A kind of cross breeding method of traditional Chinese medicine sweet wormwood
Technical field
The present invention relates to a kind of cross breeding method of traditional Chinese medicine, be specifically related to a kind of sweet wormwood cross breeding method that can improve sweet wormwood cured leaf per mu yield and artemislnin content, belong to plant genetics and breeding technical field.
Background technology
Sweet wormwood (
artemisia annual.) having another name called artemisia annua, is composite family Asteraceae artemisia Artemisia annual herb plant.Sweet wormwood is mainly distributed in the provinces and regions such as Jilin, Liaoning, Hebei, Shaanxi, Shandong, Hubei, Hunan, Sichuan, Guangdong, Guangxi, Guizhou in China, is the traditional Chinese medicine of China, cures mainly clearing away summerheat, and except steaming preventing malaria, it is used as medicine and is mainly acrial part.The seventies in last century, China scientist found qinghaosu in sweet wormwood, and confirmed that qinghaosu treatment malaria has significant curative effect, and the sweet wormwood medicinal part used as extraction qinghaosu raw material is dry blade.
According to World Health Organization's world's malaria report in 2013: in 2012, estimate to there occurs 2.07 hundred million routine case survey of malarias, cause about 62.7 ten thousand routine malaria dead, estimate to still have 3,400,000,000 people to have malariated risk.The antimalarial curative effect developed by qinghaosu is stronger than traditional anti-malarial drug quinine 16 times, and efficient, low toxicity, without pesticide resistance, particularly to encephalic malaria and anti-Chloroquine-resistant Falciparum Malaria disease curative effect more outstanding, be called " at present in the world unique effective malaria treatment medicine " by the World Health Organization.Artemisinin-based drug also has other multiple drug effects, as for clinical treatment chronic bronchitis, hyperpyrexia disease, erythematosus lupus, schistosomiasis, cancer, acquired immune deficiency syndrome (AIDS) etc., therefore along with to the developmental research of arteannuin medicine and the market expansion, the demand of qinghaosu is by increasing.
Because qinghaosu industry is sharply expanded, the wild sweet wormwood in main producing region is excessively gathered for years, wild sweet wormwood community distribution sharply reduces, dog-eat-dog between qinghaosu manufacturing enterprise, international market artemisine medicine demand progressively increases simultaneously, and wild artemisia leaf the number of acquisition and low content can not meet qinghaosu already and extract needs, and current artemisia leaf raw material is mainly from peasant planting, year plantation total scale 100,000 ~ 200,000 mu, artemisia leaf product artemislnin content in enormous quantities is generally 7 ‰ ~ 10 ‰.
Current people generally believe because sweet wormwood is cross-pollinatd plant, existence " self-incompatibillity ", by selfing, elite plant strain proterties cannot be settled out formation inbred line.Sweet wormwood breeding units is all adopt systematic breeding method to cultivate conventional variety/be, but conventional variety/be is difficult to industrialization management, breeding research is difficult to obtain corresponding continued support economically, cause main producing region sweet wormwood varieties of plant/be general homogeneity, artemislnin content and output stagnation.
Summary of the invention
The object of this invention is to provide a kind of cross breeding method of traditional Chinese medicine sweet wormwood, cultivation sweet wormwood Hybrid/be, production sweet wormwood hybrid seed, thus improve sweet wormwood cured leaf per mu yield and artemislnin content.
In order to solve the problem, the technical solution adopted in the present invention is:
A cross breeding method for traditional Chinese medicine sweet wormwood, comprises the steps:
(1) collection of germ plasm resource: collect different regions, different classes of sweet wormwood germ plasm resource, kind is distinguished from appearance character, comprise plant type, blade profile, stem look, leaf look, comprise specific general planting kind/be, germ plasm resource is wanted variation, is had obviously interspecific difference, and this successfully obtains the prerequisite of inbred line and utilizes the needs of heterosis breeding;
(2) fine individual plant isolation selfing is selected: distinguish high yield type by appearance character, measure single-strain blade artemislnin content by conventional method, under equal conditions filter out high artemisia leaf output, high artemislnin content individual plant; After pregnant flower bud completes, bloom before with breeding bag isolation selfing, until seed maturity results selfed seed, next year sowing selfed seed; Circulation like this, more than continuous selfing three generations;
(3) tissue cultures preserves inbred line individual plant: to inbred line more than continuous selfing three generations, from forth generation, carry out tissue cultures, after filtering out high artemisia leaf output, high artemislnin content individual plant, tissue cultures preserves these individual plants;
(4) hybrid combination of inbred line individual plant is selected: preserve at tissue cultures and carry out hybridization coordinate force between successful inbred line individual plant and test, select the combination that coordinate force is high in high artemisia leaf output, high artemislnin content, selected combination sweet wormwood cured leaf output more than 160 kgs/acre, artemislnin content more than 15 ‰;
(5) expand numerous inbred line individual plant plantlet in vitro isolation implant and produce hybrid seed: selected combination inbred line plantlet in vitro is carried out expansion according to seedling number needed for seed produces amount numerous, intert field planting at isolated area Parent, spontaneous pollination, Parent seed is gathered respectively.
Isolation described in step (2) isolates after 1% sodium chloride solution of spraying to individual plant.
Cultivation described in step (3), cultivate after explant is carried out disinfection, sterilization method is: tissue suds are rinsed well and first used 75% alcohol-pickled 20 seconds afterwards, then sterilize 10 minutes with saturated liquor natrii hypochloritis, finally sterilize 8 minutes with 0.05% mercuric chloride solution.
Condition of culture described in step (3) is: intensity of illumination 1500 lux, 14 hours every days of light application time, temperature 25 DEG C.
The medium of the cultivation described in step (3) is: inducing clumping bud medium is: MS+6-BA0.4 mg/litre+IAA0.5 mg/litre, differential medium is: MS+6-BA1.5 mg/litre+IAA0.3 mg/litre, and root media is: MS+ NAA0.1 mg/litre.
Beneficial effect of the present invention is:
(1) with medicament spraying process and strict isolation, can settle out formation inbred line by selfing by elite plant strain proterties, and conjunctive tissue culture technique preserves inbred line, thus makes sweet wormwood heterosis utilization become possibility.
(2) instant invention overcomes the sweet wormwood inbred line that sweet wormwood " Self-Incompatibility " property causes to cultivate, preserve a difficult problem, apply the interspecific difference of sweet wormwood " Self-Incompatibility " property innovatively, cultivate the sweet wormwood inbred line of different generations, apply this asexual reproduction method of tissue cultures innovatively and preserve the inbred line individual plant cultivated, for cultivation, the high artemisia leaf output of administration measure, high artemislnin content sweet wormwood hybrid seed provide technical guarantee.
(3) the method is simple, mild condition, is applicable to agriculture industrialization and promotes.
Embodiment
Be described in further details the present invention below by embodiment, these embodiments are only used for the present invention is described, do not limit the scope of the invention.
embodiment 1 collects different regions, different classes of sweet wormwood germ plasm resource
Within 2003,2004, from Qionghai City, Hainan Province and Tongshi City, Guangxi Zhuang Autonomous Region Bose City, Tujia-Miao Autonomous County of Youyang of Chongqing City, Shaoshan City, Hunan Province and Fengshun County In Guangdong Province, Lianping County, Wengyuan County, Shaoguan collection seeds of southernwood or plant, within 2004, introduce a fine variety and be planted in Fengshun County In Guangdong Province.Within 2004, introduction and Experiment plant appearance character differs, and stem look purple is, green, Huang mixes, and great majority are tower, internode is longer, blade is sparse, and artemislnin content is in table 1.
Table 1 separate sources breeding material artemislnin content (‰) in 2004
embodiment 2 selects fine individual plant to isolate selfing
Within 2005-2010 years, select fine individual plant, bagging selfing before blooming with high artemisia leaf output, high artemislnin content respectively, spraying 1% sodium chloride solution before bagging.Each annual bagging quantity and the average artemislnin content of individual plant are in table 2.
The each annual bagging quantity of table 2 and the average artemislnin content of individual plant
Year |
2005 |
2006 |
2007 |
2008 |
2009 |
2010 |
Bagging quantity (individual) |
841 |
407 |
248 |
200 |
51 |
24 |
Average artemislnin content (‰) |
8.1 |
9.0 |
10.4 |
12.2 |
14.6 |
15.2 |
embodiment 3 tissue cultures preserves inbred line individual plant
Within 2011, sow the selfed seed 107 parts in different year, seedling 92 parts, altogether about 2000 strains, selected to have screened 24 specificity selfing individual plant materials with high artemisia leaf output, high artemislnin content respectively and carry out tissue cultures.Clip branch is explant, explant disinfection method: rinse well with suds and first use 75% alcohol-pickled 20 seconds afterwards, then sterilize 10 minutes with saturated liquor natrii hypochloritis, finally sterilize 8 minutes with 0.05% mercuric chloride solution; Condition of culture: intensity of illumination 1500 lux, 14 hours every days of light application time, temperature 25 DEG C; Medium is selected: inducing clumping bud MS+6-BA0.4 mg/litre+IAA0.5 mg/litre, differentiation MS+6-BA1.5 mg/litre+IAA0.3 mg/litre, MS+ NAA0.1 mg/litre of taking root.
embodiment 4 selects the hybrid combination of inbred line individual plant
Bagging hybridization 26 combination within the scope of 24 specificity selfing individual plants in 2011, has 16 to combine and obtains obviously visible canescence F
1for seed, 10 combinations are separately had to fail to obtain obvious visible F
1for seed.Sowing in 2012 26 parts of hybrid seeds, wherein 14 parts of seeds do not reach in the required seedling quantity of lab scale plantation or planting process and eliminate, and 12 parts of seed lab scales plantations are carried out surveys and produced and product content detection, and concrete data are in table 3.Result shows hybrid combination F
1product content for seed is compared with the content of its Parent (inbred line) individual plant, and the former is in most cases far above the latter, and hybrid vigour is obvious.According to field observation disease resistance, survey and produce and product content testing result, wherein 2 hybrid combinations " 1110 × 1115 ", " 1102 × 1128 " Comprehensive Traits are more excellent, select this 2 combinations produce hybrid seeds.
Table 3 hybrid combination F
1for seed lab scale plantation result (Kg, ‰)
embodiment 5 expands numerous inbred line individual plant plantlet in vitro isolation implant and produces hybrid seed
Within 2013, expand numerous 1110,1115,1102,1128 plantlet in vitro 500 strains, 100 strains, 500 strains, 100 strains respectively, 2 hybrid combinations " 1110 × 1115 ", " 1102 × 1128 " point 2 isolated area Parents intert field planting, spontaneous pollination, maternal seed of gathering respectively obtains corresponding hybrid seed 8.1 kilograms (lot number 13061121), 8.6 kilograms (lot number 13071125), and these 16.7 kilograms of seeds of southernwood can popularizing planting about 20000 mu.
the pilot scale of embodiment 6 hybrid seed is promoted
2014 in Rongan County, Guangxi Zhuang Autonomous Region, Anxiang County, Hunan Province pilot scale popularizing planting, the results are shown in Table 4.
Table 4 hybrid seed pilot scale Extended Results (Kg, ‰)