CN105325291B - The method for tissue culture of F. pendula Bean - Google Patents
The method for tissue culture of F. pendula Bean Download PDFInfo
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- CN105325291B CN105325291B CN201510621194.1A CN201510621194A CN105325291B CN 105325291 B CN105325291 B CN 105325291B CN 201510621194 A CN201510621194 A CN 201510621194A CN 105325291 B CN105325291 B CN 105325291B
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- 241001065644 Ficus asperifolia Species 0.000 title claims abstract description 29
- 244000046052 Phaseolus vulgaris Species 0.000 title claims abstract description 29
- 235000010627 Phaseolus vulgaris Nutrition 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 29
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 18
- 239000005720 sucrose Substances 0.000 claims description 18
- 229920001817 Agar Polymers 0.000 claims description 17
- 239000008272 agar Substances 0.000 claims description 17
- 238000012549 training Methods 0.000 claims description 4
- 230000032696 parturition Effects 0.000 claims description 2
- 230000012010 growth Effects 0.000 abstract description 17
- 235000014001 Prunus serrulata Nutrition 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 230000006698 induction Effects 0.000 abstract description 4
- 230000032683 aging Effects 0.000 abstract description 3
- 230000004069 differentiation Effects 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 244000141698 Prunus lannesiana Species 0.000 abstract 1
- 239000000463 material Substances 0.000 description 11
- 241000392970 Prunus serrulata Species 0.000 description 10
- 238000009395 breeding Methods 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000012545 processing Methods 0.000 description 4
- 241000167854 Bourreria succulenta Species 0.000 description 3
- 235000019693 cherries Nutrition 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229930191978 Gibberellin Natural products 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 2
- 239000003448 gibberellin Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 235000003325 Ilex Nutrition 0.000 description 1
- 241000209035 Ilex Species 0.000 description 1
- 240000007189 Prunus angustifolia Species 0.000 description 1
- 235000010873 Prunus angustifolia Nutrition 0.000 description 1
- 235000005706 Prunus prostrata Nutrition 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
Classifications
-
- 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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- 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 discloses a kind of method for tissue culture of F. pendula Bean, including：Preparation, Primary culture, Multiplying culture and the culture of rootage of explant；The culture medium of the Multiplying culture contains 6 0.2~0.5mg/L of BA, NAA0.03~0.07mg/L and GA5~15mg/L.The tissue culture method of F. pendula Bean of the present invention has relatively low pollution rate; start effect good; the induction differentiation rate of Primary culture can reach 84%; the Multiplying culture stage being capable of normal jointing; tissue-cultured seedling robust growth; the problem of shortened internodes generally occurred in traditional oriental cherry tissue culture procedures, easy aging is solved, and growth coefficient can reach 7.0.
Description
Technical field
The present invention relates to field of plant tissue culture technique, more particularly to a kind of method for tissue culture of F. pendula Bean.
Background technology
The traditional modes of reproduction of oriental cherry is propagation by grafiting, but its seedling is slow, it is difficult to a large amount of quick productions.With tissue cultures
The development of technology, quick breeding and factorial praluction of the tissue cultures for oriental cherry provides an effective way.Domestic scholars pair
The research of oriental cherry is concentrated mainly in the species and kind such as underbrown japanese cherry, Tokyo oriental cherry, Cyclobanopsis chungii, wild mountain cherry.F. pendula Bean
Oriental cherry is viewed and admired for what is just grown up in recent years, is hung down because its branch is soft elegant, attractive in appearance, more and more applied to afforestation
In beautification.
Mainly have for the research in terms of F. pendula Bean tissue cultures：It is early that Nanjing Forestry University honor Chinese ilex etc. reports weeping branch
The tissue culture technology of cherry " red branch hangs down ", using stem with bud as explant, primary culture medium is MS+BA1.0mg/L+NAA0.1mg/L,
The inductivity of bud is 80%；Proliferated culture medium is MS+BA0.5mg/L+NAA0.1mg/L+ sucrose 35g/L, and growth coefficient is
4.15；Root media is 1/2MS+NAA1.0mg/L+IBA1.0mg/L, and rooting rate is up to 95% (Rong Dongqing, Wang Xian honor weeping branch
The tissue-culturing rapid propagation experiment forestry science and technology exploitations of early cherry " red branch hangs down ", 2008).
Xu Zhao ripples et al. are also studied with regard to the Introduction Observation and reproduction technique of F. pendula Bean, and training is organized in reproduction technique
Support the tissue culture only with regard to F. pendula Bean cold-resistant stock to be studied, the propagating materials used is resting bud and bud of sprouting, but research
In do not form intact plant (the F. pendula Beans Introduction Observation such as Xu Zhaobo, Chen Xiuyun and raising technology research Laiyang Agricultural Colleges
Report, 2001).
In summary, it is less for the research report of the method for tissue culture of F. pendula Bean at present, and not system, and deposit
In some not high problems of breeding coefficient.To find out its cause, in the tissue culture procedures of oriental cherry, most of species or kind all can
Run into internode not extend, easy aging, or leaf rolling occur, the phenomenon such as vitrifying, only this problem is thoroughly solved, can just made
Tissue culture reaches preferable effect.
The content of the invention
It is an object of the invention to provide a kind of method for tissue culture of F. pendula Bean, to overcome the tissue of existing F. pendula Bean
Shortened internodes present in culture, the problem of growth coefficient is low.
To reach above-mentioned purpose, the present invention is achieved by the following technical solutions：
The method for tissue culture of F. pendula Bean of the present invention, including：Preparation, Primary culture, the propagation training of explant
Support and culture of rootage；The culture medium of the Multiplying culture contains 6-BA0.2~0.5mg/L, NAA0.03~0.07mg/L and GA5
~15mg/L.
Present invention improvement proliferation culture medium formula, can solve the problem that the shortened internodes of generally existing, line of breeding in F. pendula Bean
The problem of number is low, it is preferred that the formula of the culture medium of the Multiplying culture is：MS+6-BA0.2~0.5mg/L+NAA0.03~
5~8g/L of 0.07mg/L+GA5~15mg/L+ sucrose 30~40g/L+ agar.
It is further preferred that in the culture medium of the Multiplying culture, 6-BA concentration is 0.3~0.5mg/L, and NAA's is dense
Spend for 0.05mg/L, GA concentration is 10mg/L.Further, in the culture medium of the Multiplying culture, 6-BA concentration is
0.3mg/L or 0.5mg/L.
It is preferred that, described explant is stem segment with axillary bud or stem apex, is derived from the middle and upper part for giving birth to spring slightly branch then.
After sterilization, the two ends otch of stem apex base portion, stem segment with axillary bud is trimmed, 1~1.5cm simple bud stem is trimmed to
Point or stem-segment with single bud carry out Primary culture.
It is preferred that, the formula of the culture medium of the Primary culture is：
MS+BA0.4~0.6mg/L+NAA0.03~5~8g/L of 0.07mg/L+ sucrose 30~40g/L+ agar.
It is further preferred that in the culture medium of the Primary culture, 6-BA concentration is 0.5mg/L, and NAA concentration is
0.05mg/L。
In proliferated culture medium and primary culture medium, the concentration of sucrose is specifically as follows 30g/L, and the concentration of agar specifically can be with
For 6.5g/L.
It is preferred that, the method for the Multiplying culture includes：After Primary culture 20~30 days, take 1~2cm's from tissue-cultured seedling
Stem-segment with single bud, is vertically inoculated in culture medium and is cultivated.
Compared with prior art, beneficial effects of the present invention are：The tissue culture method of F. pendula Bean of the present invention has relatively low
Pollution rate, it is good to start effect, and the induction differentiation rate of Primary culture can reach 84%, and the Multiplying culture stage being capable of normally jointing, group
Seedling robust growth is trained, the problem of shortened internodes generally occurred in traditional oriental cherry tissue culture procedures, easy aging is solved, and
Growth coefficient can reach 7.0.
Brief description of the drawings
Fig. 1 is that embodiment 1 carries out disinfection the picture of sterilizing to explant；
Fig. 2 is the picture when explant of embodiment 1 is inoculated into primary culture medium；
Fig. 3 is the Primary culture picture of 10 days of embodiment 1；
Fig. 4 is the Multiplying culture picture of 20 days of embodiment 1；
Fig. 5 is the Multiplying culture picture of 25 days of embodiment 1；
Fig. 6 is the picture of the 20 days complete stools of culture of rootage of embodiment 1；
Fig. 7 is the picture of the 20 days roots of culture of rootage of embodiment 1；
Fig. 8 is training of the embodiment 2 using only addition 6-BA0.5mg/L and the plant growth regulators of NAA0.05mg/L two
Support base and carry out the Multiplying culture picture of 20 days.
Embodiment
With reference to specific embodiment, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention, after the present invention has been read, various equivalences of the those skilled in the art to the present invention
The modification of form falls within the application appended claims limited range.
Embodiment 1
Material：Material is one of oriental cherry kind " F. pendula Bean ", chooses and gives birth to stem segment with axillary bud then.
The method for tissue culture of the present embodiment F. pendula Bean comprises the following steps：
1st, it is acquired for materials
At the noon of 3-5 month fair weather conditions, the healthy and strong disease-free spray upper position of interception F. pendula Bean is as outer
Implant, removes blade and stays petiole.It is noted that the Optimalities of selected materials, pure, accuracy when gathering explant, never at will
Take, in order to avoid cause unnecessary loss.Growth selection is healthy and strong, the spring of no disease and pests harm slightly branch section is material, to the material adopted
To be inoculated with time, prevent that dehydration from being wilted or pollution is gone rotten.
2nd, pretreatment
The oriental cherry material for gathering is repaired, removes unwanted part, cuts into 2-3cm segments, every section at least
Retain 1-2 axillary bud, then material is placed in washing powder and soaked 15 minutes, 1h is rinsed repeatedly with flowing water.
3rd, sterilization
After being pre-processed to material, it is transferred to superclean bench and carries out disinfection processing.Explant is cut into 1-1.5cm length
Stem apex or stem section with an axillary bud, with 70% ethanol postincubation 1min, then with 0.1% HgCl2Disinfect
10min, constantly rocks during processing, material is sufficiently achieved sterilization effect, then with aseptic water washing 5 times.
4th, Primary culture
It will slightly be trimmed by stem apex base portion, the stem section two ends otch of sterilization, be trimmed to 1-1.5cm simple bud stem apex
Or stem-segment with single bud.Vertical type is inoculated in primary culture medium and cultivated, and cultivation temperature is 25 DEG C, intensity of illumination 3000lx, light
According to 12h/ days time.
The formula of primary culture medium is：MS+6-BA0.5mg/L+NAA0.05mg/L+ sucrose 30g/L, and additional agar
6.5g/L, pH value is adjusted to 5.8.
5th, Multiplying culture
The tissue-cultured seedling that will be grown by Primary culture 25d, intercepts into 1.5cm or so stem-segment with single bud, subtracts bottom larger
Can touch the blade of culture medium, be vertically inoculated in proliferated culture medium and cultivated, cultivation temperature be 25 DEG C, intensity of illumination
3000lx, light application time 12h.
The formula of proliferated culture medium is：MS+6-BA0.3mg/L+NAA0.05mg/L+GA10mg/L+ sucrose 30g/L, and it is attached
Plus agar 6.5g/L, pH value is adjusted to 5.8.
6th, culture of rootage
After Multiplying culture 20d, healthy and strong tissue-cultured seedling is chosen, the callus that base portion expands is cut off, and culture can be touched
The blade of base, is transferred to the induction that root media carries out root.Root media is：
1/2MS+NAA1.0mg/L+IBA0.5mg/L+ sucrose 20g/L, additional agar 6.5g/L, pH value is adjusted to 5.8.
Fig. 1~Fig. 7 for the present embodiment F. pendula Bean tissue culture procedures in different phase picture, through statistics, this reality
The induction differentiation rate for applying exception implant is 84%, and growth coefficient is 7.0, and rooting rate is up to 93%, and tissue-cultured seedling robust growth, internode
Substantially elongation,.
The optimization of the proliferation culture medium formula of embodiment 2
In addition to proliferation culture medium formula is different from embodiment 1, remaining method and step is same as Example 1.
1st, the influence that difference 6-BA and NAA concentration is bred to F. pendula Bean
The formula of proliferated culture medium is：MS+6-BA+NAA+ sucrose 30g/L+ agar 6.5g/L, pH value is adjusted to 5.8.6-
BA and NAA concentration is referring to table 1.
Influence of the 6-BA and NAA various concentrations of table 1 to breeding in F. pendula Bean tissue cultures
As shown in table 1, the growth coefficient of all processing is relatively low, between 1.1~2.2.
2nd, the influence that minimal medium, 6-BA and NAA breed to F. pendula Bean
The formula of proliferated culture medium is：Minimal medium+6-BA+NAA+ sucrose 30g/L+ agar 6.5g/L, pH value adjustment
For 5.8.6-BA and NAA concentration, the species of minimal medium is referring to table 2.
By minimal medium 1/2MS, MS, 1/4MS, basic element of cell division BA (0.1,0.5,1.0), NAA (0.02,0.05,
0.1) it, have chosen the L of the level of three factor three9(34) orthogonal design, result of the test such as table 2, the growth coefficients of all processing still compared with
It is low.
The influence of the minimal medium of table 2,6-BA and NAA to breeding in F. pendula Bean tissue cultures
Note：Growth coefficient=propagation bud number/original inoculation bud number, propagation bud number is uniformly trimmed to 1-1.5cm height of seedling.
Above-mentioned two result of the test is found：In tested formula, growth coefficient is relatively low, main cause be internode not
Elongation, height of seedling is shorter, although MS+6-BA0.5mg/L+NAA0.1mg/L formula growth coefficient is 2.4, MS+6-BA0.5mg/L
+ NAA0.05mg/L formula growth coefficient is 2.2, but cultivation effect is still poor, and two kinds of formula Analysis of variance are not notable
Difference.
3rd, hormonal readiness is adjusted
In order to solve the problem of internode does not extend, internode can be promoted profound red mould is with the addition of on the basis of above-mentioned formula
Plain GA, and appropriate adjustment has been carried out to other hormone concentrations, formula as below is tested respectively (pH of culture medium is 5.8)：
(1) MS+6-BA0.5mg/L+NAA0.05mg/L+ sucrose 30g/L+ agar 6.5g/L；
(2) MS+6-BA0.5mg/L+NAA0.05mg/L+GA5mg/L+ sucrose 30g/L+ agar 6.5g/L；
(3) MS+6-BA0.5mg/L+NAA0.05mg/L+GA10mg/L+ sucrose 30g/L+ agar 6.5g/L；
(4) MS+6-BA0.5mg/L+NAA0.05mg/L+GA15mg/L+ sucrose 30g/L+ agar 6.5g/L；
(5) MS+6-BA0.3mg/L+NAA0.05mg/L+ sucrose 30g/L+ agar 6.5g/L；
(6) MS+6-BA0.3mg/L+NAA0.05mg/L+GA5mg/L+ sucrose 30g/L+ agar 6.5g/L；
(7) MS+6-BA0.3mg/L+NAA0.05mg/L+GA10mg/L+ sucrose 30g/L+ agar 6.5g/L；
(8) MS+6-BA0.3mg/L+NAA0.05mg/L+GA15mg/L+ sucrose 30g/L+ agar 6.5g/L.
Influence of the hormon of table 3 to breeding in F. pendula Bean tissue cultures
Experimental result such as table 3, from the above in can be seen that with the addition of gibberellin GA with weeping branch cherry can be obviously promoted
The elongation of flower internode, so as to improve height of seedling, increases growth coefficient, and seedling growth is healthy and strong.Gibberellin addition concentration is low to promote effect
Fruit is not obvious, although concentration can also promote internode elongation very well when reaching 15mg/L, easily tip easily turns yellow nursery stock, causes point
End is dead.And it is best with the effect for adding 10mg/L.Due to culture medium (3) and (7), difference is not obvious in culture effect, it is considered to
To cost-effective, the Optimum formulae that (7) are Multiplying culture is selected.
Claims (4)
1. a kind of method for tissue culture of F. pendula Bean, including：Preparation, Primary culture, Multiplying culture and the training of taking root of explant
Support；Characterized in that, the culture medium of the Multiplying culture contains 0.3~0.5mg/L of 6-BA, NAA0.05mg/L and GA10mg/
L；The method of the Multiplying culture includes：After Primary culture 20~30 days, 1~2cm stem-segment with single bud is taken from tissue-cultured seedling, vertically
It is inoculated in culture medium and is cultivated.
2. the method for tissue culture of F. pendula Bean as claimed in claim 1, it is characterised in that the culture medium of the Primary culture
Formula be：MS+BA0.4~0.6mg/L+NAA0.03~5~8g/L of 0.07mg/L+ sucrose 30~40g/L+ agar.
3. the method for tissue culture of F. pendula Bean as claimed in claim 2, it is characterised in that the culture medium of the Primary culture
In, 6-BA concentration is 0.5mg/L, and NAA concentration is 0.05mg/L.
4. the method for tissue culture of F. pendula Bean as claimed in claim 1, it is characterised in that described explant is band axillary bud
Stem section or stem apex, are derived from the middle and upper part for giving birth to spring slightly branch then.
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| CN106538392B (en) * | 2016-12-08 | 2019-04-19 | 上海杉一植物科技有限公司 | A kind of white oriental cherry tissue culture and rapid proliferation method |
| CN108271687B (en) * | 2017-01-06 | 2021-04-20 | 江苏省农业科学院宿迁农科所 | Tissue culture method of cerasus serrulata |
| CN110521595B (en) * | 2019-09-03 | 2021-11-30 | 江苏农林职业技术学院 | Method for promoting stem elongation in vitro culture of oriental cherry |
| CN110476813B (en) * | 2019-09-03 | 2021-11-30 | 江苏农林职业技术学院 | In-vitro rapid propagation method for oriental cherry |
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| CN102217534B (en) * | 2011-04-19 | 2012-07-25 | 南京林业大学 | Cerasus campanulata somatic cell embryogenesis method |
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| 垂枝早樱‘红枝垂’组培快繁试验;荣冬青等;《林业科技开发》;20081231;第22卷(第5期);第72-75页，尤其是摘要以及第73页第1.1节，第1.3节。 * |
| 福建山樱花的组织培养及植株再生;王光萍等;《南京林业大学报（自然科学版）》;20020331;第26卷(第2期);第73-75页,尤其是第74页第2.1、2.2节。 * |
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Application publication date: 20160217 Assignee: Shanghai Xuzhi Landscape Greening Co.,Ltd. Assignor: JIANGSU POLYTECHNIC College OF AGRICULTURE AND FORESTRY Contract record no.: X2023980047549 Denomination of invention: The tissue culture method of weeping cherry blossom Granted publication date: 20170811 License type: Common License Record date: 20231120 |