A method for tissue propagation of rubber grass

Abstract

The application discloses a tissue propagation method of rubber grass. The method comprises the following steps: 1) sterilizing the surface of rubber grass seeds, putting the seeds into a germination culture medium, and obtaining sterile seedlings of the rubber grass; 2) cutting the leaf blades of the sterile seedlings obtained in the step 1) into leaf discs with a length and width of about 0.5-1 cm, and connecting the leaf discs into a bud induction culture medium to induce adventitious buds; 3) picking the adventitious buds obtained in the step 2) and connecting the adventitious buds into a rooting culture medium to induce rooting; and 4) moving the adventitious buds with a root length of 2-5 cm obtained in the step 3) into a culture bottle to continue growing, and obtaining rubber grass seedlings. Compared with other asexual propagation methods, the method is suitable for rapid propagation of the rubber grass, has the characteristics of short propagation period and easy-to-obtain material, and the seedlings obtained through the tissue propagation method have the characteristics of consistent genetic background, so that the stable inheritance of excellent properties of the rubber grass is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture, a tissue propagation method of Taraxacum koksaghyz. BACKGROUND

[0002] Taraxacum koksaghyz, also known as Russian dandelion, belongs to the family of Compositae and the genus of Taraxacum. It was first discovered in the valley of the Tianshan Mountains in the southeast of Kazakhstan in 1931 (Kirschner J, et al. Available ex situ germplasm of the potential rubber crop taraxacum koksaghyz belongs to a poor rubber producer, t. brevicorniculatum (Compositae-Crepidinae). Genetic Resources & Crop Evolution, 2013). Different from other plants of the genus Taraxacum, such as medicinal dandelion (Taraxacum officinale) and short horn dandelion (Taraxacum brevicorniculatum), Taraxacum koksaghyz has a narrow geographical distribution, and the latex contains a high content of natural rubber, which is a specialized plant of the genus Taraxacum. However, due to insufficient understanding, there were a large number of studies on short horn dandelion mistaken for Taraxacum koksaghyz in the early stage. Jan, et al. Available ex situ germplasm of the potential rubber crop taraxacum koksaghyz belongs to a poor rubber producer, t. brevicorniculatum (Compositae-Crepidinae). Genetic Resources & Crop Evolution, 2013). Different from other plants of the genus Taraxacum, such as medicinal dandelion (Taraxacum officinale) and short horn dandelion (Taraxacum brevicorniculatum), Taraxacum koksaghyz has a narrow geographical distribution, and the latex contains a high content of natural rubber, which is a specialized plant of the genus Taraxacum. However, due to insufficient understanding, there were a large number of studies on short horn dandelion mistaken for Taraxacum koksaghyz in the early stage.

[0003] Natural rubber is an important industrial raw material, which is mainly derived from Hevea brasiliensis. In recent years, due to the slow progress of high-yield breeding of rubber trees, rubber allergy of rubber trees, single source of rubber production and other problems (Krotkov G. A review of literature on Taraxacum koksaghyz Rod. Botanical Review, 1945, 11 (8): 417-461), the development of other rubber-producing plants is very important. Rubber grass can synthesize natural rubber of the same high quality as rubber tree, so it has become one of the important alternative sources of commercial natural rubber (van Beilen JB, Poirier Y. Guayule and Russian dandelion as alternative sources of natural rubber. Critical reviews in biotechnology, 2007, 27 (4): 217-231). As a wild resource, rubber grass has not undergone long-term artificial selection and domestication, and the rubber content is not as high as that of Hevea brasiliensis. However, as a herbaceous plant, it has the advantages of simple genetic transformation, short growth cycle and the like. Research on it helps to deepen our understanding of the rubber production mechanism and provides guidance for high-yield breeding.

[0004] Rubber grass is an allogamous plant with self-incompatibility. Generally, self-pollination cannot produce fertile seeds, and hybridization is needed to propagate offspring (Krotkov G. A review of literature on Taraxacum koksaghyz Rod. Botanical Review, 1945, 11 (8): 417-461.). However, plants obtained by seed propagation through hybridization have different genetic backgrounds, resulting in differences in phenotype, rubber content and the like. Establishing a stable rubber grass rapid propagation system not only can preserve rubber grass germplasm resources, fix excellent traits, but also can obtain genotypically consistent strains for various experiments, and also provides a reference for the construction of a rubber grass genetic transformation system, but there is still a lack of research in this regard. SUMMARY

[0005] The purpose of the present application is to provide a rubber grass tissue culture method.

[0006] The rubber grass propagation method provided by the present application comprises the following steps:

[0007] 1) Germination culture: after disinfecting the rubber grass seeds, inoculate the germination culture medium to obtain rubber grass sterile seedlings;

[0008] 2) bud induction culture: taking the leaves of the rubber grass aseptic seedlings obtained in step 1) which are in good growth state, free of spots and browning, cutting into leaf discs with length and width of 0.5-1 cm, and then placing the leaf discs into a bud induction culture medium for induction culture of adventitious buds;

[0009] 3) rooting induction culture: placing the adventitious buds obtained in step 2) into a rooting culture medium for rooting induction culture, and obtaining the adventitious buds after rooting;

[0010] 4) obtaining of rubber grass seedlings: placing the adventitious buds with a root length of 2-5 cm obtained in step 3) into a culture bottle containing the rooting culture medium for further growth, and obtaining rubber grass seedlings; the germination culture medium is a solid culture medium prepared by adding 30 g of sucrose and 2.5 g of gel into a basic 1 / 2MS medium and adjusting the pH to 6.1;

[0011] The bud induction culture medium is a solid culture medium prepared by adding plant growth regulators and 30 g / L of sucrose into a MS medium; the plant growth regulator adding components are 0.1-1 mg / L of 6-benzyladenine (6-BA) and 0.1-0.8 mg / L of indole acetic acid (IAA), or 0.1-1 mg / L of kinetin (KT) and 0.1-0.8 mg / L of IAA;

[0012] The plant growth regulator adding components in the bud induction culture are preferably 0.8 mg / L of KT and 0.2 mg / L of IAA.

[0013] The rooting culture medium is a solid culture medium prepared by adding 30 g / L of sucrose and 2.5 g of gel into a basic 1 / 2MS medium and adjusting the pH to 5.8.

[0014] In the above method, the germination culture medium and the bud induction culture medium are cultured in 90x20 mm culture dishes; the adventitious bud rooting culture is first cultured in a 90x20 mm culture dish, and then the adventitious buds after rooting are transferred into a 500 mL transparent tissue culture bottle for culture. All the bud induction culture and the rooting induction culture are cultured under the conditions of 24-25 °C, 60-70% humidity, 16 h / 8 h light / dark, and light intensity of 60-80 μmol·m -2 ·s -1 .

[0015] In the method, the bud induction culture is replaced with fresh culture medium every 10-14 days, and the whole induction period is 30-40 days; the rooting induction culture is replaced with fresh culture medium every 7-10 days, and the rooting induction culture period is 15-20 days.

[0016] In step 1), the method for sterilizing the seeds of the rubber grass is to surface-sterilize the full rubber grass seeds with 70% ethanol for 2 minutes, soak the seeds in 0.25% sodium hypochlorite and 0.1% sodium dodecyl sulfate for 10 minutes, and then rinse the surface of the seeds with sterile water for 5 times.

[0017] In step 1), after the seeds of the rubber grass are sterilized, the seeds are inoculated on a germination medium; after 4 weeks, the obtained aseptic seedlings are transplanted into fresh germination medium, and are continuously cultured for 12 weeks to obtain aseptic seedlings.

[0018] In step 4), the adventitious buds with 2-5 cm root length are transplanted into a culture bottle containing a rooting medium, and are continuously grown for 1-3 months to obtain rubber grass seedlings.

[0019] In the method, the rubber grass seedlings after the rooting culture are transplanted; peat soil (PINDSTRUP, Denmark, 0-6 mm), vermiculite and coconut coir soil are mixed in a volume ratio of 2:1:1 to obtain a culture medium, the culture medium is poured with water, the rubber grass seedlings obtained in step 4) are transplanted into the culture medium, a transparent cover is covered, and the seedlings are cultured under the conditions of a temperature of 24-25°C, a humidity of 60-70%, a light intensity of 60-80 μmol·m -2 ·s -1 After 7 days, 5-7 air holes are punched on the transparent cover, and the seedlings are cultured for 3-5 days to adapt to the external temperature, humidity and light conditions, and finally the transparent cover is removed to restore the normal growth of the plants. The plants are watered every 10 days.

[0020] Compared with other asexual reproduction methods, the method has the advantages of controllable environmental conditions, small error, fast growth, short cycle, high induction rate and survival rate, and single source of tissue culture materials, which is beneficial to mass production, obtaining rubber grass plants with consistent genetic background, ensuring the breeding of rubber grass plants with excellent varieties, and ensuring the stable genetic characteristics of the excellent properties of the rubber grass (ensuring the stable genetic characteristics of the excellent properties of the rubber grass). BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Photos of the rubber grass seed germination culture process;

[0022] Figure 2 Photos of the explants in the bud induction culture process;

[0023] Note: Left picture: KT / IAA combination; right picture: 6-BA / IAA combination;

[0024] Figure 3 Photos of the buds differentiated in the bud induction culture process;

[0025] Note: Left picture: KT / IAA combination; right picture: 6-BA / IAA combination;

[0026] Figure 4 Fig. 2. Photos of adventitious buds in rooting culture; Note: Left photo: KT / IAA combination; Right photo: 6-BA / IAA combination;

[0027] Figure 5 Fig. 3. Photos of rubber plantlet in rooting culture;

[0028] Note: Left photo: KT / IAA combination; Right photo: 6-BA / IAA combination;

[0029] Figure 6 Fig. 4. Photos of rubber plantlet survival after transplanting. DETAILED DESCRIPTION

[0030] The following methods in tissue culture are conventional methods unless otherwise specified.

[0031] Example 1, Tissue culture of rubber plant

[0032] 1. Seed sterilization and germination culture

[0033] 1) Seed selection: Select well-grown and full rubber seeds;

[0034] 2) Seed sterilization method: Sterilize full rubber seeds on the surface with 70% ethanol for 2 min, soak them in 0.25% sodium hypochlorite and 0.1% sodium dodecyl sulfate for 10 min, then rinse the seed surface with sterile water for 5 times, inoculate them on the germination culture medium; After four weeks, the obtained sterile seedlings are moved to the bottle germination culture medium and cultured for 12 weeks to obtain sterile seedlings.

[0035] The germination medium is a solid medium obtained by adding 30 g sucrose and 2.5 g gelatin to 1 / 2MS medium, adjusting pH to 5.8; the 1 / 2MS medium is MS powder from Phytotech, USA, and 1 L of medium uses 2.2 g of the powder. The main components are: macroelements: potassium nitrate (KNO3) 850 mg / L, ammonium nitrate (NH4NO3) 825 mg / L, magnesium sulfate heptahydrate (MgSO4.7H2O) 185 mg / L, potassium dihydrogen phosphate (KH2PO4) 85 mg / L, calcium chloride dihydrate (CaCl2.2H2O) 220 mg / L; microelements: potassium iodide (KI) 0.415 mg / L, boric acid (H3BO3) 3.1 mg / L, manganese sulfate tetrahydrate (MnSO4.4H2O) 11.15 mg / L, zinc sulfate heptahydrate (ZnSO4.7H2O) 4.3 mg / L, sodium molybdate dihydrate (NaMoO4.2H2O) 0.125 mg / L, copper sulfate pentahydrate (CuSO4.5H2O) 0.0125 mg / L, cobalt chloride hexahydrate (CoCl2.6H2O) 0.0125 mg / L, iron salt disodium ethylenediaminetetraacetate (Na2-EDTA) 18.65 mg / L, ferrous sulfate tetrahydrate (FeSO4.4H2O) 13.9 mg / L; organic components: myo-inositol 50 mg / L, glycine 1 mg / L, thiamine hydrochloride 0.05 mg / L, pyridoxine hydrochloride 0.25 mg / L, nicotinic acid 0.25 mg / L.

[0036] 2. Tissue culture of rubber grass

[0037] 1) Bud induction culture

[0038] The leaves of the aseptic seedlings obtained in step 1 are used as explants, and leaves with no spots on the surface, tender green color, and good growth state are selected and cut into small pieces with a length and width of 0.5-1 cm; the pieces are placed in bud induction medium (90x20 mm culture dish) with the front side facing up, and cultured under the conditions of a temperature of 24-25°C, a humidity of 60-70%, 16h / 8h light / dark, and a light intensity of 60-80 μmol·m -2 ·s -1 The bud induction medium is replaced every 10-14 days until buds are differentiated.

[0039] The present application shows through a large number of experiments that the basic medium suitable for bud differentiation induction of rubber grass is MS medium. The MS medium selected by the laboratory is the MS powder of the United States Phytotech, and the use amount of 1L medium is 4.4g. The main components are: macroelements: potassium nitrate (KNO3) 1900mg / L, ammonium nitrate (NH4NO3) 1650mg / L, magnesium sulfate heptahydrate (MgSO4.7H2O) 370mg / L, potassium dihydrogen phosphate (KH2PO4) 170mg / L, calcium chloride dihydrate (CaCl.2H2O) 440mg / L, microelements: potassium iodide (KI) 0.83mg / L, boric acid (H3BO3) 6.2mg / L, manganese sulfate tetrahydrate (MnSO4.4H2O) 22.3mg / L, zinc sulfate heptahydrate (ZnSO4.7H2O) 8.6mg / L, sodium molybdate dihydrate (NaMoO4.2H2O) 0.25mg / L, copper sulfate pentahydrate (CuSO4.5H2O) 0.025mg / L, cobalt chloride hexahydrate (CoCl2.6H2O) 0.025mg / L, iron salt disodium ethylenediaminetetraacetate (Na2-EDTA) 37.3mg / L, ferrous sulfate tetrahydrate (FeSO4.4H2O) 27.8mg / L, organic components: myo-inositol 100mg / L, glycine 2mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, nicotinic acid 0.5mg / L.

[0040] The bud induction medium is based on the MS medium, 30g / L sucrose and 2.5g / L gel are added, the PH is adjusted to 5.8, and the medium is sterilized at 101kPa, 121℃ high temperature and high pressure for 20min, and then cooled to 50-60℃, and then plant growth regulators are added, and then poured into 90*20mm culture dishes to obtain a solid culture medium.

[0041] In order to obtain an excellent culture medium, the present application carries out a large number of screening on the induction hormone of the bud induction medium, finally uses the combination of 6-BA and IAA and KT and IAA, on this basis, designs different concentration gradient experiments, and the leaf discs are inserted into the bud induction medium added with different hormone concentrations, and the bud differentiation rate after 28 days of culture is counted, and the statistical results are shown in Tables 1 and 2.

[0042] Table 1. Bud differentiation rate of rubber grass leaf discs treated by KT and IAA

[0043]

[0044] Table 2. Bud differentiation rate of rubber grass leaf discs treated by 6BA and IAA

[0045]

[0046] The results showed that after 10-15 days of bud induction culture, buds began to emerge from the leaf disc edge, and adventitious buds grew from these buds after 20-30 days. As shown in Table 1, among the 6-BA and IAA treatments, 0.1 mg / L 6-BA and 0.1 mg / L IAA induced the highest budding rate, at 391.87%. As shown in Table 2, among the KT and IAA concentration gradients, 0.8 mg / L KT and 0.2 mg / L IAA induced the highest budding rate, at 1219.64% (budding rate = number of differentiated adventitious buds / total inoculation count × 100%). Photos of the bud induction culture process are shown below. Figure 2 , Figure 3 As shown, Figure 2 The growth status of explants on the day of bud induction culture. Figure 3 Photographs showing the growth of adventitious shoots 28 days after explant differentiation induction. Comparison of shoot emergence after treatment of leaf discs with two different hormone combinations revealed that vitrification of rubber grass explants was significantly less in the KT / IAA combination experiment compared to the 6-BA / IAA combination.

[0047] Based on the above results, the optimal bud induction medium was determined through experimental screening as follows: MS medium was used as the basal medium, with the addition of 30 g / L sucrose and 2.5 g / L gel, the pH was adjusted to 5.8, and the medium was sterilized under high temperature and high pressure conditions of 101 kPa and 121℃ for 20 min. After cooling to 50-60℃, plant growth regulators were added, and the mixture was poured into 90×20 mm petri dishes to obtain a solid medium. The plant growth regulators were 0.8 mg / L KT and 0.2 mg / L IAA.

[0048] 2) Rooting culture

[0049] When the adventitious shoots induced from the explants in step 1) grow to about 1-2 cm, break them off and transfer them to rooting medium. Inoculate 15 adventitious shoots into each rooting medium. Maintain a temperature of 24-25℃, humidity of 60-70%, and a 16h / 8h light / dark cycle with a light intensity of 60-80 μmol·m⁻². -2 ·s -1 Cultivate under the specified conditions, changing the rooting medium every 7-10 days until adventitious buds root. Figure 4 Transplant adventitious buds with roots of about 2-5 cm into 500 mL culture bottles containing 65-70 mL of rooting medium, allowing the adventitious buds to continue growing into rubber grass seedlings. Figure 5 ).

[0050] Rooting medium: Weigh 2.2g MS basal medium (M519), 30g / L sucrose, 2.5g / L gel, adjust the pH to 5.8, sterilize at 101kPa and 121℃ for 20min, cool to 50-60℃, and pour into a 90×20mm petri dish or 500mL culture flask.

[0051] The results of rooting culture showed that adventitious buds began to root after 10-14 days of culture, and all adventitious buds could root after 15-20 days of culture, with a rooting rate of 98% (rooting rate = number of rooted adventitious buds / total number of adventitious buds × 100%).

[0052] 3. Transplanting rubber grass seedlings

[0053] The rubber grass adventitious buds with roots of about 2-5 cm obtained in step 2 are grown in culture bottles containing rooting medium for 1-3 months to obtain rubber grass seedlings, which are then ready for transplanting. First, the culture bottles are placed in the following growing conditions: temperature 24-25℃, humidity 60-70%, light intensity 60-80 μmol·m⁻². -2 ·s -1 The seedlings were placed in a light-cured culture room. The caps of the culture bottles were loosened, and after 5 days of culture, the seedlings were removed. The roots of the rubber grass seedlings were gently rinsed with distilled water to remove excess culture medium before transplanting. Figure 6 ).

[0054] The specific transplanting method is as follows:

[0055] Mix peat moss (Pindstrup, 0-6mm from Denmark), vermiculite, and coconut coir in a volume ratio of 2:1:1 to obtain a culture medium. Water the medium thoroughly, then transfer the treated seedlings into it. Cover with a transparent cover and maintain the humidity for 7 days. After 7 days, make 5-7 holes in the transparent cover to gradually expose the rubber grass seedlings to outside air. Cultivate for 3-5 days, then remove the transparent cover and allow the plants to resume normal growth. Growth conditions: Temperature 24-25℃, humidity 60-70%, light intensity 60-80 μmol·m². -2 ·s -1 Water the soil thoroughly again on the 5th-7th day of plant growth. The rubber grass will fully recover its growth in about 7-10 days. Maintain the seedlings at a cultivation temperature of 24-25℃, humidity of 60-70%, and light intensity of 60-80 μmol·m⁻². -2 ·s -1 Continue growth and cultivation, with a survival rate of 90% (survival rate = number of surviving seedlings / total number of transplanted rooted seedlings × 100%). Water thoroughly every 10 days. Figure 6 This shows the growth status 30 days after transplanting.

[0056] Although the present invention has been described in detail above from three aspects: general description, specific implementation, and experiments, some modifications or improvements can be made based on the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for tissue propagation of rubber grass, comprising the following steps: 1) Germination culture: After sterilizing the rubber grass seeds, inoculate them with germination medium to cultivate sterile rubber grass seedlings; 2) Bud induction culture: Take leaves of the sterile rubber grass seedlings obtained in step 1) that are in good growth condition, without spots or browning, cut them into leaf discs with a length-to-width ratio of 0.5-1cm, and inoculate them into bud induction culture medium for adventitious bud induction culture. 3) Rooting induction culture: Place the adventitious buds obtained in step 2) on a rooting medium for rooting induction culture to obtain rooted adventitious buds; 4) Obtaining rubber grass seedlings: The adventitious buds with roots of 2-5 cm obtained in step 3) are transferred into culture bottles containing rooting medium to continue growing, thus obtaining rubber grass seedlings. The germination medium was prepared by adding 30 g of sucrose and 2.5 g of gel to 1 / 2 MS medium and adjusting the pH to 5.8 to obtain a solid medium. The bud induction medium is a solid medium obtained by adding plant growth regulators, 30 g / L sucrose, and 2.5 g / L gel to MS medium; the plant growth regulators added in the bud induction culture consist of 0.8 mg / L KT and 0.2 mg / L IAA. The rooting medium is a solid medium obtained by adding 30 g / L sugar and 2.5 g / L gel to M519 medium; Both the bud induction culture and root induction culture were conducted at 24-25℃ with a 16h / 8h light / dark cycle and a light intensity of 60-80 μmol•m. -2 •s -1 Cultured under specific conditions; The bud induction culture medium is replaced with fresh medium every 10-14 days, and the entire induction cycle is 30-40 days; the rooting induction culture medium is replaced with fresh medium every 7-10 days, and the rooting induction culture time is 15-20 days.

2. The method according to claim 1, characterized in that: In step 1), the method for disinfecting rubber grass seeds is to disinfect the surface of plump rubber grass seeds with 70% ethanol for 2 minutes, soak them in 0.25% sodium hypochlorite and 0.1% sodium dodecyl sulfate for 10 minutes, and then rinse the seed surface 5 times with sterile water.

3. The method according to claim 1, characterized in that: In step 1), after the rubber grass seeds are disinfected, they are inoculated onto the germination medium; after 4 weeks, the resulting sterile seedlings are transferred into fresh germination medium and cultured for another 12 weeks to obtain sterile seedlings that can be used for propagation.

4. The method according to claim 1, characterized in that: In step 4), the adventitious buds that have rooted to 2-5 cm are transferred into a culture bottle containing rooting medium and continue to grow for 1-3 months to obtain rubber grass seedlings.

5. The method according to any one of claims 1-4, characterized in that: The method also includes transplanting the rooted rubber grass seedlings; the rubber grass seedlings obtained in step 4) are transplanted into the culture medium, covered with a transparent cover, and kept at a suitable humidity for 7 days; after 7 days, 5-7 holes are punched in the transparent cover to allow the rubber grass seedlings to gradually come into contact with the outside air, and then the transparent cover is removed to allow normal growth to resume; growth conditions: temperature 24-25℃, humidity 60-70%, light intensity 60-80 μmol·m -2 ·s -1 The culture medium is obtained by mixing peat moss, vermiculite, and coconut coir in a volume ratio of 2:1:1.

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