| Agastache rugosa(Fisch.etMey.)O.Kuntze,Labiatae Agastache for one-year-old or perennial herbs,is a kind of traditional Chinese medicine,but also uses a wide range of spices and vegetables.Polyploidy breeding is widely used in Chinese herbal medicines plant breeding,usually can improve the content and production of medicinal ingredients. Through the establishment of the System of its regeneration will give a base for the series of working,such as breeding of fine strains,cell fusion and transformation.Transgenic technology could break through species’ reproductive isolation and make the fully use of natural gene sauces.Plant genetic engineering provides a modern and effective method for plant breeding.In this study,we obtain homozygous tetraploid of Agastache rugosa through the induction of colchicines,establishment of it is regeneration system and make an initial exploration for the Agrobacterium-mediated genetic transformation system,the main results obtained are as follows:(1) The chromosome number of Agastache rugosa was found to be 2n=2x=18 and its karyotype formula is 2n=2x=18=3sm+6m.The length of its chromosomes at metaphase in mitosis ranged from 1.34～2.81μm and the constitution of its chromosomes’ relative length is 2n=2S+2M1+4M2+1L.Its karyotype type belongs to "2B".Through the induction of colchicines,analog drug co-culture method and direct method drop wise tip and found that the former is better than the latter,The best way is Agastache rugosa seeding co-culture in 1/2MS+60mg/L colchicines for 13days.Through the plant morphology, stomata frequency and size of the observed chromosome number,policy offspring from mixed homozygous tetraploid were found with the induction rate of 23.33%.(2) The seeds of Agastache rugosa were surface sterilized by 70%ethanol for 30s and 0.1%aqueous mercuric chloride for 8min,followed by three rinses with sterilized distilled water.The sterilized seeds were placed on 1/2MS to germinate.The medium us MS(0.6%agaragar,15 g/l sucrose)as basal medium,with BA(0.2 mg/l)and 2,4-D(0.5 mg/l)for the indication of callus,and leaf as explants have higher induction rate than others,as higher as 95%.Callus after subculture in MS with BA(0.5 mg/l)and 2,4-D(0.2 mg/l)for three months,73.7%of shoot regeneration frequency and 8.4 shoots per leaf explants were achieved when cultured on MS with BA(1.5 mg/l) and NAA(0.05 mg/l). Elongated shoots were rooted on 1/2MS with NAA(0.5 mg/l),91.67%of roots regeneration frequency and 5.3 roots per shoots were achieved within 28 days,and 82.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition.(3) Detection of Agastache rugosa somaclonal variation using Morphological observation、cytogenesis and molecular markers(SRAP ).Found that Six-rowed stalk, three impeller Health and Deepening split leaf in Morphological observation,different from the normal,Schnabel stem and two leaves of Health.Choose 52 strain from somaclonal with obvious difference from normal in Morphological and Detected by SRAP,the result show that Genetic distance between 0.45～0.93and some strains have novel band.Cytogenesis observation show that there are some Variation in chromosome number of somaclonal.(4) The kanamycin resistance assay showed that 50mg/L kanamycin could completely inhibit the callus formation.200mg/L Cephalosporium ADM sufficiently inhibits Agrobacterium growth and didn’t affect the explants differentiation. Agrobacertium-mediated transformation of young stem:Adopted young stem as explants were drenched in 0.6～0.8 OD concentrations Agrobacertium about 10min,then transferred to the selective medium supplemented with 50 mg/L kanamycin and 200mg/L Cephalosporium ADM to isolate kanamycin-resistance callus.Also by PCR analysis on the kanamycin-resistance callus,80%of which were transgenic.Therefore it is concluded that most of the kanamycin-resistance callus were chimera.And check that the marker gene GFP fluorescence protein expression in kanamycin-resistance callus. |
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