CN100493345C - Method for detoxification and quick proliferation for culturing strawberry anther - Google Patents

Abstract

A detoxicating fast reproduction method of strawberry by anther culture includes such steps as taking the flower bud of strong strawberry, storing at 4 deg.C in refrigerator for 72 hr, disinfecting with the mixture of alcohol, corrosive sublimate and tween, sucking surface water by aseptic suction paper, taking anther, and sequentially culturing in colli inducing culture medium, regrowing culture medium, secondary culture medium, and rooting culture medium.

Description

A kind of strawberry anther that utilizes is cultivated the method for carrying out detoxifying fast breeding

One, technical field

The present invention's " a kind of strawberry anther that utilizes is cultivated the method for carrying out detoxifying fast breeding " belongs to crops fast breeding technique field, is exclusively used in the detoxifying fast breeding of strawberry.

Two, background technology

Strawberry (Fragaria ananassa.Duch) is the rose family (Rosaceae) Fragaria (Fragaria) herbaceos perennial, and 2n=8x=56 belongs to polyploid plant.Strawberry fruit is little berry, and is bright-coloured beautiful, and soft and succulency is sour-sweet moderate, and fragrance is strong, is one of seven big fruit in the world.Because planting strawberry has characteristics such as fruiting period is long, plant is little, breeding is fast, economic benefit height, particularly be equipped with greenhouse cultivation, for the fruit market of winter-spring season increases designs and varieties, especially at major holiday supply the markets such as New Year's Day, the Spring Festival, thereby be subjected to producers and consumers's the potentiality of liking having very much meaning promoted and development deeply.

But strawberry is easily infected virus in process of production, thereby causes the kind sexual involution of strawberry cultivars.Therefore, vegetative propagation quantity is big more, and virus disseminating is also fast more.Be prone to degradation phenomenas such as leaf-shrinkage, fruit deformity, product qualitative change are bad, output descends, plant strain growth is slow behind the strawberry infective virus.Virus is a key factor that causes that strawberry output descends.

This method obtains the strawberry regeneration plant by anther cultural method, set up a kind of convenient and practical, detoxification efficiency good, reproduction coefficient is high, genetic stability is good strawberry detoxifying fast breeding system, thereby promote the strawberry detoxification technology on producing, further to use and promote.

Three, summary of the invention

Technical problem

The objective of the invention is increases year by year at the area under cultivation and the scope of present strawberry in China, and due to illness poison infects the outstanding day by day present situation of problem that the underproduction that causes and quality descend, and proposes a kind of method that realizes detoxifying fast breeding of cultivating by strawberry anther.

Technical scheme

A kind of strawberry anther that utilizes is cultivated the method for carrying out detoxifying fast breeding, it is characterized in that:

(1) disinfects

Getting length is the strawberry bud of 4-6mm, at 4 ℃ of refrigerator cold treatment 72h;

Then bud is placed once with aseptic water washing after 70% alcohol-pickled 5s once with aseptic water washing again that to add volume ratio be that 0.1% tween and mass volume ratio are that 0.1% mercuric chloride solution soaks 8min;

Use aseptic water washing 8-10 time at last, blot the water on bud surface with aseptic blotting paper;

(2) callus induction differentiation

The bud of disinfecting is stripped flower pesticide, be inoculated into

MS+BA0.5mgL ^-1+ NAA2.0mgL ^-1+ KT0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Callus inducing medium in 30～60d;

Forward callus to MS+BA1.5mgL ^-1+ NAA0.5mgL ^-1+ ZT2.0mgL ^-1+ agar 6.5gL ^-1Differential medium in 70～90d;

Then the strawberry seedling that obtains in the differential medium is transferred to MS+BA0.5mgL ^-1+ NAA0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Subculture medium on, carry out shoot proliferation and cultivate 20～30d;

(3) rooting and transplant

The strawberry seedling forwards 1/2MS+NAA0.2gL to ^-1+ agar 6.5gL ^-1Root media take root and handle 20～30d;

After nursery stock took root, the plant that will take root added water and tempers 1-2d through tempering 7-10d under the outdoor natural conditions, uncapping during transplanting, and as transplanting medium, the temperature during transplanting is controlled at 20-25 ℃ of scope with vermiculite and perlite mixed system.

Beneficial effect

The present invention is directed to the problem that the strawberry detoxification technology exists at present on producing, on the basis of forefathers' research, set up a kind of convenient and practical, detoxification efficiency good, reproduction coefficient is high, genetic stability is good strawberry detoxifying fast breeding system first.Strawberry anther is cultivated many reports at home, but how to obtain very high regeneration rate, is a more insoluble problem all the time.

The inventive method is cultivated differentiation rate with strawberry anther and is brought up to 37.5%, and prior art has improved 28.03 percentage points, has broken through the very low predicament of traditional differentiation rate.After nursery stock took root, transplanting survival rate reached 90%.The strawberry poison-removing method that can be used as commercial use directly uses on producing, thereby promotes the strawberry detoxification technology further to use on producing and promote.

Four, description of drawings

Fig. 1 low temperature treatment different time is to the influence of callus induction

Fig. 2 avenges honey differentiation indefinite bud picture

Five, embodiment

(1) optimum processing parameter determines

1 determines the processing in early stage of best bud and determining of sterilization method

1.1 material and method that the best approach is determined

Take from the healthy and strong strawberry bud in field, developmental stage is that monokaryon keeps to the side the phase (size is about 4-6mm), at 4 ℃ of refrigerator cold treatment 0h, 48h, 72h, 96h.Cold treatment finishes the back and takes out disinfection on super-clean bench.

Disinfecting time and step are: bud places once with aseptic water washing after 70% alcohol-pickled 5,10s once with aseptic water washing that 0.1% mercuric chloride (adding tween) soaks 5,8,12min uses aseptic water washing 8-10 time (10min) at last again, specifically treatment combination (table 1).Blot the water on bud surface with aseptic blotting paper after the sterilization, the bud of disinfecting is stripped flower pesticide, be inoculated into MS+BA0.5mgL ^-1+ NAA2.0mgL ^-1+ KT0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Medium, 30d statistics pollution rate and inductivity.

Table 1 strawberry bud difference is disinfected

1.2 low temperature treatment is to the influence of callus induction

The healthy and strong strawberry plant bud of fetching from the field is removed surface impurity, and a part is placed 4 ℃ of refrigerators, carries out low temperature treatment, and the processing time is respectively 48h, 72h, 96h.During remainder directly is inoculated into No. 6 medium without low temperature treatment.Bud corolla when low temperature treatment 72h begins to become flexible, but also is in monokaryon period and keeps to the side the phase through detecting microspore development.As seen from Figure 1, in four set processing, behind the low temperature treatment 96h, most of corolla unclamps, and can not be used for inoculation.Other three processing all can induce callus, the flower pesticide of low temperature treatment 72h, snow honey (Qian Yaming when cultivating 10d, Wang Zhuanwei, Su Jiale, Zhao Mizhen. strawberry new varieties snow honey is tested with the facility cultivation of Feng Xiang. and deciduous fruit tree, 2004 (6): 20) 3.7% begins to divide callus.And low temperature treatment 48h 10d time snow honey 1.8% after inoculation has produced callus.But the flower pesticide of directly inoculating without low temperature treatment during from postvaccinal 15d snow honey 4.9% produce callus.Therefore help the startup of anther development through certain low temperature treatment, shorten the time that callus begins to divide.So the preceding bud low temperature treatment 72h. of inoculation is selected in this research

1.3 sterilization method is to the influence of callus induction

The sterilization combination that this test is chosen, in the callus induction process, the flower pesticide pollution rate is controlled at about 20%.Table 2 is the result show, increases with alcohol and mercuric chloride disinfecting time, and pollution rate is on a declining curve.Because flower pesticide wraps in the bud, and obtained examination material control bud keeps to the side the phase at monokaryon, and its development time is in petal and is also being wrapped by calyx, and the content of its assorted bacterium is relatively low, and is if disinfecting time is oversize, also bigger to the injury of flower pesticide.The flower pesticide of disinfecting is inoculated in the callus inducing medium, and the statistics disinfecting time finds that to the result that influences of inductivity inductivity is on a declining curve with the disinfecting time growth.Through 6 combination statistical datas, along with the alcohol disinfecting time lengthening, pollution rate reduces, but callus also descends thereupon, so carry out be defined as 5s, and the mercuric chloride disinfecting time being at 8min in the surface sterilization selection of service time at alcohol, pollution rate is controlled at about 14%, and inductivity snow honey is 41.7%.Take all factors into consideration and select this combination disinfection.

Table 2 difference is disinfected the influence to flower pesticide pollution rate and callus

Determining of 2 best callus inducing mediums

2.1 material and method

With MS is minimal medium, adds 6.5gL ^-1Agar strip and different hormone and variable concentrations sucrose (table 3), pH is transferred to 5.8, autoclaving 20min.Carry out the dark place reason after the inoculation, the dark place reason time is 7d, changes culturing room's cultivation that light is cultivated again over to.

Condition of culture is a temperature: 25 ℃ ± 1 ℃; Light application time: 12h light/12h is dark; Intensity of illumination: 2000Lx.

Each inducing culture is established two repetitions, three culture dishes of each repeated inoculation, 50 pieces in each culture dish inoculation flower pesticide.The callus induction rate statistics is as the criterion with the flower pesticide number that flower pesticide inoculation back 30d produces callus.

Callus induction rate %=produces flower pesticide number * 100 of the flower pesticide number/inoculation of callus.

The different inducing cultures of table 3 are handled

Annotate:,, add the compound method of IAA, KT packing again so take elder generation with behind the medium high-temperature sterilization owing to IAA, KT pyrolytic.

2.2 different hormone combinations is to the influence of callus induction rate

What this test was adopted is basic the cultivation with MS, designs 9 kinds of different hormone combinations proportionings, observes the callus induction situation behind the 20d.Brownization promptly occurred in second day behind the discovery combination 1-3 culture medium inoculated flower pesticide, and also do not divided callus through 40d.And combination 4-9 can see callus by naked eyes, and the withered phenomenon of brownization behind the inoculation flower pesticide takes place.Add up callus induction rate behind the 30d, the result shows when sucrose concentration is 30g/L, 4,6, the 9 pairs of callus of combination that improve BA concentration and reduce NAA concentration are formed with good facilitation, show as on the callus form closely, the surface yellow green has graininess, and is strong at the differentiation capability of differential period.Wherein make up 6 inductivities and be significantly higher than other several combinations, snow honey has 87.2% to split into callus (table 4), and the callus form shows as densification, yellow green, helps the differentiation of plant.To show as callus growth speed fast though make up 5, and quality is bulk, and surface color is dark, then shows as water stain shape, does not have differentiation capability.Therefore take all factors into consideration and select combination 6 (MS+BA0.5mgL for use ^-1+ NAA2.0mgL ^-1+ KT0.1mgL ^-1+ agar 6.5gL ^-1) be the prescription of best hormone kind of this research callus induction and concentration.

2.3 different sucrose combinations are to the influence of callus induction rate

Table 4 shows that sucrose concentration is 30gL aspect inductivity ^-1The inductivity of 4,6,8 pairs of callus of combination be higher than with 60gL under the hormone combinations ^-1Combination 5,7,9.On the form of callus, 30gL ^-1The combination callus growth is good, shows as yellow green, and 60gL ^-1The sucrose concentration combination then shows as the later stage brown, and then transfers water stain shape to, can not break up plant.Show 30gL ^-1Sucrose concentration starts the division of flower pesticide, and callus propagation all has good facilitation.30d statistics callus is 30gL at sucrose concentration ^-1, MS+BA0.5mgL ^-1+ NAA2.0mgL ^-1+ KT0.1mgL ^-1+ agar 6.5gL ^-1Medium combination inductivity is 87.2%.Therefore choosing the interpolation sucrose concentration in this test is 30gL ^-1

Table 4 different hormone combinations and sucrose combination are to the influence of callus induction

Annotate: English alphabet is represented different disposal difference, and on behalf of difference, capitalization in the Duncan analysis to see significance level (0.01), and lowercase is that significance level (0.05) is as follows.

The establishment of 3 best regeneration culture mediums

3.1 materials and methods

It is minimal medium that the callus of subculture 2 times is forwarded to MS, adds the differential medium of different hormones, approximately just differentiates strawberry plant (table 5) behind the 70d.

The different differential mediums of table 5 are handled

Annotate: because the ZT pyrolytic so take elder generation with behind the medium high-temperature sterilization, adds the compound method of ZT packing again.

Callus number * 100 of the callus number/inoculation of differentiation rate %=differentiation.

3.2 different hormones are to the influence of regeneration

Callus is changed in the differential medium of the additional different hormone concentrations of MS+ sucrose 30gL-1+ agar 6.5gL-1, PH=5.8 (table 6), the result shows that most callus are on differential medium, and it is green that color turns, it is solid that quality becomes, and the callus surface is graininess.Along with the prolongation of incubation time, different results has appearred in the callus on the different differential mediums, and the part callus differentiates the indefinite bud of growing thickly (Fig. 2); The easy browning of one side of the callus contact medium that has, and dead gradually.This experiment shows, is adding parahormone of the same race and concentration (BA1.5mgL with minimal medium equally ^-1+ NAA0.5mgL ^-1+ ZT2.0mgL ^-1) the situation honeywort certain kind of berries anther callus differentiation rate of snowing is higher, reaches 37.5%.Anther culture differentiation speed is slow, generally just begins to enter differentiation on the differential medium behind the 70d placing.Find out by experimental result, when NAA concentration by 0.5mgL ^-1Bring up to 1.0mgL ^-1The time, differentiation rate all reduces, and the differentiation of visible strawberry anther plant only takes place under the very low condition of external source auxin concentration.The auxin concentration height, though callus growth speed is very fast, the callus external form can show water stain shape, and passing in time, gradually becomes brown, is unfavorable for the differentiation of regeneration plant.From result of the test, when other two hormone concentrations constant, along with zeatin concentration by 1.5mgL ^-1Be increased to 2.0mgL ^-1The time, first three is handled the sweet callus differentiation rate of snow and brings up to 37.5% by 30.9%, and back three sweet callus differentiation rates of processing snow bring up to 31.7% by 28.6%, thus the explanation zeatin has significant facilitation in the bud atomization.

The different hormone concentration combinations of table 6 are to the influence of callus differentiation rate

The establishment of 4 best subculture mediums

4.1 material and method

When the tissue cultivating seedling of differentiation grows to the 1cm left and right sides, the careful cutting-out is inoculated into proliferated culture medium, proliferated culture medium is selected to handle (table 7) with the variable concentrations contrast of additional BA of MS and NAA, during enrichment culture, every kind of medium is established two repetitions, five bottles of each repeated inoculations, and every bottle of subculture tissue cultivating seedling strain number is 3 strains, the 30d subculture is 1 time in the enrichment culture process, adds up every group and handles bud propagation quantity.

Table 7 proliferated culture medium is handled

4.2 determining of subculture medium

The snow honeywort certain kind of berries healthy seedling that induces is transferred on the subculture medium, and behind the cultivation 30d, the propagation situation of investigation bud the results are shown in Table 8.The growth pattern of bud of growing thickly has two kinds, and a kind of is that foundation portion maternal plant produces callus, directly differentiates indefinite bud then on callus, and another mode is directly to grow new plant at the axillalry bud of maternal plant.In BA concentration is 0.5mgL ^-1The time, adding NAA and can increase its rate of increase, growth coefficient brings up to 4.7 by 3.9.When BA concentration increased, differentiation rate improved, and vitrification phenomenon also occurs thereupon simultaneously.It is generally acknowledged, the height of BA content in the medium, whether the generation that directly influences the vitrifying seedling has only the reasonably combined of the basic element of cell division and growth hormone, just can reach desirable Growth and Differentiation effect.This research is through taking all factors into consideration, and choosing hormone concentration BA is 0.5mgL ^-1+ NAA0.1mgL ^-1, the growth coefficient of bud is 4.7, with BA be 0.5mgL ^-1The time, NAA concentration is 0.0,0.05mgL ^-1There is significant difference.

The cultivation effect of each proliferated culture medium of table 8

The establishment of 5 rooting of vitro seedling medium and transplanting method

With subculture several times the strawberry anther tissue cultivating seedling of robust growth receive 1/2MS (macroelement reduces by half, other components unchanged) and 1/2MS+NAA0.2gL ^-1Medium, the growing state of its root of observation behind the 30d.

Strawberry seedling on proliferated culture medium generally also has a large amount of roots and grows, but has callus to occur at the root that proliferated culture medium grows in foundation portion, can influence its survival rate after the transplanting, so regeneration plant will forward the root media processing of taking root to.The result shows, 1/2MS medium root of hair evening, and can produce a large amount of callus at root, after having added growth hormone, take root early, and the rooting rate height, and the root callus obviously reduces.

Before the transplanting, the plant that will take root is through light hardening 7-10d, uncap during transplanting to add water and temper 1-2d, generally with vermiculite and perlite mixed system as being transplanting medium.Transplant back attention mouth mask and preserve moisture, the temperature during transplanting is controlled at 20-25 ℃ of scope, and transplanting survival rate reaches 90%.

(2) embodiment

Take from field healthy and strong snow honeywort certain kind of berries bud, developmental stage is that monokaryon keeps to the side the phase (size is about 4-6mm), at 4 ℃ of refrigerator cold treatment 72h.Cold treatment finishes the back and takes out disinfection on super-clean bench.

Disinfecting time and step are: bud once once places 0.1% mercuric chloride (adding 0.1% tween) to soak 8min with aseptic water washing again after 70% alcohol-pickled 5s with aseptic water washing and uses aseptic water washing 8-10 time (10min) at last.Blot the water on bud surface with aseptic blotting paper after the sterilization, the bud of disinfecting is stripped flower pesticide, be inoculated into MS+BA0.5mgL ^-1+ NAA2.0mgL ^-1+ KT0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Callus inducing medium in.

It is minimal medium that the callus of subculture 2 times (30d) is forwarded to MS, adds BA1.5mgL simultaneously ^-1, NAA0.5mgL ^-1And ZT2.0mgL ^-1Differential medium in, approximately just differentiate the strawberry plant behind the 70d.The inventive method is cultivated differentiation rate with strawberry anther and is brought up to 37.5%, than prior art 9.47% (look into middle duckweed, Liu Jun, Liu Dingfu. influence the analysis that strawberry anther is cultivated factor. the Anhui agricultural science, 2006,34 (1): 24～28) improved 28.03 percentage points.

The strawberry healthy seedling that induces is transferred to MS+BA0.5mgL-1+NAA0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1On the subculture medium, carry out shoot proliferation and cultivate 2～3 times.Forward 1/2MS+NAA0.2gL then to ^-1+ agar 6.5gL ^-1The root media processing of taking root.

After nursery stock took root, the plant that will take root was through light hardening 7-10d, uncap during transplanting to add water and temper 1-2d, generally with vermiculite and perlite mixed system as being transplanting medium.Transplant the back and note preserving moisture with the plastic film cover mouth, the temperature during transplanting is controlled at 20-25 ℃ of scope, and transplanting survival rate reaches 90%.

Claims (1)

1, a kind of strawberry anther that utilizes is cultivated the method for carrying out detoxifying fast breeding, it is characterized in that:

(1) disinfects

Getting length is the strawberry bud of 4-6mm, at 4 ℃ of refrigerator cold treatment 72h;

Then with bud with aseptic water washing once 70% alcohol-pickled 5s (second) afterwards again with aseptic water washing once place add volume ratio be 0.1% tween and mass volume ratio be 0.1% mercuric chloride solution soak 8min (minute);

Use aseptic water washing 8-10 time at last, blot the water on bud surface with aseptic blotting paper;

(2) callus induction differentiation

The bud of disinfecting is stripped flower pesticide, be inoculated into MS+BA (6-benzyl aminopurine) 0.5mgL ^-1+ NAA (methyl) 2.0mgL ^-1+ KT (cytokinin) 0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Callus inducing medium in 30～60d (my god);

Forward callus to MS+BA1.5mgL ^-1+ NAA0.5mgL ^-1+ ZT (zeatin) 2.0mgL ^-1+ agar 6.5gL ^-1Differential medium in 70～90d;

Then the strawberry seedling that obtains in the differential medium is transferred to MS+BA0.5mgL ^-1+ NAA0.1mgL ^-1+ sucrose 30gL ^-1+ agar 6.5gL ^-1Subculture medium on, carry out shoot proliferation and cultivate 20～30d;

(3) rooting and transplant

The strawberry seedling forwards 1/2MS+NAA0.2gL to ^-1+ agar 6.5gL ^-1Root media take root and handle 20～30d;

After nursery stock took root, the plant that will take root added water and tempers 1-2d through tempering 7-10d under the outdoor natural conditions, uncapping during transplanting, and as transplanting medium, the temperature during transplanting is controlled at 20-25 ℃ of scope with vermiculite and perlite mixed system.

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