CN107372124A - A kind of tissue culture culture medium prescription and cultural method for the production of American red-maple seedling - Google Patents
A kind of tissue culture culture medium prescription and cultural method for the production of American red-maple seedling Download PDFInfo
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- CN107372124A CN107372124A CN201710821960.8A CN201710821960A CN107372124A CN 107372124 A CN107372124 A CN 107372124A CN 201710821960 A CN201710821960 A CN 201710821960A CN 107372124 A CN107372124 A CN 107372124A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of tissue culture culture medium prescription and cultural method for the production of American red-maple seedling, the present invention is improved on MS medium bases, invents red maple culture medium（HF）, with reference to a 25g/L sucrose and 7g/L agar, 0.010mg/L ~ 0.003mg/L Phenyl Urea Derivatives（TDZ）Tissue cultures are carried out to American red-maple, substantially increase the reproduction speed of American red-maple, improve the numerous coefficient of expansion of American red-maple, by the incubation time of six weeks, the numerous coefficient of expansion can be made to be changed into former 2 times, the numerous coefficient of expansion is reached 6.5, the annual capacity increase of American red-maple, has very big production capacity and economic benefit.
Description
Technical field
The present invention relates to a kind of tissue culture culture medium prescription and cultural method for the production of American red-maple seedling
, belong to red maple Cultivating techniques field.
Background technology
The invention discloses a kind of American red-maple tissue culture and rapid propagation method, the tall and big fallen leaves of American red-maple system Aceraceae Acer L are tall
Wood, it is the fine tree species of afforestation the features such as adaptable strong, drought-enduring, heatproof, swift and violent growing way.
American red-maple mainly carries out seedling breeding in a manner of seed seed propagation at present, but because seed-setting rate is relatively low, because
This needs to carry out tissue cultures, and the tissue cultures of present American red-maple are mostly the MS culture mediums with improvement（In MS culture mediums
Ammonium nitrate halves）Based on culture medium, specific formula is：MS+25g/L sucrose, 7g/L agar, the 0.5mg/L cells of improvement
Mitogen, 0.01mg/L indolebutyric acids or methyl α-naphthyl acetate, still, this formula expand numerous coefficient not during squamous subculture
Height, only 3.0 or so；One bud explant is respectively the numerous coefficient of expansion with 3 and 6, exemplified by expanding numerous 1 year, is if expanding numerous coefficient
3, then the production capacity of 1 year is 0.66 ten thousand plants, and if it is 6 to expand numerous coefficient, then the production capacity of 1 year is 167.96 ten thousand plants, and this is extremely
Huge production capacity and economic benefit gap, it is therefore desirable to research and develop a kind of tissue culture culture that can be improved American red-maple and expand numerous coefficient
Base, improve the production capacity and economic benefit of American red-maple.
The content of the invention
For the problems of the prior art, the invention provides a kind of American red-maple seedling to produce tissue culture culture medium prescription, this
The formula of invention makes American red-maple reproductive capacity stronger, expands numerous coefficient and improves, so as to annual production increase.
Technical scheme is as follows, and a kind of American red-maple seedling produces tissue culture culture medium prescription, including following components：
Red maple culture medium（HF）Including a great number of elements：112.50mg/L calcium chloride, 1440.00mg/L potassium nitrate, 180.54 mg/L
Magnesium sulfate, 330.60 mg/L potassium dihydrogen phosphates, 600.00 mg/L ammonium nitrate；
Trace element：0.025 mg/L cobalt chlorides, 0.25 mg/L copper sulphate, 6.20 mg/L boric acid, 16.90 mg/L manganese sulfates,
0.25 mg/L sodium molybdates, 8.6 mg/L zinc sulfate；
Organic principle：2.00 mg/L glycine, 100.00 mg/L inositols, 1.00mg/L thiamine hydrochlorides VB, 0.50 mg/L cigarettes
Acid, 0.25 mg/L pyridoxine hydrochlorides；
Molysite：73.40mg/L iron edetates.
The culture medium prescription of the present invention is 25g/L sucrose and 7g/L agar, 0.010mg/L ~ 0.003mg/L also including concentration
Phenyl Urea Derivatives（TDZ）.
Preferably, the Phenyl Urea Derivatives in culture medium prescription of the present invention（TDZ）Concentration be 0.020mg/L.
The application method of the American red-maple seedling production tissue culture culture medium prescription of the present invention, is comprised the following steps that：
Starting culture：
（1）The selection of explant：Explant chooses the axillary bud on red maple maternal plant stem branch, is cut with scalpel；
（2）The pretreatment of explant：5min is rinsed in vibration in the aqueous solution of Tween-80, after rinsing 2h with flowing water, is placed in desinfection chamber
On superclean bench, operation backward needs to carry out sterile working on superclean bench；
（3）The sterilization of explant：First with 75% alcohol disinfecting 10s, with aseptic water washing 3 times, then it is molten with 0.8% sodium hypochlorite
Liquid disinfectant 5min, aseptic water washing 5 times, with standby after aseptic paper suck dry moisture；
（4）By step（1）Arrive（3）The explant handled well is inoculated into some of the red maple seedling production tissue culture culture medium of various concentrations
In individual bottle, every bottle of axillary bud；
（5）Step（4）In red maple production tissue culture culture medium PH be adjusted to 5.4 ~ 5.6；
（6）By above-mentioned explant culture 20 days, grow up to seedling, observation explant be axillary bud germination rate and rudiment after formed it is small
The growing way of seedling；
Squamous subculture：
（7 by step（6）In seedling be inoculated into various concentrations red maple seedling production tissue culture culture medium several bottles in, every bottle
One seedling, culture are observed after six weeks.
Above-mentioned squamous subculture is to continue to be placed into different culture media by the red maple seedling for originating culture to cultivate.
What the maternal plant used in above-mentioned steps was selected is the radiance kind in October of 4-5 lifes, and what explant was chosen is maternal plant
Axillary bud on stem branch.
Wherein, step（6）The bottle and step of middle explant access（7）In the bottle of seedling access be placed in 24 DEG C, 16 hours/
Cultivated in the environment of its illumination.
The step of the present invention（4）In the TDZ concentration of red maple seedling production tissue culture culture medium be respectively 0.01mg/L ~ 0.03mg/
L, step（7）In the TDZ concentration of red maple seedling production tissue culture culture medium be 0.015mg/L ~ 0.025mg/L.
The step of the present invention（4）In red maple seedling production tissue culture culture medium TDZ concentration be respectively 0.015mg/L ~
0.025mg/L, step（7）In the TDZ concentration of red maple seedling production tissue culture culture medium be 0.020mg/L.
Preferably, red maple of the invention is American red-maple.
Beneficial effect compared with prior art of the invention：
Utilize the red maple culture medium of the present invention（HF）The numerous coefficient of expansion of red maple plant is improved, the numerous coefficient of expansion is reached 6.5, U.S.
The annual capacity increase of the red maple of state, has very big production capacity and economic benefit.
Figure of description
Fig. 1 is germination rate and TDZ concentration gradient relations；
2. group, 3. group, the experimental data 4. organized wherein are represented, abscissa is TDZ concentration, and ordinate is germination rate.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Those skilled in the art should
It should be appreciated that the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment
The red maple culture medium used in embodiments of the invention（HF）Specific component content
Starting culture：Above-mentioned ready bud explant is inoculated into the culture medium of following ten kinds of formulas, 11 groups of experiments are as follows.
1. it is formulated one：MS culture mediums（Ammonium nitrate content is original 50%）+ 25g/L sucrose+7g/L agar+0.5mg/L cells
Mitogen+0.01mg/L indolebutyric acids.
2. it is formulated two：HF+25g/L sucrose+7g/L agar+0.01mg/L TDZ.
3. it is formulated three：HF+25g/L sucrose+7g/L agar+0.03mg/L TDZ.
4. it is formulated four：HF+25g/L sucrose+7g/L agar+0.05mg/L TDZ.
5. it is formulated five：HF+25g/L sucrose+7g/L agar+0.03mg/L TDZ+0.01mg/L methyl α-naphthyl acetates.
6. it is formulated six：HF+25g/L sucrose+7g/L agar+0.03mg/L TDZ+0.01mg/L indolebutyric acids.
7. it is formulated one：MS culture mediums（Ammonium nitrate content is original 50%）+ 25g/L sucrose+7g/L agar+0.5mg/L
The basic element of cell division+0.01mg/L indolebutyric acids.
8. it is formulated seven：HF+25g/L sucrose+7g/L agar+0.015mg/L TDZ.
9. it is formulated eight：HF+25g/L sucrose+7g/L agar+0.025mg/L TDZ.
10. it is formulated nine：HF+25g/L sucrose+7g/L agar+0.035mg/L TDZ.
Formula ten：HF+25g/L sucrose+7g/L agar+0.025mg/L TDZ+0.01mg/L indolebutyric acids.
Embodiment 1. ~ American red-maple seedling production tissue culture culture medium prescription application method, comprise the following steps that：
Starting culture
（1）The selection of explant：Explant chooses the axillary bud on American red-maple maternal plant stem branch, is cut with scalpel；
（2）The pretreatment of explant：It is put into vibration in the aqueous solution of a small amount of Tween-80 and rinses 5min, after rinsing 2h with flowing water, puts
In on desinfection chamber superclean bench, operation backward needs to carry out sterile working on superclean bench；
（3）The sterilization of explant：First with 75% alcohol disinfecting 10s, with aseptic water washing 3 times, then it is molten with 0.8% sodium hypochlorite
Liquid disinfectant 5min, aseptic water washing 5 times, with standby after aseptic paper suck dry moisture；
（4）By step（1）Arrive（3）The explant handled well be inoculated into it is above-mentioned 1. ~ 6. group of formula red maple seedling production tissue culture culture
In base, every kind of 100 bottles of culture medium, every bottle of axillary bud；
（5）Step（4）In red maple production tissue culture culture medium PH be adjusted to 5.4 ~ 5.6；
（6）By above-mentioned explant culture 20 days, grow up to seedling, observation explant be axillary bud germination rate and sprouting after formed it is small
Seedling growing way；
Squamous subculture：
（7）By step（6）In seedling that 1. tissue culture is formed be inoculated into the culture medium 7. organized, three seedlings being trained will be formulated and connect
Kind is into the red maple seedling production tissue culture culture medium of 8. ~ group of formula, totally five groups of experiments, every group of 4 bottles of culture medium inoculated, 15 every bottle
Seedling, culture are observed after six weeks.
What the maternal plant used in above-mentioned steps was selected is the radiance kind in October of 4-5 lifes, and what explant was chosen is maternal plant
Axillary bud on stem branch.
Wherein, step（6）The bottle and step of middle explant access（7）In the bottle of seedling access be placed in 24 DEG C, 16 hours/
Cultivated in the environment of its illumination.
PH is adjusted to 5.4 ~ 5.6 to the formula of 11 groups of experiments of the above before the firing.
The starting culture of the experimental procedure of embodiment cultural method, bottle should be placed in 24 DEG C, trained in the environment of 16h/d illumination
Support, bud is sprouted and grows tender seedling after 20 days.
From 1. ~ 6. the germination rate of experiment statisticses bud and from seedling growing way, the results are shown in Table one.
Table one：Influence of the different formulations to bud germination rate and upgrowth situation.
Note："+" represents sturdy degree, preferably " +++ ++ "." * " represent vitrification phenomenon, most serious for " * * * * * ", its
Middle vitrifying is distinctive a kind of physiology lesion in Plant Tissue Breeding, be some physics in culture environment, chemical factor and
Caused by Some Circulating Factors collective effect makes plant tissue metabolic disturbance, plant shows as short and small swelling, and no internode or internode are very
It is short, rosette-stape is presented, top tip and blade-section or whole chlorosis, is translucent, water soaking mode, leaf-shrinkage warp, sprout is not easy
Break up and take root.
Auxin（Indolebutyric acid or methyl α-naphthyl acetate）With the relation of sturdy degree：It will be seen that 5. group, 6. from table one
Group sturdy degree it is higher than 3. organizing, they formulas only difference is that：5. group and 6. group are to have growth in formula
Plain indolebutyric acid or methyl α-naphthyl acetate, and 3. group is no.Obviously added in the starting cultivation stage of red maple, formula appropriate
Auxin（Indolebutyric acid or methyl α-naphthyl acetate）It is required.Certainly, this not can consider simply in starting cultivation stage
Subculture expansion numerous stage is also such.
By the germination rate of accompanying drawing 1 and TDZ concentration gradient relations, we can see that following relativity：
It will be seen that with the increase of TDZ concentration, germination rate can also raise from figure one.But it is higher than in concentration
During 0.03mg/L, the rise of germination rate eases up, also, we can see that in TDZ concentration from table one 4. number experimental group
When being 0.05mg/L, vitrified phenomenon has been begun with, it is clear that 0.05mg/L concentration is above the numerical value required for us
, normal concentration should be located at 0.03mg/L or so, so in the squamous subculture stage, TDZ concentration can be used in us
Reduce, in 0.03mg/L or so.
4th, squamous subculture：
By step（7）Know, the young plant in 1. number group be inoculated into 7. number group culture medium, 3. the young plant in number group be inoculated into 8.,
9., 10., in the culture medium of number group, 7., 8., 9., 10., each group see the table below two using formula, every group of 4 bottles, every bottle 15 of inoculation
, culture environment is 24 DEG C, in the environment of 16 hour/day illumination, observes growing way after six weeks, record is performed, such as following table two.
Table two：Cultivation results of the red maple squamous subculture on different formulations
In table two, in addition to 7. number group and number experimental group, the numerous coefficient of expansion between remaining each group almost indifference is all left 6.2
It is right.
9. the numerous coefficient of expansion of number group is maximum, simply vitrifying somewhat from data.10. the vitrifying phase of number group
To serious, and 8. number group is without vitrifying, in terms of formula, their only differences in TDZ content, it is clear that TDZ concentration
It is higher, more easily cause vitrifying, so the TDZ suitable concentrations that we need should be in eight i.e. experimental group of formula seven and formula
9. 8. between the concentration used, i.e. 0.015mg/L ~ 0.025mg/L, so, the optimal TDZ concentration values that we select are
0.02mg/L。
7. the young plant growing way of number group also can, simply height is shorter, expands that numerous coefficient is low, and both have gap on seedling height.
The nursery stock of number experimental group 9. number experimental group relatively, it is relatively low on numerous coefficient is expanded, and have the phenomenon taken root, and two
On the culture medium of person, difference simply number contains auxin indolebutyric acid, and 9. number does not have auxin, this number of being also experimental group
Nursery stock the reason for taking root, so expanding in the subculture from red maple on breeding culture medium, eliminate auxin indolebutyric acid and naphthalene second
The optimization formula that 8. and 9. formula that acid, i.e. experimental group use is cultivated for the breeding of red maple.
Tested more than, on numerous coefficient is expanded, existing formula is substantially better than original formula.
Optimal case is initial medium formula：HF+25g/L sucrose+7g/L agar+0.03mg/L TDZ+0.01mg/L
Indolebutyric acid（Or methyl α-naphthyl acetate）；
Squamous subculture based formulas：HF+25g/L sucrose+7g/L agar+0.02mg/L TDZ.
Claims (9)
1. a kind of American red-maple seedling produces tissue culture culture medium prescription, including following components：
Red maple culture medium（HF）Including a great number of elements：112.50mg/L calcium chloride, 1440.00mg/L potassium nitrate, 180.54 mg/L
Magnesium sulfate, 330.60 mg/L potassium dihydrogen phosphates, 600.00 mg/L ammonium nitrate；
Trace element：0.025 mg/L cobalt chlorides, 0.25 mg/L copper sulphate, 6.20 mg/L boric acid, 16.90 mg/L manganese sulfates,
0.25 mg/L sodium molybdates, 8.6 mg/L zinc sulfate；
Organic principle：2.00 mg/L glycine, 100.00 mg/L inositols, 1.00mg/L thiamine hydrochlorides VB, 0.50 mg/L cigarettes
Acid, 0.25 mg/L pyridoxine hydrochlorides；
Molysite：73.40mg/L iron edetates.
2. American red-maple seedling according to claim 1 produces tissue culture culture medium prescription, it is characterised in that the culture medium is matched somebody with somebody
Side is 25g/L sucrose and 7g/L agar, 0.010mg/L ~ 0.003mg/L Phenyl Urea Derivatives also including concentration（TDZ）.
3. American red-maple seedling according to claim 2 produces tissue culture culture medium prescription, it is characterised in that the culture medium is matched somebody with somebody
Phenyl Urea Derivatives in side（TDZ）Concentration be 0.020mg/L.
4. a kind of application method of American red-maple seedling production tissue culture culture medium prescription, is comprised the following steps that：
Starting culture：
（1）The selection of explant：Explant chooses the axillary bud on red maple maternal plant stem branch, is cut with scalpel；
（2）The pretreatment of explant：5min is rinsed in vibration in the aqueous solution of Tween-80, after rinsing 2h with flowing water, is placed in desinfection chamber
On superclean bench, operation backward needs to carry out sterile working on superclean bench；
（3）The sterilization of explant：First with 75% alcohol disinfecting 10s, with aseptic water washing 3 times, then it is molten with 0.8% sodium hypochlorite
Liquid disinfectant 5min, aseptic water washing 5 times, with standby after aseptic paper suck dry moisture；
（4）By step（1）Arrive（3）The explant handled well is inoculated into several bottles of red maple seedling production tissue culture culture medium, often
One axillary bud of bottle；
（5）Step（4）In red maple production tissue culture culture medium PH be adjusted to 5.4 ~ 5.6；
（6）By above-mentioned explant culture 20 days, grow up to seedling, observation explant be axillary bud germination rate and rudiment after formed it is small
The growing way of seedling；
Squamous subculture：
（7 by step（6）In seedling be inoculated into several bottles of red maple seedling production tissue culture culture medium, every bottle of seedling, training
Observed after supporting six weeks.
5. according to the method for claim 4, it is characterised in that what described maternal plant was selected is the radiance in October of 4-5 lifes
Kind, what explant was chosen is the axillary bud on maternal plant stem branch.
6. according to the method for claim 4, it is characterised in that the step（6）The bottle and step of middle explant access（7）
In seedling access bottle be placed in 24 DEG C, cultivated in the environment of 16 hour/day illumination.
7. according to the method for claim 4, it is characterised in that the step（4）In red maple seedling production tissue culture culture medium
TDZ concentration is respectively 0.01mg/L ~ 0.03mg/L, step（7）In the TDZ concentration of red maple seedling production tissue culture culture medium be
0.015mg/L~0.025mg/L。
8. according to the method for claim 4, it is characterised in that the step（4）In red maple seedling production tissue culture culture medium
TDZ concentration is respectively 0.015mg/L ~ 0.025mg/L, step（7）In the TDZ concentration of red maple seedling production tissue culture culture medium be
0.020mg/L。
9. the American red-maple seedling production tissue culture culture medium prescription according to claim 1 or 2 or 3, it is characterised in that described
Red maple is American red-maple.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110839528A (en) * | 2019-11-22 | 2020-02-28 | 江苏东郁植物科技有限公司 | Tissue culture seedling breeding method for acer rubrum |
| CN115885844A (en) * | 2021-08-19 | 2023-04-04 | 大庆石油管理局有限公司 | Autumn flame rapid propagation method after northern domestication |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444187A (en) * | 2008-12-15 | 2009-06-03 | 北京林业大学 | Method for propagating American red-maple |
| CN101578963A (en) * | 2009-06-10 | 2009-11-18 | 重庆科技学院 | Tissue culture rapid propagation method for Japanese red maple |
| CN103828719A (en) * | 2014-03-13 | 2014-06-04 | 湖南天福林业科技有限公司 | Method for tissue culture of liquidambar styraciflua |
-
2017
- 2017-09-13 CN CN201710821960.8A patent/CN107372124A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101444187A (en) * | 2008-12-15 | 2009-06-03 | 北京林业大学 | Method for propagating American red-maple |
| CN101578963A (en) * | 2009-06-10 | 2009-11-18 | 重庆科技学院 | Tissue culture rapid propagation method for Japanese red maple |
| CN103828719A (en) * | 2014-03-13 | 2014-06-04 | 湖南天福林业科技有限公司 | Method for tissue culture of liquidambar styraciflua |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110839528A (en) * | 2019-11-22 | 2020-02-28 | 江苏东郁植物科技有限公司 | Tissue culture seedling breeding method for acer rubrum |
| CN115885844A (en) * | 2021-08-19 | 2023-04-04 | 大庆石油管理局有限公司 | Autumn flame rapid propagation method after northern domestication |
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