The Tissue Culture And ISSR Analysis Of Biological Diversity About Pleione

The genus Pleione, known as a group of beautiful, popular orchids, comprises about 23 species. In china, there are about 16 species of terrestrial, lithophytic Pleiones, growing in the wild in a wide variety of habitats at altitudes from about 1000m up to 4200m of the subtropical montane forests in the Himalayas. Pleione continues to be collected from the wild, especially in china, to satisfy the horticultural demand. Although most of the species are available in trade from nursery-grown, often seed-raised stock, wild-collected plants of several species are harvested in large numbers. Most of Pleione species have only one leaf, which is deciduous and plicate. Their flowers are pink or white or rarely yellow, and are characterized by an obscurely 3-lobed lip with or without spots, erose to lacerate margin and a callus consisting of several lines of lamellae or hairs. In recent years, several Pleione species have been successfully established from seed to plantlets. To our knowledge, there has been no previous systematic works on tissue culture, regeneration of Pleione genus and molecular analysisThirteen species of Pleione are studied in this paper, and they are Pleione maculata Lindl, Pleione praecox D. Don, Pleione saxicoia, Pleione hookeriana a and b, Pleione chunii C. L. Tso, Pleione aurita, Pleione forrestii Schltr, Pleione yunnanensis, Pleione grandiflora Rolfe, Pleione bulbocodioides and Pleione limprichtii Schltr. They were collected in the southern part of Yunnan Province, the People' s Republic of China and Vietnam, and were chosen to research their tissue culture and to measure their genetic diversity by ISSR analysis. The results could be summarized as follows.1. The protocorms of Pleiones were used as explants for the rapid propagations, the results showed that the budding rate of explants, which cultured on the medium of 1/2MS + 1mg/L 6-BA, was the highest compared to others.The budding rate of explants, which cultured on the medium of TH + 0.2 mg/L NAA + 1 mg/L 6-BA, was higher than the others.The explants producing clustered buds of explants must be bigger than 0.5³ cm³.Rapid propagation was induced from seed-derived protocorms cultured on half-strength of Murashing-Skoog (MS) medium with 1-naphthal-eneacetic acid (NAA, 0-1mg/L) and 6-benzylaminopurine (6-BA, l-3mg/L) at 25±2℃with the day-length of 12 hours 800-12001x.The different species of Pleiones needed to be cultured on the different media to get different high shooting frequencies.When the culture was TH medium + 0.2 mg/L NAA + 1 mg/L 6-BA, the budding formationg rate of P.maculate was 100%; on the same medium, the budding rate of P.hookeriana a was 93%, and when the medium was 1/2MS + 1 mg/L 6-BA, the rate was 80%.The shooting rate of P.chunii was 75% on 1/2MS medium + 0.2mg/L NAA +1 mg/L 6-BA.For P.forrestii, its budding rate was 100% on the both media, 1/2MS + 1 mg/L NAA + 0.2 mg/L 6-BA.When the culture was 1/2MS medium + 1 mg/L 6-BA, the shooting rate of P.grandiflora was 60%.For P.limprichtii, its budding rate was 80% on 1/2MS medium + 0.2 mg/L NAA +2 mg/L 6-BA.P.aurita, which was cultured on 1/2MS medium + 0.2mg/L NAA + 2 mg/L 6-BA, the budding rate was 75%.Seed-derived one month small protocorms of Pleione was better than the segments of tissue-cultured protocorms for shooting regeneration.The maximum number of multiple shoots generated by the Seed-derived one month small protocorms which had maximal shooting formation (20 shoots/explant).The multiple shoots were transferred to TH medium with 0.1 mg/L NAA +0.5 mg/L 6-BA + 50 mg/L tomato.TH medium containing NAA and 6-BA at 0.5 mg/L induced rooting.The "—" shape excision of protocorms could obtain more multiple shoots than "+" shape excision.2.In this study, 13 Pleione samples were evaluated using ISSR markers. The samples comprised different individuals of the same Pleione species. The sequence of the five ISSR primers which were selected from the 100 ISSR primers was respectively (AC.) ₈T, (TG) ₈C,(CT) ₈RGand so on, which showed in Pleione DNA molecular there widely existed simple sequence characterized with AC and CT or TG. Five primers generated 265 reproducible and stable DNA fragments which were during 200bp and 1500bp.Polymorpic bands were rare. The amplified result from the differet individuals of the same Pleione species was that no special bands were amplified, which showed the individuals of the same Pleione species, was highly genetic identical. The result of POPGENE analysis indicated that the distance between P. hookeriana b with P. pleionoides and P. limprichtii was the furthest and the distance between P. grandi flora and P. limprichtii was further. P. saxicoia and P. praecox or P.maculata was closer and the distance between P.maculata and P.praecox was the closest. The UPGMA dendrogram showed that two main groups could be recognized, i. e., P.maculata, P. praecox, P. saxicoia, P. bulbocodioi des P. hookeriana a&b, P. forestii, P. yunnanensis and P. grandiflora which came into one clade; P. pleionoides, P. limprichtii, P. chunii and P. aurita which came into the other clade, which was similarly accorded with morphological class. From the cluster analysis of UPGMA, its result was similarly coincided with the molecular class which was based of plastid and nuclear ribosomal ITS sequences. The phylogeny analysis of plastid, DNA and nuclear ribosomal ITS sequences and morphological data was better. The combined tree can be regarded as a good hypothesis of evolutionary relationships in Pleione because of fairly high level of congruence among all separate data sets and higher resolution and bootstrap percentages of the total evidence data set. Consequently, ISSR molecular marker could be applied in the classifying of Pleione.