CN101838630A - Method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry - Google Patents
Method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry Download PDFInfo
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- CN101838630A CN101838630A CN200910020731A CN200910020731A CN101838630A CN 101838630 A CN101838630 A CN 101838630A CN 200910020731 A CN200910020731 A CN 200910020731A CN 200910020731 A CN200910020731 A CN 200910020731A CN 101838630 A CN101838630 A CN 101838630A
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- rejuvenation
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Abstract
The invention provides a method for preparing a rejuvenation culture medium for the tissue culture of high-bush blueberry. The method comprises the following steps: (1) treating explants materials, i.e., selecting and treating explants materials, and preparing explants satisfying the requirements at the balanced point of the minimal killing rate and contamination rate; (2) inducing, i.e., inoculating the explants in step (1) on a culture medium, and inducing the explants to bud under the condition of inducing, wherein the culture medium is particularly an improved agar synthetic medium; and (3) carrying out the rejuvenation culture, i.e., carrying out the enrichment culture on the shoots of the cluster buds obtained in step (2) in a rejuvenation culture medium.
Description
Technical field:
The invention belongs to technical field of bioengineering, relate to plant tissue culture technique, be specifically related to a kind of preparation and application of industrialized tissue culture and rapid propagation of high-bush blueberry rejuvenation substratum.
Background technology:
High-bush blueberry (Blueberry), the formal name used at school cowberry is perennial shrub, originates in the North America.Generally can't form seed, adopt propagation methods such as cuttage, press strip mostly, but it is relatively stricter that high-bush blueberry requires temperature, soil, weather etc., most kinds are difficult for breeding, the cuttage maintenance is more difficult, and the degeneration of high-bush blueberry germ plasm resource is comparatively serious, and the quality of cottage propagation is very undesirable.The technology of tissue culture propagating plant is a kind of special mode of nourishing and generating, and is commonly referred to as quick breeding technology or micro-propagation technique now.It is under aseptic condition, utilizes the part of plant materials, comprises bacterium, tissue and organ, the method for breeding plant under manually operated nutrition and envrionment conditions.The difficult problem that this technology faces is that the weak phenomenon of seedling appears in succeeding transfer culture easily.Therefore, adopt the rejuvenation substratum to substitute subculture medium and can prevent deterioration of strains, guarantee seedling quality, a large amount of high-quality strong sprout can be provided again, improve fast numerous quality.Prior art is generally used traditional subculture medium, causes subculture to be grown thickly a little less than the bud; And induction time is longer, and the difficulty of taking root is big, and surviving rate is low, can not adapt to the market requirement more fast.
Summary of the invention:
Technical problem to be solved by this invention provides and a kind ofly can reduce pollution rate, improves the more strong rejuvenation culture medium for tissue culture of high-bush blueberry preparation of root survival and fast propagating seedling wood and uses.
For solving the problems of the technologies described above, the present invention adopts following technical scheme.
The high-bush blueberry quick breeding method for tissue culture comprises the steps:
(1), explant material is handled: promptly select for use explant material to handle, prepare satisfactory explant at kill ratio and the minimum trim point of pollution rate;
(2), induce: be about to the explant described in the step (1) and be inoculated on the substratum, under inductive condition, induce and sprout; Described substratum is the improvement of agar synthetic medium;
(3) rejuvenation is cultivated: be about in the step (2) the gained bud young sprout multiplication culture in the rejuvenation substratum of growing thickly;
Further, step (1) adopts gives birth to the tender stem that cuts off blade then, and tap water is rinsed well, puts into 0.1% mercury chloride thimerosal 8-30 minute that adds gac, with aseptic water washing 3-5 time, downcuts to separate and digs the bud grafting kind on aseptic worktable.
In the optimized technical scheme, described step (1) explant material is handled and is comprised cleaning, sterilization and cutting; Described cleaning will be used to make the explant material of explant and clean; Described sterilization is disinfected the explant material of cleaning; The described explant that the explant material after the sterilization is cut into suitable size of cutting out.
The prescription of substratum described in the step (2) is agar synthetic medium+zeatin 0.8-1.3mg/L+ naphthylacetic acid 0.2-1.6mg/L, explant is seeded on the inducing culture, begin after 1 week to sprout, bud grows up to the young sprout about 4-5cm after 40-52 days, and lateral bud also sprouts simultaneously 5-10 the bud of growing thickly.
Culture medium prescription described in the step (3) is agar synthetic medium+zeatin 1.0mg/L+ indolebutyric acid 0.2-0.6mg/L bud cutting 2cm left and right sides stem section young sprout of will growing thickly, change increment cultivation on the subculture medium over to, after can differentiate the 30-50 bud of growing thickly, general 54-62 days succeeding transfer culture once, appreciation rate is at 30-50 times, and strong sprout, rate reached 90%.
Described explant material is that high-bush blueberry is given birth to the tender stem of defoliation then.
Described cleaning comprises that sterile water wash and the water logging that contains liquid detergent wash.
Described sterilization comprises alcohol disinfecting and adds the mercury chloride thimerosal sterilization of gac.
Adopt the high-bush blueberry quick breeding method for tissue culture of technical solution of the present invention, the beneficial effect that is compared with the prior art is:
When the thimerosal that uses is sterilized to blueberry stem section, be advisable with 25min, pollution rate and kill ratio drop to minimum, and surviving rate reaches as high as 75%.
About first culture 50d, part stem section can directly be induced and be produced the germinating bud, and in the improvement agar synthetic medium of additional zeatin 1.0mg/L and naphthylacetic acid 1.0mg/L, induction time is shorter, and the germination rate of indefinite bud is the highest, reaches 86.67%;
Blueberry is in the improvement root media, and rooting rate reaches more than 80%, and its surviving rate can reach 95%, and strong sprout, rate reached 90%.
Comprise cleaning, sterilization and cutting because explant material is handled, can obtain fine explant more, for quick breeding method for tissue culture is identified good basis.
Because the preferred tender stem of blueberry defoliation is as explant material,, tissue-culturing quick-propagation guarantees for providing good material.
Wash owing to clean the water logging that comprises the tap water flushing and contain liquid detergent, can be so that clean more thorough.
Because sterilization comprises alcohol disinfecting and adds the thimerosal sterilization of gac that it is more thorough to sterilize.
Since with explant material remove be cut into 0.5～1cm * 0.85-1cm size after the leaf tender stem as explant, inducing culture is convenient, and better effects if.
Embodiment:
Below in conjunction with specific embodiment enforcement of the present invention is described further:
Embodiment 1:
1, explant material is handled: comprise to explant material clean, processing such as sterilization and cutting, to prepare the explant that meets the requirements.
Be specially: adopt and give birth to the tender stem that cuts off blade then, tap water is rinsed well, uses aseptic water washing 3-5 time on aseptic worktable, with washing 20～30min in the tap water, washes 20～30min with the water logging that adds liquid detergent again.The 0.1% mercury chloride thimerosal 10 minutes that adds gac is put in disinfection on Bechtop after cleaning up, and it is standby to put into aseptic bottle after sterilization is finished.
2, induce and sprout: explant is seeded in prescription in the substratum of agar synthetic medium+zeatin 0.8mg/L+ naphthylacetic acid 0.2mg/L, explant is seeded on the inducing culture, begin after 1 week to sprout, bud grows up to the young sprout about 4-5cm after 40 days, and lateral bud also sprouts simultaneously 5-10 the bud of growing thickly
3, root culture: the bud of will growing thickly cutting 2cm left and right sides stem section young sprout, change increment cultivation on the subculture medium over to, described culture medium prescription is to differentiate 30-50 the bud of growing thickly behind agar synthetic medium+zeatin 1.0mg/L+ indolebutyric acid 0.2mg/L, 54 days left and right sides succeeding transfer culture once, appreciation rate is at 30-50 doubly.
Embodiment 2:
1, explant material is handled: comprise to explant material clean, processing such as sterilization and cutting, to prepare the explant that meets the requirements.
Be specially: adopt and give birth to the tender stem that cuts off blade then, tap water is rinsed well, uses aseptic water washing 3-5 time on aseptic worktable, with washing 20～30min in the tap water, washes 20～30min with the water logging that adds liquid detergent again.The 0.1% mercury chloride thimerosal 20 minutes that adds gac is put in disinfection on Bechtop after cleaning up, and it is standby to put into aseptic bottle after sterilization is finished.
2, induce and sprout: explant is seeded in prescription in the substratum of agar synthetic medium+zeatin 1.0mg/L+ naphthylacetic acid 1.0mg/L, explant is seeded on the inducing culture, begin after 1 week to sprout, bud grows up to the young sprout about 4-5cm after 45 days, and lateral bud also sprouts simultaneously 5-10 the bud of growing thickly
3, root culture: the bud of will growing thickly cutting 2cm left and right sides stem section young sprout, change increment cultivation on the subculture medium over to, described culture medium prescription is to differentiate 30-50 the bud of growing thickly behind agar synthetic medium+zeatin 1.0mg/L+ indolebutyric acid 0.4mg/L, 58 days succeeding transfer culture once, appreciation rate is at 30-50 doubly.
Embodiment 3:
1, explant material is handled: comprise to explant material clean, processing such as sterilization and cutting, to prepare the explant that meets the requirements.
Be specially: adopt and give birth to the tender stem that cuts off blade then, tap water is rinsed well, uses aseptic water washing 3-5 time on aseptic worktable, with washing 20～30min in the tap water, washes 20～30min with the water logging that adds liquid detergent again.The 0.1% mercury chloride thimerosal 30 minutes that adds gac is put in disinfection on Bechtop after cleaning up, and it is standby to put into aseptic bottle after sterilization is finished.
2, induce and sprout: explant is seeded in prescription in the substratum of agar synthetic medium+zeatin 1.3mg/L+ naphthylacetic acid 1.6mg/L, explant is seeded on the inducing culture, begin after 1 week to sprout, bud grows up to the young sprout about 4-5cm after 52 days, and lateral bud also sprouts simultaneously 5-10 the bud of growing thickly
3, root culture: the bud of will growing thickly cutting 2cm left and right sides stem section young sprout, change increment cultivation on the subculture medium over to, described culture medium prescription is to differentiate 30-50 the bud of growing thickly behind agar synthetic medium+zeatin 1.0mg/L+ indolebutyric acid 0.6mg/L, 62 days succeeding transfer culture once, appreciation rate is at 30-50 doubly.
Claims (7)
1. a method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry comprises the steps:
(1), explant material is handled: promptly select for use explant material to handle, prepare satisfactory explant at kill ratio and the minimum trim point of pollution rate;
(2), induce: be about to the explant described in the step (1) and be inoculated on the substratum, under inductive condition, induce and sprout; Described substratum is the improvement of agar synthetic medium;
(3) rejuvenation is cultivated: be about in the step (2) the gained bud young sprout multiplication culture in the rejuvenation substratum of growing thickly.
2. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that the described material of step (1) is with 0.1% mercury chloride with contain the gac thimerosal and handle.
3. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that the described material processing time of step (1) is 8---30 minute.
4. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that the used substratum of step (2) is agar synthetic medium+zeatin 0.8-1.3mg/L+ naphthylacetic acid 0.2-1.6mg/L.
5. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that step (2) is cultivated to cut young sprout in 40-52 days again.
6. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that the used substratum of step (3) is agar synthetic medium+zeatin 1.0mg/L+ indolebutyric acid 0.2-0.6mg/L.
7. a kind of method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry as claimed in claim 1 is characterized in that step (3) cultivates root culture after 54-62 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200910020731A CN101838630A (en) | 2009-04-21 | 2009-04-21 | Method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry |
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| CN200910020731A CN101838630A (en) | 2009-04-21 | 2009-04-21 | Method for preparing rejuvenation culture medium for tissue culture of high-bush blueberry |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102461462A (en) * | 2010-11-04 | 2012-05-23 | 湖北正佳微生物工程股份有限公司 | Preparation method of culture medium suitable for rapid seedling growing tissue culture of blueberry |
| CN103222425A (en) * | 2013-02-21 | 2013-07-31 | 邱义兰 | Efficient and rapid propagation technology suitable for southern highbush blueberry |
| CN106613939A (en) * | 2016-09-30 | 2017-05-10 | 温州科技职业学院 | Culture medium for indirect regeneration of high-bush blueberry somatic embryos and culture method |
-
2009
- 2009-04-21 CN CN200910020731A patent/CN101838630A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102461462A (en) * | 2010-11-04 | 2012-05-23 | 湖北正佳微生物工程股份有限公司 | Preparation method of culture medium suitable for rapid seedling growing tissue culture of blueberry |
| CN103222425A (en) * | 2013-02-21 | 2013-07-31 | 邱义兰 | Efficient and rapid propagation technology suitable for southern highbush blueberry |
| CN106613939A (en) * | 2016-09-30 | 2017-05-10 | 温州科技职业学院 | Culture medium for indirect regeneration of high-bush blueberry somatic embryos and culture method |
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Open date: 20100922 |