CN104221882B - Explant sterilization method for ginkgo biloba tissue culture - Google Patents
Explant sterilization method for ginkgo biloba tissue culture Download PDFInfo
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- CN104221882B CN104221882B CN201410550994.4A CN201410550994A CN104221882B CN 104221882 B CN104221882 B CN 104221882B CN 201410550994 A CN201410550994 A CN 201410550994A CN 104221882 B CN104221882 B CN 104221882B
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- explant
- ginkgo biloba
- sterilization
- tissue culture
- hibernaculum
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Abstract
The invention discloses an explant sterilization method for ginkgo biloba tissue culture. The explant sterilization method for the ginkgo biloba tissue culture introduces a treatment and sterilization method for different explants in ginkgo biloba tissue culture to reduce contamination rate of ginkgo biloba tissue culture and death rate caused by improper treatment. According to the explant sterilization method for the ginkgo biloba tissue culture, common explants in the ginkgo biloba tissue culture such as stems, embryos and winter buds are utilized and partial improvement is made on a traditional tissue culture sterilization method, therefore, the survive rate of explant inoculation is improved and further the purpose of improving reproductive efficiency and yield of ginkgo biloba tissue culture is achieved. The explant sterilization method for ginkgo biloba tissue culture can be used for sterilization treatment for explants of ginkgo bilob, and is easy and feasible in operation.
Description
Technical field
The invention belongs to the technical field that arboreal growth is developed and bred, particularly one kind cultivate explant with regard to Ginkgo Biloba
The method of body sterilization.
Background technology
Ginkgo (ginkgo biloba l.) is the Relict Plant of quaternary glacier, is leaf fruit material and excellent rare tree species.
China, as ginkgo first big country in the world, has more than the 90% of world's Ginkgo Germplasm Resource, the cultivation that can investigate in China
History, for thousand, has higher economic, ecological and cultureal value.Using the excellent product of Plant Tissue Breeding Fast-propagation
Kind, be become effective most in nursery stock production, one of most widely used technology.At present, in Ginkgo Biloba culture, conventional explant has
Stem section, embryo and hibernaculum, and these explants are primarily present problem following outstanding in processing disinfecting process: (1) explant is processed
Improper, lead to survival rate low, undergrowth.(2) explant sterilization is not thorough, leads to pollution rate high, have impact on ginkgo tissue culture numerous
The efficiency grown and yield.
The present invention utilizes common explant in Ginkgo Biloba culture: stem section, embryo, hibernaculum, in traditional tissue culture sterilization method
On carried out part improvement, thus reducing the pollution rate of ginkgo tissue culture, with because dealing with the death rate causing improperly.At present also not
See that Ginkgo Biloba cultivates the report of explant sterilization method, the present invention is to propose under such technical background.
Content of the invention
It is contemplated that reducing the pollution rate in Ginkgo Biloba culture, part being done on traditional tissue culture sterilization method and has changed
Enter, introduce the sterilization method of common explant in Ginkgo Biloba culture, easy and simple to handle, pollution rate is low, improves ginkgo
The efficiency of tissue culture propagation and yield.Present invention employs technical scheme below:
(1) selection of thimerosal: carried out disinfection using 70% alcohol and 0.1% mercury chloride, 0.1% every liter of mercury chloride adds
3-4 drips polysorbas20.
(2) explant access time: in ginkgo tissue culture, conventional explant has tender stem segmentses, embryo, hibernaculum.Outside stem section culture
The implant optimal choice time is April early and middle ten dayses, takes Seedlings of Ginkgo biloba pumping to be then about the shoot of 10cm；Embryo culture explant is optimal
Access time is October early and middle ten dayses, takes Ginkgo Biloba Nut then；The hibernaculum culture explant optimal choice time sprouts for early March hibernaculum
Before sending out, the bud do not sprouted is taken on current-year branch.
(3) stem section sterilization: stem section cuts off blade, uses soap scrub water, then rinses well under running water, aseptic operating platform
Under successively with 70% alcohol disinfecting 30s, with 0.1% mercury chloride sterilization 8-10min of polysorbas20, aseptic water washing 3-5 time, then
Suck the moisture on stem section surface with aseptic filter paper, be cut into 1-2cm length, stay 1-2 axillary-bud or top-bud, inoculation.
(4) embryo sterilization: remove pericarp and plant shell, peel off kind of a skin, use soap scrub water, then rinse well under running water,
Successively with 70% alcohol disinfecting 30s under aseptic operating platform, with 0.1% mercury chloride sterilization 6-8min of polysorbas20, aseptic water washing
3-5 time, then suck the moisture on benevolence surface with aseptic filter paper, take out embryo, inoculation from endosperm.
(5) hibernaculum sterilization: clip about 1cm length, the stem section with a hibernaculum in the middle part of the branch given birth to then, use suds
Clean, then rinse well under running water, successively with 70% alcohol disinfecting 30s under aseptic operating platform, with 0.1% chlorine of polysorbas20
Change mercury sterilization 8-10min, aseptic water washing 3-5 time, suck the moisture on surface with aseptic filter paper, be then sliced out stem section, stay one
The woody part that fraction stem section is connected with hibernaculum, peels off outer layer perula, to the first spire expose when, take the wooden company with fraction
The bud connecing, as explant, is inoculated.
The invention has the beneficial effects as follows:
(1) add polysorbas20 in the thimerosal of 0.1% mercury chloride, polysorbas20 is a kind of surface reactive material, can make
Mercury chloride sterilization is more thorough.
(2) explant access time: after spring Seedlings of Ginkgo biloba pumping shoot, it is about 10cm, you can carry out, but Best Times
For April early and middle ten dayses, the tender stem growth of this period is vigorous, and is difficult to grow endophyte impact culture.Embryo culture explant is optimal
Access time is October early and middle ten dayses, and embryo now can grow to 0.5-1cm, and germination rate is high.During hibernaculum culture explant optimal choice
Between sprout for early March hibernaculum before, take a bud arriving biennial branch, now hibernaculum explant is fuller, easily inoculate, take then
Vimineous bud, so ensures that hibernaculum is leaf bud, rather than bud, impact experiment.
(3) sterilization of stem section: stem section, after rinsing, sterilizing, is cut into 1-2cm length, stays 1-2 axillary-bud or top-bud, and stem section is rushed
Wash disinfection thoroughly can reduce the pollution rate of culture.
(4) embryo sterilization: remove pericarp and plant shell, peel off kind of a skin sterilization, benevolence sterilization can be made more thorough, then from endosperm
Take out embryonic breeding kind, greatly reduce pollution rate.
(5) hibernaculum sterilization: hibernaculum explant is processed, during excision stem section, that stays that sub-fraction stem section is connected with hibernaculum is wooden
Part, peels off outer layer perula, to the first spire expose when, take with the stem apex part of the wooden connection of fraction as explant, stay
Fraction is wooden to be to protect sprout, it is to avoid destroy growing point.
Using said method, to ginkgo tissue culture, common explant carries out disinfection, and easy and simple to handle, pollution rate is low, improves ginkgo
The efficiency of tissue culture propagation and yield.
Specific embodiment
Embodiment 1
Application this method is carried out in the school district tissue culture experiments room of Shandong Province Tai'an Shandong Agricultural University south, in tradition
Tissue culture sterilization method on be partially improved, introduce Ginkgo Biloba culture in common explant sterilization method, behaviour
Make easy, pollution rate is low, improves efficiency and the yield of ginkgo tissue culture propagation.Ginkgo Biloba culture explant sterilization method is implemented
Step is as follows:
1. the selection of thimerosal
Prepare 70% alcohol and 0.1% mercury chloride carries out disinfection, 0.1% every liter of mercury chloride adds 3-4 to drip polysorbas20.
2. the selection of explant
April early and middle ten dayses, take Seedlings of Ginkgo biloba pumping to be then about the shoot of 10cm；October early and middle ten dayses, take Ginkgo Biloba Nut then；
Early March, takes the bud do not sprouted on current-year branch.
3. stem section sterilization
Stem section cuts off blade, uses soap scrub water, then rinses well under running water, successively with 70% under aseptic operating platform
Alcohol disinfecting 30s, with 0.1% mercury chloride sterilization 8-10min of polysorbas20, aseptic water washing 3-5 time, then with aseptic filter paper
Suck the moisture on stem section surface, be cut into 1-2cm length, stay 1-2 axillary-bud or top-bud, inoculation.
4. embryo sterilization
Remove pericarp and plant shell, peel off kind of a skin, use soap scrub water, then rinse well under running water, aseptic operating platform
Under successively with 70% alcohol disinfecting 30s, with 0.1% mercury chloride sterilization 6-8min of polysorbas20, aseptic water washing 3-5 time, then
Suck the moisture on benevolence surface with aseptic filter paper, take out embryo, inoculation from endosperm.
5. hibernaculum sterilization
In the middle part of the branch given birth to then, clip about 1cm length, the stem section with a hibernaculum, use soap scrub water, then certainly
Rinse well under water, successively with 70% alcohol disinfecting 30s under aseptic operating platform, with 0.1% mercury chloride sterilization 8- of polysorbas20
10min, aseptic water washing 3-5 time, suck the moisture on surface with aseptic filter paper, be then sliced out stem section, stay sub-fraction stem section
The woody part being connected with hibernaculum, peels off outer layer perula, to the first spire expose when, take the bud conduct with the wooden connection of fraction
Explant, inoculation.
The present invention is not limited to embodiment of above, as long as taking the side of above sterilization in Ginkgo Biloba culture
Method, falls within protection scope of the present invention.
Claims (1)
1. a kind of Ginkgo Biloba cultivates the method for explant sterilization it is characterised in that step is as follows:
(1) selection of thimerosal: carried out disinfection using 70% alcohol and 0.1% mercury chloride, 0.1% every liter of mercury chloride adds 3-4
Drip polysorbas20；
(2) explant access time: in ginkgo tissue culture, conventional explant has tender stem segmentses, embryo, hibernaculum；Stem section culture explant
The optimal choice time is April early and middle ten dayses, takes the shoot of Seedlings of Ginkgo biloba pumping then, is about 10cm；Embryo culture explant most preferably selects
Take the time to be October early and middle ten dayses, take Ginkgo Biloba Nut then；The hibernaculum culture explant optimal choice time sprouts for early March hibernaculum
Before, take a bud arriving biennial branch；
(3) stem section sterilization: stem section cuts off blade, uses soap scrub water, then rinses well under running water, under aseptic operating platform according to
Secondary use 70% alcohol disinfecting 30s, with polysorbas20 0.1% mercury chloride sterilization 8-10min, aseptic water washing 3-5 time, then use nothing
The filter paper of bacterium sucks the moisture on stem section surface, is cut into 1-2cm length, stays 1-2 axillary-bud or top-bud, inoculation；
(4) embryo sterilization: remove pericarp and plant shell, peel off kind of a skin, use soap scrub water, then rinse well under running water, aseptic
Successively with 70% alcohol disinfecting 30s under operating desk, with 0.1% mercury chloride sterilization 6-8min, aseptic water washing 3-5 of polysorbas20
Secondary, then suck the moisture on benevolence surface with aseptic filter paper, take out embryo, inoculation from endosperm；
(5) hibernaculum sterilization: clip about 1cm length, the stem section with a hibernaculum in the middle part of the branch given birth to then, wiped with suds
Wash, then rinse well under running water, successively with 70% alcohol disinfecting 30s under aseptic operating platform, with 0.1% chlorination of polysorbas20
Mercury is sterilized 8-10min, and aseptic water washing 3-5 time sucks the moisture on surface, is then sliced out stem section, stays one little with aseptic filter paper
The woody part that part stem section is connected with hibernaculum, peels off outer layer perula, to the first spire expose when, take the wooden connection with fraction
Bud as explant, inoculate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550994.4A CN104221882B (en) | 2014-10-17 | 2014-10-17 | Explant sterilization method for ginkgo biloba tissue culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550994.4A CN104221882B (en) | 2014-10-17 | 2014-10-17 | Explant sterilization method for ginkgo biloba tissue culture |
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| Publication Number | Publication Date |
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| CN104221882A CN104221882A (en) | 2014-12-24 |
| CN104221882B true CN104221882B (en) | 2017-01-18 |
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| CN201410550994.4A Expired - Fee Related CN104221882B (en) | 2014-10-17 | 2014-10-17 | Explant sterilization method for ginkgo biloba tissue culture |
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| CN108782239A (en) * | 2018-05-14 | 2018-11-13 | 山东组培农业发展有限公司 | A kind of ginkgo detoxification method for tissue culture |
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| US5965438A (en) * | 1995-06-07 | 1999-10-12 | Phyton, Inc. | Cryopreservation of plant cells |
| CN1167153A (en) * | 1997-06-27 | 1997-12-10 | 武汉大学 | Coversion of ginkgo rooting and establishment of clone |
| CN101130763B (en) * | 2007-08-08 | 2011-05-25 | 南京林业大学 | Rapid breeding method for gingkgo callus or suspending cell |
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