CN106258996B - Blueberry stem section rapid propagation method - Google Patents
Blueberry stem section rapid propagation method Download PDFInfo
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- CN106258996B CN106258996B CN201610921697.5A CN201610921697A CN106258996B CN 106258996 B CN106258996 B CN 106258996B CN 201610921697 A CN201610921697 A CN 201610921697A CN 106258996 B CN106258996 B CN 106258996B
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- blueberry
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention provides a kind of blueberry stem section rapid propagation method, includes the following steps：(1) selection and processing of explant：The branch of acquisition is removed into blade, washing and sterilizing, (2) blueberry stem section culture：Blueberry stem section culture is divided into two stages, in the first stage, the explant that step 1 is handled well is inoculated on MS culture mediums and cultivated 10 15 days, second stage, it selects and does not pollute, grows the stem section of new blade and be seeded on proliferated culture medium, the formula of proliferated culture medium is：WPM+0.5mg/L~1.5mg/L ZT+30g/L+6.5 8g/L agar of sugar, it is 5.4 5.8 to adjust pH, is cultivated 30 40 days.Culture medium prescription of the present invention is simple, and agents useful for same is few, easy to operate, at low cost, and reproduction speed is fast, applied widely.
Description
Technical field
The present invention relates to blueberry tissue culture technique fields, particularly blueberry stem section rapid propagation method.
Background technology
Blueberry is Ericaceae cowberry platymiscium, is the emerging fruit tree tree with higher economic value and wide development prospect
Kind.Blueberry is full of nutrition, and particularly oxidation resistance is first of all fruit and vegetables.
Blueberry is mainly bred by two methods of cuttage and tissue culture method, but conventional cultivation mode cultivation cycle is long, raw
Produce it is less efficient, and tissue culture technology is used to have breeding coefficient is high, the breeding time is not subject to seasonal restrictions, shorten cultivation cycle, just
In the factory management the advantages that, blueberry production efficiency can be improved, which, which has become, solves current blueberry industryization development to kind
The key technology of seedling demand.The selection processing of blueberry tissue culture reproductive process including explant, stem section Fiber differentiation, Multiplying culture and
The stages such as culture of rootage.Wherein, the stem section Multiplying culture stage is to increase blueberry stem section quantity to reach blueberry seeling industry demand
The committed step of plan, and this stage blueberry stem section proliferation quantity number depend on its proliferation culture medium formula if appropriate for
Stem section growth and development demand, thus stem section proliferation culture medium formula needs to obtain by test of many times.
Chinese patent CN102812905A discloses a kind of blueberry tender stem tissue culturing and rapid propagation process, it includes the following steps：
S1, the tender stem that non-lignifying is chosen from field, washing and sterilizing are cut into stem with bud；S2, Fiber differentiation, the Fiber differentiation of use
Base is MS+ZT3.0mg/L+IBA0.1mg/L+ sucrose 30g/L+ agar powders 6.5g/L；S3, squamous subculture, the shoot proliferation of use
Culture medium is MS+ZT0.5~1.5mg/L+IBA0.05~0.1mg/L+ sucrose 30g/L+ agar 6.5g/L；It is moved in blueberry bottle outlet
Nursery stock rejuvenation is carried out using MS+ZT0.3~0.7mg/L culture mediums before planting.The advantageous effect of the patent is：Breeding potential is high, numerous
It is fast to grow speed, production tissue-cultured seedling is of low cost；The seedling quality of production is good, uniformity, easily sloughs virus, well developed root system,
It lays a good foundation for later stage Growth status result.The shortcomings that patent, is the culture medium prescription used complexity, is not easy to grasp
Make, and the usage amount of three equal zeatin of cultivation stage (ZT) is larger, while indolebutyric acid is also used in incubation
(IBA), production cost is high；In addition, to pass through induction period in the patent incubation, not only increase production cost, but also
Extend cultivation cycle.
Chinese patent CN 101869078A disclose a kind of seed-cultivating method of blueberry by means of tissue cultivation and micropropagation, and the season of growth adopts from field
Collect blueberry young sprout, under aseptic condition, through surface sterilization, after being rushed with sterile water, cut into 1~2 leaf stem section of band, it is characterized in that will
Stem section with 1~2 bud is inoculated into primary inducing culture WPM+ZT3.0mg/L+NAA0.02mg/L+ sucrose 30g/L+ agar
On 6.5g/L, the medium pH 5.4；By the culture in 2~3 generations, obtain tissue culture sterile, vigorous growth, more than branch without
Property system；In blueberry tissue culture seedling shoot proliferation cultivation stage, using proliferated culture medium WPM+ZT0.5~1.5mg/L+CPPU0.05~
1.0mg/L+ sucrose 30g/L+ agar 6.5g/L, pH5.4；Every 50~60 days subculture cycles, line of breeding improve 5~10 times,
The patent shortcoming is addition forchlorfenuron (CPPU) growth regulator in culture medium, which is swelling agent, is mainly used for
Fruit etc., the rarely seen application in terms of tissue culture；In addition, to pass through induction period in the patent incubation, increase
Add production cost, cultivation cycle is long.
Chinese patent CN 103583368A disclose a kind of construction method of vaccinium ashei tissue culture rapid propagation system, are related to planting
Object method for tissue culture.The stem section that the present invention chooses Rooted Cuttings is explant, and stem shoot system is seeded in after being handled with alcohol and mercuric chloride
In inducing culture, further the stem section of stem shoot system Fiber differentiation is inserted in vertically in stem shoot system Multiplying culture, and in training of taking root
IBA concentration 2.0mgL is taken in supporting-1, rooting rate is higher than 85%.It is above-mentioned to take Rooted Cuttings pollution rate and melting brown rate are apparent as source
Less than greenhouse cultivation seedling；It is mutually promoted with alcohol and mercuric chloride disinfection；Stem shoot system induction, proliferation are consistent with Orthogonal experiment results；IBA
To the result that the influence taken root is single factor test randomized block experiment.The shortcomings that patent is that the stem section of selection Rooted Cuttings is explant
Body is cultivated using fluid nutrient medium, be easy to cause the vitrifying of blueberry, and vitrifying is that blueberry often goes out in solid medium
The problem of existing, thus culture is easier to this problem occur inside liquid；The second is be easy to causeing anoxic causes plant
It is dead；The third is the kind that above-mentioned patent is selected is Vaccinium ashei, use scope is narrow.
Invention content
It is an object of the invention to overcome deficiencies of the prior art, a kind of blueberry stem section quickly side of breeding is provided
Method, this method blueberry Stem section reproduction coefficient is high, and production cost is low, and blueberry is suitble to mass produce the demand to seedling.
Blueberry stem section rapid propagation method of the present invention includes the following steps：
(1) selection of explant is with disinfecting：
The selection of blueberry explant：The top of current-year branch and top stem section are acquired in spring.
Blueberry explant is disinfected：Freshly harvested branch is removed into blade and segment, each stem section stays 1-2 leaf
Bud is rinsed 3-5 times with tap water, then cleans 15-20min with washing powder water, and washing powder water weight concentration is 0.1-0.5%, it
It is with tap water that flush with foam is clean afterwards, then 30-40min is rinsed with circulating water, 30-40s is impregnated with 75% alcohol later, with nothing
Bacterium water rinses 4~5 times；5-10min is sterilized with 0.1% mercuric chloride again, with aseptic water washing 6~7 times, is drained away the water, it is spare.
In in this section, time and the disinfecting time of mercuric chloride that alcohol impregnates can be according to the tender journeys of children of the blueberry stem section of acquisition
Degree extends or shortens.
(2) blueberry stem section culture：
Blueberry stem section culture is divided into two stages, in the first stage, the explant that step (1) is handled well is inoculated into MS trainings
Support on base, preferably light application time be 12-15h/d, intensity of illumination 2000-3000Lx, cultivate under conditions of 25-27 DEG C of temperature
10-15 days, observation stem section growing state (observed it either with or without pollution, dead situation).According to blueberry stem section growing state,
Second stage is selected and is not polluted, grows the aseptic seedling of new blade and be seeded on proliferated culture medium, the formula of proliferated culture medium
For：WPM+0.5mg/L~1.5mg/L ZT+30g/L sugar+6.5-8g/L agar, adjusting pH be 5.4-5.8, second stage culture
30-40 days, preferably condition of culture was：25-27 DEG C of temperature, light application time 12-15h/d, intensity of illumination 2000-3000Lx.
The adjustment of pH value is adjusted with 0.1mol/LNaOH or 0.1mol/L HCl.
Sugar wherein in second stage culture medium is sucrose or white granulated sugar.
Material explanation of the present invention：
(1)MS：It is the abbreviation of name Murashige and Skoog.MS culture mediums are to use most common culture at present
Base.It has higher inorganic salt concentration, can ensure that the mineral nutrition needed for tissue growth can also accelerate the life of callus
It is long.
(2)WPM：Xylophyta is with culture medium (Woody Plant medium).
(3)ZT：Zeatin.
The beneficial effects of the invention are as follows：
(1) since the proliferation culture medium formula used in the present invention is：WPM+ZT+ white granulated sugars+agar, from culture medium prescription
From the point of view of, culture medium is simpler, and agents useful for same is less, therefore operation is comparatively simple, and at low cost, breeding coefficient can reach 5-10
Times.
(2) present invention is divided into two stages, and first stage is MS culture mediums, and second stage is proliferated culture medium.From stem
Section incubation, simpler compared to culture technique, process since the first stage is only with MS culture mediums, cost is lower,
But breeding coefficient is high, plant strain growth speed is accelerated, and specifically effect of the invention is mainly reflected in the following aspects：When
The breeding coefficient of blueberry is high, can reach 5-10 times；Second is that the speed of growth of blueberry is accelerated, show in the plant height of plant, 30
It can make plant height reach 5-6cm, and the plant than other medium cultures grows to identical height and can shorten 10-20 days, because
This greatly speeds up the reproduction speed of blueberry stem section；Third, it is relatively low since zeatin concentration is used in the present invention, thus turn out
Plant than using the plant strain growth gesture that higher concentration is turned out strong, i.e., stem is thicker, and blade is larger and quantity is more, callus
Tissue is few, can directly carry out culture of rootage without the subsequent strong seedling culture stage；Fourth, the method for the present invention does not easily cause blueberry
Vitrifying, can be seen that from more than several respects, using culture medium of the present invention, be greatly improved the reproduction speed of blueberry.
(3) propagation method of the present invention is applied widely.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment 1：Ao Ni'er's blueberry
(1) selection and processing of explant：The branch that spring acquisition has just been sprouted, removes blade by freshly harvested branch and cuts
Section, each stem section stay 1-2 leaf bud, are rinsed 3 times with tap water, then clean 15min with washing powder water, will be steeped with tap water later
Foam is rinsed well, then rinses 30min with circulating water, impregnates 30s with 75% alcohol later, with aseptic water washing 5 times；It uses again
0.1% mercuric chloride sterilizes 5-10min, with aseptic water washing 6-7 times, drains away the water, spare.
(2) blueberry stem section culture：
Blueberry stem section culture is divided into two stages, in the first stage, the explant that step 1 is handled well is inoculated into MS cultures
On base light application time be 14h/d, intensity of illumination 2500Lx, cultivate 14 days under conditions of 25 DEG C of temperature；According to blueberry stem section
Growing state is selected and is not polluted, grows the stem section of new blade and be seeded on proliferated culture medium, and the formula of proliferated culture medium is：
WPM+0.5mg/L ZT+30g/L sucrose+6g/L agar, it is 5.8 to adjust pH, second stage culture 30 days, and condition of culture is：Light
It is 14h/d according to the time, intensity of illumination 2200Lx, 25 DEG C of temperature.
By culture, breeding coefficient 5, plant strain growth height is 5-6cm；Plant strain growth gesture is strong, survival rate 100%,
The all greens of blade show as strong sprout, occur in incubation without vitrification phenomenon.
Embodiment 2：Blueberry is stepped on by U.S.A
Blueberry stem section culture stage, the formula of second stage proliferated culture medium are：WPM+1.0mg/L ZT+30g/L sucrose+
6g/L agar, other same as Example 1, breeding coefficient 8-10, plant strain growth height are 5-5.5cm；Plant strain growth gesture
By force, and uniform, all greens of blade are grown, survival rate 100% shows as strong sprout, sent out in incubation without vitrification phenomenon
It is raw.
Embodiment 3：Blue rich blueberry
Blueberry stem section culture stage, the formula of second stage proliferated culture medium are：WPM+1.5mg/L ZT+30g/L sucrose+
6.5g/L agar, other same as Example 1, breeding coefficient 6-7, plant strain growth height are 4-5cm；Plant strain growth gesture
By force, and uniform, all greens of blade are grown, survival rate 100% shows as strong sprout, sent out in incubation without vitrification phenomenon
It is raw.
Embodiment 4：All gram blueberries
Blueberry stem section culture stage, the formula of second stage proliferated culture medium are：WPM+0.5mg/L ZT+30g/L sucrose+
6g/L agar, other same as Example 1, breeding coefficient 5, plant strain growth height are 5-5.5cm；Plant strain growth gesture is strong,
And uniform, all greens of blade are grown, survival rate 100% shows as strong sprout, occurs in incubation without vitrification phenomenon.
Embodiment 5：George's blueberry
Blueberry stem section culture stage, the formula of second stage proliferated culture medium are：WPM+0.5mg/L ZT+30g/L sucrose+
6g/L agar, other same as Example 1, breeding coefficient 6, plant strain growth height are 5.5-6cm；Plant strain growth gesture is strong,
And uniform, all greens of blade are grown, survival rate 100% shows as strong sprout, occurs in incubation without vitrification phenomenon.
The present invention using solid medium using being cultivated, and without the generation of vitrifying seedling, this is trained relative to liquid
It is had the advantage that for foster base.
Comparative example 1：George's blueberry
The formula of second stage proliferated culture medium in the culture of 5 George's blueberry of embodiment is changed to：WPM+0.5mg/L ZT+
30g/L sucrose+6g/L agar+0.5mg/LCPPU, other same as Example 5, breeding coefficient 4-5, plant strain growth height
For 5.5-6cm；The phenomenon that survival rate 85%, stem section base portion callus is more, and falling leaves occurs in plant or radical leaves turn to be yellow.
Comparative example 2：
The formula of second stage proliferated culture medium in the culture of 5 George's blueberry of embodiment is changed to：WPM+0.5mg/L ZT+
30g/L sucrose+6g/L agar+0.1mg/L IBA, other same as Example 5, breeding coefficient 4-5, plant strain growth height
For 5.0-5.5cm；The phenomenon that survival rate 86%, stem section base portion callus is larger, and blade yellow occurs or comes off.
Comparative example 3：
The formula of second stage proliferated culture medium in the culture of 5 George's blueberry of embodiment is changed to：WPM+3mg/L ZT+
30g/L sucrose+6g/L agar, other same as Example 5, breeding coefficient 6, plant strain growth height are 4.5-5cm；It survives
Rate 95% occurs without vitrification phenomenon in incubation.
More than content describes the basic principle and main feature of the present invention, and the present invention is not limited to the above embodiments,
Without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements are all
It falls into scope of the claimed invention.
Claims (3)
1. a kind of blueberry stem section rapid propagation method, it is characterised in that include the following steps：(1) at the selection of explant and disinfection
Reason；(2) blueberry stem section culture：Blueberry stem section culture is divided into two stages, in the first stage, the explant that step (1) is handled well
Body stem section is inoculated on MS culture mediums and cultivates 10-15 days, second stage, selects the stem section inoculation for not polluting, growing new blade
On proliferated culture medium, the formula of proliferated culture medium is：WPM+0.5mg/L~1.5mg/LZT+30g/L sugar+6.5-8g/L fine jades
Fat, adjusting pH are 5.4-5.8, second stage culture 30-40 days and
Step (1) condition of disinfecting is that the branch of acquisition is removed blade, and branch is cut into the stem with 1-2 leaf bud
Section is rinsed 3-5 times with tap water, then cleans 15-20min with washing powder water, and washing powder water weight concentration is 0.1-0.5%, it
It is with tap water that flush with foam is clean afterwards, then 30-40min is rinsed with circulating water, after moisture is drained 30- is impregnated with 75% alcohol
40s, with aseptic water washing 4~5 times；5-10min is sterilized with 0.1% mercuric chloride again, with aseptic water washing 6~7 times, is drained away the water,
It is spare；
Step (2) the first stage condition of culture is：25-27 DEG C of temperature, light application time 12-15h/d, intensity of illumination 2000-
3000Lx；
Step (2) the second stage condition of culture is：25-27 DEG C of temperature, light application time 12-15h/d, intensity of illumination 2000-
3000Lx；
The blueberry is Ao Ni'er's blueberry, blue rich blueberry, all any one of gram blueberry and George's blueberry.
2. blueberry stem section rapid propagation method according to claim 1, it is characterised in that the explant described in step (1) is
Spring just sprouted branch top or close to top part.
3. blueberry stem section rapid propagation method according to claim 1, it is characterised in that step (2) the second stage training
It is sucrose or white granulated sugar to support the sugar in base.
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| CN201610921697.5A CN106258996B (en) | 2016-10-21 | 2016-10-21 | Blueberry stem section rapid propagation method |
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| CN201610921697.5A CN106258996B (en) | 2016-10-21 | 2016-10-21 | Blueberry stem section rapid propagation method |
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| CN106258996B true CN106258996B (en) | 2018-06-29 |
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| CN114287341A (en) * | 2021-11-15 | 2022-04-08 | 安徽师范大学 | Method for culturing blueberry tissue |
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| CN101069487A (en) * | 2007-06-20 | 2007-11-14 | 金陵科技学院 | Labbiteye blueberry tissue culturation method |
| CN101869078A (en) * | 2010-07-14 | 2010-10-27 | 长春百瑞蓝莓科技发展有限公司 | Seed-cultivating method of blueberry by means of tissue cultivation and micropropagation |
| CN102812905A (en) * | 2012-09-14 | 2012-12-12 | 四川立益生态农业开发有限公司 | Blueberry tender stem tissue culturing and rapid propagation process |
| CN103583368A (en) * | 2013-11-22 | 2014-02-19 | 西南林业大学 | Construction method for vaccinium ashei tissue culture rapid propagation system |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103222425A (en) * | 2013-02-21 | 2013-07-31 | 邱义兰 | Efficient and rapid propagation technology suitable for southern highbush blueberry |
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2016
- 2016-10-21 CN CN201610921697.5A patent/CN106258996B/en not_active Expired - Fee Related
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| CN102812905A (en) * | 2012-09-14 | 2012-12-12 | 四川立益生态农业开发有限公司 | Blueberry tender stem tissue culturing and rapid propagation process |
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