CN103636496B - A kind of Zhegucai artificial raise seedling method - Google Patents
A kind of Zhegucai artificial raise seedling method Download PDFInfo
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- CN103636496B CN103636496B CN201310612301.5A CN201310612301A CN103636496B CN 103636496 B CN103636496 B CN 103636496B CN 201310612301 A CN201310612301 A CN 201310612301A CN 103636496 B CN103636496 B CN 103636496B
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Abstract
The present invention relates to a kind of Zhegucai artificial culture method.The steps include: the Zhegucai that growth selection is vigorous, the attachment on frond surface and assorted algae are removed; Sterilizing 20-40 minute in the solution containing Benzylpenicillin sodium salt 20-60TU/L; Zhegucai is cut into the tissue block of 1-3 mm in size, move and be put on 200-300 object bolting silks, add culture fluid, be 20-25 DEG C, intensity of illumination 1500-2000Lux in temperature, periodicity of illumination 12:12 hour, every day at least reveals dry 3-5 hour, every 4-5 days changes a culture fluid, with aseptic distilled water washing frond while replacing culture fluid, cultivates 15-20 days visible seedlings afterwards.The artificial breeding technique of Zhegucai can shorten growth cycle greatly, can produce seed throughout the year, increases the output that cultivation frequency increases Zhegucai then.Meet the demand of producing and then the needs meeting patient, there is huge social benefit.
Description
Technical field
The present invention relates to a kind of Zhegucai tissue cultures artificial raise seedling method.
Background technology
Zhegucai Caloglossaleprieurii (Montagne) J.I belongs to Ceramiale Ceramiales, Delesseriaceae Delesseriaceae, is warm warm nature marine alga, is distributed in China Zhejiang, Fujian and Coast of Guangdong Province.Because Zhegucai contains kainic acid (degenicacid) and digeneaside (degeneaside), there is anthelmintic action, be used for treating the roundworm parasitized in human body large intestine by the traditional Chinese medical science, it is that primary raw material is made that China's Chinese patent drug compound Zhegucai falls apart with Zhegucai.It is also often added to some and treats in the Chinese medicine preparation of the disease caused by malnutrition due to parasitic infestation or insect bite.As treated parasitic infestation type acute pancreatitis, treatment parasitic infestation type insomnia and treatment insect bite type ecthyma etc.In addition, also have scholar to inquire into its hypotensive effect mechanism, research shows that Zhegucai has the effect reduced blood pressure.Along with the further investigation to Zhegucai, it will become widely used sea-plant medicine resource.But due to the reason such as environmental change and artificial destruction, Zhegucai stock number but gradually reduces, and cannot satisfy social needs.Although Chinese scholar once carried out artificial cultivation research, because the problems such as the production cycle is long, cost is high, seedling development is uneven can not promoted, current medicinal herb grower still to gather Zhegucai by self-sow.
The cultivation of current Zhegucai does not cultivate seedling growing process in a organized way, still the traditional mode that nature is collected seedling is continued to use, annual seven, August is about to seedl-picking rope or other attachment to be placed at Zhegucai the tetraspore or carpospore attachment that district of collecting seedling allows frond diffuse out old and feeble season, again the rope being attached with spore is transplanted to culturing area to the Second Year January, along with weather slowly warms, attachment spore will germinate, four, the May be Zhegucai growth the most vigorous period just can gather in Zhegucai.
The aquaculture model of self-sow exists the production cycle to be fixed and long, the problems such as cost is high, seedling development is uneven.The time of collecting seedling is fixed, and is just difficult to adopt seed once miss the time of collecting seedling.Because spore can find rapidly the attachment of suitable attachment after diffusing, if can not find attachment will death rapidly.Owing to being self-sow pattern, self law of development is followed in its growth, within 1 year, can only gathering in the crops frond, therefore can only expand farming site to obtain enough fronds in fixing month.The expansion of farming site must increase cost.Its developmental potency of spore that traditional approach gathers is uncontrollable, causes the seedling after growing may occur uneven phenomenon.Moreover the spore density on attachment can not control accurately, so just occurred that place that Spore adhesion is many is with regard to riotous growth, the place that Spore adhesion is few just grows sparse situation.
Summary of the invention
The object of the invention is: the object of the invention is " totipotency " that utilize plant cell to have, a kind of Zhegucai seedling artificial culture method is provided.Method of the present invention is with short production cycle, cost is low, seedling is neatly healthy and strong.
Technical scheme provided by the invention
Zhegucai seedling of the present invention is manually cultivated step by the following method and is carried out:
The first step: Zhegucai frond tissue block prepares
A. select materials: the Zhegucai that namely growth selection animated period gathers, the attachment on Zhegucai frond surface and assorted algae are removed;
B. sterilizing: Zhegucai frond is immersed in sterilizing 20-40 minute in the solution containing Benzylpenicillin sodium salt 20-60TU/L;
C. stripping and slicing: the tissue block Zhegucai frond through sterilizing being cut into 1-3 mm in size, then uses rinsed with sterile water;
D. adhere to: moved by the Zhegucai tissue block cut and be put on 200-300 object bolting silks that sterilizing is good, bolting silk is as the adherance of tissue block;
Second step: Zhegucai uterus tissue pieces
E. seed attachment: the bolting silk being paved with Zhegucai tissue block is kept moistening 3-4 hour with culture fluid at normal temperatures, and the addition of culture fluid is as the criterion with the dipped bolting silk of culture fluid, ensures the not positioning of Zhegucai tissue block, makes Zhegucai tissue block be attached on bolting silk;
F. seedling fostering: move into incubator after Zhegucai tissue block adhesion-tight, suitably strengthen culture fluid consumption simultaneously, be limited to flood tissue block, cultivation temperature is 20-25 DEG C, intensity of illumination 1500-2000Lux, periodicity of illumination 12:12 hour, every day at least reveals dry 3-5 hour, every 4-5 days changes a culture fluid, with the washing of aseptic distilled water while replacing culture fluid, to remove the salinity be accumulated on frond surface;
G. emerge: Zhegucai tissue block is shown in seedling after cultivating 15-20 days, and namely the sea area that the bolting silk of emerging is placed directly into cultivation grows into Zhegucai;
The preparation of culture fluid: above-mentioned culture fluid sterilization seawater and distilled water are allocated in proportion, and specific gravity control is between 1.005-1.010, and deployed seawater adds following component, and its content is mass ratio:
Beneficial effect of the present invention
The artificial breeding technique of Zhegucai can shorten growth cycle greatly, and tissue block just can be emerged at about 15-20 days, can produce seed so throughout the year, increases the output that cultivation frequency increases Zhegucai then.Like this when constant or reduce cultivation land used considerably increase biomass, for raiser brings considerable economic benefit.Artificial breeding avoids the uncontrollability that nature is collected seedling, and can accomplish optimized operation.Tissue block donor optimization, tissue block layout density optimization, rearing condition optimization ... the seedling brought out like this is healthy and strong neat.The seed of neat stalwartness ensure that the quality of Zhegucai, also for lay a good foundation with the quality assurance of its product being raw material.The raising of output can meet the demand of production and then meet the needs of patient, is also recent studies on, the place mat that done newly developed, has huge social benefit.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
The tissue cultures key step of Zhegucai seedling of the present invention divides two steps: Zhegucai tissue block prepares, Zhegucai uterus tissue pieces.
First step raw material is selected: the selection of Zhegucai tissue block and cutting are the most important condition determining that Zhegucai seed grows, and the excellent Zhegucai tissue block of a unsoundness just can cultivate healthy and strong Zhegucai seed.To gather Zhegucai at Zhegucai growth animated period, the attachment on Zhegucai frond surface and assorted algae are removed.It should be noted that reduce the damage to Zhegucai frond as far as possible when cleaning attachment and assorted algae, keep the complete of Zhegucai frond, be convenient to the carrying out of subsequent step.By wash clean Zhegucai frond carry out sterilization treatment, Zhegucai frond is immersed in sterilizing 20-40 minute in the solution containing Benzylpenicillin sodium salt 20-60TU/L.The Zhegucai frond processed is selected under anatomical lens, and the complete frond that selection and comparison is tender carries out tissue block cutting, is cut into the tissue block of 1-3 mm in size, uses Aseptic sterilisation water rinse.Moved by Zhegucai tissue block subsequently and be put on 200-300 good object bolting silks of sterilizing, bolting silk is as the adherance of tissue block.
Second step uterus tissue pieces: the bolting silk being paved with Zhegucai tissue block is kept moistening 3-4 hour, so that the attachment of Zhegucai tissue block.Between the Zhegucai tissue block setting stage, culture fluid addition should not add too much, and to guarantee the dipped culture fluid of bolting silk, but the not positioning of Zhegucai tissue block is limited, and object allows Zhegucai tissue block be attached to better on bolting silk.Proceed to incubator after Zhegucai tissue block adhesion-tight to cultivate, at this moment can suitably strengthen culture fluid consumption, be limited to flood tissue block.Cultivation temperature is 20-25 DEG C, intensity of illumination 1500-2000Lux, periodicity of illumination 12:12 hour, i.e. illumination every day and each 12 hours of interlunation, every day at least reveals dry 3-5 hour, every 4-5 days changes a culture fluid, with the washing of aseptic distilled water while replacing culture fluid, to remove the salinity be accumulated on frond surface.See seedling after 15-20 days, namely the sea area that the bolting silk of emerging is placed directly into cultivation grows into healthy healthy and strong Zhegucai.
The preparation of culture fluid, culture fluid is the key determining seed quality.Culture fluid sterilization seawater and distilled water are allocated in proportion, and specific gravity control is between 1.005-1.010, and deployed seawater adds NaNO
3its concentration in culture fluid is made to be 5-10ppm (mass ratio, lower with), to add KH
2p0
4make its concentration in culture fluid be 0.5-1.0ppm, add the concentration that 6-benzyl aminopurine makes it in culture fluid and be 0.05-0.1ppm, add that the concentration that dichlorphenoxyacetic acid makes it in culture fluid is 0.05-0.1ppm, to add the concentration that methyl α-naphthyl acetate makes it in culture fluid be 0.05-0.1ppm, be made into final culture fluid.Due to NaNO
3, KH
2p0
4, 6-benzyl aminopurine (6-BA), dichlorphenoxyacetic acid (2,4-D), methyl α-naphthyl acetate consumption be less, can be configured to concentrate respectively, be placed in refrigerator and cooled and hide, the used time gets corresponding amount and adds in culture fluid.If find that concentrate has precipitation, suspension or microbial contamination at process for preparation, this concentrate should be eliminated immediately, re-start preparation.
In the present invention, frond sterilization can adopt its broadspectrum antibiotic to substitute Benzylpenicillin sodium salt can reach frond sterilisation purpose of the present invention equally.
Embodiment
Embodiment 1:
By the sterilizing 25 minutes in containing the solution of Benzylpenicillin sodium salt 25TU/L of the Zhegucai of wash clean, under anatomical lens, cutting into the Zhegucai tissue block of 1.2 millimeters, keeping moistening 3 hours with culture fluid with moving to after rinsed with sterile water on bolting silk; The bolting silk of attachment Zhegucai tissue block is put into incubator cultivate, cultivation temperature is 20 DEG C, and intensity of illumination is 1500Lux, photoperiod 12:12 hour, and every day, dry 4 hours of dew, changed a culture fluid in every four days, cultivates seedling after 15 days and moves to sea area and put in a suitable place to breed.
It is 6.5 ‰ (proportions 1.005) that culture fluid sterilization seawater and distilled water are adjusted to salinity, adds NaNO
3its concentration in culture fluid is made to be 6ppm (mass ratio, lower with), to add KH
2p0
4make its concentration in culture fluid be 0.6ppm, add the concentration that 6-benzyl aminopurine makes it in culture fluid and be 0.06ppm, add that the concentration that dichlorphenoxyacetic acid makes it in culture fluid is 0.06ppm, to add the concentration that methyl α-naphthyl acetate makes it in culture fluid be 0.06ppm, be mixed with culture fluid.
Embodiment 2:
By the sterilizing 25 minutes in containing the solution of Benzylpenicillin sodium salt 25TU/L of the Zhegucai of wash clean, under anatomical lens, cutting into the tissue block of 1.2 millimeters, keeping moistening 3 hours with culture fluid with moving to after rinsed with sterile water on bolting silk.The bolting silk of adhering tissue block is put into incubator cultivate, cultivation temperature is 25 DEG C, and intensity of illumination is 2000Lux, photoperiod 12:12 hour, and every day, dry 5 hours of dew, changed a culture fluid in every four days, cultivates seedling after 15 days and moves to sea area and put in a suitable place to breed.
It is 12.8 ‰ (proportions 1.010) that culture fluid sterilization seawater and distilled water are adjusted to salinity, adds NaNO
3its concentration in culture fluid is made to be 8ppm (mass ratio, lower with), to add KH
2p0
4make its concentration in culture fluid be 0.8ppm, add the concentration that 6-benzyl aminopurine makes it in culture fluid and be 0.06ppm, add that the concentration that dichlorphenoxyacetic acid makes it in culture fluid is 0.06ppm, to add the concentration that methyl α-naphthyl acetate makes it in culture fluid be 0.06ppm, be mixed with culture fluid.
The tissue block that the present invention chooses Zhegucai optimum is bred as new seed by artificial cultivation technique of nourishing and generating, and experimental result shows that tissue block just can be emerged at about 15 to 20 days, substantially reduces the production cycle.As long as frond well-grown just can, as tissue block donor, not need to carry out at a fixed time, ensure can cultivate seedling in any time of frond growth.The shortening of production cycle repeatedly can cultivate Zhegucai in 1 year, greatly improves the output of Zhegucai and without the need to expanding farming site, reducing cost and dropping into.Seed due to this technology is grown by tissue block instead of naturally gathers, and can ensure that seedling is healthy and strong neat, finished product cultivation high and stable yields.
Claims (1)
1. a Zhegucai artificial raise seedling method, carries out according to the following steps:
The first step: Zhegucai frond tissue block prepares
A. select materials: the Zhegucai that namely growth selection animated period gathers, the attachment on Zhegucai frond surface and assorted algae are removed;
B. sterilizing: Zhegucai frond is immersed in sterilizing 20-40 minute in the solution containing Benzylpenicillin sodium salt 20-60TU/L;
C. stripping and slicing: the tissue block Zhegucai frond through sterilizing being cut into 1-3 mm in size, then uses Aseptic sterilisation water rinse;
D. adhere to: moved by the Zhegucai tissue block cut and be put on 200-300 object bolting silks that sterilizing is good, bolting silk is as the adherance of tissue block;
Second step: Zhegucai uterus tissue pieces
E. seed attachment: the bolting silk being paved with Zhegucai tissue block is kept moistening 3-4 hour at normal temperatures, and the addition of culture fluid is as the criterion with dipped bolting silk, ensures the not positioning of Zhegucai tissue block, makes Zhegucai tissue block be attached on bolting silk;
F. seedling fostering: proceed to incubator after Zhegucai tissue block adhesion-tight, suitably strengthen culture fluid consumption simultaneously, be limited to flood tissue block, cultivation temperature is 20-25 DEG C, intensity of illumination 1500-2000Lux, periodicity of illumination 12:12 hour, every day at least reveals dry 3-5 hour, every 4-5 days changes a culture fluid, with the washing of aseptic distilled water while replacing culture fluid, to remove the salinity be accumulated on frond surface;
G. emerge: Zhegucai tissue block is shown in seedling after cultivating 15-20 days, and namely the sea area that the bolting silk of emerging is placed directly into cultivation grows into Zhegucai;
The preparation of culture fluid: above-mentioned culture fluid sterilization seawater and distilled water are allocated in proportion, and specific gravity control is between 1.005-1.010, and deployed seawater adds following component, and its content is mass ratio:
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| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN101018480A (en) * | 2004-04-20 | 2007-08-15 | 国立大学法人鹿儿岛大学 | Manual intensive cultivation apparatus and cultivating method for algae |
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| KR20120079067A (en) * | 2009-08-13 | 2012-07-11 | 트리프리 바이오메스 솔루션스, 인코포레이티드 | Methods for vegetative propagation of grass plants |
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|---|---|---|---|---|
| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN101018480A (en) * | 2004-04-20 | 2007-08-15 | 国立大学法人鹿儿岛大学 | Manual intensive cultivation apparatus and cultivating method for algae |
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| Regeneration Studies in Grateloupia filicina (J.V. Lamouroux) C. Agardh – An Important Carrageenophyte and Edible Seaweed;Pooja Baweja et al.;《Algae》;20091231;第24卷(第3期);第163-168页 * |
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