CN107439378A - A kind of method of tomato test tube seedling high efficiently multiplying - Google Patents
A kind of method of tomato test tube seedling high efficiently multiplying Download PDFInfo
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- CN107439378A CN107439378A CN201710764345.8A CN201710764345A CN107439378A CN 107439378 A CN107439378 A CN 107439378A CN 201710764345 A CN201710764345 A CN 201710764345A CN 107439378 A CN107439378 A CN 107439378A
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- test tube
- tomato
- tube seedling
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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
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a kind of method of tomato test tube seedling high efficiently multiplying, belong to field of plant tissue culture technique, be related to processing, fluid nutrient medium addition, axillary bud sprouting culture and the new test tube seedling incubation step of tomato test tube seedling mother's bottle.The present invention can substantially speed up tomato test tube seedling axillary bud sprouting; shorten the tomato sprout seedling time; a large amount of tomato test tube seedlings are quickly obtained in the short period of time; the tomato test tube seedling obtained has growing power strong and the advantages of high quality; and this method can reduce the subculture number of tomato test tube seedling; delay the increase of tomato test tube seedling aberration rate, so as to provide technical support using plant tissue culture technique large-scale production tomato seedling.
Description
Technical field
The invention belongs to field of plant tissue culture technique, more particularly to a kind of method of tomato test tube seedling high efficiently multiplying.
Background technology
Tomato (Lycopersicum esculentum Mill) is the annual herb plant of Solanaceae tomato genus tomato species,
It is main culturing vegetable or Vegetable-type fruit.In production, tomato seeds be almost F1 generation hybrid, it is necessary to the annual production of hybrid seeds or from
External import seed, seed costs can be in any more, increase production cost so that many breedings can not be widely applied.Utilize group
Knit culture technique and carry out quick breeding tomato seedling, production cost can be reduced, keep its merit, in a short time to market
A large amount of seedlings are provided, there is very high economic value.Seldom produced but the apical dominance of tomato test tube seedling is very strong, during propagation
Multiple Buds sprout from section, generally with stem section expand it is numerous so that breeding coefficient is relatively low, it is impossible to reach the upper fast numerous mesh of production
's.In tomato test tube seedling Multiplying culture, typically the whole strain of aseptic seedling is taken out, cuts the sprout of top stem section, and by base portion
Abandon, this causes waste of material to a certain extent.The applicant has found to utilize scissors clip plant stem section in pre-stage test,
Complete base portion 1~2 axillary bud stem section of band that retains is replaced in culturing room and cultivated, after a few days, base thereof is in female bottle
Can sprout new stem section, but due in old culture medium nutrient consumption excessively not enough support the stem section newly sprouted to grow, therefore
Base portion stem section is promoted to sprout, growth is key technology.
The present invention takes the mother for leaving them tissue of sprout away to tomato test tube seedling on the basis of early-stage Study
Bottle is used by certain technological means, can significantly improve the speed of tomato test tube seedling axillary bud sprouting seedling, while can
To reduce the subculture number of test tube seedling, delay the increase of tomato test tube seedling aberration rate, improve the efficiency for producing high-quality tomato seedling,
To provide technical support using plant tissue culture technique large-scale production tomato seedling.
The content of the invention
The technical problem to be solved in the present invention, it is to provide a kind of method of tomato test tube seedling high efficiently multiplying.
What the present invention was realized in：
A kind of method of tomato test tube seedling high efficiently multiplying of the present invention, comprises the following steps：
(1) processing of tomato test tube seedling mother bottle：It is sterile from tomato test tube seedling to cut for expanding numerous sprout, stay in female bottle
Interior base portion retains 1~2 axillary bud, and the stem section that 0.3~0.5cm grows is stayed on axillary bud top, then wipes out the blade on axillary bud,
The petiole for staying 0.3~0.5cm to grow；
(2) fluid nutrient medium adds：By tomato test tube seedling mother's bottle after step (1) processing, under aseptic condition, liquid is added
Body culture medium, wherein Liquid Culture based component are additional 0.1-1.5mg/L 6-benzyladenines and 0.1-0.5mg/L indoles fourths
The MS fluid nutrient mediums of acid, pH are 5.8~6.2；
(3) axillary bud sprouting culture：Step (2) is added to tomato test tube seedling mother's bottle of fluid nutrient medium, first in low light condition
2~3d of lower culture, intensity of illumination is then gone to as under the conditions of 3000~5000lx, daily 10~12h of illumination, it is further cultured for 23~
28d, obtain the tomato test tube seedling formed after axillary bud sprouting；
(4) new test tube seedling culture：By the tomato test tube seedling described in step (3), repeat step (1)-(3) are cultivated,
To produce new sprout.
It is highly preferred that the composition of step (2) described fluid nutrient medium be additional 1.0mg/L 6-benzyladenines and
The MS fluid nutrient mediums of 0.5mg/L indolebutyric acids, pH are 5.8~6.2.
Step (3) described low light condition is daily 10~12h of illumination in the case where intensity of illumination is less than 500lx.
According to the method for above-mentioned steps, the culture of sustainable 5~6 wheel.
The beneficial effects of the present invention are：Tomato test tube seedling axillary bud sprouting can be substantially speeded up, when shortening tomato sprout seedling
Between (in tomato test tube seedling Multiplying culture, traditional tomato test tube seedling Multiplying culture need 35~40d, using the present invention method,
As long as 25~30d, shortening 10~15d), a large amount of tomato test tube seedlings, the tomato obtained are quickly obtained in the short period of time
Test tube seedling has growing power strong and the advantages of high quality, and this method can reduce the subculture number of tomato test tube seedling (for control kind
Eggplant test tube seedling aberration rate, the general generation of tomato Plantlet subculture culture 13~15 will from new materials, using the method for the present invention, by
Then from the direct axillary bud sprouting of female bottle, the problem of without considering subculture number, so within the same time, can significantly subtract
Few subculture number), delay the increase of tomato test tube seedling aberration rate, so as to be to utilize plant tissue culture technique large-scale production kind
Eggplant seedling provides technical support.
Embodiment
A kind of method of tomato test tube seedling high efficiently multiplying, the present invention is specifically described with reference to embodiment, implements
Without specified otherwise it is prior art in example, blake bottle volume is 240mL.Comprise the following steps that：
First, the processing of tomato Shoots in vitro mother bottle
Taking the tomato test tube seedling mother bottle of robust growth, (initial materials can use kind obtained through the culture of tomato seed asepsis
Eggplant test tube seedling), under conditions of sterile, with scissors from test tube seedling stem section base portion (staying 1~2 axillary bud) clip test tube seedling sprout in
On aseptic filter paper, the sprout cut is used for Multiplying culture or culture of rootage.Axillary bud top stays 0.3~0.5cm to grow in cutting process
Stem section, and the blade on axillary bud is wiped out, the petiole for staying 0.3~0.5cm to grow.
2nd, fluid nutrient medium adds
1st, operating method
In the female bottle for taking out test tube seedling, the base portion stem section of 1~2 axillary bud of band is left, 7 kinds of different liquid are added in female bottle
Body culture medium, culture medium prescription are as follows：
Y1：MS+BA 0.1mg/L+IBA 0.1mg/L
Y2：MS+BA 0.2mg/L+IBA 0.1mg/L
Y3：MS+BA 0.5mg/L+IBA 0.1mg/L
Y4：MS+BA 1.0mg/L+IBA 0.1mg/L
Y5：MS+BA 1.5mg/L+IBA 0.1mg/L
Y6：MS+BA 1.0mg/L+IBA 0.2mg/L
Y7：MS+BA 1.0mg/L+IBA 0.5mg/L
Every liter of culture medium adds sucrose 30g/L, pH value 5.8~6.2.Each fluid nutrient medium adds 9 bottles respectively, each
Contain three base portion stem sections in female bottle, totally 63 bottles, every bottle is added 3mL fluid nutrient mediums.
The MS culture mediums specifically include following composition：Ammonium nitrate (NH4NO3) 1650mg/L, potassium nitrate (KNO3)
1900mg/L, calcium chloride (CaCl22H2O) 440mg/L, magnesium sulfate (MgSO4·7H2O) 370mg/L, potassium dihydrogen phosphate
(KH2PO4) 170mg/L, manganese sulfate (MnSO4·H2O) 22.3mg/L, zinc sulfate (ZnSO4·7H2O) 8.6mg/L, cobalt chloride
(CoCl2·6H2O) 0.025mg/L, copper sulphate (CuSO4·5H2O) 0.02mg/L, boric acid (H3BO3) 6.2, sodium molybdate
(Na2MoO4·2H2O) 0.25mg/L, KI (KI) 0.83mg/L, ferrous sulfate (FeSO4·7H2O) 28.7mg/L, ethylenediamine
Tetraacethyl disodium (Na2- EDTA) 37.3mg/L, nicotinic acid 0.5mg/L, vitamin B60.5mg/L, vitamin B10.1mg/L, flesh
Alcohol 100mg/L, glycine 2mg/L；Described BA is 6-benzyladenine；The IBA is indolebutyric acid.
2nd, fluid nutrient medium optimizes
1) fluid nutrient medium of different BA concentration is added
BA can induced dormancy bud sprout, promote stem, leaf elongation growth.In the tomato test tube seedling proliferation test of early stage
In, it is found that additional BA cultivation effect is substantially better than other class basic elements of cell division, therefore this experiment is carried out to BA concentration
Optimization.By be separately added into tomato test tube seedling mother's bottle of the base portion stem section of the axillary bud of band one 3ml add different BA concentration liquid increase
Grow culture medium Y1~Y5.Its sprout growing state is observed after culture 10d, records the data of each observation item.
2) influence that different BA concentration grow to female bottle tomato stem section
As can be seen that being 0.1~1.5mg/ in BA concentration from female bottle them stem segment with axillary buds germination rate (being shown in Table 1)
During L, with the increase of BA concentration, the change of the germination rate of tomato stem segment with axillary buds is turned to first to reduce and raised again.BA concentration is 0.1mg/L
When, tomato mother's bottle base portion stem section germination rate is up to 88.89%.Tomato test tube seedling apical dominance is obvious, and the incidence of clump bud is low,
Propagation is realized mainly by section, therefore this experiment is counted to effective joint number, plant height and the number of blade of tomato test tube seedling
Analyze (being shown in Table 2).Have in data analysis comparative result and can be seen that：Y1 average plant height highest, it is 1.96cm.But Y1 is averagely saved
Number is minimum, significant difference be present between other several processing in average effective joint number, is 1.11.Y1 mean leaf
Number leaf is also minimum.But there was no significant difference for average plant height and average leaf number between each processing.The shoot proliferation of tomato is main
It is that in all processing, the growth trend of average effective joint number is reduced afterwards first to increase by the axillary bud in stem section.Y4 average effective
Joint number 1.81 is highest.Axillary bud sprouting can be promoted by illustrating the BA of low concentration, and be advantageous to the elongation growth behind stem section, but low
Concentration BA is unfavorable for the differentiation of axillary bud in stem section.Also the differentiation of axillary bud can be suppressed during BA excessive concentrations.
Influences of the BA of table 1 to tomato mother's bottle them stem segment with axillary buds germination rate
Influences of the BA of table 2 to tomato test tube seedling plant height, effective joint number and the number of blade
Note：Different letters represent the Deng Kenshi multiple range test significances of difference in table, and wherein capitalization is difference pole
Significantly (P<0.01), lowercase is significant difference (P<0.05).Similarly hereinafter.
3) fluid nutrient medium of different IBA concentration is added
The mutual cooperation of auxin and the basic element of cell division, be advantageous to the Growth and Differentiation of Tissue-cultured Tomato Plants.It is raw in experimentation
Long plain IBA can promote to effectively facilitate the propagation of Tissue-cultured Tomato Plants.During adding liquid medium culture, suitable IBA is dense
Spend most important.Three concentration of this Setup Experiments are carried out on the basis of previous experiments.Its concentration is respectively 0.1mg/
L、0.2mg/L、0.5mg/L.Corresponding culture medium prescription is Y4, Y6, Y7.By the tomato test tube seedling of the base portion stem section of the axillary bud of band one
Liquid proliferated culture medium Y4, Y6, Y7 that 3mL adds different IBA concentration are separately added into female bottle.Its sprout is observed after culture 10d
Growing state, record the data of each observation item.
4) influence that different IBA concentration grow to female bottle tomato stem section
From table 3 it can be seen that influence of two processing of Y4 and Y6 to tomato mother's bottle them stem segment with axillary buds germination rate
It is similar, germination rate is 81.48%.Y4 and Y5 is compared in Y7 processing, and it promotes base portion stem segment with axillary buds sprouting effect preferable, reaches
To 88.89%.In the growth in later stage, as can be seen from Table 4,3 concentration processing, to tomato test tube seedling plant height, effective joint number
Influence with the number of blade does not have significant difference.Analyzed from average value；Y7 processing average plant height, average effective joint number,
And numerical value is all highest on average leaf number, it may be said that bright first than in the IBA of low concentration, the IBA of higher concentration is to tomato
The growth of tissue-cultured seedling has certain facilitation.
Influences of the IBA of table 3 to tomato mother's bottle them stem segment with axillary buds germination rate
Influences of the IBA of table 4 to tomato test tube seedling plant height, effective joint number and the number of blade
5) most suitable fluid nutrient medium recipe determination
With reference to tomato mother's bottle base portion stem section growing state statistics after addition Y1~Y7, further analyze, compare, so as to
Filter out the culture medium prescription of most suitable base portion stem section growth.Y4 processing is one processing of average effective joint number value highest.Cause
6-BA concentration 1.0mg/L during this Y6/Y7 is handled using Y4 is used as fixed concentration, the change of progress IBA concentration.It can be seen by table 5
Go out：Simultaneously significant difference is not present in average plant height between 7 processing, Y1 highests in average plant height, is 1.96cm, Y7 next,
For 1.81cm.Y7 is 2.15 in average effective joint number, most for mean number in 7 processing.And exist between Y2, Y3 aobvious
Write between sex differernce, with Y1 and pole significant difference be present.Also it is not present finally again on average leaf number, between 7 processing notable
Sex differernce.From average, Y6 and Y7 average leaf number are most, reach 3.15.
The situation of change of the tomato test tube seedling plant height of table 5, effective joint number and the number of blade
In summary tomato test tube seedling indices are analyzed, binding tube seedling stem thickness, the color of blade, final to determine
The Liquid Culture based formulas for being adapted to tomato test tube seedling fast breeding is Y7：MS+BA 1.0mg/L+IBA 0.5mg/L are best suitable for.
3rd, axillary bud sprouting culture
By the female bottle for leaving 1~2 axillary bud base portion of tomato test tube seedling band of the addition fluid nutrient medium described in step (2),
2~3d is first cultivated under low light condition (intensity of illumination is less than 500lx, daily 10~12h of illumination), then going to intensity of illumination is
3000~5000lx LED, daily 10~12h of illumination, is further cultured for 23~28d or so, can obtain formed after axillary bud sprouting it is new
Tomato Shoots in vitro；It is (25 ± 1) DEG C to cultivate room temperature.Found in experiment, dim light 2~3d of culture, be advantageous to tomato base portion
The recovery of cut out portion, while be advantageous to the sprouting of axillary bud.
4th, new Shoots in vitro is cut
By the new tomato Shoots in vitro described in step (3), under aseptic condition, the method as described in step (1) operates,
Take new sprout away, female bottle continuously adds 3mL fluid nutrient mediums and cultivated, and to produce new sprout, is so repeated, can
Continue the culture of 5~6 wheels.More than the tomato them of 6 wheels, the speed and germination rate of its axillary bud sprouting can decline, Ying Chong
New materials.
In tomato test tube seedling Multiplying culture, traditional tomato test tube seedling Multiplying culture wants 35~40d, using the side of the present invention
Method, as long as 25~30d, it can so shorten 10~15d of tomato test tube seedling seedling time.In tomato test tube seedling incubation,
To control tomato test tube seedling aberration rate, in the general generation of tomato Plantlet subculture culture 13~15, will be from new materials, using the present invention
Method, due to being from the direct axillary bud sprouting of female bottle, 5~6 wheels are from female bottle axillary bud sprouting, the problem of without considering subculture number,
So within the identical time, subculture number can be greatly reduced, so as to delay the increase of tomato test tube seedling aberration rate.
Although the foregoing describing the embodiment of the present invention, those familiar with the art should manage
Solution, the specific embodiment described by us are merely exemplary, rather than for the restriction to the scope of the present invention, are familiar with this
The equivalent modification and change that the technical staff in field is made in the spirit according to the present invention, should all cover the present invention's
In scope of the claimed protection.
Claims (3)
- A kind of 1. method of tomato test tube seedling high efficiently multiplying, it is characterised in that：Comprise the following steps：(1) processing of tomato test tube seedling mother bottle：It is sterile from tomato test tube seedling to cut for expanding numerous sprout, stay in female bottle Base portion retains 1~2 axillary bud, and the stem section that 0.3~0.5cm grows is stayed on axillary bud top, is then wiped out the blade on axillary bud, is stayed The petiole of 0.3~0.5cm length；(2) fluid nutrient medium adds：By tomato test tube seedling mother's bottle after step (1) processing, under aseptic condition, liquid training is added Base is supported, wherein Liquid Culture based component is additional 0.1-1.5mg/L 6-benzyladenine and 0.1-0.5mg/L indolebutyric acids MS fluid nutrient mediums, pH are 5.8~6.2；(3) axillary bud sprouting culture：Step (2) is added to tomato test tube seedling mother's bottle of fluid nutrient medium, first trained under low light condition 2~3d is supported, then goes to intensity of illumination under the conditions of 3000~5000lx, daily 10~12h of illumination, to be further cultured for 23~28d, obtaining The tomato test tube seedling formed after to axillary bud sprouting；(4) new test tube seedling culture：By the tomato test tube seedling described in step (3), repeat step (1)-(3) are cultivated, with production Raw new sprout.
- 2. the method for tomato test tube seedling high efficiently multiplying according to claim 1, it is characterised in that：Step (2) described liquid The composition of culture medium is the MS fluid nutrient mediums of additional 1.0mg/L 6-benzyladenines and 0.5mg/L indolebutyric acids, pH 5.8 ~6.2.
- 3. the method for tomato test tube seedling high efficiently multiplying according to claim 1, it is characterised in that：Step (3) described dim light Condition is daily 10~12h of illumination in the case where intensity of illumination is less than 500lx.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108541574A (en) * | 2018-05-04 | 2018-09-18 | 孟静 | A kind of cultural method improving Lycopene in Tomatoes content |
| CN110358790A (en) * | 2019-07-12 | 2019-10-22 | 河南科技学院 | The method of one plant fast genetic transformation or virus infection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105145360A (en) * | 2015-09-17 | 2015-12-16 | 福建省农业科学院农业工程技术研究所 | Rapid propagation method for tomato test tube seedling capable of improving proliferation rate |
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2017
- 2017-08-30 CN CN201710764345.8A patent/CN107439378A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105145360A (en) * | 2015-09-17 | 2015-12-16 | 福建省农业科学院农业工程技术研究所 | Rapid propagation method for tomato test tube seedling capable of improving proliferation rate |
Non-Patent Citations (1)
| Title |
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| 赖呈纯等: "夏日阳光’番茄侧芽离体培养快繁技术", 《中国农学通报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108541574A (en) * | 2018-05-04 | 2018-09-18 | 孟静 | A kind of cultural method improving Lycopene in Tomatoes content |
| CN110358790A (en) * | 2019-07-12 | 2019-10-22 | 河南科技学院 | The method of one plant fast genetic transformation or virus infection |
| CN110358790B (en) * | 2019-07-12 | 2023-02-24 | 河南科技学院 | Method for quickly genetically transforming or infecting plant with virus |
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