L 6-BA, 0.2mg/L NAA, 28g/L sucrose, 6g/L fine jade
Rouge, pH 5.7) carry out rooting induction.The condition of culture of Multiplying culture and rooting induction is to be placed at a temperature of 23-27 DEG C often
Its illumination 14 hours, intensity of illumination are cultivated under conditions of being 1800lx.
Embodiment 3
A kind of fresh water Chinese pear germplasm Plantlet in vitro provided in this embodiment and restoration methods, include the following steps:
(1) in vitro cuttings are induced: being rushed from the tender young sprout of fresh water Chinese pear children of a length of 5-10cm of field acquisition with tap water
The sundries such as wash clean surface smut, dust, silt, then impregnated 10 minutes with saturation washing powder solution, after changing water-removing rinsing 5 times
It is placed in flowing running water to rinse 12 hours, be saved backup until rinsing well and being placed in the environment that temperature is 4-10 DEG C.It takes light
The stem with bud that water sand pears refrigerator saves backup, through 0.3% mercuric chloride solution sterilize 10 minutes, then superclean bench without
It is washed 6 times under collarium border with sterile pond, after aseptic filter paper suck dry moisture, is cut into the stem segment with bud with 1-3 bud with scalpel
Section (a length of 0.5-2.0cm) is inoculated into induced medium, daily illumination 16 hours is placed at a temperature of 23-27 DEG C, illumination is strong
Degree is cultivated under conditions of being 2100 lx, and the bud in stem section is sprouted and growth obtains in vitro cuttings.
(2) succeeding preservation test tube seedling: the in vitro cuttings for taking step (1) to cultivate are used under superclean bench gnotobasis
After surgical scissors wipe out lower end blade, test tube seedling is cut into a height of 1-3cm, the stem section with 1-4 piece leaflet takes stem section morphology
Storaged media (1/3MS, 1.5mg/L 6-BA, 0.30mg/L NAA, 12mg/L PP333,30g/L sucrose, 7g/ are accessed in lower end
L agar, pH5.8), daily illumination 12 hours is placed at a temperature of 27 DEG C, intensity of illumination is saved under conditions of being 1500lx
Culture.
(3) it renewal cultivation test tube seedling: takes step (2) to save culture up to 7 months fresh water Chinese pear test tube seedlings, cuts examination
Pipe seedling stem-segment with node, a length of 0.5-1.0cm are inoculated in recovery media, and it is small that daily illumination 16 is placed at a temperature of 23-27 DEG C
When, intensity of illumination carries out renewal cultivation under the conditions of being 2100lx.Renewal cultivation 30-50 days renewal cultivation test tube seedlings can be inoculated with
It is transferred to Storaged media to carry out continuing to save, inoculation is transferred to proliferated culture medium (MS, 1.5mg/L 6- if being proliferated
BA, 0.10mg/L IBA, 0.10mg/L GA3,30g/L sucrose, 7g/L agar, pH5.8) it is proliferated, if to induce life
Root, which is then inoculated with, is transferred to rooting induction culture medium (1/4MS, 0.6mg/L 6-BA, 0.2mg/L NAA, 30g/L sucrose, 7g/L fine jade
Rouge, pH5.8) carry out rooting induction.The condition of culture of Multiplying culture and rooting induction is placed in every daylight at a temperature of being 23-27 DEG C
According to 16 hours, intensity of illumination was cultivated under conditions of being 2100lx.
A kind of fresh water Chinese pear germplasm Plantlet in vitro provided by the present invention and restoration methods make fresh water sand under normal temperature conditions
Pears germplasm is slowly grown, and it is that a kind of consumption electric energy is few, minimum living is saved as this that extension subculture cycle is simultaneously standby without special ultralow temperature
Preserving seed method, and test tube seedling does not need to carry out switching squamous subculture every month in the method for the present invention, saves a large amount of people
Power material resources reduce because transferring often due to bring opportunities for contamination, while simple to operate in the method for the present invention, process
Few, the inoculation technique for grasping tissue cultures can be carried out the operation of germplasm Plantlet in vitro, in addition, culture medium in the method for the present invention
For formula using MS culture medium as minimal medium, MS is the culture medium that uses in most plants tissue cultures, preparation and user
Just, be conducive to production application and Technique Popularizing, the method for the present invention saves the high survival rate of fresh water Chinese pear germ plasm resource, restores training
Motility rate is formed up to 90% or more.
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 change, modification, substitution, combination, letter
Change, should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. a kind of fresh water Chinese pear germplasm Plantlet in vitro and restoration methods, which comprises the steps of:
Step (1) induces in vitro cuttings: choosing the tender pretreated stem with bud of young sprout of fresh water Chinese pear children is explant, to institute
It states explant to sterilize 10-16 minutes using the mercuric chloride solution of 0.1%-0.3%, then uses nothing under superclean bench gnotobasis
Bacterium pond is washed 5-6 times, then after aseptic filter paper suck dry moisture, and the explant is cut into the length with 1-3 bud with scalpel
It for the stem with bud of 0.5-2.0cm, and is inoculated into induced medium, is in daily illumination 13-15 hours, intensity of illumination
1500-2100lx, temperature are cultivated under the conditions of being 23-27 DEG C, and the bud on the stem with bud is sprouted and growth acquisition is sterile
Test tube seedling;
Step (2) succeeding preservation test tube seedling: the in vitro cuttings that will be obtained in step (1), under superclean bench gnotobasis
Lower end blade is wiped out with surgical scissors, and the in vitro cuttings are cut into a height of 1-3cm, the sterile test tube with 1-4 piece leaflet
The stem-segment with node of seedling takes in vitro cuttings stem section morphology lower end access Storaged media, daily illumination 8-12 hours,
It is saved under the conditions of intensity of illumination is 1000-1500lx, temperature is 23-27 DEG C fresh water Chinese pear test tube seedling 6-7 months;
Step (3) renewal cultivation test tube seedling: cutting the stem-segment with node of fresh water Chinese pear test tube seedling obtained in step (2), a length of
0.5-1.0cm is inoculated in recovery media, is in daily illumination 13-15 hours, intensity of illumination 1500-2100lx, temperature
Renewal cultivation is carried out under conditions of 23-27 DEG C, renewal cultivation 30-50 days renewal cultivation test tube seedlings, which can be inoculated with, is transferred to preservation culture
Base, which continue to save or be transferred to proliferated culture medium and is proliferated or is transferred to rooting induction culture medium, carries out rooting induction.
2. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(1) the tender young sprout pretreatment of fresh water Chinese pear children described in, the method for processing is: by newly sprouting from a length of 5-10cm of field acquisition
The tender young sprout of fresh water Chinese pear children of hair is rinsed with water clean surface dirt, dust, silt and other sundries, then molten with saturation washing powder
Liquid impregnates 5-10 minutes, changes water-removing rinsing 3-5 times and is placed on flowing water purification flushing 5-12 hours, is placed on temperature until rinsing well
It is saved backup in the environment that degree is 4-10 DEG C.
3. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(1) component of the induced medium described in includes MS, 0.5-1.0mg/L 6-BA/6- benzyl aminoadenine, 0.10-
The combination of one or more of 0.20mg/L IBA/ heteroauxin, 25-30g/L sucrose, 4-7g/L agar, the induction training
Support base pH 5.6-5.8.
4. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(2) the Storaged media component described in includes 1/2-1/3MS, 1.0-1.5mg/L 6-BA, 0.1-0.30mg/L NAA/ naphthalene second
The combination of one or more of acid, 4-12mg/L PP333/ paclobutrazol, 25-30g/L sucrose, 4-7g/L agar, the guarantor
Deposit medium pH 5.6-5.8.
5. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(3) the recovery media component described in includes for MS, 1.0-1.5mg/L 6-BA, 0.05-0.10mg/L IBA, 0.05-
The combination of one or more of 0.10mg/L GA3/ gibberellin, 25-30g/L sucrose, 4-7g/L agar, the recovery training
Support base pH 5.6-5.8.
6. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(3) component of the proliferated culture medium described in includes MS, 2.0-4.0mg/L 6-BA, 0.1-0.30mg/L NAA, 25-30g/L
The combination of one or more of sucrose, 4-7g/L agar, the proliferated culture medium pH 5.6-5.8.
7. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(3) the rooting induction nutrient media components described in include 1/2-1/4MS, 0.4-0.6mg/L 6-BA, 0.1-0.2mg/L NAA,
The combination of one or more of 25-30g/L sucrose, 4-7g/L agar, the rooting induction medium pH 5.6-5.8.
8. a kind of fresh water Chinese pear germplasm Plantlet in vitro according to claim 1 and restoration methods, it is characterised in that: step
(3) condition of culture of Multiplying culture and rooting induction described in is daily illumination 13-15 hours, intensity of illumination 1500-
2100lx, temperature are cultivated under conditions of being 23-27 DEG C.
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Application publication date: 20190611 |