CN110419447A - A kind of blueberry tissue cultural method - Google Patents
A kind of blueberry tissue cultural method Download PDFInfo
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- CN110419447A CN110419447A CN201910818126.2A CN201910818126A CN110419447A CN 110419447 A CN110419447 A CN 110419447A CN 201910818126 A CN201910818126 A CN 201910818126A CN 110419447 A CN110419447 A CN 110419447A
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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
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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
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Abstract
The invention discloses a kind of blueberry tissue cultural method, this method is to choose the disinfection of blueberry explant, is put into culture medium and is cultivated through evoked callus growth phase, the induction of bud and its growth phase and the stage of taking root.Of the invention cultural method reproduction speed of knitting is fast, is not limited by the geographical time, and plant susceptible gene is low, improves and takes root and survival rate, the tissue cultures breeding nursery stock industrialized development suitable for blueberry.
Description
Technical field
The present invention relates to blueberry cultivation field, especially a kind of blueberry tissue cultural method.
Background technique
Blueberry (scientific name: Blueberry) belongs to the perennial fallen leaves xylophyta of Ericaceae Vaccinium.Its fruit is small slurry
Fruit, fruit concern by people with plurality of health care functions such as improve the immunity of the human body, anti-aging, softening blood vessel, improve
Eyesight, anticancer and anti-cardiovascular disease function；Rich in nutriment abundant, such as protein, fat, fiber, SOD, vitamin
A, vitamin B, vitamin C, vitamin E and a variety of mineral elements, such as iron, zinc, calcium, magnesium, copper and anthocyanidin, flavonol,
The special nutrient compositions such as tartaric acid.Due to blueberry have more than nutritional ingredient and healthcare function, occur in the market and blueberry phase
The product of pass is increasing, for example, blueberry fruit wine, jam, can, preserved fruit, fruit juice and beverage etc. or cake, jelly, Yoghourt,
The raw material of candy etc., blueberry gradually have certain position in the market, this is also the key that blueberry rapidly develops.
Blueberry is distributed mainly on North America, also has point of wild blueberry on the ground such as the Daxing'an Mountainrange of northern China and the Xiaoxinanlin Mountains
Cloth.The history of blueberry cultivation is shorter, and carrying out introducing and planting to blueberry earliest is the U.S., takes the lead in from the nineteen thirties U.S.
Since realizing blueberry commodity industrialization production, more than 30 countries such as Canada, Germany, Australia, Holland, Japan start in succession
Introducing and planting is rapidly progressed blueberry.Currently, the ground such as the U.S., Europe, Japan have carried out blueberry industry metaplasia production.
But worldwide, also much supply falls short of demand for Blueberry, shows according to data, and American blueberry total output is only 350,000 within 2012
Ton, accounts for about the 60% of global total output, and present blueberry yield is significantly larger than this number.Study on tissue culture is carried out to blueberry
That earliest is Niekerson, he carries out callus tissue culture with blueberry branch and succeeds, this indicates that blueberry production enters
New era carries out efficient blueberry nursery stock production, lays a good foundation to commercially produce.Currently, plant tissue culture technique is good
Kind is cultivated, quickly breeding, detoxification breeding etc. are preferably developed, in this technical application to blueberry, progress blueberry seedling
Wood is cultivated and fine breeding, provides technical support to commercially produce, blueberry is made to obtain good and fast development.
Blueberry introducing and planting work in China's starts from nineteen eighty-three, being taken the lead in by Jilin Province, China agriculture university.Blueberry industry exists
The development in China is later, and many people do not hear this noun also.Cultivation history is short at home for blueberry, and Technical comparing falls behind, to indigo plant
The habit of the certain kind of berries and Environmental Studies are not thorough enough, mainly introduce a fine variety and cuttage on seedling propagation, usually hardwood cutting, but
Also there is green wood cutting, this is all traditional modes of reproduction, is had the shortcomings that many.To adapt to Times ' Demand, on seedling propagation
It must carry out tissue cultures mode and carry out seedling propagation.Cheng Shuyun etc. carries out outside sprout-cultivating-bottle culture to blueberry tissue culture seedling and succeeds,
This is developed China's blueberry tissue culture also, supports for industrialization production blueberry add-on technology.Blueberry industry existed in recent years
China rapidly develops and comes, and sees in cultivated area, and China's blueberry area is more and more big, and by East China, southwester area extends,
Guizhou, which is just particularly suitable for the place of blueberry plantation, especially Qiandongnan Prefecture of Guizhou Province, has the unique advantage for developing blueberry, this is blueberry
One of the key greatly developed and promoted.
Blueberry belongs to emerging fruit, the deep favor by numerous people in China.All parts of the country start to carry out to blueberry one after another
Introducing and planting is spread by north orientation south, and especially south is more suitble to the growth of blueberry, it is possible thereby to see, blueberry has very big
Develop scene.In recent years, people's living standard is increasingly promoted, and is increasingly stringenter to the fruit pursuit of high-quality, more emphasis fruit
The nutrition and health of product, this is applied with huge pressure to blueberry development.The production of the Blueberry of high-quality needs excellent
Seedling and its good growing environment.But there are many problems for current China's blueberry development, are that cultivating and growing history compares first
Short, the 1980s just starts to carry out introducing and planting from foreign countries；Secondly planting technology relatively falls behind, it is main using introducing a fine variety and
The domestication of wild blueberry；Production technology is not high to cause yield and quality low, and the growing environment of blueberry and growth are practised in production base
Property it is fuzzy, be suitble to which type of growing environment unclear, it is not known that how to produce the Blueberry of high yield, high-quality；Blueberry
Main problem is seedling-wood breeding problem, and conventional blueberry modes of reproduction is mainly introduced a fine variety with cuttage etc., cannot quickly be bred in this way
A large amount of nursery stock, and disease can occur in reproductive process, make nursery stock itself disease carrying germ, secondly cuttage seedling rooting is difficult, this
The phenomenon that modes of reproduction seriously affects seedling quality, and especially blueberry will appear variety deterioration and low quality after introducing a fine variety.Cause
This, it is fast to invent a kind of reproduction speed, is not limited by the geographical time, reduces that plant is susceptible, and raising is taken root and the indigo plant of survival rate
Certain kind of berries tissue culture technique and the development of nontoxic seedling technology are very urgent.
Summary of the invention
The object of the present invention is to provide a kind of blueberry tissue cultural methods.Of the invention knits cultural method reproduction speed
Fastly, it is not limited by the geographical time, plant susceptible gene is low, improves and takes root and survival rate, promotes the tissue cultures of blueberry
Breeding and Seedling Industrialization Development.
Technical solution of the present invention: a kind of blueberry tissue cultural method, this method are to choose the disinfection of blueberry explant, are put into
It is cultivated in culture medium through evoked callus growth phase, the induction of bud and its growth phase and the stage of taking root.
In a kind of blueberry tissue cultural method above-mentioned, the method is to choose blueberry explant, washes away table with tap water
Face dirt impurity sterilizes explant then at superclean bench, is cut into 1.5 centimeter lengths, the explant that will be cut above alcolhol burner
Body is put into culture medium, and sealing treatment is carried out after the completion of inoculation, in the growth phase of evoked callus, selects WPM+6- benzyl
Adenine phosphate selects WPM+6- benzylaminopurine+methyl α-naphthyl acetate+kinetin culture medium culture, induction and its growth step in bud
Duan Xuanyong WPM+6- benzylaminopurine+methyl α-naphthyl acetate culture medium culture selects WPM+6- benzylaminopurine+naphthalene in the stage of taking root
Acetic acid+kinetin culture medium culture.
In a kind of blueberry tissue cultural method above-mentioned, blueberry explant is the blueberry branch of robust growth in the method
Item.
In a kind of blueberry tissue cultural method above-mentioned, the sterilization method of explant is the alcohol with 75% in the method
30s is impregnated, then impregnates 8min with 0.1% mercuric chloride again.
In a kind of blueberry tissue cultural method above-mentioned, WPM is calculated by parts by volume in the method, by 50 parts of a great number of elements
Partially, 10 parts of calcium salt parts, 5 parts of microelement parts, 5 parts of molysite parts and 9 parts of organic substance parts are formulated, in which:
A great number of elements part is the KNO by 3.8/L3The MgSO of+7.4g/L4﹒ 7H2The KH of O+3.4g/L2PO4+ 8.0g/L's
NH4NO3It is formulated with water；
Calcium salt part is the Ca (NO by 55.6g/L3)2﹒ 4H2O and water are formulated；
Microelement part is the MnSO by 4.5g/L4﹒ 4H2The ZnSO of O+1.72g/L4﹒ 7H2The H of O+1.24g/L3BO3+
The CuSO of 0.05g/L4﹒ 5H2The Na of O+0.05g/L2MoO4﹒ 2H2O and water are formulated；
Molysite part is the Na by 7.45g/L2The FeSO of-EDTA+5.57g/L4·7H2O and water are formulated；
Organic substance part is by the Vb6+ of the Vb1+0.1g/L of the glycine+0.2g/L of the inositol+0.4g/L of 40g/L
The Vb5 and water of 0.1g/L is formulated.
In a kind of blueberry tissue cultural method above-mentioned, the pH value of culture medium is 6.2 in the method, the culture medium
PH value be to be adjusted with the sodium hydroxide solution of 1mol/L and 1mol/L hydrochloric acid solution.
Inventor verifies effect of the invention and has carried out following experiment:
Experimental example:
1 materials and methods
1.1 test material
Material for this test is the Vaccinium ashei branch of triennial, its stem section is mainly taken to be cultivated.Material source is in expensive
State agricultural college, university vegetables sample plot.
1.2 test apparatuses and reagent
2.2.1 test apparatus
This test apparatus mainly has: sterilizing installation (high-pressure steam sterilizing pan)；Inoculating facility (transfer room, ultra-clean work
Platform, tweezers, alcolhol burner, absorbent cotton, vaccinating lancet)；Culture device (constant temperature incubation room, constant incubator, triangular flask, plastic closures
Film, wiring)；Storage facilities (refrigerator, Brown Glass Brown glass bottles and jars only, baking oven)；It is other: iron cylinder, electromagnetic oven, label paper, camera, pH test paper
Deng.
1.2.2 experiment reagent
Reagent includes sucrose, agar, 6- benzylaminopurine (6-BA), methyl α-naphthyl acetate (NAA), zeatin (ZT), cell point
Split element, kinetin (KT), 75% alcohol, 0.1% mercuric chloride (mercury chloride), sodium bicarbonate etc..
1.3 test method
1.3.1 experimental design
1.3.1.1 the configuration of mother liquor
Mother liquor including MS culture medium and improvement WPM culture medium, a great number of elements mother liquor, calcium salt, microelement mother liquor, iron
Salt, organic matter mother liquor, hormone mother liquor etc. are configured to certain density mother liquor, and the labelled and date is put into 4 DEG C of refrigerator
It saves.Mother liquor including MS culture medium and improvement WPM culture medium, a great number of elements mother liquor, microelement mother liquor, molysite, have calcium salt
Machine matter mother liquor, hormone mother liquor etc. are configured to certain density mother liquor, and the labelled and date is put into 4 DEG C of refrigerator and saves.
It is used for the later period, when in use as precipitating occurs in fruit mother liquor or crystal is precipitated, then cannot use, preparation mother liquor need to be reappeared.
1 MS culture medium prescription of table
Table 2 improves WPM culture medium prescription
1.3.1.2 the configuration of culture medium
Corresponding mother liquor is drawn by the ingredient of each culture medium and is trained culture medium, is adjusted pH value to 6.2, is generally used 1mol/L
Sodium hydroxide solution and 1mol/L hydrochloric acid solution to be adjusted pH value too low, and the amount of agar is a timing, and culture medium is not
It is fixed easily, pH value is excessively high, then is unfavorable for blueberry growth, so adjusting suitable pH value has vital effect to test.
1.3.1.3 sterilizing
Configured culture medium, the distilled water of bottled sealing, culture dish (containing filter paper), filter and filter paper is (close
Envelope), size a pair of glass beaker etc. of sealing be put into 121 DEG C together, sterilize in the high-pressure steam sterilizing pan of 0.1kpa
20min。
1.3.1.4 inoculation
The blueberry stem section tissue that explant is 3 years, including spray and stiff wood (complete lignifying and semi-lignified), it is then right
Explant washes away surface smut impurity with tap water, with the suitable size of scissors clip, after the sterilized sterilizing of superclean bench
About 1.5 centimeter lengths are cut into, then the stem section cut are put into culture medium above alcolhol burner, then cover bottle cap.It has been inoculated with
At rear carry out sealing treatment, label and date are posted.
1.3.1.5 culture
Including several stages such as Initial culture, squamous subculture, culture of rootage.Initial culture is the culture after stem section inoculation,
Including long callus and bud；Squamous subculture refers to be further cultured on the basis of Initial culture, the Multiplying culture including stem section and
Stem section growth etc.；Culture of rootage, which refers to the process of, induces its long root.Inoculated triangular flask is put into constant temperature illumination box
Cultivated, temperature be 25 ± 1 DEG C, intensity of illumination 2000lx, before 5 days progresss dark cultures, the later period carry out brightness combine train
It supports, it is general to carry out optical culture 14h, dark culture 10h daily.
1.3.1.6 acclimatization and transplants
After the long budding of explant and root, first slowly bottleneck is opened, is fully opened bottle after 3d, in tap water undershoot
Then the culture medium of wash clean tissue-cultured seedling base portion is transplanted in the hole tray equipped with matrix.Matrix uses turf, perlite, rotted leaf
Soil, organic fertilizer etc. are uniformly mixed to be configured.It is sprinkled profoundly water after moving into hole tray, place culture first weaker in illumination, after 5d again
It goes under general illumination condition, allows its normal growth.
1.3.1.7 observation and record
It is taken pictures after inoculation and writing record, the explant cut surface situation after physical record inoculation, if occur
The data such as color change.Also the growing state of explant is recorded during dark culture, if having callus generation, if having bud long
Out and color change.After carrying out illumination cultivation, every the growing state of 5d observation explant, and take pictures and writing record.
2.4 data processing
Test data is handled with EXCEL software, analyzes the reasonability etc. of data.
3 results and analysis
The influence of 3.1 different thimerosals and processing time to blueberry stem section
In the case where 0.1% mercuric chloride disinfection 8min processing time is constant, change 75% alcohol to the place of blueberry stem section
It manages the time, the pollution condition and survival rate after record inoculation carry out table 3 obtained from single-factor control.It can by the result of table 3
Know, in the case where the mercuric chloride disinfection treatment time is constant, with the increase of the processing time of alcohol, pollution rate is successively reduced, at
Motility rate is first to increase to reduce afterwards.Pollution rate is highest be 0s 75% ethanol postincubation time, that is, when not having to ethanol postincubation, pollution rate
It is 53.3%；Pollution rate it is minimum be the ethanol postincubation time be mono- group of 50s, pollution rate is almost 0.From survival rate interpretation of result,
Survival rate is highest be mono- group of 30s 75% ethanol postincubation time, up to 67.7%, followed by mono- group of 20s survive for 60%
Rate is successively 10s, 0s, 40s, 50s thereafter.It obtains from Mortality data when the mercuric chloride processing time is identical, with ethanol postincubation
The increase of time, survival rate are gradually increased, and the death rate is up to 80%.From these data it is known that 75% alcohol different disposal
Time blueberry stem section it is polluted and survival rate from the point of view of, the optimization process time is mono- group of 30s, and pollution rate is lower and survival rate for reorganization
Higher, 75% alcohol most preferably disinfects the time.
Influence of the 3 75% alcohol different disposal time of table to blueberry stem section
Note: after survival rate is 30d, stem section does not change colour and necrosis, is denoted as and survives.
Table 4 is to change 0.1% mercuric chloride to blueberry stem section in the case where the 75% alcohol disinfecting 30s processing time is constant
The time is handled, pollution condition and survival rate after record inoculation carry out obtained from single-factor control.By table 4 the result shows that,
When 0.1% mercuric chloride disinfecting time is 4min, highest is polluted, with the increase of disinfecting time, pollution rate is gradually decreased, pollution rate
Minimum is 0.1% mercuric chloride disinfection treatment 10min and 12min (for 6.7%).From survival rate, as 0.1% mercuric chloride sterilizes
Liquid handle the time increase, survival rate is first rises to decline afterwards, up to 0.1% mercuric chloride processing 8min, up to 73.3%,
It is 8min > 10min > 6min > 4min > 12min that 0.1% mercuric chloride, which disinfects influence size of the blueberry stem section to its survival rate,.From
Mortality results are learnt, when the ethanol postincubation time is identical, when mercuric chloride handles 4min, 6min, 8min, the death rate is lower, is
In incremental trend, the death rate of 12min processing is up to 60.0% for 13.3%, 10min and the death rate of 12min processing.So
Comprehensively consider pollution rate and survival rate, the more appropriate mercuric chloride disinfection treatment time is 8min, the period pollution rate it is low and at
Motility rate is relatively high.
It can be concluded that, considered in conjunction with pollution rate and survival rate by table 3 and table 4, more appropriate disinfection combination are as follows: 30s
75% alcohol disinfecting processing+10min, 0.1% mercuric chloride, the combination pollution rate is low and survival rate is higher.
Influence of the 4 0.1% mercuric chloride different disposal time of table to blueberry stem section
Note: after survival rate is 30d, stem section does not change colour and necrosis, is denoted as and survives.
Influence of 3.2 different culture mediums to blueberry stem section
Under conditions of hormone 6-BA and NAA concentration is constant, in five kinds of bases such as MS, 1/2MS, 1/4MS, WPM and 1/2WPM
Hormone 1mg/L 6-BA and 0.5mg/L NAA is added on basal culture medium, is counted the generation of callus and is sprouted, situation of taking root,
Screen the minimal medium for being suitble to the growth of blueberry stem section.As shown in Table 5, WPM culture medium callus production rate highest,
It is 67.7%, followed by MS culture medium is 60%, and minimum is that 1/4MS culture medium is 13.3%.On induction stem section sprouts,
It is the long bud rate of WPM culture medium is 76.7% that long bud rate is highest, and followed by the long bud rate of MS culture medium is 63.3%, minimum
The long bud rate for being 1/4MS culture medium is 26.7%.
By the production of stem section evoked callus and from the aspect of sprouting, more appropriate culture is improvement WPM culture medium, it
It is not only higher in the generation rate of callus, still higher at the induction stem section aspect that sprouts, followed by MS culture medium.Later period
Both culture mediums for being all in experiment filter out suitable blueberry stem section tissue training in conjunction with other hormones and its concentration
Feeding formula.
Influence of 5 different culture medium of table to blueberry stem section
The influence that 3.3 plant growth regulator types and concentration grow blueberry stem section
In growing process, growth regulatory substance is played a crucial role.During plant tissue culture, these
Although the dosage of substance is seldom, to obvious effects such as induction, the dedifferentiations of calli induction, bud.
As shown in Table 6, in any growth regulating material of no addition, without the generation of callus and root,
Only a small amount of bud generates.There is the formation of callus and bud in each group of addition hormone, callus formation rate is higher
It is two groups of 1.0mg/L 6-BA and (0.5+1.0+0.5mg/L) 6-BA+NAA+KT, the inductivity of callus is
73.3%, it is secondly the combination such as 2mg/L 6-BA, (1+0.5mg/L) 6-BA+NAA, (1.0+1.0mg/L) ZT+NAA, it is poor
It is 3mg/L and 4mg/L 6-BA processing, the inductivity of the higher callus of 6-BA concentration is lower.The induction of callus and swash
Plain 6-BA has close relationship, and the combination for having it to participate in, the inductivity of callus is higher, the callus group participated in without it
It is lower to knit inductivity.
As can be seen that these hormones are less obvious to the induction of bud from the growing state result of bud, have a certain amount of
Bud generates, and has plenty of the generation of a little bud, preferably (0.5+1.0mg/L) 6-BA+NAA combination, and the formation rate of bud is higher.
Other combination differences are little, can form the differential growth of bud.
It is recognised that the formation rate of root is lower from root growth situation result, the generation of most of not root, or generate few
Perhaps a root, but (0.5+1.0+0.5mg/L) 6-BA+NAA+KT processing has two roots to be formed, it can be seen that and the combination is advantageous
It takes root in blueberry stem section tissue cultures.It is relatively difficult that root is formed from the point of view of the growing state of root, in blueberry stem section bottle, only a little
1 to 2 roots are formed, this may be the suitable hormone combinations that do not explore, this needs the research and probe in later period.
The case where 6 hormon type of table and concentration grow blueberry stem section table
Note: CK is indicated without addition hormone；
Indicate that no callus (bud or root) generates；
+ indicating that a small amount of callus (bud or root) generates, callus production quantity is only one on a small quantity within diameter 0.5cm
Root；
++ indicate that a certain amount of callus (bud or root) generates, callus production quantity is certain in diameter 0.5cm to 1.0cm
Amount, there is two roots；
+++ indicate a large amount of callus (bud or root) generate, callus production quantity diameter 1.0cm to 1.5cm be largely,
Root has three；
Callus (bud or root) production quantity is very big for ++++indicate, callus production quantity is callus production more than diameter 1.5cm
It measures very big.
3.4 blueberry tissue culture situations observe and record
Blueberry tissue culture is as shown in Figure 1, under the regulation of hormone and culture medium, and calli induction rate is higher, In
When culture medium and the more suitable formula combination of hormone, obviously there is callus to be formed it can be seen that blade and stem section have after a week,
Agglomerating to be gathered in incision, the callus of stem section appears in the stem section bottom inside culture medium, and the callus of blade appears in blade
Around, including with inside culture medium intersection and culture medium, some is also exposed, is creamy white.Callus group after cultivating 2 weeks
Knitting growing way does not have significant change, then carries out switching and continues to cultivate, and callus is in faint yellow after switching 1 time, and figure A is stem section switching 1
The growing state of secondary callus, particulate matter become larger, in yellow green and slightly brownish red.After scheming B for stem section 2 times switchings
The growing state of callus, in flaxen, some is in brownish red.Scheme the growth feelings that C is blade 3 times switching callus
Condition, callus periphery callus are in the particle of yellow green, browning occur at centre, it may be possible to the browning of itself secretion
Caused by substance.These callus are taken out, can gently be crumbed with hand, be had a little moisture on finger, illustrate its quality
It is loose, it is interior to contain a certain amount of moisture.
After stem section inoculation, usually stem section first sprouts and sprouts, then long callus again, but also has callus earlier than bud
Appearance.When culture environment is proper, it is seen that axillary bud expands after 3 days, blade starts to burst forth long after a week, is tender
Green.Figure G is to sprout to come into leaves after stem section is inoculated with 1 week, and blade is in peak green.Scheme D and scheme H to be the growth that stem section is inoculated with 2 Zhou Houya
Situation, the blade grown earliest have two panels browning occur, other blades are peak green, and bud is up to 3 centimetres.(scheming I is by figure E and figure I
The picture taken pictures after being inoculated with 3 weeks from culture basal part, the area of callus are gradually increased) it is the life that stem section is inoculated with 3 Zhou Houya
Long situation, original stem section top browning is dead, and callus generation is arranged at bottom, and bud is up to 6-8 centimetres.Scheming F is that stem section is inoculated with 4 weeks
, there is apparent branch phenomenon in the growing state of bud afterwards, and blade is green.
Blueberry tissue culture is taken root relatively slowly, also more difficult.This research culture discovery, callus is by culture, after 50 days
Just it can be seen that having the generation of a little root, the appearance browning that callus at this time has.Scheming J is after blade inoculation 8 weeks
The long root situation of callus has two roots to occur, is creamy white, and one layer of white powder is with outside callus at this time and is occurred.
Figure K is to take root after blade inoculation 9 weeks, has two roots to occur, and is up to 3 centimetres, browning obviously occurs in callus centre.
Scheming L is long root after blade inoculation 10 weeks, has three roots to occur, and longer to be up to 5 centimetres, bifurcation also occur in some roots, when
Root long starts bifurcated afterwards to a certain extent, grows in 2 bifurcated forms, and the callus browning area of center is more and more big.
4 discussion and conclusion
4.1 discussing
This experimental study blueberry stem section tissue cultures and fast breeding technique, to obtain high-quality indigo plant by way of in vitro culture
Certain kind of berries seedling.This test carries out each condition optimizing and finally obtains tissue-cultured seedling by improved culture medium and plant growth regulating substance
Process.
Firstly, screening suitable thimerosal and disinfecting time.Single-factor is carried out by two factors of control alcohol and mercuric chloride
Test, screens best sterilization conditions.Test result obtains 75% ethanol postincubation 30s, and the 0.1% mercuric chloride processing time is that 8min is most
Good sterilization conditions, then use aseptic water washing 3-5 times, pollution rate can be made to substantially reduce, at the same do not influence stem section growth,
Differentiation.The probability for going out to show notch browning phenomenon after inoculation, and occurring in triennial stem section material (complete lignifying) compared with
It is high.Stem section should be immediately placed in culture medium after cutting, and prevent its browning.Activated carbon material is added in preliminary experiment and is not played changes
The effect of kind brown stain, makes culture medium become black, is unfavorable for observing instead.
Secondly, screening the minimal medium of suitable blueberry stem section tissue culture.By configuring MS, 1/2MS, 1/4MS, improvement
Five culture mediums such as WPM, improvement 1/2WPM, add corresponding hormone, count the growing state of stem section callus and bud.Synthesis result
Learn that improvement WPM culture medium evoked callus rate and long bud rate highest, callus rate are 67.7%, long bud rate is 76.7%, secondary
More appropriate is MS culture medium, respectively 60.0% and 63.3%.
Third compares test to the type of plant growth regulating material, concentration and combinations thereof, screens most suitable induction stem
Segment length's callus, the condition for sprouting, taking root.Growth regulatory substance has mainly used 6-BA, NAA, KT, ZT etc., wherein 6-BA+NAA+
KT (0.5+1.0+0.5mg/L) is best with the use of effect, and its growth high to the inductivity of callus is put up the best performance,
In the yellowish bulk particle of peak green and part.The using effect of single HORMONE TREATMENT 6-BA as the result is shown is preferable, callus
Inductivity it is higher and growth is very fast, after a week it can be seen that apparent callus is formed, later callus is obviously grown up within 3 weeks.
Secondly, the color of callus growth is preferable in 6-BA+NAA processing, most of is in peak green；Induced bud growth is fast, bud after 3d
Start to sprout, be sprouted completely once all, blade tip and it is thin, greeny, bud is obviously grown up after three weeks, up to 5cm, some buds also companion
There is branch.
The color change of main detection stem section notch during tissue culture.Notch after explant inoculation generally will become brown
Color gradually becomes peak green after 1 week, and then grows callus.Callus early period is flaxen particle, then outer
It encloses and slowly becomes peak green, the presentation white powder being exposed on culture medium.Bud generally occurs earlier than callus, the stem with axillary bud
Duan Qiya 3d begins to sprout, and expands gradually, significantly opens after 1 week, grows the leaf of peak green, some culture mediums are in bud
Leaf whitens when longer, this should be a lack of caused by nutrition, lacks difficult mobile mineral element, besides when leaf is long
Out 7 to 8 when, several leaf yellowing of lower part bleach, this be mainly a lack of free nutrient (such as N P and K) or training
Caused by the condition of supporting is not suitable for.Observe the growth of root, 30d or so can see the tiny milky being differentiated to form by callus
Status, then slowly long length is thicker, is accompanied by bifurcated, i.e., so-called formation.
During the test, it there is also some problems, as being easy browning after explant inoculation, by adding active carbon object
Matter not can solve the problem.Explant, which is not inoculated with, occurs as soon as browning, may because after cutting when exposed on superclean bench
Between it is too long, lead to notch and the external world react extremely.
4.2 conclusion
Plant Tissue Breeding is in vitro in gnotobasis by several stages such as Initial culture, squamous subculture, culture of rootage
The process of culture, the requirement in each period difference, such as illumination, hormone, sterilizing difference.In blueberry stem section tissue cultures
In, the branch of robust growth is first chosen as explant material, and carry out disinfection to it after tap water is rinsed well processing, through this
Test the results show that the disinfection treatment of explant (blueberry stem section) be 75% alcohol impregnate 30s, then again with 0.1%
Mercuric chloride is dipped into 8min, reduces pollution rate with this.Under identical hormone and external condition same case, five culture mediums are sieved
It chooses, discovery improvement WPM culture medium is relatively suitble to the tissue cultures of blueberry stem section.In the growth of the induction long callus of stem section,
Optimal medium is WPM+2mg/L 6-BA and WPM+0.5mg/L 6-BA+1.0mg/L NAA+0.5mg/L KT, at this two
Reason callus induction rate is up to 73.3%, and early period is milky, then gradually becomes the particulate matter of peak green, periphery coating white
Particulate matter.
In terms of the induction of bud and its growth, optimal culture medium and hormone combinations are WPM+0.5mg/L 6-BA+
1.0mg/L NAA, processing bud growth is very fast, and leaf growth is more healthy and stronger and is in peak green；It newly sprouts and grows rapidly, stem section was at 4 weeks
After can grow to 6-9cm long, bottom callus is in faint yellow and peak green, and callus area is larger, newly to sprout
Growth elongation provides sufficient nutrition.
Callus is taken root quickly in blueberry stem section tissue culture, and the formula of taking root is improvement WPM+ (0.5+1.0+0.5mg/L)
6-BA+NAA+KT, it is high which combines rooting rate, has two root longs to go out, and root is more sturdy, is up to 4cm, some there are also bifurcated,
This makes root growth more flourishing, provides nutrition for upper part in lower absorbent sufficient nutrient, can generate complete plant
Strain.
The tissue cultures of blueberry are the bases of nontoxic nursery stock, this test is the infrastest of blueberry tissue culture, to be blueberry
Tissue cultures breeding nursery stock lay the foundation.
Compared with prior art, technology of the invention obtains high-quality blueberry seedling, by changing in a manner of through in vitro culture
Good culture medium and plant growth regulating substance, and carry out the process that each condition optimizing finally obtains tissue-cultured seedling.
Firstly, screening suitable thimerosal and disinfecting time.Single-factor is carried out by two factors of control alcohol and mercuric chloride
Test, screens best sterilization conditions.Test result obtains 75% ethanol postincubation 30s, and the 0.1% mercuric chloride processing time is that 8min is most
Good sterilization conditions, then use aseptic water washing 3-5 times, pollution rate can be made to substantially reduce, at the same do not influence stem section growth,
Differentiation.The probability for going out to show notch browning phenomenon after inoculation, and occurring in triennial stem section material (complete lignifying) compared with
It is high.Stem section should be immediately placed in culture medium after cutting, and prevent its browning.Activated carbon material is added in preliminary experiment and is not played changes
The effect of kind brown stain, makes culture medium become black, is unfavorable for observing instead.
Secondly, screening the minimal medium of suitable blueberry stem section tissue culture.By configuring MS, 1/2MS, 1/4MS, improvement
Five culture mediums such as WPM, improvement 1/2WPM, add corresponding hormone, count the growing state of stem section callus and bud.Synthesis result
Learn that improvement WPM culture medium evoked callus rate and long bud rate highest, callus rate are 67.7%, long bud rate is 76.7%, secondary
More appropriate is MS culture medium, respectively 60.0% and 63.3%.
Third compares test to the type of plant growth regulating material, concentration and combinations thereof, screens most suitable induction stem
Segment length's callus, the condition for sprouting, taking root.Growth regulatory substance has mainly used 6-BA, NAA, KT, ZT etc., wherein 6-BA+NAA+
KT (0.5+1.0+0.5mg/L) is best with the use of effect, and its growth high to the inductivity of callus is put up the best performance,
In the yellowish bulk particle of peak green and part.The using effect of single HORMONE TREATMENT 6-BA as the result is shown is preferable, callus
Inductivity it is higher and growth is very fast, after a week it can be seen that apparent callus is formed, later callus is obviously grown up within 3 weeks.
Secondly, the color of callus growth is preferable in 6-BA+NAA processing, most of is in peak green；Induced bud growth is fast, bud after 3d
Start to sprout, be sprouted completely once all, blade tip and it is thin, greeny, bud is obviously grown up after three weeks, up to 5cm, some buds also companion
There is branch.
The color change of main detection stem section notch during tissue culture.Notch after explant inoculation generally will become brown
Color gradually becomes peak green after 1 week, and then grows callus.Callus early period is flaxen particle, then outer
It encloses and slowly becomes peak green, the presentation white powder being exposed on culture medium.Bud generally occurs earlier than callus, the stem with axillary bud
Duan Qiya 3d begins to sprout, and expands gradually, significantly opens after 1 week, grows the leaf of peak green, some culture mediums are in bud
Leaf whitens when longer, this should be a lack of caused by nutrition, lacks difficult mobile mineral element, besides when leaf is long
Out 7 to 8 when, several leaf yellowing of lower part bleach, this be mainly a lack of free nutrient (such as N P and K) or training
Caused by the condition of supporting is not suitable for.Observe the growth of root, 30d or so can see the tiny milky being differentiated to form by callus
Status, then slowly long length is thicker, is accompanied by bifurcated, i.e., so-called formation.
In conclusion of the invention cultural method reproduction speed of knitting is fast, do not limited by the geographical time, plant susceptible gene
It is low, it improves and takes root and survival rate, the tissue cultures breeding nursery stock industrialized development suitable for blueberry.
Detailed description of the invention
Fig. 1 is that blueberry tissue culture situation in experimental example observes and records figure.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawings and examples, but be not intended as to the present invention limit according to
According to.
Embodiment 1.1. a kind of blueberry tissue cultural method, this method is to choose the blueberry branch conduct of blueberry robust growth
Explant washes away surface smut impurity with tap water, sterilizes then at superclean bench to explant, 1.5 centimeter lengths is cut into, in wine
The explant cut is put into culture medium above smart lamp, sealing treatment is carried out after the completion of inoculation, in the life of evoked callus
The long stage selects WPM+6- benzylaminopurine or selects WPM+6- benzylaminopurine+methyl α-naphthyl acetate+kinetin culture medium training
It supports, WPM+6- benzylaminopurine+methyl α-naphthyl acetate culture medium culture is selected in the induction of bud and its growth phase, in stage choosing of taking root
With WPM+6- benzylaminopurine+methyl α-naphthyl acetate+kinetin culture medium culture.
The sterilization method of explant is to impregnate 30s with 75% alcohol in the method, is then soaked again with 0.1% mercuric chloride
Steep 8min；WPM is calculated by parts by volume in the method, by 50 parts of a great number of elements parts, 10 parts of calcium salt parts, 5 parts of microelements
Partially, 5 parts of molysite parts and 9 parts of organic substance parts are formulated, in which:
A great number of elements part is the KNO by 3.8/L3The MgSO of+7.4g/L4﹒ 7H2The KH of O+3.4g/L2PO4+ 8.0g/L's
NH4NO3It is formulated with water；
Calcium salt part is the Ca (NO by 55.6g/L3)2﹒ 4H2O and water are formulated；
Microelement part is the MnSO by 4.5g/L4﹒ 4H2The ZnSO of O+1.72g/L4﹒ 7H2The H of O+1.24g/L3BO3+
The CuSO of 0.05g/L4﹒ 5H2The Na of O+0.05g/L2MoO4﹒ 2H2O and water are formulated；
Molysite part is the Na by 7.45g/L2The FeSO of-EDTA+5.57g/L4·7H2O and water are formulated；
Organic substance part is by the Vb6+ of the Vb1+0.1g/L of the glycine+0.2g/L of the inositol+0.4g/L of 40g/L
The Vb5 and water of 0.1g/L is formulated.
The pH value of above-mentioned culture medium is 6.2, the pH value of the culture medium be with the sodium hydroxide solution of 1mol/L and
1mol/L hydrochloric acid solution is adjusted.
Claims (6)
1. a kind of blueberry tissue cultural method, it is characterised in that: this method is to choose the disinfection of blueberry explant, is put into culture medium
It is cultivated through evoked callus growth phase, the induction of bud and its growth phase and the stage of taking root.
2. a kind of blueberry tissue cultural method according to claim 1, it is characterised in that: the method is chosen outside blueberry
Implant washes away surface smut impurity with tap water, sterilizes then at superclean bench to explant, 1.5 centimeter lengths is cut into, in alcohol
The explant cut is put into culture medium above lamp, sealing treatment is carried out after the completion of inoculation, in the growth of evoked callus
Stage selects WPM+6- benzylaminopurine or selects WPM+6- benzylaminopurine+methyl α-naphthyl acetate+kinetin culture medium culture,
WPM+6- benzylaminopurine+methyl α-naphthyl acetate culture medium culture is selected in the induction of bud and its growth phase, is selected in the stage of taking root
WPM+6- benzylaminopurine+methyl α-naphthyl acetate+kinetin culture medium culture.
3. a kind of blueberry tissue cultural method according to claim 1, it is characterised in that: blueberry explant in the method
It is the blueberry branch of robust growth.
4. a kind of blueberry tissue cultural method according to claim 1, it is characterised in that: explant disappears in the method
Malicious method is to impregnate 30s with 75% alcohol, then impregnates 8min with 0.1% mercuric chloride again.
5. a kind of blueberry tissue cultural method according to claim 2, it is characterised in that: WPM presses volume in the method
Part calculates, by 50 parts of a great number of elements parts, 10 parts of calcium salt parts, 5 parts of microelement parts, 5 parts of molysite parts and 9 parts of organic matters
Matter part is formulated, in which:
A great number of elements part is the KNO by 3.8/L3The MgSO of+7.4g/L4﹒ 7H2The KH of O+3.4g/L2PO4The NH of+8.0g/L4NO3
It is formulated with water；
Calcium salt part is the Ca (NO by 55.6g/L3)2﹒ 4H2O and water are formulated；
Microelement part is the MnSO by 4.5g/L4﹒ 4H2The ZnSO of O+1.72g/L4﹒ 7H2The H of O+1.24g/L3BO3+
The CuSO of 0.05g/L4﹒ 5H2The Na of O+0.05g/L2MoO4﹒ 2H2O and water are formulated；
Molysite part is the Na by 7.45g/L2The FeSO of-EDTA+5.57g/L4·7H2O and water are formulated；
Organic substance part is by the Vb6+0.1g/L of the Vb1+0.1g/L of the glycine+0.2g/L of the inositol+0.4g/L of 40g/L
Vb5 and water be formulated.
6. a kind of blueberry tissue cultural method according to claim 2, it is characterised in that: the pH of culture medium in the method
Value is 6.2, and the pH value of the culture medium is adjusted with the sodium hydroxide solution of 1mol/L and 1mol/L hydrochloric acid solution.
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