CN1273007C - Method for quickly reproducing engineering seedling of myriophyllum - Google Patents

Abstract

The present invention belongs to the field of a biological technique. High quality engineering seedlings are obtained by a watermifoil tissue culture technique and propagated in a large-scale and expansile mode to overcome problems that seed sources of species are limited in a natural environment, and the species are polluted seriously. The present invention satisfies urgent requirement that every high quality engineering seedling is used as a water purifying tool and a vegetation recovery pioneer species in lake ecological remediation engineering at present, as a place for baits, refuge and spawning in the process of the cultivation of fishes, prawns and crabs in a pond and as a scenery material in the process of the cultivation of indoor ornamental aquatic animals.

Description

The watermifoil engineering seedling fast reproducing method

One, technical field

The invention belongs to biological technical field, relate to a kind of method of quick production watermifoil high grade project seedling.

Two, background technology

Watermifoil (Myriophyllum spicatum Linn.) is a kind of aquatic herbaceous plant of Haloragaceae fox-brush Trentepohlia, is grown in pond, lakes and marhshes or the paddy field.This plant has higher tolerance to water pollution, is often used as water purification instrument kind and revegetation pioneer in eutrophication water restoration of the ecosystem engineering.This plant is also extensively planted in fish, shrimp and the crab pool, not only can also can provide necessary taking refuge and the spawning place for it for above-mentioned aquatic products provide bait.In addition, in indoor appreciation aquiculture process, this plant also be use always make the scape material.

In the market the watermifoil seedling is in great demand.But about the research of watermifoil all concentrate on its natural population distribution, its resistance to soiling and purifying water effect, with and application in fish shrimp crab is cultured.In practice process, the watermifoil provenance all is collected in natural environment.This axis softness and multi-branched, many subjects to severe risks of damage of root system in the salvaging process, and not of uniform size between plant are unfavorable for that it successfully transplants.In addition, the watermifoil of natural source has the assorted bacterium of various assorted algaes, is unfavorable for the safe handling in the fish shrimp crab pool and the indoor appreciation aquarium.The watermifoil provenance is limited in the last natural environment, and excessive collection causes the destruction in its primary habitat easily.

Do not see the research and the report of the cultivation and the extensive artificial propagation thereof of relevant watermifoil aseptic seedling so far both at home and abroad.

Three, summary of the invention

The objective of the invention is: utilize plant tissue culture technique, obtain and at indoor large-scale breeding watermifoil engineering seedling, and keep it aseptic as far as possible, to overcome open-air limited and with serious pollution drawback, the needs of the freshwater lake revegetation of adaptation China, open-air fish shrimp crab aquaculture and indoor appreciation Shui nationality setting collected of these species.

Technical scheme of the present invention is:

1. the acquisition of the aseptic bud of watermifoil

Gather the watermifoil plant from open-air natural habitat, clip children tender shoots section is soaked more than the 2h in suds, rinses well with running water.In the ultra-clean operating room, with 70% alcohol immersion certain hour, soak certain hour with the NaClO bleaching agent of debita spissitudo and a certain proportion of Tween-80 again, with being inoculated in after the thorough rinsing of sterile water during 1. culture fluid (see Table 1), and place tissue culture room to cultivate.Indoor average intensity of illumination 2000lx, illumination every day 10h, temperature is about 25 ℃.Note during this time observing, reject the material that still pollutes at any time, in order to avoid cross-infection.

2. the amplification of watermifoil clump bud

After the aseptic individual that is obtained by above-mentioned steps was cultivated through 2 weeks, most cauline leafs obviously extended, and have lateral bud to grow.In the ultra-clean operating room, all lateral buds more than the 2cm are downcut, long stem section is cut little again with every 2cm length, with every section be transferred to new culture fluid 1. in.Repeat cutting as stated above every the material that will grow up in 2 weeks later on, to obtain abundant aseptic bud.Above-mentioned amplification cultivation process is still carried out illumination and temperature-resistant in group training chamber.

3. watermifoil takes root and the inducing of complete seedling

Get above-mentioned steps 2 and obtain aseptic lateral bud, remain on equally about 2cm, be transferred to during 2. culture fluid (see Table 1), continue to cultivate more than the 10d in group training chamber, induced material is taken root and is formed complete seedling.Illumination and temperature-resistant in the group training chamber.

Table 1. watermifoil culture fluid is 1., 2. prescription is:

The culture fluid type	①	②
			Basis	1 * Ms basic element	(1/2-1) * the Ms basic element
Hormone addition 6-BA 2,4-D IAA NAA	0.00-4.00mg/L 0.00-2.00mg/L 0.00-3.00mg/L -	0.00-2.00mg/L 0.00-2.00mg/L - 0.00-2.00mg/L

^*All adjust pH to 5.7 after above-mentioned culture fluid prepares by concentration, divide to install in the 100mL triangular flask, with sealing film wrapping back in 121 ℃, 0.1MPa left and right sides moist heat sterilization 18min with rare HCl or NaOH.

4. strong sprout of watermifoil engineering seedling

With above-mentioned culture fluid 2. in more than the long 6cm of stem, and have the watermifoil seedling taking-up of 2 above roots of 2cm at least, be covered with bottom being transplanted in the big glass jar of sandstone of the bacterium of going out.Keep changing water weekly and adding a certain amount of Hoagland nutrient solution more than the depth of water 50cm to promote its growth.When outdoor daytime, temperature was more than 20 ℃, glass jar is placed outdoor, directly utilize solar irradiation; Otherwise, glass jar is placed the greenhouse, diurnal temperature keeps respectively more than 18 ℃ and 25 ℃, and suitably replenishes artificial lighting by day.Above-mentioned cultivation remains to plant and grows to more than the 40cm.

5. the application of watermifoil engineering seedling

The above-mentioned long 6cm of stem by step 3 cultivation back results is above, the healthy watermifoil seedling of well developed root system, directly applies to indoor appreciation Shui nationality setting.Concrete operation method is for rinsing culture fluid residual on the plant well with running water earlier, in case adhere to culture fluid to viewing and admiring the pollution of the Shui nationality, then according to the needs of setting, transplanting is in the aquarium bed-sit.Above-mentioned plant height 40cm by step 4 cultivation back acquisition is above, the healthy watermifoil plant of well developed root system, is applied in the breed and large-size lake water ecology recovery project on the fish shrimp crab pool.Concrete operation method is for taking healthy watermifoil plant from big glass jar, note keeping its root system complete, after adopting the dark and the mode of preserving moisture to transport target ground rapidly to, root a tree name fishes and shrimps juvenile crab injected volume or water eutrophication and vegetation degeneration degree decision watermifoil plantation density, and according to actual needs, adopt individual plant or clump strain to transplant, and single species is transplanted or is mixed transplanting with other species.

Effect of the present invention is:

1) obtained the watermifoil aseptic seedling.The watermifoil raw material of field acquisition itself are seriously polluted, and this pasture and water cauline leaf delicacy has absorbing capacity again concurrently, makes its sterilization process have suitable difficulty.Present technique adopts relatively mild suds to embathe earlier, after preliminary reduction is polluted, and again with ethanol, NaClO bleaching agent and Tween-80 acting in conjunction, thoroughly sterilization.By this program, on the basis that guarantees average aseptic rate 60%, the material survival rate can guarantee more than 50%.Utilize suitable culture fluid and hormone combination again, induce clump bud and root respectively, and finally obtain complete aseptic seedling.The aseptic bud of cultivating through domestication of watermifoil, culture fluid 1. in the rate of increase in per 2 weeks (be each simple bud after 2 weeks cultivated, can obtain clump bud more than 30 at least) 1: 30 or more, the while culture fluid 2. in, the rooting rate of material is also more than 80%.

2) set up watermifoil engineering seedling fast speed breeding approach.Present technique directly utilizes its lateral bud to cut apart breeding on the basis that obtains the aseptic bud of watermifoil, and step is simple, and is easy to operate.Agents useful for same is the chemical product that routinizes, and is with low cost, is convenient to its large-scale industrialized production.The dedifferentiation and the atomization that do not relate to callus have during this time been avoided the possibility of genetic variation in the tissue culture procedures as far as possible, in case obtain no bacterial classification bud, the breeding of can increasing for a long time.

This invention can be regardless of season, views and admires setting for lake revegetation, the breed of the fish shrimp crab pool and the indoor Shui nationality at any time enough watermifoil high grade project seedlings are provided.

Four, embodiment

Example 1. is gathered the watermifoil provenance in October, 2003 from Jiangpu, Nanjing county, 5 of the bud sections of clip 2cm length are used the running water rinsing behind the immersion 4h in suds.Enter in the ultra-clean operating room, with 70% alcohol immersion several seconds, soaked several minutes with dilution proper proportion and the NaClO bleaching agent that contains a certain amount of Tween-80 again, after the thorough rinsing of sterile water, each bud is inoculated in separately contains 1. also sterilized the sealing in the triangular flask of culture fluid, in group training chamber, cultivate.Average intensity of illumination 2000lx, illumination every day 10h, about 25 ℃ of temperature.This finds that wherein 2 bottles are still polluted after observe, and in addition 3 bottles aseptic and after several days, begin obviously to return green.2 week backs in the ultra-clean operating room, each lateral bud more than the 2cm in 3 bottles of materials downcut and is inoculated in separately new culture fluid 1. in, altogether 11 bottles.Continue to cultivate cutting once more after 2 weeks, and be one bottle with per 5 buds, be inoculated into culture fluid 2. in, be 62 bottles altogether.Behind the 20d more than the results stem long 6cm, and at least with complete watermifoil aseptic seedling 272 strains of 2 above roots of 2cm.Be used to view and admire Shui nationality setting with these healthy seedlings in January, 2004.

Example 2. in November, 2003 from Suzhou Taihu Lake, east adopt the watermifoil provenance, 20 of the long bud sections of clip 2cm are used the running water rinsing soak 4h in suds after.Enter in the ultra-clean operating room,, continue sterilization with dilution proper proportion and the NaClO bleaching agent that contains a certain amount of Tween-80 again with 70% alcohol immersion several seconds.After the thorough rinsing of sterile water, with each bud be inoculated in separately culture fluid 1. in, in group training chamber, cultivate.Average intensity of illumination 2000lx, illumination every day 10h, about 25 ℃ of temperature.Reject 11 parts of contaminated materials in 2 weeks altogether, have 5 parts to rise appreciably in 9 parts of materials in addition.Obtain 48 lateral buds altogether after the first cutting, continue to be incubated at culture fluid 1. in.After this every cut apart once in 2 weeks and be transferred to new culture fluid 1. in to induce the buds of growing thickly more.After 2 months, will newly sprout be transferred to culture fluid 2. in root induction.Behind the 15d complete seedling is taken out, be transplanted in the big glass jar of the sandstone that are covered with the bacterium of going out, and in the greenhouse, cultivate.Change water and additional Hoagland nutrient solution weekly with strong sprout.This batch of material will be applied in the Taihu Lake eutrophication water restoration of the ecosystem engineering.

Claims (1)

1. a watermifoil aseptic seedling is cultivated and the numerous method of engineering seedling fast speed expansion, it is characterized by this method is made up of following steps: 1) gather the watermifoil plant from open-air natural habitat, clip children tender shoots section, in suds, soak more than the 2h, rinse well with running water, with 70% alcohol immersion certain hour, NaClO bleaching agent and a certain proportion of Tween-80 with debita spissitudo soaks certain hour again, obtains the aseptic bud of watermifoil; 2) primary aseptic bud being inoculated in Ms is basis and additional 0.00-4.00mg/L 6-BA, 0.00-2.00mg/L 2, sprout with induced bundle in the culture fluid of 4-D and 0.00-3.00mg/L IAA hormone, and every 2 weeks the sprouting of the above length of 2cm being downcut and was inoculated in the above-mentioned culture fluid with continuous amplification up to obtaining abundant material; 3) being transferred to Ms after sprouting is cut down equally is basis and the 0.00-2.00mg/L 6-BA that comprises, 0.00-2.00mg/L 2, in 4-D and the 0.00-2.00mg/L NAA culture fluid with root induction; 4) will cultivate more than the long 6cm of stem, the seedling of well developed root system forwards in the big glass jar of the sandstone that are covered with the bacterium of going out, and keeps more than the depth of water 50cm, replenishes the Hoagland nutrient solution and continues strong sprout, grows to more than the 40cm up to plant.