CN102160524A

CN102160524A - Separation culture and plant regeneration method for kappaphycus seaweed protoplast

Info

Publication number: CN102160524A
Application number: CN 201110003481
Authority: CN
Inventors: 刘涛; 张偲; 金月梅
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2011-01-10
Filing date: 2011-01-10
Publication date: 2011-08-24

Abstract

The invention discloses a separation culture and plant regeneration method for kappaphycus seaweed protoplast. The method mainly comprises the following steps of: performing zymolysis on kappaphycus tissues by using a seaweed tool according to the totipotency of somatic cell development of seaweeds; freeing somatic cell protoplast to obtain somatic cells and cell systems of regenerated cell walls; and culturing the somatic cells and the cell systems to obtain regenerated plants. By the method, the somatic cell protoplast is directly subjected to seedling culture, maturely reproductive seaweeds for kappaphycus are not required to be cultured, and the regenerated cell walls of the somatic cell protoplast are converted into the somatic cells. Compared with a sexual reproduction seedling technology, the method has the advantages that: a sexual reproduction process of germ cells is not needed, the seedling cycle is shortened, the seedling culturing efficiency is high, and the method is suitable for large-scale seedling culture production.

Description

A kind of method of blocking handkerchief Trentepohlia marine alga protoplast separation and Culture and regeneration plant

Technical field

The present invention relates to a kind of algae culture method, be specifically related to a kind of separation and Culture of handkerchief Trentepohlia marine alga protoplast and method of regeneration plant of blocking.

Background technology

Breeding naturally of card handkerchief Trentepohlia marine alga mainly is that mode by tetrasporangium and carpospore cyst is carried out.At present, in card handkerchief Trentepohlia algae culturing is produced, mainly be to adopt the mode of segment regeneration to cultivate breed.But since this mode to take coverage of water big, and summer frond water temperature is too high because of being subjected to, precipitation makes seawater salinity cross environmental influences such as low greatly, cause extremely instability of seed breeding production.In addition, card handkerchief Trentepohlia marine alga body wall has thicker gelatinous layer, thereby genetic transformation technology such as directly application cell fusion, electricity conversion are carried out the improvement of kind.

Summary of the invention

At the problems referred to above, the purpose of this invention is to provide the cultural method of the card handkerchief Trentepohlia marine alga that a kind of growing-seedling period is short, seed breeding efficient is high, it can solve card handkerchief Trentepohlia marine alga seed and protect the problem of planting and breeding, and can be used for yielding ability and grows seedlings.

A kind of method of blocking handkerchief Trentepohlia marine alga protoplast separation and Culture and regeneration plant, it mainly is the totipotency according to the marine alga somatocyte development, utilize the marine alga toolenzyme to divide unfreezing handkerchief algae tissue, the somatoplasm body dissociates, obtain the somatic cell and the cell-line of regenerative cell's wall, then its cultivation is obtained regeneration plant.Concrete steps are as follows:

(1) utilizes seashells to extract purifying and make the marine alga toolenzyme, add bleeding agent then;

(2) will block the handkerchief algae and grind or cut into tiny piece of tissue or little branch, and be immersed in the marine alga toolenzyme solution and digest, obtain card handkerchief frond cellular plasm;

(3) protoplast is cultivated the further cultivation of cell that obtains regenerative cell's wall and can obtain regeneration plant.

Described seashells is a Bao.

Described bleeding agent is a kind of among mannitol, sorbierite, glucose, sucrose, KCl, the NaCl.

The condition of culture of protoplast is in the described step (3): adopt the PES medium to be made into culture fluid, and 20 ℃ of following dark culturing 3-4 of temperature days, transfer illumination cultivation to, light application time is 12h/d, light intensity 1500-2000lx, periodic replacement culture fluid.

The present invention utilizes the somatoplasm body directly to grow seedlings, and need not cultivate the frond of card handkerchief algae reproduction maturation; Somatoplasm body regenerative cell wall is converted into somatic cell, with respect to the sexual reproduction seedling growing process, its advantage be need not reproductive cell the sexual reproduction process, shortened growing-seedling period; And seed breeding efficient height is suitable for the production of growing seedlings on a large scale.

Description of drawings

Fig. 1 is the fluoroscopic examination figure (* 10 times) of long heart card handkerchief algae protoplast.

Fig. 2 is the blue coloration result of long heart card handkerchief algae protoplast Ai Wensi.

Wherein, arrow 1 is depicted as living cells, and coloration result shows glassy yellow; Arrow 2 is depicted as dead cell, and coloration result shows navy blue.

Fig. 3 is that long heart card handkerchief algae protoplast is cultivated each stage diagram.

Wherein, (a) cultivate 7d; (b) cultivate 10d; (c) cultivate 25d; (d) cultivate 40d; (e) (f) cultivate 45d; (g) cultivate 55d; (h) cultivate 60d; (i) cultivate 65d.

Embodiment

Further specify the present invention below in conjunction with accompanying drawing and by specific embodiment.

The present embodiment experiment material be long heart card handkerchief algae ( Kappaphycus alvarezii).

1. protoplast separating method

Get the long heart card handkerchief algae of 1-2 gram, with its grinding or cut into tiny piece of tissue or little branch; Utilize the Bao glandula digestive to extract purifying, make the marine alga toolenzyme according to the method for biochemistry protein purification (enzyme), the glucose that adds 2 M is as bleeding agent; Under 20-25 ℃, piece of tissue that long heart card handkerchief algae is tiny or little branch are immersed in the marine alga toolenzyme solution 2-3 hour, during stir with glass bar; With 300 mesh sieve thin,tough silk filtration cell mixed liquors, remove indigested cell, cell mass, fragment; It is centrifugal to get supernatant, the centrifugal 10min of 800rpm, unicellular (protoplast) sunk, cell fragment is stayed in the supernatant, abandoning supernatant, with height ooze sterilization seawater (containing 1mol/L respectively, O.5mol/L glucose) wash repeatedly, centrifugal, abandon process such as supernatant 2-3 time, i.e. the comparatively pure long heart card handkerchief frond cell protoplast suspension of acquisition.

2. protoplast authentication method

The fluorescent whitening agent decoration method of employing 0.1% is identified the gained cell.At first adopt 0.1% fluorescent whitening agent to the gained sample 5min that dyes, observe down in the fluorescence microscope mirror, excitation wavelength is the 370nm ultraviolet light, detects shown in red as shown in Figure 1.This is that blue-green fluorescence display fibers is plain to be existed because lepocyte has, and protoplast does not have blue-green fluorescence, and it is rubescent look fluorescence owing to the existence of chloroplast, shows the existence of cellulose-less.

With blood counting chamber statistics protoplast quantity, add up its output and average survival under the different enzymolysis times, see Table 1 and table 2.

Protoplast output under the different enzymolysis times of table 1

Enzymolysis time (h)	2.0	2.5	3.0
				Protoplast output (individual/ml)	（16.4±3.6）×10 ⁴	（23.8±1.5）×10 ⁴	（35.1±2.6）×10 ⁴

Protoplast average survival under the different enzymolysis times of table 2

Enzymolysis time (h)	2.0	2.5	3.0
				Protoplast average survival (%)	71.43±0.20	62.50±0.19	56.41±0.78

Adopt 0.25% Evans Blue decoration method to protoplast vigor dyeing mensuration, on slide, drip the gained sample, drip dye liquor again, observe counting in microscopically, the protoplast that survives presents glassy yellow, and dead cell is navy blue, as shown in Figure 2.

3. protoplast cultural method

Culture fluid: medium adds the PES medium with the filter-sterilized seawater and is made into, and adds the growth that germanium dioxide suppresses diatom.

The PES culture medium prescription is for to add NaNO in 100 ml distilled water ₃350mg, Na ₂Glycerophosphate ^.5 H ₂O(five water phosphoglycerin disodiums) 50 mg, Fe-solution (ferrous solution) 25 mL, PII molten metal 25 mL, Cobastab ₁₂10 μ g, thiamines 0.5 mg, vitamin h 5 μ g, Tris buffer (Tris-HCl buffer solution) 500 mg regulate pH 7.8, and every 20ml PES culture fluid adds aseptic seawater and is settled to 1000ml.

Condition of culture: in culture dish, cultivate, 20 ℃ of temperature, dark culturing 3-4 days, transfer illumination cultivation to, light application time is 12h/d, light intensity 1500-2000lx, periodic replacement culture fluid.

Cultivate after 7 days cell wall and observe unintelligible (Fig. 3-a); Cultivating after 10 days cell culture observes cell and circular contour (Fig. 3-b) occurs; Cultivate after 25 days the clear (Fig. 3-c) of cell outline; Cultivate and occur cell division (Fig. 3-d), arrow indication place after 40 days; Cultivate after 45 days, observe cell division and become bigger cell mass, (visible cell unity structure among Fig. 3-e), in the visual field, also there are some small cell clusters, (there are several cellules in Fig. 3-f) around being depicted as a maxicell, and (arrow indication place is that cell interior is full of the round granular material among Fig. 3-f); Cultivate after 55 days, observe cell and constantly carry out cell division, and visible cell unity structure (Fig. 3-g); Cultivate after 60 days piece of tissue and carry out microscopy, can see that pointed projections is arranged, the doubtful outstanding (Fig. 3-h) of budlet that has; Cultivate and observed the regeneration seedling after 65 days and grow (Fig. 3-i).

Claims

1. a method of blocking handkerchief Trentepohlia marine alga protoplast separation and Culture and regeneration plant is characterized in that it comprises the steps:

2. according to the method for the described card handkerchief of claim 1 Trentepohlia marine alga protoplast separation and Culture and regeneration plant, it is characterized in that described seashells is a Bao.

3. according to the method for the described card handkerchief of claim 1 Trentepohlia marine alga protoplast separation and Culture and regeneration plant, it is characterized in that described bleeding agent is a kind of among mannitol, sorbierite, glucose, sucrose, KCl, the NaCl.

4. according to the method for the described card handkerchief of claim 1 Trentepohlia marine alga protoplast separation and Culture and regeneration plant, the condition of culture that it is characterized in that protoplast in the step (3) is: adopt the PES medium to be made into culture fluid, 20 ℃ of following dark culturing 3-4 of temperature days, transfer illumination cultivation to, light application time is 12h/d, light intensity 1500-2000lx.