CN102626050B - Preservation, activation and rapid propagation method of extreme-drought-tolerant syntrichia caninervis single plant cloning system - Google Patents
Preservation, activation and rapid propagation method of extreme-drought-tolerant syntrichia caninervis single plant cloning system Download PDFInfo
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Abstract
The invention relates to a preservation, activation and rapid propagation method of extreme-drought-tolerant syntrichia caninervis single plant cloning system, the method allows peeled rehydrated leaves to be inoculated to an improved Knop solid medium, and then switched to a fresh improved Knop solid medium to generate a large number of single plant cloning regenerated plantlets; the regenerated plantlets are put in an ultra low temperature refrigerator for long-term preservation after being slowly dried; after the preserved materials undergo slow rehydration, enlargement culture is carried out to obtain large batches of pure material. The method in the invention assists in rapidly cloning large batches of single plant cloning systems in 1-2 months from a leaf or explant, providing continuous pure materials for syntrichia caninervis as a drought resisting research mode plant, and simultaneously providing methods for "desert carpet engineering". A sustaining obtain and enlargement of syntrichia caninervis single plant cloning system materals is realized. The method in the invention has the advantages of simple method, low cost and short period, and growth degrees of the obtained single plant cloning system are consistent.
Description
Technical field
The preservation, activation and the fast-propagation method that the present invention relates to the red moss individual plant clone of a kind of extremely drought-enduring tooth rib system, belong to technical field of bioengineering.
Background technology
Climate arid is the global Tough questions facing.Arid not only causes agriculture heavy losses, has also aggravated deterioration and desertification and the water and soil loss of ecotope.The direct way of head it off is effective water saving.Plant is the basis that the mankind depend on for existence, how can just make plant drought water saving? this need to be just point of penetration from drought-enduring plant, understands drought-enduring mechanism and the regulatory mechanism of plant in depth, thereby improves the drought-resistance ability of plant.Along with developing rapidly with perfect of gene engineering and Protocols in Molecular Biology, the search of anti-drought gene resource and drought resisting Study on Molecular Mechanism deepen continuously, and make some progress.，Cong moss section plant due to outstanding anti-adversity and special aquation-rehydration process and concerned, becomes the material of molecular biology research field (especially plant stress-resistance molecular mechanism aspect) tremendous potential in recent years.
Liver moss is aquatic a kind of interim form to Lu Sheng, is monoid the most original in higher plant, is a kind of small-sized non-vascular plant simple in structure.It can growth procreation under the severe natural environmental condition such as high temperature, high and cold, arid and the low light level, and has extremely strong vitality.And liver moss has a lot of similar features to high terrestrial plant, for the research of gene function provides good material.As far back as the seventies in last century, scientist just be take small liwan moss (Physcomitrella patens) as molecular biology of plants research material has carried out a lot of correlative studys, has obtained certain progress.During epoch constantly advance, in the beginning of this century (2000), liver moss expert Oliver proposes the model plant that drought-enduring gable moss (Tortula ruralis) is research stress biology, and carries out preliminary genomics analysis.In the research in the present invention early stage, comprise the physiology of the red moss of tooth rib and the result of study of ecological aspect, and and oliver seminar result of study relatively in, we show that the red moss of tooth rib is better than gable moss as molecular biology material.And solve first and cultivation, preservation, the activation of the perfect red moss individual plant clone of tooth rib pure lines materials and expand traditional font system.Can be used for the research of Physiology and biochemistry, degeneration-resistant molecular biology and environment remediation aspect.
The red moss of tooth rib (Syntrichia caninervis) is under the jurisdiction of the red Rhodobryum of Cong Xian section (Pottiaceae) (Syntrichia sp.), is distributed in the Desert Area of arid and semi-arid.China is found in Gurbantunggut Desert, xinjiang, china first.It is one of Major Members in desert muskeg, as pionner main in environment During Succession, participates in Soil Biological Crusts and forms, and in checking winds and fixing drifting sand, plays an important role.It or a kind of poikilohydric plants, under extreme drought, the natural conditions that barren, high wind is husky, be resting state, like " shell " of one deck grey black, covers desert surface, is formed with the liver moss biological breadcrust of important ecological significance; Once run into precipitation, can " recovery " be fresh and alive green rapidly by rehydration process, and start to carry out photosynthesis.Because these special biological phenomenas receive much concern, make it be expected to become the model plant of degeneration-resistant molecular biology research.
Through literature search to existing technology, Chinese Patent Application No. provides the little plumage moss of a kind of spire gametophytic quick-breeding method for 200910054354.3, utilizes pinniform tip for material, the newborn gametophyte of induction through cutting shred expand numerous.This patent can be applicable to the improvement aspect of atmosphere heavy metal pollution; Chinese Patent Application No. is material for 200810035554.x provides the red moss protonema of tooth rib, in sandy soil, expands and cultivates (needing 4 months altogether), and this patent is that desert ecological recovers and environmental greening supplying method foundation; Yet, in the present invention's experiment in early stage, find that protonema expands cultivation and easily pollutes green alga in sandy soil, thereby the expansion that affects the red moss of tooth rib is numerous, compare with 200810035554.x, this patent is to be cloned on sandy soil and to expand with individual plant, or directly in medium, expand to cultivate, save incubation time (only need 1-2 each and every one month), and be not easy by green alga pollution.The patent that number of patent application is 201110084559.3 provides the store method of protonema; Yet previous experiments shows that the activation of protonema is not as explant easy (supposition is because explant has possessed characters of drought resistance and the rehydration phenomenon of the red moss of tooth rib), therefore, this patent improves on this basis, preserves material and clones for growing complete individual plant.And the Chinese Patent Application No. patent that is 201110116117.2 the red moss individual plant clone's of tooth rib medium is provided is BBM (pH=8.0); In the example of this patent, show, the Knop medium component of improvement is simpler, more economical, and is more suitable for the red moss individual plant clone's of tooth rib growth.Further retrieval does not have the report of preservation, activation and the fast-propagation aspect of the red moss individual plant clone of relevant tooth rib system.Yet, in view of the red moss of tooth rib will become model plant, set up the preservation of the red moss individual plant clone of the tooth rib of quickness and high efficiency system and fast numerously seem particularly important, therefore, present invention focuses on to provide a kind of economy, efficiently, the red moss of tooth rib is organized cultivation, preservation, activation and fast-propagation method fast, for the foundation of the red moss genetic conversion system of tooth rib lays the foundation, for utilizing the red moss of tooth rib to build biological breadcrust, carry out desertification treatment this " desert carpet engineering " technical support is provided simultaneously.
Summary of the invention
The object of the invention is to, for the state of the art of cultivating and preserving about the red moss of tooth rib both at home and abroad, the red moss individual plant clone of a kind of extremely drought-enduring tooth rib is provided preservation, activation and the fast-propagation method of system, the method is by the bud green blade of the red moss of wild tooth rib separating after rehydration, be seeded on the Knop solid culture medium of improvement and cultivate and produce protonema, then be forwarded in the Knop solid culture medium of fresh improvement and can generate a large amount of individual plants clone regrowths; After regrowth is slowly dried, be placed in ultra low temperature freezer long preservation; The material of preserving, after slow rehydration, then expands the large batch of pure lines material of cultivation acquisition.The red moss monoclonal of the tooth rib strain of activation has than wild explant differentiation capability faster more by force.The method of the invention can go out large batch of monoclonal system from a blade or explant quick clone in 1-2 month, for the red moss of tooth rib provides lasting pure lines material as the model plant of drought resisting research, is " desert carpet engineering " supplying method foundation simultaneously.For adversity gene group, forward, reverse genetics research provide pure lines material, advance the red moss of tooth rib as the process of degeneration-resistant molecular biology research type material.Realizing the forever continuous of the red moss monoclonal pure lines of tooth rib material obtains and expands.The individual plant clone that the method for the invention is easy, cost is low, the cycle is short, obtain is that development degree is consistent.
Cultivation, preservation, activation and the fast-propagation method of the red moss individual plant clone of a kind of extremely drought-enduring tooth rib of the present invention system, follow these steps to carry out:
The tissue of the red moss of tooth rib is cultivated:
A, the red moss of tooth rib of field acquisition is cleaned after, peel off rhizoid, stem and leaf, separate the bud green blade of the red moss of wild tooth rib after rehydration, sterile water concussion washing 5 times, is then seeded on the Knop solid culture medium of improvement, is placed in illumination box and cultivates, cultivation temperature is 25 ℃ of daytimes, in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, cultivates 2-3 week, a large amount of protonemas of generation;
B, by the protonema subculture producing in step a to continuing to cultivate 7-10 days on fresh Knop solid culture medium, cultivation temperature is 25 ℃ of daytimes, 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, and humidity is 50%, produces the red moss individual plant clone of a large amount of tooth ribs regrowth;
The preservation of the red moss of tooth rib:
C, by the red moss individual plant of step b tooth rib clone regrowth, through room temperature, be slowly loaded in the 1.5mLEP pipe of sterilizing after dry 24 hours, be placed in ultra low temperature freezer long preservation;
The red moss individual plant clone's of tooth rib activation:
The red moss individual plant clone of tooth rib that d, taking-up ultralow temperature are preserved, be placed in room temperature or place after 30min on ice, be seeded in the knop solid culture medium of improvement, be placed in illumination box and recover 1 day, then whole plant cutting is shredded or complete plant is seeded to the Knop solid culture medium of improvement or complete plant is expanded numerous in sandy soil matrix;
The red moss individual plant clone's of tooth rib fast-propagation:
E, the individual plant clone system that whole plant cutting is shredded to activation inoculate the fragment of regrowth by the mode of spraying, within 10 days, just start to occur that a large amount of protonemas and individual plant clone seedling blank in the Knop solid culture medium of improvement; Or complete plant is seeded to subculture on the Knop solid culture medium of improvement and cultivates, every 10-15 days subcultures once, cultivation temperature is 25 ℃ of daytimes, 15 ℃ of evenings, periodicity of illumination are 14h on daytime, evening 10h, intensity of illumination is 4000lx, and humidity is 50%, can obtain a large amount of monoclonal strains; The numerous cultivation of expansion of earth culture seedling: first sandy soil are soaked with the Knop liquid nutrient medium of improvement, the complete plant after activation is sprayed in sandy soil medium to incubated at room temperature, illumination 12h on daytime, evening 12h, humidity is 50%, light intensity is 4000lx, can obtain a large amount of monoclonal strains.
The Knop solid culture medium of the improvement relating in described method is 0.25g/L KH
2pO
4, KCl and MgSO
47H2O, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o forms, and pH value is 6.5.
Cultivation, preservation, activation and the fast-propagation method of the red moss individual plant clone of a kind of extremely drought-enduring tooth rib of the present invention system, the method has been passed through a large amount of screening tests:
The red moss Screening of Media of tooth rib
The red moss sample of tooth rib of field acquisition is peeled off green blade after cleaning, and with aqua sterilisa concussion washing 5 times, inoculates respectively 60 leaves to the Knop medium of BCD, ppNH4, BBM, BG11, Knop and improvement.Each culture medium prescription is in Table 1, being placed in illumination box cultivates, condition of culture: temperature is 25 ℃ of daytimes, 15 ℃ of evenings, photoperiod are 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, after 10 days, add up protonema incidence, the area of protonema and density are shown in microexamination Fig. 1, and result shows that protonema incidence, area and the density of Knop medium of improvement is the highest.
The medium component table that table 1 the present invention is used
Note: MS is for conventional medium, unlisted herein.
The red moss individual plant clone's of tooth rib the suitableeest explant:
Peel off respectively rhizoid, stem and the leaf of the red moss of tooth rib after cleaning, after aqua sterilisa concussion sterilization, be seeded on the Knop medium of improvement, being placed in illumination box cultivates, condition of culture temperature is 25 ℃ of daytimes, 15 ℃ of evenings, photoperiod are 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, after 15 days, a situation arises (seeing Fig. 2) to observe protonema, and result shows: rhizoid, stem and blade can produce protonema, but the protonema incidence of blade is 95% left and right, and all likely produce protonema at rachis and leaf margin and blade tip place; Caulonema body incidence is 50% left and right, and rhizoid protonema incidence is minimum.Therefore, green blade is the best explant of the red moss Regeneration System of tooth rib.
The red moss individual plant clone's of tooth rib store method comparison:
Be captured in the red moss individual plant clone of tooth rib (seeing Fig. 3) who grows 1-2 month on the Knop solid culture medium of improvement or sandy soil, after being slowly dried 24 hours, room temperature is loaded in the 1.5mLEP pipe of sterilizing, be placed in respectively temperature-80 ℃ ,-20 ℃ and 4 ℃ of refrigerators and room temperature preservation, preserve the chlorophyll content (seeing Fig. 4) of the individual plant clone system that measures respectively these four kinds of preservations after 2 months.From Fig. 4,5, the individual plant clone who preserves 2 months tie up to four kinds of store methods on chlorophyll content to affect difference not remarkable, and can well growth (Fig. 5 d, e) on the Knop medium of improvement.Yet in previous experiments, destroyed the explant (fragment) of whole plant and be kept in low temperature or ultra low temperature freezer after dry, room temperature preservation easily causes explant yellow (Fig. 5 e).Therefore, the red moss individual plant clone of tooth rib is that store method is preferably low temperature or ultralow temperature is preserved.
The activation of the red moss individual plant clone of tooth rib system:
Take out the red moss individual plant clone of the tooth rib of preserving based material, be placed in room temperature or place after 30min on ice, add aqua sterilisa recovery material (Fig. 5 a, b, c is the material of-80 ℃ of preservations, see Fig. 5 a), be seeded in the knop solid culture medium of improvement, be placed in illumination box recover 1 day (Fig. 5 b), visible regrowth is after ultralow temperature is preserved, still there is vitality (Fig. 5), then directly or this material of fragmentation, be seeded in the Knop solid culture medium of improvement or sandy soil matrix and expand and cultivate, Fig. 5 c is the photo that solid culture medium is cultivated 7 days, visible regeneration strain can produce a large amount of protonemas and regeneration plant again.
The expansion of the red moss individual plant clone of tooth rib system is cultivated:
The red moss individual plant clone of tooth rib of activation ties up on Knop solid culture medium and cultivates after 10 days, (Fig. 6 a) to have grown a large amount of protonemas and regrowth, subculture is to fresh Knop medium, continue to cultivate and within 7-10 days, can generate a large amount of regrowth (Fig. 6 b), every 10 days subcultures once, it is aseptic seedling material that repetition like this can obtain a large amount of individual plants clones, earth culture expands: be cut into fragment or whole plant and be transferred in sandy soil matrix, the fragment that fragment is inoculated regrowth by the mode of spraying is to sandy soil medium, when fragment mode is inoculated, guard against green alga pollution and cause the inhibitory action to the red moss plant strain growth of tooth rib, therefore sandy soil should first thoroughly sterilizing, condition of culture is incubated at room temperature, illumination 12h on daytime, evening 12h, humidity is 50%, light intensity is 4000lx, can obtain the red moss individual plant clone of tooth rib of a large amount of earth cultures.
Cultivation, preservation, activation and the fast-propagation method of the red moss individual plant clone of a kind of extremely drought-enduring tooth rib of the present invention system, the method compared with prior art its feature is:
The knop medium of improvement provided by the invention is better than other medium, is particularly useful for the cultivation of the red moss of tooth rib, is characterized in that nutrient component is only conventional mineral salt and component few (economical and practical, easy and simple to handle).
The present invention in protonema subculture, not only make protonema as far as possible differentiation produce regeneration strain, and cutting to shred regeneration strain be to take full advantage of liver moss vegetative propagation characteristic, improved the reproductive output of single explant blade.
The present invention just can obtain the red moss individual plant clone of a large amount of tooth ribs material in 2 months.Can be used for the group research that environment stress is processed.Can provide material for desert carpet engineering simultaneously.
The invention provides the red moss preservation of tooth rib, activation and expanding propagation method.Having realized the forever continuous of the red moss monoclonal pure lines of tooth rib materials obtains and expands.The degeneration-resistant molecular biology research type material that becomes extensive use for the red moss of tooth rib provides safeguard.
Accompanying drawing explanation
Fig. 1 is the growth picture of the present invention on different culture media, improvement Knop medium used in the present invention wherein, and the photo that a is magnify 20, b is for amplifying the photo of 40 times.
Fig. 2 is the comparison diagram of the red moss explant of tooth rib of the present invention different parts generation protonema, and wherein a is the protonema that rhizoid produces, and b is the protonema that stem produces, and c is the protonema that leaf margin and petiole produce.
Fig. 3 is the red moss individual plant clone figure of system of tooth rib of the present invention, and wherein a is the Knop solid culture medium relaying culture of improvement, and b is that individual plant clone strain is amplified the photo of 10 times, and c is that sandy soil expand the red moss of cultivation tooth rib.
Fig. 4 is under the different store methods of the present invention, the chlorophyll content comparison diagram of the red moss individual plant clone of tooth rib system, and wherein RT is room temperature preservation, 4 ℃ ,-20 ℃ and-80 ℃ storage temperatures that are respectively the red moss individual plant clone of tooth rib system.
Fig. 5 is the red moss individual plant of tooth rib of the present invention clone's activation figure, and wherein a is the red moss dry sample of tooth rib that takes out in the ultra low temperature freezer photo after rehydration; The state that b grows 1 day in the Knop solid culture medium of improvement for preserving sample; The state that c grows 7 days in the Knop solid culture medium of improvement for preserving sample; D, represent respectively the cultivation photo after the activation of the red moss individual plant clone of the tooth rib of normal temperature, 4 ℃ of preservations system; E represents that respectively-20 ℃ are cloned the cultivation photo after system's activation with the red moss individual plant of tooth rib of-80 ℃ of preservations; F represents fragment room temperature preservation yellow after 1 month of the red moss individual plant clone of tooth rib system, not resuscitable photo in the Knop solid culture medium of improvement.
Fig. 6 is the growth conditions figure that the red moss individual plant clone of tooth rib of the present invention is, wherein a is the red moss individual plant clone of the tooth rib after activating system's cultivation photo of 10 days, and b is the red moss individual plant clone of the tooth rib after activating system's cultivation photo of 20 days.
Embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated: the present embodiment is to implement under prerequisite in technical solution of the present invention, provided detailed embodiment and specific operation process, but the scope that the present invention protects is not limited to following embodiment.
Embodiment 1
The tissue of the red moss of tooth rib is cultivated:
A, the red moss of tooth rib of field acquisition is cleaned after, peel off rhizoid, stem and leaf, separate the bud green blade of the red moss of wild tooth rib after rehydration, sterile water concussion washing 5 times, the Knop solid culture based component that is then seeded to improvement is 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o forms, and pH value is 6.5, is placed in illumination box and cultivates, and cultivation temperature is 25 ℃ of daytimes, and in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, cultivates 2-3 all, a large amount of protonemas of generation;
B, by the protonema subculture producing in step a to the Knop solid culture medium of fresh improvement, be 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/LFeSO
47H
2o is upper, and pH value is 6.5, and 7-10 days is cultivated in upper continuation, and cultivation temperature is 25 ℃ of daytimes, and in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, produces the red moss individual plant clone of a large amount of tooth ribs regrowth;
The preservation of the red moss of tooth rib:
C, by the red moss individual plant of step b tooth rib clone regrowth, through room temperature, be slowly loaded in the 1.5mLEP pipe of sterilizing after dry 24 hours, be placed in ultra low temperature freezer long preservation;
The red moss individual plant clone's of tooth rib activation:
The red moss individual plant clone of tooth rib that d, taking-up ultralow temperature are preserved, is placed in room temperature or places on ice after 30min, and the knop solid culture medium that is seeded to improvement is 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2in O, pH value is 6.5, is placed in illumination box and recovers 1 day, then whole plant cutting is shredded or complete plant is seeded to the Knop solid culture medium of improvement or complete plant is expanded numerous in sandy soil matrix;
The red moss individual plant clone's of tooth rib fast-propagation:
E, the individual plant clone system that whole plant cutting is shredded to activation inoculate the fragment of regrowth by the mode of spraying, at the Knop solid culture medium of improveing, be 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/LCa (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2in O, pH value is within 6.5,10 days, just to start to occur a large amount of protonemas and individual plant clone seedling blank;
Or be 0.25g/L KH by the Knop solid culture medium that complete plant is seeded to improvement
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o is upper, and pH value is 6.5, and subculture is cultivated, and every 10-15 days subcultures once, cultivation temperature is 25 ℃ of daytimes, and 15 ℃ of evenings, periodicity of illumination are 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, can obtain a large amount of monoclonal strains.
Embodiment 2
The tissue of the red moss of tooth rib is cultivated:
A, the red moss of tooth rib of field acquisition is cleaned after, peel off rhizoid, stem and leaf, separate the bud green blade of the red moss of wild tooth rib after rehydration, sterile water concussion washing 5 times, the Knop solid culture based component that is then seeded to improvement is 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o forms, and pH value is 6.5, is placed in illumination box and cultivates, and cultivation temperature is 25 ℃ of daytimes, and in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, cultivates 2-3 all, a large amount of protonemas of generation;
B, by the protonema subculture producing in step a to the Knop solid culture medium of fresh improvement, be 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/LFeSO
47H
2o is upper, and pH value is 6.5, and 7-10 days is cultivated in upper continuation, and cultivation temperature is 25 ℃ of daytimes, and in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, produces the red moss individual plant clone of a large amount of tooth ribs regrowth;
The preservation of the red moss of tooth rib:
C, by the red moss individual plant of step b tooth rib clone regrowth, through room temperature, be slowly loaded in the 1.5mLEP pipe of sterilizing after dry 24 hours, be placed in ultra low temperature freezer long preservation;
The red moss individual plant clone's of tooth rib activation:
The red moss individual plant clone of tooth rib that d, taking-up ultralow temperature are preserved, is placed in room temperature or places on ice after 30min, and the knop solid culture medium that is seeded to improvement is 0.25g/L KH
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2in O, pH value is 6.5, is placed in illumination box and recovers 1 day, then whole plant cutting is shredded or complete plant is seeded to the Knop solid culture medium of improvement or complete plant is expanded numerous in sandy soil matrix;
The red moss individual plant clone's of tooth rib fast-propagation:
The numerous cultivation of expansion of e, earth culture seedling: be first 0.25g/LKH by sandy soil with the Knop liquid nutrient medium of improvement
2pO
4, KCl and MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o soaks, and pH value is 6.5, by activation after complete plant be sprayed in sandy soil medium, incubated at room temperature, illumination 12h on daytime, evening 12h, humidity is 50%, light intensity is 4000lx, can obtain a large amount of monoclonal strains.
After the red moss individual plant of tooth rib clonal activation, its protonema generation and seedling generation time are than the red moss explant of the tooth rib of field acquisition more Zao (few about 2 weeks of whole cycle), and just protonema seems thinner.This may be because individual plant clone is stronger than the differentiation capability of field sample, but nutrient accumulation is shorter and few than the time in field.
Claims (1)
1. the red moss individual plant clone of extremely drought-enduring tooth rib is cultivation, preservation, activation and a fast-propagation method, is characterized in that following these steps to carrying out:
The tissue of the red moss of tooth rib is cultivated:
A, the red moss of tooth rib of field acquisition is cleaned after, peel off rhizoid, stem and leaf, separate the bud green blade of the red moss of wild tooth rib after rehydration, sterile water concussion washing 5 times, then be seeded on the Knop solid culture medium of improvement, being placed in illumination box cultivates, cultivation temperature is 25 ℃ of daytimes, and in 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, and humidity is 50%, cultivates 2-3 week, produce a large amount of protonemas, wherein the Knop solid culture medium of improvement is 0.25g/LKH
2pO
4, 0.25g/L KCl and 0.25g/L MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/LFeSO
47H
2o forms, and pH value is 6.5;
B, the protonema subculture producing in step a is continued to cultivate 7-10 days to the Knop solid culture medium of fresh improvement, cultivation temperature is 25 ℃ of daytimes, 15 ℃ of evenings, periodicity of illumination is 14h on daytime, evening 10h, intensity of illumination is 4000lx, humidity is 50%, producing the red moss individual plant clone of a large amount of tooth ribs is regrowth, and wherein the Knop solid culture medium of improvement is 0.25g/L KH
2pO
4, 0.25g/L KCl and 0.25g/L MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o forms, and pH value is 6.5;
The preservation of the red moss of tooth rib:
C, by the individual plant clone who produces in step b, be regrowth, through room temperature, be slowly loaded in the 1.5mL EP pipe of sterilizing after dry 24 hours, be placed in ultra low temperature freezer long preservation;
The red moss individual plant clone's of tooth rib activation:
The red moss individual plant clone of the tooth rib system that d, taking-up ultralow temperature are preserved, be placed in room temperature or place after 30min on ice, be seeded in the knop solid culture medium of improvement, being placed in illumination box recovers 1 day, then whole plant cutting is shredded or complete plant is seeded to the Knop solid culture medium of improvement or complete plant is expanded in sandy soil matrix numerous, wherein the Knop solid culture medium of improvement is 0.25g/L KH
2pO
4, 0.25g/L KCl and 0.25g/L MgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/LFeSO
47H
2o forms, and pH value is 6.5;
The red moss individual plant clone's of tooth rib fast-propagation:
E, the whole plant cutting that is by the individual plant clone of activation shred, and inoculate the fragment of regrowth by the mode of spraying, and cultivate and within 10 days, just start to occur a large amount of protonemas and individual plant clone seedling blank in the Knop solid culture medium of improvement; Or complete plant is seeded to subculture on the Knop solid culture medium of improvement and cultivates, every 10-15 days subcultures once, cultivation temperature is 25 ℃ of daytimes, 15 ℃ of evenings, periodicity of illumination are 14h on daytime, evening 10h, intensity of illumination is 4000lx, and humidity is 50%, can obtain a large amount of monoclonal strains; The numerous cultivation of expansion of earth culture seedling: first sandy soil are soaked with the Knop liquid nutrient medium of improvement, complete plant after activation is sprayed in sandy soil medium, incubated at room temperature, illumination 12h on daytime, evening 12h, humidity is 50%, light intensity is 4000lx, can obtain a large amount of monoclonal strains, wherein the Knop solid culture medium of improvement is 0.25g/L KH
2pO
4, 0.25g/L KCl and 0.25g/LMgSO
47H
2o, 1g/L Ca (NO
3)
2.4H
2o and 0.0125g/L FeSO
47H
2o forms, and pH value is 6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210123284.4A CN102626050B (en) | 2012-04-25 | 2012-04-25 | Preservation, activation and rapid propagation method of extreme-drought-tolerant syntrichia caninervis single plant cloning system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101268739A (en) * | 2008-04-03 | 2008-09-24 | 上海交通大学 | Desert muskeg and biological breadcrust quick proliferation method thereof |
| CN101606486A (en) * | 2009-07-03 | 2009-12-23 | 上海师范大学 | A kind of method of fast propagating gametophyte of haplocladium microphyllum by tissue culture |
| CN102257955A (en) * | 2011-05-06 | 2011-11-30 | 中国科学院新疆生态与地理研究所 | Method for establishing syntrichia caninervis mitt. individual plant cloning system through asexual propagation |
| CN102257954A (en) * | 2011-04-06 | 2011-11-30 | 中国科学院新疆生态与地理研究所 | Methods for culturing and preserving syntrichia caninervis mitt. protonemata |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101268739A (en) * | 2008-04-03 | 2008-09-24 | 上海交通大学 | Desert muskeg and biological breadcrust quick proliferation method thereof |
| CN101606486A (en) * | 2009-07-03 | 2009-12-23 | 上海师范大学 | A kind of method of fast propagating gametophyte of haplocladium microphyllum by tissue culture |
| CN102257954A (en) * | 2011-04-06 | 2011-11-30 | 中国科学院新疆生态与地理研究所 | Methods for culturing and preserving syntrichia caninervis mitt. protonemata |
| CN102257955A (en) * | 2011-05-06 | 2011-11-30 | 中国科学院新疆生态与地理研究所 | Method for establishing syntrichia caninervis mitt. individual plant cloning system through asexual propagation |
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