A method for quickly cultivating a mature sporophyte from a Dicranopteris dichotoma prothallus

Abstract

The present invention discloses a method for rapidly cultivating mature sporophytes from Dicranopteris dichotoma prothalli. Using Dicranopteris dichotoma prothalli as the breeding basic material, the cultivation process combines tissue culture and facility cultivation and is divided into 4 processes, namely: prothallus development and germination, prothallus development and maturity, artificial environment to promote the generation of young sporophytes from prothalli, and facility cultivation to promote the development of young sporophytes into mature sporophytes. By using tissue culture induction technology and facility cultivation, the present invention can obtain a large number of Dicranopteris dichotoma seedlings with strong stress resistance that can be directly planted in difficult sites such as mines in a relatively short time, without being affected by seasonal climate changes and natural disasters, providing technical support for the factory cultivation of Dicranopteris dichotoma and the cultivation of its clones. The present invention shortens the cultivation time of Dicranopteris dichotoma plants, saves the cost of Dicranopteris dichotoma seedling cultivation, and can provide plant planting materials for ecological restoration of difficult sites such as mines, slopes, tailing waste dumps, and strongly acidic soils.

Description

Technical Field

[0001] The present invention belongs to the technical field of Dicranopteris pedata propagation, and particularly relates to a method for rapidly cultivating mature sporophytes from Dicranopteris pedata prothalli. Background Art

[0002] Dicranopteris pedata belongs to the Gleicheniaceae family and the Dicranopteris genus. It is a fern clonal plant, an indicator plant for subtropical and tropical acidic soils, and a pioneer fern plant.

[0003] Dicranopteris pedata is evergreen, erect or trailing; its rhizome is slender, brownish - brown, densely covered with brown scales, creeping and growing in the surface soil layer, constantly branching. The rhizomes of the Dicranopteris pedata population are intertwined in a net - like pattern, forming a root mat layer together with the root system, i.e., the root - system soil and litter. The apical bud is ovoid. The leaves are sparsely arranged, papery, yellow - green on the surface and gray - white on the back. The petiole is cylindrical, of varying lengths, brownish - brown; the leaf blade has repeated pseudo - dichotomous branching, with pinnae attached at each intersection, and two - branched pinnae at the last bifurcation; the sporangium groups are round, borne in the middle of the fine veins; the leaf layer and root mat layer of the Dicranopteris pedata population form a double - layer structure, which has a good carbon sequestration function. The leaf layer reduces the erosion of the ground surface by rainfall, and the root mat layer has a strong soil - fixing function.

[0004] Dicranopteris pedata can also adsorb heavy metals such as lead (Pb), zinc (Zn), tungsten (W), manganese (Mn), cadmium (Cd), rare earth elements such as lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), as well as radioactive elements such as radium (Ra) and thorium (Th) in the soil.

[0005] Dicranopteris pedata likes sunlight, is acid - tolerant, barren - tolerant, drought - tolerant, and has strong adaptability. It grows well under moist and fertile conditions, and can also grow and reproduce in difficult sites such as rare - earth tailings and highway slopes. It has good heavy - metal enrichment ability and soil and water conservation ability.

[0006] As a pioneer fern plant, after the surface soil layer in tropical and subtropical regions is damaged by production activities such as mining and road construction, under natural conditions, the spores of Dicranopteris pedata float onto the ground surface, and under suitable conditions, the spores germinate and start the developmental generation of fern plants.

[0007] After the mature sporophyte is settled, the rhizome creeps on the ground surface and grows clonally to form a population, which has ecological functions such as soil and water conservation, ecological restoration, heavy - metal enrichment, and carbon sequestration.

[0008] During the 15 - year period of natural vegetation restoration after the formation of rare - earth tailings in the subtropics, the importance value of Dicranopteris pedata ranks first in the shrub - grass - fern layer.

[0009] At present, Dicranopteris pedata is planted by spore sowing and rhizome transplantation. Under natural conditions, it takes 2 to 3 years for Dicranopteris pedata to germinate from spores into prothalli, form sporophytes, and then form plants; while field experiments show that using the rhizomes of Dicranopteris pedata for asexual reproduction has a low survival rate and is difficult to be directly applied to the ecological restoration of mines and tailings.

[0010] Applying the ecological function of the clonal plant population of Dicranopteris dichotoma and its heavy metal enrichment function can well achieve the purpose of ecological restoration of difficult sites such as tailings, mines, and slopes. The first problem to be solved is the cultivation of Dicranopteris dichotoma seedlings, and the breeding and proliferation problems such as artificially promoting the rapid growth of rhizomes and the rapid spread of populations. Summary of the Invention

[0011] Aiming at the deficiencies and problems in the prior art, the present invention aims to provide a method for quickly cultivating mature sporophytes from the prothalli of Dicranopteris dichotoma. The present invention provides a method for cultivating Dicranopteris dichotoma plants that artificially induces the development of Dicranopteris dichotoma prothalli, accelerates the maturation of prothalli in an artificial cultivation environment, and artificially promotes the formation and growth of sporophytes. Through artificial induction and cultivation methods, the smooth progress of the prothallial generation and sporophyte generation of Dicranopteris dichotoma is promoted, the survival rate of each generation is increased, the success rate of the transformation of prothalli into the sporophyte generation is increased, and the development duration of the two generations is shortened, which can effectively improve its reproduction speed and efficiency, shorten its reproduction cycle, and provide technical support for realizing the cultivation of Dicranopteris dichotoma on difficult sites such as tailings.

[0012] The present invention is realized through the following technical solutions:

[0013] A method for quickly cultivating mature sporophytes from the prothalli of Dicranopteris dichotoma, which uses the prothalli of Dicranopteris dichotoma as the basic breeding material, and the cultivation process adopts a combination of tissue culture and facility cultivation, and is divided into 4 processes, namely: the germination of prothalli development, the maturation of prothalli development, the formation of young sporophytes from prothalli, and the development of young sporophytes into mature sporophytes.

[0014] Specifically, it includes the following processes:

[0015] 1. Inducing the germination of Dicranopteris dichotoma prothalli development, including the following steps:

[0016] Using the prothalli formed by the germination of Dicranopteris dichotoma spores, or further obtaining a large number of green globular bodies (GGB) of prothalli through tissue culture as the propagation material;

[0017] Dispersing the green globular bodies of Dicranopteris dichotoma prothalli into fine particles of 0.5 mm to 1.0 mm, namely rice grain-shaped prothallial masses, in a sterile environment;

[0018] Collecting the rice grain-shaped prothallial masses in a sterile environment, mixing them with sterile water, and evenly spreading them on the development induction medium, and culturing them in a sterile environment for 3-4 weeks; the culture light intensity is 2000-3000 LX, the light time is 12 h / d, and the temperature is 23 ± 3 °C;

[0019] The development induction medium is 1 / 8MS + 15 g / L of sucrose + 5.6 g / L of agar, pH 5.4-5.8;

[0020] The culture product is a germinating prothallus mass, about 1 cm in diameter, with small laminae rich in chloroplasts;

[0021] 2. Promote the rapid maturation of germinating prothalli, including the following steps:

[0022] Under sterile conditions, transfer the germinating prothallus mass to continue dispersing, transfer it to the prothallus development culture medium, and culture it in the culture room for one week; the culture light intensity is 2000 - 3000 LX, the light time is 12 h / d, and the temperature is 23 ± 3 °C;

[0023] The formula of the prothallus development medium is: 1 / 2 MS + 1 mg / L NAA + 15 g / L sucrose + 5.6 g / L agar, pH 5.4 - 5.8;

[0024] The culture product is: a mature prothallus mass with a diameter of 1 - 3 cm, the laminae grow larger and brown rhizoids grow out;

[0025] 3. Artificially promote the formation of young sporophytes from Dicranopteris dichotoma prothalli, including the following steps:

[0026] Use the methods of twisting with hands and tearing with hands to disperse the mature prothallus mass into single mature prothallus pieces or small groups composed of 2 to 3 mature prothalli, and sow them on a specific induction culture substrate;

[0027] Cover with a heat preservation film to keep the temperature Cultivate for 2 weeks, and apply a specific nutrient solution once during this period;

[0028] During the cultivation period, water with a watering can to keep the culture substrate moist, so that there are water droplets or small water flows on the prothalli on the culture substrate every 3 to 5 days, and the environment is 50% shaded and at room temperature;

[0029] The specific culture substrate is V 草炭 ∶V 蛭虫 ∶V 腐殖土 =1∶1∶1;

[0030] The culture product is Dicranopteris dichotoma young sporophytes;

[0031] The formula of the specific nutrient solution is: 1 / 2 MS + 1 mg / L NAA + 30 g / L sucrose, pH 5.4 - 5.8.

[0032] 4. Artificially accelerate the development of Dicranopteris dichotoma young sporophytes into mature spores, including the following steps:

[0033] Cultivate Dicranopteris dichotoma young sporophytes through facility cultivation or field cultivation, using a specific culture substrate;

[0034] According to the growth potential of the sporophyte, apply the leachate of wild Dicranopteris dichotoma roots and soil;

[0035] Water the specific Dicranopteris linearis root mixture once a month, shade by 25%, and fertilize regularly according to the growth potential of the sporophyte.

[0036] The specific culture medium is V 草炭 ∶V 蛭虫 ∶V 腐殖土 =1∶1∶1;

[0037] The specific Dicranopteris linearis root mixture water refers to the mixed water of wild Dicranopteris linearis root soil and crushed Dicranopteris linearis roots;

[0038] The wild Dicranopteris linearis root and soil leachate refers to the mixture of the root soil, humus layer and water of well - grown Dicranopteris linearis plants in the wild.

[0039] Compared with the prior art, the beneficial effects of the present invention include:

[0040] (1) The present invention utilizes tissue culture induction technology and facility cultivation, and can obtain a large number of Dicranopteris linearis seedlings with strong stress resistance that can be directly planted in difficult sites such as mines in a relatively short time, without being affected by seasonal climate changes and natural disasters, providing technical support for the factory - scale seedling raising of Dicranopteris linearis and the cultivation of its clones.

[0041] (2) The present invention promotes the success rate of prothallus development and can obtain a large number of mature prothalli in a relatively short time.

[0042] (3) The present invention promotes the success rate of mature prothalli generating zygotes.

[0043] (4) The present invention accelerates the rate of zygotes developing into young sporophytes and the rate of young sporophytes developing into mature sporophytes.

[0044] (5) The present invention promotes the rate of mature sporophytes growing into transplantable Dicranopteris linearis plants.

[0045] (6) The present invention shortens the cultivation time of Dicranopteris linearis plants, saves the cost of Dicranopteris linearis seedling raising, and can provide plant planting materials for ecological restoration of difficult sites such as mines, slopes, tailing waste dumps, and strongly acidic soils. Detailed implementation mode

[0046] Example 1

[0047] Process 1, adopt the cultivation method for artificially inducing the development of Dicranopteris linearis prothalli, including the following steps: The cultivation and propagation material is GGB. Under a sterile environment, disperse GGB into small particles, the size of rice grains, that is, rice - grain - shaped prothallus clusters; collect the rice - grain - shaped prothallus clusters in a sterile environment and mix them with sterile water, and make them evenly scattered on a specific culture medium, and culture them in a culture room. Week; the culture light intensity is 2000 - 3000 LX, the light duration is 12 h / d, and the temperature is 23 ± 3 °C; the development induction medium used is 1 / 8 MS + 15 g / L sucrose + 5.6 g / L agar, pH 5.4 - 5.8; it takes 0.5 months for the prothallus to develop into a visible prothallus, about 0.5 mm * 0.5 m.

[0048] Process 2, adopt a cultivation method for artificially promoting the rapid maturation of the small prothallus of Dicranopteris dichotoma. It includes the following steps: transfer the germinated prothallus mass to a specific medium under sterile conditions and culture it in a culture room Week; the culture light intensity is 2000 - 3000 LX, the light duration is 12 h / d, and the temperature is 23 ± 3 °C; the specific medium formula is: 1 / 2 MS + 1 mg / L NAA + 15 g / L sucrose + 5.6 g / L agar, pH 5.4 - 5.8; it takes 1.0 month for the prothallus to develop into about 4 mm * 4 mm.

[0049] Process 3, adopt a cultivation method for artificially promoting the generation of young sporophytes of Dicranopteris dichotoma, including the following steps: use the methods of twisting and tearing by hand to break up the mature prothallus mass into individual mature prothallus pieces, mix the mature prothallus with pure water, sprinkle it on a specific culture medium, sprinkle water on the culture medium to make water droplets or small water flows flow on the culture medium. Cover with a heat preservation film to keep the temperature Cultivate for 2 weeks. The specific culture medium used is V 草炭 :∶ 蛭虫 ∶V 腐殖土 =1∶1∶1. It takes 0.5 months for the 4 mm * 4 mm prothallus to develop into a young sporophyte.

[0050] Process 4, adopt a method for artificially accelerating the growth and development of the sporophyte of Dicranopteris dichotoma, including the following steps: cultivate with a specific young sporophyte culture medium, keep warm, moisturize and fertilize, spray with a specific mixed water, and shade 25%; the young sporophyte culture medium used is V 草炭 ∶V 蛭石 ∶V 腐殖土 =1∶1∶1. It takes 6 months for the young sporophyte to develop into a mature sporophyte.

[0051] Comparative Example 1

[0052] Adopt the simulation of the spore reproduction process in nature as a comparative example:

[0053] Process 1, in the steps, use the spores of Dicranopteris dichotoma as the direct culture material; sprinkle the spores on the outdoor natural humus soil; it takes 2 months for the spores of Dicranopteris dichotoma to germinate into visible prothalli to the naked eye;

[0054] Process 2, without artificial measures, under the conditions of natural water, fertilizer, light, and temperature, the prothalli grow naturally; it takes 5 months for the Dicranopteris dichotoma prothalli to develop into prothalli of 3 mm * 5 mm in size and 9 months for those of 5 mm * 6 mm in size;

[0055] Process 3, without artificial measures, under the conditions of natural water, fertilizer, light, and temperature, the prothalli develop into young sporophytes naturally; it takes 1 month for the Dicranopteris dichotoma prothalli of 5 mm * 6 mm in size to develop into young sporophytes;

[0056] Process 4, without artificial measures, under the conditions of natural water, fertilizer, light, and temperature, the young sporophytes develop into mature sporophytes naturally; it takes 8 months for the young sporophytes to develop into mature young sporophytes.

[0057] As shown in Table 1, compared with Comparative Example 1, the induction and development time of the Dicranopteris dichotoma prothalli under the operation of Example 1 are greatly reduced:

[0058] Table 1 Comparison of the time consumed in each stage between Comparative Example 1 and Example 1

[0059]

[0060] The above only expresses the preferred embodiments of the present invention, and the description is relatively specific and detailed. However, it should not be construed as a limitation to the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several variations, improvements, and substitutions can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be subject to the appended claims.

Claims

1. A method for quickly cultivating a mature sporophyte from a prothallus of Dicranopteris dichotoma, characterized in that, The method uses the prothallus material of Dicranopteris dichotoma as the breeding basic material, and the cultivation process adopts the form of combining tissue culture and facility cultivation. The method includes the following processes: (1) Prothallus development and germination: Induce the development of the Dicranopteris dichotoma prothallus material to form a germinated prothallus. Specifically, use the globular gemmae bodies (GGB) of the Dicranopteris dichotoma prothallus as the starting cultivation material for obtaining a large number of mature sporophyte plants through Dicranopteris dichotoma cloning. Under a sterile environment, disperse the GGB into fine particles of 0.5 mm to 1.0 mm, that is, rice-grain-shaped prothallus clusters. Transfer the rice-grain-shaped prothallus clusters to the prothallus development induction medium, and culture for 3 - 4 weeks in a sterile environment. The culture light intensity is 2000 - 3000 LX, the light time is 12 h / d, and the temperature is 23 ± 3 °C to conduct the development induction of the prothallus, obtaining a germinated prothallus cluster; the development induction medium is 1 / 8 MS + 15 g / L sucrose + 5.6 g / L agar, pH 5.4 - 5.8; (2) Prothallus development to maturity: Transfer the germinated prothallus mass after development induction to a prothallus development medium for prothallus development culture, and culture it in a sterile culture environment. After culturing for weeks, a mature prothallus mass is obtained. The diameter of the developed and mature prothallus is 1 - 3 cm, heart-shaped leaf-like, with fibrous roots; the culture light intensity is 2000 - 3000 LX, the light duration is 12 h / d, and the temperature is 23 ± 3 °C; the prothallus development medium is 1 / 2 MS + 1 mg / L NAA + 15 g / L sucrose + 5.6 g / L agar, pH 5.4 - 5.8; (3) Mature prothalli generate young sporophytes: Disperse the mature prothallus clusters into single mature prothalli, or small clusters composed of 2 to 3 mature prothalli; Inducing the generation of young sporophytes under protected cultivation conditions: Scatter single mature prothalli of Dicranopteris dichotoma or small groups composed of 2 to 3 mature prothalli onto the induction medium; cover with a film to keep warm and culture at 35°C to 40°C for 2 weeks, and apply a specific nutrient solution once during this period; water with a watering can to ensure that water droplets flow on the prothalli in the culture medium every 3 to 5 days, and the environment is 50% shaded and at room temperature; the formula of the specific nutrient solution is: 1 / 2MS + 1 mg / L NAA + 30 g / L sucrose, pH 5.4 - 5.8; the induction medium is V 草炭 : V 蛭石 : V 腐殖土 = 1:1:1; (4) Young sporophytes develop into mature sporophytes: Cultivate the young sporophytes through facility cultivation or field cultivation, using a specific culture substrate; Apply the leachate of wild Dicranopteris dichotoma roots and soil according to the growth potential of the sporophyte; Pour the specific Dicranopteris dichotoma root mixture water once a month, and at the same time apply regular fertilizers according to the growth potential of the sporophyte; wherein the specific culture medium is V 草炭 : V 蛭石 : V 腐殖土 = 1:1:1; The specific Dicranopteris dichotoma root mixture water refers to the mixed water of wild Dicranopteris dichotoma root soil and crushed Dicranopteris dichotoma roots; The leachate of wild Dicranopteris dichotoma roots and soil refers to the mixture of the roots, humus layer and water of well-grown wild Dicranopteris dichotoma plants.

Images