CN110419447B - A kind of blueberry tissue culture method - Google Patents

Abstract

The invention discloses a blueberry tissue culture method. The method comprises the steps of selecting blueberry explants to sterilize, putting them into a culture medium, and culturing them through inducing callus growth stage, bud induction, growth stage and rooting stage. The tissue culture method of the invention has fast propagation speed, is not limited by geographical time, has low plant disease susceptibility rate, improves rooting and survival rate, and is suitable for the industrialization development of blueberry tissue culture seedlings.

Description

A kind of blueberry tissue culture method

technical field

The invention relates to the field of blueberry cultivation, in particular to a blueberry tissue culture method.

Background technique

Blueberry (scientific name: Blueberry) is a perennial deciduous woody plant of the Rhododendron family bilberry. Its fruit is a small berry, which has a variety of health care functions and has attracted much attention from people, such as improving human immunity, anti-aging, softening blood vessels, improving eyesight, anti-cancer and anti-cardiovascular disease functions; rich in nutrients, Such as protein, fat, fiber, SOD, vitamin A, vitamin B, vitamin C, vitamin E and various mineral elements, such as iron, zinc, calcium, magnesium, copper, etc. and special nutrients such as anthocyanins, flavonoids, and fruit acids Element. Due to the above nutritional components and health care functions of blueberries, more and more blueberry-related products have appeared on the market, such as blueberry wine, jam, canned food, preserves, juice and beverages, etc. Blueberries have gradually gained a certain position in the market, which is also the key to the rapid development of blueberries.

Blueberries are mainly distributed in North America, and wild blueberries are also distributed in Daxing'anling and Xiaoxing'anling in northern my country. The history of blueberry cultivation is short. The earliest introduction and cultivation of blueberry was in the United States. Since the United States took the lead in realizing the industrialized production of blueberry in the 1930s, more than 30 countries such as Canada, Germany, Australia, the Netherlands, and Japan have successively introduced it. Cultivation, the rapid development of blueberries. At present, the United States, Europe, Japan and other places have carried out blueberry industrial production. But in the world, blueberry fruit is far from being in short supply. According to data, the total blueberry output in the United States in 2012 was only 350,000 tons, accounting for about 60% of the global total. Now the blueberry output is much higher than this figure. The earliest research on blueberry tissue culture was Niekerson, who successfully used blueberry shoots for callus culture, which marked the entry of a new era in blueberry production, and the efficient production of blueberry seedlings, which laid the foundation for commercial production. At present, the plant tissue culture technology has been well developed in the cultivation of improved varieties, rapid propagation, and detoxification. This technology is applied to blueberries for the cultivation of blueberry seedlings and the propagation of improved varieties, providing technical support for commercial production and making blueberries possible. Get good and fast development.

The introduction and cultivation of blueberry in my country began in 1983, and it was first started by Jilin Agricultural University in China. The development of the blueberry industry in my country is relatively late, and many people have not heard of this term. Blueberry has a short history of cultivation in China, the technology is relatively backward, and the research on the growth habit and environment of blueberry is not thorough enough. The propagation of seedlings is mainly introduction and cutting, usually hard branch cutting, but there are also green branch cuttings, which are all traditional propagation. way, has many disadvantages. In order to meet the needs of the times, it is necessary to carry out tissue culture method for seedling propagation in seedling propagation. Cheng Shuyun et al. successfully carried out rooting culture of blueberry tissue culture seedlings outside the bottle, which led to the development of blueberry tissue culture in my country and added technical support for the industrialized production of blueberry. The blueberry industry has developed rapidly in my country in recent years. In terms of planting area, the area of blueberries in my country is getting larger and larger, extending from east China to southwest China. Guizhou is a particularly suitable place for blueberry planting, especially Qiandongnan Prefecture has the ability to develop blueberries. This is one of the keys to the vigorous development and promotion of blueberries.

Blueberries are an emerging fruit in my country and are favored by the majority of people. Blueberries have been introduced and cultivated all over the country, spreading from north to south, especially the south is more suitable for the growth of blueberries. It can be seen that blueberries have a great development scenario. In recent years, people's living standards have been improving, the pursuit of high-quality fruits has become more and more strict, and more attention has been paid to the nutrition and health of fruits, which has put enormous pressure on the development of blueberries. The production of high-quality blueberry fruit requires excellent seedlings and a good growing environment. But at present, there are many problems in the development of blueberry in my country. First of all, the history of cultivation and planting is relatively short, and the introduction and cultivation from abroad began in the 1980s; secondly, the planting technology is relatively backward, mainly using introduction and domestication of wild blueberries; production technology is not high Causes low yield and quality, the production base is ambiguous about the growth environment and growth habit of blueberries, what kind of growth environment is suitable for it is unclear, and they do not know how to produce high-yield and high-quality blueberry fruits; the main problem of blueberries is seedling breeding. Propagation methods are mainly introduction and cuttings, etc., which can not quickly propagate a large number of seedlings, and diseases will occur during the breeding process, which will cause the seedlings to carry pathogens themselves, and secondly, it is difficult for cuttings to take root, which seriously affects the quality of seedlings, especially blueberries. Variety degradation and poor quality will appear after introduction. Therefore, it is urgent to invent a blueberry tissue culture technology and non-toxic seedling technology with fast reproduction speed, which is not limited by geographical time, reduces plant susceptibility, and improves rooting and survival rate.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a blueberry tissue culture method. The tissue culture method of the invention has fast reproduction speed, is not limited by geographical time, and has low plant disease susceptibility rate, improves rooting and survival rate, and promotes tissue culture reproduction and seedling industrialization development of blueberries.

The technical scheme of the present invention: a blueberry tissue culture method, the method is to select blueberry explants to sterilize, put them into a medium, and culture them through the induction of callus growth stage, bud induction, growth stage and rooting stage.

In the aforementioned blueberry tissue culture method, the method is to select blueberry explants, wash off surface dirt and impurities with tap water, disinfect the explants on an ultra-clean workbench, cut them into 1.5 cm long, and place them on an alcohol lamp. The cut explants are put into the culture medium, and after the inoculation is completed, the sealing process is carried out. In the growth stage of the callus induction, WPM+6-benzylaminopurine or WPM+6-benzylaminopurine+naphthaleneacetic acid are selected for use. +Kinetin medium culture, select WPM+6-benzylaminopurine+naphthaleneacetic acid medium for bud induction and growth stage, select WPM+6-benzylaminopurine+naphthaleneacetic acid+kinetin for rooting stage base culture.

In the aforementioned blueberry tissue culture method, the blueberry explants in the method are blueberry shoots with strong growth.

In the aforementioned blueberry tissue culture method, the explants are sterilized by soaking in 75% alcohol for 30s, and then soaking in 0.1% mercuric chloride for 8min.

In the aforementioned a kind of blueberry tissue culture method, in the described method, WPM is calculated by volume parts, by 50 parts of macroelement parts, 10 parts of calcium salt parts, 5 parts of trace element parts, 5 parts of iron salt parts and 9 parts of organic matter parts. formulated, wherein:

Major elements are composed of 3.8/L KNO ₃ +7.4g/L MgSO ₄ ﹒ 7H ₂ O + 3.4g/L of KH ₂ PO ₄ +8.0g/L of NH ₄ NO ₃ and water;

The calcium salt part is composed of 55.6g/L of Ca(NO ₃ ) ₂ ﹒ 4H ₂ O and water are prepared;

The trace element part is composed of 4.5g/L MnSO ₄ ﹒ 4H ₂ O+1.72g/L of ZnSO ₄ ﹒ 7H ₂ O+1.24g/L of H ₃ BO ₃ +0.05g/L of CuSO ₄ ﹒ 5H ₂ O+0.05g/L of Na ₂ MoO ₄ ﹒ 2H ₂ O and water are prepared;

The iron salt part is prepared from 7.45g/L Na ₂ -EDTA+5.57g/L FeSO ₄ ·7H ₂ O and water;

The organic matter part is made up of 40g/L inositol+0.4g/L glycine+0.2g/L Vb1+0.1g/L Vb6+0.1g/L Vb5 and water.

In the aforementioned blueberry tissue culture method, the pH value of the medium in the method is all 6.2, and the pH value of the medium is adjusted with 1 mol/L sodium hydroxide solution and 1 mol/L hydrochloric acid solution.

The inventor has carried out the following experiments to verify the effect of the present invention:

Experimental example:

1 Materials and methods

1.1 Test material

The materials for this experiment were 3-year-old rabbit eye blueberry branches, and the stem segments were mainly used for cultivation. Materials were obtained from the vegetable experimental field of the College of Agriculture, Guizhou University.

1.2 Test instruments and reagents

2.2.1 Test equipment

The main test instruments are: sterilization equipment (autoclave); inoculation equipment (inoculation room, ultra-clean workbench, tweezers, alcohol lamp, absorbent cotton, inoculation knife); culture equipment (incubation room, incubator with constant temperature) , triangular bottle, plastic sealing film, wiring); storage equipment (refrigerator, brown glass bottle, oven); other: iron cylinder, induction cooker, label paper, camera, pH test paper, etc.

1.2.2 Experimental reagents

Reagents include sucrose, agar, 6-benzylaminopurine (6-BA), naphthalene acetic acid (NAA), zeatin (ZT), cytokinin, kinetin (KT), 75% alcohol, 0.1% mercuric (chlorine) mercury), sodium bicarbonate, etc.

1.3 Test method

1.3.1 Experimental Design

1.3.1.1 Configuration of mother liquor

Including the mother liquor of MS medium and modified WPM medium, macroelement stock solution, calcium salt, trace element stock solution, iron salt, organic matter stock solution, hormone stock solution, etc., prepared to a certain concentration of stock solution, labelled and dated, and placed at 4°C stored in the refrigerator. Including the mother liquor of MS medium and modified WPM medium, macroelement stock solution, calcium salt, trace element stock solution, iron salt, organic matter stock solution, hormone stock solution, etc., prepared to a certain concentration of stock solution, labelled and dated, and placed at 4°C stored in the refrigerator. For later use, if precipitation or crystal precipitation occurs in the mother liquor during use, it cannot be used, and the mother liquor needs to be reconstituted.

Table 1 MS medium formulation

Table 2 Modified WPM medium formula

1.3.1.2 Configuration of culture medium

According to the composition of each medium, absorb the corresponding mother liquor and cultivate it into a medium, adjust the pH value to 6.2, generally use 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution to adjust the pH value is too low, and the amount of agar is At a certain time, the medium is not easy to fix, and the pH value is too high, which is not conducive to the growth of blueberries, so adjusting the appropriate pH value plays a crucial role in the experiment.

1.3.1.3 Sterilization

Put the prepared culture medium, bottled and sealed distilled water, petri dish (including filter paper), filter and filter paper (sealed), and a pair of sealed glass beakers together in a 121°C, 0.1kpa high pressure steam sterilizer Sterilize within 20 min.

1.3.1.4 Vaccination

The explants are 3-year-old blueberry stem tissue, including twigs and hard branches (completely lignified and semi-lignified), and then the explants were washed with tap water to remove the surface dirt and impurities, cut to a suitable size with scissors, and then super-lignified. After disinfection and sterilization, the clean workbench is cut into about 1.5 cm long, and then the cut stems are placed in the culture medium above the alcohol lamp, and then the bottle cap is closed. After the inoculation is completed, it is sealed, labelled and dated.

1.3.1.5 Culture

It includes several stages such as primary culture, subculture, and rooting culture. Primary culture refers to the culture after inoculation of stem segments, including long callus and buds; subculture refers to re-cultivation on the basis of primary culture, including the proliferation culture of stem segments and the growth of stem segments; rooting culture refers to the induction of The process of rooting. Put the inoculated triangular flask into a constant temperature light incubator for cultivation, the temperature is 25 ± 1 ℃, the light intensity is 2000lx, the dark cultivation is carried out for the first 5 days, and the light and dark combined cultivation is carried out in the later period. Incubate for 10h.

1.3.1.6 Refining and transplanting

After the explants grew sprouts and roots, the bottle mouth was slowly opened, and after 3 days, the bottle was completely opened, and the medium at the base of the tissue culture seedlings was rinsed with tap water, and then transplanted into a plug tray with matrix. The substrate is prepared by mixing peat, perlite, humus, organic fertilizer, etc. evenly. After moving into the plug, water it thoroughly, first cultivate it in a place with weak light, and then transfer it to normal light conditions for 5 days to allow it to grow normally.

1.3.1.7 Observation and recording

Photographs and text records were taken after inoculation, and data such as the surface condition of the explant incision after inoculation and whether there was any color change were recorded. Explant growth, callus formation, shoot growth, and color changes were also recorded during dark incubation. After light culture, the growth of explants was observed every 5 d, and pictures and texts were recorded.

2.4 Data processing

Use EXCEL software to process the test data and analyze the rationality of the data.

3 Results and Analysis

3.1 Effects of different disinfectants and treatment time on blueberry stems

Under the condition that the treatment time of 0.1% mercuric chloride for 8 minutes is unchanged, the treatment time of 75% alcohol on blueberry stems is changed, the pollution situation and survival rate after inoculation are recorded, and the single factor control is obtained in Table 3. It can be seen from the results in Table 3 that under the condition that the mercuric chloride disinfection treatment time remains unchanged, with the increase of the treatment time of alcohol, the pollution rate decreases in turn, and the survival rate first increases and then decreases. The highest pollution rate is 75% alcohol treatment time of 0s, that is, when no alcohol treatment is used, the pollution rate is 53.3%; the lowest pollution rate is the alcohol treatment time of 50s, and the pollution rate is almost 0. From the analysis of the survival rate results, the highest survival rate was the group of 75% alcohol treated for 30s, up to 67.7%, followed by the group of 20s, with a survival rate of 60%, followed by 10s, 0s, 40s, and 50s. From the mortality data, at the same time of mercury chloride treatment, the survival rate gradually increased with the increase of alcohol treatment time, and the mortality rate was as high as 80%. From these data, it can be seen that the contamination and survival rate of blueberry stems with different treatment time of 75% alcohol, the best treatment time is 30s, the shuffling pollution rate is lower and the survival rate is higher, 75% alcohol is the best Disinfection time.

Table 3 Effects of different treatment times of 75% alcohol on blueberry stems

Note: After the survival rate was 30 days, the stem segment had no discoloration and necrosis, which was recorded as survival.

Table 4 is obtained by changing the treatment time of 0.1% mercuric chloride on blueberry stems under the condition that the treatment time of 75% alcohol disinfection is unchanged for 30s, recording the pollution situation and survival rate after inoculation, and performing single-factor control. The results in Table 4 show that when the disinfection time of 0.1% mercuric chloride is 4min, the pollution is the highest. With the increase of disinfection time, the pollution rate gradually decreases. The lowest pollution rate is 0.1% mercuric chloride for 10min and 12min (6.7%). . From the perspective of survival rate, with the increase of the treatment time of 0.1% mercuric chloride solution, the survival rate first increased and then decreased. The effect on the survival rate was 8min>10min>6min>4min>12min. From the mortality results, when the alcohol treatment time was the same, the mortality rate was lower at 4 min, 6 min and 8 min with mercuric chloride treatment, which was 13.3%. up to 60.0%. Therefore, considering the contamination rate and the survival rate comprehensively, a more suitable mercuric chloride disinfection treatment time is 8min, the contamination rate is low and the survival rate is relatively high during this time period.

From Table 3 and Table 4, it can be concluded that considering the pollution rate and survival rate, a more suitable disinfection combination is: 30s 75% alcohol disinfection treatment + 10min 0.1% mercury chloride, the combination has a low pollution rate and a high survival rate.

Table 4 Effects of different treatment times of 0.1% mercury chloride on blueberry stems

Note: After the survival rate was 30 days, the stem segment had no discoloration and necrosis, which was recorded as survival.

3.2 Effects of different media on blueberry stems

The hormones 1 mg/L 6-BA and 0.5 mg/L were added to five basic media including MS, 1/2MS, 1/4MS, WPM and 1/2WPM under the condition of constant hormone 6-BA and NAA concentrations. NAA counts callus formation, budding, and rooting, and selects the basic medium suitable for blueberry stem growth. As can be seen from the results in Table 5, the WPM medium had the highest callus formation rate of 67.7%, followed by the MS medium with 60%, and the 1/4MS medium with the lowest rate of 13.3%. In the induction of long buds in stem segments, the highest bud growth rate was 76.7% in WPM medium, followed by MS medium with 63.3%, and the lowest in 1/4MS medium with 26.7%. %.

In terms of callus production and bud induction from stem segments, the most suitable culture is modified WPM medium, which not only has a higher callus production rate, but also induces stem segment bud growth, followed by MS medium. In the later experiments, these two media were all used, and combined with other hormones and their concentrations, a formula suitable for blueberry stem tissue culture was screened.

Table 5 Effects of different media on blueberry stems

3.3 Effects of types and concentrations of plant growth regulators on the growth of blueberry stems

In the process of plant growth, its growth regulators play a crucial role. In the process of plant tissue culture, although the amount of these substances is very small, they have obvious effects on the formation of callus, the induction of buds, and the dedifferentiation.

As can be seen from the results in Table 6, when no growth regulator was added, no callus and roots were formed, and only a small amount of shoots were produced. Callus and buds were formed in each group added with hormones, and the callus formation rate was higher in 1.0mg/L 6-BA and (0.5+1.0+0.5mg/L) 6-BA+NAA+KT In both groups, the callus induction rate was 73.3%, followed by 2mg/L 6-BA, (1+0.5mg/L)6-BA+NAA, (1.0+1.0mg/L)ZT+NAA and other combinations , the worse is 3mg/L and 4mg/L 6-BA treatment, the higher the 6-BA concentration, the lower the callus induction rate. The induction of callus has a close relationship with the hormone 6-BA. With the combination of 6-BA, the induction rate of callus is higher, and the induction rate of callus without it is lower.

From the results of the growth of buds, it can be seen that the induction of buds by these hormones is not obvious, and there is a certain amount of bud formation, and some buds are formed. The better one is (0.5+1.0mg/L) 6-BA+ NAA combination, the formation rate of shoots was higher. Other combinations have little difference and can form the differentiation and growth of buds.

From the results of root growth, it can be known that the root formation rate is low, most of them do not produce roots, or a few roots are produced, but (0.5+1.0+0.5mg/L) 6-BA+NAA+KT treatment has Two roots were formed, which shows that the combination is beneficial to the rooting of blueberry stem tissue culture. From the perspective of root growth, it is difficult to form roots in the bottle of blueberry stem segments, and only a few 1 to 2 roots are formed. This may be an unexplored suitable hormone combination, which needs to be explored in later research.

Table 6 The effect of different hormone types and concentrations on the growth of blueberry stems

Note: CK means no hormone added;

- Indicates that no callus (shoots or roots) is formed;

+ means that a small amount of callus (bud or root) is generated, and the callus generated within 0.5cm in diameter is a small amount, and there is only one root;

++ means that a certain amount of callus (bud or root) is generated, and the amount of callus generation is a certain amount when the diameter is 0.5cm to 1.0cm, and there are two roots;

+++ indicates that a large amount of callus (bud or root) is generated, and the amount of callus generated is a large amount of 1.0cm to 1.5cm in diameter, and there are three roots;

++++ indicates that the callus (bud or root) is produced in a large amount, and callus production with a diameter of more than 1.5 cm is a large amount of callus production.

3.4 Observation record of blueberry tissue culture

Blueberry tissue culture is shown in Figure 1. Under the control of hormones and medium, the formation rate of callus is relatively high. When the medium and hormones are more suitable formulas, it can be seen that both leaves and stems have Callus is obviously formed, and it gathers in clusters near the incision. The callus of the stem segment appears at the bottom of the stem segment in the medium, and the callus of the leaf appears around the leaf, including the junction with the medium and inside the medium. There are also exposed, milky white. After 2 weeks of culture, the growth of the callus did not change significantly, and then the transfer was carried out to continue the culture. After one transfer, the callus was pale yellow. Figure A shows the growth of the callus after one transfer of the stem segment, and the granular material gradually became larger. Yellowish green and slightly brownish red. Figure B shows the growth of the callus after the second transfer of the stem segment, which was pale yellow, and some were brown-red. Figure C shows the growth of the leaf transfer callus for three times. The callus outside the callus has yellow-green granules, and the central part appears browning, which may be caused by the browning substances secreted by itself. Take out the callus and crush it gently with your hands. There is a little water on your fingers, which means that it has a loose texture and contains a certain amount of water.

After the stem section is inoculated, the stem section usually germinates and grows buds first, and then callus grows, but there are also callus that appear earlier than buds. When the culture environment is more suitable, the axillary buds can be seen swelled after 3 days, and the leaves begin to bloom after a week, which is tender green. Figure G shows buds and long leaves after stem inoculation for 1 week, and the leaves are tender green. Figures D and H show the growth of buds 2 weeks after stem inoculation. Two of the earliest leaves were browned, and the other leaves were bright green, and the buds were 3 cm long. Figure E and Figure 1 (Figure 1 is the picture taken from the bottom of the medium after inoculation 3 weeks, and the area of the callus gradually increases) is the growth situation of the stem section inoculation after 3 weeks, and the top of the original stem section is browned and died, and the bottom has Callus is formed and shoots are 6-8 cm long. Figure F shows the growth of buds 4 weeks after stem inoculation, with obvious branching and green leaves.

Blueberry tissue culture is slower and more difficult to root. In this study, it was found that after the callus was cultured, a little root formation could be seen after 50 days, and some callus at this time appeared browning. Figure J shows the root growth of the callus after 8 weeks of leaf inoculation. Two roots appeared, which were milky white. At this time, a layer of white powder appeared on the periphery of the callus. Figure K shows that the leaves took root 9 weeks after inoculation, two roots appeared, up to 3 cm long, and the central part of the callus was obviously browned. Figure L shows that the leaves grow roots 10 weeks after inoculation, three roots appear, the longer one is 5 cm long, and some roots also appear bifurcation. , the callus browning area in the central position is getting bigger and bigger.

4 Discussion and Conclusion

4.1 Discussion

In this experiment, blueberry stem tissue culture and rapid propagation technology were studied, in order to obtain high-quality blueberry seedlings by in vitro culture. In this experiment, the process of obtaining tissue culture seedlings was finally obtained by improving the medium and plant growth regulating substances, and optimizing each condition.

First, screen the appropriate disinfectant and disinfection time. The best sterilization conditions were screened by controlling the two factors of alcohol and mercuric chloride to conduct a single factor test. The test results show that 75% alcohol treatment for 30s, 0.1% mercuric chloride treatment time for 8min is the best sterilization condition, and then use sterile water to rinse 3-5 times, which can greatly reduce the pollution rate, while not affecting the growth of stem segments, differentiation. Incision browning occurs after inoculation and occurs at a higher rate in 3-year-old stem material (completely lignified). The stem sections should be placed in the medium immediately after being cut to prevent them from browning. The addition of activated carbon in the pre-experiment did not improve browning, but instead made the medium black, which was not conducive to observation.

Secondly, the basic medium suitable for tissue culture of blueberry stems was screened. By configuring MS, 1/2MS, 1/4MS, modified WPM, modified 1/2WPM and other five media, adding the corresponding hormones, the growth of stem callus and buds was counted. The comprehensive results showed that the improved WPM medium induced the highest callus rate and bud growth rate, the callus rate was 67.7%, and the bud growth rate was 76.7%. The second most suitable medium was MS medium, which were 60.0% and 63.3%, respectively.

Thirdly, comparative experiments were carried out on the types, concentrations and combinations of plant growth regulators, and the optimal conditions for inducing callus, bud and rooting of stem segments were screened. Growth regulators mainly use 6-BA, NAA, KT, ZT, etc. Among them, 6-BA+NAA+KT (0.5+1.0+0.5mg/L) has the best effect when used together, and the induction rate of callus is extremely high Its growth performance is the best, with tender green and partly yellowish granules. The results of single hormone treatment showed that the effect of 6-BA was better, the induction rate of callus was higher and the growth rate was faster, obvious callus formation could be seen after one week, and callus grew significantly after 3 weeks. Secondly, in the 6-BA+NAA treatment, the color of callus growth was better, and most of them were bright green; the induced buds grew fast, buds began to germinate after 3 days, and germinated completely within a week, and the leaves were thin and green, and three After a week, the buds grew significantly, up to 5cm long, and some buds were also accompanied by branches.

In the process of tissue culture, the color change of the incision of the stem segment was mainly observed. After explant inoculation, the incision will generally turn brown, and gradually turn bright green after 1 week, and then callus will grow. The callus is pale yellow in the early stage, and then the periphery gradually turns bright green, and the callus is white powder when exposed on the medium. The buds generally appear earlier than the callus. The buds of the stem segments with axillary buds begin to sprout in 3 days, and gradually expand. After 1 week, they open obviously and grow tender green leaves. In some media, the leaves turn white when the buds grow. This should be caused by lack of nutrition, lack of mineral elements that are difficult to move, and when the leaves grow 7 to 8, the lower leaves turn yellow and white, which is mainly due to the lack of free nutrients (such as nitrogen, phosphorus and potassium) etc.) or the culture conditions are not suitable. The growth of the root was observed, and after about 30 days, the small milky white formed by the differentiation of callus can be seen, and then it gradually grows and becomes thicker, and it is also accompanied by bifurcation, that is, the so-called root formation.

During the experiment, there were still some problems, such as easy browning of explants after inoculation, which could not be solved by adding activated carbon. The browning of the explants before they were inoculated may be caused by the long exposure time on the ultra-clean workbench after cutting, which may cause the incision to react with the outside world.

4.2 Conclusion

Plant tissue culture is a process of in vitro culture in a sterile environment from several stages such as primary culture, subculture, and rooting culture. The requirements of each period are different, such as differences in light, hormones, and sterilization. In the tissue culture of blueberry stem segments, first select the robust branches as explant materials, rinse them with tap water, and then carry out disinfection treatment. The results of this experiment show that the disinfection treatment of explants (blueberry stem segments) is as follows: Soak in 75% alcohol for 30s, and then soak in 0.1% mercuric chloride for 8min to reduce the pollution rate. Under the same hormones and the same external conditions, five mediums were screened, and it was found that the improved WPM medium was more suitable for the tissue culture of blueberry stems. In inducing the growth of callus with long stem segments, the best mediums were WPM+2mg/L 6-BA and WPM+0.5mg/L 6-BA+1.0mg/L NAA+0.5mg/L KT. The callus induction rate of each treatment was as high as 73.3%. The early stage was milky white, and then gradually turned into tender green granular matter, and the periphery was coated with white granular matter.

In terms of bud induction and growth, the best combination of medium and hormones is WPM+0.5mg/L 6-BA+1.0mg/L NAA, the buds grow faster, and the leaves grow stronger and green; The new shoots grow rapidly, the stem segment can grow to 6-9cm long after 4 weeks, the bottom callus is pale yellow and bright green, and the callus area is large, which provides sufficient nutrition for the growth and elongation of the new shoots.

The callus in the tissue culture of blueberry stems quickly took root. The rooting formula was improved WPM+ (0.5+1.0+0.5mg/L) 6-BA+NAA+KT. This treatment combination had a high rooting rate and two roots grew out. , the roots are relatively strong, up to 4cm long, and some have bifurcations, which makes the root system more developed, absorbs sufficient nutrients in the lower part to provide nutrients to the upper part, and can generate a complete plant.

The tissue culture of blueberry is the basis of non-toxic seedlings. This experiment is the basic experiment of tissue culture of blueberry, in order to lay a foundation for the tissue culture of blueberry to breed seedlings.

Compared with the prior art, the technology of the present invention obtains high-quality blueberry seedlings by in vitro culture, and finally obtains tissue culture seedlings by improving the medium and plant growth regulating substances, and optimizing various conditions.

First, screen the appropriate disinfectant and disinfection time. The best sterilization conditions were screened by controlling the two factors of alcohol and mercury chloride to conduct a single factor test. The test results show that 75% alcohol treatment for 30s, 0.1% mercuric chloride treatment time for 8min is the best sterilization condition, and then use sterile water to rinse 3-5 times, which can greatly reduce the pollution rate, and does not affect the growth of stem segments, differentiation. Incision browning occurs after inoculation and is more frequent in 3-year-old stem material (completely lignified). The stem sections should be placed in the medium immediately after they are cut to prevent them from browning. The addition of activated carbon in the pre-experiment did not improve browning, but instead made the medium black, which was not conducive to observation.

Secondly, the basic medium suitable for tissue culture of blueberry stems was screened. By configuring MS, 1/2MS, 1/4MS, modified WPM, modified 1/2WPM and other five media, adding the corresponding hormones, the growth of stem callus and buds was counted. The comprehensive results showed that the improved WPM medium induced the highest callus rate and bud growth rate, the callus rate was 67.7%, and the bud growth rate was 76.7%. The second most suitable medium was MS medium, which were 60.0% and 63.3%, respectively.

Thirdly, comparative experiments were carried out on the types, concentrations and combinations of plant growth regulators, and the optimal conditions for inducing callus, bud and rooting of stem segments were screened. Growth regulators mainly use 6-BA, NAA, KT, ZT, etc. Among them, 6-BA+NAA+KT (0.5+1.0+0.5mg/L) has the best effect when used together, and the induction rate of callus is extremely high Its growth performance is the best, with tender green and partly yellowish granules. The results of single hormone treatment showed that the effect of 6-BA was better, the induction rate of callus was higher and the growth rate was faster, obvious callus formation could be seen after one week, and callus grew significantly after 3 weeks. Secondly, in the 6-BA+NAA treatment, the color of callus growth was better, and most of them were bright green; the induced buds grew fast, buds began to germinate after 3 days, and germinated completely within a week, and the leaves were thin and green, and three After a week, the buds grew significantly, up to 5cm long, and some buds were also accompanied by branches.

In the process of tissue culture, the color change of the incision of the stem segment was mainly observed. After explant inoculation, the incision will generally turn brown, and gradually turn bright green after 1 week, and then callus will grow. In the early stage of callus, the granules were light yellow, and then the periphery gradually turned bright green, and the ones exposed on the medium appeared as white powder. The buds generally appear earlier than the callus. The buds of the stem segments with axillary buds begin to sprout in 3 days, and gradually expand. After 1 week, they open obviously and grow tender green leaves. In some media, the leaves turn white when the buds grow out. This should be caused by lack of nutrients, lack of mineral elements that are difficult to move, and when the leaves grow 7 to 8, the lower leaves turn yellow and white, which is mainly due to the lack of free nutrients (such as nitrogen, phosphorus and potassium). etc.) or the culture conditions are not suitable. The growth of the root was observed, and the small milky white formed by the differentiation of the callus could be seen in about 30 days, and then gradually grew and thickened, and was accompanied by bifurcation, that is, the so-called root formation.

To sum up, the tissue culture method of the present invention has fast propagation speed, is not limited by geographical time, has low plant disease susceptibility rate, improves rooting and survival rate, and is suitable for the industrialization development of blueberry tissue culture seedlings.

Description of drawings

Fig. 1 is an observation record diagram of the blueberry tissue culture in the experimental example.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments, but not as a basis for limiting the present invention.

Embodiment 1. 1. A blueberry tissue culture method, the method is to select the blueberry branches with strong blueberry growth as explants, wash off the surface dirt and impurities with tap water, then disinfect the explants on an ultra-clean workbench, cut into 1.5 Centimeter long, put the cut explants into the medium above the alcohol lamp, and seal the inoculation after the completion of the inoculation. In the growth stage of inducing callus, select WPM+6-benzylaminopurine or select WPM+6 - Benzylaminopurine + naphthalene acetic acid + kinetin medium for cultivation, select WPM + 6-benzyl aminopurine + naphthalene acetic acid medium for bud induction and growth stage, select WPM + 6-benzylamino in the rooting stage Purine + naphthalene acetic acid + kinetin medium.

In the method, the explants are sterilized by soaking in 75% alcohol for 30s, and then soaking in 0.1% mercuric chloride for 8min; in the method, WPM is calculated by volume, consisting of 50 parts of macroelements and 10 parts of calcium salts. part, 5 parts trace element part, 5 parts iron salt part, and 9 parts organic matter part, where:

Major elements are composed of 3.8/L KNO ₃ +7.4g/L MgSO ₄ ﹒ 7H ₂ O + 3.4g/L of KH ₂ PO ₄ +8.0g/L of NH ₄ NO ₃ and water;

The calcium salt part is composed of 55.6g/L of Ca(NO ₃ ) ₂ ﹒ 4H ₂ O and water are prepared;

The trace element part is composed of 4.5g/L MnSO ₄ ﹒ 4H ₂ O+1.72g/L of ZnSO ₄ ﹒ 7H ₂ O+1.24g/L of H ₃ BO ₃ +0.05g/L of CuSO ₄ ﹒ 5H ₂ O+0.05g/L of Na ₂ MoO ₄ ﹒ 2H ₂ O and water are prepared;

The iron salt part is prepared from 7.45g/L Na ₂ -EDTA+5.57g/L FeSO ₄ ·7H ₂ O and water;

The organic matter part is made up of 40g/L inositol+0.4g/L glycine+0.2g/L Vb1+0.1g/L Vb6+0.1g/L Vb5 and water.

The pH values of the above-mentioned culture media were all 6.2, and the pH values of the culture media were adjusted with 1 mol/L sodium hydroxide solution and 1 mol/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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