Freshwater reeds non-callus culture method
One, technical field
The invention belongs to field of plant tissue culture technique.
Two, background technology
The natural population of reed (Phragmites australis) is mostly occurred at the shallow water wetland, is common in pond, river alongshore, beach, many water area, He Xi limit.Reed is as economic crops, and the reed stalk can be used for papermaking and artificial silk, establishment reed mat, straw mattress; The reed in vegetative period contains amounts of protein and sugar, is good feed; The leaf of reed, flower, stem, root, bamboo shoot all can be used as medicine.Simultaneously, reed has important ecological functions, protects native dyke strengthening, the wave unhurried current that disappears, short silt corrosion protection etc.; In recent years, reed resistant, decontamination, the ability of improving eutrophication water quality enjoys attention, and in lakeside band, river sloping bank and marsh wetland recovered and rebuild, reed became essential plant, by establishing in large scale.Along with a large amount of enforcements that China's improvement of the ecological environment, the water environment comprehensive regulation and wetland recover engineering, seedling of reed and provenance demand can be more and more urgent, and current technology will be difficult to satisfy the market demand that grows to even greater heights; Therefore, a kind of method that high-quality reed seedling can be provided fast of invention needs that are inevitable.
Through Chinese patent net and relevant paper retrieved web, find at present more existing reports about the reed tissue culture, cultivate by callus with seed in " formation of reed embryo callus and the regeneration of plant " as Wu Guoliang etc. and to obtain whole plant, but the cycle of callus is longer, and process is complicated; Wang Xiyou has only studied callus Growth in " reed embryo callus Study on Growth ", do not grow whole plant; Guo Rui also is to obtain plant by callus culture in " research of blast generation of salt tolerant reed stipes organizer and plant regeneration ", has long problem of cycle equally; In addition, in the patent " asexual reproduction method of salt tolerant reed " of Yang Yingen etc., also describe the process that obtains whole plant by callus culture, had long problem of cycle equally.
Three, summary of the invention
The problem that the present invention need solve is: invent the method for a kind of quick in-vitro propagate reed, make to obtain a large amount of high-quality same specification reed seedlings in a short time, satisfy the needs in wetland recovery and the reconstruction engineering, promote the recovery of environment.
Technical scheme of the present invention is:
(1) the tall and big reed of Luoma Lake growing way is selected in open-air seed selection;
(2) sterilization, the reed stem that will fetch from the field is cut into segment, every section 2-3cm is long, under distilled water, clean, then on super-clean bench with 70% alcohol immersion 30s, rinse with distilled water and to wash 2-3 time, use 0.1%HgCl thereafter
2Aqueous solution sterilization 5min is again with sterile distilled water washing 4-5 time;
(3) the stem section of the band bud original hase of the reed after will sterilizing is inoculated into evoking adventive bud on the inducing culture, has added polyvinylpyrrolidone (PVP), 6-benzyladenine (BA) and methyl (NAA) in the inducing culture; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h; Cultivate 7-23d, the bud of most stem sections can be grown 3cm about two weeks;
(4) indefinite bud that produces is transferred to carried out successive transfer culture on the subculture medium, added PVP, BA and NAA in the subculture medium; Temperature is that 25 2 ℃ in soil, light intensity are to cultivate 11-24d under 2000lx, the illumination every day 14h condition;
(5) lure root on the root media when bud length moves on to during to 4-5cm, lure and added PVP, IBA and NAA in the root medium; Temperature is that 25 2 ℃ in soil, light intensity are 2000lx, illumination every day 14h;
(6) when the test tube shoot root grows to about 2cm, open bottleneck and add a small amount of distilled water hardening 2-3d, then root agar is cleaned, move into transition in the perlite, move into soil behind the 3-5d.
The culture medium prescription of each cultivation stage (hormone combination) is as following table:
The culture medium prescription in each stage
The group training stage |
The medium constituent |
Adventitious bud inducing |
MS medium+3% sucrose+0.43% agar+BA 6mg/L+NAA 0.2mg/L+PVP 50mg/L |
The indefinite bud subculture |
MS medium+3% sucrose+0.43% agar+BA 5mg/L+NAA 0.2mg/L+PVP 50mg/L |
Plant takes root |
1/2MS medium+3% sucrose+0.43% agar+IBA 1.0mg/L+NAA 1.0mg/L+PVP 50mg/L |
Annotate: because pH can slightly descend behind the medium sterilization, so the pH of medium transfers to 6.1 when preparing; All medium are 121 ℃ of sterilization 20min in high-pressure sterilizing pot, and culture medium after sterilization is placed on the super-clean bench behind the 1d usefulness again, to dry moisture film.
The present invention has set up the integral framework of a quick in-vitro propagate reed, compared with prior art its beneficial effect is: (1) obtains complete reed tissue cultivating seedling by non-callus culture method, its process is simple, the cycle is short, and growing complete test-tube plantlet by explant only need about 35-65d; (2) inductivity is up to 92.3%, and rooting rate is also up to 95.8%; (3) transplanting survival rate height is 90.2%; (4) eliminated browning by adding 50mg/L PVP, stoped the influence of brownization incubation; (5) material therefor is taken at Luoma Lake among the present invention, and plant is tall and big, and on average about 6m can be widely used in shore, river course bank and lakeside band and recover.
Four, embodiment
1. explant sterilization
The reed stem that to fetch from the field is cut into segment, and every section 2-3cm is long, under distilled water, clean, then on super-clean bench with 70% alcohol immersion 30s, rinse with distilled water and to wash 2-3 time, use 0.1%HgCl thereafter
2Aqueous solution sterilization 5min is again with sterile distilled water washing 4-5 time;
2. indefinite bud induces
On super-clean bench, stem section after the sterilization is inoculated on the inducing culture, being MS medium+3% sucrose+0.43% agar+BA6mg/L+NAA0.2mg/L+PVP 50mg/L, is that 25 2 ℃ in soil, light intensity are to cultivate under the condition of 2000lx, illumination every day 14h in temperature.Cultivate 7-23d, the bud of most stem sections can be grown 3cm about two weeks; Best when BA is 6mg/l, inductivity is up to 92.3%.
3. successive transfer culture
The bud that induces is transferred on the subculture medium, i.e. MS medium+3% sucrose+0.43% agar+BA5mg/L+NAA0.2mg/L+PVP 50mg/L is that 25 2 ℃ in soil, light intensity are to cultivate 11-24d under the condition of 2000lx, illumination every day 14h in temperature.
4. culture of rootage
Clump shape indefinite bud is separated into single bud, move on on the root media, be 1/2MS medium+3% sucrose+0.43% agar+IBA1.0mg/L+NAA1.0mg/L+PVP50mg/L, temperature is that 25 2 ℃ in soil, light intensity are that 2000lx, every sight are according under the condition of 14h, turn out complete regeneration plant, the rooting of vitro seedling rate reaches 95.8%.
5, transplant
When the test tube shoot root grows to the 2cm left and right sides, open bottleneck and add a small amount of distilled water hardening 2-3d, then root agar is cleaned, move into transition in the perlite, move into soil behind the 3-5d, survival rate reaches 90.2%.