A method for tissue culture and propagation of Axonopus compressus

Abstract

The present invention discloses a method for tissue culture and propagation of Axonopus compressus, which comprises the following steps: taking the stem segments of Axonopus compressus as explants, placing them in an induction medium for induction culture after disinfection to obtain adventitious buds; transferring the adventitious buds to a proliferation medium for proliferation culture to obtain clustered buds; transferring the clustered buds to a rooting medium for rooting culture to obtain tissue culture seedlings, and transplanting the tissue culture seedlings to a substrate after strengthening and acclimatizing to obtain Axonopus compressus seedlings. By using the technology of the present invention, a large number of Axonopus compressus seedlings can be obtained in a short period, and high-quality seedlings with consistent growth can be provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant cultivation, and particularly relates to a method for tissue culture and propagation of Axonopus compressus (Swartz) Beauv. Background Art

[0002] Axonopus compressus (Swartz) Beauv., also known as Axonopus affinis Chase, is a perennial herbaceous plant of the genus Axonopus in the family Poaceae. It is native to Brazil and Mexico in South America and has now been introduced to many tropical and subtropical countries and regions. Due to its low height, good elasticity, easy reproduction, fast spread and good lawn-forming property, it is also an excellent warm-season lawn grass. It belongs to the warm-season lawn grass in the classification of lawn grasses. In the tropical and subtropical regions of China, Axonopus compressus (Swartz) Beauv. is currently widely used. The stolons of this grass species spread rapidly, and roots and new plants can be produced at each node. The plant body spreads on the ground to form a carpet-like lawn. Axonopus compressus (Swartz) Beauv. is a C4-type positive warm-season lawn grass with strong shade tolerance. In addition to being widely used as a lawn ground cover in gardens, it can also be used for greening and protection in some relatively shady and humid special areas. It also has the advantages of heat resistance, waterlogging tolerance, barren tolerance, heavy metal pollution tolerance and low management requirements. Therefore, it is widely used in public green space lawns, soil and water conservation and highway slope protection lawns. Axonopus compressus (Swartz) Beauv. may form different ecotypes after long-term natural adaptation in different habitats, and there may be ecotypes with better resistance among them. Axonopus compressus (Swartz) Beauv. can be propagated by seeds and also by conventional division methods. However, Axonopus compressus (Swartz) Beauv. is not drought-tolerant and is also infected by viruses and fungi. It is necessary to establish a biotechnology breeding and regeneration system to strengthen the work of biotechnology breeding. So far, there has been no report on propagation by tissue culture technology. Summary of the Invention

[0003] The object of the present invention is to provide a method for tissue culture and propagation of Axonopus compressus (Swartz) Beauv. in view of the above deficiencies.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A method for tissue culture and propagation of Axonopus compressus (Swartz) Beauv. includes the following steps: using the stem segments of Axonopus compressus (Swartz) Beauv. as explants, after disinfection, placing them in an induction medium for induction culture to obtain adventitious buds; transferring the adventitious buds to a proliferation medium for proliferation culture to obtain clustered buds; transferring the clustered buds to a rooting medium for rooting culture to obtain tissue culture seedlings, and transplanting the tissue culture seedlings to a substrate after strengthening and acclimatization to obtain Axonopus compressus (Swartz) Beauv. seedlings.

[0006] Preferably, the disinfection step of the explant is as follows: using the stem segment of Axonopus compressus as the explant, removing the old leaves, rinsing in clean water for 10 min, disinfecting with 75% ethanol for 30 s, then washing three times in sterile water, putting the stem segment into 0.1% mercuric chloride for disinfection for 15 min, and finally washing five times with sterile water, 3 min each time.

[0007] Preferably, the composition of the induction medium is: MS medium + 30 g / L sucrose + 3 g / L phytagel + 1.0 mg / L 6-BA, pH 6.0; the temperature of the induction culture is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s -1 PPFD.

[0008] Preferably, the time of the induction culture is 30 - 50 days.

[0009] Preferably, the composition of the proliferation medium is: MS medium + 30 g / L sucrose + 3 g / L phytagel + 1.0 - 3.0 mg / L 6-BA + 0.01 - 0.1 mg / L NAA, pH 5.8 - 6.0; the conditions of the proliferation culture are: the temperature of the culture is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s -1 PPFD.

[0010] Preferably, the time of the proliferation culture is 30 days.

[0011] Preferably, the rooting medium is MS medium + 30 g / L sucrose + 3 g / L phytagel + IAA or IBA or NAA 0 - 2 mg / L, pH 5.8 - 6.0; the conditions of the rooting culture are: the temperature of the culture is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s -1 PPFD.

[0012] Preferably, the time of the rooting culture is 15 - 30 days.

[0013] Preferably, the composition of the substrate is peat soil, the volume ratio of peat soil to vermiculite = 1:1 or the volume ratio of peat soil to sand = 1:1.

[0014] Preferably, the strong seedling culture lasts for 1 - 2 months and the acclimatization lasts for 2 days.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention uses the stem segments of Axonopus compressus as explants and successfully establishes a multiple shoot and regeneration system. During the process of multiplying multiple shoots with different cytokinins, we found that 6-BA has a better effect on adventitious bud induction than kinetin (KIN). Axonopus compressus can produce a large number of clustered seedlings on the MS medium supplemented with 3.0 mg / L 6-benzyladenine (6-BA) and 0.1 mg / L naphthaleneacetic acid (NAA), and the monthly multiplication coefficient can reach 12.88. Transferring the seedlings to the MS medium without plant growth regulators for 1 to 2 months can induce root formation. The rooted seedlings are then transplanted into the peat soil substrate, and the survival rate can reach 100% after one month. Using the technology of the present invention, a large number of Axonopus compressus seedlings can be obtained in a short period, which can provide high-quality and uniformly growing seedlings.

[0017] The technology of the present invention is simple, affordable, practical, and has high application value, laying a foundation for the large-scale propagation of Axonopus compressus and its biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It shows the proliferation of Axonopus compressus buds cultured in different proliferation media for 30 days. a-d: MS media containing (from left to right) 0.5, 1.0, 2.0, and 3.0 mg / L 6-BA; e-h: MS media containing (from left to right) 0.5, 1.0, 2.0, and 3.0 mg / L KIN; Note: 6 axillary bud bodies proliferate in all media. Scale bar = 1.0 cm.

[0019] Figure 2 It shows the proliferation, plant regeneration, and transplantation of Axonopus compressus buds. a: Proliferation on the MS medium containing 3.0 mg / L 6-BA and 0.1 mg / L NAA; b: Rooting for 12 days on the MS medium containing 0.5 mg / L IBA; c: Transplanting the rooted seedlings into plastic bags filled with peat soil with different substrates (volume ratio) and growing for 60 days. Scale bar = 1.0 cm. DETAILED DESCRIPTION OF THE INVENTION

[0020] The following examples are further illustrations of the present invention rather than limitations thereof.

[0021] Example 1:

[0022] 1. Obtaining and disinfecting the explants:

[0023] The Axonopus compressus materials are from the lawn of the South China Botanical Garden in Guangzhou, China, and are currently stored in the seedling breeding base of the South China Botanical Garden, Chinese Academy of Sciences. Using the stem segments of Axonopus compressus as explants, removing the old leaves, after rinsing in clean water for 10 min, disinfecting with 75% ethanol for 30 s, then washing three times in sterile water, and then putting the stem segments into 0.1% mercuric chloride for 15 min, and finally washing five times in sterile water, 3 min each time.

[0024] 2. Induction of cluster buds:

[0025] Inoculate the sterilized stem segments of Axonopus compressus into the induction medium and carry out induction culture for 30 - 50 days under the conditions of a temperature of 25 ± 1 °C, a light time of 12 h / 12 h L / D, and a light intensity of 80 μmol m -2 s -1 PPFD to obtain adventitious buds. The composition of the induction medium is: MS medium + 30 g / L sucrose + 3 g / L phytagel + 1.0 mg / L 6 - BA, with a pH of 6.0.

[0026] 3. Effect of plant growth regulators on the proliferation effect of cluster buds:

[0027] Set four concentration gradients of 0.5, 1.0, 2.0, and 3.0 mg / L for two cytokinins, 6 - BA and kinetin (KIN), and add them to MS medium + 30 g / L sucrose + 3 g / L phytagel (pH 6.0) for adventitious bud proliferation experiments. Select the most suitable cytokinin type and concentration and add 0.01 or 0.1 mg / L NAA (naphthaleneacetic acid) for adventitious bud proliferation experiments. Use adventitious buds of 2 - 3 cm as explants, with 30 explants for each treatment, and count the number of adventitious buds after 30 days of culture. Culture conditions: temperature of 25 ± 1 °C, light time of 12 h / 12 h L / D, and light intensity of 80 μmol m -2 s -1 PPFD. The results are shown in Table 1 and Figure 1 .

[0028] The results show that 6 - BA has a better effect on inducing adventitious bud proliferation, with uniform overall size and healthy adventitious buds ( Figure 1 a - d). KIN has a secondary effect on inducing adventitious bud proliferation, but the plants are stronger and have a large number of adventitious roots ( Figure 1 e - h). The higher the concentration, the higher the propagation coefficient. After adding a small amount of NAA based on 1.0 mg / L or 3.0 mg / L 6 - BA, it is found that a small amount of NAA can promote adventitious bud proliferation. Among the many experimental groups, the effect of 3.0 mg / L 6 - BA + 0.1 mg / L NAA on inducing adventitious bud proliferation is the best, with healthy plants and a monthly proliferation coefficient of up to 12.88 (Table 1, Figure 2 a).

[0029] Table 1 Effect of plant growth regulators on the induction of cluster buds in Axonopus compressus

[0030]

[0031] 4. Rooting culture:

[0032] After 30 days of proliferative culture of adventitious buds, the clustered buds obtained were cut into 1 - 3 buds and then inoculated into different rooting media (as shown in Table 2, the basic medium was MS medium + 30 g / L sucrose + 3 g / L phytagel, pH 6.0) and cultured for one month. Culture conditions: temperature was 25 ± 1 °C, light time was 12 h / 12 h L / D, and light intensity was 80 μmol m -2 s -1 PPFD. During this period, the rooting situation at the base of the buds was observed. The results showed that Axonopus compressus was relatively easy to root. Whether there was no auxin or different concentrations of auxin, it could induce rooting. Its rooting rate reached over 86% within half a month and 100% within one month (Table 2, Figure 2 b).

[0033] Table 2. Effects of different auxins and concentrations on the rooting of Axonopus compressus

[0034]

[0035]

[0036] 5. Seedling strengthening, acclimatization and transplantation:

[0037] The newly differentiated seedlings were transferred to MS medium without plant growth regulators for seedling strengthening culture for 1 - 2 months. Culture conditions: temperature was 25 ± 1 °C, light time was 12 h / 12 h L / D, and light intensity was 80 μmol m -2 s -1 PPFD. Select the robust tissue - cultured seedlings that had grown to 3 - 5 cm, place them under natural light conditions for acclimatization for 2 days. Carefully wash the agar medium on the roots with tap water, cut off the old leaves at the bottom, and only retain 1 to 3 leaves at the top. Then plant them in the following transplanting substrates respectively: (1) peat soil (100%); (2) peat soil: vermiculite = 1:1 (v:v); (3) peat soil: sand = 1:1 (v:v), (4) yellow clay (100%); (5) yellow clay (100%): peat soil (100%) = 1:1. Water thoroughly. Water once every three days to keep the soil moist. Transplant 40 plants for each substrate, and count the survival rate after 30 days. The results are shown in Table 3 and Figure 2 .

[0038] Table 3. Effects of different substrates on the survival of transplanted Axonopus compressus

[0039]

[0040] The results showed that after culturing the newly differentiated seedlings for 1 month in the MS medium without plant growth regulators for strong seedling cultivation, they could grow to 5-6 cm and could take root. Although there were no obvious growth differences among the various substrates for the surviving Axonopus compressus, most were dark green and a few leaves were yellow. However, from the data, it could be seen that the survival rate of Axonopus compressus seedlings was the highest in peat soil or peat soil:sand (or vermiculite) = 1:1 (v:v), and the lowest in yellow clay (100%) (Table 3). After continuing the management for two months, the small seedlings grew very well on the substrates and some had already grown and spread. Figure 2 c).

[0041] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations on the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and retouches can also be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A method for tissue culture and propagation of Axonopus compressus, characterized in that, It includes the following steps: Using Axonopus compressus stem segments as explants, after disinfection, they are placed in an induction medium for induction culture to obtain adventitious buds; the adventitious buds are transferred to a proliferation medium for proliferation culture to obtain clustered buds; the clustered buds are transferred to a rooting medium for rooting culture to obtain tissue culture seedlings, and the tissue culture seedlings are transplanted into a substrate after strengthening and acclimatization to obtain Axonopus compressus seedlings. The composition of the induction medium is: MS medium + 30 g / L sucrose + 3 g / L phytagel + 1.0 mg / L 6-BA, with a pH of 6.0; the temperature for induction culture is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s - 1 PPFD; The composition of the proliferation medium is: MS medium + 30 g / L sucrose + 3 g / L phytagel + 1.0 - 3.0 mg / L 6-BA + 0.01 - 0.1 mg / L NAA, pH 5.8 - 6.0; the conditions for the proliferation culture are: the culture temperature is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s -1 PPFD; The rooting medium is MS medium + 30 g / L sucrose + 3 g / L phytagel + 0 - 2 mg / L of IAA or IBA or NAA, with a pH of 5.8 - 6.0; the conditions for rooting culture are: the culture temperature is 25 ± 1 °C, the light time is 12 h / 12 h L / D, and the light intensity is 80 μmol m -2 s -1 PPFD; The strong seedling and hardening processes involve culturing the seedlings in an MS medium without plant growth regulators for 1-2 months. The culture conditions are as follows: temperature is 25±1°C, light duration is 12h / 12h L / D, and light intensity is 80μmol m -2 s -1 PPFD. Select healthy tissue culture seedlings that have grown to 3-5 cm and place them under natural light conditions for 2 days of hardening; The composition of the substrate is peat soil, with a volume ratio of peat soil:vermiculite = 1:1 or a volume ratio of peat soil:sand = 1:

1.

2. The method for tissue culture and breeding of Axonopus compressus according to claim 1, characterized in that, The specific steps for disinfecting the explants are as follows: Using Axonopus compressus stem segments as explants, removing the old leaves, rinsing in clean water for 10 minutes, and then disinfecting with 75% ethanol for 30 seconds. Subsequently, wash three times in sterile water, then place the stem segments in 0.1% mercuric chloride for 15 minutes, and finally wash five times with sterile water, 3 minutes each time.

3. The method for tissue culture and propagation of Axonopus compressus according to claim 1, characterized in that, The time for the induction culture is 30 - 50 days.

4. The tissue culture and propagation method of Axonopus compressus according to claim 1, characterized in that, The time for the proliferation culture is 30 days.

5. The method for tissue culture and propagation of Axonopus compressus according to claim 1, characterized in that, The time for the rooting culture is 15 - 30 days.

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