Study On Embryogenesis And Plant Regeneration Of Hairy Roots In Panax Ginseng And Panax Quinquefolium

Both Panax ginseng C.A.Mey and Panax quinquefolium L. belong to Araliaceae, are perennial herb, and have been extensively used in medicine, food and cosmetics. Although culture of Panax ginseng and Panax quinquefolium has a long history, their products can not meet the market demand, due to their long growth cycle, sensitivity to pests, disease, climate and enviromental conditions. Therefore,it has become an important issue that improve Panax ginseng and Panax quinquefolium by cell and genetic engineering. Studies on embryogenesis of Panax ginseng and Panax quinquefolium hairy roots not only play an important role in their germplasm conservation,breeding,fast micropropagation and transgenetic, but also could provide experimental systems for researches on their embryogenesis mechanism. Additionally, characteristics of hairy roots make it possible to overcome the difficulty in traditional plant regeneration of Panax ginseng and Panax quinquefolium. Hairy roots-regenerated plants present an effective alternative for breeding and fast micropropagation of Panax ginseng and Panax quinquefolium.In the present study, firstly, callus induction were performed with explant of hairy Panax ginseng and Panax quinquefolium, effects of various influencing factors on callus indction were also investigated. It is found that, compared with B5 and MSB medium, MS medium is more suitable for callus induction with a induction rate of 98.7% and 95.6% for Panax ginseng and Panax quinquefolium hairy roots, respectively.. The callus induction rate is the highest for Panax ginseng hairy roots sbucultured for 7 days, more or less than which the induction rate will decrease, while 11 days was optimal for callus induction of Panax quinquefolium hairy roots. Callus could be induced from Panax ginseng and Panax quinquefolium hairy roots either under illumination or in darkness; however callus induction rate (95%) under illumination were 6% more than that in darkness. The optimal medium conditions for callus induction and growth of Panax ginseng hairy roots were MS+2,4-D 1mg/L+BA 0.1mg/L and MS+2,4-D 1.5mg/L+KT 1.5mg/L, respectively; while the optimal medium conditions for induction and growth of Panax ginseng hairy roots callus were MS+1mg/L 2,4-D+1mg/L NAA.Calluses with fast growth and compact texture were used for somatic embryogenesis studies. 2,4-D play an important role in somatic embryogenesis of both Panax ginseng and Panax quinquefolium hairy root calluses. Coupled with 2,4-D, BA could enhance the embryogenesis of Panax ginseng hairy root calluses. While, 2,4-D can significantly enhance the embryogenesis of Panax quinquefolium hairy root calluses only under the presence NAA. The optimal medium for embryogenesis of Panax ginseng hairy root calluses was MS+2,4-D 1mg/L+BA 0.5mg/L with an induction rate of 43.5%; while for Panax quinquefolium hairy root calluses, it is MS+2,4-D 0.5mg/L+NAA 0.5mg/L with an induction rate of 66.2%. Embryogenesis frequencies for Panax ginseng and Panax quinquefolium hairy root calluses under illumination were significantly different from those in darkness. The embryogenesis induction frequency for Panax ginseng hairy root calluses was 20% higher under illumination than in darkness, while for Panax ginseng hairy root calluses, it was 16.7% higher under illumination than in darkness.The embryogenesis began as soon as the embryoid of callus was transferred in medium containing low level of hormones. Regenerated plants were generated by embryogenesis through several stages, including globular embryo, heart-shape embryo, torpedo embryo and cotyledon embryo. Somatic embryogenesis was also affected by Osmotic pressures caused by sucrose of different concentration in the medium. Our observation showed that somatic embryogenesis frequency increased with increasing of osmotic pressure, and reached a plateau when the sucrose concentration were 50g/L and 30g/L for Panax ginseng and Panax quinquefolium hairy root calluses.Moreover the effects of carbon source, organic compound, illumination and generations of subculture on somatic embryo propagation were investigated. For Panax ginseng hairy root calluses, embryo proliferated efficiently in medium with glucose of 40g/L, casein hydrolysate of 300mg/L, and illumination had no obvious effects on embryo proliferation. On the other hand, for Panax quinquefolium hairy root calluses, embryo proliferated efficiently in medium with glucose of 40g/L without addition of organic compound; however illumination had a positive effect on embryo propagation. In addition, regardless of the generations of subculture, the embryogenic capability of the established somatically-embryogenic system by hairy roots was relatively stable, thus the system is genetically-stable and possesses stronger capability of embryogenesis. Of the 5 tested medium including MS,1/2MS,1/4MS,B5,1/2B5, MS mediums was the most suitable for germination of somatic embryos from Panax ginseng hairy roots; while 1/2MS was the most suitable for that of Panax quinquefolium hairy roots. Cytokinin and auxin could enhance the germination of somatic embryos, at the same time reduced the regeneration frequency of abnormal seedling. In medium containing BA and IBA, germination frequencies of Panax ginseng and Panax quinquefolium hairy root somatic embryos were relatively high (57.5% and 85.3% respectively), and the formation frequencies for both somatic embryos were relatively low (44.7% and 29.6% respectively). ABA also had the similar effects on embryogenesis. GA of suitable concentration could improve the germination frequencies of the embryos, decrease the formation frequencies of abnormal seedling, also promote the greening of the seedling. Addition of active carbon had no significantly effects on germination of the embryos.The cytological observations of embryogenic calluses by paraffin section and hand section showed that the cell shape is normal, nucleus is large and situated at the cell center, cytoplasm is dense. While, the un-embryogenic calluse cell volume is large, nucleus is small, and the cytoplasm is thin. Embryogenesis of Panax ginseng and Panax quinquefolium hairy root calluse went sequentially through the stages of formative cell clump, globular embryo, heart-shape embryo, torpedo-shape embryo and cytyledon.mRNA Differential Expression Analysis of embryogenesis was performed with globular embryo, heart-shape embryo, torpedo embryo and cotyledon embryo. Guanidine thiocyanate and kit were used to isolate total RNA from cells of different embryogenic stage. The results suggested that kit was suitable for total RNA extraction. DDRT-PCR were performed with the following system: dNTP, 100μmol/L; one 5'random primer;renaturation temperature, 40℃.There were more amplified nucleic acid segments in PCR using Oligo-p(dT)11G and Oligo-p(dT)11C as primers than using Oligo-p(dT)11 A as primer. Eight bands were observed in the electrophoresis with length 0f 250-1000bp. For embryogenesis of Panax ginseng hairy callus, the difference at transcript level was mainly observed among the stages of globular, heart-shape and torpedo embryo, while for embryogenesis of Panax quinquefolium hairy callus, the difference was mainly notable between globular and torpedo embryo stages. Induction-specific expression difference is one of the main kind of gene differential expression, which mainly occurred at the stages of globular, heart-shape and torpedo embryo in this study. Another main kind of gene differential expression is quantitatively-differential expression, which could be observed at the stages of globular and heart-shape embryo.