CN102257964B - Method for cultivating triploid dendrobium officinale - Google Patents
Method for cultivating triploid dendrobium officinale Download PDFInfo
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Abstract
The invention relates to a method for cultivating triploid dendrobium candidum. The method comprises the following steps of: (1) firstly widely collecting dendrobium candidum seed resources in which good dendrobium candbium is selected; (2) carrying out artificial mutagenesis on the selected dendrobium candbium to obtain autotetraploid dendrobium candbium; (3) carrying out sexual hybridization based on the autotetraploid dendrobium candbium as a female parent and diploid dendrobium candbium as a male parent to obtain triploid dendrobium candbium seeds; (4) carrying out non symbiosis germination culture on the triploid dendrobium candbium seeds; (5) carrying out induction and differentiation culture on the protocorms (PLBs) of the triploid dendrobium candbium seeds; (6) carrying out differentiation on triploid dendrobium candbium mature seedling; (7) transplanting triploid dendrobium candbium seedlings in bottles; and (8) carrying out cultivation management on the triploid dendrobium candbium mature seedling. In the invention, a triploid having the advantages of a polyploid and a hybrid cultivar can be obtained through genome triplication, and the triploid is giant and non substitutable; the yield of dendrobium candbium medicinal material can be greatly improved; and meanwhile, the triploid dendrobium candbium is a permanent hybrid, and hybrid vigor can be fixed.
Description
Technical field:
The present invention relates to a kind of breeding method of triploid dendrobium officinale.
Background technology:
Dendrobium candidum (Dendrobium candidum Wall.ex Lindl) claim again ribbed hedyotis herb, it be a kind ofly grow nonparasitically upon another plant, the perennial herb orchid.Mainly be distributed in the ground such as Yunnan Province of China, Guizhou, Guangxi, Taiwan.Average temperature of the whole year 12-18 degree, relative moisture 60-75%, woodland light transmittance about 60%, annual rainfall 1100-1500mm.Be grown in the evergreen broad-leaved forest, grow nonparasitically upon another plant on the trunk or on the limestone.Wild Seeds of Dendrobium Candidum survival rate is extremely low, and poor growth just can be gathered to entering commodity stage need 5-7 from seed germination, causes its output seldom.In recent years, supply falls short of demand owing to city's field boundary, and people excessively excavate, and add environmental deterioration, and the dendrobium candidum natural resources is increasingly exhausted.Dendrobium candidum is commonly called as Tiepi Fengdou after the processing, be traditional rare traditional Chinese medicine, has the immunity of enhancing, antitumor, tonifying-Yin and nourishing-stomach, clearing heat and promoting fluid, the effect that moistens the lung and relieve the cough.The prediction dendrobium candidum will become the senior throat health beverages that protects of 21 century.Modern age, the pharmacology medicine studies show that, dendrobium candidum pharmaceutical chemistry composition mainly is polysaccharose, alkaloid and anti-tumor active substance.Polyoses content reaches 18.2%-22.27%, and these polysaccharoses are immunopotentiators, has the T cell of enhancing and macrophage immunity active function.Dendrobium candidum contains two kinds of anticancer luxuriant and rich with fragrance compounds, has the inhibitory action to liver cancer and ehrlich ascites cell.The effect of promoting the production of body fluid that dendrobium candidum is nourishing Yin and clearing heat mainly with-atpase activity is relevant.
Country's Eleventh Five-Year Plan and " 12 " plan all propose the task of " directive breeding of the excavation of germ plasm resource, preservation, innovation and new varieties ".For implementing, implement this task, we propose to use the method for " the genome polyploidization combines with hybridization ", can realize the purpose of the directive breeding of the innovation of germ plasm resource and new varieties.
Summary of the invention:
The breeding method that the purpose of this invention is to provide a kind of triploid dendrobium officinale is used the method for " the genome polyploidization combines with hybridization ", and the cultivation triploid dendrobium officinale can be realized the purpose of the directive breeding of the innovation of germ plasm resource and new varieties.It is that the dliploid dendrobium candidum induced mutations that at first natural world is existed becomes autotetraploid, then, tetraploid dendrobium candidum and diploid hybrid produce triploid dendrobium officinale, that is: 4x ♀ * 2x ♂ → 3x, hence one can see that, and triploid dendrobium officinale is produced by hybridization, has heterosis, hybrid vigor, simultaneously, triploid is that polyploid also has polyploid advantage, so triploid has crossbreed and double dominancy of polyploid.Simultaneously, behind the formation triploid, formed the reproduction isolation in " on the space " with other species, can not form the offspring, do not had the offspring to separate, its first filial generation is that permanent hybrid (can be designated as: permanent F1), fixed hybrid vigour.Therefore, triploid dendrobium officinale root yield and main content of active component aspect have superpower advantage, have significant application value.
The breeding method of triploid dendrobium officinale of the present invention may further comprise the steps:
1) at first from germ plasm resource, selects good dendrobium candidum kind;
2) the good dendrobium candidum of selecting is carried out induced mutations, obtain the autotetraploid dendrobium candidum;
3) carry out sexual hybridization with the dliploid dendrobium candidum as male parent as maternal take the tetraploid dendrobium candidum, obtain the triploid dendrobium officinale seed;
4) the triploid dendrobium officinale seed being carried out non-symbiosis germination cultivates;
5) triploid dendrobium officinale seed protocorm (PLBs) is induced and break up cultivation;
6) triploid dendrobium officinale seedling differentiation;
7) transplantation of seedlings of triploid dendrobium officinale bottle is carried out the seedling cultivation management with dendrobium candidum routinely.
The breeding method of a kind of triploid dendrobium officinale provided by the invention may further comprise the steps:
1) the dliploid dendrobium candidum carries out cultivation management according to a conventional method, carries out geitonogamy in the 5-10 after blooming days, and the dendrobium candidum plant after the pollination is placed under ventilation, the semi-transparent condition, keeps 30-50% humidity, 15-20 ℃ of temperature.
2) after artificial pollination 120-160 days, cut the dendrobium candidum capsule, in superclean bench with 75% alcohol disinfecting 1 minute, again with 0.1% mercuric chloride aqueous solution sterilization 10 minutes, aseptic water washing 5 times cuts along the capsule y direction with the aseptic operation cutter, and faint yellow Seeds of Dendrobium Candidum is seeded in the 1/2MS liquid nutrient medium, 60 times/minutes vibrations or static non-symbiosis germination were cultivated about 40-50 days, and (PLBs) develops into sphere to the seed protocorms.
3) carry out tetraploid dendrobium candidum artificial induction, change in the 1/2MS liquid nutrient medium that contains 0.02% colchicin developing into spherical seed protocorm (former embryonic stage), mutagenic treatment 150-180 hour,
4) the dendrobium candidum protocorm after the mutagenic treatment is changed on the 1/2MS solid culture medium and to continue to cultivate, until the dendrobium candidum seedling forms, break up to seedling; Getting the tip of a root of seedling, carry out first the chromosome pre-treatment, then carry out Methods of Ploidy Identification, choose the seedling of chromosome number 2n=76, is the autotetraploid dendrobium candidum that doubles;
It is under illumination that described continuation is cultivated, 14 hours/day photoperiods, luminous intensity 3000Lux, 25 ℃ of room temperatures;
Described dyeing times gonosome authentication method: by the strain numbering, get the tip of a root of the new growth of seedling, put into p-dichlorobenzene saturated aqueous solution pre-treatment 3-4h, then put into 3: 1 methyl alcohol of volume ratio and glacial acetic acid fixer, save backup in 4 ℃ of refrigerators; With the dyeing of carbolfuchsin dyeing liquor, pressed disc method prepares chromosome specimen; Microscopic examination chromosome number more than 400 times is chosen the seedling that chromosome number is 2n=76, is the autotetraploid dendrobium candidum that doubles;
5) cultivation management of tetraploid dendrobium candidum is transplanted doubling successful dendrobium candidum seedling, and dendrobium candidum routinely carries out cultivation management, until bloom;
6) after the tetraploid dendrobium candidum is bloomed, take the autotetraploid dendrobium candidum of induced mutations as maternal, the dliploid dendrobium candidum is male parent, carries out sexual hybridization, and the Seeds of Dendrobium Candidum of tying all is triploid dendrobium officinale, that is: 4x ♀ * 2x ♂ → 3x;
Triploid dendrobium officinale is compared with the dliploid dendrobium candidum, and its main morphological traits of tea plant is basic identical, but its stem, leaf, flower show obvious increase, and root yield and dendrobium polysaccharide content significantly improve.Triploid has polyploid and crossbreed is dual excellent, and its first-filial generation is " permanent F1 ", and namely permanent hybrid has been fixed hybrid vigour.
7) triploid iron sheet dry measure used in former times seed is carried out non-symbiosis germination is cultivated, protocorm is induced, seedling is broken up, become transplantation of seedlings, dendrobium candidum carries out cultivation management routinely.
The invention provides the triploid breeding method of a kind of breeding method of dendrobium candidum new varieties, particularly dendrobium candidum, can realize the purpose of the directive breeding of the innovation of germ plasm resource and new varieties.By the genome tripling, can obtain to have the triploid of polyploid and crossbreed double dominant, its Heterosis goes out to have huge property and irreplaceability, can increase substantially the dendrobium candidum root yield, simultaneously, triploid dendrobium officinale is permanent hybrid, can hybrid vigor fixing.Triploid dendrobium officinale has crossbreed and double dominancy of polyploid.Simultaneously, behind the formation triploid, formed the reproduction isolation in " on the space " with other species, can not form the offspring, do not had the offspring to separate, its first filial generation is that permanent hybrid (can be designated as: permanent F1), namely fixed hybrid vigour.Therefore, triploid dendrobium officinale root yield and main content of active component aspect have superpower advantage, have significant application value.
Description of drawings
Fig. 1: dliploid dendrobium candidum parent, chromosome number is 2n=2x=38.
Fig. 2: tetraploid dendrobium candidum parent, chromosome number is 2n=4x=76.
Fig. 3: dliploid and the positive and negative friendship fruit of tetraploid dendrobium candidum hybridization F1.
Fig. 4: dliploid and Tetraploid F1 Seeds of Dendrobium Candidum non-symbiosis germination are cultivated, the sprouting PLBs of haircut for amplifying.
Fig. 5: the seedling differentiation of triploid dendrobium officinale is cultivated.
Fig. 6: triploid dendrobium officinale bottle seedling is cultivated, the bottle seedling of arrow for amplifying.
Fig. 7: the Ploidy Identification of triploid dendrobium officinale, 2n=3x=57.
Fig. 8: hardening (a) is cultivated (b) with the rear seedbed of transplanting before the transplantation of seedlings of triploid dendrobium officinale bottle.
Embodiment
The characteristics that the present invention gives prominence to and marked improvement can be embodied from following example, but can not impose any restrictions the present invention.
The breeding method of triploid dendrobium officinale of the present invention may further comprise the steps:
1) at first from germ plasm resource, selects good dliploid dendrobium candidum kind;
2) the good dliploid dendrobium candidum of selecting is carried out induced mutations, obtain the autotetraploid dendrobium candidum, induce tetraploid method with reference to ZL200710061225.8;
3) take the tetraploid dendrobium candidum as maternal, dliploid dendrobium candidum carry out sexual hybridization as male parent, obtain the triploid dendrobium officinale seed;
4) the triploid dendrobium officinale seed being carried out non-symbiosis germination cultivates;
5) triploid dendrobium officinale seed protocorm (PLBs) is induced and break up cultivation;
6) triploid dendrobium officinale seedling induction is cultivated;
7) transplantation of seedlings of triploid dendrobium officinale bottle is carried out the seedling cultivation management with dendrobium candidum routinely.
Concrete Application Example
Introduce dliploid dendrobium candidum (2n=38) from Yunnan Province's mountain of papers in April, 2005, as: Fig. 1, carried out the different colored artificial pollination of homophyletic on May 20 in then, dendrobium candidum after the pollination is at 25 ℃, under the 50% natural lighting condition, on November 22nd, 2005, cut capsule, in superclean bench, use first 75% alcohol disinfecting 1 minute, again with 0.1% mercuric chloride sterilization 10 minutes, aseptic water washing 5 times, cut along the capsule y direction with aseptic scalpel, Seeds of Dendrobium Candidum is seeded in the 1/2MS liquid nutrient medium shaken cultivation under 25 ℃ of conditions, after about 30 days, change in the 1/2MS liquid nutrient medium that contains 0.01% colchicin, mutagenic treatment 130 hours, after change on the 1/2MS solid culture medium, 14 hours/day photoperiods, luminous intensity 3000Lux continues under 25 ℃ of conditions of room temperature to cultivate, until the differentiation of dendrobium candidum seedling.
When the bottle transplantation of seedlings, get respectively the tip of a root of new growth by strain, prepare chromosome specimen with pressed disc method, choose and double successful dendrobium candidum seedling (2n=76), transplant, the cultivation condition of dendrobium candidum carries out cultivation management routinely.
On May 30th, 2008, the tetraploid dendrobium candidum was bloomed for the first time, as: Fig. 2.Within the week of blooming, take former mountain of papers dliploid dendrobium candidum as male parent, the tetraploid dendrobium candidum of mutagenesis success is maternal, carry out artificial pollination, to hybridize the maturescent triploid dendrobium officinale capsule (143 days) that connects that obtains on October 20th, 2008 cuts, as: Fig. 3, in superclean bench, use first 75% alcohol disinfecting 1 minute, again with 0.1% mercuric chloride sterilization 10 minutes, aseptic water washing 5 times cuts along the capsule y direction with aseptic scalpel, and Seeds of Dendrobium Candidum is seeded in the 1/2MS liquid nutrient medium, shaken cultivation 3-4 week under 25 ℃ of conditions, change over to and leave standstill cultivation, until seed germination forms the seed protocorm, as: Fig. 4; Then change in the 1/2MS solid culture medium, 14 hours/day photoperiods, luminous intensity 3000Lux carried out the differentiation of triploid dendrobium officinale seedling and cultivates under 25 ℃ of conditions of room temperature, as: Fig. 5; Then continue the bottle seedling and cultivate, as: Fig. 6; Grow to bottleneck 1/2-2/3 height, blade 3-4 sheet, when new root is vigorous, opens bottleneck and carry out hardening, such as Fig. 8 a at the bottle seedling; Carry out Methods of Ploidy Identification before the transplanting, as: Fig. 7,2n=57 can carry out a bottle transplantation of seedlings for the triploid dendrobium officinale that the hybridization transformation becomes, as: Fig. 8 b.
Triploid dendrobium officinale seedling cultivation management is basically identical with general dliploid officinal dendrobium stem plantation management condition, and seedling is transplanted officinal dendrobium stem plantation management condition routinely, gathers with medicinal material until bloom.
Claims (3)
1. the breeding method of a triploid dendrobium officinale is characterized in that may further comprise the steps:
1) the dliploid dendrobium candidum carries out cultivation management according to a conventional method, carries out geitonogamy in the 5-10 after blooming days, and the dendrobium candidum plant after the pollination is placed under ventilation, the semi-transparent condition, keeps 30-50% humidity, 25 ℃ of temperature;
2) after artificial pollination 180 days, cut the dendrobium candidum capsule, in superclean bench with 75% alcohol disinfecting 1 minute, with 0.1% mercuric chloride aqueous solution sterilization 10 minutes, aseptic water washing 5 times cut along the capsule y direction with the aseptic operation cutter again, faint yellow Seeds of Dendrobium Candidum is seeded in the 1/2MS liquid nutrient medium, 60 rev/mins, the vibration non-symbiosis germination was cultivated 30 days, and (PLBs) develops into sphere to the seed protocorms;
3) carry out tetraploid dendrobium candidum artificial induction, change in the 1/2MS liquid nutrient medium that contains 0.01% colchicin developing into spherical seed protocorms, mutagenic treatment 130 hours,
4) protocorms of dendrobium candidum after the mutagenic treatment is changed on the 1/2MS solid culture medium and to continue to cultivate, until the dendrobium candidum seedling forms, break up to seedling; Getting the tip of a root of seedling, carry out first the chromosome pre-treatment, then carry out Methods of Ploidy Identification, choose the seedling of chromosome number 2n=76, is to double successful autotetraploid dendrobium candidum;
5) cultivation management of tetraploid dendrobium candidum is transplanted doubling successful dendrobium candidum seedling, and dendrobium candidum routinely carries out cultivation management, until bloom;
6) after the tetraploid dendrobium candidum is bloomed, take the tetraploid dendrobium candidum as maternal, the dliploid dendrobium candidum is male parent, carries out sexual hybridization, and the Seeds of Dendrobium Candidum of tying all is triploid dendrobium officinale, that is: 4x ♀ * 2x ♂ → 3x;
Triploid dendrobium officinale is compared with the dliploid dendrobium candidum, and the Main Morphology shape is basic identical, but the stem of triploid dendrobium officinale, leaf, flower show obvious increase, and root yield and dendrobium polysaccharide content significantly improve; Triploid has polyploid and crossbreed double dominant, and its first-filial generation is " permanent F1 ", and namely permanent hybrid has been fixed hybrid vigour;
7) the triploid dendrobium officinale seed is carried out non-symbiosis germination is cultivated, protocorms is induced, seedling break up, the one-tenth transplantation of seedlings, dendrobium candidum carries out cultivation management routinely.
2. according to breeding method claimed in claim 1, it is characterized in that step 4) described continuation is cultivated is under illumination, 14 hours/day photoperiods, luminous intensity 3000Lux, 25 ℃ of room temperatures.
3. according to breeding method claimed in claim 1, it is characterized in that step 4) described chromosome pre-treatment and Methods of Ploidy Identification method: by the strain numbering, get the tip of a root of the new growth of seedling, put into p-dichlorobenzene saturated aqueous solution pre-treatment 3-4h, then put into 3: 1 methyl alcohol of volume ratio and glacial acetic acid fixer, save backup in 4 ℃ of refrigerators; With the dyeing of carbolfuchsin dyeing liquor, pressed disc method prepares chromosome specimen; Microscopic examination chromosome number more than 400 times is chosen the seedling that chromosome number is 2n=76, is the autotetraploid dendrobium candidum that doubles.
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| CN103535274A (en) * | 2012-07-13 | 2014-01-29 | 宁波洛祥药业有限公司 | Method of largely breeding homologous homozygous tetraploid dendrobium officinale |
| CN103718948B (en) * | 2013-12-06 | 2016-08-17 | 广西壮族自治区林业科学研究院 | View and admire the breeding method of Herba Dendrobii new germ plasm |
| CN103828705B (en) * | 2014-01-13 | 2016-03-30 | 陈瑞阳 | The breeding method of the genuine red sage root |
| CN104396722B (en) * | 2014-07-18 | 2017-03-08 | 皖西学院 | A kind of method that utilization tetraploid plant cultivates triploid Herba Dendrobii with liploid plant breeding by crossing |
| CN104429939B (en) * | 2014-07-18 | 2016-12-14 | 皖西学院 | A kind of method utilizing diploid cell to cultivate triploid Herba Dendrobii with pollen cell Cell-fusion breeding method |
| CN104429916B (en) * | 2014-07-18 | 2017-04-05 | 安徽斛生记生物科技有限公司 | A kind of method that gamete breeding by crossing using multiplication chromosome cultivates triploid Dendrobidium huoshanness |
| CN105010149B (en) * | 2015-08-23 | 2017-07-07 | 安徽科技学院 | A kind of breeding method of root-mustard triploid hydrid kind |
| CN107360968A (en) * | 2017-07-26 | 2017-11-21 | 海南大学 | A kind of breeding method of the polyploid China stem of noble dendrobium |
| CN107801633A (en) * | 2017-11-07 | 2018-03-16 | 安徽同济生生物科技有限公司 | A kind of stem of noble dendrobium polyploid seedling abductive approach |
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| 李秀兰.石斛属种质资源的遗传改良与创新研究.《中国植物学会七十五周年年会论文摘要汇编（1933-2008）》.2008, * |
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