CN104041405B - A kind of method of rapid screening high yield sea-tangle strain - Google Patents

A kind of method of rapid screening high yield sea-tangle strain Download PDF

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CN104041405B
CN104041405B CN201410317512.0A CN201410317512A CN104041405B CN 104041405 B CN104041405 B CN 104041405B CN 201410317512 A CN201410317512 A CN 201410317512A CN 104041405 B CN104041405 B CN 104041405B
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tangle
sea
gametophyte
sporophyte
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CN104041405A (en
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徐东
叶乃好
张晓雯
范晓
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Abstract

A method for rapid screening high yield sea-tangle strain, belongs to kelp seedling breeding technique field, and it adopts chlorophyll fluorescence techniques detect and contrast different lines kelp gametophyte photosynthetical system II photosynthetical system parameter, and the strain that prioritizing selection parameter values is large is reserved seed for planting.The chlorophyll fluorescence techniques that the present invention adopts is a kind of efficient, undamaged detection technique, fluoroscopic examination is required gametophyte sample size few (0.05g fresh weight) not only, and can not have a negative impact to gametophyte, the sample after detection can continue on for gametophyte clone breeding; The present invention is by detecting the height screening high yield sea-tangle new lines of gametophyte photosynthetic parameters level, and compare traditional seed selection and greatly save the seed selection time, seed selection result is with strong points.

Description

A kind of method of rapid screening high yield sea-tangle strain
Technical field:
The invention belongs to kelp seedling breeding technique field, relate to a kind of method of rapid screening high yield sea-tangle strain particularly.
Background technology:
Sea-tangle (Saccharina japonica) is under the jurisdiction of Phaeophyta (Phaeophyta), Phaeophyceae (Phaeosporae), Laminariales (Laminariales), Laminariaceae (Laminariaceae), sugar maple genus (Saccharina) in classification.Be a kind of cold aqueous large-scale economical alga, being mainly distributed in the Pacific Ocean and Atlantic Ocean north Coast, is the important component part of littoral marine ecosystems.Sea-tangle is not only a kind of ocean food vegetables of rich in nutrition content, and be rich in the economic sectors such as algin, mannitol, iodine, polysaccharide from Dunaliella salina, be the important source material of the industries such as seaweed chemical, medicines and health protection and agricultural fertilizer simultaneously.
China former without sea-tangle distribution, until nineteen twenty-seven sea-tangle just successfully introduce China.The fifties in last century starts, China carries out the research of sea-tangle genetic improvement, about more than 20 improved seeds (being) are cultivated altogether, it applies the development greatly facilitating sea-tangle industry, and make China become maximum in the world sea-tangle cultivation state, from Liaoning in the north, all there is commerial growing in Shandong to southern Jiangsu, Zhejiang and Fujian.But due to the restriction of conventional manufacturing techniques, the problems such as deterioration of variety, heterozygosis, resistance difference are more and more outstanding.To cultivate and the demand of seed selection good quality and high output, the sea-tangle new lines of strong stress resistance and new varieties is day by day urgent.
At present, the seed selection of sea-tangle mainly comprises land for growing field crops seed selection and hybridization cultivation two kinds of modes.These two kinds of modes are cultivated all needs experience indoor raising of seedling and sea area to form two vegetative stages.Therefore, existing selection not only experimental period long, need to go through 10 months, and in experimentation, need a large amount of human and material resources.In addition, owing to being subject to the impact of sea environment, there is certain blindness and randomness in seed selection result, and not all seed selection result can reach ideal character.
Summary of the invention:
The technical problem to be solved in the present invention is to provide a kind of method of rapid screening high yield sea-tangle strain.The present invention, by detecting and contrasting different lines kelp gametophyte photosynthetical system II photosynthetical system parameter size, reaches the high yield sea-tangle new lines that rapid screening possesses high photosynthetic capacity, greatly saves the time of traditional sea-tangle seed selection, reduce seed selection cost.
The present invention is achieved through the following technical solutions:
A method for rapid screening high yield sea-tangle strain, it adopts chlorophyll fluorescence techniques detect and contrast the photosynthetical system parameter of different lines kelp gametophyte photosynthetical system II, and the strain that prioritizing selection parameter values is large is reserved seed for planting.
Further, the maximum photosynthesis amount suboutput F of gametophyte of strain of reserving seed for planting described in v/ F mbe greater than 0.55, actual photosynthesis quantum yield Y (II) is greater than 0.50.
The concrete steps of the inventive method are as follows:
A method for rapid screening high yield sea-tangle strain, concrete steps are as follows:
1) final trip sporophyte water obtains: collection possesses ripe sporangial different sea-tangle strain and plants vegetables, and after low temperature process is transported to laboratory, adopts indoor method of making herbs dry gradually in the shade to obtain the final trip sporophyte water of different lines sea-tangle;
Further, sea-tangle refers to the transhipment of described low temperature process, and after possessing of gathering, ripe sporangial Laminaria Japonica adopts gauze parcel, be placed in incubation chamber on the rocks and be transported to laboratory, incubation chamber temperature on the rocks controls at 2 ~ 5 DEG C, and haulage time is within 6 hours.
Further, the method that described indoor method of making herbs dry gradually in the shade acquisition sea-tangle finally swims sporophyte water is divided into following three steps: 1) adopt low temperature sterilization seawater flushing to possess sporangial Laminaria Japonica; 2) clean possessing in sporangial Laminaria Japonica dark is placed in 10 DEG C of illumination boxs to dry in the shade; 3) will possess sporangial Laminaria Japonica as 1 ~ 3h in 10 ~ 12 DEG C of sterilizing seawater after drying in the shade, period adopts glass bar to stir seawater, obtains sea-tangle and initially swims sporophyte water, detects trip sporophyte density and is greater than 3 × 10 5cells/ml; 4) sporophyte water filtration will initially be swum in the container of sterilizing, trip sporophyte water in the middle of obtaining; 5) by the PES medium of mesospore body water and sterilizing by volume 1:1 mix, obtain final trip sporophyte water.
(2) gametophytic acquisition: the final trip sporophyte Aquaponic of acquisition 28 ~ 35 days, trip sporophyte is divided into cotton-shaped gametophyte, and gametophyte density is 3 ~ 6g fresh weight/L;
Further, the condition of culture of described step (2) is intensity of illumination is 20 μm of ol photons m -2s -1, temperature is 10 ± 1 DEG C, and light dark period is 12h (L): 12h (D), i.e. illumination 12h, dark 12h.
(3) chlorophyll fluorescence detects: after 10 ~ 20 minutes, adopt PAM chlorophyll fluorescence techniques to detect the maximum photosynthesis amount suboutput F of its photosynthetical system II the cotton-shaped gametophyte of different lines obtained dark adaptation in 10 ± 1 DEG C of incubators v/ F m, actual photosynthesis quantum yield Y (II).
Further, described PAM chlorophyll fluorescence techniques detects its photosynthetical system II photochemistry parameter concrete operation method, 1) carry out photosynthetic parameters setting to binary channels modulated chlorophyll fluorescence instrument Dual-PAM-100, arranging photochemical light intensity is 22 μm of ol photons m -2s -1, the saturation pulse time is 300ms, and saturated light intensity is 1000 μm of ol photons m -2s -1; 2) draw 1ml density be the kelp gametophyte of the dark adaptation process of 5g fresh weight/L on slide, slide is placed in the middle of optic probe, opens and detect light and obtain minimum fluorescent value F 0, then use strong saturation pulse light (1000 μm of ol photons m -2s -1, 300ms) excite, obtain dark lower maximum fluorescence parameter F m, then open actinic light, minimum fluorescence F under acquisition light 0', open saturation pulse light after 5min, maximum fluorescence F under acquisition light m'; 3) according to the maximum photosynthesis amount suboutput F of formulae discovery v/ F m=(F m-F 0)/F mwith actual photosynthesis quantum yield Y (II)=(F m' – F 0')/F m'.
(4) according to the maximum photosynthesis amount suboutput F of gametophyte v/ F mbe greater than 0.55, actual photosynthesis quantum yield Y (II) is greater than 0.50, selects high yield sea-tangle strain.
The present invention's beneficial effect compared with prior art:
1) the present invention is by detecting the height screening high yield sea-tangle new lines of gametophyte photosynthetic parameters level, compares traditional seed selection and greatly saves the seed selection time; 2) chlorophyll fluorescence techniques adopted is a kind of efficient, undamaged detection technique, fluoroscopic examination is required gametophyte sample size few (0.05g fresh weight) not only, and can not have a negative impact to gametophyte, the sample after detection can continue on for gametophyte clone breeding; 3) seed selection result is with strong points, the level height of gametophyte photosynthetic parameters is greater than 0.8 with sporophyll weight in wet base size relative coefficient, therefore, the present invention is undertaken selecting the technical scheme of high yield sea-tangle breed breeding to be feasible by the height of gametophyte photosynthetic parameters level; 4) technical scheme of the present invention can be implemented on a large scale, meets the needs of kelp seedling seed selection.
Accompanying drawing illustrates:
The photosynthetic parameters size of Fig. 1 different lines kelp gametophyte photosynthetical system II
Fig. 2 different lines sea-tangle sea area cultivation weight in wet base size
Fig. 3 different lines sea-tangle per mu yield size
Embodiment:
Below by embodiment, the technical solution of the present invention is further explained, but protection scope of the present invention is not by any pro forma restriction of embodiment.
Embodiment 1
A method for rapid screening high yield sea-tangle strain, it adopts chlorophyll fluorescence techniques detect and contrast the photosynthetical system parameter of different lines kelp gametophyte photosynthetical system II,
1) experiment different lines sea-tangle plants vegetables and picks up from laminaria culture district, Sang Gou gulf, Rongcheng City of Shandong Province, is labeled as ZS respectively, BN, JA and JB.
2) in experimentation, the composition of PES medium used and final concentration are: sodium nitrate (NaNO 3) 46mg/L, glycerophosphate (C 3h 7na 2o 6p5H 2o) 6.66mg/L, boric acid (H 3bO 3) ethylenediamine tetra-acetic acid (C of 3.8mg/L, 0.5M pH=8 10h 16n 2o 8) 18.2ml/L, Trismabase (C 4h 11nO 3) 66.6mg/L, six water ammonium ferric sulfate ((NH 4) 2fe (SO 4) 26H 2o) 2.34mg/L, iron chloride (FeCl 3) 100 μ g/L, manganese sulfate monohydrate (MnSO 4h 2o) 546 μ g/L, white vitriol (ZnSO 47H 2o) 73.4 μ g/L, cobalt sulfate (CoSO 47H 2o) 16 μ g/L, cobalamin (C 63h 88coN 14o 14p) 1.34 μ g/L, vitamin B1 (C 12h 16n 4oSHCl) 66 μ g/L, vitamin h (C 10h 16n 2o 3s) 0.66 μ g/L.Indoor incubation adopts constant indoor temperature cultivation, carries out in plant incubator (HP1000G-D type, the auspicious China in Wuhan).Arranging intensity of illumination is 20 μm of ol photons m -2s -1, temperature is 10 ± 1 DEG C, and light dark period is 12h (L): 12h (D).
3) concrete implementation method
(1) final trip sporophyte water obtains: on June 18th, 2012 gathers four different lines sea-tangles in sea area, Sang Gou gulf, Rongcheng City of Shandong Province and plants vegetables, and is labeled as ZS, BN, JA and JB respectively.Have sporangial blade with scissors clip, put into the incubation chamber on the rocks prepared in advance after 10 layers of gauze parcel, after about 4 hours, sample is transported to Huanghai Sea aquatic products research institute algae laboratory.In transportation, incubation chamber temperature on the rocks controls at 2 ~ 5 DEG C; Adopt method of making herbs dry gradually in the shade to obtain different lines trip sporophyte water, having operating procedure is: a) adopt low temperature 10 DEG C of sterilizing seawater flushings to possess sporangial Laminaria Japonica; B) possessing in sporangial Laminaria Japonica dark of cleaning is placed in 10 DEG C of illumination boxs to dry in the shade 60min; C) will possess sporangial Laminaria Japonica as 2h in 10 DEG C of sterilizing seawater after drying in the shade, period adopts glass bar to stir seawater 3 times, obtains sea-tangle and initially swims sporophyte water.Drawing the initial sporophyte water of 10 μ l is placed in after slide adds a cover cover glass, detects trip spore density and be greater than 3 × 10 under light microscope four times of object lens 5cells/ml; D) be filtered in the conical flask of sterilizing by initially swimming the 20 mesh sieve thin,tough silk of sporophyte water by high-temperature sterilization, trip sporophyte water in the middle of obtaining; E) pipetting 50ml sporophyte water is in the sterilizing conical flask of 200ml in volume, supplements the PES medium adding 50ml sterilizing, obtains final trip sporophyte water.
(2) gametophytic acquisition: the final trip sporophyte water of acquisition is being 20 μm of olphotons m in intensity of illumination -2s -1, temperature is 10 ± 1 DEG C, and light dark period is cultivate 30 days under the condition of 12h:12h, and trip sporophyte is divided into cotton-shaped gametophyte, and gametophyte density is 5g fresh weight/L.
(3) chlorophyll fluorescence detects: after 15 minutes, adopt PAM chlorophyll fluorescence techniques to detect the maximum photosynthesis amount suboutput F of its photosynthetical system II the cotton-shaped gametophyte of different lines obtained dark adaptation in 10 DEG C of incubators v/ F m, actual photosynthesis quantum yield Y (II).Concrete operation method is, 1) photosynthetic parameters setting is carried out to binary channels modulated chlorophyll fluorescence instrument Dual-PAM-100 (Heinz Walz, Germany), and arranging actinic light intensity is 22 μm of ol photons m -2s -1, the saturation pulse time is 300ms, and light intensity is 1000 μm of ol photonsm -2s -1; 2) draw 1ml density be the kelp gametophyte of the dark adaptation process of 5g fresh weight/L on slide, slide is placed in the middle of optic probe, opens and detect light and obtain minimum fluorescent value F 0, then use strong saturation pulse light (1000 μm of ol photons m -2s -1, 300ms) excite, obtain dark lower maximum fluorescence parameter F m, then open actinic light, minimum fluorescence F under acquisition light 0', open saturation pulse light after 5min, maximum fluorescence F under acquisition light m'; 3) according to the maximum photosynthesis amount suboutput F of formulae discovery v/ F m=(F m-F 0)/F mwith actual photosynthesis quantum yield Y (II)=(F m' – F 0')/F m'.
(4) the maximum photosynthesis amount suboutput F of different lines sea-tangle v/ F mwith actual photosynthesis quantum yield Y (II), as shown in Figure 1, intend selecting maximum photosynthesis amount suboutput F v/ F mbe greater than 0.55, the JA strain that actual photosynthesis quantum yield Y (II) is greater than 0.50 to be reserved seed for planting strain as selection high yield sea-tangle.
Embodiment 2 swims spore breeding and sporophyte sea area cultivates:
Traditional trip spore breeding technique is adopted to cultivate the different lines sea tangle sporophyte seedling of embodiment 1, reach (being about 2cm) after nursery outbound specification, be transferred to laminaria culture district, Sang Gou gulf, Rongcheng, Shandong October, support temporarily after one month, divide seedling to be cultured to gather May in next year, during in January, 2013 to May, detect sea-tangle weight in wet base size month by month.The maximum photosynthesis amount suboutput F of different lines kelp gametophyte v/ F mwith actual amount suboutput Y (II) as shown in Figure 1.As shown in Figure 2, different lines sea-tangle weight in wet base all significantly increases (p<0.05) with culturing time, and the average weight in wet base of different lines sea-tangle in May is 1.448 ± 0.010kg, 1.063 ± 0.054kg, 1.949 ± 0.045kg, 1.250 ± 0.053kg.One-way analysis of variance result shows, and the weight in wet base of strain JA is significantly higher than other three strains (p<0.05).According to often restricting 40, every mu 400 rope calculates different lines per mu yield as shown in Figure 3, by May, the per mu yield of ZS, BN, JA and JB 4 different lines sea-tangles is respectively 23.168 ± 0.153 tons/mu, 17.013 ± 0.870 tons/mu, 31.189 ± 0.721 tons/mu and 20.000 ± 0.847 tons/mu.Wherein the per mu yield of JA strain is significantly higher than other three strains (p<0.05), and per mu yield, higher than 30 tons/mu, is high yield sea-tangle strain.
One-way analysis of variance result shows, the F of strain JA v/ F mother three strain ZS, BN and JB (p<0.05) are significantly higher than with Y (II).
Adopt SPSS17.0 statistical software, to the maximum photosynthesis amount suboutput F of different lines kelp gametophyte v/ F m, Y (II) carries out correlation regression analysis from different month sea-tangle weight in wet base, result is respectively as shown in Table 1 and Table 2.Result display F v/ F m, Y (II) all with sea-tangle month by month weight in wet base correlation significantly (p<0.05), relative coefficient R 2all be greater than 0.8.
The correlation of the maximum photosynthesis amount suboutput Fv/Fm of table 1 and sea-tangle weight in wet base month by month
Date Linear equation Relative coefficient (R 2)
20130107 Y＝0.9081X-0.2483 0.9119
20130123 Y＝4.3808X-1.6366 0.8052
20130222 Y＝3.5602X-1.0096 0.8560
20130315 Y＝5.0935X-1.4360 0.8587
20130421 Y＝4.5813X-0.9732 0.9054
20130517 Y＝5.0402X-0.9664 0.9608
The correlation of table 2 actual photosynthesis quantum yield YII and sea-tangle weight in wet base month by month
Date Linear equation Relative coefficient (R 2)
20130107 Y＝0.8819X-0.1895 0.8985
20130123 Y＝4.2880X-1.3671 0.8060
20130222 Y＝3.4736X-0.7858 0.8513
20130315 Y＝4.9706X-1.1163 0.8543
20130421 Y＝4.4196X-0.6640 0.8803
20130517 Y＝4.8355X-0.6149 0.9239
This experiment, from 18 days June in 2012, stops on May 17th, 2013.By measuring different lines kelp gametophyte photosynthetic parameters F v/ F mfound that with Y (II) size and sea area cultivation, F v/ F mwith Y (II) all with fresh weight size significant correlation.By this experimental technique screening high yield sea-tangle new lines (JA) 1, per mu yield is higher than 30 tons.Therefore adopt this experimental technique to reach high yield sea-tangle new lines that rapid screening possesses high photosynthetic capacity, greatly saves the time of traditional sea-tangle seed selection, reduces seed selection cost.

Claims (3)

1. a method for rapid screening high yield sea-tangle strain, it is characterized in that it adopts chlorophyll fluorescence techniques detect and contrast the photosynthetical system parameter of different lines kelp gametophyte photosynthetical system II, the strain that prioritizing selection parameter values is large is reserved seed for planting, and concrete steps are as follows:
1) final trip sporophyte water obtains: collection possesses ripe sporangial different sea-tangle strain and plants vegetables, and after low temperature process is transported to laboratory, adopts indoor method of making herbs dry gradually in the shade to obtain different lines sea-tangle and finally swims sporophyte water;
2) gametophytic acquisition: the final trip sporophyte Aquaponic of acquisition 28 ~ 35 days, condition of culture is intensity of illumination is 20 μm of ol photons m -2s -1, temperature is 10 ± 1 DEG C, and light dark period is 12h:12h; Trip sporophyte is divided into cotton-shaped gametophyte, and gametophyte density is 3 ~ 6g fresh weight/L;
3) chlorophyll fluorescence detects: after 10 ~ 20 minutes, adopt PAM chlorophyll fluorescence techniques to detect the maximum photosynthesis amount suboutput F of its photosynthetical system II the cotton-shaped gametophyte of different lines obtained dark adaptation in 10 ± 1 DEG C of incubators v/ F m, actual photosynthesis quantum yield Y (II);
4) according to the maximum photosynthesis amount suboutput F of gametophyte v/ F mbe greater than 0.55, actual photosynthesis quantum yield Y (II) is greater than 0.50, selects high yield sea-tangle strain;
Step 1) described in indoor method of making herbs dry gradually in the shade obtain the method that different lines sea-tangle finally swims sporophyte water and be divided into following three steps, (1) adopts low temperature sterilization seawater flushing to possess sporangial Laminaria Japonica; (2) clean possessing in sporangial Laminaria Japonica dark is placed in 10 DEG C of illumination boxs to dry in the shade; (3) will possess sporangial Laminaria Japonica as 1 ~ 3h in 10 ~ 12 DEG C of sterilizing seawater after drying in the shade, period adopts glass bar to stir seawater, obtains sea-tangle and initially swims sporophyte water, detects trip spore density and is greater than 3 × 10 5cells/ml; (4) sporophyte water filtration will initially be swum in the container of sterilizing, trip sporophyte water in the middle of obtaining; (5) the PES medium of centre being swum sporophyte water and sterilizing by volume 1:1 mixes, and obtains final trip sporophyte water.
2. the method for a kind of rapid screening high yield sea-tangle strain according to claim 1, it is characterized in that step 1) described step low temperature process transhipment sea-tangle refers to, after possessing of gathering, ripe sporangial Laminaria Japonica adopts gauze parcel, be placed in incubation chamber on the rocks and be transported to laboratory, incubation chamber temperature on the rocks controls at 2 ~ 5 DEG C, and haulage time is within 6 hours.
3. the method for a kind of rapid screening high yield sea-tangle strain according to claim 1, it is characterized in that step 3) described in PAM chlorophyll fluorescence techniques detect its photosynthetical system II photochemistry parameter concrete operation method and be, (1) carry out photosynthetic parameters setting to binary channels modulated chlorophyll fluorescence instrument Dual-PAM-100, arranging photochemical light intensity is 22 μm of ol photons m -2s -1, the saturation pulse time is 300ms, and saturated light intensity is 1000 μm of olphotons m -2s -1; (2) draw 1ml density be the kelp gametophyte of the dark adaptation process of 5g fresh weight/L on slide, slide is placed in the middle of optic probe, opens and detect light and obtain minimum fluorescent value F 0, then use strong saturation pulse light 1000 μm of ol photons m -2s -1, 300ms excites, and obtains dark lower maximum fluorescence parameter F m, then open actinic light, minimum fluorescence F under acquisition light 0', open saturation pulse light after 5min, maximum fluorescence F under acquisition light m'; 3) according to the maximum photosynthesis amount suboutput F of formulae discovery v/ F m=(F m-F 0)/F mwith actual photosynthesis quantum yield Y (II)=(F m' – F 0')/F m'.
CN201410317512.0A 2014-07-04 2014-07-04 A kind of method of rapid screening high yield sea-tangle strain Expired - Fee Related CN104041405B (en)

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