CN103270948B - Two polyploid centella asiatica varieties and cultivation method thereof - Google Patents
Two polyploid centella asiatica varieties and cultivation method thereof Download PDFInfo
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Abstract
The invention discloses two polyploid centella asiatica varieties and a cultivation method thereof. The cultivation method comprises the following steps of 1, selecting terminal buds of a diploid centella asiatica adult plant, and carrying out breeding of a tissue cultured seedling or callus tissue, 2, carrying out chromosome doubling induction culture and differentiation culture of tissue subcultured seedling or callus tissue by oryzalin, 3, cutting down novel buds obtained by the differentiation culture and carrying out cluster bud culture, 4, carrying out chromosome doubling identification of the cultured complete plants having roots, stems and leaves, screening the tetraploid plant, and planting the tetraploid plant to obtain the tetraploid centella asiatica variety, 5, carrying out artificial emasculation and pollination hybridization of the tetraploid plant and the diploid plant to obtain hybrid seeds (female parent 4N*male parent 2N or male parent 4N* female parent 2N, wherein F1 is 3x), and 6, breeding the hybrid seeds into complete plants, planting the plants, carrying out chromosome number detection and screening the triploid centella asiatica variety.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of Si Bei Ti ﹑ triploid centella kind and breeding method thereof.
Background technology
Samphire centella Centela asiatica (L.) Urban calls Gotu Kola, collapses jorum etc., for liploid plant, main product in Guangxi, Guizhou, Yunnan, the ground such as Guangdong, be the conventional Chinese medicine that version over the years " Chinese Pharmacopoeia " is recorded, all herbal medicine.Its bitter, pungent, cool in nature, there is effect of clearing heat and promoting diuresis, removing toxicity for detumescence.Be the core material of the Chinese patent drug such as production three gold plaque, beautiful leaf removing toxic substances syrup, tcm clinical practice is used for the treatment of jaundice with damp-heat pathogen, and heatstroke is suffered from diarrhoea, and urolithiasis blood drenches, carbuncle sore tumefacting virus, the diseases such as injury from falling down.Chang Zuowei cold tea among the people, vegetables eat.In recent years, all multiple enterprises are for extracting Asiaticoside, asiatic acid etc., and the market demand constantly increases.For a long time, centella breed of variety, seedling breeding, artificial planting are still in blank, only rely on wild resource at present.But the distribution of wild centella is scattered, plant is little, yield poorly, difficulty of gathering in a large number is large.Even if carry out artificial planting, its input is greater than output, nobody shows any interest in, and causes centella raw material supply nervous.
Make medicinal plant genome polyploidization by ploidy breeding technique, plant type is large, active constituent content is high kind can be obtained, significantly improve the yield and quality of medicinal plant, artificial planting is become a reality.And triploid medicinal plant is spatially isolated with other breed formation reproductions in heredity, infertility is separated, and is permanent hybrid, can hybrid vigor fixing.
Summary of the invention
For improving the yield and quality of centella, the invention provides two polyploid centella asiatica kind and breeding method thereof.The present invention is clever in derivant with ammonia sulphur, carries out artificial induction, successfully obtains a kind of centella autotetraploid kind, then can cultivate triploid centella kind with conventional wild diploid species screening by hybridization by liquid processes or solid process.
Two polyploid centella asiatica breed of variety side provided by the invention comprises the following steps:
1) from dliploid centella germ plasm resource, excellent centella plant is filtered out;
2) choose the terminal bud of excellent centella adult plants, differential medium induction produces indefinite bud and callus routinely after pretreatment, is then transferred to routine propagation medium and carries out squamous subculture;
3) by step 2) in the strong group training Regenerated plant of growth potential or the strong callus light culture of meristematic capacity after 5 days, with the spirit of ammonia sulphur for mutagen, liquid or solid is adopted to mix training method, mutagenesis centella group training Regenerated plant or callus produce the change of chromogene group ploidy, proceed in general differentiation medium and carry out illumination cultivation, obtain Multiple Buds, proceed to after plant division in root media and be trained the complete plantlet in vitro of root, stem and leaf;
4) the complete plantlet in vitro of root, stem and leaf that step 3) obtains is contrasted with dliploid plantlet in vitro, the candidate plant having the plant of significant change to elect genome as leaf blade size, color may to double;
5) meristematic tissue of the candidate plant in clip step 4), carries out Methods of Ploidy Identification, chooses the seedling of chromosome number 2n=4x=36, its hardening, plantation is survived, and namely obtains tetraploid centella kind.
Can also continue to comprise the following steps:
6) by tetraploid centella and dliploid centella artificial pollination hybridization, hybridization centella seed is obtained;
7) hybrid seed is bred into whole plant plantation, then carry out chromosome number detection to its meristematic tissue, screening obtains triploid centella kind.
Enumerate the preferred method of operation of above key step below:
In step 1), the excellent centella plant as original parent refers to: be select to have that stem section internode is long, stem is thick, blade is large and abundant, that active constituent content is high adult plants the wild centella of 2n=18 from karyotype index.
Described in step 3), method of mutagenesis is: solid mixes training method and refers to aseptically by group strong for growth potential training Regenerated plant or the strong callus light culture 5-7 days of meristematic capacity, then be transferred to and be added with in 10-12 μm of ol.L-1 ammonia sulphur spirit solution MS solid culture medium light culture 2-5d under 5-10 DEG C of low temperature, then proceed to and do not carry out illumination cultivation containing in ammonia sulphur spirit solution general differentiation medium; Liquid processes refers to the material covers Regenerated plant terminal bud or callus of aseptically first using good water-retaining property, after cotton is soaked by 8-10 μm of ol.L-1 ammonia sulphur spirit solution completely that then instill filtration sterilization under 5-10 DEG C of low temperature light culture 10-12 hour, sterile water is cleaned, and proceeds to not containing illumination cultivation in the general differentiation medium of ammonia sulphur spirit solution;
Illumination cultivation described in step 3) is illumination every day 10 hours, and intensity of illumination is 1000-2000lx, and cultivation temperature is 25 DEG C;
Culture of rootage described in step 3) is: medium is 1/2MS+NAA0.3mg/L+IAA0.3mg/L, and condition of culture is carry out illumination cultivation after light culture 7d, and intensity of illumination is 1000-2000lx, and incubation time is 10h ∕ d, and cultivation temperature is 25 DEG C.
Methods of Ploidy Identification method described in step 5) is: the tender tip of a root 0.5cm of clip target strain children, clear water soaks, through absolute ethyl alcohol: more than 15min fixed by the Kano fixer of glacial acetic acid=3:1, clear water rinses 15min, concentrated hydrochloric acid: the dissociation solution of methyl alcohol=1:1 is dissociated 2-30min, and clear water rinses 15min, cuts tip of a root 0.1mm, with the dyeing of carbolfuchsin dyeing liquor, statistics root tip chromosomes observed by compressing tablet.
Triploid hydrid centella seed described in step 6) is: ♀ 4N × ♂ 2N or ♂ 4N × ♀ 2N → F1 is 3x.
The tetraploid centella kind that two polyploid centella asiatica kind provided by the invention is said method cultivation and obtains or triploid centella kind.
Advantage of the present invention:
The centella tetraploid of the invention, triploid new germ plasm, its root, stem, leaf, flower and active ingredient thereof are obviously greater than or higher than diploid parents, overcome the deficiency of existing wild species.Wherein triploid centella also has hybrid vigour and polyploid advantage, and triploid centella is because spatially isolating with other breed formation reproductions in heredity, and infertility is separated, can hybrid vigor fixing by vegetative manner; In addition triploid centella blooms but does not bear seeds, and decreases the nutrient consumption that seminal propagation produces, thus realizes the high yield and high quality of centella medicinal material.
Embodiment
Content of the present invention can be had a clear understanding of further by specific embodiment given below, but not be limitation of the invention.
Embodiment 1
1) the centella adult plants terminal bud 2cm that clip growth potential is strong, healthy and strong, after being pushed aside by outer blade, rinses 2-4 time with clear water, then aseptically first 20min is soaked with 0.3% liquor potassic permanganate, sterile water is cleaned, then uses 0.2% mercuric chloride process 5min, aseptic washing 6 times.Receive induction in general differentiation medium respectively and produce indefinite bud and callus, be then transferred to routine propagation medium and carry out squamous subculture;
2) the group training Regenerated plant that growth potential is strong is chosen, aseptically first cover Regenerated plant terminal bud or callus with sterilizing cotton is soft, then 10 μm of ol.L-1 ammonia sulphur spirit (chemical names: 3 of filtration sterilization are drawn with sterilizing dropper, 5-dinitro-N4, N4-propyl group sulfanilamide (SN)) solution cotton is soaked completely after under 5-10 DEG C of low temperature light culture 12 hours, sterile water is cleaned, forward in the general differentiation medium not containing ammonia sulphur spirit solution and carry out illumination cultivation, illumination every day 10 hours, intensity of illumination is 1000-2000lx, cultivation temperature is 25 DEG C, until grow sprouting,
3) cut and cultivate through differentiation the sprouting that grows and proceed in general differentiation medium and induce it to produce Multiple Buds, then be trained the complete plantlet in vitro of root, stem and leaf by proceeding to after Multiple Buds plant division in root media 1/2MS+NAA0.3mg/L+IAA0.3mg/L;
4) from 3) plantlet in vitro that obtains contrasts with dliploid plantlet in vitro, the candidate plant having the plant of significant change to elect genome as leaf blade size, color may to double;
5) clip 4) in the tip of a root 0.5cm of plant, clear water soaks, and through Kano, more than 15min fixed by fixer, and clear water rinses 15min, to dissociate 20-30min by dissociation solution again, clear water rinses 15min, cuts tip of a root 0.1mm, dyes with carbolfuchsin dyeing liquor, statistics root tip chromosomes number observed by compressing tablet, filter out tetraploid plant, plantation survives, and obtains tetraploid centella kind.
6) by tetraploid centella and dliploid centella artificial pollination hybridization (♀ 4N × ♂ 2N), hybridization centella seed is obtained;
7) hybrid seed is bred into whole plant plantation, then carry out chromosome number detection to its tip of a root, screening obtains triploid centella kind.
Embodiment 2
1) the centella adult plants terminal bud 2cm that clip growth potential is strong, healthy and strong, after being pushed aside by outer blade, rinses 2-4 time with clear water, then aseptically first 20min is soaked with 0.3% liquor potassic permanganate, sterile water is cleaned, then uses 0.2% mercuric chloride process 5min, aseptic washing 6 times.Receive induction in general differentiation medium respectively and produce indefinite bud and callus, squamous subculture in routine propagation medium of then transferring;
2) after choosing the strong group training Regenerated plant light culture 5d of growth potential, aseptically peel off outer blade gently with tweezers, then be transferred to and be added with in 12 μm of ol.L-1 ammonia sulphurs spirit solution MS solid culture mediums light culture 2-5d under 5-10 DEG C of low temperature, proceed to again and do not carry out illumination cultivation containing in ammonia sulphur spirit solution general differentiation medium, illumination every day 10 hours, intensity of illumination is 1000-2000lx, and cultivation temperature is 25 DEG C, until grow sprouting;
3) cut and cultivate through differentiation the sprouting that grows and proceed in general differentiation medium and induce it to produce Multiple Buds, then be trained the complete plantlet in vitro of root, stem and leaf by proceeding to after Multiple Buds plant division in root media 1/2MS+NAA0.3mg/L+IAA0.3mg/L;
4) from 3) plantlet in vitro that obtains contrasts with dliploid plantlet in vitro, the candidate plant having the plant of significant change to elect genome as leaf blade size, color may to double;
5) clip 4) in the tip of a root 0.5cm of candidate plant, clear water soaks, and through Kano, more than 15min fixed by fixer, and clear water rinses 15min, to dissociate 2-30min by dissociation solution again, clear water rinses 15min, cuts tip of a root 0.1mm, dyes with carbolfuchsin dyeing liquor, statistics root tip chromosomes observed by compressing tablet, filter out tetraploid plant, plantation survives, and obtains tetraploid centella kind;
6) by tetraploid centella and dliploid centella artificial pollination hybridization (♂ 4N × ♀ 2N), hybridization centella seed is obtained;
7) hybrid seed is bred into whole plant plantation, then carry out chromosome number detection to its stem apex, screening obtains triploid centella kind.
Claims (3)
1. two polyploid centella asiatica breed of variety method, is characterized in that, comprises the following steps:
1) from dliploid centella germ plasm resource, excellent centella plant is filtered out;
2) choose the terminal bud of excellent centella adult plants, differential medium induction produces indefinite bud and callus routinely after pretreatment, is then transferred to routine propagation medium and carries out squamous subculture;
3) by step 2) in the strong group training Regenerated plant of growth potential or the strong callus light culture of meristematic capacity after 5 days, with the spirit of ammonia sulphur for mutagen, liquid or solid is adopted to mix training method, mutagenesis centella group training Regenerated plant or callus produce the change of chromogene group ploidy, proceed in general differentiation medium and carry out illumination cultivation, obtain Multiple Buds, proceed to after plant division in root media and be trained the complete plantlet in vitro of root, stem and leaf; Described illumination cultivation is illumination every day 10 hours, and intensity of illumination is 1000-2000lx, and cultivation temperature is 25 DEG C; Described culture of rootage is: medium is 1/2MS+NAA0.3mg/L+IAA0.3mg/L, and condition of culture is carry out illumination cultivation after light culture 7d, and intensity of illumination is 1000-2000lx, and incubation time is 10h/d, and cultivation temperature is 25 DEG C; Described method of mutagenesis is: solid mixes training method and refers to aseptically by group strong for growth potential training Regenerated plant or the strong callus light culture 5-7 days of meristematic capacity, then be transferred to and be added with in 10-12 μm of ol.L-1 ammonia sulphur spirit solution MS solid culture medium light culture 2-5d under 5-10 DEG C of low temperature, then proceed to and do not carry out illumination cultivation containing in ammonia sulphur spirit solution general differentiation medium; Liquid processes refers to the material covers Regenerated plant terminal bud or callus of aseptically first using good water-retaining property, after cotton is soaked by 8-10 μm of ol.L-1 ammonia sulphur spirit solution completely that then instill filtration sterilization under 5-10 DEG C of low temperature light culture 10-12 hour, sterile water is cleaned, and proceeds to not containing illumination cultivation in the general differentiation medium of ammonia sulphur spirit solution;
4) by step 3) plantlet in vitro that the root, stem and leaf that obtains is complete contrasts with dliploid plantlet in vitro, the candidate plant having the plant of significant change to elect genome as leaf blade size, color may to double;
5) clip step 4) in the meristematic tissue of candidate plant, carry out Methods of Ploidy Identification, choose the seedling of chromosome number 2n=4x=36, its hardening, plantation are survived, namely obtain tetraploid centella kind; Described Methods of Ploidy Identification method is: the tender tip of a root 0.5cm of clip target strain children, clear water soaks, through absolute ethyl alcohol: more than 15min fixed by the Kano fixer of glacial acetic acid=3:1, clear water rinses 15min, concentrated hydrochloric acid: the dissociation solution of methyl alcohol=1:1 is dissociated 2-30min, and clear water rinses 15min, cuts tip of a root 0.1mm, with the dyeing of carbolfuchsin dyeing liquor, statistics root tip chromosomes observed by compressing tablet.
2. breeding method according to claim 1, is characterized in that, further comprising the steps of:
6) by tetraploid centella and dliploid centella artificial pollination hybridization, hybridization centella seed is obtained; Described triploid hydrid centella seed is: ♀ 4N × ♂ 2N or ♂ 4N × ♀ 2N → F1 is 3x;
7) hybrid seed is bred into whole plant plantation, then carry out chromosome number detection to its meristematic tissue, screening obtains triploid centella kind.
3. breeding method according to claim 1 and 2, it is characterized in that: step 1) in, the excellent centella plant as original parent refers to: be select to have that stem section internode is long, stem is thick, blade is large and abundant, that active constituent content is high adult plants the wild centella of 2n=18 from karyotype index.
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| CN105359898B (en) * | 2014-08-13 | 2018-04-10 | 衢州市优德工业设计有限公司 | A kind of snow removing equipment of the plant with accumulated snow detection means |
| CN105794632B (en) * | 2014-12-31 | 2017-11-24 | 桂林三金药业股份有限公司 | A kind of centella polyploid and its induced breeding method |
| CN105699141B (en) * | 2016-02-16 | 2017-12-19 | 中国林业科学研究院热带林业研究所 | A kind of tabletting method of eucalyptus chromosome |
| CN108849496A (en) * | 2017-05-12 | 2018-11-23 | 王元龙 | The raising technology of triploid Radix Angelicae Sinensis |
| CN106973693B (en) * | 2017-06-05 | 2020-07-14 | 株洲市农业科学研究所 | Method for rapidly recovering fertility of lily distant hybrid |
| CN115918537B (en) * | 2022-12-13 | 2024-01-26 | 广西壮族自治区药用植物园 | Centella tissue culture rapid propagation method |
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