| Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma is one main original plants ofthe traditional Chinese materia medica "Shanmaidong". Its tuberous roots are usedmedicinally, with effects of nourishing Yin to promote the production of body fluid andmoistening the lung to clear away heart-fire. L. spicata var. prolifera is one geoherb fromHubei province and the main variety of common traditional Chinese materia medica"Maidong". It has no seeds after efflorescence and reproduces only by vegetativepropagation. Because of long-term vegetative propagation, its germplasm degenerated andproduction and quality were influenced severely. In this paper the resource-quality and themechanism of sterility were studied by using the method of bencaology, plant morphology,cell biology, plant embryology, pharmacognosy and molecular biology. It providesavailable data and theoretical foundation for breeding. Our findings have importantpractical significance for increasing production and quality and raising industry of L.spicata var. prolifera. The results were as following.1 Studies on bencaology, pharmacognosy and RAPD analysis of L. spicata var.prolifera.The research on bencaology indicated that Chinese materia medica "Maidong" wasused medicinally for long history. Suizhou Maidong was recorded in Classified MateriaMedica for Emergency in Song Dynasty, which was similar with plants of the genusLiriope. Its shapes and place of produce were consistent with L. spicata var. prolifera.In order to provide the scientific evidence for identification and estimation, L. spicatavar. prolifera from Xiangfan city, Hubei province was studied in pharmacognosy. Theresults show that there exists certain difference in morphology and cytology between L.spicata var. prolifera and other original plants of "Maidong". L. spicata var. prolifera, L.spicata, Ophiopogon japonicus and L. muscari Bailey can be distinguished by lengths offilaments of flowers, place of ovary, blunt-end or sharp-end anthers, lenth of scape and littlebuds after efflorescency. On the other hand, L. spicata var. prolifera and O. japonicus canbe distinguished by the number of phloem bunch in cross sections of root tubers.To analyze the genetic relationships and diversity, random amplified polymorphic DNA (RAPD) was applied to germplasm accessions in 22 cultivated and wild medicinalbotanic samples of L. spicata var. prolifera, L. spicata and O. japonicus. 10 random primersof high stable quality were screened from 100 primers. 10 arbitrary primers produced 75distinctive bands in total and 58 of them were found to be polymorphic, which accountedfor 77.3%. Cluster analysis showed that histological culture and cultivated methods couldn'tobviously affect the genetic relationships and there are obvious genetic differences amongOphiopogon japonicus in the different areas of zhejiang, Sichuan and Hubei provinces. Thephylogenetic relationships of them are basically accordant with the results of classical taxology.2 The relation between pollination biology and sterility of L. spicata var. prolifera was studied.The morphological character, florescence, pollen viability, stigma receptivity,pollen-ovule ratio and pollinating agent of flower of L. spicata var. prolifera were estimated.L. spicata var. prolifera bloomed from late June to early August. The anthesis was only oneday. It bloomed at around 09:20 and finished at around 16:30. Wind pollination did notexist in this area. The flowers of this species were pollinated by insects. The stigma of L.spicata var. prolifera could only be receptive 1 to 2 hours after blooming, although stigmahad catalase activity in the whole anthesis. Pollen in the anthesis had no viability and theiraberration rate was 95.55%. The pollen-ovule ratio, ranging from 4 296±1 614, reflected asan obligate xenogamy system. Conclusion: Its high aberration rate, pollen without viabilityand low pollen number were sterility of pollination biology.3 The karyotype of L. spicata var. prolifera was examined.The results are as follow: L. spicata var. prolifera is a triploid plant. The karyotypeformula is 2n=3x=54=30m+24sm(1SAT), which belongs to symmetrical 2B type. Becauseof triploid plants being naturally sterile, the sterility of L. spicata var. prolifera was inducedby its triploid. Karyotype of L. spicata var. prolifera was reported for the first time. Theresults agree with the views treating them as separate species. In addition, our findingsindicate that L. spicata var. prolifera might originate from hybridization between tetraploidand diploid species. Conclusion: the discovery of triploid L. spicata var. prolifera revealedthe cytogenetic mechanism of its sterility.4 Meiotic behavior and pollen development of L. spicata var. prolifera were describedin detail for the first time. A study on anther and pollen development process was carried out in L. spicata var.prolifera by means of paraffin section under a microscope. Meiotic behavior was alsoobserved. During the meiosis of microspore mother cells (MMC), most of cells displayedabnormally. The abnormity was showed that some chromosomes or their fragments movedout of the spindle of the cell at metaphase. Some chromosome bridges, fragments andlagging ones were formed at anaphase, and many microspores displayed the micronucleusat the telophase. The pollen development was abnormal and with high aberration rate ofpollen. The degradation of the tapetum was taken place at the early time of meiosis stageand tapetum cells inflated like vacuolization. The mononuclear pollens were abortive,which eventually formed abnormal pollens.Conclusion: These results indicate that someabnormalities of meiotic behavior and pollen development are significant reasons inducingmicrospore abortion during its development. It also supports the viewpoint that L. spicatavar. prolifera belongs to a triploid plant. The pollen abortion of L. spicata var. prolifera isassociated with tapetum abnormalities and degradation in advance.5 To study the technique of the callus induction from anther and plant regeneration ofL. spicata var. prolifera.Callus was induced from anther of L. spicata var. prolifera on a MS mediumsupplemented with different hormones. The squash methods combined with a microscopewere used to analyse chromosomes of regenerated plantlets. Result: MS+2, 4-D 1.0mg·L-1+KT 2.0 mg·L-1 gave the highest induction ratio which was 41.07%. MS+6-BA1.5-2.0 mg·L-1 +NAA 0.1-0.3 mg·L-1 was suitable for the induction and proliferation ofindefinite buds. The buds were transferred to 1 / 2 MS medium supplemented with NAA0.1-0.3 mg·L-1 for rooting. The shoots produced roots of culture and formed completeplantlets. The regenerated plantlets originated from somatic cells. At the same time, theeffects of pretreatment of low temperature at 4℃on the callus induction were studied anddiscussed. Conclusion: This paper sets up the method of tissue culture of anthersomatic-cells and intermediate propagation of L. spicata var. prolifera. |
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