CN110419447A - A kind of blueberry tissue cultural method - Google Patents

Abstract

The invention discloses a blueberry tissue culture method. The method comprises the steps of selecting blueberry explants for disinfection, putting them into a culture medium, and cultivating through the stage of inducing callus growth, bud induction, growth stage and rooting stage. The weaving 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 industrialized development of blueberry tissue culture breeding 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) belongs to the perennial deciduous woody plant of the Rhododendronaceae Vaccinium. Its fruit is a small berry, and the fruit has a variety of health functions and has attracted much attention, such as improving human immunity, anti-aging, softening blood vessels, improving vision, 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., as well as special nutrients such as anthocyanins, flavonoids, and fruit acids Element. Due to the above nutritional and health functions of blueberries, there are more and more products related to blueberries on the market, such as blueberry wine, jam, canned food, candied fruit, juice and beverages, etc., or raw materials for pastries, jellies, yogurt, candies, 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'an Mountains and Xiaoxing'an Mountains in northern my country. The history of blueberry cultivation is relatively short. The United States was the first to introduce and cultivate blueberries. Since the United States took the lead in realizing the commercial production of blueberries in the 1930s, more than 30 countries including Canada, Germany, Australia, the Netherlands, and Japan have successively introduced blueberries. Cultivated to make blueberries develop rapidly. At present, the United States, Europe, Japan and other places have carried out industrialized production of blueberries. But in the world, the supply of blueberry fruit is still far short of demand. According to statistics, the total output of blueberries in the United States in 2012 was only 350,000 tons, accounting for about 60% of the total global output. Now the output of blueberries is much higher than this figure. Niekerson was the first to conduct tissue culture research on blueberries. He succeeded in using blueberry shoots for callus culture, which marked that blueberry production has entered a new era, and efficient blueberry seedling production has laid the foundation for commercial production. At present, plant tissue culture technology has been well developed in the fields of improved seed cultivation, rapid propagation, and virus-free propagation. This technology is applied to blueberries to carry out blueberry seedling cultivation and improved seed propagation, providing technical support for commercial production, and making blueberry Get good and fast development.

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

Blueberry is an emerging fruit in my country, and is deeply favored by the people. Blueberries have been introduced and cultivated across 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 improved day by day, the pursuit of high-quality fruit has become more and more stringent, and more attention has been paid to the nutrition and health of the fruit, which has put enormous pressure on the development of blueberries. The production of high-quality blueberry fruit requires excellent seedlings and a good growth environment. But at present, there are many problems in the development of blueberries in my country. First, the cultivation history is relatively short, and the introduction and cultivation from abroad was only started in the 1980s; second, the planting technology is relatively backward, and the introduction and domestication of wild blueberries are mainly used; the production technology is not high. The production base is unclear about the growth environment and growth habits of blueberries. It is not clear what kind of growth environment is suitable for it. It does not know how to produce high-yield and high-quality blueberry fruits. The main propagation methods are introduction and cuttings, etc. In this way, a large number of seedlings cannot be propagated quickly, and diseases will occur during the propagation process, which will cause the seedlings to carry pathogens. Secondly, it is difficult for cutting seedlings to take root. This propagation method seriously affects the quality of seedlings, especially for blueberries. Variety degeneration and poor quality will occur after the introduction. Therefore, it is urgent to invent a blueberry tissue culture technology and non-toxic seedling technology development that have a fast propagation speed, are not limited by geographical time, reduce plant susceptibility, and improve rooting and survival rates.

Contents of the invention

The object of the present invention is to provide a blueberry tissue culture method. The weaving 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 promotes tissue culture 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 for disinfection, put them into the culture medium, and cultivate through the stage of induced callus growth, induction of buds and its growth stage and rooting stage.

In the aforesaid blueberry tissue culture method, the method is to select blueberry explants, wash away surface dirt and impurities with tap water, then sterilize the explants on an ultra-clean workbench, cut them into 1.5 cm long, and put them on the top of the alcohol lamp. The cut explants are placed in the culture medium, and sealed after the inoculation is completed. During the growth stage of the induced callus, WPM+6-benzylaminopurine or WPM+6-benzylaminopurine+naphthaleneacetic acid are selected. + kinetin medium culture, choose WPM+6-benzylaminopurine+naphthalene acetic acid medium for bud induction and growth stage, choose WPM+6-benzylaminopurine+naphthalene acetic acid+kinetic acid medium for rooting stage base culture.

In the aforesaid blueberry tissue culture method, the blueberry explant is a robust blueberry branch in the described method.

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

In the aforesaid blueberry tissue culture method, WPM is calculated in parts by volume in the method, consisting of 50 parts of macroelements, 10 parts of calcium salts, 5 parts of trace elements, 5 parts of iron salts and 9 parts of organic matter formulated in which:

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

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

The trace elements are made 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 Na ₂ MoO ₄ . 2H ₂ O and water are prepared;

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

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

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

The inventor has carried out following experiment for verifying the effect of the present invention:

Experimental example:

1 Materials and methods

1.1 Test material

The materials used in this test are 3-year-old Rabbiteye blueberry branches, and the stems are mainly taken for cultivation. The materials come from the vegetable experiment field of the Agricultural College of Guizhou University.

1.2 Test equipment and reagents

2.2.1 Test equipment

The test instruments mainly include: sterilization equipment (high-pressure steam sterilizer); inoculation equipment (inoculation room, ultra-clean workbench, tweezers, alcohol lamp, absorbent cotton, inoculation knife); cultivation equipment (constant temperature cultivation room, constant temperature incubator) , triangular bottle, plastic sealing film, wiring); storage equipment (refrigerator, brown glass bottle, oven); others: iron cylinder, induction cooker, label paper, camera, pH test paper, etc.

1.2.2 Experimental reagents

Reagents include sucrose, agar, 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), zeatin (ZT), cytokinin, kinetin (KT), 75% alcohol, 0.1% mercuric chloride (chloro Mercury), sodium bicarbonate, etc.

1.3 Test method

1.3.1 Experimental design

1.3.1.1 Mother liquor configuration

Including the mother liquor of MS medium and improved WPM medium, macroelement mother liquor, calcium salt, trace element mother liquor, iron salt, organic matter mother liquor, hormone mother liquor, etc., prepare mother liquor with a certain concentration, label and date, put in 4 ℃ Store in the refrigerator. Including the mother liquor of MS medium and improved WPM medium, macroelement mother liquor, calcium salt, trace element mother liquor, iron salt, organic matter mother liquor, hormone mother liquor, etc., prepare mother liquor with a certain concentration, label and date, put in 4 ℃ Store in the refrigerator. For later use, if the mother liquor precipitates or crystals are precipitated during use, it cannot be used, and the mother liquor needs to be prepared again.

Table 1 MS medium formula

Table 2 Modified WPM medium formula

1.3.1.2 Configuration of medium

According to the composition of each culture medium, absorb the corresponding mother liquor to cultivate the culture 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 vital 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), a pair of sealed glass beakers, etc. into a 121°C, 0.1kpa high-pressure steam sterilizer Sterilize within 20 minutes.

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 are washed with tap water to remove surface dirt and impurities, cut to a suitable size with scissors, and placed in super- The clean workbench is sterilized and cut into about 1.5 cm long, then put the cut stems into the culture medium above the alcohol lamp, and then cover the bottle cap. After the inoculation is completed, it is sealed, and the label and date are affixed.

1.3.1.5 Cultivation

Including primary culture, subculture, rooting culture and other stages. Primary culture is the cultivation after inoculation of stem segments, including long calli and buds; subculture refers to recultivation on the basis of primary culture, including stem segment proliferation and stem growth; rooting culture refers to the induction of other The process of growing roots. Put the inoculated flask into a constant temperature light incubator for cultivation, the temperature is 25±1°C, the light intensity is 2000lx, the dark cultivation is carried out in the first 5 days, and the combination of light and dark cultivation is carried out in the later stage. Generally, light cultivation is carried out for 14 hours a day, and dark Cultivate for 10h.

1.3.1.6 Seedling hardening and transplanting

After the explants grow buds and roots, slowly open the mouth of the bottle, and then fully open the bottle after 3 days, rinse the culture medium at the base of the tissue culture seedlings under tap water, and then transplant them into a plug with a matrix. The substrate is evenly mixed with peat, perlite, leaf humus, organic fertilizer, etc. After moving into the hole tray, water it thoroughly, and cultivate it in a place with weak light first, and then transfer it to normal light conditions after 5 days to allow it to grow normally.

1.3.1.7 Observation and recording

After the inoculation, take photos and record in writing, specifically record the surface condition of the incision of the explant after inoculation, whether there is a color change and other data. The growth of the explants, the presence or absence of callus formation, the emergence of shoots, and the color change of the explants were also recorded during the dark culture period. After light culture, the growth of the explants was observed every 5 days, and photographed and written records were taken.

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

Table 3 was obtained by changing the treatment time of 75% alcohol on blueberry stems with 0.1% mercuric chloride disinfection for 8 minutes, and recording the pollution and survival rate after inoculation, and performing single factor control. From the results in Table 3, it can be seen that when the treatment time of mercuric chloride disinfection remains unchanged, as the treatment time of alcohol increases, the pollution rate decreases in turn, and the survival rate first increases and then decreases. The highest pollution rate is 75% alcohol treatment time is 0s, that is, when no alcohol treatment, the pollution rate is 53.3%; the lowest pollution rate is a group of alcohol treatment time is 50s, the pollution rate is almost 0. From the analysis of the survival rate results, the group with the highest survival rate was 75% alcohol treatment time of 30s, up to 67.7%, followed by the group of 20s, with a survival rate of 60%, followed by 10s, 0s, 40s, 50s. According to the mortality data, when the mercury treatment time is the same, the survival rate gradually increases with the increase of the alcohol treatment time, and the mortality rate is as high as 80%. From these data, it can be known that, in terms of the pollution and survival rate of blueberry stems in different processing times of 75% alcohol, the best processing time is 30s, the pollution rate of reorganization is low and the survival rate is high, and 75% alcohol is the best Disinfection processing time.

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

Note: After 30 days of survival rate, if the stem segment has no discoloration and necrosis, it is recorded as survival.

Table 4 is obtained by changing the treatment time of 0.1% mercuric chloride on blueberry stems, recording the pollution situation and survival rate after inoculation, and performing single factor control under the condition that the treatment time of 75% alcohol disinfection for 30 seconds remains unchanged. The results in Table 4 show that when the 0.1% mercury liter disinfection time is 4 minutes, the pollution is the highest, and as the disinfection time increases, the pollution rate decreases gradually, and the lowest pollution rate is 0.1% mercury liter disinfection treatment for 10 minutes and 12 minutes (6.7%) . From the perspective of survival rate, with the increase of 0.1% mercury liter disinfectant treatment time, the survival rate first increased and then decreased, the highest was 0.1% mercury liter treatment for 8 minutes, up to 73.3%, 0.1% mercury liter disinfection treatment of blueberry stems The impact on the survival rate was 8min>10min>6min>4min>12min. From the results of the mortality rate, when the alcohol treatment time was the same, the mortality rate was lower at 13.3% when the mercuric chloride was treated for 4 minutes, 6 minutes, and 8 minutes. up to 60.0%. Therefore, considering the pollution rate and survival rate comprehensively, the more appropriate mercury disinfection treatment time is 8 minutes, and the pollution 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, the more suitable disinfection combination is: 30s75% alcohol disinfection treatment + 10min 0.1% mercury chloride, which 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 30 days of survival rate, if the stem segment has no discoloration and necrosis, it is recorded as survival.

3.2 Effects of different culture media on blueberry stems

Under the condition of constant concentration of hormone 6-BA and NAA, hormone 1mg/L 6-BA and 0.5mg/L NAA, counting the formation of callus, bud growth and rooting, and screening the basic medium suitable for the growth of blueberry stems. From the results in Table 5, it can be seen that the callus formation rate of WPM medium is the highest, which is 67.7%, followed by MS medium, which is 60%, and the lowest is 1/4MS medium, which is 13.3%. On inducing long buds in the stem section, the highest bud growth rate is 76.7% in WPM medium, followed by 63.3% in MS medium, and the lowest is 26.7% in 1/4MS medium. %.

From the perspective of the production and bud growth of stem induced callus, the more suitable culture is the improved WPM medium, which not only has a higher callus production rate, but also has a higher induction of stem bud growth, followed by MS medium. In the later experiments, these two media were used, combined with other hormones and their concentrations, to screen out the formula suitable for blueberry stem tissue culture.

The influence of table 5 different media on blueberry stem section

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

In the process of plant growth, its growth regulating substances play a vital role. In the process of plant tissue culture, although the amount of these substances is very small, the effects on callus formation, bud induction, dedifferentiation, etc. are more obvious.

From the results in Table 6, it can be seen that when no growth-regulating substances were added, no callus and roots were formed, and only a small amount of shoots were produced. Callus and bud formation were found in all hormone-added groups, and the higher rate of callus formation was 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 , 3mg/L and 4mg/L 6-BA treatments were worse, the higher the concentration of 6-BA, the lower the induction rate of callus. The induction of callus is closely related to the hormone 6-BA. The combination with its participation has a higher callus induction rate, and the callus induction rate without it is lower.

It can be seen from the growth results of the buds that the induction of these hormones to the buds is not obvious, and a certain amount of buds are produced, and some of them are a little buds, preferably (0.5+1.0mg/L) 6-BA+ NAA combination, the rate of bud formation is higher. There is little difference in other combinations, all of which can form the differentiation and growth of buds.

Can know from the result of root growth situation, the formation rate of root is lower, and most of them do not have the generation of root, or produce a little root, but (0.5+1.0+0.5mg/L) 6-BA+NAA+KT process 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 for blueberry stems to form roots in the bottle, and only a few 1 to 2 roots are formed. This may be a suitable hormone combination that has not been explored, which needs to be explored in later research.

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

Note: CK means no hormone added;

- indicates that no callus (shoot or root) is formed;

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

++ means that there is a certain amount of callus (bud or root) generation, 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 number of calluses (buds or roots) are generated, and the amount of callus generation is 1.0cm to 1.5cm in diameter. There are three roots;

++++ means that the callus (bud or root) has a large amount of generation, and the callus with a diameter of more than 1.5 cm is a large amount of callus.

3.4 Observation records of blueberry tissue culture

Blueberry tissue culture is shown in Figure 1. Under the control of hormones and medium, the callus formation rate is relatively high. When the combination of medium and hormones is more suitable, it can be seen that both leaves and stems have callus after one week. There are obvious callus formation, clusters gather 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 in the medium, some There are also exposed ones, which are 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 the transfer once, the callus was light yellow. Figure A is the growth of the callus after the stem segment was transferred once, and the granular matter gradually became larger. It is yellow-green and slightly brownish-red. Figure B shows the growth of the callus after the second transfer of the stem segment, which is light yellow, and some are brownish red. Figure C shows the growth of the callus transferred from the leaves three times. The callus on the periphery of the callus is yellow-green particles, and browning appears in the center, which may be caused by the browning substances secreted by itself. Take out these calluses and crush them gently with your hands. There is a little water on your fingers, indicating that it is loose in texture and contains a certain amount of water.

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

Blueberry tissue culture rooting is slow and difficult. This study found that after the callus was cultured, a few roots could be seen after 50 days, and some of the callus at this time appeared browning. Figure J is the long root situation of the callus after 8 weeks of leaf inoculation. Two roots appeared, which were milky white, and a layer of white powder appeared on the periphery of the callus at this time. Figure K shows that the leaves took root 9 weeks after inoculation, and two roots appeared, up to 3 cm long, and the center of the callus was obviously browned. Figure L shows the long roots after 10 weeks of leaf inoculation. There are three roots, the longest one is 5 cm long, and some roots also appear forked. When the root grows to a certain extent, it begins to fork and grows in the form of two forks. , the browning area of callus in the center is getting larger and larger.

4 Discussion and conclusion

4.1 Discussion

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

First, screen the appropriate disinfectant and disinfection time. By controlling the two factors of alcohol and mercury chloride, the single factor test was carried out to screen the best bactericidal conditions. The test results show that 75% alcohol treatment for 30 seconds and 0.1% mercury chloride treatment time of 8 minutes are the best sterilization conditions, followed by rinsing with sterile water for 3-5 times, which can greatly reduce the pollution rate without affecting the growth of stem segments. differentiation. Notch browning occurs after inoculation and is more frequent in 3-year-old stem material (completely lignified). After the stem segment is cut, it should be placed in the medium immediately to prevent it from browning. The addition of activated carbon substances in the pre-experiment did not improve browning, but instead made the medium black, which was not conducive to observation.

Secondly, screen the basic medium suitable for the tissue culture of blueberry stems. By configuring five media including MS, 1/2MS, 1/4MS, improved WPM, and improved 1/2WPM, and adding corresponding hormones, the growth of stem callus and buds were counted. According to the comprehensive results, the improved WPM medium induced the highest rate of callus and shoot growth, the callus rate was 67.7%, and the rate of shoot growth was 76.7%. The next most suitable medium was MS medium, which were 60.0% and 63.3% respectively.

Thirdly, comparative experiments are carried out on the types, concentrations and combinations of plant growth regulating substances to screen the most suitable conditions for inducing callus growth, bud growth and rooting of stem segments. The growth regulator substances mainly use 6-BA, NAA, KT, ZT, etc., among which 6-BA+NAA+KT (0.5+1.0+0.5mg/L) has the best effect when used in combination, and the induction rate of callus is extremely high And its growth performance is the best, it is light green and partly light yellow agglomerated particles. The results of single hormone treatment showed that the use of 6-BA had a better effect, and the induction rate of callus was higher and the growth was faster. After one week, obvious callus formation could be seen, and the callus grew significantly after 3 weeks. Secondly, the color of the callus growth in the 6-BA+NAA treatment is better, most of which are tender green; the induced buds grow fast, and after 3 days, the buds begin to germinate, and they germinate completely in one week, with thin and green leaf tips. After a week, the buds grow up obviously, up to 5cm, and some buds are accompanied by branches.

During the tissue culture process, the color change of the cut of the stem segment was mainly observed. The incision after explant inoculation generally turns brown, and gradually turns green after 1 week, and then grows callus. The early stage of the callus was light yellow granules, and then the periphery gradually turned into light green, and the ones exposed on the medium were in the form of white powder. Buds generally appear earlier than the callus, and the buds of the stem segment with axillary buds start to germinate in 3 days, gradually expand, and open obviously after 1 week, and grow tender green leaves. In some cultures, the leaves turn white when the buds grow out. This should be caused by lack of nutrients, lack of hard-to-move mineral elements, and when the leaves grow 7 to 8 pieces, the lower leaves turn yellow and white, which is mainly due to the lack of free nutrients (such as nitrogen, phosphorus, potassium) etc.) or inappropriate culture conditions. Observing the growth of root, about 30d can see the present situation of the fine milky white that is formed by the differentiation of callus, then grows and becomes thicker slowly, is also accompanied by bifurcation, promptly so-called root formation.

During the test, there were still some problems, such as the explants were easy to brown after inoculation, and this problem could not be solved by adding activated carbon substances. The browning of the explants before inoculation may be caused by the reaction between the incision and the outside world due to the long exposure time on the ultra-clean workbench after cutting.

4.2 Conclusion

Plant tissue culture is a process of in vitro culture in a sterile environment in several stages such as primary culture, subculture, rooting culture, etc. The requirements of each period are different, such as differences in light, hormones, and sterilization. In the tissue culture of blueberry stem section, first select the vigorous branch as the explant material, rinse it with tap water and carry out disinfection treatment. The results of this test show that the disinfection treatment of the explant (blueberry stem section) is Soak in 75% alcohol for 30s, and then soak in 0.1% mercuric chloride for 8 minutes, so as to reduce the pollution rate. Under the condition of the same hormone and the same external conditions, the improved WPM medium was found to be more suitable for the tissue culture of blueberry stems in the screening of five mediums. In terms of inducing the growth of long stem segment callus, the best medium is WPM+2mg/L 6-BA and WPM+0.5mg/L 6-BA+1.0mg/L NAA+0.5mg/L KT, the two The induced callus rate of each treatment was as high as 73.3%. It was milky white in the early stage, and then gradually turned into light green granular matter, and the periphery was covered with white granular matter.

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

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

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 the foundation for the tissue culture of blueberry seedlings.

Compared with the prior art, the technology of the present invention obtains high-quality blueberry seedlings through in vitro culture, improves the medium and plant growth regulating substances, and optimizes various conditions to finally obtain the process of tissue culture seedlings.

First, screen the appropriate disinfectant and disinfection time. By controlling the two factors of alcohol and mercuric chloride, the single factor experiment was carried out to screen the best bactericidal conditions. The test results show that 75% alcohol treatment for 30 seconds and 0.1% mercury chloride treatment time of 8 minutes are the best sterilization conditions, followed by rinsing with sterile water for 3-5 times, which can greatly reduce the pollution rate without affecting the growth of stem segments. differentiation. Incision browning occurs after inoculation and is more frequent in 3-year-old stem material (completely lignified). After the stem segment is cut, it should be placed in the medium immediately to prevent it from browning. Adding activated carbon substances in the pre-experiment did not improve browning, but instead made the medium black, which was not conducive to observation.

Secondly, screen the basic medium suitable for the tissue culture of blueberry stems. By configuring five media including MS, 1/2MS, 1/4MS, improved WPM, and improved 1/2WPM, and adding corresponding hormones, the growth of stem callus and buds were counted. According to the comprehensive results, the improved WPM medium induced the highest rate of callus and shoot growth, the callus rate was 67.7%, and the rate of shoot growth was 76.7%. The next most suitable medium was MS medium, which were 60.0% and 63.3% respectively.

Thirdly, comparative experiments are carried out on the types, concentrations and combinations of plant growth regulating substances to screen the most suitable conditions for inducing callus growth, bud growth and rooting of stem segments. The growth regulator substances mainly use 6-BA, NAA, KT, ZT, etc., among which 6-BA+NAA+KT (0.5+1.0+0.5mg/L) has the best effect when used in combination, and the induction rate of callus is extremely high And its growth performance is the best, it is light green and partly light yellow agglomerated particles. The results of single hormone treatment showed that the use of 6-BA had a better effect, and the induction rate of callus was higher and the growth was faster. After one week, obvious callus formation could be seen, and the callus grew significantly after 3 weeks. Secondly, the color of the callus growth in the 6-BA+NAA treatment is better, most of which are tender green; the induced buds grow fast, and after 3 days, the buds begin to germinate, and they germinate completely in one week, with thin and green leaf tips. After a week, the buds grow up obviously, up to 5cm, and some buds are accompanied by branches.

During the tissue culture process, the color change of the cut of the stem segment was mainly observed. The incision after explant inoculation generally turns brown, and gradually turns green after 1 week, and then grows callus. The early stage of the callus was light yellow granules, and then the periphery gradually turned into light green, and the ones exposed on the medium were in the form of white powder. Buds generally appear earlier than the callus, and the buds of the stem segment with axillary buds start to germinate in 3 days, gradually expand, and open obviously after 1 week, and grow tender green leaves. In some cultures, the leaves turn white when the buds grow out. This should be caused by lack of nutrients, lack of hard-to-move mineral elements, and when the leaves grow 7 to 8 pieces, the lower leaves turn yellow and white, which is mainly due to the lack of free nutrients (such as nitrogen, phosphorus, potassium) etc.) or inappropriate culture conditions. Observing the growth of root, about 30d can see the present situation of the fine milky white that is formed by the differentiation of callus, then grows and becomes thicker slowly, is also accompanied by bifurcation, promptly so-called root formation.

In summary, the tissue culture method of the present invention has a fast propagation speed, is not limited by geographical time, has a low plant disease susceptibility rate, improves rooting and survival rates, and is suitable for the industrial development of blueberry tissue culture seedlings.

Description of drawings

Fig. 1 is the observation record diagram of the blueberry tissue culture situation 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 kind of blueberry tissue culture method, this method is to choose the blueberry branch that blueberry grows vigorously as explant, wash away surface dirt impurity with tap water, explant is sterilized in ultra-clean workbench again, cut into 1.5 cm long, put the cut explants into the culture medium above the alcohol lamp, and seal them after inoculation. During the growth stage of the induced callus, choose WPM+6-benzylaminopurine or WPM+6 - Benzylaminopurine + naphthalene acetic acid + kinetin culture medium, choose WPM + 6-benzylaminopurine + naphthalene acetic acid medium for cultivation in the bud induction and growth stage, and choose WPM + 6-benzylaminopurine in the rooting stage Cultured in purine + naphthaleneacetic acid + kinetin medium.

The disinfection method of the explant in the described method is to soak with 75% alcohol for 30s, and then soak with 0.1% mercuric chloride for 8min; in the described method, WPM is calculated by volume, consisting of 50 parts of macroelements, 10 parts of calcium salt 1 part, 5 parts trace elements, 5 parts iron salts and 9 parts organic matter, where:

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

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

The trace elements are made 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 Na ₂ MoO ₄ . 2H ₂ O and water are prepared;

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

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

The pH value of the above medium is 6.2, and the pH value of the medium is adjusted with 1mol/L sodium hydroxide solution and 1mol/L hydrochloric acid solution.

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.