Artificial rapid breeding and efficient cultivation method for cibotium barometz

Abstract

The invention belongs to the technical field of artificial breeding of plants, and discloses an artificial rapid breeding and efficient cultivation method of cibotium barometz, which comprises the following steps: spore disinfection and germination: collecting mature spores of cibotium barometz, performing surface disinfection, inoculating into a first culture medium, and performing germination culture to obtain gametophytes; seedling induction and rooting: transferring the gametophyte into a second culture medium, inducing the gametophyte to develop into a seedling and root, and culturing until the seedling has a robust root system; acclimatization and transplanting: transplanting the rooted seedlings into an acclimatization matrix, performing acclimatization culture in an acclimatization environment with the humidity controlled to be 88-92% and the temperature controlled to be 18-22 DEG C, and then transplanting the seedlings into natural soil. According to the method, the pollution rate is reduced from 50% to 15%, and the disinfection success rate of the cibotium barometz spores is greatly improved; growth hormone is added, so that the germination period of spores and the rooting period of seedlings are shortened; the survival rate of the seedlings can reach 80%, and the success rate of seedling hardening is increased.

Description

Technical Field

[0001] This invention belongs to the field of plant artificial propagation technology, specifically relating to a method for rapid artificial propagation and efficient cultivation of Cibotium barometz. Background Technology

[0002] Cibotium barometz, a Class II protected wild plant in China, has significant ecological and ornamental value. Its wild populations grow slowly and are strictly protected by law, prohibiting commercial harvesting. To meet market demand and protect wild populations, artificial propagation techniques, especially tissue culture techniques, have become crucial for achieving large-scale, sustainable production.

[0003] Currently, artificial tissue culture technology for cultivating Cibotium barometz has significantly increased seedling yield and shortened its growth cycle, representing a relatively advanced advancement in this technological aspect. However, several technical bottlenecks still exist throughout the tissue culture and transplantation process, severely impacting its industrialization efficiency and economic benefits. These bottlenecks mainly focus on the following two aspects: Firstly, spore disinfection and germination are challenging: the initial material for tissue culture—spores—is collected from the wild environment, and its surface is often covered with spore powder from other ferns, fungi, and other microorganisms, resulting in complex sources of contamination. Disinfection is extremely crucial but also exceptionally difficult: if the disinfectant concentration is too low, it cannot completely sterilize the spores, leading to contamination in subsequent cultures; if the concentration is slightly too high, it can easily damage or kill the spores, severely affecting the germination rate. Maintaining this disinfection balance is difficult and has become the primary technical challenge limiting the success rate of tissue culture. Simultaneously, spores germinate slowly in the wild, and the artificial disinfection process further impacts their viability; therefore, shortening their artificial germination cycle remains a problem to be solved.

[0004] Secondly, the survival rate during the hardening-off stage is low: Seedlings obtained through tissue culture face severe environmental stress during the transition from a sterile, temperature- and humidity-controlled tissue culture environment to a microbial environment in the soil (i.e., "hardening-off"). During this stage, seedlings must adapt to multiple challenges, including changes in microorganisms, humidity, and light intensity. Current cultivation techniques are insufficient to address these challenges, resulting in extremely high seedling mortality rates, generally exceeding 50%, ultimately causing the overall survival rate to remain persistently low at 30%-40%. This low survival rate during hardening-off has become a major obstacle to the transformation of laboratory tissue culture results into large-scale cultivation.

[0005] In conclusion, although fundamental breakthroughs have been achieved in tissue culture technology for *Cibotium barometz*, efficient spore sterilization and germination techniques at the front end, and seedling hardening and domestication cultivation techniques at the back end, remain two key weaknesses restricting its overall production efficiency and success rate. There is an urgent need to develop more reliable and efficient processing methods and cultivation techniques to address these bottlenecks and improve the stability and economics of the entire breeding system. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for the rapid artificial propagation and efficient cultivation of Cibotium barometz, so as to reduce the microbial contamination rate during the spore disinfection process; shorten the cycle of spore germination and seedling rooting; and effectively improve the survival rate of tissue culture seedlings during the hardening and transplanting stage.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: The rapid artificial propagation and efficient cultivation methods of Cibotium barometz include the following: (I) Spore disinfection and germination: Mature spores of Cibotium barometz were collected, disinfected, and then inoculated into the first culture medium for germination culture to obtain gametophytes; (II) Seedling induction and rooting: The gametophytes are transferred to the second culture medium to induce them to develop into seedlings and root, and cultured until the seedlings have a robust root system; (III) Hardening and transplanting: After the seedlings have taken root, they are transplanted into hardening substrate and acclimatized in a hardening environment with humidity of 88% to 92% and temperature of 18 to 22°C. After that, they are transplanted into natural soil.

[0008] This invention removes bacteria and contaminants attached to spores in the wild by surface disinfection, and then inoculates them in a first culture medium (such as MS medium) rich in nutrients and germination inducing substances, providing them with all the energy and substances required to break dormancy, initiate cell division and develop into gametophytes; Transferring gametophytes to a second culture medium (such as 1 / 2 MS medium) with adjusted nutrient composition and hormone ratios reduces inorganic salt concentration (osmotic pressure), which is more conducive to root differentiation. Adding specific plant growth regulators (such as auxins) can directly stimulate the formation of root primordia, thereby completing the key transformation from gametophytes to sporophyte seedlings with roots, stems, and leaves. Sterile seedlings were transplanted into a hardening substrate that simulated the characteristics of natural soil (water retention and aeration), and acclimatized in a transitional environment with controlled temperature and humidity (humidity 88%–92%, temperature 18–22℃). This process allowed the seedlings to gradually adapt to the following key changes: from heterotrophic (dependent on the sugar content of the culture medium) to autotrophic (performing photosynthesis), and from sterile to seroactive (microbial environment). By buffering the sudden change in environment, the transplanting stress was greatly alleviated, resulting in a leap in the survival rate of the seedlings.

[0009] In some optional instances, the humidity may be 88%, 89%, 90%, 91% or 92%, but is not limited to the listed values; other unlisted values ​​within this range also apply.

[0010] The temperature can be 18℃, 19℃, 20℃, 21℃ or 22℃, but is not limited to the listed values. Other unlisted values ​​within this range also apply.

[0011] As a preferred technical solution of the present invention, in content (I), the surface disinfection includes the following: First, soak the sample in an ethanol solution for 10–30 seconds, then disinfect it by soaking it in a mercuric chloride solution for 10–20 minutes.

[0012] In this invention, alcohol can quickly wet and remove hydrophobic impurities and some microorganisms on the surface of spores; mercuric chloride, as a powerful surface disinfectant, binds to proteins and enzymes on the cell membrane of microorganisms through mercury ions, causing them to denature and become inactive, thereby rapidly killing most fungi, bacteria and other microorganisms attached to the surface of spores. The combined use of the two achieves highly efficient sterilization.

[0013] In some optional instances, the volume percentage concentration of alcohol is 70% to 80%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, but is not limited to the listed values; other unlisted values ​​within this range also apply.

[0014] The concentration of mercuric chloride is 0.08% to 0.12%, for example, it can be 0.08%, 0.09%, 0.1%, 0.11% or 0.12%, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0015] As a preferred embodiment of the present invention, in content (I), after disinfection by soaking in mercuric chloride solution, the following content is also included. Antibiotic solutions are used for auxiliary disinfection.

[0016] In this invention, the use of antibiotic solution can effectively eliminate specific microorganisms (such as endophytes) that are latent inside spores or are insensitive to chemical disinfectants (alcohol, mercuric chloride). The use of antibiotic solution can achieve deeper and more thorough sterilization without excessively damaging spore viability, and control the initial contamination rate.

[0017] As a preferred technical solution of the present invention, in content (I), the first culture medium is MS culture medium, and a first plant growth regulator for promoting spore germination is added thereto; in content (I), the cycle from spore germination to the formation of gametophytes is 35 to 45 days.

[0018] In this invention, the high content of inorganic salts and sucrose in MS medium provides sufficient substances and energy for spores to break dormancy and initiate cell division. The first plant growth regulator directly stimulates spore cell activation and division, thereby shortening the cycle from spore germination to gametophyte formation.

[0019] In some optional instances, the first plant growth regulator is a growth hormone.

[0020] As a preferred technical solution of the present invention, in content (II), the second culture medium is 1 / 2MS culture medium, and a second plant growth regulator for promoting seedling rooting is added thereto. In content (II), the gametes are cultured in the second culture medium for 30 to 40 days.

[0021] In this invention, the salt concentration (osmotic pressure) of the 1 / 2MS medium is reduced, which can alleviate the physiological stress of seedlings and is more conducive to root differentiation and elongation. The addition of a second plant growth regulator can directly stimulate the formation and growth of root primordia and shorten the culture cycle.

[0022] As a preferred technical solution of the present invention, in content (III), the seedling hardening substrate is peat soil.

[0023] In this invention, peat soil is both water-retaining and aerated, mimicking the soil characteristics of the original habitat under the golden dog fern forest.

[0024] The beneficial effects of this invention are: 1. By disinfecting the spores of *Ceratophyllum demersum* with alcohol, mercuric chloride, and antibiotics, the contamination rate was reduced from 50% to 15%, which greatly improved the success rate of spore disinfection and increased the yield of seedlings. 2. By adding growth hormone, the germination cycle of spores and the rooting cycle of seedlings are shortened. The germination cycle is shortened from more than three months to 35-45 days, and the rooting cycle of seedlings is shortened from the usual two months to about 30-40 days. 3. The survival rate of seedlings can reach 80%, which improves the success rate of hardening off seedlings. Attached Figure Description

[0025] The present invention can be further illustrated by the non-limiting embodiments given in the accompanying drawings; Figure 1 This is a diagram illustrating the cultivation process of Cibotium barometz in an embodiment of the present invention. Detailed Implementation

[0026] The technical solutions of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described herein are specific implementations of the present invention, used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary, and should not be construed as limiting the implementation methods or the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can employ other obvious technical solutions based on the content disclosed in the claims and specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein. Example

[0027] like Figure 1 As shown, this embodiment provides a method for rapid artificial propagation and efficient cultivation of Cibotium barometz, including the following: (1) Spore disinfection and germination: Mature spores of Cibotium barometz were collected, and after surface disinfection, they were inoculated into MS medium for germination culture to obtain gametophytes. The MS medium contained growth hormones to promote spore germination. The cycle from spore germination to gametophyte formation was 40 days. The specific content of surface disinfection was as follows: first, soaking in 75% ethanol solution for 15 seconds, then soaking in 0.1% mercuric chloride solution for 15 minutes, and finally using antibiotic solution for auxiliary disinfection. (2) Seedling induction and rooting: The gametophytes were transferred to 1 / 2 MS medium to induce them to develop into seedlings and root. The seedlings were cultured until they had a strong root system. The 1 / 2 MS medium contained growth hormones to promote seedling rooting. The gametophytes were cultured in the second medium for 35 days. (3) Hardening and transplanting: After the seedlings have taken root, they are transplanted into peat soil and acclimatized in an environment with controlled humidity of 90% and temperature of 20℃. After that, they are transplanted into natural soil.

[0028] In this embodiment, the disinfection effect of using ethanol solution, mercuric chloride solution, and antibiotic solution was significant, reducing the high contamination rate from 50% to 15%, greatly improving the success rate of spore disinfection for *Cibotium barometz* and increasing seedling yield. Adding growth hormone shortened the spore germination cycle, avoiding the impact of artificial tissue culture disinfection on spore germination rate. Adding growth hormone to the 1 / 2 MS medium to promote seedling rooting shortened the gametophyte culture time in the second medium to 35 days, significantly reducing the seedling rooting cycle. In this embodiment, the seedling survival rate reached 80%, significantly improving the success rate of hardening off. In contrast, in conventional cultivation processes using existing technologies, *Cibotium barometz* seedlings typically die from various problems during the transition from artificial tissue culture to soil cultivation, with a mortality rate reaching 50%. In actual cultivation, 200,000 tissue culture seedlings have been produced in batches, and large-scale understory planting has been achieved through the above-mentioned technology.

[0029] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A rapid artificial propagation and efficient cultivation method for Cibotium barometz, characterized by: The method comprises the following steps, Spore disinfection and germination, mature spores of the Stenochlaena palustris are collected, surface-disinfected, inoculated into a first culture medium for germination culture, and gametophytes are obtained; Induction of seedlings and rooting, the gametophytes are transferred into a second culture medium to induce development of seedlings and rooting, and the seedlings are cultured until they have robust root systems; Hardening-off and transplanting, the seedlings with roots are transplanted into a hardening-off substrate, acclimated and cultured in a hardening-off environment with humidity of 88%-92% and temperature of 18-22℃, and then transplanted into natural soil.

2. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 1, characterized in that: In the content (I), the surface disinfection comprises the following steps, firstly, the spores are soaked in an ethanol solution with a volume percentage concentration of 70%-80% for 10-30 seconds, and then soaked in a mercury chloride solution with a concentration of 0.08%-0.12% for 10-20 minutes.

3. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 2, characterized in that: In the content (I), after the soaking in the mercury chloride solution, the method further comprises the following steps, auxiliary disinfection is performed using an antibiotic solution.

4. The rapid artificial propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 1, characterized in that: In the content (I), the first culture medium is a MS culture medium, and a first plant growth regulator for promoting spore germination is added into the medium.

5. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 4, characterized in that: In the content (I), the period for spore germination to form gametophytes is 35-45 days.

6. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 1, characterized in that: In the content (II), the second culture medium is a 1 / 2MS culture medium, and a second plant growth regulator for promoting seedling rooting is added into the medium.

7. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 6, characterized in that: In the content (II), the culture time of the gametophytes in the second culture medium is 30-40 days.

8. The artificial rapid propagation and efficient cultivation method of the Matteuccia struthiopteris according to claim 1, characterized in that: In the content (III), the hardening-off substrate is selected from peat soil.

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