Rapid propagation method for six-fold tissue culture

Abstract

The invention discloses a hexapole tissue culture rapid propagation method, and belongs to the technical field of plant propagation. The method comprises the following steps: by taking hexapoil seeds as explants, performing disinfection and sterilization treatment on the explants, and sequentially performing callus induction culture, adventitious bud differentiation culture, multiplication culture and rooting culture to obtain hexapoil seedlings with a good condition state; the optimal culture conditions are obtained by screening a disinfection and sterilization mode, a callus culture medium, an adventitious bud differentiation culture medium, a proliferation culture medium and a rooting culture medium, so that technical support is provided for tissue culture and propagation of hexapole, and a feasible scheme is provided for large-scale commercial hexapole breeding; the method can meet the requirements of batch breeding and planting, can significantly improve the six-fold breeding speed, and has a high commercial value.

Description

Technical Field

[0001] The invention belongs to the technical field of plant propagation, and in particular relates to a six-fold tissue culture rapid propagation method. Background Art

[0002] Lobelia is a perennial or biennial herb of the genus Lobelia in the Campanulaceae family. It is native to South Africa. It needs long daylight and low temperature environment to bloom. It is not cold-resistant and avoids extreme heat. It likes loam rich in humus and loose soil.

[0003] The flowers of Liubeili are small, blue or blue-purple, and the flowering period is from April to June. Because of its small and cute flower shape, it is an excellent garden ornamental plant. It can be planted in pots, or planted in vertical greening in parks and courtyards. It is also widely used in flower beds and flower borders. At the same time, the whole plant of Liubeili can be used as medicine, which has certain medicinal value. It has certain effects on detoxification, anti-inflammatory and swelling, carbuncle, furuncle, etc. The seeds of Liubeili are small, and the weight of 1,000 seeds is 0.03g.

[0004] The traditional sowing method of Liubeili has some disadvantages, such as low germination rate and slow growth. The main reason is that the seeds of Liubeili are very small, and the size of a single seed is smaller than sesame. It is often difficult to accurately control the distribution and coverage depth of seeds using traditional sowing methods, resulting in low and uneven germination rates. In this case, even if pelletized seeds are used to assist, the germination rate still depends on many factors, including temperature, humidity and sowing technology, making the whole process more complicated and unpredictable.

[0005] Secondly, it is dependent on the environment. The germination and growth of Liubeili seeds are very sensitive to temperature and light conditions. The optimal germination temperature of Liubeili seeds is usually around 25°C, so the sowing time is usually chosen in autumn to avoid the adverse effects of high temperature on seed germination, and sufficient light is also a key factor in promoting seed germination and healthy growth of seedlings. Traditional sowing methods often have difficulty in providing uniform and stable environmental conditions, which may lead to differences in germination rate and growth rate, thus affecting the final flowering effect.

[0006] The traditional sowing process requires complicated management methods, and the management of the six-beli seedlings requires great care and patience, especially in the transplanting and planting stages. During the transplanting stage, it is necessary to provide loose and well-drained soil conditions for the six-beli, and a bright, ventilated and stable growth environment for its growth needs. During the planting stage, it is also necessary to provide it with sufficient nutritional support. The six-beli seedlings are also very susceptible to diseases and insect pests. In the traditional sowing process, because the density of the plants is high, once diseases and insect pests appear, this dense planting method will quickly aggravate the spread and spread of diseases and insect pests, affecting the growth quality of the six-beli seedlings and the later flowering effect.

[0007] To sum up, the traditional way of planting Liubei Li requires a lot of manpower in the breeding and seedling cultivation of Liubei Li. Therefore, a new sowing technology is needed to reduce the burden of Liubei Li seedling cultivation and planting. Without affecting the flower ball effect, a new method can meet the needs of mass breeding and planting and significantly improve the reproduction speed of Liubei Li. Summary of the invention

[0008] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a method for rapid propagation of Hexaploidy by tissue culture, which utilizes tissue culture technology to carry out tissue culture and expansion propagation technology on Hexaploidy, which will greatly reduce the shortcomings of traditional sowing methods, meet a large number of application needs, and improve the propagation speed of Hexaploidy plants.

[0009] In order to solve the above problems, the technical solution adopted by the present invention is as follows: A six-fold tissue culture rapid propagation method comprises the following steps: Step 1, selecting hexapod seeds as explants; Step 2, sterilizing the hexapod explants; Step 3, placing the explants on a primary culture medium for culturing to produce primary sterile seedlings; Step 4, cutting the stem segments of the primary sterile seedlings cultured in step 3, and placing them on a callus induction medium for callus induction culture; Step 5, transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture; Step 6, transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture; Step seven, harden and transplant the seedlings after the rooting culture in step six.

[0010] Preferably, the screening process in step 1 is: selecting seeds with full grains and maturity, removing dead seeds, diseased seeds and seeds with sclerotia, and then soaking the seeds in warm water to allow them to fully absorb water.

[0011] Preferably, the disinfection process in step 2 is: using a 0.1% concentration of mercuric chloride solution for disinfection for 12 minutes.

[0012] Preferably, the primary culture medium in step three is an MS culture medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.6 mg / L NAA (naphthaleneacetic acid) and 0.6 mg / L 6-BA (6-benzyladenine), and the pH of the primary culture medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0013] Preferably, the callus induction medium in step 4 is an MS medium added with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the callus induction medium is adjusted to pH 5.6 by adding 0.1 mg / L NaOH solution.

[0014] Preferably, the differentiation and proliferation medium in step five is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the pH of the differentiation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0015] Preferably, the differentiation and proliferation medium in step five is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA and 2 mg / L 6-BA, and the pH of the proliferation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0016] Preferably, the rooting medium in step six is ​​an MS medium added with 30 g / L sucrose, 7 g / L agar and 0.05 mg / L NAA, and the pH of the rooting medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0017] Preferably, the treatment methods used for the callus induction culture in step 4 and the adventitious bud differentiation culture in step 5 are both dark treatment.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses six-fold loquat seeds as explants, performs disinfection and sterilization treatment on the explants, and sequentially performs callus induction culture, adventitious bud differentiation culture, proliferation culture and rooting culture to obtain six-fold loquat seedlings with good appearance conditions, and screens the disinfection and sterilization methods, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium and rooting culture medium to obtain the best culture conditions, thereby providing technical support for the tissue culture and propagation of six-fold loquat, and providing a practical and feasible solution for large-scale commercial six-fold loquat breeding, which can meet the needs of batch breeding and planting, and can also significantly improve the reproduction speed of six-fold loquat, and has high commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is an implementation flow chart of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments.

[0021] The present invention provides a method for rapid propagation of hexaploid tissue culture, the method comprising the following steps: Step 1, selecting hexapod seeds as explants; Step 2, sterilizing the hexapod explants; Step 3, placing the explants on a primary culture medium for culturing to produce primary sterile seedlings; Step 4, cutting the stem segments of the primary sterile seedlings cultured in step 3, and placing them on a callus induction medium for callus induction culture; Step 5, transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture; Step 6, transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture; Step seven, harden and transplant the seedlings after the rooting culture in step six.

[0022] Among them, the disinfection process in step 2 is: use 0.1% concentration of mercuric chloride solution for disinfection for 12 minutes.

[0023] The primary culture medium in step three is MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.6 mg / L NAA and 0.6 mg / L 6-BA, and the pH of the primary culture medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0024] The callus induction medium in step 4 is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the callus induction medium is adjusted to pH 5.6 by adding 0.1 mg / L NaOH solution.

[0025] The differentiation medium in step five is MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the pH of the differentiation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0026] The proliferation medium in step six is ​​MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA and 2 mg / L 6-BA, and the pH of the proliferation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0027] The rooting medium in step seven is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar and 0.05 mg / L NAA, and the pH of the rooting medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

[0028] The treatment methods used for the callus induction culture in step 4 and the adventitious bud differentiation culture in step 5 are both dark treatment.

[0029] The above specific data were compared through a series of orthogonal experiments to analyze the effects of different disinfection and sterilization methods and culture medium ratios on the six-fold tissue culture rapid propagation system. The specific experimental process and results are shown in the examples.

[0030] The range of culture medium components used in the control experiment in this example is as follows: Initial culture medium: MS+NAA0~0.6mg / L+6-BA0~0.6mg / L.

[0031] Callus induction medium: MS+6-BA1~1.5mg / L+NAA0.1~0.15mg / L.

[0032] Differentiation medium: MS+6-BA1~2mg / L+NAA0.1~0.2mg / L.

[0033] Proliferation medium: MS+6-BA1~2mg / L+NAA0.1~0.2mg / L.

[0034] Rooting medium: MS+NAA 0.05~0.15mg / L.

[0035] The above culture media are all expressed in a way that is easy to visually distinguish. The meaning of the expression refers to the basic culture medium and the different concentrations of hormones added to it. For example, MS represents MS culture medium, NAA0.6 mg / L represents NAA added with a concentration of 0.6 mg / L, and 6-BA0.6 mg / L represents 6-BA added with a concentration of 0.6 mg / L. In addition, in the above culture media, all prepared culture media are added with sucrose at a concentration of 30 g / L and agar at a concentration of 7 g / L, and the pH is adjusted to about 5.6 by adding NaOH solution at a concentration of 0.1 mg / L.

[0036] During the different stages of cultivation, the culture medium was placed in a culture room under the conditions of a temperature of 25°C and a light intensity of 3000lx for cultivation.

[0037] First, select the explants. Choose Liubei seeds as the explants. The selection criteria for seeds are full-grained and mature seeds. After removing dead seeds, diseased seeds and seeds with sclerotia, soak the seeds in warm water to allow them to fully absorb water.

[0038] After the explants are selected, the six-fold explants need to be disinfected to establish a sterile seedling system.

[0039] Based on the seed screening in the previous step, 0.1% concentration of mercuric chloride solution was used for disinfection for different lengths of time to obtain the disinfection effects of different disinfection durations. A total of four disinfection durations were compared, namely: 3min, 6min, 9min, and 12min.

[0040] After the disinfection, the explants were inoculated on MS medium to compare the disinfection effect. Ten bottles of medium were selected for each disinfection time, and five seeds were placed in each bottle of medium. The experimental process was as follows: Figure 1 As shown in the figure, the contamination rate was observed and recorded during the disinfection process. If the seeds were found to have grown bacteria, they were immediately cleaned. After 10 days, the total contamination rate of each seed with different disinfection times was calculated. The impact of different disinfection times on the Liubeili seeds was recorded to obtain the optimal disinfection time.

[0041] Table 1 serial number Disinfection time (min) Contamination rate (%) 1 3 60 2 6 30 3 9 30 4 12 20 The contamination rate in the above table is calculated as: number of contaminated bottles / total number of inoculated bottles × 100%.

[0042] The test results are shown in Table 1. It can be seen that the bacterial growth rate is 60% in 3 minutes. As the disinfection time increases, the contamination rate decreases. When the disinfection time is 12 minutes, the contamination rate is controlled, and the disinfection time is too long to damage the seeds and affect their germination. Therefore, it is concluded that the best effect is to use 0.1% mercuric chloride solution for disinfection for 12 minutes.

[0043] After determining the optimal disinfection time, the primary culture medium with the optimal hormone ratio is then determined.

[0044] The configuration of the culture medium mainly lies in the addition ratio of the two hormones, auxin NAA and cytokinin 6-BA. The culture medium base selected is MS culture medium. The comparison method adopts 2-factor 3-level orthogonal method for experimental comparison. The results are shown in Table 2.

[0045] Table 2 Hormone concentration (mg / L) Seedling height Growth 6-BA 0+NAA 0 5-6cm Rapid growth, but with leggy growth and spindly leaves 6-BA 0.3+NAA 0 3-5cm Fast growth, leggy growth, slender leaves 6-BA 0+NAA 0.3 1.5-2cm Seed germination is slightly slow, and no leggy growth occurs. 6-BA 0.3+NAA 0.3 1.5-2cm Seed germination is slightly slow, no leggy growth 6-BA 0.6+NAA 0.3 2-2.5cm Seeds germinate normally without leggy growth 6-BA 0+NAA 0.6 2-3cm Seeds germinate slightly faster, without leggy growth 6-BA 0.3+NAA 0.6 2.5-3cm Seeds germinate slightly faster, without leggy growth 6-BA 0.6+NAA 0.6 3-4cm The seeds germinate quickly, without any leggy growth or elongated leaves. As shown in Table 2, when no hormones were added, the plant grew fastest, but it also grew too long and had thin leaves; When only cytokinin was added, the plant grew faster as the concentration of cytokinin 6-BA increased sixfold, and the leaves became slender and elongated. When only the auxin NAA is added without cytokinin, the growth rate becomes faster with the increase of auxin concentration; when both hormones are added at the same time, the growth rate of Hexaphylla increases with the increase of cytokinin 6-BA concentration, and Hexaphylla grows fast without the phenomenon of excessive growth.

[0046] Different hormone concentrations have different effects on hexachlorobenzene. According to this experiment, it can be seen that in the primary culture medium of MS+NAA0.6mg / L+6-BA0.6mg / L, hexachlorobenzene seeds germinate rapidly, the sterile seedlings are strong, and there is no excessive growth.

[0047] Therefore, it is believed that the culture medium of MS+NAA0.6mg / L+6-BA0.6mg / L is the best culture medium, which is more conducive to the subsequent experiments. Based on this primary culture medium, sterile seedlings are obtained and a sterile seedling system is established.

[0048] After the sterile seedling system was established, the stem segments and leaf parts of the six-fold seedlings were cut from the sterile seedlings obtained from the primary culture medium for callus induction and adventitious bud differentiation.

[0049] In the whole process of callus induction and adventitious bud differentiation, it is divided into dark treatment induction method and light treatment induction method. In addition, the cut parts include stem segments and leaves, so it is necessary to carry out dark treatment and light treatment control experiments on the two cut parts respectively.

[0050] First, stem segments were selected for callus induction, and control experiments were conducted under two different induction conditions, dark treatment and light treatment, with different hormone concentrations. Table 3 shows the induction effects of different hormone concentrations on the six-fold sharp stem segments under dark treatment conditions, and Table 4 shows the induction effects of different hormone concentrations on the six-fold sharp stem segments under light treatment conditions.

[0051] The induction rate in the table was calculated as: number of calli / number of inoculated seedlings × 100%.

[0052] Table 3 Hormone concentration (mg / L) Induction rate (%) 6-BA 1.0+NAA 0.1 88 6-BA 1.0+NAA 0.15 100 6-BA 1.0+NAA 0.2 92 6-BA 1.5+NAA 0.1 28 6-BA 1.5+NAA 0.15 68 6-BA 1.5+NAA 0.2 40 6-BA 2.0+NAA 0.1 64 6-BA 2.0+NAA 0.15 56 6-BA 2.0+NAA 0.2 85 Table 4 Hormone concentration (mg / L) Induction rate (%) 6BA 1.0+NAA0.1 84 6-BA1.0+NAA0.15 84 6-BA1.0+NAA0.2 84 6-BA1.5+NAA0.1 90 6-BA1.5+NAA0.15 60 6-BA1.5+NAA0.2 40 6-BA2.0+NAA0.1 76 6-BA2.0+NAA0.15 68 6-BA2.0+NAA0.2 84 According to Table 3, under dark treatment, when the concentration of cytokinin 6-BA was 1.0 mg / L, as the concentration of auxin increased, the rate of callus induction was higher or even all callus was induced; when the concentration of cytokinin 6-BA increased to 1.5 mg / L and 2 mg / L, the induction rate of six-fold induction began to decrease. The entire induction rate of six-fold induction showed a single peak phenomenon. It can be concluded that the induction rate was the highest when MS+6-BA1 mg / L+NAA0.15 mg / L, and the induction rate was 100%.

[0053] According to Table 4, under light treatment, callus tissue can be induced in stem segments regardless of the concentration of hormone, but the induction effect is different when the concentration is different. The best effect is MS+6-BA1.5mg / L+NAA0.1mg / L, with an induction rate of 90%.

[0054] Then, leaves were selected for callus induction, and control experiments were carried out under two different induction conditions, dark treatment and light treatment, and different hormone concentrations. Table 5 shows the induction effects of different hormone concentrations on the leaves of Hexaphylla under dark treatment conditions, and Table 6 shows the induction effects of different hormone concentrations on the leaves of Hexaphylla under light treatment conditions.

[0055] When the leaves were inducing callus tissue, they also showed the phenomenon of leaf whitening.

[0056] The albino rate in the table was calculated as: number of albino seedlings / number of seedlings inoculated × 100%.

[0057] Table 5 Hormone concentration (mg / L) Induction rate (%) Albinism rate (%) 6BA 1.0+NAA0.1 45 25 6-BA1.0+NAA0.15 30 25 6-BA1.0+NAA0.2 12 36 6-BA1.5+NAA0.1 28 16 6-BA1.5+NAA0.15 48 20 6-BA1.5+NAA0.2 50 40 6-BA2.0+NAA0.1 32 40 6-BA2.0+NAA0.15 40 20 6-BA2.0+NAA0.2 5 15 Table 6 Hormone concentration (mg / L) Induction rate (%) Albinism rate (%) 6BA 1.0+NAA0.1 32 36 6-BA1.0+NAA0.15 0 100 6-BA1.0+NAA0.2 36 20 6-BA1.5+NAA0.1 38 44 6-BA1.5+NAA0.15 36 20 6-BA1.5+NAA0.2 40 44 6-BA2.0+NAA0.1 35 28 6-BA2.0+NAA0.15 3 15 6-BA2.0+NAA0.2 25 65 According to Table 5, it can be seen that the induction of callus tissue from leaves under dark treatment conditions is more difficult than that from stem segments, and whitening may occur. The highest induction rate is MS+6-BA1.5g / L +NAA0.2mg / L, and the callus induction rate using six-fold leaves is low, and the effect is not ideal.

[0058] According to Table 6, the leaves treated under light had a lower induction rate and also showed bleaching.

[0059] Based on the above control experiments, it can be considered that stem segments are more suitable as explants for inducing callus than leaves.

[0060] Under dark treatment, the induction rate of MS+6-BA1mg / L+NAA0.15mg / L medium was the highest, with an induction rate of 100%. Under light treatment, the induction rate of MS+6-BA1.5mg / L+NAA0.1mg / L medium was the highest, with an induction rate of 90%.

[0061] After the callus induction of the explants is completed, the explants are transferred to the differentiation medium for differentiation of adventitious buds. The same two types of explants, stem segments and leaves, are used, and control experiments are carried out with light treatment and dark treatment culture methods respectively to test the effects of different auxin concentrations and treatment methods on adventitious bud differentiation.

[0062] First, the differentiation of stem segment explants was carried out, and control experiments with different hormone concentrations were conducted under two different treatment conditions: dark treatment and light treatment. Table 7 shows the effects of different hormone concentrations on the differentiation of six-fold stem segments under dark treatment conditions, and Table 8 shows the effects of different hormone concentrations on the differentiation of six-fold stem segments under light treatment conditions.

[0063] The differentiation rate in the table is calculated as follows: Differentiation rate = number of adventitious buds / number of inoculated seedlings × 100%.

[0064] Table 7 Hormone concentration (mg / L) Differentiation rate (%) 6-BA 1.0+NAA 0.1 32 6-BA 1.0+NAA 0.15 100 6-BA 1.0+NAA 0.2 72 6-BA 1.5+NAA 0.1 28 6-BA 1.5+NAA 0.15 60 6-BA 1.5+NAA 0.2 40 6-BA 2.0+NAA 0.1 100 6-BA 2.0+NAA 0.15 24 6-BA 2.0+NAA 0.2 25 Table 8 Hormone concentration (mg / L) Differentiation rate (%) 6-BA 1.0+NAA 0.1 40 6-BA 1.0+NAA 0.15 28 6-BA 1.0+NAA 0.2 40 6-BA 1.5+NAA 0.1 55 6-BA 1.5+NAA 0.15 40 6-BA 1.5+NAA 0.2 52 6-BA 2.0+NAA 0.1 16 6-BA 2.0+NAA 0.15 24 6-BA 2.0+NAA 0.2 25 According to Table 7, it can be seen that under dark treatment, all stem segments can differentiate into adventitious buds, but the degree of differentiation is different at different hormone concentrations. Under MS+6-BA1mg / L+NAA0.15mg / L and MS+6-BA2mg / L+NAA0.1mg / L, 100% of the stem segments can differentiate into adventitious buds.

[0065] Therefore, it is believed that MS+6-BA1 mg / L+NAA0.15mg / L and MS+6-BA2mg / L+NAA0.1mg / L are the optimal hormone ratios for differentiation of six-fold stem segments under dark conditions.

[0066] According to Table 8, it can be seen that under light treatment, all stem segments can also differentiate into adventitious buds, and the degree of differentiation is proportional to the concentration of 6-BA. The best differentiation effect is achieved when 6-BA1.5mg / L+NAA0.1mg / L is added to the MS medium, and the differentiation rate is 55%.

[0067] First, the differentiation of leaf explants was carried out, and control experiments with different hormone concentrations were conducted under two different treatment conditions: dark treatment and light treatment. Table 9 shows the effects of different hormone concentrations on the differentiation of hexaphylla leaves under dark treatment conditions, and Table 10 shows the effects of different hormone concentrations on the differentiation of hexaphylla leaves under light treatment conditions.

[0068] Table 9 Hormone concentration (mg / L) Differentiation rate (%) 6-BA 1.0+NAA 0.1 0 6-BA 1.0+NAA 0.15 0 6-BA 1.0+NAA 0.2 0 6-BA 1.5+NAA 0.1 0 6-BA 1.5+NAA 0.15 0 6-BA 1.5+NAA 0.2 0 6-BA 2.0+NAA 0.1 0 6-BA 2.0+NAA 0.15 0 6-BA 2.0+NAA 0.2 0 Table 10 Hormone concentration (mg / L) Differentiation rate (%) 6-BA 1.0+NAA 0.1 0 6-BA 1.0+NAA 0.15 0 6-BA 1.0+NAA 0.2 0 6-BA 1.5+NAA 0.1 0 6-BA 1.5+NAA 0.15 0 6-BA 1.5+NAA 0.2 0 6-BA 2.0+NAA 0.1 0 6-BA 2.0+NAA 0.15 0 6-BA 2.0+NAA 0.2 0 It can be seen from Tables 9 and 10 that under dark treatment conditions, the differentiation of leaves is more difficult than that of stem segments. Although the effect of using hexachlorobenzene leaves to differentiate adventitious buds is not ideal, according to Table 9, it can be found that when the ratio of cytokinin 6-BA and auxin NAA is 10:1, adventitious buds can be differentiated. The best differentiation effect is the culture medium MS+6-BA1.5mg / L+NAA0.15mg / L, with a differentiation rate of 12%.

[0069] The leaves treated under light were induced to differentiate, but the induction rate was low, no differentiation occurred, the differentiation rate was all 0, and all showed albinism.

[0070] Therefore, according to the results of the above experiments, it can be concluded that stem segments are more suitable for adventitious bud differentiation explants than leaves. Under dark conditions, the culture medium with the two hormone combinations of MS+6-BA1.0 mg / L+NAA0.15 mg / L and MS+6-BA2.0 mg / L+NAA0.1 mg / L was used for differentiation and proliferation. The stem segments can 100% differentiate into adventitious buds, and can also effectively promote the growth and differentiation of young tissues. Under the action of this culture medium, the stems are thick, the buds are many, and the proliferation effect is the best, which can effectively increase the number and yield of seedlings in tissue culture, as well as enhance the quality and growth rate of seedlings.

[0071] After the differentiation of adventitious buds is completed, it can be subjected to rooting culture. During the rooting culture stage, Hexalobium is very easy to take root. Even in MS culture medium without adding hormones, Hexalobium can take root quickly. However, after adding a certain amount of NAA, the growth and differentiation of the root system of Hexalobium is more obvious, as shown in Table 11, which shows the growth of the root system after adding different concentrations of NAA in MS culture medium.

[0072] Table 11 Hormone concentration (mg / L) Root length Growth NAA 0 1-2cm The main root system develops normally, without additional lateral roots NAA 0.01 1-3cm The main root system develops normally, without additional lateral roots NAA 0.03 2-3cm The main root system develops normally, without additional lateral roots NAA 0.05 3-5cm The main root system develops normally, with a small number of lateral roots NAA 0.07 3-5cm The main root system develops normally, with many lateral roots NAA 1 2-4cm The main root system sprouts more and the lateral roots appear more NAA 1.2 1-3cm The main root system sprouts more and the lateral roots appear more NAA 1.5 1-2cm The main root system sprouts more and the lateral roots appear more It can be seen from Table 11 that MS+NAA 0.05 mg / L has the best effect, which can promote the root growth and differentiation of seedlings and will not have a negative impact on the health of the seedlings.

[0073] When the concentration of NAA is lower than 0.05 mg / L, the effect of promoting growth is not obvious. When the concentration of NAA is higher than 0.05 mg / L, the growth of the main root system will be inhibited. The number of main roots will be more but the length will be shorter. There will be more lateral roots, which are thinner than the main root system. In the subsequent growth process, some lateral roots will gradually atrophy. More lateral roots will consume more nutrients, which is not conducive to the normal growth of the main root system. The germination of more main roots will make it difficult to take root after transplanting.

[0074] After the rooting culture is completed, the six-fold seedlings can be hardened and transplanted. The six-fold seedlings are easier to survive and do not require additional hardening. When the roots grow to 3-4cm, the six-fold sterile seedlings can be taken out. Just clean the culture medium at the roots, plant them in a greenhouse with the temperature adjusted, and cultivate them under light conditions. Planting should be carried out within an appropriate temperature range, usually around 20-25℃.

[0075] By comparing the induction of callus and differentiation of adventitious buds between hexavalent stem segments and leaves, it was found that the effect of stem segments, whether it is differentiation of adventitious buds or induction of callus, is better than that of leaves as explants, and under MS+6-BA1mg / L+NAA0.15mg / L and MS+6-BA2mg / L+NAA0.1mg / L, 100% of stem segments can differentiate into adventitious buds, which is most suitable for stem segment differentiation of adventitious buds and has the best effect. MS+6-BA1.0 mg / L+NAA0.15mg / L and MS+6-BA2.0mg / L+NAA0.1mg / L were determined to be the optimal differentiation culture media.

[0076] The present invention uses six-fold seeds as explants, performs disinfection and sterilization treatment on the explants, and sequentially performs callus induction culture, adventitious bud differentiation culture, proliferation culture and rooting culture. The present invention obtains the best culture conditions by screening the disinfection and sterilization method, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium and rooting culture medium. Experimental results show that when six-fold seeds are used as explants, the callus induction rate is 90% under light treatment, and 55% of the adventitious buds are differentiated; the callus induction rate is 100% under dark treatment, and 100% of the adventitious buds are differentiated.

[0077] The tissue culture method and the optimal culture medium for each step determined through the above-mentioned experiments provide technical support for the tissue culture expansion of Liubeili, and provide a practical solution for large-scale commercial breeding of Liubeili, which can meet the needs of mass breeding and planting, and can also significantly improve the reproduction speed of Liubeili, and has high commercial value.

Claims

1. A method for rapid propagation of hexapod tissue culture, characterized in that: The following steps are involved: Step 1, selecting hexapod seeds as explants; Step 2, sterilizing the hexapod explants; Step 3, placing the explants on a primary culture medium for culturing to produce primary sterile seedlings; Step 4, cutting the stem segments of the primary sterile seedlings cultured in step 3, and placing them on a callus induction medium for callus induction culture; Step 5, transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture; Step 6, transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture; Step seven, harden and transplant the seedlings after the rooting culture in step six.

2. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that: The screening process in step 1 is: selecting seeds with full grains and maturity, removing dead seeds, diseased seeds and seeds with sclerotia, and then soaking the seeds in warm water to allow them to fully absorb water.

3. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that: The disinfection process in step 2 is: using a 0.1% concentration of mercuric chloride solution for disinfection for 12 minutes.

4. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that: The primary culture medium in step three is an MS culture medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.6 mg / L NAA and 0.6 mg / L 6-BA, and the pH of the primary culture medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

5. The method for rapid propagation of hexapod tissue culture according to claim 1, characterized in that: The callus induction medium in step 4 is an MS medium added with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the callus induction medium is adjusted to pH 5.6 by adding 0.1 mg / L NaOH solution.

6. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that: The differentiation and proliferation medium in step 5 is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.15 mg / L NAA and 1 mg / L 6-BA, and the pH of the differentiation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

7. The method for rapid propagation of hexapod tissue culture according to claim 1, characterized in that: The differentiation and proliferation medium in step 5 is an MS medium supplemented with 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA and 2 mg / L 6-BA, and the pH of the proliferation medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

8. The method for rapid propagation of hexapod tissue culture according to claim 1, characterized in that: The rooting medium in step six is ​​a medium containing 30 g / L sucrose, 7 g / L agar and 0.05 mg / L NAA MS, and the pH of the rooting medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution.

9. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that: The treatment methods used for the callus induction culture in step 4 and the adventitious bud differentiation culture in step 5 are both dark treatment.

Images