ABSTRACT
ObjectiveAgrobacterium tumefaciens-mediated transformation （ATMT） of Clonostachys rosea， an endophytic fungus of Glycyrrhiza uralensis seeds， was established and optimized， and orthogonal test was designed to optimize the colonization conditions of C. rosea for G. uralensis seeds， so as to lay foundation for the development of biofertilizer and the breeding of high-quality G. uralensis. MethodThe conditions of ATMT were optimized from three aspects， including the concentration of acetosyringone， co-culture time and the concentration of conidia of recipient fungi. Then， high-quality transformants were selected. Orthogonal test was used to optimize the colonization conditions by taking co-culture temperature， co-culture time and spore concentration as factors and colonization rate as index. ResultWhen spore concentration was 1×107 cfu·mL-1， acetosyringone concentration was 150 μmol·L-1 and the co-culture time was 60 h， the transformation efficiency of C. rosea was the highest， which was 135 transformants per 1×107 recipient fungal spores. The accuracy and stability of the transformations were tested by cloning the marker gene green fluorescent protein （GFP） and β-glucuronidase （GUS） staining. When co-culture temperature was 25 ℃， co-culture time was 36 h and the spore concentration was 1×106 cfu·mL-1， the colonizing rate for C. rosea back dyeing into G. uralensis seeds by seed soaking method was the highest， which was 71.11%. ConclusionThis study successfully establishes stable and efficient technical systems not only of ATMT in C. rosea， but also of colonization of the transformants into G. uralensis seeds， which can lay a foundation for the development of biofertilizer of G. uralensis.
ABSTRACT
Licorice is one of the most frequently used Chinese herbs, mainly containing triterpenoids and flavonoids. Three original plants, Glycyrrhiza glabra L., Glycyrrhiza uralensis Fisch., and Glycyrrhiza inflata Bat., are defined as licorice in Chinese pharmacopeia. In this study, 40 G. uralensis samples [Group A], 60 G. glabra samples [Group B, C and D] and 40 G. inflata samples [Group E and F], were used as plant materials, the genetic diversity of samples were determined by gene sequencing technology and the chemotypic diversity were detected by HPLC. The chemotypic diversity analysis showed that contents of triterpenoids in G. glabra [isoglycyrrhizin: 2.483 +/- 0.0671 mg·g-1, glycyrrhizin: 34.660 +/- 0.8591 mg·g-1] were obviously higher than that in G. uralensis and G. inflata. However, the contents of flavonoids [liquiritin: 21.996 +/- 0.6396 mg·g-1, isoliquiritin: 4.556 +/- 0.1252 mg.g-1, liquiritigenin: 0.623 +/- 0.0200 mg·g-1, isoliquiritigenin: 0.281 +/- 0.008 mg·g-1] in G. uralensis were higher than that in G. glabra and G. inflata. And contents of triterpenoids and flavonoids were both lowest in G. inflata. The genetic diversity analysis showed that the psbA-trnH intergenic regions on chloroplast DNA sequences were same in the same species, and significantly different between any two species. These findings will lay a solid foundation for the identification and quality control of licorice. Furthermore, recently the activity of isoglycyrrhizin has attracted more and more attentions and researches. The HPLC method established in this paper for the simultaneous assay of isoglycyrrhizin and glycyrrhizin will be helpful for the screening of superior quality licorice with a high content of isoglycyrrhizin