CN110419447B - Blueberry tissue culture method - Google Patents
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Abstract
The invention discloses a blueberry tissue culture method, which comprises the steps of selecting a blueberry explant for disinfection, and putting the blueberry explant into a culture medium to be cultured in a callus induction growth stage, a bud induction stage, a growth stage and a rooting stage. The tissue culture method disclosed by the invention is high in propagation speed, not limited by geographical time, low in plant infection rate, capable of improving rooting and survival rate, and suitable for the industrialized development of blueberry tissue culture breeding seedlings.
Description
Technical Field
The invention relates to the field of blueberry cultivation, in particular to a blueberry tissue culture method.
Background
Blueberries (academic name: Blueberry) belong to the perennial deciduous woody plant of the genus Vaccinium of the family Ericaceae. The fruit is small berry, and has multiple health care functions, such as improving human immunity, resisting aging, softening blood vessels, improving vision, resisting cancer and cardiovascular diseases; is rich in nutrients such as protein, fat, fiber, SOD, vitamin A, vitamin B, vitamin C, vitamin E, various mineral elements such as iron, zinc, calcium, magnesium, copper, etc., and special nutrients such as anthocyanin, flavone, fruit acid, etc. Because the blueberries have the nutritional ingredients and the health-care function, products related to the blueberries are increasing on the market, such as blueberry fruit wine, jam, cans, preserves, fruit juice, beverages and the like, and are also raw materials of cakes, jelly, yoghourt, candies and the like, and the blueberries gradually have a certain position in the market, which is also the key for the rapid development of the blueberries.
Blueberries are mainly distributed in North America, and wild blueberries are also distributed in great Khingan mountains, lesser Khingan mountains and the like in the North of China. The history of blueberry cultivation is short, the introduction and cultivation of blueberries is in the United states at the earliest, and since the commercial production of blueberries is first realized in the United states in the thirties of the twentieth century, more than 30 countries such as Canada, Germany, Australia, the Netherlands, Japan and the like start the introduction and cultivation one after another, so that the blueberries are rapidly developed. At present, blueberry industrial production is carried out in the United states, Europe, Japan and the like. However, the blueberry fruits are far from being in short supply worldwide, and data show that the total yield of the blueberries in the united states in 2012 is only 35 ten thousand tons, which accounts for about 60% of the total global yield, and the yield of the blueberries is far higher than the number at present. The earliest research on tissue culture of blueberries is Niekerson, and the success of callus culture of other blueberry branches indicates that the production of blueberries enters a new era, high-efficiency blueberry seedling production is carried out, and a foundation is laid for commercial production. At present, the plant tissue culture technology is well developed in the aspects of fine seed cultivation, rapid propagation, detoxification propagation and the like, and the technology is applied to blueberries to carry out blueberry seedling cultivation and fine seed propagation, so that technical support is provided for commercial production, and the blueberries are well and rapidly developed.
The blueberry introduction and cultivation work in China starts in 1983 and is started by the initiative of Jilin agriculture university in China. The blueberry industry has developed relatively late in China, and many people have not heard the term. The blueberry has short cultivation history in China, laggard technology, and is not thorough in research on growth habits and environment of the blueberry, and seedling propagation mainly comprises introduction and cuttage, usually hardwood cuttage, but also green branch cuttage, which are traditional propagation modes and have many defects. In order to meet the demand of the times, the tissue culture mode is also carried out on the seedling propagation for the seedling propagation. Cheng shuyun and the like successfully perform ex-vitro rooting culture on blueberry tissue culture seedlings, so that the blueberry tissue culture in China is also developed, and technical support is added for industrial production of blueberries. The blueberry industry is developed rapidly in China in recent years, the blueberry area in China is getting bigger and bigger in view of planting area, the blueberry area extends from the east to the southwest area in China, Guizhou is a place particularly suitable for blueberry planting, and particularly Guizhou has the unique advantage of developing blueberries, which is one of the keys for vigorous development and popularization of the blueberries.
Blueberries belong to emerging fruits in China and are deeply favored by people. The introduction and cultivation of the blueberries are started in many places in China, the blueberries spread from north to south, and particularly the blueberries are suitable for growing in the south, so that the blueberries have great development scenes. In recent years, the living standard of people is increasingly improved, the pursuit of high-quality fruits is stricter and stricter, the nutrition and the health of the fruits are more emphasized, and great pressure is exerted on the development of blueberries. Production of high quality blueberry fruit requires good seedlings and a good growing environment. However, the development of the blueberries in China has many problems at present, firstly, the cultivation and planting history is short, and the introduction and cultivation from abroad only in the eighties of the twentieth century begin; secondly, the planting technology is laggard, and introduction and domestication of wild blueberries are mainly adopted; the production technology is not high, so that the yield and the quality are low, the production base is fuzzy to the growth environment and the growth habit of the blueberries, the suitable growth environment is not clear, and how to produce the high-yield and high-quality blueberry fruits is unknown; the main problem of blueberry is seedling breeding, and conventional blueberry breeding methods mainly include introduction, cuttage and the like, so that a large number of seedlings cannot be rapidly bred, diseases can occur in the breeding process, the seedlings carry germs, and the cuttage seedlings are difficult to root, so that the quality of the seedlings is seriously affected by the breeding method, and particularly, the phenomena of variety degradation and poor quality of the blueberries after introduction can occur. Therefore, the development of the blueberry tissue culture technology and the non-toxic seedling technology which have the advantages of high propagation speed, no geographic time limit, reduction of plant diseases, and improvement of rooting and survival rate is not slow.
Disclosure of Invention
The invention aims to provide a blueberry tissue culture method. The tissue culture method disclosed by the invention is high in propagation speed, is not limited by geographical time, is low in plant infection rate, improves the rooting and survival rate, and promotes the tissue culture propagation and seedling industrialization development of the blueberries.
The technical scheme of the invention is as follows: a blueberry tissue culture method comprises the steps of selecting a blueberry explant for disinfection, and putting the blueberry explant into a culture medium to be cultured in an induced callus growth stage, a bud induction stage, a growth stage and a rooting stage.
In the blueberry tissue culture method, blueberry explants are selected, tap water is used for washing away dirt and impurities on the surfaces of the blueberry explants, then the explants are disinfected on an ultraclean workbench and cut into the length of 1.5cm, the cut explants are placed in a culture medium above an alcohol burner, sealing treatment is carried out after inoculation is finished, WPM + 6-benzylaminopurine or WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium is selected for culture in the growth stage of callus induction, WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium is selected for culture in the bud induction and growth stage, and WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium is selected for culture in the rooting stage.
In the blueberry tissue culture method, the blueberry explants in the method are strong blueberry branches.
In the blueberry tissue culture method, the method for disinfecting the explant comprises the steps of soaking the explant in 75% alcohol for 30s, and then soaking the explant in 0.1% mercuric chloride for 8 min.
In the blueberry tissue culture method, the WPM is prepared from 50 parts by volume of a macroelement part, 10 parts by volume of a calcium salt part, 5 parts by volume of a trace element part, 5 parts by volume of an iron salt part and 9 parts by volume of an organic matter part, wherein:
the macroelement part is KNO of 3.8/L3+7.4g/L MgSO4﹒7H2KH of O +3.4g/L2PO4+8.0g/L NH4NO3And water;
the calcium salt portion is composed of 55.6g/L Ca (NO)3)2﹒4H2O and water;
the trace element part is composed of 4.5g/L MnSO4﹒4H2O +1.72g/L ZnSO4﹒ 7H2O +1.24g/L of H3BO3+0.05g/L of CuSO4﹒5H2O +0.05g/L of Na2MoO4﹒ 2H2O and water;
the iron salt fraction is composed of 7.45g/L Na2-EDTA +5.57g/L of FeSO4·7H2O and water;
the organic matter portion was composed of 40g/L of inositol +0.4g/L of glycine +0.2g/L of Vb1+0.1g/L of Vb6+0.1g/L of Vb5 and water.
In the blueberry tissue culture method, the pH value of the culture medium in the method is 6.2, and the pH value of the culture medium is adjusted by 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution.
The inventors carried out the following experiments to verify the effects of the present invention:
experimental example:
1 materials and methods
1.1 test materials
The material for this test was 3-year old branches of rabbit-eye blueberries, whose stem sections were mainly taken for culture. The material is from vegetable experimental land of agricultural college of Guizhou university.
1.2 test instruments and reagents
2.2.1 test apparatus
The test instrument mainly comprises: sterilization equipment (autoclave); inoculation equipment (inoculation chamber, super clean bench, tweezers, alcohol lamp, absorbent cotton, inoculation knife); culture equipment (constant temperature culture room, constant temperature incubator, triangular flask, plastic sealing film, wiring); storage facilities (refrigerator, brown glass bottle, oven); and (3) the other: iron jar, electromagnetism stove, label paper, camera, pH test paper etc..
1.2.2 Experimental reagents
The reagent comprises sucrose, agar, 6-benzylaminopurine (6-BA), naphthylacetic acid (NAA), Zeatin (ZT), cytokinin, Kinetin (KT), 75% alcohol, 0.1% mercuric chloride (mercuric chloride), sodium bicarbonate, etc.
1.3 test methods
1.3.1 Experimental design
1.3.1.1 mother liquor preparation
The method comprises the steps of preparing mother liquor of an MS culture medium and an improved WPM culture medium, preparing the mother liquor with a certain concentration from macroelement mother liquor, calcium salt, microelement mother liquor, ferric salt, organic matter mother liquor, hormone mother liquor and the like, sticking a label and date, and storing in a refrigerator at 4 ℃. The method comprises the steps of preparing mother liquor of an MS culture medium and an improved WPM culture medium, preparing the mother liquor with a certain concentration from macroelement mother liquor, calcium salt, microelement mother liquor, ferric salt, organic matter mother liquor, hormone mother liquor and the like, sticking a label and date, and storing in a refrigerator at 4 ℃. The mother liquor can not be used if precipitation or crystal precipitation occurs during use, and the mother liquor needs to be prepared again.
TABLE 1 MS culture Medium formulation
TABLE 2 modified WPM media formulation
1.3.1.2 preparation of the culture Medium
Absorbing corresponding mother liquor according to the components of each culture medium to culture the mother liquor into the culture medium, adjusting the pH value to 6.2, generally using 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution to adjust the pH value too low, and when the amount of agar is constant, the culture medium is not easy to fix, and the growth of blueberries is not facilitated if the pH value is too high, so that the adjustment of a proper pH value plays a vital role in the test.
1.3.1.3 Sterilization
The prepared culture medium, bottled and sealed distilled water, culture dish (containing filter paper), filter and filter paper (sealing), and sealed pair of glass beakers are placed into a high-pressure steam sterilizer at 121 deg.C and 0.1kpa for sterilization for 20 min.
1.3.1.4 inoculation
The explant is 3 years blueberry stem tissue comprising twigs and hard branches (completely lignified and semi-lignified), then the explant is washed away with tap water to remove dirt and impurities on the surface, cut to a proper size by scissors, cut into about 1.5cm in length after being sterilized on an ultra-clean workbench, and then the cut stem is put into a culture medium above an alcohol lamp and covered with a bottle cap. And sealing after inoculation, and sticking a label and a date.
1.3.1.5 cultivation
Comprises several stages of primary culture, secondary culture, rooting culture and the like. The primary culture is culture after stem section inoculation, including long callus and bud; subculture refers to subculture based on primary culture, including multiplication culture and stem growth of stem segments; the rooting culture refers to a process of inducing the rooting of the plant. And (3) placing the inoculated triangular flask into a constant-temperature illumination incubator for culture, wherein the temperature is 25 +/-1 ℃, the illumination intensity is 2000lx, dark culture is carried out in the first 5 days, light and dark combined culture is carried out in the later period, and light culture is generally carried out for 14h and dark culture is carried out for 10h every day.
1.3.1.6 hardening and transplanting seedlings
After the explant buds and roots, the bottle mouth is opened slowly, the bottle is opened completely after 3d, the culture medium at the base part of the tissue culture seedling is washed clean under tap water, and then the tissue culture seedling is transplanted into a plug tray filled with the matrix. The matrix is prepared by uniformly mixing turf, perlite, leaf mold, organic fertilizer and the like. And (5) moving into a plug tray, watering thoroughly, culturing in a place with weak illumination, and transferring to a common illumination condition after 5 days to allow the plug tray to grow normally.
1.3.1.7 Observation and recording
And (4) photographing and recording characters after inoculation, and specifically recording the cut surface condition of the inoculated explant, whether color change and other data occur. The growth of the explants, whether callus was formed, whether shoots were growing, and the color change were also recorded during the dark culture period. After the cultivation in the light, the explants were observed for growth every 5 days, and photographed and recorded in text.
2.4 data processing
And processing the test data by using EXCEL software, analyzing the reasonability of the data and the like.
3 results and analysis
3.1 Effect of different disinfectants and treatment time on blueberry Stem segments
Under the condition that the treatment time of 0.1% mercuric chloride disinfection is not changed for 8min, the treatment time of 75% alcohol on the blueberry stem is changed, the pollution condition and the survival rate after inoculation are recorded, and single-factor control is performed to obtain table 3. From the results in table 3, it can be seen that the contamination rate decreases with the increase of the alcohol treatment time and the survival rate decreases after increasing, without changing the mercuric chloride disinfection treatment time. The highest pollution rate is that the treatment time of 75 percent alcohol is 0s, namely the pollution rate is 53.3 percent when the alcohol is not used for treatment; the lowest contamination rate was 50s group alcohol treatment time, and the contamination rate was almost 0. From the results of the survival rate analysis, the highest survival rate was 30s group of 75% alcohol treatment time, up to 67.7%, followed by 20s group of 60% survival rate, followed by 10s, 0s, 40s, and 50s in this order. The mortality data shows that when the mercury-raising treatment time is the same, the survival rate gradually increases along with the increase of the alcohol treatment time, and the mortality rate reaches 80 percent. From the data, the optimal treatment time is 30s in one group from the viewpoint of pollution and survival rate of the blueberry stems with different treatment time of 75% alcohol, the pollution rate of the group change is low, the survival rate is high, and the optimal disinfection treatment time of the 75% alcohol is obtained.
TABLE 375% alcohol Effect of different treatment times on blueberry Stem segments
Note: after the survival rate is 30 days, the stem segments have no color change and necrosis and are marked as survival.
Table 4 shows that under the condition that the treatment time of 75% alcohol disinfection is not changed for 30s, the treatment time of 0.1% mercury bichloride on the stem of blueberry is changed, the pollution condition and the survival rate after inoculation are recorded, and single-factor control is carried out to obtain the blueberry. The results in Table 4 show that the contamination is highest when the 0.1% mercuric chloride disinfection time is 4min, the contamination rate gradually decreases with the increase of the disinfection time, and the contamination rate is lowest when the 0.1% mercuric chloride disinfection treatment is performed for 10min and 12min (6.7%). From the aspect of survival rate, with the increase of the treatment time of the 0.1% mercury-mercuric disinfection solution, the survival rate is increased firstly and then decreased, the maximum mercury-mercuric treatment is 0.1% for 8min and is up to 73.3%, and the influence of the stem segments of the blueberries sterilized by 0.1% mercury-mercuric disinfection treatment on the survival rate is 8min >10min >6min >4min >12 min. From the mortality results, when the alcohol treatment time is the same, the mortality is lower and is 13.3% when the mercuric chloride is treated for 4min, 6min and 8min, the mortality of the mercuric chloride treated for 10min and 12min is in an increasing trend, and the mortality of the mercuric chloride treated for 12min is up to 60.0%. Therefore, the pollution rate and the survival rate are comprehensively considered, the proper mercury mercuric chloride disinfection treatment time is 8min, the pollution rate in the time period is low, and the survival rate is relatively high.
From tables 3 and 4, it can be seen that, in combination with the contamination rate and the survival rate, a suitable disinfection combination is: 30s and 75 percent alcohol disinfection treatment and 10min and 0.1 percent mercury liter, the combined pollution rate is low and the survival rate is higher.
TABLE 40.1 Effect of different treatment times on blueberry Stem segments
Note: after the survival rate is 30 days, the stem segments have no color change and necrosis and are marked as survival.
3.2 Effect of different media on blueberry Stem segments
Under the condition that the concentrations of the hormones 6-BA and the NAA are not changed, 1 mg/L6-BA and 0.5mg/L NAA are added into five basic culture media such as MS, 1/2MS, 1/4MS, WPM and 1/2WPM, the generation, the bud growth and the rooting conditions of the callus are counted, and the basic culture media suitable for the growth of the blueberry stem segments are screened. As is clear from the results in Table 5, the callus formation rate was 67.7% in the WPM medium, 60% in the MS medium, and 13.3% in the 1/4MS medium. On the induction stem section bud growth, the highest bud growth rate is 76.7% of the WPM culture medium, the next highest bud growth rate is 63.3% of the MS culture medium, and the lowest bud growth rate is 26.7% of the 1/4MS culture medium.
From the viewpoint of production and shoot growth of stem-induced callus, a preferable culture is a modified WPM medium which is not only higher in the production rate of callus but also higher in inducing stem-induced shoot growth, followed by an MS medium. In later experiments, the two culture media are used, and the formula suitable for blueberry stem tissue culture is screened by combining other hormones and the concentrations of the hormones.
TABLE 5 Effect of different media on blueberry Stem segments
3.3 Effect of plant growth regulator type and concentration on growth of blueberry Stem
During the growth of plants, growth regulating substances play a crucial role. In the process of plant tissue culture, although the dosage of the substances is small, the influence on the formation of callus, the induction of buds, dedifferentiation and the like is obvious.
As is clear from the results in Table 6, no callus and roots were produced and only a small number of shoots were produced without adding any growth regulator. Each group added with hormone has callus and bud, the formation rate of the callus is 1.0 mg/L6-BA and (0.5+1.0+0.5mg/L)6-BA + NAA + KT, the induction rate of the callus is 73.3%, then the combination of 2 mg/L6-BA, (1+0.5mg/L)6-BA + NAA, (1.0+1.0mg/L) ZT + NAA, and the like, the worse is the treatment of 3mg/L and 4 mg/L6-BA, and the higher the concentration of 6-BA is, the lower the induction rate of the callus is. The induction of the callus has close relation with the hormone 6-BA, the combination with the participation of the hormone has higher induction rate of the callus, and the induction rate of the callus without the participation of the hormone is lower.
As can be seen from the results of the growth of shoots, these hormones induced shoots less significantly, and all had a certain amount of shoots, some had a few shoots, and the combination of (0.5+1.0mg/L)6-BA + NAA was preferred, and the rate of shoot formation was high. The other combinations have small differences and can form the differentiation and growth of buds.
From the root growth results, it can be known that the formation rate of roots is low, most roots are not generated, or a few roots are generated, but two roots are formed in the treatment of (0.5+1.0+0.5mg/L)6-BA + NAA + KT, so that the combination is beneficial to blueberry stem tissue culture rooting. From the root growth aspect, root formation in the blueberry stem section bottle is difficult, and only a few 1 to 2 roots are formed, which may be an unexplored proper hormone combination and is yet to be explored in later research.
TABLE 6 Table of growth conditions of blueberry stems according to different types and concentrations of hormones
Note: CK means no hormone added;
-means no callus (shoot or root) is produced;
+ represents a small amount of callus (bud or root) generation, the amount of callus generation is a small amount within 0.5cm of diameter, and only one root is generated;
+ indicates that a certain amount of callus (buds or roots) is generated, the generation amount of the callus is a certain amount of callus with two roots, and the diameter of the callus is 0.5cm to 1.0 cm;
+ + + indicates that there are a large number of calli (buds or roots) formed, the number of calli formed is large between 1.0cm and 1.5cm in diameter, and there are three roots;
the expression of ++++ indicates that the amount of callus (shoot or root) produced is large, and that the amount of callus produced is large when the diameter of the callus is 1.5cm or more.
3.4 Observation record of blueberry tissue culture conditions
Blueberry tissue culture is shown in figure 1, under the regulation and control of hormone and a culture medium, the formation rate of callus is high, when the culture medium and the hormone are combined in a proper formula, after one week, obvious callus formation can be seen on leaves and stem sections, conglobation is gathered near an incision, the callus of the stem sections appears at the bottom of the stem sections in the culture medium, the callus of the leaves appears around the leaves, including the junction with the culture medium and the culture medium, and some of the callus is exposed and is milky white. After 2 weeks of culture, the growth of the callus did not change significantly, then the callus was cultured continuously by grafting, the callus was light yellow after 1-time grafting, and FIG. A shows the growth of the callus after 1-time stem grafting, and the granular substances gradually increased, and appeared yellow green and slightly reddish brown. FIG. B shows the growth of calli after 2 stem transfers, which are pale yellow, and some are reddish brown. And the figure C shows the growth condition of the callus transferred by the leaf for 3 times, the callus at the periphery of the callus is yellow green particles, and the browning phenomenon appears at the central part of the callus and is probably caused by self-secreted browning substances. The callus was removed and gently crushed by hand, and the fingers had a little moisture, indicating that it was loose and contained a certain amount of moisture.
After stem inoculation, the stem typically sprouts to grow buds first, and then grows callus, but there is also callus that appears earlier than the buds. When the culture environment is appropriate, the axillary buds can be seen to be enlarged after 3 days, and the leaves begin to bloom in a long time after one week, so that the leaves are light green. FIG. G shows that the stem section grows shoots and leaves 1 week after inoculation, and the leaves are light green. And the graph D and the graph H show the growth condition of the bud after the stem section is inoculated for 2 weeks, two leaves which grow out at the earliest appear browning, other leaves are light green, and the bud length reaches 3 cm. FIGS. E and I (FIG. I is a photograph taken from the bottom of the culture after 3 weeks of inoculation, the area of the callus gradually increased) show the growth of the shoots after 3 weeks of stem inoculation, the top of the original stem died due to browning, the bottom of the original stem had callus formed, and the shoots reached 6-8 cm in length. FIG. F shows the growth of shoots 4 weeks after stem inoculation, with obvious branching and green leaves.
The blueberry tissue culture has slow and difficult rooting. The research and the culture show that after the callus is cultured, a few roots can be generated after 50 days, and the callus at the moment has a browning phenomenon. FIG. J shows callus growth of two roots appearing in milk white, after 8 weeks of leaf inoculation, and a layer of white powder appeared on the periphery of the callus. FIG. K shows that two roots appear after 9 weeks of leaf inoculation, the length of the roots reaches 3 cm, and brownish cheeks appear in the center of the callus. FIG. L shows that after 10 weeks of leaf inoculation, three roots appear, the longer one reaches 5cm, and some roots also have bifurcation phenomena, when the roots grow to a certain degree, the roots begin to bifurcate and grow in a 2-bifurcation mode, and the callus browning area at the central position is larger and larger.
4 discussion and conclusions
4.1 discussion
The experiment researches the blueberry stem tissue culture and rapid propagation technology to obtain high-quality blueberry seedlings in an in vitro culture mode. The experiment finally obtains the process of tissue culture seedlings by improving the culture medium and the plant growth regulating substances and optimizing each condition.
First, the appropriate disinfecting solution and disinfecting time are screened. And (3) carrying out single factor test by controlling two factors of alcohol and mercury bichloride, and screening the optimal sterilization condition. The test result shows that the treatment with 75% alcohol for 30s and the treatment time of 0.1% mercury bichloride for 8min are the best sterilization conditions, and then the sterile water is used for washing for 3-5 times, so that the pollution rate can be greatly reduced, and the growth and differentiation of stem segments are not influenced. Incision browning occurs after inoculation and occurs with a higher rate in 3 year old stem material (fully lignified). The cut stem sections should be placed in the culture medium immediately to prevent browning. The addition of the activated carbon material in the preliminary experiment does not play a role in improving browning, but turns the culture medium black, which is not favorable for observation.
And secondly, screening a suitable basic culture medium for tissue culture of the blueberry stem sections. The growth conditions of stem callus and buds are counted by preparing five culture media such as MS, 1/2MS, 1/4MS, improved WPM, improved 1/2WPM and the like and adding corresponding hormones. The comprehensive results show that the improved WPM culture medium has the highest callus induction rate and the highest bud growth rate, the callus rate is 67.7 percent, the bud growth rate is 76.7 percent, and the improved WPM culture medium is an MS culture medium which is 60.0 percent and 63.3 percent respectively.
Thirdly, the species, the concentration and the combination of the plant growth regulator substances are compared and tested, and the conditions which are most suitable for inducing the stem sections to grow callus, bud and root are screened. The growth regulating substance mainly uses 6-BA, NAA, KT, ZT and the like, wherein the 6-BA + NAA + KT (0.5+1.0+0.5mg/L) has the best matching use effect, has extremely high inductivity on callus and the best growth performance, and is a light green and partially light yellow bulk particle. The result of single hormone treatment shows that the use effect of 6-BA is better, the induction rate of the callus is higher and the callus grows faster, obvious callus formation can be seen after one week, and the callus grows obviously after 3 weeks. Secondly, the callus in the 6-BA + NAA treatment has good growth color, and most of the callus is light green; the induced buds grow fast, the buds begin to sprout after 3 days, completely sprout in one week, the leaf tips are thin and green, the buds grow obviously after three weeks, the length reaches 5cm, and some buds are accompanied by branches.
The color change of the stem section cut is mainly observed in the tissue culture process. The cut after explant inoculation typically turns brown, gradually turning to a bright green color after 1 week, and callus grows. The early stage of the callus is light yellow granules, and then the periphery of the callus slowly becomes light green and is exposed on the culture medium to be white powder. The bud generally appears earlier than the callus, the bud 3d of the stem segment with axillary buds begins to sprout and gradually expands, the stem segment is obviously opened after 1 week to grow light green leaves, some culture mediums have whitened leaves when the bud grows out, which is caused by lack of nutrition and lack of mineral elements which are difficult to move, and when the leaves grow out 7 to 8 leaves, the lower leaves turn yellow and white, which is mainly caused by lack of free nutrient elements (such as nitrogen, phosphorus, potassium and the like) or unsuitable culture conditions. The growth of roots was observed, and the fine milky white state formed by callus differentiation was observed for about 30 days, and then the growth gradually became thick, accompanied by branching, that is, so-called root formation.
In the experimental process, there were some problems such as easy browning of explants after inoculation, which could not be solved by adding activated carbon substance. The browning phenomenon appears when the explants are not inoculated, and the cut possibly reacts with the outside because the cut is exposed on a clean bench for too long time.
4.2 conclusion
The plant tissue culture is a process of in vitro culture in a sterile environment by a plurality of stages of primary culture, secondary culture, rooting culture and the like, and the requirements of each stage are different, such as the difference of illumination, hormone, sterilization and the like. In the blueberry stem tissue culture, the strongly growing branches are selected as explant materials, and are disinfected after being washed clean by tap water, and the results of the test show that the disinfection treatment of the explants (blueberry stem) is that the explants are soaked in 75% alcohol for 30s and then soaked in 0.1% mercuric chloride for 8min, so that the pollution rate is reduced. Under the condition of the same hormone and the same external conditions, five culture media are screened, and the improved WPM culture medium is found to be more suitable for tissue culture of blueberry stem sections. In the process of inducing the growth of the stem section long callus, the optimal culture medium is WPM +2 mg/L6-BA and WPM +0.5 mg/L6-BA +1.0mg/L NAA +0.5mg/L KT, the callus induction rate of the two treatments reaches 73.3 percent, the early stage is milky white, and then the callus gradually becomes light green granular substances, and the periphery is coated with white granular substances.
In the aspects of bud induction and growth, the optimal combination of the culture medium and the hormone is WPM +0.5 mg/L6-BA +1.0mg/L NAA, the treated bud grows faster, and the leaves grow more robustly and are light green; the new buds grow rapidly, the stem segments can grow to 6-9cm after 4 weeks, the bottom callus is light yellow and light green, the area of the callus is large, and sufficient nutrition is provided for the growth and the extension of the new buds.
The callus in the blueberry stem tissue culture rapidly takes root, the rooting formula is improved WPM + (0.5+1.0+0.5mg/L)6-BA + NAA + KT, the rooting rate of the treatment combination is high, two roots grow out, the roots are thick and strong and grow to 4cm, and branches exist in some cases, so that the root system grows more well, sufficient nutrients are absorbed at the lower part for providing nutrition for the upper part of the root system, and a complete plant can be generated.
The tissue culture of the blueberries is the basis of nontoxic seedlings, and the test is a basic test for tissue culture of the blueberries and is expected to lay a foundation for tissue culture and breeding of the seedlings of the blueberries.
Compared with the prior art, the technology of the invention obtains high-quality blueberry seedlings by in vitro culture, and finally obtains the tissue culture seedlings by improving the culture medium and the plant growth regulating substances and optimizing various conditions.
First, the appropriate disinfecting solution and disinfecting time are screened. And (3) carrying out single factor test by controlling two factors of alcohol and mercury bichloride, and screening the optimal sterilization condition. The test result shows that the treatment with 75% alcohol for 30s and the treatment time of 0.1% mercury bichloride for 8min are the best sterilization conditions, and then the sterile water is used for washing for 3-5 times, so that the pollution rate can be greatly reduced, and the growth and differentiation of stem segments are not influenced. Incision browning occurs after inoculation and occurs with a higher rate in 3 year old stem material (fully lignified). The cut stem sections should be placed in the culture medium immediately to prevent browning. The addition of the activated carbon material in the preliminary experiment does not play a role in improving browning, but turns the culture medium black, which is not favorable for observation.
And secondly, screening a suitable basic culture medium for tissue culture of the blueberry stem sections. The growth conditions of stem callus and buds are counted by preparing five culture media such as MS, 1/2MS, 1/4MS, improved WPM, improved 1/2WPM and the like and adding corresponding hormones. The comprehensive results show that the improved WPM culture medium has the highest callus induction rate and the highest bud growth rate, the callus rate is 67.7 percent, the bud growth rate is 76.7 percent, and the improved WPM culture medium is an MS culture medium which is 60.0 percent and 63.3 percent respectively.
Thirdly, comparing the species, the concentration and the combination of the plant growth regulating substances, and screening the conditions which are most suitable for inducing stem sections to grow callus, bud and root. The growth regulating substance mainly uses 6-BA, NAA, KT, ZT and the like, wherein the 6-BA + NAA + KT (0.5+1.0+0.5mg/L) has the best matching use effect, has extremely high inductivity on callus and the best growth performance, and is a light green and partially light yellow bulk particle. The result of single hormone treatment shows that the use effect of 6-BA is better, the induction rate of the callus is higher and the callus grows faster, obvious callus formation can be seen after one week, and the callus grows obviously after 3 weeks. Secondly, the callus in the 6-BA + NAA treatment has good growth color, and most of the callus is light green; the induced buds grow fast, the buds begin to sprout after 3 days, completely sprout in one week, the leaf tips are thin and green, the buds grow obviously after three weeks, the length reaches 5cm, and some buds are accompanied by branches.
The color change of the stem section cut is mainly observed in the tissue culture process. The cut after explant inoculation typically turns brown, gradually turning to a bright green color after 1 week, and callus grows. The early stage of the callus is light yellow granules, and then the periphery of the callus slowly becomes light green and is exposed on the culture medium to be white powder. The bud generally appears earlier than the callus, the bud 3d of the stem segment with axillary buds begins to sprout and gradually expands, the stem segment is obviously opened after 1 week to grow light green leaves, some culture mediums have whitened leaves when the bud grows out, which is caused by lack of nutrition and lack of mineral elements which are difficult to move, and when the leaves grow out 7 to 8 leaves, the lower leaves turn yellow and white, which is mainly caused by lack of free nutrient elements (such as nitrogen, phosphorus, potassium and the like) or unsuitable culture conditions. The growth of roots was observed, and the fine milky color formed by differentiation of callus was observed for about 30 days, and then the growth and thickening were gradually progressed, and the branching, so-called root formation, was also accompanied.
In conclusion, the tissue culture method disclosed by the invention is high in propagation speed, not limited by geographical time, low in plant infection rate, capable of improving rooting and survival rate, and suitable for the industrialized development of blueberry tissue culture breeding seedlings.
Drawings
FIG. 1 is a recorded image of the observation of the tissue culture of blueberry in the experimental example.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Example 1. 1. A blueberry tissue culture method comprises the steps of selecting strong blueberry branches as explants, washing off dirt impurities on the surfaces of the blueberry branches by using tap water, disinfecting the explants on an ultra-clean workbench, cutting the explants into 1.5cm long, putting the cut explants above an alcohol lamp into a culture medium, sealing after inoculation is finished, selecting WPM + 6-benzylaminopurine or WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium for culture in a callus induction and growth stage, selecting WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium for culture in a bud induction and growth stage, and selecting WPM + 6-benzylaminopurine + naphthylacetic acid + kinetin culture medium for culture in a rooting stage.
The method for disinfecting the explant comprises the steps of soaking the explant in 75% of alcohol for 30s, and then soaking the explant in 0.1% of mercuric chloride for 8 min; the WPM in the method is prepared from 50 parts of macroelement part, 10 parts of calcium salt part, 5 parts of trace element part, 5 parts of iron salt part and 9 parts of organic matter part by volume part, wherein:
the macroelement part is KNO of 3.8/L3+7.4g/L of MgSO4﹒7H2KH of O +3.4g/L2PO4+8.0g/L NH4NO3And water;
the calcium salt portion is composed of 55.6g/L Ca (NO)3)2﹒4H2O and water;
the trace element part is composed of 4.5g/L MnSO4﹒4H2O +1.72g/L ZnSO4﹒ 7H2O +1.24g/L of H3BO3+0.05g/L of CuSO4﹒5H2O +0.05g/L of Na2MoO4﹒ 2H2O and water;
the iron salt fraction is composed of 7.45g/L Na2-EDTA +5.57g/L of FeSO4·7H2O and water;
the organic matter portion was composed of 40g/L of inositol +0.4g/L of glycine +0.2g/L of Vb1+0.1g/L of Vb6+0.1g/L of Vb5 and water.
The pH values of the culture media are all 6.2, and the pH values of the culture media are adjusted by 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution.
Claims (2)
1. A blueberry tissue culture method is characterized by comprising the following steps:
selecting a blueberry explant, washing off dirt and impurities on the surface of the blueberry explant by using tap water, soaking the blueberry explant in 75% alcohol for 30s on a superclean bench, then soaking the blueberry explant in 0.1% mercury bichloride for 8min for disinfection, cutting the disinfected explant into 1.5cm long, putting the cut explant above an alcohol lamp into a culture medium, and sealing after inoculation is finished;
the growth stages of callus induction and growth, bud induction and growth and rooting culture can be completed on the improved WPM +1 mg/L6-BA or the improved WPM +0.5 mg/L6-BA +1.0mg/L NAA +0.5mg/L KT or the MS +1 mg/L6-BA or the MS +0.5 mg/L6-BA +1.0mg/L NAA +0.5mg/L KT;
wherein, in the method, each 1 liter of improved WPM culture medium is prepared from 50ml of macroelement mother liquor, 10ml of calcium salt mother liquor, 5ml of microelement mother liquor, 5ml of iron salt mother liquor and 9ml of organic substance mother liquor;
wherein the macroelement mother liquor is composed of KNO of 3.8g/L3+7.4g/L MgSO4·7H2KH of O +3.4g/L2PO4+8.0g/L NH4NO3And water;
the calcium salt mother liquor is prepared from 55.6g/L of Ca (NO)3)2·4H2O and water;
the microelement mother liquor is composed of 4.5g/L MnSO4·4H2O +1.72g/L ZnSO4·7H2O +1.24g/L of H3BO3+0.05g/L of CuSO4·5H2O +0.05g/L of Na2MoO4·2H2O and water;
the mother liquor of iron salt is prepared from 7.45g/L of Na2-EDTA +5.57g/L of FeSO4·7H2O and water;
the organic mother liquor is composed of 40g/L inositol, 0.4g/L glycine and 0.2g/L VB1+0.1g/L of VB6VB of +0.1g/L5And water;
in the method, the blueberry explant is a strong rabbit eye blueberry branch.
2. The blueberry tissue culture method of claim 1, which is characterized in that: in the method, the pH value of the culture medium is 6.2, and the pH value of the culture medium is adjusted by 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution.
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