CN107306796A - One grows tobacco vitrifying callus and adventitious bud goes vitrified method - Google Patents
One grows tobacco vitrifying callus and adventitious bud goes vitrified method Download PDFInfo
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- CN107306796A CN107306796A CN201710673926.0A CN201710673926A CN107306796A CN 107306796 A CN107306796 A CN 107306796A CN 201710673926 A CN201710673926 A CN 201710673926A CN 107306796 A CN107306796 A CN 107306796A
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- vitrifying
- callus
- adventitious bud
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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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- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
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- Botany (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to biological technical field, specifically one grows tobacco vitrifying callus and adventitious bud goes vitrified method, it is characterized in that：The vitrifying callus and adventitious bud that are produced in Tissues of Tobacco incubation are separated in superclean bench with normal structure, is transferred in vacant sterile tissue culture bottle, is covered bottle cap, be placed in illumination box（It is consistent with normal tissue culture）Middle progress Nature enemy 17 days.After after vitrifying organized renewing normal condition, differential medium is transferred to（Callus）Or elongation medium（Adventitious bud）It is middle to continue to cultivate.Using this technology the vitrifying callus or adventitious bud that are produced in Tissues of Tobacco incubation can be made to go vitrifying, then normal regeneration plant can be formed through squamous subculture.The advantage of the invention is that：The hungry cultural method of empty bottle of no culture medium is employed, without increasing any culture composition, it is not required that increase other special equipments, easy to operate, effect is notable.
Description
Technical field
Grown tobacco vitrifying callus and/or adventitious bud the present invention relates to one（Two objects are all applicable）Remove glass
Change method, belongs to biological technical field.The vitrifying callus group that can make to produce in Tissues of Tobacco incubation using this method
Knit or adventitious bud goes vitrifying, and continue normal differentiation or elongation, ultimately form intact plant.
Background technology
Tobacco belongs to tubular flower subject, and Solanaceae is annual or limited herbaceos perennial, it has now been found that cigarette category
There are 66 kinds.Traditionally tobacco is a kind of important industrial crops, has important effect in national economy, while being also to plant
Important pattern species in thing subject.Since being come out from first plant of transgene tobacco, by stronger cytothesis ability and
The ease for operation of tissue cultures and genetic transformation, tobacco turn into biotechnology research field research material the most active it
One.In pharmaceutical engineering field, by the use of tobacco as bioreactor, have been widely used for production transgenic tobacco medicine,
Tobacco medical albumen, tobacco antibody and tobacco vaccine etc..Have many on Tissues of Tobacco culture and transgenosis at present
Report, but vitrifying is explant phenomenon common in tissue culture procedures, belong to Plant Tissue Breeding Three Difficult Issues it
One.Vitrification Occurred also very severe in Tissues of Tobacco incubation, it would be highly desirable to solve.
Vitrification phenomenon is distinctive a kind of physiological disorder in plant tissue culture course, is referred in Plant Tissue Breeding
There is growth failure in Cheng Zhong, part test tube seedling, and transparent or semitransparent water stain shape, the short and small swelling chlorosis of sprout, leaf is presented in tissue
Piece is crimped to longitudinal direction volume and stretched, frangible, also known as overly hydrated's phenomenon.It is low that vitrifying tissue often shows differentiation capability, it is difficult to
It is proliferated into bud, it is also difficult to seedling of taking root.Current vitrifying formation mechanism study is concentrated mainly on the too high influence of the flow of water, growth and adjusted
Agent equilibrium problem and mineral element balance three aspects of supply problem are saved, being total to for each side factor in culture environment is widely considered to be
Same-action causes plant tissue metabolic disturbance, so as to cause the appearance of vitrification phenomenon.Existing solution mainly has drop
Low 6-BA concentration and increase culture curing agent（Agar or plant gel）Concentration, but these methods can not all prevent glass completely
Change the generation of phenomenon, vitrifying tissue can not be saved.
The content of the invention
The purpose of the present invention is based on above-mentioned prior art situation and provides one kind and promote vitrified tobacco callus group
Knit and go vitrified method with adventitious bud.This method is simple and easy to apply, easy to operate, can effectively promote vitrified tobacco callus
Tissue and adventitious bud go vitrifying, and effect is notable.The tobacco healing tissue of bare glass or adventitious bud after this method is handled,
Normal differentiation or elongation can be continued, intact plant is ultimately formed.Extra cost, which need not be increased, can promote vitrifying tobacco
Callus and adventitious bud, which are reversed, turns into normal structure.
The purpose of the present invention is achieved through the following technical solutions：
One grows tobacco vitrifying callus and adventitious bud goes vitrified method, comprises the following steps：
(1) tobacco vitrifying callus and adventitious bud processing：When occurring vitrified cigarette on the differential medium normally cultivated
When careless callus or adventitious bud, vitrified portion is separated with normal structure in super-clean bench, if vitrifying callus
Tissue volume is larger, is divided into suitable size using aseptic nipper, and is taken out from culture medium, is transferred to the vacant drying of sterilizing
In tissue culture bottle, bottleneck is sealed.It is placed in normal illumination incubator 1-7 days, until vitrifying organized renewing normal condition, if during which
Excess moisture in tissue culture bottle, can change tissue culture bottle；
（2）Callus and Adventitious bud culture after processing：By step（1）Recover the tobacco healing tissue of normal condition after processing
Or adventitious bud is transferred to squamous subculture in differential medium or elongation medium in super-clean bench, after 5-8 days, bare glass tobacco
Callus or adventitious bud can normal differentiation or elongations；
（3）If by step（2）Culture processing go vitrifying tobacco healing tissue and adventitious bud in Subculture again
It is secondary vitrifying sign, repeat step occur（1）With（2）.
In the present invention, the condition of the normal illumination incubator is：Relative humidity 60%, h/28 DEG C of illumination 16 is dark
8 h/25 DEG C, the Lx of light intensity 4.
The differential medium composition：4.4g/L MS inorganic salts（Sigma MS basal mediums）, 30g/L sucrose, 1.0
Mg/L 6-BA, 0.1 mg/L NAA, 2.5 g/L plant gels, surplus is water, pH 5.8-5.9.
The elongation medium composition：4.4g/L MS inorganic salts（Sigma MS basal mediums）, 30g/L sucrose, 0.1
μ g/mL 6-BA, 2.5 g/L plant gels, surplus is water, pH 5.8-5.9.
The invention has the advantages that and effect：It is any without increasing using the hungry cultural method of the empty bottle without culture medium
Cultivate composition, it is not required that increase other special equipments, easy to operate, effect is notable.By the tobacco glass of empty bottle Nature enemy
Glass callus and adventitious bud can effectively go vitrifying to revert to normal condition, and continuation is broken up in the culture medium of script
Or elongation, ultimately form intact plant.The inventive method has important reality for Tissues of Tobacco culture and Transgenic Tobacco
Meaning and prospect is widely applied, the planting percent of tissue cultures can be greatly improved, therefore improve operating efficiency.
Brief description of the drawings
The vitrifying tobacco healing tissue of Fig. 1 present invention and the process state display figure of adventitious bud, wherein：
A：Vitrifying tobacco healing tissue and adventitious bud,
B：Vitrifying tobacco healing tissue and adventitious bud pass through the h of empty bottle Nature enemy 24,
C：Vitrifying tobacco healing tissue and adventitious bud pass through the h of empty bottle Nature enemy 48,
D：The tissue for recovering normal condition after empty bottle Nature enemy moves into culture in differential medium,
E：20 d cultivate in differential medium in bare glass tobacco healing tissue,
F：Bare glass tobacco healing tissue is through squamous subculture formation intact plant.
Embodiment：
The present invention is described in further detail with reference to embodiments.
1. the culture of aseptic seedling：The K326 seeds of 30-50 μ l volumes are taken in 1.5ml EP pipes, with 75% alcohol
After outer spray disinfectant is managed containing seed-bearing EP, superclean bench is put into（Advance ultraviolet irradiation 15-30min）.Add in EP pipes
Enter 850 μ l sterilized water, add 150 μ l liquor natrii hypochloritis（Active Chlorine>6.0%）, 15min is sterilized, does not during which stop to shake
Shake, enable all seed sufficiently steriliseds.With 1ml aseptic water washing 5-6 times, dry, MS is connected to sterilized toothpick point
Culture medium（Tissue culture bottle）In, light culture two days then goes under 28 DEG C, 16h/8h illumination conditions and cultivates 30 d or so.
2. the cutting of explant：The aseptic seedling grown fine is taken, the tender full blade of children is cut into 0.5-1cm2Pros
Shape blade.When cutting blade, with big flat board, a little sterilized water is added, in case blade is withered；Blade is sharp, during section, will
Blade edge and vein are cut away；It is rapid during section, prevent from tearing, extrude, blade injury is avoided as far as possible.
3. the induction of callus and adventitious bud：The blade of cutting is moved into differential medium, 28 DEG C are inserted, RH=
In between 60% illumination cultivation.It is within 7-10 days a cycle, changes fresh culture medium（At least twice）.It can grow within 10 days or so
Plant callus, and adventitious bud is differentiated, adventitious bud is cut from callus, is inoculated in elongation medium, treats indefinite
Bud grows to after suitable size and moved into root media.Wherein, differential medium composition：4.4g/L MS inorganic salts（Sigma's
MS basal mediums）, 30g/L sucrose, 1.0 mg/L 6-BA, 0.1 mg/L NAA, 2.5 g/L plant gels, surplus is water,
pH 5.8-5.9；Elongation medium composition：4.4g/L MS inorganic salts（Sigma MS basal mediums）, 30g/L sucrose, 0.1
μ g/mL 6-BA, 2.5 g/L plant gels, surplus is water, pH 5.8-5.9；Culture of rootage based component：4.4g/L MS are inorganic
Salt（Sigma MS basal mediums）, 30g/L sucrose, 0.02 mg/L NAA, 2.5 g/L plant gels, surplus is water, pH
5.8-5.9。
4. the explant cultivated by step 3 can form callus, and then differentiate adventitious bud, but a portion
Callus and adventitious bud are vitrifying shapes, and the later stage is difficult to be regenerated as normal plant.At the right time, to these glass
The callus and adventitious bud of change take empty bottle Nature enemy, and vitrifying tissue can be reversed as normal structure, continue normal
Differentiation extends and forms intact plant.
5. tobacco vitrifying callus and adventitious bud processing：By vitrified tobacco healing tissue or adventitious bud super
Vitrified portion is separated with normal structure in net platform, if vitrifying callus volume is larger, using aseptic nipper
Suitable size is separated into, and is taken out from culture medium, is transferred in vacant sterile dry tissue culture bottle, seals bottleneck.It is placed in just
Normal illumination box（Relative humidity 60%, h/28 DEG C of illumination 16, dark 8 h/25 DEG C, the Lx of light intensity 4）Middle 1-7 days, until glass
Glass organized renewing normal condition, you can terminate Nature enemy.If excess moisture in period tissue culture bottle, tissue culture bottle can be changed.
6. callus and Adventitious bud culture after processing：By 5 handle after recover normal condition tobacco healing tissue or
Adventitious bud is transferred to squamous subculture in differential medium or elongation medium in super-clean bench.Bare glass can be found within one week or so
Tobacco healing tissue or adventitious bud normal differentiation or elongation.If going vitrifying tobacco callus group by step 5 culture processing
Knit with adventitious bud in Subculture and occur vitrifying sign again, repeat step 5 and 6, bare glass callus and not
Normal bud can be with normal differentiation and elongation.
7. adventitious bud rooting：Adventitious bud after elongation is transferred in root media, after root system is more flourishing, moved into
It is potted plant in soil.
8. tissue-cultured seedling buries potted plant：, it is necessary to tissue culture bottle be opened into lid hardening 2-3 days, then by group before tissue-cultured seedling buries
Training seedling takes out from tissue culture bottle, is rinsed after the agar that root is adhered to, is transplanted into the small basin of plastics full of matrix with running water.First
After being cultivated 2-3 days under dim light, growth between culture is placed in.
It is demonstrated experimentally that the present invention by vitrified tobacco healing tissue and adventitious bud in sterile empty bottle is dried at starvation
Reason is reversed as normal structure after 1-7 days, and can form intact plant after normally cultivating.
Claims (4)
1. one grows tobacco vitrifying callus and adventitious bud goes vitrified method, it is characterised in that：Comprise the following steps：
(1) tobacco vitrifying callus and adventitious bud processing：When occurring vitrified cigarette on the differential medium normally cultivated
When careless callus or adventitious bud, vitrified portion is separated with normal structure in super-clean bench, if vitrifying callus
Tissue volume is larger, is divided into suitable size using aseptic nipper, and is taken out from culture medium, is transferred to the vacant drying of sterilizing
In tissue culture bottle, bottleneck is sealed；It is placed in normal illumination incubator 1-7 days, until vitrifying organized renewing normal condition, if during which
Excess moisture in tissue culture bottle, can change tissue culture bottle；
（2）Callus and Adventitious bud culture after processing：By step（1）Recover the tobacco healing tissue of normal condition after processing
Or adventitious bud is transferred to squamous subculture on differential medium or elongation, culture medium in super-clean bench, after 5-8 days, bare glass cigarette
Careless callus or adventitious bud can normal differentiation or elongations；
（3）If by step（2）Culture processing go vitrifying tobacco healing tissue and adventitious bud in Subculture again
It is secondary vitrifying sign, repeat step occur（1）With（2）.
2. tobacco vitrifying callus according to claim 1 and adventitious bud go vitrified method, it is characterised in that：
The condition of the normal illumination incubator is：Relative humidity 60%, h/28 DEG C of illumination 16, dark 8 h/25 DEG C, the Lx of light intensity 4.
3. tobacco vitrifying callus according to claim 1 and adventitious bud go vitrified method, it is characterised in that：
The differential medium composition：4.4g/L MS inorganic salts（Sigma MS basal mediums）, 30g/L sucrose, 1.0 mg/L 6-
BA, 0.1 mg/L NAA, 2.5 g/L plant gels, surplus is water, pH 5.8-5.9.
4. tobacco vitrifying callus according to claim 1 and adventitious bud go vitrified method, it is characterised in that
It is described：The elongation medium composition：4.4g/L MS inorganic salts（Sigma MS basal mediums）, 30g/L sucrose, 0.1 μ
G/mL 6-BA, 2.5 g/L plant gels, surplus is water, pH 5.8-5.9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710673926.0A CN107306796B (en) | 2017-08-09 | 2017-08-09 | A kind of tobacco vitrifying callus and adventitious bud go vitrified method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710673926.0A CN107306796B (en) | 2017-08-09 | 2017-08-09 | A kind of tobacco vitrifying callus and adventitious bud go vitrified method |
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| CN107306796A true CN107306796A (en) | 2017-11-03 |
| CN107306796B CN107306796B (en) | 2019-04-12 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113170733A (en) * | 2021-05-31 | 2021-07-27 | 上海应用技术大学 | Culture medium and method for vitrifying day lily callus and unglassing adventitious buds |
| CN116034872A (en) * | 2022-11-18 | 2023-05-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Vitrification callus and adventitious bud removal vitrification method for glabrous tarragon |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102047845A (en) * | 2010-12-13 | 2011-05-11 | 湖北省烟草科研所 | Method for in-vitro liquid culture of tobacco pollen with high efficiency |
| CN103250641A (en) * | 2013-05-13 | 2013-08-21 | 江苏省农业科学院 | Devitrification method for vitrified regeneration seedlings of cabbage type rape |
-
2017
- 2017-08-09 CN CN201710673926.0A patent/CN107306796B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102047845A (en) * | 2010-12-13 | 2011-05-11 | 湖北省烟草科研所 | Method for in-vitro liquid culture of tobacco pollen with high efficiency |
| CN103250641A (en) * | 2013-05-13 | 2013-08-21 | 江苏省农业科学院 | Devitrification method for vitrified regeneration seedlings of cabbage type rape |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113170733A (en) * | 2021-05-31 | 2021-07-27 | 上海应用技术大学 | Culture medium and method for vitrifying day lily callus and unglassing adventitious buds |
| CN116034872A (en) * | 2022-11-18 | 2023-05-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Vitrification callus and adventitious bud removal vitrification method for glabrous tarragon |
| CN116034872B (en) * | 2022-11-18 | 2024-03-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Vitrification callus and adventitious bud removal vitrification method for glabrous tarragon |
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| CN107306796B (en) | 2019-04-12 |
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