Fig two-step subculture tissue culture medium and tissue culture rapid propagation method thereof

Abstract

The invention discloses a fig two-step subculture tissue culture medium and a tissue culture rapid propagation method thereof, and belongs to the technical field of biological tissue culture seedling culture. The invention provides a two-step subculture tissue culture medium which comprises a proliferation medium and a seedling strengthening medium, induction and proliferation are carried out by adopting the proliferation medium containing TDZ, explosive growth of adventitious buds is realized, the proliferation coefficient is improved, cluster buds are transferred into the seedling strengthening medium added with 6-BA and IBA for seedling strengthening culture, internode elongation and stalk thickening are promoted, and the survival rate of seedlings is increased. High-quality tissue culture seedlings which are normal in form and strong in growth are obtained. Meanwhile, the two-step subculture tissue culture seedling method for the figs, provided by the invention, can be used for remarkably improving the breeding efficiency and the survival rate and quality of fig seedlings.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological tissue culture, and particularly relates to a two-step subculture medium for figs and a tissue culture and rapid propagation method thereof. BACKGROUND

[0002] Plant tissue culture technology is a key biological technology for realizing efficient and rapid propagation of forest tree varieties. Figs (Ficus carica L.) are usually propagated by using semi-lignified or tender stem segments as explants. During the induction and proliferation stage of growth regulators, the hormone regulation of synergistic action of cytokinin and auxin is generally adopted; the combination of 6-benzylaminopurine (6-BA) and naphthalene acetic acid (NAA) has no obvious effect on the proliferation of the vigorous fig variety "Jinaofen", and the proliferation coefficient is about 1.9-2.8, the seedling quality is poor, the bud seedlings grow slowly, and it is difficult to meet the demand for efficient and rapid propagation. The proliferation and seedling strengthening are difficult to be considered at the same time in one-time culture, and it is difficult to obtain high proliferation coefficient while ensuring the robustness of the bud seedlings and the elongation of the internodes. At present, there are very limited research reports on the application of thidiazuron (TDZ) in fig tissue culture in China. The existing technical system ignores the potential value of TDZ in inducing fig axillary bud germination and promoting the proliferation of multiple shoots.

[0003] At present, there are few research reports on the inoculation density per bottle, culture conditions and light quality in the subculture process of figs. Usually, the inoculation density is 3-5 per bottle, and the culture efficiency is low. This leads to low utilization efficiency of the culture medium and culture space, and increases the production cost of single seedling. Indole-3-butyric acid (IBA) is often used for rooting in the rooting culture, and the relative effect is better when the IBA concentration is 1.0 mg / L, but the rooting rate is low. For "Jinaofen" fig tissue culture seedlings, there is no clear quantitative standard for the optimal transplanting physiological state (including seedling height, leaf number and root system morphology, etc.), which leads to excessive dependence on personal experience in the domestication and transplanting link, and the survival rate of transplanting and the subsequent growth stability of seedlings are insufficient. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a two-step subculture medium for figs and a tissue culture and seedling raising method, which improves the propagation efficiency, seedling survival rate and quality of vigorous fig varieties such as "Jinaofen" fig with low propagation coefficient.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The application provides a two-step subculture tissue culture medium for figs, which comprises a proliferation culture medium and a strong seedling culture medium; components of the proliferation culture medium comprise: MS culture medium, additional TDZ 0.65-0.85 mg / L, 6-BA 0.05-0.15 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L; components of the strong seedling culture medium comprise: MS culture medium, additional 6-BA 0.5-1.5 mg / L, IBA 0.15-0.25 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L; and the pH value of the proliferation culture medium and the strong seedling culture medium is 5.8-6.0.

[0006] The application provides a two-step subculture tissue culture rapid propagation method for figs, which comprises the following steps: cutting figs with axillary buds and stem segments as explants, disinfecting and pre-treating before inoculation; inoculating the treated explants on a primary culture medium to induce sprouting; inoculating the fig test-tube seedlings cultured in the primary culture on the proliferation culture medium to carry out cluster bud proliferation culture, and then inoculating on the strong seedling culture medium to carry out strong seedling culture; cutting the single bud seedlings cultured in the strong seedling culture to transfer to a rooting culture medium for rooting culture; and acclimatizing and transplanting the rooted tissue culture seedlings.

[0007] Preferably, the axillary bud stem segments are from annual branches of figs.

[0008] Preferably, the disinfecting solution is 1-2% NaClO solution, and the disinfection time is 9-11 min.

[0009] Preferably, components of the primary culture medium comprise: MS culture medium, additional 6-BA 1.0-3.0 mg / L, NAA 0.5-1.5 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L, and the pH value is 5.8-6.0.

[0010] Preferably, the inoculation density in the proliferation culture and the strong seedling culture is 6-8 stem segments per bottle.

[0011] Preferably, components of the rooting culture medium comprise: 1 / 2MS rooting culture medium, additional sucrose 10-30 g / L, agar 5.5-7.5 g / L and NAA 0.2-0.4 mg / L, and the pH value is 5.8-6.0.

[0012] Preferably, when the adventitious buds in the strong seedling culture medium grow to 3-4 cm high, the strong single bud seedlings are selected to transfer to the rooting culture medium.

[0013] Preferably, the conditions of the primary culture, the proliferation culture, the strong seedling culture and the rooting culture all comprise: 14-18 h of light per day, light intensity 2200-2700 lx, temperature 20-26 DEG C, and culture for 25-35 days.

[0014] Preferably, the rooting tissue culture seedlings are selected healthy seedlings, stem height ≥ 6 cm, stem diameter ≥ 1.2 mm, leaf number ≥ 4, root length ≥ 6 cm, and root number ≥ 5, and are transplanted after hardening.

[0015] The present application has the following beneficial effects: In the subculture, the first stage uses the proliferation culture medium containing TDZ for induction and proliferation, and the explosive growth of adventitious buds is successfully realized, and the highest proliferation coefficient can reach 5.72, which is much higher than that of the traditional one-step culture method (such as 6-BA+NAA combination, usually the proliferation coefficient is about 1.9.0-2.8), which greatly improves the yield of unit explant and the propagation speed. In the second stage of subculture, the chrysanthemum buds are transferred to the seedling culture medium added with 6-BA and IBA for seedling culture, which effectively promotes the internode elongation and stem thickening, and obtains high-quality tissue culture seedlings with normal morphology and healthy growth, and the seedling height can reach 29.94 mm after 35 days of inoculation. It lays a solid foundation for subsequent efficient rooting and transplanting survival.

[0016] Further, the two-step subculture seedling method of fig provided by the present application can significantly improve the propagation efficiency, and the survival rate and quality of fig seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Effects of different hormone ratios on the primary culture of 'Jin'aofen' fig tissue culture seedlings. Among them, A1, A2, A3, A4 represent different hormone treatments.

[0018] Figure 2 Effects of different hormone ratios on the subculture of 'Jin'aofen' (proliferation). Among them, A, B, C represent different hormone treatments; numbers 1, 2, 3, 4, 5 represent the 1st to 5th weeks after culture.

[0019] Figure 3 Dynamic effects of different hormone ratios on the subculture of 'Jin'aofen' (proliferation).

[0020] Figure 4 Effects of different hormone ratios on the subculture of 'Jin'aofen' (seedling strengthening). Among them, A, B, C represent different hormone treatments; numbers 1, 2, 3, 4, 5 represent the 1st to 5th weeks after culture.

[0021] Figure 5 Dynamic effects of different hormone ratios on the subculture of 'Jin'aofen' (seedling strengthening).

[0022] Figure 6 Effects of inoculation density on the growth effect of 'Jin'aofen' tissue culture seedlings. Among them, a, b, c, d correspond to 3, 5, 7, and 9 bottles.

[0023] Figure 7 Effects of different temperature and light quality on growth of tissue culture seedlings, wherein a, b and c represent different light and temperature combinations.

[0024] Figure 8 Effects of different hormone ratios on rooting culture of Jinaofen (rooting), wherein a, b and c represent different hormone ratios.

[0025] Figure 9 Growth state of fig tissue culture seedlings before domestication and transplantation, wherein a, b, c and d represent different sizes of transplanted seedlings.

[0026] Figure 10 Growth dynamic of fig tissue culture seedlings five months after domestication and transplantation, wherein a, b, c and d represent different sizes of transplanted seedlings, and the numbers 1, 2, 3, 4 and 5 correspond to the first to fifth months after transplantation.

[0027] Figure 11 Effects of different treatments on dynamic development of leaf and growth of Jinaofen transplanted seedlings, wherein a, b, c and d represent different sizes of transplanted seedlings. DETAILED DESCRIPTION

[0028] The application provides a two-step subculture medium for fig, which comprises a proliferation medium and a seedling strengthening medium; components of the proliferation medium comprise: MS medium, additional TDZ 0.65-0.85 mg / L, 6-BA 0.05-0.15 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L; components of the seedling strengthening medium comprise: MS medium, additional 6-BA 0.5-1.5 mg / L, IBA 0.15-0.25 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L; the pH value of the proliferation medium and the seedling strengthening medium is 5.8-6.0. Preferably, components of the proliferation medium comprise: MS medium, additional TDZ 0.75 mg / L, 6-BA 0.1 mg / L, sucrose 30 g / L and agar 6.5 g / L; components of the seedling strengthening medium comprise: MS medium, additional 6-BA 1.0 mg / L, IBA 0.20 mg / L, sucrose 30 g / L and agar 6.5 g / L. The proliferation medium containing TDZ is used for induction and proliferation, and the explosive growth of adventitious buds of the fig variety 'Jinaofen' which is difficult to proliferate is successfully realized, and the proliferation coefficient is improved. The seedling strengthening medium containing 6-BA and IBA is used for seedling strengthening culture, and the internode elongation and stem thickening are effectively promoted, and high-quality tissue culture seedlings with normal morphology and healthy growth are obtained, and the seedling height is significantly improved.

[0029] The application also provides a two-step subculture tissue culture rapid propagation method of figs, comprising the following steps: cutting figs with axillary buds as explants, disinfecting and pre-treating before inoculation; inoculating the treated explants on a primary culture medium to induce sprouting; inoculating the fig test-tube seedlings cultured in the primary culture on a proliferation culture medium for proliferation culture of cluster buds, and then inoculating on a robust seedling culture medium for robust seedling culture; cutting the single bud seedlings after robust seedling culture and transferring to a rooting culture medium for rooting culture; and acclimatizing and transplanting the rooted tissue culture seedlings.

[0030] In the application, the axillary bud stem segment is from annual branches of figs; the disinfecting solution is 1-2% NaClO solution, and the disinfection time is 9-11 min, preferably the disinfecting solution is 2% NaClO solution, and the disinfection time is 10 min. The disinfection mode set in the application improves the survival rate, reduces the browning rate and the contamination rate.

[0031] In the application, the primary culture medium comprises: MS medium, 6-BA 1.0-3.0 mg / L, NAA 0.5-1.5 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L, and the pH value is 5.8-6.0; preferably the primary culture medium comprises: MS medium, 6-BA 2.0 mg / L, NAA 0.1 mg / L, sucrose 30 g / L and agar 6.5 g / L, and the pH value is 5.8-6.0. The setting of the primary culture medium improves the sprouting initiation rate and the height of new sprouts.

[0032] In the application, the inoculation density in the proliferation culture and robust seedling culture is 6-8 stem segments per bottle, preferably 7 stem segments per bottle. The setting of the inoculation density in the proliferation culture and robust seedling culture in the application guarantees the growth space of single seedlings, greatly improves the output efficiency of unit space, and improves the propagation coefficient and the height of seedlings.

[0033] In the application, the rooting culture medium comprises: 1 / 2MS rooting culture medium, sucrose 10-30 g / L, agar 5.5-7.5 g / L and NAA 0.2-0.4 mg / L, and the pH value is 5.8-6.0; preferably the rooting culture medium comprises: 1 / 2MS rooting culture medium, sucrose 20 g / L, agar 6.5 g / L and NAA 0.3 mg / L, and the pH value is 5.8-6.0. Adding NAA 0.3 mg / L to the 1 / 2MS culture medium has the best effect on inducing figs to root, and improves the rooting rate and the root system quality.

[0034] In the application, when the adventitious buds in the robust seedling culture medium grow to 3-4 cm high, the robust single bud seedlings are selected and transferred to the rooting culture medium.

[0035] In the present application, the conditions of the primary culture, the proliferation culture, the seedling culture and the rooting culture include: 14-18h light per day, 2200-2700lx light intensity, 20-26℃ temperature, and 25-35 days of culture. The culture conditions determined under the LED lamp culture condition improve the proliferation coefficient and the seedling height.

[0036] In the present application, the healthy seedlings with stem height ≥6cm, stem thickness ≥1.2mm, leaf number ≥4, root length ≥6cm and root number ≥5 are selected for the acclimatization and transplanting. The present application first explicitly proposes the stem height ≥6cm, stem thickness ≥1.2mm, leaf number ≥4, root length ≥6cm and root number ≥5 as the objective and measurable physiological indexes of the high-quality transplanting seedlings, completely changes the previous judgment mode depending on subjective experience, makes the transplanting link controllable and predictable, and ensures the survival rate and uniformity of the end link of the factory seedling.

[0037] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0038] In the following examples, the conventional methods are used unless otherwise specified.

[0039] In the following examples, the materials and reagents used are commercially available unless otherwise specified.

[0040] Example 1 1. Explant treatment (plant variety: ‘Jin'aofen’ fig) The natural germination branches of one-year-old branches were used as explant materials. The materials were first pruned, the semi-lignified shoots were cut to 1-2cm stem segments with one axillary bud, the leaves were removed, the leaf stalks were reserved for 0.5cm, then the materials were washed with flowing water to remove the dust and sundries on the surface of the materials, the surface of the explants was washed with a soft hair brush in the washing liquid, the treated materials were moved to a sterile clean bench, the stem segments were placed in a sterile bottle in the clean bench, the bottle was sterilized with 75% alcohol for 30s, then sterilized with 2% NaClO for 10min. During the period, the sterile bottle was shaken, and washed with sterile water for 3 times.

[0041] 2. Primary culture The above treated fig material is cut into 1.5 cm stem segments with one axillary bud using a scalpel, and inoculated into a tissue culture bottle using sterile tweezers, 2-3 plants per bottle. The illumination time is 16 hours per day, the illumination intensity is 2500 lx, the culture temperature is 24±2℃, and the culture time is 30 days. Among them, the initial culture in the tissue culture bottle uses MS medium (Solebo, item number: M8521) as the basic culture medium, the basic culture medium is added with 30 g / L sucrose, 6.5 g / L agar, 2.0 mg / L 6-BA and 1.0 mg / L NAA, and the pH is adjusted to 5.8-6.0.

[0042] 3. Subculture (double-step culture method) The fig test-tube seedlings obtained in the initial culture are transferred to MS medium containing 0.75 mg / L TDZ and 0.1 mg / L 6-BA at a planting density of 7 plants per bottle, and the medium is added with 30 g / L sucrose and 6.5 g / L agar, and the pH is adjusted to 5.8-6.0. The culture conditions are as follows: illumination time, 16 hours of illumination and 8 hours of dark culture; illumination intensity, 2500 lx; temperature, 24±2℃; and culture time, 28-30 days.

[0043] The test-tube seedlings obtained in the above proliferation are transferred to MS medium containing 1.0 mg / L 6-BA and 0.2 mg / L IBA at a planting density of 7 plants per bottle, and the other medium components and culture conditions are the same as in the proliferation stage, and the culture time is 28-30 days.

[0044] 4. Rooting culture When the adventitious buds in the subculture medium grow to a height of 3-4 cm, healthy single bud seedlings are selected, cut off and transferred to 1 / 2MS rooting medium (Solebo, item number: M8527) containing 0.3 mg / L NAA, and the medium is additionally added with 20 g / L sucrose and 6.5 g / L agar, and the pH is adjusted to 5.8-6.0. The culture conditions are as follows: illumination time, 16 hours per day; illumination intensity, 2500 lx; culture temperature, 24±2℃; and culture time, 30 days.

[0045] 5. Hardening and transplanting The rooting tissue culture seedlings with the specifications of healthy, stem height ≥6 cm, stem diameter ≥1.2 mm, leaf number ≥4, root length ≥6 cm and root number ≥5 are placed in a greenhouse for 1-2 weeks of hardening, and then uncovered for one week of acclimatization. At the end of the day, the seedlings are taken out, and the residual culture medium in the root system is washed off with clean water, and then the seedlings are soaked in 1000 times of carbendazim solution for 1-2 minutes for sterilization. Before transplanting, the tissue culture seedlings are placed on wet newspaper and covered with moisture retention. The next day, the seedlings are transplanted into a mixture of grass charcoal and perlite (volume ratio 1:1), and placed in a plastic film-covered greenhouse, and watered twice a day to maintain humidity. After one week, the film is removed, and after 30 days, the survival rate and plant growth are counted.

[0046] Example 2 1. Explant treatment (plant variety: ‘Jin'aofen’ fig) The natural germination branches of one-year-old branches were used as explant materials. The materials were first pruned, and the semi-lignified shoots were cut to 1-2 cm long with one axillary bud, the leaves were removed, and the 0.5 cm petiole was retained. Then the materials were washed with flowing water to remove dust and debris on the surface of the materials, and the surface of the explants was washed with a soft brush in the washing liquid. The treated materials were moved to a sterile clean bench, and the stem segments were placed in a sterile bottle in the clean bench. The stem segments were sterilized with 75% alcohol for 30 seconds, and then sterilized with 1% NaClO for 9 minutes. During the sterilization, the sterile bottle was shaken, and washed with sterile water for 3 times.

[0047] 2. Primary culture The treated fig materials were cut into 1.5 cm long stem segments with one axillary bud using a scalpel, and inoculated into a tissue culture bottle with 2-3 plants per bottle using sterile tweezers. The culture was performed under light for 14 hours per day, with an illumination intensity of 2200 lx and a culture temperature of 24±2℃, for 28 days. The MS medium (Solebao, item number: M8521) was used as the basic medium for primary culture in the tissue culture bottle, with 25 g / L sucrose, 5.5 g / L agar, 1.0 mg / L 6-BA, and 0.05 mg / L NAA, and the pH was adjusted to 5.8-6.0.

[0048] 3. Subculture (two-step culture method) Proliferation stage: the fig test-tube seedlings obtained by primary culture were transferred to a MS medium containing 0.65 mg / L TDZ and 0.05 mg / L 6-BA, with 25 g / L sucrose and 5.5 g / L agar added to the medium, and the pH was adjusted to 5.8-6.0. The culture conditions were as follows: light cycle of 14 h / 10 h, light intensity of 2200 lx, and temperature control at 24±2℃, for 28 days.

[0049] Strong seedling stage: the test-tube seedlings obtained by proliferation were transferred to a MS medium containing 0.5 mg / L 6-BA and 0.15 mg / L IBA, with the same medium components and culture conditions as the proliferation stage, for 28 days.

[0050] 4. Rooting culture When the adventitious buds in the subculture medium grow to 3-4 cm in height, select healthy single bud seedlings, cut them off and transfer them to 1 / 2MS rooting medium (Solebao, item number: M8527) containing NAA 0.2 mg / L, and meanwhile add sucrose 10 g / L and agar 5.5 g / L to the medium, and adjust the pH value to 5.8-6.0. The culture conditions are: light time 14 h / day, light intensity 2200 lx, culture temperature 24±2℃, and culture for 28 days.

[0051] 5. The seedling hardening and transplanting step is the same as in Example 1.

[0052] Example 3 1. Explant treatment (plant variety: Fuyu Ficus carica) The natural germination branch of one-year-old branches is used as the explant material. The material is first subjected to pruning treatment, the semi-lignified tender stems are cut to remove leaves and cut into 1-2 cm stem segments with one axillary bud, the leaves are removed, the leaf stalks are retained for 0.5 cm, then the material is washed with flowing water to remove dust and sundries on the surface of the material, and then the surface of the explant is washed with a soft-bristled brush in a washing liquid. The treated material is moved to a sterile clean bench, the stem segments are placed in a sterile bottle in the clean bench, and then the material is sterilized with 75% alcohol for 30 s, and then sterilized with 3% NaClO for 11 min. During the sterilization, the sterile bottle is shaken, and washed with sterile water for 3 times.

[0053] 2. Primary culture The above treated Ficus carica material is cut into 1.5 cm stem segments with one axillary bud using a scalpel, and then inoculated into a tissue culture bottle using sterile tweezers, 2-3 plants per bottle. The light time is 18 h per day, the light intensity is 2700 lx, the culture temperature is 24±2℃, and the culture is performed for 30 days. Among them, MS medium (Solebao, item number: M8521) is used as the basic medium for primary culture in the tissue culture bottle, sucrose 35 g / L, agar 7.5 g / L, 6-BA 3.0 mg / L and NAA 0.15 mg / L are added to the basic medium, and the pH is adjusted to 5.8-6.0.

[0054] 3. Subculture (two-step culture method) The proliferation stage: the Ficus carica test tube seedlings obtained by primary culture are transferred to MS medium containing TDZ 0.85 mg / L and 6-BA 0.15 mg / L at an inoculation density of 8 plants per bottle, and sucrose 35 g / L and agar 7.5 g / L are added to the medium, and the pH is adjusted to 5.8-6.0. The culture conditions are: light cycle light 18 h and dark 6 h, light intensity 2700 lx, temperature control at 24±2℃, and culture for 35 days.

[0055] Stage of strong seedling: the test tube seedlings obtained by the above proliferation were transferred to MS medium containing 6-BA 1.5 mg / L and IBA 0.25 mg / L at an inoculation density of 8 per bottle, and the other medium components and culture conditions were the same as those in the proliferation stage, and the culture was performed for 35 days.

[0056] 4. Rooting culture When the adventitious buds in the subculture medium grew to 3-4 cm in height, healthy single bud seedlings were selected, cut off and transferred to 1 / 2MS rooting medium (Solabio, item number: M8527) containing NAA 0.4 mg / L, and the medium was additionally supplemented with sucrose 30 g / L and agar 7.5 g / L, and the pH value was adjusted to 5.8-6.0. The culture conditions were as follows: light time 18 h / day, light intensity 2700 lx, culture temperature 24±2℃, and culture for 30 days.

[0057] 5. The seedling hardening and transplanting steps were the same as in Example 1.

[0058] Experimental Example 1 1. Optimization of disinfection time of explants The disinfection time was set to 6 min, 8 min and 10 min, respectively, and the other steps were the same as the explant treatment process in Example 1. After 30 days of culture, the contamination rate, survival rate and browning rate were investigated. It was found that when the disinfection time was 10 min, the survival rate was the highest, reaching 66.67%, the browning rate was the lowest, reaching 9.09%, and the contamination rate reached 24.24%. Considering the contamination rate, browning rate and survival rate, the most suitable disinfection time for fig explants was 10 min.

[0059] 2. Optimization of hormone ratio in primary culture The different hormone ratios are shown in Table 1, and the other steps were the same as the primary culture process in Example 1. After 30 days of culture, the initiation rate and the height of new buds were investigated. Table 1 shows that when the hormone concentration was 6-BA 2.0 mg / L + NAA 1.0 mg / L, the initiation rate reached 65.27%, which was significantly different from other treatments, and the initiation effect was the best. Although the height of new buds was slightly lower, the height and size had already reached the conditions for transfer and multiplication.

[0060] Table 1 Effect of different hormones on primary culture of Jinaofen at 30 days

[0061] 3. Optimization of subculture conditions (1) Optimization of hormone ratio in proliferation stage The different hormone ratios are shown in Table 2, and the other steps were the same as the subculture proliferation stage process in Example 1. Thirty stem segments were inoculated in each treatment, and the experiment was repeated three times. The height of new buds was counted every week after inoculation, and the multiplication coefficient was calculated. The medium ratio most suitable for bud proliferation was screened by continuously counting for 5 weeks. Table 2, Figures 2-3It is shown that, in the first step of proliferation induction stage, the use of medium containing TDZ can effectively promote the explosive growth of adventitious buds; when the hormone ratio is TDZ 0.75 mg / L+6-BA 0.1 mg / L, the proliferation coefficient is the highest, reaching 5.72, which is significantly better than the traditional one-step culture method (such as the proliferation coefficient of 6-BA and NAA combination is usually only 2.0-3.0), greatly improving the yield of seedlings per explant and the speed of propagation.

[0062] At the same time, the results also show that different plant growth regulator ratios have a significant effect on the proliferation effect of fig tissue culture seedlings. Under the condition of fixed 6-BA concentration (0.1 mg / L), with the increase of TDZ concentration (0.5, 0.75, 1.0 mg / L), the proliferation coefficient shows a trend of first rising and then falling. Among them, the proliferation effect is the best when the TDZ concentration is 0.75 mg / L, and the proliferation coefficient reaches 5.72; when the TDZ concentration rises to 1.0 mg / L, the proliferation coefficient decreases significantly.

[0063] Table 2 Influence of different hormone ratios on the subculture of Jinaofen on the 35th day (proliferation)

[0064] (2) Optimization of hormone ratio in the seedling stage Different hormone ratios are shown in Table 3, and the rest of the steps are the same as in Example 1. Each treatment is inoculated with 30 stem segments, repeated 3 times, and the height of new buds is measured every week after inoculation, continuously measured for 5 weeks, to evaluate the seedling effect and select the most suitable medium. Table 3 and Figures 4-5 It is shown that, in the second step (proliferation and seedling stage), the cluster buds are transferred to the medium added with 6-BA and IBA for seedling culture, which effectively promotes the internode elongation and stem thickening, and obtains high-quality tissue culture seedlings with normal morphology and healthy growth. When the hormone concentration is 6-BA 1.0 mg / L+IBA 0.2 mg / L, the seedling height can reach 29.94 mm, which performs the best, effectively solving the problems of bud cluster, malformation and glassification caused by single TDZ culture, and laying a solid foundation for subsequent efficient rooting and transplanting survival.

[0065] Table 3 Influence of different hormone ratios on the subculture of Jinaofen on the 35th day (seedling)

[0066] Based on the above proliferation stage and seedling stage condition optimization results, it is recommended to use two-step culture method: first, the stem segments are inoculated in the induction medium containing TDZ to promote the occurrence of a large number of adventitious buds, and then the shoots are transferred to the seedling medium containing 6-BA 1.0 mg / L and IBA 0.2 mg / L to promote the elongation of internodes and the growth of seedling height. Under this optimized condition, the proliferation coefficient is 3.70, and the seedling height is 29.94 mm, showing that the tissue culture seedlings have a strong morphology and good growth.

[0067] (3) Optimization of inoculation density in the proliferation and seedling stage Different inoculation densities are shown in Table 4, and the rest of the steps are the same as in Example 1 in the process of subculture and proliferation and seedling stage. 90 stem segments were inoculated for each treatment, and repeated 3 times. After 30 days, the height of the new shoots was counted, and the proliferation coefficient was calculated. Table 4 and Figure 6 It is shown that the inoculation density has a significant effect on the growth of tissue culture seedlings. When the inoculation density is 7 per bottle, the proliferation coefficient is the highest (3.30), and the height of the new shoots is also the largest (25.09 mm), showing that the plant growth state is optimal under this density condition.

[0068] Table 4 Effect of inoculation density on the growth of Jinaofen tissue culture seedlings on the 30th day

[0069] (4) Optimization of different light periods and temperature combinations in the proliferation and seedling stage Different temperature light periods are shown in Table 5, and the rest of the steps are the same as in Example 1 in the process of subculture and proliferation and seedling stage. 90 stem segments were inoculated for each treatment, and repeated 3 times. After 30 days of culture, the height of the new shoots was counted and the proliferation coefficient was calculated. Table 5 and Figure 7 It is shown that different light and temperature combinations have a significant effect on the growth of tissue culture seedlings. In the c treatment condition (light period 16h / 8h, temperature 24±2℃), the proliferation coefficient of tissue culture seedlings is the highest (3.30), and the height of the new shoots is also the largest (26.09 mm), indicating that this light and temperature combination is most beneficial to the growth of tissue culture seedlings.

[0070] Table 5 Effect of different temperature light periods on the growth of tissue culture seedlings on the 30th day

[0071] 4. Optimization of plant growth regulator ratio in rooting culture Different plant growth regulator ratios are shown in Table 6, and the rest of the steps are the same as in Example 1 in the process of rooting culture stage. After 30 days of culture, the rooting rate and average root number were counted. Table 6 and Figure 8The results showed that different plant growth regulator combinations had significant effects on rooting of F. communis plantlets. Among them, the NAA concentration of 0.3 mg / L (treatment a) was the best, with an average of 4.09 roots and a rooting rate of 90.00%, which were significantly higher than those of other treatments, and the rooting condition was the best, with fast root initiation, rich root quantity, developed fibrous roots, and thick main roots.

[0072] Table 6 Effects of different hormone combinations on rooting culture of Jiaofen

[0073] 5. Results of seedling hardening and transplanting The rooting plantlets of different sizes and growth states are shown in Table 7 and their corresponding morphologies are shown in Figure 9 The rest of the steps were the same as the seedling hardening and transplanting process of Example 1. After 30 days, the survival rate and plant growth of each treatment were counted, and the results are shown in Table 8. After further cultivation, the growth dynamics of F. communis plantlets five months after domestication and transplanting were counted, and the results are shown in Figure 10 and Figure 11 .

[0074] Table 7 Growth state and classification of F. communis plantlets before domestication and transplanting

[0075] Table 8 Effects of different categories on F. communis plantlets on the 30th day

[0076] According to Table 8, different categories of fig tissue culture seedlings have significant differences in various growth indicators after 30 days of transplanting. Among them, treatment a performs best in survival rate and all growth indicators, while treatment d performs worst. Specifically, the survival rate of treatment a is as high as 93.47%, which is significantly higher than that of other treatments. With the category decreasing from a to d, the survival rate shows a significant downward trend, and the survival rates of treatments b, c and d are 80.95%, 72.27% and 44.47% respectively. In terms of plant height, treatment a reaches 86.12 mm, which is significantly better than other categories; there is no significant difference between treatments b and c, and between treatments c and d, but treatment b is significantly higher than treatments c and d, and treatment d has the lowest plant height of only 40.74 mm. The stem thickness shows relatively small differences among the treatments, only treatment a (1.71 mm) is significantly higher than treatment d (1.49 mm), indicating that this indicator is less affected by the category of seedlings. The leaf-related indicators (leaf amount, leaf length, leaf width) show a consistent trend: a > b > c > d, and treatment a is significantly the highest in all leaf indicators. In summary, the a category of tissue culture seedlings with standard initial specifications (i.e. healthy, stem height ≥ 6 cm, stem thickness ≥ 1.2 mm, leaf number ≥ 4, root length ≥ 6 cm, root number ≥ 5) is the key to successfully cultivating fig virus-free seedlings, this specification can achieve the highest transplanting survival rate and the best comprehensive growth traits, and also provides a reliable guarantee for the optimal nutrient growth of plants in the later stage, which can be used as the preferred category for large-scale production of fig tissue culture seedlings.

[0077] According to Figure 10 and 11 different categories of tissue culture seedlings have a significant impact on growth indicators (including leaf width, leaf number, leaf length, plant height and stem thickness) after transplanting. In terms of leaf traits, by 90 days after transplanting, the leaf width, leaf length and leaf number of treatment a are significantly higher than those of other treatments, among which the leaf length is 145.80 mm, the leaf width is 137.61 mm, and the leaf number is 11.16. In terms of plant height, treatment a still performs best at 90 days, which is significantly higher than the rest of the treatments, with a plant height of 222.33 mm. At the same time, treatment a is also significantly better than other treatments in stem thickness, reaching 4.18 mm at 90 days. Based on the analysis of various growth indicators, treatment a (healthy, stem height ≥ 6 cm, stem thickness ≥ 1.2 mm, leaf number ≥ 4, root length ≥ 6 cm, root number ≥ 5) shows the best comprehensive growth traits, which can be used as the best choice for large-scale cultivation and transplanting of fig virus-free seedlings.

[0078] The above description is only a preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. A two-step subculture culture medium for figs, characterized in that, Including proliferation culture medium and seedling strengthening culture medium; The proliferation medium components include: MS medium supplemented with TDZ 0.65-0.85 mg / L, 6-BA 0.05-0.15 mg / L, sucrose 25-35 g / L and agar 5.5-7.5 g / L; The seedling growth medium consists of: MS medium supplemented with 0.5-1.5 mg / L 6-BA, 0.15-0.25 mg / L IBA, 25-35 g / L sucrose and 5.5-7.5 g / L agar; The pH value of the proliferation medium and the seedling strengthening medium is 5.8-6.

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2. A two-step rapid propagation method for figs via tissue culture, characterized in that, Includes the following steps: Fig stem segments with axillary buds were cut as explants and disinfected and treated before inoculation. The treated explants were inoculated onto primary culture medium to induce shoot growth; The first-generation cultured fig test-tube seedlings were inoculated onto the proliferation medium described in claim 1 for bud proliferation culture, and then inoculated onto the seedling strengthening medium described in claim 1 for seedling strengthening culture; After cutting off strong seedlings and culturing individual buds, they were transferred to rooting medium for rooting culture. Harden the rooted tissue culture seedlings and transplant them.

3. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The axillary bud stem segments are derived from one-year-old branches of fig trees.

4. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The disinfectant used for disinfection is a 1-2% NaClO solution, and the disinfection time is 9-11 minutes.

5. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The primary culture medium components include: MS medium supplemented with 1.0-3.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 25-35 g / L sucrose and 5.5-7.5 g / L agar, with a pH of 5.8-6.

0.

6. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The inoculation density for the propagation culture and seedling culture is 6-8 stem segments per bottle.

7. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The rooting medium comprises: 1 / 2 MS rooting medium, supplemented with 10-30 g / L sucrose, 5.5-7.5 g / L agar and 0.2-0.4 mg / L NAA, with a pH of 5.8-6.

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8. The two-step subculture rapid propagation method as described in claim 2, characterized in that, When the adventitious buds in the seedling culture medium grow to a height of 3-4 cm, select healthy single seedlings and transfer them to the rooting culture medium.

9. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The conditions for the initial culture, proliferation culture, seedling culture and rooting culture all include: 14-18 hours of light per day, 2200-2700 lx light intensity, 20-26℃ temperature, and 25-35 days of culture.

10. The two-step subculture rapid propagation method as described in claim 2, characterized in that, The rooted tissue culture seedlings were selected from healthy seedlings with stem height ≥6cm, stem diameter ≥1.2mm, number of leaves ≥4, root length ≥6cm, and root coefficient ≥5, and were hardened off and transplanted.

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