CN103283595A - Primary culture method of stem tip tissue of strawberry - Google Patents
Primary culture method of stem tip tissue of strawberry Download PDFInfo
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- CN103283595A CN103283595A CN2013101827089A CN201310182708A CN103283595A CN 103283595 A CN103283595 A CN 103283595A CN 2013101827089 A CN2013101827089 A CN 2013101827089A CN 201310182708 A CN201310182708 A CN 201310182708A CN 103283595 A CN103283595 A CN 103283595A
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Abstract
The invention relates to a primary culture method of stem tip tissue of strawberry. The method comprises the following steps of: (1) collecting stolon, wherein the stolon collecting time is 4-7 months, and the stolon of which the tip is between the un-spread leaf and a first spread leaf and which robustly grows is selected; (2) treating the stolon: cleaning and sterilizing the collected stolon, and carrying out low-temperature treatment under 3-8 DEG C, wherein the treatment time is 72h; (3) sterilizing and inoculating in a bechtop. The primary culture method provided by the invention has the advantages that (1) the generation of the brown stain of an explant can be effectively changed, and the survival rate of the explant can be more than 90%; (2) the explant tissue can directly form into buds by an adopted culture medium, so that the happening of heteromorphosis can be avoided, and the purity of the strain characters of the later generation can be guaranteed; (3) the diameter of the explant can be effectively controlled to be less than 0.5mm, the explant can be free from virus, and the bred offspring is completely free from virus.
Description
Technical field
The present invention relates to the first breeding method of being commissioned to train of a kind of strawberry stem tip tissue.
Background technology
Plant Tissue Breeding refers to isolate tissue, organ or the cell that suits the requirements from plant corpus, protoplast etc., by sterile working, under the manual control condition, cultivate the whole plant that obtains to regenerate or the technology of producing other products of economically valuable.This technology has wide practical use at the aspects such as preservation of breeding plant, plant corpus virus-free and the plant germplasm resource of rare plant or high economic worth fast.
Strawberry is asexually propagated plant, and maternal plant easily produces stolon, can produce new plant on the joint of stolon, breeds the offspring.But strawberry is subjected to virus infection easily, and the virus of susceptible parent can infect by stolon, makes offspring plant band poison, causes the yield and quality of offspring plant to descend.Utilize the strawberry stem tip tissue to carry out the artificial culture and propagation offspring, can effectively prevent the infection of virus, guarantee that the offspring is not malicious in spite of illness.Foster, the propagation of being commissioned to train at the beginning of stem-tip tissue cultivate to divide is cultivated, and 4 steps such as culture of rootage and domestication just are commissioned to train wherein that to support be the important step that obtains virus-free plant, also are the key technologies of strawberry stem tip tissue cultivation.
The strawberry stem tip tissue just is commissioned to train to support and is referred to the strawberry stem tip tissue is inoculated into the process that obtains virus-free plant on the specific medium, and the problem that present strawberry stem tip exists in just being commissioned to train and supporting is:
1, stem-tip tissue explant (by cutting that part of stem-tip tissue that gets off to cultivate on the living plant body) survival rate is low, and inoculation is difficult to successfully.
2, the plant aberration rate height that goes out of stem-tip tissue explant induction can not guarantee the purity of kind.
3, the plant that breeds can not be realized anosis poisoning.
Summary of the invention
The purpose of this invention is to provide a kind of strawberry stem tip breeding method of just being commissioned to train, this method can make the explant survival rate of strawberry stem tip top tissue inoculation reach more than 90%, the complete detoxification of offspring plant, does not have variation and takes place.
The applicant finds after deliberation:
1, the explant generation brown stain of strawberry stem tip tissue inoculation back is to cause survival rate to hang down main cause.A large amount of through the applicant
Research and experiment are found before the inoculation stolon to be handled 72h and the dark 72h of cultivation in inoculation back down at 5-8 ℃, can reduce the generation of brown stain, and the survival rate of explant can be brought up to more than 90%.
The formation approach of the generation of variation and bud was closely related when 2, strawberry stem tip just was commissioned to train and is supported.The approach that explantation tissue forms sprout has two kinds, and the one: the dedifferentiation of elder generation of postvaccinal explantation tissue forms callus, is differentiated to form bud by callus then; The 2nd: explantation tissue directly forms bud; The former can produce variation, and the latter does not have variation and produces.Realize not having variation and produce, key is medium.The applicant is through a large amount of research and experiment, developed that the good medium of a kind of effect: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+sucrose (3%)+agar (8%) pH is 5.8-6.0, this medium can make explantation tissue directly form bud, prevent the generation that makes a variation, guaranteed the purity of sprout kind
3, just the be commissioned to train height of the sprout virus elimination rate that support to form is inoculated the influence of explant size, and explant just can reach the effect of complete detoxification less than 0.5mm.
Based on above-mentioned research, the present invention is by low temperature treatment, and dark cultured method prevents explantation tissue's brown stain; By adopting explant is seeded on the specific medium, makes its direct development become the method for bud to prevent the generation that makes a variation; Size by the control explant improves virus elimination rate, and its concrete technical scheme is as follows:
1, the first breeding method of being commissioned to train of a kind of strawberry stem tip tissue is characterized in that comprising the steps:
1) collection of stolon: the stolon acquisition time is the 4-7 month, choose the top be in do not open up between leaf and first the exhibition leaf, the stolon of robust growth;
2) stolon is handled: after the stolon of gathering is cleaned, sterilizes, and 3-8 ℃ of following low temperature treatment, 72 hours processing times;
3) sterilization and inoculation: sterilization and inoculation are all carried out in superclean bench, and be specific as follows:
3.1 sterilization:
3.1.1 with the stolon of above-mentioned processing, begin clip 5-8cm from the top.
3.1.2 the alcohol disinfecting 15-20 second with 75%;
3.1.3 effective chlorine density is 1% liquor natrii hypochloritis, sterilizes 5-10 minute;
It is after 3.1.4 aqua sterilisa cleans, standby;
3.2 inoculation:
3.2.1 winning of explant: ecto-entad divests the blade of the peripheral parcel of growing point successively under anatomical lens, exposes up to growing point.
3.2.2 with dissecting needle needle point picking growing point top tissue, be inoculated in the medium:
Cultivate: the good explant of inoculation was secretly cultivated 72 hours down under 20 ℃, and then was positioned over and cultivates 25 ℃ of condition of culture, 2000lx, 16hr under the light; Cultivate 10-15d and form sprout, 20-30d exhibition leaf, take root, when 50-60d plant size be 3-5cm, finish first be commissioned to train foster; The composition of described medium is: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+and sucrose (3%)+agar (8%), pH is 5.8-6.0;
In the step 1), in order to guarantee the purity of kind, the stolon collection will be carried out in the kind garden, and the acquisition length of stolon is 10-15cm, to guarantee the quality of stolon.
Step 2) sterilization in is with 400 times of 84 medicining liquid dipping stolon 10-15 minute, uses distilled water flushing 10-again
15 minutes, aforementioned 400 times referred to that water dilutes 84 thimerosals by the volume ratio of 400:1.
In the step 3) from the head the reason of clip stolon 5-8cm be that to be convenient to when inoculation sterilization easy to operate;
Clean 3-5 time with aqua sterilisa in the step 3).
The multiplication factor of anatomical lens described in the step 3) is 10-40x, so that clear observation growing point, convenient sampling.
In the step 3), for guaranteeing explant size ﹤ 0.5mm, described dissecting needle pin rugosity is Φ=0.2-0.4mm, and as contrast, needle point is got the rugosity that the growing point tissue is no more than pin during operation.
In the step 3), describedly just be commissioned to train that culture vessel is test tube Φ=2.5cm when supporting, every pipe medium addition is 20-25ml.
The present invention has following advantage:
1, low temperature treatment stolon before the inoculation is secretly cultivated after the inoculation, can effectively prevent the generation of explant brown stain, guarantees that explant survives and reaches more than 90%.
2, the medium of Cai Yonging can make explantation tissue directly form sprout, prevents the generation that makes a variation, has guaranteed the purity of offspring's kind property.
3, adopt the dissecting needle of rugosity Φ=0.2-0.4mm, as contrast, the diameter that can effectively control explant has guaranteed that explant is not have virus, offspring's totally nontoxicization that it breeds less than 0.5mm during sampling.
Embodiment
1, the collection of stolon: will carry out in the kind garden for the purity stolon collection that guarantees kind, the stolon acquisition time is the 4-7 month, choose the top be in do not open up between leaf and first the exhibition leaf, the stolon of robust growth, acquisition length 10-15cm.
2, stolon is handled: with the stolon gathered with after washing 15-20 minute from the beginning, with 400 times 84 medicining liquid dipping 10-15 minute, use distilled water flushing 10-15 minute again, put into freshness protection package, to put into 5-8 ℃ refrigerator low temperature treatment, 72 hours processing times after the freshness protection package sealing.
3, sterilization and inoculation: sterilization and inoculation are all carried out in superclean bench
3.1 sterilization:
3.1.1 with the stolon of above-mentioned processing, begin clip 5-8cm from the top.
3.1.2 the alcohol disinfecting 15-20 second with 75%;
3.1.3 effective chlorine density is 1% liquor natrii hypochloritis, sterilizes 5-15 minute
3.1.4 it is standby that aqua sterilisa cleans 3-5 back.
3.2 inoculation:
3.2.1 winning of explant: ecto-entad divests the blade of the peripheral parcel of growing point successively under the 10-40x anatomical lens,
Expose up to growing point.
3.2.2 be the dissecting needle needle point picking growing point top tissue of 0.2-0.4mm with pin rugosity Φ, be inoculated in the medium, be contrast with the dissecting needle during operation, needle point is got the rugosity that the growing point tissue is no more than pin:
Cultivate: the good explant of inoculation was secretly cultivated 72 hours down under 20 ℃, and then was positioned over and cultivates 25 ℃ of condition of culture, 2000lx, 16hr under the light; Cultivate 10-15d and form sprout, 20-30d exhibition leaf, take root, when 50-60d plant size be 3-5cm, finish first be commissioned to train foster; The composition of described medium is: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+and sucrose (3%)+agar (8%), pH is 5.8-6.0; Just being commissioned to train, culture vessel is test tube Φ=2.5cm when supporting, and every pipe medium addition is 20-25ml.
Application examples 1
Strawberry cultivars: " Feng Xiang "
To infect the stolon of virus disease plant, after employing the method for the invention inoculated and cultured, the survival rate of inoculation explant is 97.5%.Adopt multiple RT-PCR detection method to detect virus, the result shows strawberry mottle virus (SMOV), strawberry yellow edge poison (SMYEV), strawberry crinkle virus, (SCV), the viral disease poison testing result of 4 kinds of harm such as strawberry veinbanding virus (SVBV) virus strawberry is all negative, and comparing virus elimination rate with plant before the inoculation is 100%.Breed seedling in Yangzhou, the result of ground test plantations such as Zhenjiang, Lianyun Harbour, Suqian, it is pure to show kind of property, blooms, uniformity as a result, no variation plant produces, and compares output raising 50-60% with the seedling of local conventional breeding.
Application examples 2
Strawberry cultivars: " red cheek "
Gather the stolon that two kinds infect the virus disease plant respectively, the survival rate of explant is 100%.Adopt multiple RT-PCR detection method to detect virus, the result shows strawberry mottle virus (SMOV), strawberry yellow edge poison (SMYEV), strawberry crinkle virus, (SCV), the viral disease poison testing result of 4 kinds of harm such as strawberry veinbanding virus (SVBV) virus strawberry is all negative, and comparing virus elimination rate with plant before the inoculation is 100%.Seedling is in more than 10 cities and counties in Jiangsu and ground test plantations such as Shaanxi, Beijing, Shanghai, Shandong, Anhui, the Inner Mongol, the result shows: various places all do not have variation and take place, bloom, uniformity as a result, compare with the seedling of local conventional breeding, the output increase is respectively Jiangsu 35-65%, Shaanxi 50-7-%, Beijing 50-70%, Shanghai 40-50%, Shandong 50-60%, Anhui 40-70%, Inner Mongol 50-70%.
Claims (7)
1. the first breeding method of being commissioned to train of strawberry stem tip tissue is characterized in that comprising the steps:
1) collection of stolon: the stolon acquisition time is the 4-7 month, choose the top be in do not open up between leaf and first the exhibition leaf, the stolon of robust growth;
2) stolon is handled: after the stolon of gathering is cleaned, sterilizes, and 3-8 ℃ of following low temperature treatment, 72 hours processing times;
3) sterilization and inoculation: sterilization and inoculation are all carried out in superclean bench, and be specific as follows:
3.1 sterilization:
3.1.1 with the stolon of above-mentioned processing, begin clip 5-8cm from the top;
3.1.2 the alcohol disinfecting 15-20 second with 75%;
3.1.3 effective chlorine density is 1% liquor natrii hypochloritis, sterilizes 5-10 minute;
It is after 3.1.4 aqua sterilisa cleans, standby;
3.2 inoculation:
3.2.1 winning of explant: ecto-entad divests the blade of the peripheral parcel of growing point successively under anatomical lens, exposes up to growing point;
3.2.2 with dissecting needle needle point picking growing point top tissue, be inoculated in the medium:
Cultivate: the good explant of inoculation was secretly cultivated 72 hours down under 20 ℃, and then was positioned over and cultivates 25 ℃ of condition of culture, 2000lx, 16hr under the light; Cultivate 10-15d and form sprout, 20-30d exhibition leaf, take root, when 50-60d plant size be 3-5cm, finish first be commissioned to train foster; The composition of described medium is: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+and sucrose (3%)+agar (8%), pH is 5.8-6.0.
2. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1, it is characterized in that: the acquisition length of stolon is 10-15cm in the step 1).
3. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1, it is characterized in that: step 2) in sterilization be with 400 times of 84 medicining liquid dipping stolon 10-15 minute, use distilled water flushing 10-15 minute again, aforementioned 400 times refer to that water dilutes 84 thimerosals by the volume ratio of 400:1.
4. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1 is characterized in that: clean 3-5 time with aqua sterilisa in the step 3).
5. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1, it is characterized in that: in the step 3), the multiplication factor of described anatomical lens is 10-40x.
6. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1, it is characterized in that: in the step 3), described dissecting needle pin rugosity Φ is 0.2-0.4mm, and needle point is got the rugosity that the growing point tissue is no more than pin.
7. the strawberry stem tip tissue breeding method of just being commissioned to train according to claim 1 is characterized in that: in the step 3), describedly just be commissioned to train that culture vessel is test tube Φ=2.5cm when supporting, every pipe medium addition is 20-25ml.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN105325298A (en) * | 2015-11-18 | 2016-02-17 | 扬州大学 | Primary culture method of lotus root stem tip tissue |
| CN105969794A (en) * | 2016-06-06 | 2016-09-28 | 江苏农林职业技术学院 | Transgenic method adopting strawberry stem tip growth point |
| CN107135949A (en) * | 2017-06-16 | 2017-09-08 | 黔东南民族职业技术学院 | Strawberry adventitious bud induction culture base and preparation method and application |
| CN112586350A (en) * | 2020-12-02 | 2021-04-02 | 蚌埠海上明珠农业科技发展有限公司 | Strawberry virus-free seedling culture method |
| CN114788495A (en) * | 2022-04-07 | 2022-07-26 | 南充市农业科学院 | Hormone-free strawberry stem tip culture method |
| CN114885837A (en) * | 2022-04-07 | 2022-08-12 | 南充市农业科学院 | Hormone-free culturing method for citrus stem tips |
| CN116686707A (en) * | 2023-06-12 | 2023-09-05 | 江苏省农业科学院 | Pretreatment method for plant stem tip tissue culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN105325298A (en) * | 2015-11-18 | 2016-02-17 | 扬州大学 | Primary culture method of lotus root stem tip tissue |
| CN105969794A (en) * | 2016-06-06 | 2016-09-28 | 江苏农林职业技术学院 | Transgenic method adopting strawberry stem tip growth point |
| CN107135949A (en) * | 2017-06-16 | 2017-09-08 | 黔东南民族职业技术学院 | Strawberry adventitious bud induction culture base and preparation method and application |
| CN112586350A (en) * | 2020-12-02 | 2021-04-02 | 蚌埠海上明珠农业科技发展有限公司 | Strawberry virus-free seedling culture method |
| CN114788495A (en) * | 2022-04-07 | 2022-07-26 | 南充市农业科学院 | Hormone-free strawberry stem tip culture method |
| CN114885837A (en) * | 2022-04-07 | 2022-08-12 | 南充市农业科学院 | Hormone-free culturing method for citrus stem tips |
| CN114788495B (en) * | 2022-04-07 | 2023-04-14 | 南充市农业科学院 | Hormone-free strawberry stem tip culture method |
| CN114885837B (en) * | 2022-04-07 | 2023-04-25 | 南充市农业科学院 | Hormone-free culture method for citrus stem tip |
| CN116686707A (en) * | 2023-06-12 | 2023-09-05 | 江苏省农业科学院 | Pretreatment method for plant stem tip tissue culture |
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