A six-fold rapid propagation method for tissue culture

Abstract

The present invention discloses a method for rapid propagation of six-fold loquat through tissue culture, belonging to the field of plant propagation technology. The present invention uses six-fold loquat seeds as explants, disinfects and sterilizes the explants, and sequentially performs callus induction culture, adventitious bud differentiation culture, proliferation culture, and rooting culture to obtain six-fold loquat seedlings in good condition. The method further screens the disinfection and sterilization methods, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium, and rooting culture medium to obtain optimal culture conditions. This method provides technical support for the tissue culture and propagation of six-fold loquat, and provides a practical and feasible solution for large-scale commercial six-fold loquat breeding, which can meet the needs of batch breeding and planting, significantly improve the propagation speed of six-fold loquat, and has 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 sempervirens 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.

[0003] The flowers of the plant are small, blue or purple, and bloom from April to June. Its small, lovely blooms make it an excellent ornamental plant for gardens. It can be grown in pots or vertically planted in trellises in parks and courtyards. It is also widely used in flower beds and borders. The entire plant is medicinal and has significant benefits, including detoxification, anti-inflammatory, and swelling reduction, as well as for treating carbuncles, boils, and boils. The seeds are small, weighing 0.03g per thousand.

[0004] Traditional sowing methods for Liubeili have several drawbacks, including low germination rates and slow growth. This is primarily due to the extremely small size of Liubeili seeds, with individual seeds smaller than sesame seeds. This makes it difficult to precisely control seed distribution and coverage depth using traditional sowing methods, resulting in low and uneven germination rates. Even with the aid of pelletized seeds, germination rates still depend on multiple factors, including temperature, humidity, and sowing technique, making the process more complex and unpredictable.

[0005] Secondly, there's the issue of environmental dependence. The germination and growth of Liubeili seeds are very sensitive to temperature and light conditions. The optimal germination temperature for Liubeili seeds is typically around 25°C, so sowing is usually done in autumn to avoid the adverse effects of high temperatures on seed germination. Adequate light is also a key factor in promoting seed germination and healthy seedling growth. Traditional sowing methods often struggle to provide uniform and stable environmental conditions, which can lead to variations in germination rates and growth rates, thus affecting the final flowering effect.

[0006] The traditional sowing process is complex to manage, and the management of the six-benefit seedlings requires great care and patience, especially during the transplanting and planting stages. During the transplanting stage, the six-benefit seedlings need to be provided with loose and well-drained soil conditions, and a bright, ventilated and stable growth environment for their growth needs. During the planting stage, they also need to be provided with sufficient nutritional support. In addition, the six-benefit seedlings are also very susceptible to diseases and pests. In the traditional sowing process, the density of the plants is high. Once diseases and pests appear, this dense planting method will quickly aggravate the spread of diseases and pests, affecting the growth quality of the six-benefit seedlings and the later flowering effect.

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

[0008] In response to 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 through tissue culture. By using tissue culture technology, Hexaploidy can be cultured and propagated, 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 solutions adopted by the present invention are as follows:

[0010] A six-fold tissue culture rapid propagation method comprises the following steps:

[0011] Step 1, selecting hexapod seeds as explants;

[0012] Step 2, sterilizing the hexapod explants;

[0013] Step 3: placing the explant on a primary culture medium for culturing to produce primary sterile seedlings;

[0014] 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;

[0015] Step 5: transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture;

[0016] Step 6: transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture;

[0017] Step seven, hardening and transplanting the seedlings after the rooting culture in step six is ​​completed.

[0018] Preferably, the screening process in step 1 is: selecting seeds with full grains and mature, removing dead seeds, diseased seeds and seeds with sclerotia, and then soaking the seeds in warm water to allow them to fully absorb water.

[0019] Preferably, the disinfection process in step 2 is: using a 0.1% concentration of mercuric chloride solution for disinfection for 12 minutes.

[0020] Preferably, the primary culture medium in step 3 is an MS 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 a NaOH solution with a concentration of 0.1 mg / L.

[0021] Preferably, 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.

[0022] 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.

[0023] 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 a NaOH solution with a concentration of 0.1 mg / L.

[0024] Preferably, the rooting medium in step 6 is 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.

[0025] 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.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention uses hexagram seeds as explants, disinfects and sterilizes the explants, and sequentially performs callus induction culture, adventitious bud differentiation culture, proliferation culture and rooting culture to obtain hexagram seedlings with good appearance. The invention also screens the disinfection and sterilization methods, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium and rooting culture medium to obtain optimal culture conditions, thereby providing technical support for the tissue culture and propagation of hexagram, and providing a practical and feasible solution for large-scale commercial hexagram breeding, which can meet the needs of batch breeding and planting, and can also significantly improve the reproduction speed of hexagram, and has high commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is an implementation flow chart of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] The present invention provides a method for rapid propagation of hexaploid tissue culture, the method comprising the following steps:

[0031] Step 1, selecting hexapod seeds as explants;

[0032] Step 2, sterilizing the hexapod explants;

[0033] Step 3: placing the explant on a primary culture medium for culturing to produce primary sterile seedlings;

[0034] 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;

[0035] Step 5: transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture;

[0036] Step 6: transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture;

[0037] Step seven, hardening and transplanting the seedlings after the rooting culture in step six is ​​completed.

[0038] Among them, the disinfection process in step 2 is: use 0.1% concentration of mercuric chloride solution for disinfection for 12 minutes.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The rooting medium in step seven is 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.

[0044] The treatment methods adopted for the callus induction culture in step 4 and the adventitious bud differentiation culture in step 5 are both dark treatment.

[0045] 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.

[0046] The range of culture medium components used in the control experiment in this example is as follows:

[0047] Initial culture medium: MS+NAA0~0.6mg / L+6-BA0~0.6mg / L.

[0048] Callus induction medium: MS+6-BA1~1.5mg / L+NAA0.1~0.15mg / L.

[0049] Differentiation medium: MS+6-BA1~2mg / L+NAA0.1~0.2mg / L.

[0050] Proliferation culture medium: MS+6-BA1~2mg / L+NAA0.1~0.2mg / L.

[0051] Rooting medium: MS+NAA 0.05~0.15mg / L.

[0052] The above culture media are expressed in a way that is easy to visually distinguish. The meaning of the expression refers to the basal culture medium and the different concentrations of hormones added to it. For example, MS represents MS medium, NAA0.6 mg / L represents NAA added at a concentration of 0.6 mg / L, and 6-BA0.6 mg / L represents 6-BA added at 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 approximately 5.6 by adding NaOH solution at a concentration of 0.1 mg / L.

[0053] 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 3000 lx for cultivation.

[0054] First, select the explant. Choose Liubeili seeds as the explant. 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.

[0055] After the explants are selected, the six-fold explants need to be disinfected to establish a sterile seedling system.

[0056] 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 times. A total of four disinfection time lengths were compared, namely: 3min, 6min, 9min, and 12min.

[0057] After the disinfection was completed, the explants were inoculated on MS medium to compare the disinfection effect. Ten bottles of culture medium were selected for each type of disinfection time, and 5 seeds were placed in each bottle of culture 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 fungi, they were immediately cleaned. After 10 days, the total contamination rate of each seed with different disinfection time was calculated. The effect of different disinfection time on Liubeili seeds was recorded to obtain the optimal disinfection time.

[0058] Table 1

[0059] serial number Disinfection time (min) Contamination rate (%) 1 3 60 2 6 30 3 9 30 4 12 20

[0060] The contamination rate in the above table is calculated as: number of contaminated bottles / total number of inoculated bottles × 100%.

[0061] The test results are shown in Table 1. It can be seen that the bacterial growth rate was 60% after 3 minutes. As the disinfection time increased, the contamination rate decreased. At 12 minutes, the contamination rate was under control, and prolonged disinfection did not damage the seeds and affect germination. Therefore, disinfection with 0.1% mercuric chloride solution for 12 minutes was the most effective.

[0062] After determining the optimal disinfection time, the primary culture medium with the optimal hormone ratio is then determined.

[0063] The configuration of the culture medium mainly depends on 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 the 2-factor 3-level orthogonal method for experimental comparison. The results are shown in Table 2.

[0064] Table 2

[0065] Hormone concentration (mg / L) seedling height Growth 6-BA 0+NAA 0 5-6cm It grows rapidly, but it becomes elongated and has thin leaves. 6-BA 0.3+NAA 0 3-5cm Fast growth, leggy growth, and 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, and there is no leggy growth phenomenon 6-BA 0.6+NAA 0.3 2-2.5cm Seeds germinate normally without excessive growth 6-BA 0+NAA 0.6 2-3cm Seeds germinate slightly faster, without excessive growth 6-BA 0.3+NAA 0.6 2.5-3cm Seeds germinate slightly faster, without excessive growth 6-BA 0.6+NAA 0.6 3-4cm The seeds germinate quickly, without any elongation or spindling of the leaves.

[0066] As shown in Table 2, when no hormones were added, the plant grew fastest, but it also had elongated growth and thin leaves.

[0067] 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.

[0068] When only auxin NAA is added without cytokinin, the growth rate becomes faster as the auxin concentration increases; when both hormones are added at the same time, the growth rate of Hexaphyllum accelerates as the cytokinin 6-BA concentration increases, and Hexaphyllum grows fast without becoming too tall.

[0069] Different hormone concentrations have different effects on hexachlorocyclohexane. According to this experiment, it can be seen that under the primary culture medium of MS+NAA0.6mg / L+6-BA0.6mg / L, hexachlorocyclohexane seeds germinate rapidly, the sterile seedlings are strong, and there is no excessive growth.

[0070] 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.

[0071] After the sterile seedling system is established, the stem segments and leaf parts of the six-fold seedlings are cut from the sterile seedlings obtained from the primary culture medium for callus induction and adventitious bud differentiation.

[0072] In the entire process of callus induction and adventitious bud differentiation, there are two ways of induction: dark treatment and light treatment. In addition, the cut parts include stem segments and leaves, so it is necessary to conduct control experiments of dark treatment and light treatment for the two cut parts respectively.

[0073] First, stem segments were selected for callus induction, and control experiments were conducted under two different induction conditions, dark treatment and light treatment, and different hormone concentrations. Table 3 shows the induction effects of different hormone concentrations on six-fold sharp stem segments under dark treatment conditions, and Table 4 shows the induction effects of different hormone concentrations on six-fold sharp stem segments under light treatment conditions.

[0074] The induction rate in the table was calculated as: number of calli / number of inoculated seedlings × 100%.

[0075] Table 3

[0076] 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

[0077] Table 4

[0078] 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

[0079] Table 3 shows that under dark conditions, when the cytokinin 6-BA concentration was 1.0 mg / L, the callus induction rate increased, and even complete callus induction occurred, as the auxin concentration increased. When the cytokinin 6-BA concentration increased to 1.5 mg / L and 2 mg / L, the induction rate of hexachlorobenzene began to decline. The overall induction rate of hexachlorobenzene showed a single peak. Therefore, the highest induction rate, reaching 100%, was achieved with MS, 1 mg / L 6-BA, and 0.15 mg / L NAA.

[0080] Table 4 shows that under light treatment, callus tissue can be induced in stem segments regardless of the hormone concentration, but the induction effect varies with the concentration. The best effect is MS + 6-BA 1.5mg / L + NAA 0.1mg / L, with an induction rate of 90%.

[0081] 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.

[0082] When the leaves were inducing callus tissue, they also showed the phenomenon of leaf whitening.

[0083] The albino rate in the table was calculated as: number of albino plants / number of seedlings inoculated × 100%.

[0084] Table 5

[0085] 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

[0086] Table 6

[0087] 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

[0088] 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. The induction rate of callus induction using six-fold leaves is low, and the effect is not ideal.

[0089] According to Table 6, the leaves treated under light had a lower induction rate and also showed bleaching.

[0090] Based on the above control experiments, it can be considered that stem segments are more suitable than leaves as explants for inducing callus tissue.

[0091] Under dark treatment, the induction rate of MS+6-BA1mg / L+NAA0.15mg / L medium was the highest, with an induction rate of 100%.

[0092] 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%.

[0093] 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.

[0094] 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 sharp stem segments under dark treatment conditions, and Table 8 shows the effects of different hormone concentrations on the differentiation of six-fold sharp stem segments under light treatment conditions.

[0095] The calculation method of the differentiation rate in the table is: differentiation rate = number of adventitious buds / number of inoculated seedlings × 100%.

[0096] Table 7

[0097] 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

[0098] Table 8

[0099] 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

[0100] 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.

[0101] 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 sharp stem segments under dark conditions.

[0102] 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 6-BA concentration. The best differentiation effect is achieved when 6-BA1.5mg / L+NAA0.1mg / L is added to MS medium, with a differentiation rate of 55%.

[0103] 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 hexaphyllous leaves under dark treatment conditions, and Table 10 shows the effects of different hormone concentrations on the differentiation of hexaphyllous leaves under light treatment conditions.

[0104] Table 9

[0105] 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

[0106] Table 10

[0107] 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

[0108] It can be seen from Tables 9 and 10 that under dark treatment conditions, the differentiation of leaves is more difficult than the differentiation of stem segments. Although the effect of using hexachlorobenzene leaves for the differentiation of 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%.

[0109] 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 of them showed albinism.

[0110] Therefore, based on the results of the above experiments, it can be concluded that stem segments are more suitable explants for adventitious bud differentiation than leaves. Under dark conditions, using culture media containing two hormone combinations, MS + 1.0 mg / L 6-BA + 0.15 mg / L NAA and MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA, 100% of stem segments differentiated into adventitious buds and effectively promoted the growth and differentiation of young tissues. This culture medium produced thicker stems, more buds, and optimal proliferation, effectively increasing the number and yield of seedlings in tissue culture, as well as enhancing their quality and growth rate.

[0111] After the differentiation of adventitious buds is completed, it can be subjected to rooting culture. During the rooting culture stage, Liubeili is very easy to take root. Even in MS culture medium without adding hormones, Liubeili can take root quickly. However, after adding a certain amount of NAA, the growth and differentiation of the Liubeili root system 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.

[0112] Table 11

[0113] 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 germinates more and more lateral roots appear NAA 1.2 1-3cm The main root system germinates more and more lateral roots appear NAA 1.5 1-2cm The main root system germinates more and more lateral roots appear

[0114] 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 without having a negative impact on the health of the seedlings.

[0115] 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 germinate more but the length will become shorter, and there will be more lateral roots. The lateral roots 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.

[0116] 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 and plant them in a greenhouse with adjusted temperature. Cultivate them under light conditions. Planting should be carried out within the appropriate temperature range, usually around 20-25℃.

[0117] By comparing the induction of callus tissue and differentiation of adventitious buds by six-fold stem segments and leaves, it was found that the effect of stem segments in both differentiation of adventitious buds and induction of callus tissue was better than that when leaves were used as explants. Stem segments under MS+6-BA1mg / L+NAA0.15mg / L and MS+6-BA2mg / L+NAA0.1mg / L could differentiate into adventitious buds 100%, which was most suitable for stem segment differentiation of adventitious buds and had 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.

[0118] The present invention uses hexapod seeds as explants, disinfects and sterilizes the explants, and sequentially performs callus induction culture, adventitious bud differentiation culture, proliferation culture, and rooting culture. The present invention obtains optimal culture conditions by screening the disinfection and sterilization methods, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium, and rooting culture medium. Experimental results show that when hexapod seeds are used as explants, and the optimal disinfection and sterilization methods, callus culture medium, adventitious bud differentiation culture medium, proliferation culture medium, and rooting culture medium components and ratios are used, the callus induction rate under light treatment is 90%, with 55% of adventitious buds differentiated; and the callus induction rate under dark treatment is 100%, with 100% of adventitious buds differentiated.

[0119] The tissue culture method and the optimal culture medium for each step determined through the above experiments provide technical support for the tissue culture expansion of Liubeili, and provide a practical solution for large-scale commercial Liubeili breeding, which can meet the needs of batch 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 explant on a primary culture medium for culturing to produce primary sterile seedlings; The primary culture medium in step 3 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; 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; 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 pH of the callus induction medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution; Step 5: transplanting the callus cultured in step 4 into a differentiation and proliferation medium for adventitious bud differentiation and proliferation culture; The differentiation and proliferation medium in step 5 includes a differentiation medium and a proliferation medium. The differentiation medium 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 a 0.1 mg / L NaOH solution. The proliferation medium was 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 was adjusted to 5.6 by adding 0.1 mg / L NaOH solution; Step 6: transplanting the seedlings after differentiation and proliferation culture in step 5 into a rooting medium for rooting culture; The rooting medium in step 6 is a medium containing 30 g / L sucrose, 7 g / L agar, and 0.05 mg / L NAAMS, and the pH of the rooting medium is adjusted to 5.6 by adding 0.1 mg / L NaOH solution; Step 7: hardening and transplanting the seedlings after the rooting and cultivation in step 6; Furthermore, the treatment methods adopted for the callus induction culture in step 4 and the adventitious bud differentiation culture in step 5 are both dark treatment.

2. A six-fold tissue culture rapid propagation method according to claim 1, characterized in that, The screening process in step 1 is as follows: 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.

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