CN107079804A - A kind of in-vitro conservation method of Sargassum horneri germ plasm resource - Google Patents
A kind of in-vitro conservation method of Sargassum horneri germ plasm resource Download PDFInfo
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- CN107079804A CN107079804A CN201710518427.4A CN201710518427A CN107079804A CN 107079804 A CN107079804 A CN 107079804A CN 201710518427 A CN201710518427 A CN 201710518427A CN 107079804 A CN107079804 A CN 107079804A
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- sargassum horneri
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G33/00—Cultivation of seaweed or algae
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Abstract
The present invention provides a kind of method of Sargassum horneri germ plasm resource Plantlet in vitro, wherein more practical preserving seed nutrient solution is the NaNO that 0.1mol/L is added in sterilizing seawater3, 0.1mol/L NaH2PO4With 1mg/L uniconazole Ps.Method provided by the present invention carries out Sargassum horneri Plantlet in vitro, can substantially prolongs the holding time of Sargassum horneri algae strain major branch.Saved material can quickly restoration ecosystem, and amount reproduction can be carried out, the seedling that amount reproduction is obtained is compared with the seedling of not saved mistake, its morphological feature and growing way no significant difference.The method of the present invention can provide substantial amounts of Sargassum horneri high quality seedling in a short time, efficiently solve the problems, such as Sargassum horneri Germ-plasma resources protection and scale breeding, be with a wide range of applications.
Description
Technical field
The invention belongs to algae technical field of tissue culture, and in particular to a kind of Plantlet in vitro side of Sargassum horneri germ plasm resource
Method.
Background technology
Sargassum horneri Sargassum horneri (Turner) C.Agardh is under the jurisdiction of Sargassum (Sargassum), is coastal waters
The important component of the marine eco-environment, can not only provide for economic fish and look for food and habitat, in also adsorbable seawater
Eutrophication materials and heavy metal etc., be the important economic brown alga of a class as the instrument marine alga for repairing Offshore Ecology environment.
In recent years, as what people were studied it gradually gos deep into, algin, fucoxanthin, dietary fiber, polysaccharide for being rich in Sargassum horneri etc.
Bioactive substance causes common concern both domestic and external, is widely applied to the side such as medicine, food, feed and organic fertilizer
Face.With the exploitation of Sargassum horneri resource, adding to be developed and polluted by immediate offshore area is influenceed, under causing its stock number continuous
Drop, therefore while existing wild resource is protected, using algae method for tissue culture to the Sargassum horneri germplasm with merit
Resource progress Plantlet in vitro is bred to the artificial seed for carrying out it and sea area propagation has important meaning.
Conservation in vitro is increasingly becoming the conventional means of large-scale economical alga Germ-plasma resources protection, but generally existing
Problem is exactly that subculture is frequent, and subculture is all likely to result in cross pollution each time, and when reserve capacity is larger, squamous subculture is consumed
Man power and material's cost it is very high.In addition, Sargassum horneri frond is larger, fast growth is difficult to keep its algae for a long time in laboratory
The normal activity of body, the Plantlet in vitro to Sargassum horneri germ plasm resource brings very big difficulty.At present, the research both at home and abroad to Sargassum horneri is main
Concentrate in terms of ecological, artificial increasing cultivation and reproductive biology, but it is also fewer about the research of its Germ-plasma resources protection.
It is public in Chinese invention patent " a kind of method of utilization Cord blood Sargassum horneri embryonated egg " (publication No. is the A's of CN 104145946)
The method using Cord blood Sargassum horneri embryonated egg is opened, this method is, in being preserved at 4-8 DEG C, it is extended in rhizoid by embryonated egg
The trans-regional transport in strange land is completed before so as to carry out seed rearing, and it is current still for the indoor conservation in vitro of Sargassum horneri germplasm
Without effective store method.
The content of the invention
A kind of Sargassum horneri germ plasm resource Plantlet in vitro is provided the invention aims to solve the deficiency of above-mentioned technology
Method, so as to effectively carry out the Plantlet in vitro of Sargassum horneri germ plasm resource.
The method of the Sargassum horneri germ plasm resource Plantlet in vitro of the present invention, including the steps：
1) the Sargassum horneri germ plasm resource that will be preserved is cleaned；
2) cleaned Sargassum horneri is taken to alcohol-pickled 25~30s of algae strain major branch volumetric concentration 75%, then with sterilization
Seawater is got express developed 3~4 times, and algae strain major branch tip is cut as the object of Plantlet in vitro under sterilising conditions after flushing；
Described algae strain major branch tip, its length is preferably 8~12cm
3) algae cut strain major branch tip is put into Storaged media, 5~8 DEG C, intensity of illumination be 2000~
2500lx, the photoperiod is 10L:Preserved under 14D, venting condition；Regularly replace culture medium
The described preserving seed nutrient solution stated is the NaNO that 0.1mol/L is added in sterilizing seawater3, 0.1mol/L's
NaH2PO4With 1mg/L uniconazole Ps；
Described nutrient solution is changed once for preferably every 10 days；
Described illumination is provided by white fluorescent lamp；
4) when algae strain major branch to be saved grows into length and is not shorter than 15 centimetres, remove the base portion of algae strain major branch, will retain
Algae strain tip continue to be put into preserving seed nutrient solution progress preserving seed culture.
(5) restoration ecosystem step is after the preservation：The Plantlet in vitro frond of 6 months is taken out, it is length to be transferred to outside dimension
In 80cm × wide 45cm × high 40cm cyclic water tank, nutrient solution is normal seawater.Condition of culture is：10~15 DEG C of temperature, light
According to 4000~5000lx of intensity, photoperiod 12L:12D, aeration quantity is larger, makes to roll above and below frond, every 7 days quantity of exchanged water are total water
The 50% of body, frond keeps vigorous growth, and by the culture of about 15~20 days, frond length was to more than 20 centimetres, you can after being used for
Continuous nursery is used.
Method provided by the present invention carries out Sargassum horneri Plantlet in vitro, when can substantially prolongs the preservation of Sargassum horneri algae strain major branch
Between.Saved material can quick restoration ecosystem, and can carry out amount reproduction, the seedling that amount reproduction is obtained with it is not saved
The seedling crossed is compared, its morphological feature and growing way no significant difference.The method of the present invention can provide substantial amounts of Sargassum horneri in a short time
High quality seedling, efficiently solves the problems, such as Sargassum horneri Germ-plasma resources protection and scale breeding, is with a wide range of applications.
Brief description of the drawings
Fig. 1：Add influence figure of the nutrient solution of various concentrations uniconazole P to Chlorophyll-a Content；
Fig. 2：Add influence figure of the nutrient solution of various concentrations uniconazole P to carotenoid content.
Embodiment
Found in applicant's research, culture preservation is carried out to Sargassum horneri major branch tip with sterilizing seawater, occurs frond during 30d
Color substantially shoals, the Apparent character such as tip is turned white, and is found by the measure to its chlorophyll a and carotenoid content bright
Aobvious reduction.So, the purpose that the application prepares Storaged media is exactly to increase the dry weight of frond in preservation process, and keep
The content of chlorophyll a and carotenoid.
The method to the present invention is described in detail below.
Embodiment 1
The preserving seed nutrient solution that the present embodiment is prepared, its component is as follows：Add 0.1mol/L NaNO3, 0.1mol/L
NaH2PO4, the sterilizing seawater of 1mg/L uniconazole Ps.Said components and proportioning, speed of growth when can both delay the Sargassum horneri to preserve, again
The restoration ecosystem after its preservation is not influenceed.
Preservation step to Sargassum horneri is as follows：
(1) Sargassum horneri germplasm of the selection with merit, the aufwuch on surface is removed with hairbrush and tweezers, then with sea
Water cleans removal silt and impurity repeatedly；
(2) Sargassum horneri handled well is placed on superclean bench, takes the alcohol-pickled of frond major branch volumetric concentration 75%
25~30s, is then got express developed 3~4 times with a large amount of sterilization seawater, 10 centimetres of frond major branch tip is cut with the scalpel of sterilizing
Fragment as Plantlet in vitro object；
(3) condition of preserving seed is：Carried out in volume is 1000mL triangular flask, each bottled 800mL trainings of triangle
Nutrient solution, places two plants of fronds, and micro inflation makes nutrient solution surface have minute bubbles to emerge；Storage temperature is 5~8 DEG C, intensity of illumination
For 2000~2500lx, the photoperiod is 10L:14D, is provided by white fluorescent lamp.
(4) routine observation preserves the growing state of frond, compared with control (being cultivated in normal sterilizing seawater), frond growth
Slowly, length substantially shortens, the obvious thickening of major branch, and about 30 days or so, frond length length was cut off to 15 cms with scalpel
Base portion, only retains the frond fragment at 10 centimetres of tip, is further continued for being put into progress preserving seed culture in preserving seed nutrient solution.
The influence of different nutrient solutions, temperature, intensity of illumination, inflated size to preservation effect is as follows, the growth of measure
Index includes Fresh Yuxincao (fresh weight, FW), frond length and apparent symptom of frond etc., the wherein increase of Fresh Yuxincao
Amount represents that calculation formula is as follows with specific growth rate (relative growth rate, RGR)：RGR=[(Wt/W0)1/t-1]×
10
W in formulatFor experiment mid-term or at the end of frond fresh weight (g), W0Frond fresh weight (g) when starting for experiment, t is culture
Time (d).
(1) influence of the nutrient solution to Sargassum horneri Plantlet in vitro
Test material is put into the nutrient solution of various concentrations uniconazole P and preserved, three parallel, preservation conditions of every group of processing：
10 DEG C of temperature, intensity of illumination 2500lx, photoperiod 12L:12D, micro inflation.As a result show, Sargassum horneri is in uniconazole P concentration
The 1.0mg/L holding times are most long, are 182 days, concrete condition is shown in Table 1.
The different nutrient solutions of table 1 are to Sargassum horneri Germ-plasma resources protection effect
(2) influence of the temperature to Sargassum horneri Plantlet in vitro
In the Storaged media that test material is put into the present invention, 5 DEG C of temperature is respectively placed in, 8 DEG C, 10 DEG C, 15 DEG C
Illumination box is preserved, and every group of processing three is parallel, other preservation conditions：Intensity of illumination 2500lx, photoperiod 12L:12D, it is micro-
Amount inflation.As a result show, Sargassum horneri holding time in temperature is 5 DEG C of incubators is most long, is 187 days.
Concrete condition is shown in Table 2.
The different temperatures of table 2 is to Sargassum horneri Germ-plasma resources protection effect
(3) influence of the intensity of illumination to Sargassum horneri Plantlet in vitro
In the Storaged media that test material is put into the present invention, 1000lx, 1500lx, 2000lx are respectively placed in,
Preserved under 2500lx, 3000lx intensity of illumination, every group of processing three is parallel, other preservation conditions：10 DEG C of temperature, photoperiod 12L:
12D, micro inflation.As a result show, Sargassum horneri is that the 2000lx holding times are most long in intensity of illumination, is 187 days, is specifically shown in Table 3.
The different illumination intensity of table 3 is to Sargassum horneri Germ-plasma resources protection effect
(4) influence of the aeration quantity to Sargassum horneri Plantlet in vitro
Test material is respectively placed in non-aerating, micro inflation (nutrient solution surface there are minute bubbles to emerge), aeration quantity is big
Culture, 10 DEG C of temperature, intensity of illumination 2500lx, photoperiod 12L are preserved under (being rolled above and below frond):12D.As a result show, Sargassum horneri
The holding time is most long under the conditions of aeration quantity is less, is 157 days, is specifically shown in Table 4.
The inflated size of table 4 is to Sargassum horneri Germ-plasma resources protection effect
(5) influence of the nutrient solution to the main Contents of Photosynthetic Pigments of Sargassum horneri
The assay method of chlorophyll a and carotenoid content is as follows：Take the fresh fronds of 0.1g to grind to form homogenate shape, add
The acetone of 8mL 80% is placed at 4 DEG C of dark and extracts 24h.4000r/min, 4 DEG C of centrifugation 10min abandon precipitation, 80% acetone of supernatant
It is settled to 10mL.Using 80% acetone as blank control, determine 665,652,510, the light absorption value at 480nm wavelength.It is repeated 3 times
More than, calculate average value.The content of chlorophyll a is according to formula w (Chl-a)=(16.29OD665- 8.54OD652)×V/W/
1000 calculate, and the content of carotenoid is according to formula w (Car)=7.6 × (OD480- 1.49 × OD510) × V/W/1000, formula
Middle V is the volume (mL) of extraction acetone, and W is frond quality (g), and unit is mg/g.
Test material is put into the nutrient solution of various concentrations uniconazole P and preserves 30d, chlorophyll a and class Hu trailing plants are determined respectively
Bu Su content, control is used as to preserve the material value of the 0th day.As a result show, 30d, chlorophyll are cultivated in sterilizing seawater naturally
The content of a and carotenoid is remarkably decreased to the 58.43% of control and 73.36, with the addition of different quality concentration (0.1~
2.0mg/L) difference of the main Contents of Photosynthetic Pigments of Sargassum horneri is shown in accompanying drawing after the nutrient solution culture 30d of uniconazole P.As seen from Figure 1, exist
In the range of 0.1~2.0mg/L mass concentrations, with the raising of uniconazole P mass concentration, the Chlorophyll-a Content of Sargassum horneri frond gradually increases
It is high.Wherein, 0.1 and 0.5mg/L experimental groups frond Chlorophyll-a Content is substantially less than control, and relatively control reduces 29.4% respectively
With 6.5%, and 1.0 and 2.0mg/L experimental group is with compareing no significant difference.The content of carotenoid is also 0.1 and 0.5mg/
L experimental groups are substantially less than control；The class Hu Luosu content highests of 1.0mg/L experimental groups, are 0.092 ± 0.007mg/g fresh weights, slightly
Higher than control group (0.090 ± 0.002mg/g fresh weights)；The class Hu Luosu contents of 2.0mg/L experimental group are 0.090 ± 0.008,
With compareing no significant difference.It can be seen that, appropriate uniconazole P is added in nutrient solution can significantly improve the chlorophyll a of Sargassum horneri frond
With class Hu Luosu content, and with 1.0mg/L mass concentrations most useful for chlorophyll a and class Hu Luosu synthesis and accumulation.
The above results show that the culture liquid energy of the present invention effectively carries out the in vitro long-term preservation of Sargassum horneri.
(6) the Sargassum horneri growth recovery situation of Plantlet in vitro
Taken out after Sargassum horneri germplasm with merit is preserved 182 days in the nutrient solution of the present invention and carry out recovery life
Long, restoration ecosystem step is as follows after the preservation：Take out Plantlet in vitro frond, be transferred to outside dimension for long 80cm × wide 45cm ×
In high 40cm cyclic water tank, nutrient solution is normal seawater.Condition of culture is：10~15 DEG C of temperature, intensity of illumination 4000~
5000lx, photoperiod 12L:12D, aeration quantity is larger, makes to roll above and below frond, every 7 days quantity of exchanged water are the 50% of total water body.
Sargassum horneri grows 30d comparison before the Sargassum horneri recovered after the Plantlet in vitro of table 5 and preservation
| Material | Specific growth rate | Length (cm) |
| Before preservation | 2.43±0.05 | 25.11±1.03 |
| After preservation | 2.41±0.16 | 24.89±1.77 |
Material is preserved after culture, growth is vigorous, color is normal, the life of Sargassum horneri after the measure display through growth indexes is preserved
It is long not to be adversely affected because of long-term preserve.By the culture of about 15~20 days, to more than 20 centimetres, i.e., frond can be grown
Used available for follow-up nursery.
Claims (6)
1. a kind of method of Sargassum horneri germ plasm resource Plantlet in vitro, it is characterised in that described method includes the steps：
1) Sargassum horneri that will be preserved is cleaned；
2) cleaned Sargassum horneri is taken to alcohol-pickled 25~30s of algae strain major branch volumetric concentration 75%, then with sterilization seawater
Get express developed 3~4 times, algae strain major branch tip is cut as the object of Plantlet in vitro under sterilising conditions after flushing；
3) algae cut strain major branch tip is put into Storaged media, is 2000~2500lx, light in 5~8 DEG C, intensity of illumination
Cycle is 10L:Preserved under 14D, venting condition；Regularly replace culture medium
4) when algae strain major branch to be saved grows into length and is not shorter than 15 centimetres, the base portion of algae strain major branch is removed, by the algae of reservation
Continue to be put into progress preserving seed culture in preserving seed nutrient solution in strain tip.
2. the method as described in claim 1, it is characterised in that described step 2) algae strain major branch tip, its length is preferred
For 8~12cm.
3. the method as described in claim 1, it is characterised in that described step 3) preserving seed nutrient solution be in sterilizing sea
0.1mol/L NaNO is added in water3, 0.1mol/L NaH2PO4Prepared with 1mg/L uniconazole Ps.
4. the method as described in claim 1, it is characterised in that described step 3) nutrient solution change one within preferably every 10 days
It is secondary.
5. the method as described in claim 1, it is characterised in that described step 3) illumination provided by white fluorescent lamp.
6. make the method for the Sargassum horneri restoration ecosystem that the method described in claim 1 preserves, it is that will take out the frond of Plantlet in vitro, puts
Enter in seawater and cultivate, 10~15 DEG C of the temperature of culture, 4000~5000lx of intensity of illumination, photoperiod 12L:12D, changes water in every 7 days
Measure as the 50% of total water body.
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Cited By (2)
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| CN114051921A (en) * | 2021-11-16 | 2022-02-18 | 温州大学 | Method for in vitro preservation of germplasm resources of sargassum fusiforme rhizoid |
| CN117136833A (en) * | 2023-08-18 | 2023-12-01 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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Cited By (3)
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| CN114051921A (en) * | 2021-11-16 | 2022-02-18 | 温州大学 | Method for in vitro preservation of germplasm resources of sargassum fusiforme rhizoid |
| CN117136833A (en) * | 2023-08-18 | 2023-12-01 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
| CN117136833B (en) * | 2023-08-18 | 2024-05-24 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | Sargassum periwinkle germplasm preservation method |
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