CN106258996A - Blue berry stem section method for quickly breeding - Google Patents
Blue berry stem section method for quickly breeding Download PDFInfo
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- CN106258996A CN106258996A CN201610921697.5A CN201610921697A CN106258996A CN 106258996 A CN106258996 A CN 106258996A CN 201610921697 A CN201610921697 A CN 201610921697A CN 106258996 A CN106258996 A CN 106258996A
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- stem section
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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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- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention provides a kind of blue berry stem section method for quickly breeding, comprise the following steps: the selection of (1) outer implant and process: the branch of collection is removed blade, washing and sterilizing, (2) blue berry stem section culture: blue berry stem section culture is divided into two stages, in the first stage, outer implant step 1 handled well is inoculated in MS culture medium cultivation 10 15 days, second stage, select and do not pollute, the stem section growing new blade is seeded on proliferated culture medium, the formula of proliferated culture medium is: WPM+0.5mg/L～1.5mg/L ZT+30g/L sugar+6.5 8g/L agar, regulation pH is 5.4 5.8, cultivate 30 40 days.Culture medium prescription of the present invention is simple, and agents useful for same is few, simple to operate, low cost, and reproduction speed is fast, applied widely.
Description
Technical field
The present invention relates to blueberry tissue culture technique field, particularly blue berry stem section method for quickly breeding.
Background technology
Blue berry is Ericaceae Vaccinium plant, is the emerging fruit tree tree with higher economic worth and wide DEVELOPMENT PROSPECT
Kind.Blueberry is nutritious, and particularly oxidation resistance is first of all fruit and vegetables.
Blue berry is mainly bred by cuttage and group two kinds of methods of training method, but conventional cultivation mode cultivation cycle is long, raw
Produce inefficient, and breeding coefficient is high, the time of breeding is not subject to seasonal restrictions to use tissue culture technology to have, shorten cultivation cycle, just
In advantages such as factory managements, can improve blue berry production efficiency, this technology has become the current blueberry industryization of solution and has developed kind
The key technology of Seedling demand.Blueberry tissue culture reproductive process include the selection process of outer implant, stem section inducing culture, enrichment culture and
The stages such as root culture.Wherein, the stem section enrichment culture stage is to make blue berry stem section increasing number reach blue berry seeling industry demand
Plan committed step, and this stage blue berry stem section propagation quantity number depend on its proliferation culture medium formula if appropriate for
Stem section growth promoter demand, thus stem section proliferation culture medium formula needs can obtain through test of many times.
Chinese patent CN102812905A discloses a kind of blueberry tender stem tissue culturing and rapid propagation process, and it comprises the following steps:
S1, choose the most lignified tender stem, washing and sterilizing from field, be cut into stem with bud；S2, inducing culture, the inducing culture of employing
Base is MS+ZT3.0mg/L+IBA0.1mg/L+ sucrose 30g/L+ agar powder 6.5g/L；S3, successive transfer culture, the subculture multiplication of employing
Culture medium is MS+ZT0.5～1.5mg/L+IBA0.05～0.1mg/L+ sucrose 30g/L+ agar 6.5g/L；Move at blue berry bottle outlet
MS+ZT0.3～0.7mg/L culture medium is used to carry out nursery stock rejuvenation before cultivation.This patent provides the benefit that: breeding potential is high, numerous
Grow speed fast, produce tissue cultured seedling with low cost；The seedling quality produced is good, and uniformity easily sloughs virus, well developed root system,
Lay a good foundation for later stage Growth status result.The shortcoming of this patent is that the culture medium prescription used is complicated, is difficult to behaviour
Make, and the usage amount of three the equal zeatin of cultivation stages (ZT) is relatively big, indolebutyric acid to be used in incubation simultaneously
(IBA), production cost is high；It addition, this patent incubation not only to increase production cost through induction period, and
Extend cultivation cycle.
Chinese patent CN 101869078A discloses a kind of seed-cultivating method of blueberry by means of tissue cultivation and micropropagation, and the season of growth adopts from field
Collection blue berry young sprout, under aseptic condition, through surface sterilization, after rushing with sterilized water, cuts into 1～2 leaf stem section of band, it is characterized in that by
Stem section with 1～2 bud is inoculated into just for inducing culture WPM+ZT3.0mg/L+NAA0.02mg/L+ sucrose 30g/L+ agar
On 6.5g/L, this medium pH 5.4；Cultivation through 2～3 generations, it is thus achieved that the group training nothing aseptic, vigorous growth, branch is many
Property system；At blueberry tissue culture Seedling subculture multiplication cultivation stage, use 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 improves 5～10 times,
This patent shortcoming is in culture medium to add forchlorfenuron (CPPU) growth regulator, and this regulator is swelling agent, is mainly used in
The aspects such as Fruit, rarely seen application in terms of group training；It addition, this patent incubation to increase through induction period
Adding production cost, cultivation cycle is long.
Chinese patent CN 103583368A discloses the construction method of a kind of vaccinium ashei tissue culture rapid propagation system, relates to planting
Fabric texture cultural method.It is outer implant that the present invention chooses the stem section of Rooted Cuttings, is seeded in stem shoot system after processing with ethanol and mercuric chloride
In inducing culture, further the stem section of stem shoot system inducing culture is vertically inserted in stem shoot system enrichment culture, and in training of taking root
IBA concentration 2.0mgL is taken in Yanging-1, rooting rate is higher than 85%.Above-mentioned water intaking seedlings cultivating is as source, and its pollution rate and melting brown rate are obvious
Less than greenhouse cultivation Seedling；Sterilize with ethanol and mercuric chloride and mutually promote；Stem shoot system is induced, propagation is consistent with Orthogonal experiment results；IBA
On the result that impact is single factor test randomized block experiment taken root.The shortcoming of this patent is that to choose the stem section of Rooted Cuttings be outer planting
Body, uses fluid medium to cultivate, easily causes the vitrification of blue berry, and vitrification is that blue berry often goes out in solid medium
Existing problem, thus cultivate inside liquid and be easier to that this problem occurs；It two easily causes anoxia and causes plant
Dead；Its three be above-mentioned patent select kind be Vaccinium ashei, range is narrow.
Summary of the invention
It is an object of the invention to overcome deficiencies of the prior art, it is provided that a kind of blue berry stem section Fast-propagation side
Method, the method blue berry Stem section reproduction coefficient is high, and production cost is low, is suitable for the blue berry large-scale production demand to seedling.
Blue berry stem section method for quickly breeding of the present invention comprises the following steps:
(1) selection of outer implant with disinfect:
The selection of the outer implant of blue berry: gather top and the top stem section of current-year branch spring.
Disinfecting of the outer implant of blue berry: the branch newly gathered removes blade cutting, and each stem section stays 1-2 leaf
Bud, rinses 3-5 time with tap water, then cleans 15-20min with detergent water, and detergent water weight concentration is 0.1-0.5%, it
With tap water, flush with foam is clean afterwards, then rinse 30-40min with circulating water, afterwards with 75% alcohol-pickled 30-40s, by nothing
Bacterium water rinses 4～5 times；Again with 0.1% mercuric chloride sterilization 5-10min, with aseptic water washing 6～7 times, drain away the water, standby.
In in this section, alcohol-pickled time and the disinfecting time of mercuric chloride can be according to the tender journeys of children of the blue berry stem section gathered
Degree extends or shortens.
(2) blue berry stem section culture:
Blue berry stem section culture is divided into two stages, in the first stage, the outer implant that step (1) is handled well is inoculated into MS training
Support on base, preferably light application time be 12-15h/d, intensity of illumination be 2000-3000Lx, cultivate under conditions of temperature 25-27 DEG C
10-15 days, observe stem section growing state (i.e. observing it either with or without pollution, dead situation).According to blue berry stem section growing state,
Second stage, selects and does not pollute, 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, regulation pH is 5.4-5.8, and second stage is cultivated
30-40 days, preferably condition of culture was: temperature 25-27 DEG C, and light application time is 12-15h/d, intensity of illumination is 2000-3000Lx.
The adjustment 0.1mol/LNaOH or 0.1mol/L HCl of pH value adjust.
Wherein the sugar in second stage culture medium is sucrose or white sugar.
Material of the present invention illustrates:
(1) MS: be the abbreviation of name Murashige and Skoog.MS culture medium is currently used most common cultivation
Base.It has higher inorganic salt concentration, it is possible to the mineral nutrition needed for ensureing tissue growth can also accelerate the life of callus
Long.
(2) WPM: xylophyta is with culture medium (Woody Plant medium).
(3) ZT: zeatin.
The invention has the beneficial effects as follows:
(1) proliferation culture medium formula owing to using in the present invention is: WPM+ZT+ white sugar+agar, from culture medium prescription
From the point of view of, culture medium is relatively simple, and agents useful for same is less, therefore operates simple comparatively speaking, and low cost, breeding coefficient can reach 5-10
Times.
(2) present invention is divided into two stages, and first stage is MS culture medium, and second stage is proliferated culture medium.From stem
Section incubation, owing to the first stage is only with MS culture medium, therefore compares culture technique, process simpler, and cost is lower,
But breeding coefficient is high, plant strain growth speed is accelerated, and the effect of the present invention is mainly reflected in the following aspects specifically: one is
The breeding coefficient of blue berry is high, can reach 5-10 times；Two is the speed of growth quickening of blue berry, shows on the plant height of plant, 30
It can make plant height reach 5-6cm, grows to identical height than the plant of other culture medium culturing and can shorten 10-20 days, because of
This greatly speeds up the reproduction speed of blue berry stem section；Three is owing to using zeatin concentration relatively low in the present invention, thus turns out
Plant stronger than using the plant strain growth gesture turned out of higher concentration, i.e. stem is relatively thicker, and blade is compared with big and quantity is more, wound healing
Tissue is few, can directly carry out root culture without the follow-up strong seedling culture stage；Four is that the inventive method does not easily cause blue berry
Vitrification, can be seen that from above several respects, use culture medium of the present invention, be greatly improved the reproduction speed of blue berry.
(3) propagation method of the present invention is applied widely.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but those skilled in the art will
Understanding, 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 blue berry
(1) selection of outer implant and process: spring gathers the branch just sprouted, and the branch newly gathered is removed blade and cuts
Section, each stem section is stayed 1-2 leaf bud, is rinsed 3 times with tap water, then clean 15min with detergent water, will steep with tap water afterwards
Foam is rinsed well, then rinses 30min with circulating water, afterwards with 75% alcohol-pickled 30s, with aseptic water washing 5 times；Use again
0.1% mercuric chloride sterilization 5-10min, with aseptic water washing 6-7 time, drains away the water, standby.
(2) blue berry stem section culture:
Blue berry stem section culture is divided into two stages, and in the first stage, outer implant step 1 handled well is inoculated into MS and cultivates
On base light application time be 14h/d, intensity of illumination be 2500Lx, under conditions of temperature 25 DEG C cultivate 14 days；According to blue berry stem section
Growing state, selects and does not pollute, 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, regulation pH is 5.8, and second stage is cultivated 30 days, and condition of culture is: light
According to the time be 14h/d, intensity of illumination be 2200Lx, temperature 25 DEG C.
Through cultivating, its breeding coefficient is 5, and 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 without vitrification phenomenon in incubation.
Embodiment 2: blue berry is stepped on by U.S.A
In the blue berry stem section culture stage, the formula of second stage proliferated culture medium is: WPM+1.0mg/L ZT+30g/L sucrose+
6g/L agar, other is same as in Example 1, and its breeding coefficient is 8-10, and plant strain growth height is 5-5.5cm；Plant strain growth gesture
By force, and grow homogeneous, all greens of blade, survival rate 100%, show as in strong sprout, incubation being sent out without vitrification phenomenon
Raw.
Embodiment 3: blue rich blue berry
In the blue berry stem section culture stage, the formula of second stage proliferated culture medium is: WPM+1.5mg/L ZT+30g/L sucrose+
6.5g/L agar, other is same as in Example 1, and its breeding coefficient is 6-7, and plant strain growth height is 4-5cm；Plant strain growth gesture
By force, and grow homogeneous, all greens of blade, survival rate 100%, show as in strong sprout, incubation being sent out without vitrification phenomenon
Raw.
Embodiment 4: all gram blue berries
In the blue berry stem section culture stage, the formula of second stage proliferated culture medium is: WPM+0.5mg/L ZT+30g/L sucrose+
6g/L agar, other is same as in Example 1, and its breeding coefficient is 5, and plant strain growth height is 5-5.5cm；Plant strain growth gesture is strong,
And grow homogeneous, and all greens of blade, survival rate 100%, show as strong sprout, incubation occurs without vitrification phenomenon.
Embodiment 5: George's blue berry
In the blue berry stem section culture stage, the formula of second stage proliferated culture medium is: WPM+0.5mg/L ZT+30g/L sucrose+
6g/L agar, other is same as in Example 1, and its breeding coefficient is 6, and plant strain growth height is 5.5-6cm；Plant strain growth gesture is strong,
And grow homogeneous, and all greens of blade, survival rate 100%, show as strong sprout, incubation occurs without vitrification phenomenon.
The present invention uses and utilizes solid medium to cultivate, and does not has the generation of vitrification Seedling, and this is to train relative to liquid
Have the advantage that for supporting base.
Comparative example 1: George's blue berry
The formula of second stage proliferated culture medium in the cultivation of embodiment 5 George's blue berry is changed to: WPM+0.5mg/L ZT+
30g/L sucrose+6g/L agar+0.5mg/LCPPU, other is same as in Example 5, and its breeding coefficient is 4-5, plant strain growth height
For 5.5-6cm；Survival rate 85%, stem section base portion callus is more, and fallen leaves or the phenomenon of radical leaves jaundice occurs in plant.
Comparative example 2:
The formula of second stage proliferated culture medium in the cultivation of embodiment 5 George's blue berry is changed to: WPM+0.5mg/L ZT+
30g/L sucrose+6g/L agar+0.1mg/L IBA, other is same as in Example 5, and its breeding coefficient is 4-5, plant strain growth height
For 5.0-5.5cm；Survival rate 86%, stem section base portion callus is relatively big, and yellow or the phenomenon come off occurs in blade.
Comparative example 3:
The formula of second stage proliferated culture medium in the cultivation of embodiment 5 George's blue berry is changed to: WPM+3mg/L ZT+
30g/L sucrose+6g/L agar, other is same as in Example 5, and its breeding coefficient is 6, and plant strain growth height is 4.5-5cm；Survive
Rate 95%, occurs without vitrification phenomenon in incubation.
Above content describes ultimate principle and the principal character of the present invention, and the present invention is not restricted to the described embodiments,
Without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these changes and improvements are all
Fall in scope of the claimed invention.
Claims (6)
1. a blue berry stem section method for quickly breeding, it is characterised in that comprise the following steps: at the selection of (1) outer implant and sterilization
Reason；(2) blue berry stem section culture: blue berry stem section culture is divided into two stages, in the first stage, the outer planting that step (1) is handled well
Body stem section is inoculated in MS culture medium cultivation 10-15 days, second stage, selects the stem section inoculation not polluting, growing new blade
On proliferated culture medium, the formula of proliferated culture medium is: WPM+0.5mg/L～1.5mg/L ZT+30g/L sugar+6.5-8g/L fine jade
Fat, regulation pH is 5.4-5.8, and second stage is cultivated 30-40 days.
Blue berry stem section method for quickly breeding the most according to claim 1, it is characterised in that the outer implant described in step (1) is
Just sprouted spring branch top or near the part on top.
Blue berry stem section method for quickly breeding the most according to claim 1, it is characterised in that step disinfects bar described in (1)
Part is that the branch of collection removes blade, and branch is cut into the stem section of 1-2 leaf bud of band, rinses 3-5 time with tap water, then uses
Detergent water cleans 15-20min, and detergent water weight concentration is 0.1-0.5%, with tap water, flush with foam is clean afterwards,
30-40min is rinsed again, with 75% alcohol-pickled 30-40s after being drained by moisture, with aseptic water washing 4～5 times with circulating water；Again
With 0.1% mercuric chloride sterilization 5-10min, with aseptic water washing 6～7 times, drain away the water, standby.
Blue berry stem section method for quickly breeding the most according to claim 1, it is characterised in that step (2) the described first stage trains
Foster condition is: temperature 25-27 DEG C, light application time 12-15h/d, intensity of illumination 2000-3000Lx.
Blue berry stem section method for quickly breeding the most according to claim 1, it is characterised in that step (2) described second stage is trained
Foster condition is: temperature 25-27 DEG C, light application time 12-15h/d, intensity of illumination 2000-3000Lx.
Blue berry stem section method for quickly breeding the most according to claim 1, it is characterised in that step (2) described second stage is trained
The sugar supported in base is sucrose or white sugar.
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| CN201610921697.5A CN106258996B (en) | 2016-10-21 | 2016-10-21 | Blueberry stem section rapid propagation method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114287341A (en) * | 2021-11-15 | 2022-04-08 | 安徽师范大学 | Method for culturing blueberry tissue |
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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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