CN107079804A - A kind of in-vitro conservation method of Sargassum horneri germ plasm resource - Google Patents

Abstract

The invention provides a method for in vitro preservation of copper algal germplasm resources, wherein the more practical germplasm preservation culture solution is to add 0.1mol/L NaNO ₃ , 0.1mol/L NaH ₂ PO ₄ and 1mg/L uniconazole. The method provided by the invention preserves the copper algae in vitro, which can greatly prolong the preservation time of the main branch of the copper algae strain. The preserved materials can recover quickly and reproduce in large quantities. Compared with the unpreserved seedlings, the seedlings obtained by mass propagation have no obvious difference in morphological characteristics and growth. The method of the invention can provide a large amount of high-quality copper algae seedlings in a short period of time, effectively solves the problems of copper algae germplasm resource preservation and large-scale seedling cultivation, and has wide application prospects.

Description

A kind of in vitro preservation method of copper algae germplasm resources

technical field

The invention belongs to the technical field of algae tissue culture, and in particular relates to an in vitro preservation method of copper algae germplasm resources.

Background technique

The copper algae Sargassum horneri (Turner) C.Agardh belongs to the genus Sargassum and is an important part of the offshore marine ecological environment. It can not only provide foraging and habitat for economic fish, but also absorb eutrophication in seawater Chemical substances and heavy metals, etc., seaweed as a tool for restoring the offshore ecological environment is an important class of economical brown algae. In recent years, with the gradual deepening of people's research on copper algae, the biologically active substances such as algin, fucoxanthin, dietary fiber, and polysaccharides rich in copper algae have attracted widespread attention at home and abroad, and have been widely used in medicine, food, etc. , feed and organic fertilizers. With the development and utilization of copper algae resources, coupled with the impact of the development and pollution of coastal waters, the amount of its resources has been continuously declining. The in vitro preservation of germplasm resources is of great significance to its artificial seed breeding and sea area multiplication.

In vitro culture preservation is increasingly becoming a common method for the preservation of large-scale economical seaweed germplasm resources, but the common problem is that the subculture is frequent, and each subculture may cause cross-contamination. The cost of manpower and material resources consumed is very high. In addition, copper algae are large and grow fast, so it is difficult to maintain the normal activity of copper algae for a long time in the laboratory, which brings great difficulties to the in vitro preservation of copper algae germplasm resources. At present, domestic and foreign studies on copper algae mainly focus on ecology, artificial breeding and reproductive biology, etc., but there are relatively few studies on the preservation of its germplasm resources. The Chinese invention patent "A Method for Preserving Fertilized Eggs of Copper Algae at Low Temperature" (publication number CN 104145946 A) discloses a method for preserving fertilized eggs of copper algae at low temperature. The method is to store the fertilized eggs at 4-8°C Preservation, so that it can be transported across different regions before rhizoid elongation for seed cultivation, but there is no effective preservation method for indoor in vitro culture preservation of copper algae germplasm.

Contents of the invention

The object of the present invention is to provide a method for in vitro preservation of copper algae germplasm resources in order to solve the deficiencies of the above technologies, so as to effectively carry out in vitro preservation of copper algae germplasm resources.

The method for in vitro preservation of copper algal germplasm resources of the present invention comprises the following steps:

1) cleaning the germplasm resources of copper algae to be preserved;

2) Take the main branch of the cleaned copper algae and soak it in alcohol with a volume concentration of 75% for 25-30 seconds, then quickly rinse it with sterilized seawater for 3-4 times, cut off the main branch tip of the algae strain under sterilized conditions after washing and store it in vitro Object;

The tip of the main branch of the algae strain preferably has a length of 8-12 cm

3) Put the cut tip of the main branch of the algae strain into the preservation medium, and store it at 5-8°C, with a light intensity of 2000-2500lx, a photoperiod of 10L:14D, and aerated conditions; replace the medium regularly

The above germplasm preservation culture solution is to add 0.1mol/L NaNO ₃ , 0.1mol/L NaH ₂ PO ₄ and 1mg/L uniconazole to sterilized seawater;

The culture medium is preferably replaced every 10 days;

The illumination is provided by white fluorescent lamps;

4) When the main branch of the algae strain to be preserved grows to a length of not less than 15 cm, remove the base of the main branch of the algae strain, and continue to put the tip of the retained algae strain into the germplasm preservation culture medium for germplasm preservation and cultivation.

(5) The step of restoring growth after storage is as follows: take out the algae that have been stored in vitro for 6 months, and transfer them to a circulating water tank whose external dimensions are 80 cm long x 45 cm wide x 40 cm high, and the culture medium is normal sea water. The culture conditions are: temperature 10-15°C, light intensity 4000-5000lx, photoperiod 12L:12D, a large amount of aeration to make the algae roll up and down, and the amount of water change every 7 days is 50% of the total water body, and the algae maintain vigorous growth , After about 15-20 days of cultivation, the algae grow to more than 20 cm, and can be used for subsequent seedling cultivation.

The method provided by the invention preserves the copper algae in vitro, which can greatly prolong the preservation time of the main branch of the copper algae strain. The preserved materials can recover quickly and reproduce in large quantities. Compared with the unpreserved seedlings, the seedlings obtained by mass propagation have no obvious difference in morphological characteristics and growth. The method of the invention can provide a large amount of high-quality copper algae seedlings in a short period of time, effectively solves the problems of copper algae germplasm resource preservation and large-scale seedling cultivation, and has wide application prospects.

Description of drawings

Figure 1: The effect diagram of the culture solution with different concentrations of uniconazole on the content of chlorophyll a;

Figure 2: Effect diagram of the content of carotenoids in the culture solution with different concentrations of uniconazole added.

detailed description

In the applicant's research, it was found that the tip of the main branch of copper algae was cultivated and preserved in sterilized seawater, and the color of the algae body became lighter and the tip was white after 30 days. It was found to be significantly reduced. Therefore, the purpose of preparing the preservation medium in this application is to increase the dry weight of the algae during the preservation process and maintain the content of chlorophyll a and carotenoids.

The method of the present invention is described in detail below.

Example 1

The germplasm preservation culture solution prepared in this example has the following components: add 0.1mol/L NaNO ₃ , 0.1mol/L NaH ₂ PO ₄ , and 1mg/L uniconazole to sterilized seawater. The above-mentioned components and proportions can not only delay the growth rate of copper algae during preservation, but also not affect its recovery after preservation.

The preservation steps of copper algae are as follows:

(1) Select the copper algae germplasm with excellent characters, remove the attached organisms on the surface with a hairbrush and tweezers, then repeatedly clean with seawater to remove silt and impurities;

(2) Put the treated copper algae on the ultra-clean workbench, take the main branches of the algae and soak them in alcohol with a volume concentration of 75% for 25-30s, then quickly wash them with a large amount of sterilized seawater for 3-4 times, and use sterilized A 10-centimeter segment of the tip of the main branch of the algae is cut with a scalpel as an object for in vitro preservation;

(3) The condition of germplasm preservation is: carry out in the Erlenmeyer flask with volume of 1000mL, each Erlenmeyer flask is filled with 800mL culture solution, place two strains of algae, slightly inflate, so that there are small bubbles on the surface of the culture solution; the storage temperature is 5-8°C, light intensity of 2000-2500lx, photoperiod of 10L:14D, provided by white fluorescent lamps.

(4) Regularly observe the growth of the preserved algae. Compared with the control (cultivated in normal sterilized seawater), the algae grows slowly, the length is obviously shortened, and the main branch is obviously thickened. About 30 days, the length of the algae grows to About 15 centimeters, use a scalpel to cut off the base, and only keep the 10 centimeters of algae body fragments at the tip, and then continue to put them into the germplasm preservation culture medium for germplasm preservation and culture.

The effects of different culture medium, temperature, light intensity, and aeration on the preservation effect are shown below. The measured growth indicators include the fresh weight (FW), the length of the algae, and the apparent symptoms, among which the fresh weight is The increase is represented by relative growth rate (RGR), and the calculation formula is as follows: RGR=[(W _t /W ₀ ) ^1/t -1]×10

In the formula, W _t is the fresh weight of algae at the middle or end of the experiment (g), W ₀ is the fresh weight of algae at the beginning of the experiment (g), and t is the culture time (d).

(1) Effect of culture medium on copper algae preservation in vitro

The test materials were stored in the culture solution of different concentrations of uniconazole, and each group was treated in triplicate. The storage conditions were: temperature 10°C, light intensity 2500lx, photoperiod 12L:12D, and aerated slightly. The results showed that copper algae had the longest preservation time at a concentration of 1.0 mg/L of uniconazole, which was 182 days. See Table 1 for details.

Table 1 Effects of different culture solutions on the preservation of copper algae germplasm resources

(2) Effect of temperature on the in vitro preservation of copper algae

Put the test materials into the preservation medium of the present invention, place them in light incubators with a temperature of 5°C, 8°C, 10°C, and 15°C for preservation, and each group is treated in three parallels. Other preservation conditions: light intensity 2500lx, light Cycle 12L:12D, micro inflation. The results showed that copper algae had the longest storage time in the incubator at 5℃, which was 187 days.

See Table 2 for details.

Table 2 Effects of different temperatures on the preservation of copper algae germplasm resources

(3) Influence of light intensity on in vitro preservation of copper algae

Put the test materials into the preservation medium of the present invention, place them respectively under the light intensity of 1000lx, 1500lx, 2000lx, 2500lx, and 3000lx for preservation, each group is processed in three parallels, other preservation conditions: temperature 10°C, photoperiod 12L: 12D, micro inflation. The results showed that copper algae had the longest storage time of 187 days when the light intensity was 2000lx, see Table 3 for details.

Table 3 Effects of different light intensities on the preservation of copper algae germplasm resources

(4) Influence of aeration volume on in vitro preservation of copper algae

The test materials were stored and cultivated under the condition of no air, slight air (small air bubbles appear on the surface of the culture solution), large amount of air (the algal body rolls up and down), the temperature is 10°C, the light intensity is 2500lx, and the photoperiod is 12L:12D. The results showed that copper algae had the longest storage time under the condition of a small amount of aeration, which was 157 days, see Table 4 for details.

Table 4 The size of aeration has the effect on the preservation of copper algae germplasm resources

(5) Effect of culture medium on content of main photosynthetic pigments in copper algae

The determination method of chlorophyll-a and carotenoid content is as follows: take 0.1 g of fresh algae body and grind it into a homogenous slurry, add 8 mL of 80% acetone and extract in a dark place at 4°C for 24 hours. Centrifuge at 4000r/min for 10min at 4°C to discard the precipitate, and dilute the supernatant to 10mL with 80% acetone. With 80% acetone as a blank control, the absorbance values at wavelengths of 665, 652, 510, and 480 nm were measured. Repeat more than 3 times and calculate the average value. The content of chlorophyll a is calculated according to the formula w(Chl-a)=(16.29OD ₆₆₅ －8.54OD ₆₅₂ )×V/W/1000, and the content of carotenoids is calculated according to the formula w(Car)=7.6×(OD ₄₈₀ －1.49× OD ₅₁₀ )×V/W/1000, where V is the volume of acetone extracted (mL), W is the mass of algae (g), and the unit is mg/g.

The test materials were stored in the culture solution of different concentrations of uniconazole for 30 days, and the contents of chlorophyll a and carotenoids were measured respectively, and the values of the stored materials on the 0th day were used as controls. The results showed that after 30 days of culture in naturally sterilized seawater, the contents of chlorophyll a and carotenoids were significantly reduced to 58.43% and 73.36% of the control. The differences in the content of main photosynthetic pigments in copper algae after 30 days of culture are shown in the attached figure. It can be seen from Figure 1 that within the range of 0.1-2.0 mg/L mass concentration, with the increase of uniconazole mass concentration, the chlorophyll a content of copper algae algae gradually increased. Among them, 0.1 and 0.5mg/L experimental group algae chlorophyll a content was significantly lower than the control, respectively decreased by 29.4% and 6.5% compared with the control, while the 1.0 and 2.0mg/L experimental group had no significant difference from the control. The content of carotenoids in the 0.1 and 0.5 mg/L experimental groups was also significantly lower than that of the control group; the content of carotenoids in the 1.0 mg/L experimental group was the highest, which was 0.092 ± 0.007 mg/g fresh weight, slightly higher than that of the control group (0.090 ± 0.002mg/g fresh weight); The carotenoid content of the experimental group of 2.0mg/L is 0.090 ± 0.008, no significant difference with the contrast. It can be seen that adding an appropriate amount of uniconazole in the culture solution can significantly increase the content of chlorophyll a and carotenoids in the copper algae, and it is most beneficial to the synthesis and accumulation of chlorophyll a and carotenoids at a mass concentration of 1.0mg/L .

The above results show that the culture solution of the present invention can effectively carry out long-term preservation of copper algae in vitro.

(6) Growth recovery of copper algae preserved in vitro

The germplasm of copper algae with excellent properties is preserved in the culture solution of the present invention for 182 days and then taken out to restore growth. The steps of restoring growth after the preservation are as follows: take out the algae preserved in vitro, and transfer the outer dimension to be 80cm long × wide In a circulating water tank of 45cm x 40cm in height, the culture medium is normal seawater. The culture conditions are: temperature 10-15°C, light intensity 4000-5000lx, photoperiod 12L:12D, large amount of aeration to make the algae roll up and down, and the amount of water exchanged every 7 days is 50% of the total water body.

The copper algae that recovers after the in vitro preservation of table 5 and the comparison of the copper algae growth 30d before preservation

Material specific growth rate Length (cm) before saving 2.43±0.05 25.11±1.03 after saving 2.41±0.16 24.89±1.77

After being cultured, the preserved material grows vigorously and has a normal color, and the measurement of the growth index shows that the growth of the copper algae after preservation is not adversely affected by long-term preservation. After about 15 to 20 days of cultivation, the algal body can grow to more than 20 centimeters, which can be used for subsequent seedling cultivation.

Claims (6)

1. a method for in vitro preservation of copper algae germplasm resources, is characterized in that, described method comprises the steps: 1) cleaning the copper algae to be preserved; 2) Take the main branch of the cleaned copper algae and soak it in alcohol with a volume concentration of 75% for 25-30 seconds, then quickly rinse it with sterilized seawater for 3-4 times, cut off the main branch tip of the algae strain under sterilized conditions after washing and store it in vitro Object; 3) Put the cut tip of the main branch of the algae strain into the preservation medium, and store it at 5-8°C, with a light intensity of 2000-2500lx, a photoperiod of 10L:14D, and aerated conditions; replace the medium regularly 4) When the main branch of the algae strain to be preserved grows to a length of not less than 15 cm, remove the base of the main branch of the algae strain, and continue to put the tip of the retained algae strain into the germplasm preservation culture medium for germplasm preservation and cultivation. 2. The method according to claim 1, characterized in that the length of the main branch tip of the algae strain in the step 2) is preferably 8-12 cm. 3. The method according to claim 1, characterized in that, the germplasm preservation culture solution of the step 3) is to add 0.1mol/L NaNO ₃ , 0.1mol/L NaH ₂ PO in sterilized seawater ₄ and 1mg/L uniconazole prepared. 4. The method according to claim 1, characterized in that the culture medium in step 3) is preferably replaced every 10 days. 5. The method according to claim 1, characterized in that the illumination in step 3) is provided by a white fluorescent lamp. 6. The method for recovering the copper algae preserved by the method according to claim 1 is to take out the algae preserved in vitro and put it into seawater for cultivation, the temperature of cultivation is 10～15°C, the intensity of light is 4000～5000lx, and the light intensity is 4000～5000lx. The cycle is 12L:12D, and the water exchange volume is 50% of the total water body every 7 days.