CN1148115C - Gametophyte cloning method of breeding heterotic kelp - Google Patents

Abstract

The present invention relates to a gametophyte cloning method for breeding kelp hybrid dominant seedlings, which comprises a seedling breeding step, a temporary culture step in the sea and a seedling separating and culturing step. The method is characterized in that before the seedlings are bred, cloned cells of female gametophytes and male gametophytes are respectively amplified and bred, the cloned cells of the gametophytes are attached to a substrate, and then, the cloned cells of the female and male gametophytes are hybridized. The cloned cells of the dominant strain female and male kelp gametophytes are respectively amplified by the present invention, and the biphyletic hybridization is carried out to produce hybrid seedlings. A process for breeding seed kelp on the sea is dismissed, and the indoor seedling breeding time is shortened. The method integrates dominant seedling cultivation and dominant seedling production into a whole. The method not only can stably produce heterosis seedlings, but also can greatly reduce the production cost of the seedling cultivation.

Description

Cultivate the method for sea-tangle hybrid vigour seed with gametophyte clone

The present invention relates to a kind of seedling cultivation method for sea-tangle, particularly relate to a kind of method of cultivating sea-tangle hybrid vigour seed with gametophyte clone.

It is the seedling raising process that present China sea-tangle total man worker cultures common employing that sea-tangle summer sporelings (of laminaria) natural daylight is cultivated, and it comprises the selection main laminaria, and indoor spore and the seedling culture adopted plunged into the commercial sea and supported temporarily and divide step such as seedling breed.At present, though this seedling-cultivating method generally adopts in China unit of growing seedlings, also exist the problem of the following aspects: the seed selection and the marine cultivating and managing of (1) main laminaria is numerous and diverse, labour intensity and risk are bigger; In south, the main laminaria indoor low temperature is cultivated and to be added that the stage of growing seedlings reaches half a year, easier increasing production cost and cause that disease takes place; (2) because the restriction of marine natural temperature factor, for preventing that water temperature from raising main laminaria is grown suitable water temperature unfavorable and that seedling is plunged into the commercial sea, therefore should early collect seedling (adopting spore), again in prometaphase control light, the temperature of growing seedlings, cause the seedling phase to cultivate overlong time, not only cause improper growth and the morbidity chance of seedling, but also strengthened the production cost of growing seedlings; (3) can't really realize the cultivation of breeding seedling.During kelp seedling cultivation was produced, main laminaria consumption big (produce 500,000,000 young plants and need 30,000 main laminarias) mixed and adopts spore, and the filial generation proterties is separated bigger, can't keep single.Therefore, after several culture-cycles, kind is prone to proterties and degenerates, and output descends, and causes damage to laminaria culture.

The purpose of this invention is to provide a kind of method of cultivating sea-tangle hybrid vigour seed with gametophyte clone, it can remedy the above-mentioned deficiency of prior art.

A kind of method of cultivating sea-tangle hybrid vigour seed with gametophyte clone, comprise seedling culture, plunge into the commercial sea and support temporarily and the breed of branch seedling, it is characterized in that before seedling culture, earlier with female and male gametophytes clone cell difference amplification cultivation, again with gametophyte clone attached on the matrix, allow female and male gametophytes clone hybridize then.

The present invention increases the male and female laminaria gametophytic clone of excellent strain respectively, carries out double-line hybrid, produces the hybrid seedling, and the process of having removed marine cultivation main laminaria from has shortened the indoor raising of seedling time.Prevalent variety cultivation and the production of breeding seedling are integrated, and both the seedling of production hybrid vigour stably can reduce the production cost of growing seedlings again greatly.

Further specify the present invention below by embodiment.

The female and male gametophytes clone that present embodiment is used has been the part material of inventor's institute's separation and Culture since 1978.Wherein, female sex clone has: C _11-1(1982), C _15-1(1982), C _26-2(1982), TPJ _-1(1994);

Earlier gametophyte clone is ground into the individuality of a spot of cell, carries out amplification cultivation again.The present invention utilizes the ultrasonic crusher will clone bunch shape body and pulverizes.Beginning is cloned bulk with 400W, 30S earlier and bunch is smashed, and with 200W, 120S the clone is decomposed into the segment branch of 5～10 cells then.Also can utilize the method for mechanical crushing, the clone is ground into the filamentous of 20～50 cells.

Clone cell after pulverizing is added culture fluid (amounting to the 2L volume).Begin to cultivate, move into again after water colour is deepened in the container of 20L and cultivate with the 3L triangular flask.The amplification cultivation condition is a temperature: 8～18 ℃; Intensity of illumination: 40～60 μ molm ^-2s ^-1(be equivalent to 2000～3000Lux), light application time 12h; Culture fluid: the natural sea-water scalding adds nutritive salt (NO ₃-N 16mg/L, PO ₄-P 1mg/L); 24h inflation, the purpose one of inflation are to replenish CO ₂, the 2nd, prevent that clone cell from sinking and accumulation.Change water: except that need fall bottle or weigh and change the water, generally seal cultivation, add new culture fluid on time, and replenish nutritive salt in right amount.In the inoculum concentration problem about the laminaria gametophytic clone cell amplification initial stage, according to day increasing speed, from comparison of daily gain effect and saving provenance, (culture fluid 1～2L) suits experiment initial inoculation amount 1～2g.

About gametophyte clone attached to the clone of the female and male gametophytes before on matrix ratio, according to experiment, under similarity condition, male and female clone's growth rate and daily gain effect are basic identical, for helping fertilization, gametophyte clone attached to matrix on before 5～12 days just can be with the male and female clone cell by 1: 1 ratio Mixed culture to promote to work in coordination with growth.

Again with gametophyte clone attached on the matrix.Attaching substratum adopts brown curtain or dimension Buddhist nun curtain two seedling curtains.Available gametophyte clone directly adheres to and also can use curing materials to promote its adhesion effect.Fixing agent uses the CaCl of 3% (w/v) sodium alginate solution and 1mol/L ₂Solution.According to female and male gametophytes clone cell ratio 1: 1, make the clone cell suspension, be sprinkled upon on the seedling curtain with sprayer then, the seedling curtain promptly moves on under the low temperature (8～10 ℃) and cultivates, and slowly adds the sterilization seawater after keeping seedling humidity lucifuge to place 24h.Nutritive salt (NO ₃-N16mg/L, PO ₄-P 1mg/L), light intensity 20～30 μ ml1m ^-2s ^-1, photoperiod 12h, 8～10 ℃ of temperature.Before using the seedling curtain is immersed in 60 ℃ the curing agent solution, takes out behind the 5min, it is standby to drain the back.Find in the experiment that the vinylon curtain interweaves closeer and smooth surface, be sprayed onto on the seedling curtain as directly cloning suspension that most of clone's suspension is slipped in the dissecting pan, difficulty is adhered to.Add the vinylon curtain of overcuring agent, clone's adhesion effect is good, but clone's major part is to anchor at the curing agent surface, and there is certain hidden danger in this spore set for the clone seedling breeding later stage.Later stage uses brown curtain to add curing agent, clone's suspension (3～5 cell/branches, cell density 1.0 * 10 that the excusing from death ripple is pulverized ⁵) be sprayed onto on the brown curtain, the sprinkling amount (is divided 2～3 sprinklings) about 40ml.The discovery fixed effect is better, can reach * adhesion effect of 80 branches in the every visual field of 150 microscopicallies.And brown curtain silk is looser, the later stage sporophyte is adhered to have certain effect.

After clone cell adhered to, gametophyte clone cell development in 7～20 days formed fertilized egg and juvenile sporophyte, promptly obtains the hybrid vigour seed, carries out seedling culture then, plunged into the commercial sea and supported temporarily and the breed of branch seedling, finished the whole technology of this project.

Choose C _11-1And D _4-4Carry out the clone seedling breeding experiment.Control group is for leaving standstill spatfall group (the seedling curtain being placed in clone's the cell suspension).At the 30th day that cultivates, cut a small amount of palm fiber and observe, find that sporophyte appears in the sprinkling group, have only a small amount of ovum, and control group is very few because of the cell attachment amount, can't add up developmental rate.Proceed on January 24th, 2000 in experiment, the sea-tangle seedling is long to 2～3cm on the brown curtain, and average 6/cm has reached the standard of commercial seedling outbound.

Claims (4)

1, a kind of method of cultivating sea-tangle hybrid vigour seed with gametophyte clone, comprise seedling culture, plunge into the commercial sea and support temporarily and the breed of branch seedling, it is characterized in that before seedling culture, earlier with after the female and male gametophytes clone cell difference amplification cultivation, gametophyte clone attached to matrix on before 5-12 days, the male and female clone cell is cultivated in 1: 1 ratio, again with gametophyte clone attached on the matrix, allow the female and male gametophytes clone hybridization then.

2, the method for claim 1 is characterized in that described amplification cultivation condition is a temperature: 8-18 ℃; Intensity of illumination: 40-60 μ molm ^-2s ^-1Light application time 12h; The natural sea-water scalding adds nutritive salt; The 24h inflation.

3, the method for claim 1 is characterized in that described attaching substratum adopts brown curtain or dimension Buddhist nun curtain.

4, method as claimed in claim 3 is characterized in that described curtain is immersed in the curing agent solution before use, drains after the taking-up.