A tissue culture propagation method of eriochloa villosa

Abstract

The application discloses a tissue culture propagation method of eriochloa contracta. The creeping stem section of the eriochloa contracta is collected, washed, and then sterilized and dried; the sterilized and treated stem section is inoculated into a bud induction medium of MS+1-1.5 mg / L 6-BA+0.1-0.5 mg / L NAA+30 mg / L sucrose+6.5 mg / L agar to perform bud induction, and after 15-20 days of culture, the adventitious bud seedling is obtained; the adventitious bud of about 1-1.5 cm induced in the primary culture medium is inoculated into a proliferation culture medium to perform subculture, and the next step can be divided into single bud expansion or direct transplanting domestication. The two-step method only needs primary culture-proliferation and rooting to be simultaneously performed, and the transplanting survival rate is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology and biotechnology, and more particularly, the present application relates to a tissue culture propagation method of Eremochloa ophiuroides. BACKGROUND

[0002] Eremochloa ophiuroides is a perennial rhizomatous broadleaf turfgrass plant of the family Poaceae, which is widely distributed in the Yangtze River Basin and the south of China, and is one of the most widely distributed warm-season turfgrass in China. Eremochloa ophiuroides has many advantages, such as low plant height, fast turfing, thick grass layer, long green period, tolerance to poor soil and trampling, strong spreading and regenerative ability, strong disease and pest resistance, strong soil fixation, extensive management, easy maintenance, etc., and is particularly known for obtaining high-quality turf with low maintenance management, and is an excellent soil and water conservation plant. Eremochloa ophiuroides has outstanding advantages in preventing and controlling soil and water loss of embankment (mainly the backwater side), reducing the daily management cost of embankment, and facilitating the inspection and risk removal during flood season, and has broad application prospects in ecological embankment construction.

[0003] The traditional propagation methods of Eremochloa ophiuroides seedlings mainly include sowing, cutting, and division. However, Eremochloa ophiuroides has low seed production, inconsistent maturation period, and low germination rate, making it difficult to collect and obtain batch seeds. At present, there is still a lack of batch supply of high-quality Eremochloa ophiuroides seeds in China, and the price of Eremochloa ophiuroides seeds in the United States is 70 US dollars per pound (900 grams). The efficiency of cutting and division of Eremochloa ophiuroides is also low. The low efficiency of the propagation method limits the popularization and application of Eremochloa ophiuroides. At the same time, the production of turf using traditional methods requires a large amount of non-agricultural land, and there is also a lack of a large amount of land for seedling production in the current environment of emphasizing the protection of agricultural land in China. Therefore, although Eremochloa ophiuroides has great space for application in green space turf, especially in water conservancy slope protection, it lacks the ability to supply grass and seedlings on a large scale. SUMMARY

[0004] In order to improve the propagation efficiency of Eremochloa ophiuroides, the present application provides a tissue culture propagation method of Eremochloa ophiuroides, which adopts a two-step method, and only needs primary culture-propagation and rooting to be carried out at the same time, and has high survival rate after transplanting.

[0005] The present application is realized by the following technical scheme. A tissue culture propagation method of Eremochloa ophiuroides, comprising:

[0006] Collecting Eremochloa ophiuroides stolon segments, washing and disinfecting, and absorbing water;

[0007] Primary culture: the stems after disinfection and treatment are inoculated into MS+1-1.5mg / L 6-BA+0.1-0.5mg / L NAA+30mg / L sucrose+6.5mg / L agar bud induction medium for bud induction, and after inoculation, they are cultured under the culture conditions of light intensity of 2500-3000LUX, light duration of 14-18h / d, and temperature of 24-26℃; and after 15-20 days of culture, the adventitious bud seedlings are obtained.

[0008] Proliferation and rooting culture: the 1-1.5cm adventitious buds induced in the primary culture medium are inoculated into the proliferation medium for subculture, MDR2+1-1.5mg / L KT+0.4-0.8mg / L IBA+3-6mg / L Put+0.5-2mg / L SA+30mg / L sucrose+6.5mg / L agar, and the next step can be to continue amplification of single bud or direct transplantation and domestication.

[0009] The transplantation and domestication is that the rooting tissue culture seedlings are gently taken out from the bottle, the agar on the roots is washed away with water, and the seedlings are transplanted into the mixed substrate of peat soil, perlite and vermiculite at a ratio of 1:1:1, covered with plastic film to maintain humidity of more than 80%, and after 10 days, ventilation and humidity reduction are paid attention to; and after 20 days, the seedlings can be transplanted into the field, and the survival rate reaches more than 98%.

[0010] Further preferably, the washing refers to washing with washing powder water and then rinsing with running water.

[0011] Further preferably, the disinfection refers to soaking in 75% alcohol on the super-clean bench, then rinsing with sterile water, and then disinfecting with sodium hypochlorite and rinsing with sterile water.

[0012] Further preferably, in the primary culture, 10 explants are inoculated in each culture dish.

[0013] Further preferably, in the proliferation and rooting culture, 15-20 buds are inoculated in each bottle.

[0014] Further preferably, subculture is carried out once every 25 days.

[0015] The present application develops the tissue culture and asexual propagation technology of eriochloa contracta, uses the tissue culture technology to carry out factory seedling raising of eriochloa contracta, utilizes the characteristics of high expansion coefficient of tissue culture to shorten the seedling raising time, can solve the problems of small amount of conventional seeds, difficulty in collection, high price of imported seeds, etc., and can produce a large amount of transplanted seedlings with high quality and stable genetic traits in a short time. Meanwhile, the tissue culture seedling raising is carried out in facilities, can utilize the three-dimensional space, and save open land. Therefore, the establishment of the tissue culture and propagation technology system of eriochloa contracta has important significance for the large-scale popularization and application of eriochloa contracta in greening lawn, water conservancy slope protection and the like. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Effect picture of case one conventional culture-propagation proliferation;

[0017] Figure 2 Effect picture of case one conventional culture-rooting culture;

[0018] Figure 3 Effect picture of case one application-propagation proliferation rooting culture.

[0019] Figure 4 Effect picture of different differentiation proliferation culture formula test comparison group 1;

[0020] Figure 5 Effect picture of different differentiation proliferation culture formula test comparison group 2;

[0021] Figure 6 Effect picture of different differentiation proliferation culture formula test comparison group 3;

[0022] Figure 7 Effect picture of different differentiation proliferation culture formula test application group;

[0023] Figure 8 Adventitious shoots germinated on explants after 15 days of primary culture;

[0024] Figure 9 Adventitious shoots transferred from primary culture to proliferation medium for about 10 days;

[0025] Figure 10 Bud cluster after elongation of adventitious shoots after 20 days of conventional proliferation culture;

[0026] Figure 11 Bud cluster after 15 days of single bud growth on proliferation medium;

[0027] Figure 12 Rooted seedling (single plant) of single bud in rooting medium for 15 days;

[0028] Figure 13 Rooted seedling of single bud in rooting medium for 15 days;

[0029] Figure 14 Rooted bud cluster after elongation of adventitious shoots after 20 days of application proliferation rooting culture.

[0030] Figure 15 Bud cluster and root after elongation of adventitious shoots after 20 days of application proliferation rooting culture.

[0031] Figure 16 Acclimatized seedling after 30 days of tissue culture rooted seedling transplanted into substrate. DETAILED DESCRIPTION

[0032] The application will be further illustrated in detail in the following with respect to the implementation process.

[0033] A tissue culture propagation method of eriochloa villosa, comprising:

[0034] Disinfection of explants: Collect the creeping stem segments of eriochloa villosa, wash them with detergent water, and then rinse them with running water for about 10-15 minutes; first immerse them in 75% alcohol for 60 seconds on the clean bench, rinse them with sterile water once, then disinfect them with 10% sodium hypochlorite for 10-12 minutes, and rinse them with sterile water four times; dry the water on filter paper, and cut off the damaged and whitened parts with a sterile blade.

[0035] Primary culture: inoculate the disinfected and treated stem segments into bud induction medium for bud induction MS+1-1.5 mg / L 6-BA+0.1-0.5 mg / L NAA+30 mg / L sucrose+6.5 mg / L agar, inoculate 10 explants in each culture dish; after inoculation, place them in a culture condition with light intensity of 2500-3000 LUX, light duration of 16 h / d, and temperature of 24-26℃; after 15-20 days of culture, obtain adventitious bud seedlings.

[0036] Proliferation and rooting culture: inoculate the 1-1.5 cm adventitious buds induced in the primary culture medium into proliferation medium MDR2+1-1.5 mg / L KT+0.4-0.8 mg / L IBA+3-6 mg / L Put+0.5-2 mg / L SA+30 mg / L sucrose+6.5 mg / L agar, inoculate 15-20 buds in each bottle, subculture every 25 days; the proliferation buds can reach about 5-9 and elongate to more than 3 cm, the seedlings are very uniform in height, the bud clumps are all provided with roots, each plant has more than 5 roots, the rooting rate is more than 95%, the next step can be to separate single buds for further expansion, or directly transplant and acclimate; this culture medium not only can proliferate and expand, but also can root, thus reducing the step of rooting culture in the conventional process, saving time and reducing labor and material resources.

[0037] Transplanting and acclimatization: gently take out the tissue culture seedlings with roots cultured for about 25 days from the bottle, wash off the agar on the roots with water, and transplant them into a substrate mixed at a ratio of 1:1:1 of peat soil, perlite and vermiculite, cover them with plastic film to maintain humidity of more than 80%, and pay attention to ventilation and humidity reduction after 10 days. After 20 days, they can be transplanted into the field, and the survival rate of transplantation reaches more than 98%.

[0038] Example 1

[0039] Disinfection of explants: Collect the creeping stem segments of Eleusine indica, wash them with detergent water, then rinse them with running water for about 10 minutes; first immerse them in 75% alcohol for 60 seconds on the clean bench, rinse them with sterile water once, then disinfect them with 10% sodium hypochlorite for 10 minutes, rinse them with sterile water four times; dry the water on filter paper, cut off the damaged and whitened parts with a sterile knife.

[0040] Primary culture: inoculate the disinfected and treated stem segments into MS + 1 mg / L 6-BA + 0.1 mg / L NAA + 30 mg / L sucrose + 6.5 mg / L agar bud induction medium for bud induction, 10 explants per culture dish; after inoculation, place them in a culture condition with light intensity of 2500 LUX, light duration of 16 h / d, and temperature of 24°C; after 15 days of culture, obtain adventitious bud seedlings.

[0041] Proliferation and rooting culture: inoculate the 1-1.5 cm adventitious buds induced in the primary culture medium into the proliferation medium MDR2 + 1 mg / L KT + 0.4 mg / L IBA + 3 mg / L Put + 0.5 mg / L SA + 30 mg / L sucrose + 6.5 mg / L agar, 15-20 buds per bottle, subculture every 25 days; the proliferation buds can reach 5-9 and grow to more than 3 cm in length, the seedlings are very uniform in height, the bud clumps are all with roots, each plant has more than 5 roots, the rooting rate is more than 95%, the next step can be to separate the single buds for further expansion, or directly transplant and acclimate; this medium not only proliferates and proliferates, but also roots, which can reduce the step of rooting culture in the conventional process, saving time and reducing manpower and material resources.

[0042] Transplanting and acclimatization: gently take out the rooting seedlings from the bottle after 25 days of culture, wash off the agar on the roots with water, and transplant them into a mixture of peat soil, perlite, and vermiculite at a ratio of 1:1:1, cover them with plastic film to maintain humidity above 80%, and pay attention to ventilation and humidity reduction after 10 days. After 20 days, they can be transplanted into the field, and the survival rate reaches more than 98%.

[0043] Example 2

[0044] Disinfection of explants: Collect the creeping stem segments of Eleusine indica, wash them with detergent water, then rinse them with running water for about 10 minutes; first immerse them in 75% alcohol for 60 seconds on the clean bench, rinse them with sterile water once, then disinfect them with 10% sodium hypochlorite for 10 minutes, rinse them with sterile water four times; dry the water on filter paper, cut off the damaged and whitened parts with a sterile knife.

[0045] Primary culture: the stems after disinfection and treatment, inoculated to MS + 1.2 mg / L 6-BA + 0.3 mg / L NAA + 30 mg / L sucrose + 6.5 mg / L agar bud induction medium for bud induction, 10 explants were inoculated in each culture dish; after inoculation, they were placed in a culture condition of light intensity 2700LUX, light duration 16h / d, temperature 25℃; after 20 days of culture, adventitious bud seedlings were obtained.

[0046] Proliferation culture rooting: the 1-1.5 cm adventitious buds induced in the primary culture medium were inoculated into the proliferation medium MDR2 + 1.2 mg / L KT + 0.6 mg / L IBA + 5 mg / L Put + 1 mg / L SA + 30 mg / L sucrose + 6.5 mg / L agar, 15-20 buds were inoculated in each bottle, and the proliferation was subcultured every 25 days; the proliferation buds could reach 5-9 and grow to more than 3 cm, the seedlings were very uniform in height, the buds had roots below, each plant had more than 5 roots, and the rooting rate was more than 95%; the next step could be to separate single buds for further expansion, or to directly transplant and acclimate; this medium not only proliferates and proliferates, but also roots, which can reduce the step of rooting culture in the conventional process, saving time and reducing manpower and material resources.

[0047] Transplanting and acclimatization: the tissue culture seedlings with roots after 25 days of rooting were gently taken out of the bottle. The agar on the roots was washed off with water, and the seedlings were transplanted into a mixture of peat soil, perlite and vermiculite at a ratio of 1:1:1, covered with plastic film to maintain humidity above 80%, and ventilated and dehumidified after 10 days. After 20 days, they could be transplanted into the field, and the survival rate reached more than 98%.

[0048] Example 3

[0049] Explant disinfection: the creeping stems of Eragrostis curvula were collected, washed with laundry detergent water, and then rinsed with running water for about 15 minutes; in the clean bench, first soaked in 75% alcohol for 60 seconds, rinsed with sterile water once, then disinfected with 10% sodium hypochlorite for 12 minutes, and rinsed with sterile water 4 times; the damaged and whitish parts were cut off with a sterile blade on filter paper.

[0050] Primary culture: the stems after disinfection and treatment, inoculated to MS + 6-BA1.5mg / l + NAA0.5mg / l + sucrose30mg / l + agar6.5mg / l bud induction medium for bud induction, 10 explants were inoculated in each culture dish; after inoculation, they were placed in a culture condition of light intensity 3000LUX, light duration 16h / d, temperature 26℃; after 20 days of culture, adventitious bud seedlings were obtained.

[0051] Proliferation and rooting culture: the 1-1.5 cm or so adventitious buds induced in the primary culture medium were inoculated into the proliferation medium MDR2+1.5 mg / L KT+0.8 mg / L IBA+6 mg / L Put+2 mg / L SA+ sucrose 30 mg / L+agar 6.5 mg / L, 15-20 buds were inoculated into each bottle, and the subculture was carried out once every 25 days; the proliferation buds could reach 5-9 or more and were elongated to more than 3 cm, the height of the seedlings was very uniform, the bud clumps were all with roots, each plant had more than 5 roots, and the rooting rate was more than 95%, the next step could be to separate the single buds for further expansion, or the seedlings could be directly transplanted and acclimated; this culture medium not only proliferated and proliferated, but also rooted, which could reduce the step of rooting culture in the conventional process, saving time and reducing labor and material resources.

[0052] Transplanting and acclimation: the tissue culture seedlings with roots cultured for about 25 days were gently taken out of the bottle, the agar on the roots was washed off with water, and the seedlings were transplanted into a substrate mixed at a ratio of 1:1:1 of peat soil, perlite and vermiculite, covered with plastic film to maintain humidity of more than 80%, and ventilated to reduce humidity after 10 days. After 20 days, the seedlings could be transplanted into the field, and the survival rate reached more than 98%.

[0053] Comparative Example 1

[0054] The method of establishing a high-efficiency tissue culture regeneration system of Eragrostis teff in Molecular Plant Breeding, 2019, Vol. 17, No. 19, pp. 6461-6468 was used, and the technical parameters are shown in Table 1. From the culture process, it is a three-step method.

[0055] Comparative Example 2

[0056] The method of inducing callus and plant regeneration from lateral buds of Eragrostis teff disclosed in Patent No. CN101194595A was used, which was a three-step method, and the technical parameters are shown in Table 1. The tender stems of Eragrostis teff were selected in the field, the leaf sheaths and leaves were removed, and the stems were cut into 2 cm long sections with lateral buds after being washed clean, soaked in 0.2% laundry detergent for 20 min, and then washed with running water for 1 h. The stems were then soaked in 75% ethanol for 50 s, 0.2% mercury for 15 min, and then washed with sterile water for 4-5 times in a clean bench. The surface-sterilized stem sections were first inoculated into lateral bud growth medium, and the lateral buds produced callus on the callus induction medium. Yellow, tight, granular callus was selected for subculture and proliferation at 28 days, and the callus was subcultured twice before being moved to green seedling differentiation medium. After 4-5 weeks, the regenerated plants were transferred to test-tube seedling rooting medium, and the regenerated plants rooted in the test-tube seedling rooting medium. After 2 weeks, the test-tube plants were transplanted to pots and the field; the temperature in the culture room during in vitro culture was 25±1℃, the callus induction and proliferation culture was carried out under scattered light, and the green seedling differentiation, growth and rooting culture was carried out under light conditions.

[0057] Comparative Example 3

[0058] The method for establishing a high-efficiency regeneration system of eriochloa using a young ear as an explant disclosed in CN106538382A is a four-step method, and the technical parameters are shown in Table 1.

[0059] (1) Fresh young ears (inflorescences) of eriochloa at the initial flowering stage were selected, the outermost leaf sheath was peeled off, and then the disinfection treatment was performed in a clean bench, and then the bract outside the young ear was peeled off to obtain the sterile young ear;

[0060] (2) The sterile young ear explant was inoculated on the callus induction medium (MS+2-4mg / L 2,4-D

[0061] +0.1mg / L BAP) to perform induction culture to obtain callus with an induction rate of 76,7%;

[0062] (3) The callus was peeled off from the explant and transferred to the subculture medium (MS+1.5mg / L 2,4-D+0.1mg / L BAP) to perform proliferation subculture;

[0063] (4) The callus after subculture was transferred to the green seedling differentiation medium (MS+2.0mg / L KT+0.1mg / L NAA) to perform induction of adventitious bud differentiation (100% differentiation rate) to obtain test tube seedlings;

[0064] (5) The differentiated test tube seedlings were transferred to the rooting medium (MS+0.2mg / L KT+0.6mg / L NAA) to culture to root;

[0065] (6) The regenerated plants after rooting were hardened;

[0066] (7) After the hardening was completed, the regenerated seedlings were taken out, the root culture medium was washed off, and were transplanted into a seedling raising substrate and placed in a greenhouse for culture.

[0067] Table 1. Comparison of tissue culture seedling raising technologies of eriochloa

[0068]

[0069] Compared with the comparative examples, the present application has the following differences:

[0070] On the path: other methods are basically regeneration methods, and at least experience three steps of primary culture (callus induction)-bunch bud differentiation (proliferation)-rooting; the present application adopts a two-step method, and only primary culture-proliferation and rooting are performed at the same time.

[0071] Efficiency: the previous method only showed the differentiation rate (100% of the multiplication rate) in the comparative example 3, and the other did not show the proliferation efficiency. The transplant survival rate of the method of the present application is also the highest, and the transplant rate mainly depends on the influence of the culture medium of the previous culture on the root quality.

[0072] Case 1:

[0073] Table 1: Time statistics table of two culture methods:

[0074]

[0075] • The conventional culture needs two different culture media for propagation and rooting, that is, it wastes a rooting medium once, needs to transfer the bottle seedlings manually once, and takes an additional half month for the seedling period, which is time-consuming and labor-intensive and material-consuming.

[0076]

[0077]

[0078] • The propagation and rooting culture of the present application only needs one culture medium, roots while propagating, reduces the rooting step, saves 15 days of time, reduces the labor cost of transferring the bottle seedlings once, and also saves the cost of the rooting medium.

[0079]

[0080] Case 2:

[0081] Table 2: Statistics table of three culture media

[0082]

[0083] • The basic medium MDR2 is beneficial to rooting, because the large amount of elements of MDR2 is lower than that of MS medium, and the rooting rate of the culture medium B is increased from 3% of the culture medium A to 50%.

[0084] • The culture medium C changes the basic medium on the basis of the original culture medium A, and additionally adds putrescine PUT and salicylic acid SA which are beneficial to rooting, and not only does not greatly reduce the number of bud propagation, but also almost all of the tissue culture propagation seedlings have 3-5 roots, and the rooting rate is increased to more than 98%.

[0085]

[0086] Case 3:

[0087] Table 3: Statistics table of different differentiation and proliferation culture formula tests:

[0088]

[0089] • Through the test comparison, it is found that the tissue culture seedlings of the present application not only root, but also have very neat seedling height, which is beneficial to the subsequent transplanting and transplanting survival rate. ​​​​

[0090] to transplant domestication;

[0091] The other three comparative groups are not uniform in size, and need rooting culture before transplanting;

[0092] The cluster propagation rate of the application is slightly higher than that of the three comparative groups, but there is no significant difference.

[0093] It should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for tissue culture propagation of *Pseudolarix amabilis*, characterized in that, include: Collect the creeping stem segments of *Phyllostachys edulis*, wash and disinfect them, and then dry them. Primary culture: Disinfected and treated stem segments were inoculated into MS medium containing 1-1.5 mg / L 6-BA, 0.1-0.5 mg / L NAA, 30 mg / L sucrose, and 6.5 mg / L agar for bud induction. After inoculation, the medium was placed under the following conditions: light intensity of 2500-3000 LUX, light duration of 14-18 h / d, and temperature of 24-26℃. Adventitious shoots were obtained after 15-20 days of culture. Proliferation and rooting culture: Adventitious shoots induced in the shoot induction medium (1-1.5 cm) are inoculated into a proliferation and rooting medium for subculture. The proliferation and rooting medium is MDR2 + 1-1.5 mg / L KT + 0.4-0.8 mg / L IBA + 3-6 mg / L Put + 0.5-2 mg / L SA + 30 mg / L sucrose + 6.5 mg / L agar. The next step is to divide them into single shoots for further amplification or directly transplant them for acclimatization.

2. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, The transplanting and acclimatization process is as follows: gently remove the rooted tissue culture seedlings from the bottle; wash off the agar on the roots with water; transplant them into a substrate of peat moss, perlite, and vermiculite in a 1:1:1 ratio; cover with plastic film to maintain a humidity of over 80%; and ventilate to reduce humidity after 10 days. Transplanted to the field 20 days later, the survival rate of transplanted seedlings reached over 98%.

3. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, The cleaning refers to washing with laundry detergent and water, followed by rinsing with running water.

4. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, The disinfection process involves first soaking the surface in 75% alcohol on a clean bench, then rinsing it with sterile water, followed by disinfection with sodium hypochlorite and rinsing with sterile water.

5. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, During the initial culture, 10 explants were inoculated into each culture dish.

6. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, When propagating and rooting culture, 15-20 buds are inoculated into each bottle.

7. The method for tissue culture propagation of *Pseudolarix amabilis* according to claim 1, characterized in that, Subculture is performed every 25 days.

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