Gracilaria lemaneiformis cell engineering seedling raising method

Abstract

The invention belongs to the field of cell engineering seedling culture of algae, and particularly relates to a cell engineering seedling culture method of gracilaria lemaneiformis. The gracilaria lemaneiformis frond is constructed into a miniaturized form, and high-density intensive culture is carried out by utilizing a photobioreactor. According to the method, the microminiaturized asparagus frond obtained by a cell engineering means can be subjected to vegetative propagation, efficient propagation is carried out by utilizing the algae photobioreactor, and the microminiaturized asparagus frond is further cultured into seedlings for artificial cultivation.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering seedling cultivation of algae, and specifically relates to a method for cell engineering seedling cultivation of Asparagus officinalis. Background Technology

[0002] Seaweed cultivation and seedling propagation generally involve two methods: spore propagation (sexual reproduction), such as kelp, nori, and wakame; and vegetative propagation (asexual reproduction), such as Gracilaria, Glechoma, and Euphorbia. Vegetative propagation is easier to operate, requires less technical training, and allows for rapid biomass growth, which helps maintain stable genetic traits and is more readily accepted. Currently, the artificial cultivation of Gracilaria mainly relies on vegetative propagation through branching and seedling grafting, typically using thallium harvested directly from floating rafts, accounting for about 20%-30% of total production. However, this method is susceptible to genetic variation due to environmental influences during long-term cultivation, leading to the degeneration of desirable traits. Furthermore, vegetative propagation requires a large number of seedlings, which traditional natural propagation methods cannot meet. Large-scale cultivation also faces unavoidable challenges such as long cultivation cycles, high energy consumption, and high risks, and is difficult to prevent from disease outbreaks.

[0003] Currently, the use of photobioreactors to cultivate algae is mainly focused on microalgae, with very few large algae. There are no publicly available reports on the cultivation of Gracilaria in a photobioreactor.

[0004] Gracilaria is a large red algae with a large thallus and numerous branches. In reactors with limited volume, it easily becomes entangled and accumulates, affecting mass transfer and light uniformity. Furthermore, Gracilaria growth is highly dependent on water flow, substrate, and specific light spectrum; static or simple agitation cultivation methods cannot simulate the hydrodynamic conditions of its natural habitat. Existing photobioreactor systems for the large-scale cultivation of large seaweeds often suffer from high equipment investment, high energy consumption, and high algal damage rates. Therefore, developing a seedling cultivation method suitable for the morphological and physiological characteristics of Gracilaria, addressing its unique growth requirements and the shortcomings of existing cultivation methods, is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a cell engineering seedling cultivation method for Gracilaria salsa, based on the growth characteristics of Gracilaria salsa thallus, so as to achieve rapid large-scale cultivation of Gracilaria salsa.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for cultivating *Gnaphalium affine* using cell engineering involves constructing miniaturized *Gnaphalium affine* thallus and then conducting high-density intensive cultivation using a photobioreactor.

[0007] Furthermore, based on the morphological characteristics of Gracilaria, cell engineering techniques were used to construct Gracilaria thallus into a miniaturized form suitable for cultivation in a photobioreactor, thereby achieving high-density intensive cultivation of Gracilaria thallus in the photobioreactor.

[0008] The thallus of Gracilaria was first cultured incrementally in a photobioreactor, and then cultured for further growth.

[0009] The miniaturized form of Gracilaria is constructed by forming 1-5 mm segments from the germination of Gracilaria spores or the newly formed thallus of Gracilaria.

[0010] The construction method is cutting or breaking.

[0011] The photobioreactor is an externally illuminated, internally guided airlift type. Its design takes into account the growth and morphogenesis characteristics of large seaweeds, and can maintain the regular and orderly circulation of Gracilaria segments in the reactor; aeration culture helps the shedding of new algae, thereby continuously increasing the number of Gracilaria seedlings in the reactor.

[0012] During incremental culture, the external light bar of the photobioreactor is set to a light intensity of 60-120 μEm. -2 s -1 Photoperiod 10:14 L / D - 24:0 L / D; During growth culture, the external light bar of the photobioreactor is: light intensity 20-80 μEm. -2 s -1 μEm -2 s -1 ; Photoperiod 10:14L / D-24:0L / D.

[0013] Preferably, during incremental culture, the external light bar of the photobioreactor has a light intensity of 60-100 μEm. -2 s -1 Photoperiod 10:14 L / D - 24:0 L / D; Preferably, during growth culture, the external light bar of the photobioreactor has a light intensity of 30-56 μEm. -2 s -1 μEm -2 s -1 ; Photoperiod 10:14L / D-24:0L / D.

[0014] During incremental culture, the aeration rate within the photobioreactor is 0.25-0.5 m³. 3 / h; During growth culture, the aeration rate in the photobioreactor is 0.1-0.25 m³ / h. 3 / h.

[0015] During incremental culture, the culture medium in the photobioreactor is PES medium; During the growth and cultivation process, the culture medium in the photobioreactor is prepared from sterilized seawater, with added nitrate nitrogen of 10-500 mg / L and phosphate of 2-60 mg / L. The culture medium contains trace elements such as Ca, Mg, Fe, and Zn.

[0016] The culture temperature inside the photobioreactor is 15-30℃.

[0017] After cultivation in the photobioreactor, the algae are extended through a secondary expansion culture to reach the specifications required for production.

[0018] Advantages of this invention: 1. The present invention obtains miniaturized algae of Gracilaria through cell engineering, which can be propagated vegetatively and efficiently expanded using an algal photobioreactor, and further cultivated into seedlings that can be used for artificial cultivation.

[0019] 2. The cell engineering seedling technology of Gracilaria of the present invention adopts the method of cell isolation to first dedifferentiate it, and then provides it with suitable conditions to form explants, thereby achieving the miniaturization of Gracilaria and enabling its asexual reproduction. Combined with the culture conditions of algal photobioreactor, it realizes the large-scale propagation of Gracilaria seedlings, obtains sufficient seedling source, and achieves the purpose of high-quality seedling cultivation.

[0020] 3. This invention utilizes the morphological characteristics of seaweed to construct a miniaturized form of Gracilaria suitable for photobioreactor cultivation through cell engineering. By using an algal bioreactor to cultivate the Gracilaria thallus, seasonal limitations are eliminated, enabling high-density intensive cultivation year-round and solving the problem of seedling source and supply. Considering the miniaturized characteristics of Gracilaria cultivated in an algal bioreactor, secondary expansion cultivation can be carried out in conjunction with seedling ponds until the thallus grows to the required seedling size for production. Attached Figure Description

[0021] Figure 1 This is the reactor culture of Example 1 of the present invention.

[0022] Figure 2 The scale bar is 5cm, showing the incremental culture of Gracilaria in the reactor of Example 1 of this invention.

[0023] Figure 3 The image shows the growth and cultivation of *Asparagus setaceus* seedlings in a reactor according to Example 1 of this invention. Scale bar: 1 cm. Detailed Implementation

[0024] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0025] Example 1 Choose dragon's beard vegetable ( Gracilariopsis lemaneiformis 6.2g of newly formed, healthy, and uncontaminated algae were chopped into 3mm segments using a sterilized scalpel, rinsed three times repeatedly with sterilized seawater, and then placed in a 10L algal photobioreactor (see...). Figure 1 The algae were cultured (incremental culture) in a PES medium. The light intensity on the surface of the algae bioreactor was 70 μEm. -2 s -1 The light-dark cycle is 12h:12h. Filtered air is continuously introduced into the reactor at a flow rate of 0.3m³. 3 / h to control the Gracilaria to move steadily on the surface of the culture medium with a small number of broken bubbles; cultured at 23℃, with 1 / 3 of the culture medium replaced every 3 days; after 21 days, the Gracilaria seedling density in the reactor reached 3.4 g FW / L, the algal length reached 5 mm, and the number of newly sprouted branches increased significantly. Figure 2 .

[0026] Then replace with fresh seawater, add 100 mg / L nitrate nitrogen and 15 mg / L phosphate, and continue aeration culture (growth culture) at an aeration rate of 0.15 m³ / s. 3 / h, light intensity is 50μEm -2 s -1 The light-dark cycle was 12h:12h, and the culture was carried out at 23℃. Fresh culture medium was added every 3 days. The culture was terminated after 14 days, yielding 56g of fresh algae. The average length of the algae reached 3cm. (See attached image.) Figure 3 The length of the reactor seedlings increased significantly.

[0027] Comparative Example 1 The newly formed Gracilaria segments were broken down to less than 1 mm in length using a stirrer. The algae were collected using a 200-mesh sieve and rinsed three times with sterilized seawater. They were then transferred to a 2L algal bioreactor for cultivation. The initial inoculation density was 0.45 g FW / L, and the cultivation conditions were the same as in Example 1 above. During the cultivation process, contamination phenomena such as whitening of the algae occurred. After 21 days of cultivation, the average length of the algae was 1.8 mm, with no obvious branching. The cultivation density was 0.79 g FW / L.

[0028] As shown in Comparative Example 1, algal segments smaller than 1 mm are more prone to contamination during cultivation due to greater damage, and the growth retardation period is increased, reducing cultivation efficiency. Furthermore, when newly formed *Gracilaria fusiforme* algal segments were processed to be larger than 5 mm, and cultivation conditions were the same as in Example 1, it was found that the algae were not easily separated to produce new algal branches, and problems such as entanglement between algal cells also easily occurred, significantly reducing cultivation efficiency. Therefore, this invention constructs newly formed *Gracilaria fusiforme* algal segments of 1-5 mm, which can improve cultivation efficiency.

[0029] Comparative Example 2 The difference from Example 1 is that the light intensity for both the incremental culture stage and the growth culture stage is 70 μEm. -2 s -1 The ventilation rate was 0.3 m³ / s. 3 The culture was completed in 14 days, and 57.4g of fresh thallium was harvested. The average length of the thallium was 1.5cm, and there were many new twigs.

[0030] As can be seen from Comparative Example 2, if the light and ventilation conditions are not changed during the growth cultivation stage, the length of the Gracilaria thallus will not increase while the branching density will increase, making it unsuitable as a seedling for production. Therefore, the present invention sets different cultivation conditions for different cultivation stages to achieve large-scale propagation of Gracilaria seedlings.

[0031] In summary, this invention miniaturizes Gracilaria and implements high-density cultivation. Then, to adapt to production needs, the cultivation conditions are changed, increasing the length of the algae in the reactor stage, maximizing the efficiency of the photobioreactor, and realizing the reactor seedling cultivation of Gracilaria.

Claims

1. A method for cell-engineered seedling cultivation of Gracilaria, characterized in that: The thallus of Gracilaria was constructed into a miniaturized form and then intensively cultured at high density using a photobioreactor.

2. The method according to claim 1, characterized in that: Based on the morphological characteristics of Gracilaria fusiforme, cell engineering techniques were used to construct the Gracilaria fusiforme thallus into a miniaturized form suitable for cultivation in a photobioreactor, thereby achieving high-density intensive cultivation of Gracilaria fusiforme thallus in the photobioreactor.

3. The method according to claim 2, characterized in that: The thallus of Gracilaria was first cultured incrementally in a photobioreactor, and then cultured for further growth.

4. The method according to claim 2, characterized in that: The miniaturized form of Gracilaria is constructed by forming 1-5 mm segments from the germination of Gracilaria spores or the newly formed thallus of Gracilaria.

5. The method according to claim 3, characterized in that: During incremental culture, the external light bar of the photobioreactor is set to a light intensity of 60-120 μEm. -2 s -1 Photoperiod 10:14 L / D - 24:0 L / D; During growth culture, the external light bar of the photobioreactor is: light intensity 20-80 μEm. -2 s -1 ; Photoperiod 10:14L / D-24:0L / D.

6. The method according to claim 3, characterized in that: During incremental culture, the aeration rate within the photobioreactor is 0.25-0.5 m³ / h. 3 / h; During growth culture, the aeration rate in the photobioreactor is 0.1-0.25m³ / h. 3 / h.

7. The method according to claim 3, characterized in that: During incremental culture, the culture medium in the photobioreactor is PES medium; During growth culture, the culture medium in the photobioreactor is prepared with sterilized seawater, with nitrate nitrogen of 10-500 mg / L and phosphate of 2-60 mg / L added.

8. The method according to claim 3, characterized in that: The culture temperature inside the photobioreactor is 15-30℃.

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