Tissue Culture And Molecular Systemetics Studies On Paris

There are 24 species in the Genus Paris of Family Trilliaceae in all, 19 of which have distribution in China, with the south-western region as the distributing center. They are impotant Chinese medicine and significant for phylogenetic research of Angiosperm. Our research work focused on three aspects: the investigation of its tissue culture conditions, the clarification of its genetic diversity and interspecies relationship based on RAPD analysis and the phylogenetic research through the analysis of ITS sequence.(1) Tissue Culture: Taking MS as the basic medium, we tried to culture different organs of P. polyphylla var. chinensis under a series of NAA and 6BA concentration gradients. The results indicated that its stem and root part could not dedifferentiate into callus, the node of stem could produce protrusion and some callus under induction, but the growth rate was quite slow, while the latent buds could develop hi vitro but grew into abnormal seedling; callus of the rhizome part is inducible but easy to be polluted by the symbiotic bacteria, leaving alone its slow growth rate and tendency of browning; the vitality of spire can maintain vigorous for long, but was hard to dedifferentiate into callus; the ovary was highly vigorous and swellable, a small quantity of callus could be produced from it.(2) RAPD analysis:Using RAPD as molecular marker, we analyzed the genetic diversity and interspecies relationship among 10 species and 1 variant of Genus Paris. We got 86 gel bands in total through the PCR amplification of all the 12 random primers, 82 of which, that is, 95.3%, showed the polymorphism. P. cronquist showed the highest polymorphism (90.2%) while P. forrestii showed thelowest (79.0%) . Systematic tree constructed by UPGMA didn't support the dividence of Genus Paris into Subgenus Daiswa and Subgenus Paris. We divided the 11 species into 7 sections, which is different from the traditional classification in that the 5 species of Sect. Euthyra were set in four branches. P. crongquist clustered with P. polyphylla var. chinensis while P. daliensisis formed a separate branch. P. undulates clustered with Sect. Fargesianae while P. mairei clustered with Sect. Marmorata.(3) ITS sequence analysis: We sequenced the ITS region of 15 species or variants in Genus Paris and a species Trillium tschonoskii. The ITS sequence of the rest 5 Paris species and Trillium govanianum used in the article were quoted from Genbank. The ITS sequence length of these 22 species or variant after alignment ranged from 631bp to 640bpf in which the lengths of ITS1 vary from 242bp to 250bp, the lengths of 5.8sDNA were exactly all 164bp, and the lengths of ITS2 varied from 225bp to 227bp. 76.4% sites were conservative while 22.8% sites showed variation and 14.1% sites were informative. The GC content of the sequences was 54.3%. The ratio of transition to transvertion is 2.9. The analysis showed that the variation of ITS region is concentrated in ITS1 and ITS2 and ITS2 had a much higher GC content transition/transvertion value than ITS 1.Trillium tschonoskii and Trillium govanianum taken as outgroup, we carried through the clustering analysis of ITS sequences under the environment of MEGA2.1 and came to the following systematic treats: I Paris should be divided into three separate genera^orm, Kinugasa, Daiswa); II For that P. polyphylla var. minor and P. thibetica have the same ITS sequence, and that the distributing area of the prior is completely included in the later's where many intermediates could be found, as well that they have no great difference in configration characters, we assumed that the later is not a variant of P. polyphylla but a variant of P. thibetica; III The phylogenetic tree showed that P. polyphylla var. yunnanensis, P. polyphylla var. chiensis and P. polyphylla var. stenophylla didn't cluster with P. polyphylla. They had special morphologic characters respectively, but to determine whetherthey should be upgraded to species or not, we need more evidence; IV P. daliensis and P. polyphy...