CN101717748B - Method for generating macrogametocyte by somatic cell of inducing undaria pinnatifida parthenogenesis for juvenile sporophyte - Google Patents
Method for generating macrogametocyte by somatic cell of inducing undaria pinnatifida parthenogenesis for juvenile sporophyte Download PDFInfo
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- CN101717748B CN101717748B CN2009102202613A CN200910220261A CN101717748B CN 101717748 B CN101717748 B CN 101717748B CN 2009102202613 A CN2009102202613 A CN 2009102202613A CN 200910220261 A CN200910220261 A CN 200910220261A CN 101717748 B CN101717748 B CN 101717748B
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Abstract
The invention discloses a method for generating macrogametocytes by somatic cells of inducing undaria pinnatifida parthenogenesis juvenile sporophytes, which has easy operation, low cost and high maturation rate of the generated macrogametocytes and can provide a large quantity of the macrogametocytes for the large-scale seedling production of undaria pinnatifida. The method comprises the following steps of: cutting undaria pinnatifida macrogametocytes stored and cultured in a room into pieces and then culturing under the conditions of temperature suitable for the undaria pinnatifida macrogametocytes to grow to be mature, illumination and nutritive salt, wherein when the undaria pinnatifida macrogametocytes grow to be mature, form oocysts in large quantities and ovulate with ovums, a small quantity of the fertilized ovums are germinated to seedlings through parthenogenesis because unfertilized ovums are died by being unfertilized by spermatozoa; and when the somatic cells of the parthenogenesis seedlings are grown to 0.4-0.6 cm, adopting a rhizoid cutting method to enable fronds to lose polarity and constraint on the somatic cells, and inducing the somatic cells of the seedlings to generate the macrogametocytes.
Description
Technical field:
The present invention relates to a kind of working method of wakame megagametophyte, especially a kind of megagametophyte maturing rate of simple to operate, with low cost, institute's output is high, can be the method that wakame mass-producing seed produces the generating macrogametocyte by somatic cell of inducing undaria pinnatifida gynecogenic juvenile sporophyte that a large amount of megagametophytes are provided.
Background technology:
Wakame (Undaria pinnatifida) belongs to phaeophyta, brown sub-guiding principle, Laminariales, Alariaceae, undaria; Belong to the eurythermic algae, its delicious flavour, nutritious; Contain 19 seed amino acids, protein, sugar part and the VITAMINs that comprise whole essential amino acids; Because the content of cellulose of wakame is merely about 3% of dry weight, therefore be easy to by human consumption, be very popular heath food.
The large-scale frond of wakame is a sporophyte, and begin seedling and occur at natural waters annual autumn, through winter be grown to serve as the frond of 1～2m spring; Spring frond germinate, form special reproductive organ sporophyll in stem, form sporocyst on sporophyll surface; Spring and summer the sporocyst maturation emit the trip spore; Get into nursery stage, diffuse that frond is dead behind the spore runs off, finish the life cycle in 1 year by a definite date.Have 50% to form megagametophyte in the spore that kind of algae is emitted approximately, 50% forms microgametophyte, and female and male gametophytes is spent the pliotherm period in summer during above 23 ℃ with the state of dormancy in water temperature through growing after.Female and male gametophytes was recovered from dormancy and is also reached maturity gradually after water temperature descended in the fall; Megagametophyte forms egg capsule and discharges ovum; Microgametophyte forms antheridium and emits sperm; Sprouting after the feritilization of ovum is juvenile sporophyte, and juvenile sporophyte is grown to serve as the wakame seedling very soon under suitable water temperature condition.
At present; The mode of collecting seedling in the production of wakame artificial breeding mainly contains spore and collects seedling and the gametophyte dual mode of collecting seedling; The shortcoming that spore is collected seedling is to adopt the spore time to receive kind of an algae propagation season limit, and seedling raise period reaches more than 3 months in the area, Dalian, and seedling cost is high, have a big risk.It is to develop in recent years and a kind of new wakame that grows up is collected seedling and method for culturing seedlings that gametophyte is collected seedling; Its principle is the wakame spore to be captured in petridish or the flask in a large number in nursery stage at wakame; Under indoor suitable condition, cultivate into the female and male gametophytes of some amount; Adopt the isolating method of male and female can female and male gametophytes be separated the back single culture, after water temperature descended in the fall, the method for employing gametophyte chopping made gametophyte go up the directly entering stage of maturity attached to the seedling rope; The sperm fertilization that ovum that megagametophyte forms and microgametophyte are emitted forms the wakame seedling; Seedling raise period had only about 1 month, had reduced the risk of growing seedlings, and had increased substantially the success ratio of growing seedlings.The gametophyte another one advantage of collecting seedling is to use the megagametophyte of separation and Culture to carry out different strains and different local race intermolecular hybrids with microgametophyte, cultivates the good quality and high output seed.
But; Because the female and male gametophytes limited amount after separating; Especially the microgametophyte cell quantity is many; A cell can form a plurality of antheridiums and compare, and the megagametophyte cell quantity is few and have only a terminal cell can form egg capsule, produces the requirement to its quantity though therefore still be difficult to reach the mass-producing seed through indoor multiplication culture megagametophyte.
Summary of the invention:
The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, provide a kind of megagametophyte maturing rate simple to operate, with low cost, institute's output high, can be the method that wakame mass-producing seed produces the generating macrogametocyte by somatic cell of inducing undaria pinnatifida gynecogenic juvenile sporophyte that a large amount of megagametophytes are provided.
Technical solution of the present invention is: a kind of method of generating macrogametocyte by somatic cell of inducing undaria pinnatifida gynecogenic juvenile sporophyte, carry out according to following steps:
A. the wakame megagametophyte 10mg that goes bail for and deposit cultivation; Plant a megagametophyte body fluid with organizing stirrer chopping to 190～210 μ m algae sections to form after adding 300ml sterilization seawater; To plant megagametophyte body fluid moves in a plurality of petridish by the 1ml/ petridish; Adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are that 2000Lx, light application time are to cultivate nutrient solution full dose exchange in per 3 days 1 time 12 days under 12 hours/day the condition;
B. the petridish that growth is had a parthenogenesis seedling is that 15 ℃, intensity of illumination are that 2000Lx, light application time are to continue to be cultured to parthenogenesis seedling length under 12 hours/day the condition to reach 0.4～0.6cm, nutrient solution full dose exchange in per 3 days 1 time in temperature;
C. the parthenogenesis seedling is peeled from the petridish bottom surface; Be layered on through on the slide glass of sterilization; The seedling rhizoid is removed from the position of handle; To remain blade and move on in the beaker, adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are that 2000Lx, light application time are to cultivate nutrient solution full dose exchange in per 7 days 1 time 2 months under 12 hours/day the condition;
D. the blade of a plurality of growth megagametophyte algaes being rolled into a ball moves on in the tissue mashing machine; Smash algae section formation megagametophyte body fluid after adding nutrient solution to 490～510 μ m; Blade weight in wet base and nutrient solution volume ratio are 10mg: 300ml; Draw megagamete body fluid and move in a plurality of petridish by the 1ml/ petridish, adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are cultivation 2 months 2000Lx, 24 hours/degree/day illumination condition under;
E. the megagametophyte algae in petridish group is placed the flask after the sterilization; Add behind the nutrient solution under the said culture condition of d step propagation and preserve and cultivate, nutrient solution exchange in per 10 days half amount was shaken in per 10 days bottle for 1 time; Cultivated 2 months; Megagametophyte in the flask is moved on in 2～3 flasks together with nutrient solution, add nutrient solution again and carry out branch bottle propagation, obtain megagametophyte;
Said nutrient solution adds NaNO for being heated in 80 ℃ of nature seawaters after filtering to 1L
3100mg, NaH
2PO
412H
2O 20mg and PI solution 1ml.
The wakame megagametophyte that the said preservation of above-mentioned a step is cultivated is to obtain through following steps:
F. in the nursery stage of wakame; Get the ripe sporophyll of sophisticated kind of algae of buoyant raft cultivation; Behind dry in the shade stimulation, put into the beaker that adds the sterilization seawater, when the seawater in the beaker is light brown, pull sporophyll out, get spore fluid with suction pipe and move on in the petridish that adds nutrient solution; Is that 20 ℃, intensity of illumination are to cultivate nutrient solution full dose exchange in per 10 days 1 time 1 month under the condition of 2000Lx with petridish in temperature;
G. after microscopically is distinguished megagametophyte and microgametophyte; Megagametophyte is moved on in the other petridish; Add nutrient solution and carried out wakame megagametophyte separation and Culture 2 months, group peels from the petridish bottom surface with the megagametophyte algae, moves on in the flask; Preserve cultivation after adding nutrient solution, nutrient solution full dose exchange in 1 month 1 time.
The present invention is that the unfertilized egg parthenogenesis that utilizes the wakame megagametophyte to discharge forms haploid seedling; The induction method that rhizoid is removed in employing makes its generating macrogametocyte by somatic cell; The ovum of discharging after this megagametophyte maturation can be sprouted into the wakame seedling with the sperm fertilization that normal microgametophyte maturation is emitted, and is simple to operate, with low cost.Because the kind megagametophyte in its source of megagametophyte of institute's output has identical inherited character, can prolonged preservation cultivate equally with normal female and male gametophytes, can be widely used in wakame cross-breeding research and seed and produce; The megagametophyte of institute of the present invention output; Through cultivating a large amount of high-quality (fast growth, quality good) high-yield hybrid seedling with the hybridization of other strains and local race microgametophyte; Fundamentally solve megagametophyte quantity not sufficient problem in the production of wakame cross hybrid seedling, reached mass-producing commercial application level.
Embodiment:
Embodiment 1:
Can male and female wakame gametophyte be separated the back according to the isolating method of prior art male and female and preserve cultivation separately, carry out as follows afterwards:
A. wakame megagametophyte (weight in wet base) 10mg that goes bail for and deposit cultivation; Using behind the interpolation sterilization 300ml seawater and organizing the stirrer chopping is 190～210 μ m algae sections; Form kind of a megagametophyte body fluid; To plant megagametophyte body fluid with suction pipe and move to a plurality of petridish (diameter 9cm by the 1ml/ petridish; Down with) in, add that to move on to temperature behind the nutrient solution (adding the sterilization seawater of nutritive salt) be that 20 ℃, intensity of illumination are that 2000Lx, light application time are to cultivate nutrient solution full dose exchange in per 3 days 1 time 12 days under 12 hours/day the condition; The weight in wet base of wakame megagametophyte can be adjusted in the ratio of above-mentioned 10mg: 300ml with the volume that adds the sterilization seawater.
Cultivate after 7 days and observe; Visible part wakame megagametophyte germinates and ripe form egg capsule and discharge ovum; Hang over and wait for fertilization on the egg capsule bag; Cultivate after 12 days 80% megagametophyte and discharge ovum, the visible most ovum of microscopically is fallen on the petridish bottom surface deadly because of no sperm fertilization, has the unfertilized egg parthenogenesis about 10% to sprout the formation seedling approximately.
B. having the petridish of parthenogenesis seedling to move on to temperature growth is that 15 ℃, intensity of illumination are that 2000Lx, light application time are to continue to be cultured to parthenogenesis seedling length under 12 hours/day the condition to reach 0.4～0.6cm, nutrient solution full dose exchange in per 3 days 1 time;
C. with the sterilization tweezers parthenogenesis seedling is peeled from the petridish bottom surface; Be layered on through on the slide glass of sterilization; The sterilization blade is removed the seedling rhizoid from the position of handle; To remain blade and move on in the 300ml beaker, add that to move on to temperature behind the nutrient solution be that 20 ℃, intensity of illumination are that 2000Lx, light application time are to cultivate nutrient solution full dose exchange in per 7 days 1 time 2 months under 12 hours/day the condition.
The frond (blade) of excision behind the rhizoid loses polarity, and to multi-direction growth, blade is polygon after the constraint of cell detachment frond, and microscopically visible part cell volume expands, and protoplasma increases and is persimmon in the cell.Subsequently, visible cystite produces projection and stretches out gradually to elongate and forms megagametophyte.Prolongation along with incubation time; The quantity that megagametophyte forms increases gradually; Cultivate after 1 month and observe; The rate of formation of megagametophyte reaches more than 10% of somatocyte sum on the partial blade, induces the megagametophyte of generation to carry out cell fission fast in the suitable culture condition, and cultivating has become macroscopic many cells branch megagametophyte algae group after 2 months.
D. the blade of a plurality of growth megagametophyte algaes being rolled into a ball moves on in the tissue mashing machine; Add the algae section of smashing behind the nutrient solution to 490～510 μ m and form megagametophyte body fluid; Blade weight in wet base and nutrient solution volume ratio are 10mg: 300ml; Draw megagamete body fluid and move in a plurality of petridish, add that to move on to temperature behind the nutrient solution be that 20 ℃, intensity of illumination are that 2000Lx, 24 hours/degree/day illumination condition were cultivated 2 months down by the 1ml/ petridish.
Cultivate after 1 month, the visible megagametophyte of naked eyes has been covered with the petridish bottom surface, cultivates after 2 months, and megagametophyte has covered with petridish.
E. use the sterilization hairbrush down, move on in the 5000ml flask after the sterilization, add behind the nutrient solution under the said condition of d step propagation and preserve and cultivate the group of the megagametophyte algae in petridish brush; Nutrient solution exchange in per 10 days half amount; Shook bottle in per 10 days 1 time, cultivate after 2 months, visible megagametophyte algae is rolled into a ball diameter and reaches more than the 1mm; Be brown in the flask; Can move on to the megagametophyte in the flask in 2～3 flasks together with nutrient solution this moment, carries out branch bottle propagation behind the interpolation nutrient solution, thereby obtain a large amount of megagametophytes.
Embodiment 2:
Other steps are all like embodiment 1, and the wakame megagametophyte that the said preservation of a step is cultivated can obtain in such a way:
F. in the nursery stage of wakame; Get the sophisticated sporophyll of buoyant raft cultivar algae; Behind dry in the shade stimulation, put into the beaker that adds the sterilization seawater, diffuse and pull sporophyll when spore fluid is light brown out, move on in the petridish that adds nutrient solution (adding the sterilization seawater of nutritive salt) with the suction pipe spore fluid that takes a morsel when having observed a large amount of spores; After 2 hours; It is thus clear that the petridish bottom surface has circular sporoblast to adhere to, it is that 20 ℃, illumination are to cultivate nutrient solution full dose exchange in per 10 days 1 time 1 month under the condition of 2000Lx that petridish is moved on to temperature;
Cultivate after 2 days, sprout the formation female and male gametophytes, cultivate after 1 month the female and male gametophytes algae group of naked eyes visible growth in petridish attached to the wakame sporoblast in the petridish.
E. after microscopically is distinguished megagametophyte and microgametophyte, megagametophyte is moved on in the other petridish, carry out wakame gametophyte separation and Culture behind the interpolation nutrient solution with the sterilization tweezers; Cultivate after 2 months; The wakame megagametophyte algae group that is grown in the petridish has reached about 1.0mm, and group peels from the petridish bottom surface with the megagametophyte algae, moves on in the 300ml flask; Add nutrient solution and preserve cultivation, nutrient solution full dose exchange in 1 month 1 time.
A～said nutrient solution of g step is to be heated in 80 ℃ of nature seawaters after filtering to 1L and adds NaNO
3100mg, NaH
2PO
412H
2O 20mg and PI solution 1ml.
PI solution is disclosed in the damp person of outstanding talent in west, and thousand former light are male.Algae organon [M] Shu Jing: upright altogether Co., Ltd., 1979:281～293. of publishing
Experiment: in mid or late August; The megagametophyte after the embodiment of the invention propagation and the normal microgametophyte of different strain wakames of indoor cultivation pressed 2: 1 mixed; With being sprinkling upon on the seed rope of seedling curtain after the tissue mashing machine chopping algae section to the 200 μ m; Leave standstill and treated that female and male gametophytes behind the adhere firmly moved on to the seedling curtain 2 tons and cultivates in the tanks and cultivate in 3 days on seed rope, be set under temperature, the illumination condition 20 ℃, 2000Lx, 12 hours/day, cultivating water is that local seawater is behind sedimentation and filtration; Condition SODIUMNITRATE 20mg, potassium primary phosphate 5mg in the seawater per ton cultivate water full dose exchange in per 2 days 1 time.
The result: the megagametophyte of inducing acquisition and the normal microgametophyte maturation that after one week of chopping, germinates successively, megagametophyte forms egg capsule and also discharges ovum, and microgametophyte forms antheridium and also emits sperm, sprouts after the feritilization of ovum to be the wakame seedling.Through about 1 month cultivation, seedling length reached more than the 1mm, and the seedling curtain of the seedling that before and after September 20, will grow moves on on the buoyant raft of sea area supports temporarily.Support temporarily through about 1 month sea area; Seedling length reaches more than the 0.5cm; The vinylon rope of growth seedling is cut into the 3cm segment; Be clipped in the cultivation Miao Shengshang of long 8m, diameter 3cm with the 35cm spacing, will cultivate Miao Shengping and hang on the wakame cultivation buoyant raft and get into the cultivation stage, be the commodity wakame in March, second cultivation.
Claims (1)
1. the method for a generating macrogametocyte by somatic cell of inducing undaria pinnatifida gynecogenic juvenile sporophyte is characterized in that carrying out according to following steps:
A. the wakame megagametophyte 10mg that goes bail for and deposit cultivation; Plant a megagametophyte body fluid with organizing stirrer chopping to 190～210 μ m algae sections to form after adding 300ml sterilization seawater; To plant megagametophyte body fluid moves in a plurality of petridish by the 1ml/ petridish; Adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are that 2000Lx and light application time are to cultivate nutrient solution full dose exchange in per 3 days 1 time 12 days under 12 hours/day the condition;
B. the petridish that growth is had a parthenogenesis seedling is that 15 ℃, intensity of illumination are that 2000Lx and light application time are to continue to be cultured to parthenogenesis seedling length under 12 hours/day the condition to reach 0.4～0.6cm, nutrient solution full dose exchange in per 3 days 1 time in temperature;
C. the parthenogenesis seedling is peeled from the petridish bottom surface; Be layered on through on the slide glass of sterilization; The seedling rhizoid is removed from the position of handle; To remain blade and move on in the beaker, adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are that 2000Lx and light application time are to cultivate nutrient solution full dose exchange in per 7 days 1 time 2 months under 12 hours/day the condition;
D. the blade of a plurality of growth megagametophyte algaes being rolled into a ball moves on in the tissue mashing machine; Smash algae section formation megagametophyte body fluid after adding nutrient solution to 490～510 μ m; Blade weight in wet base and nutrient solution volume ratio are 10mg: 300ml; Draw megagamete body fluid and move in a plurality of petridish by the 1ml/ petridish, adding behind the nutrient solution in temperature is that 20 ℃, intensity of illumination are cultivation 2 months 2000Lx and 24 hours/degree/day illumination condition under;
E. the megagametophyte algae in petridish group is placed the flask after the sterilization; Add behind the nutrient solution under the said culture condition of d step propagation and preserve and cultivate, nutrient solution exchange in per 10 days half amount was shaken in per 10 days bottle for 1 time; Cultivated 2 months; Megagametophyte in the flask is moved on in 2～3 flasks together with nutrient solution, add nutrient solution again and carry out branch bottle propagation, obtain megagametophyte;
Said nutrient solution adds NaNO for being heated in 80 ℃ of nature seawaters after filtering to 1L
3100mg, NaH
2PO
412H
2O 20mg and PI solution 1ml;
It is to obtain through following steps that said a step is preserved the wakame megagametophyte of cultivating:
F. in the nursery stage of wakame; Get the ripe sporophyll of sophisticated kind of algae of buoyant raft cultivation; Behind dry in the shade stimulation, put into the beaker that adds the sterilization seawater, when the seawater in the beaker is light brown, pull sporophyll out, get spore fluid with suction pipe and move on in the petridish that adds nutrient solution; It is to cultivate 1 month under the condition of 2000Lx with intensity of illumination that petridish is 20 ℃ in temperature, nutrient solution full dose exchange in per 10 days 1 time;
G. after microscopically is distinguished megagametophyte and microgametophyte; Megagametophyte is moved on in the other petridish; Add nutrient solution and carried out wakame megagametophyte separation and Culture 2 months, group peels from the petridish bottom surface with the megagametophyte algae, moves on in the flask; Preserve cultivation after adding nutrient solution, nutrient solution full dose exchange in 1 month 1 time.
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| CN103651094B (en) * | 2012-09-13 | 2015-04-15 | 中国科学院海洋研究所 | Undariapinnatifida seedling cultivation method through parthenogenesis |
| CN104094832B (en) * | 2014-06-30 | 2017-02-01 | 中国科学院海洋研究所 | Method for crossbreeding of undaria pinnatifida by using unisexual planospores |
| CN109601365B (en) * | 2018-12-27 | 2021-01-26 | 浙江海洋大学 | Cultivation method for seaweed |
| CN110771495B (en) * | 2019-11-27 | 2022-02-15 | 山东省海洋生物研究院 | Method for reducing aberration rate of parthenogenesis sporophyte of kelp germplasm |
| CN112956413B (en) * | 2021-01-28 | 2022-07-05 | 大连海宝渔业有限公司 | Method for preventing benthic diatom pollution in undaria pinnatifida gametophyte seedling culture production |
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| 张栩等.裙带菜配子体和幼孢子体的光合作用特性.<<海洋科学>>.2004,(第11期),20-27. * |
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