|Plant latin name||Panax ginseng C. A. Mey.|
|Reference||Shu W et al., Bull., Natl., Health Sci., 117: 140-147 (1999)|
|Summary||Embryogenic cultures of Panax ginseng were established without using phytohormones. Somatic embryos developed from the roots of an in vitro seedling and from excised leaf and petiole segments
cultured in half-macro-salt strength Murashige and Skoog medium. Excised leaf and petiole segments were obtained from in vitro germinated seedlings. Plantlets were subsequently obtained from developing somatic embryos in phytohormone-free media. Shoot formation from somatic embryos was influenced by
light intensity. The rate of growth and frequency of embryogenesis were improved when cut-up embryogenic tissues were inoculated into liquid media in the dark. The ginsenoside contents of a 4 yea-rold field-cultivated root, seedlings from zygotic embryos, somatic embryos and embryogenic tissues were determined and compared. Somatic embryos contained 1.7 timcs the amount of ginsenoside Rb1 and 2.3 times the amount of ginsenoside Re compared to seedlings from zygotic embryos. Ginsenoside Rd, which was absent in the seedlings derived from zygotic embryos, was detected in somatic embryos. Higher ginsenosides Rd and Rg1 levels were found in embryogenic tissues grown on solid media than in tissues grown in liquid media. The total ginsenoside yields, including the ginsenosides Rb1 and Rg1 levels of cut-up embryogenic tissues, were higher than those of clump tissues.|
|Objectives||Establishment of clonal propagation method through somatic embryogenesis|
|Materials||Seeds of Panax ginseng C.A.Meyer field-cultivated at Tsukuba Medicinal Plant Station, National Institute of Health Sciences|
|Explant||Immature seeds of Panax ginseng C.A.Meyer|
|Initial culture||Immature seeds were surface sterilized in 75% ethanol for 30 seconds and then in 2% (vlv) sodium hypochlorite solution with Tween 20 ( I drop / 40 mL) for 10 min. Seeds were rinsed three times with sterile deionized water and cultured on 0.5% agar medium containing 0.5% sucrose and incubated in the dark at 25 ± 2 ℃. After 9 to 10 months of culture, germination occurred. Seedlings removed of leaves, excised leaf and petiole segments from seedlings were transferred to phytohormone-free (HF) a half macro salt strength Murashige and Skoog (1/2 MS) solid medium supplemented with 3% sucrose and 0.2% Gelrite and cultured under dim light (16 h photoperiod, 9 µEm-2s-1) at 25 ± 2 ℃. After 1 month, embryogenic tissues formed on the surface of the root of the seedling. Leaf and petiole segments excised from seedlings gave rise to embryogenic tissues on HF 1/2 MS solid medium. Embryogenic cultures from root of the seedling (Pg) , leaf (Pga) and petiole (Pgb) could be maintained via secondary embryogenesis on HF 1/2 MS solid medium at 25 ± 2'℃ under dim light (16 h photoperiod, 9 µEm-2s-1). Approximately 0.4 g of embryogenic tissues was inoculated into culture tubes (40 i.d. x 130 mm, 30 ml solid medium) and transferred to fresh HF 1/2 MS medium every 8 weeks. The frequency of embryogenesis was highest in the tissues derived from the seedlings (Pg), followed by the tissues derived from the petiole (Pgb) and leaf (Pga). A total of 82 somatic embryos could develop via secondary embryogenesis from a piece of embryogenic tissue (Pg) of 0.4 g cultured for 6 weeks under dim light. Subsequently plantlet formation via shool formation in germinating somatic embryos occurred on phytohormone-free medium. Highest number of shoots per culture was obtained from somatic embryos of petiole tissues (Pgb). All further experiments were carried out using Pg tissues as this source of embryogenic tissue produced the highest number of somatic embryos.|
|Shoot multiplication||Embryogenic tissues (Pg) were inoculated into culture tubes containing HF 1/2 MS solid medium as described above. Cultures were subjected to full light at 57 µEm-2s-1, dim light at 9 µEm-2s-1 or complete darkness. After 6 weeks, frequency of somatic embryogenesis and shoot formation were determined. Number of somatic embryos formed per culture was optimum when embryogenic tissues were cultured on 1/2 MS solid medium in the dark. Full light intensity at 57 µEm-2s-1 was found to be beneficial for the shoot formation in somatic embryos.
Embryogenic tissues (Pg) grown on HF 1/2 MS solid medium were prepared into clumps (10-20 mm) or into small pieces (1-2 mm, cut-up using a scalpel). On solid media (0.2% Gelrite), 0.4 g of tissues was inoculated into a culture tube (40 i.d. x 130 mm, 30 ml medium). In liquid media, 0.5 g of tissues was inoculated into a 100 ml Erlenmeyer flask containing 50 ml liquid media. Culture tubes were incubated as described above and flasks were incubated at 25 ± 2℃ in the dark on a gyratory shaker at 100 rpm. After 6 weeks,
growth (fresh weight) and somatic embryogenesis frequency were detennined. Growth of embryogenic tissues cultured in 1/2 MS liquid medium was approximately 2.2 times higher than that on solid medium. An increase in fresh weight was also observed in the tissues which had been cut-up compared to the tissues in clumps. The number of somatic embryos formed in liquid medium was twice the number formed on solid medium. The number of somatic embryos formed per culture in liquid medium increased from 88 in clump tissues to 211 when tissues were cut-up. These results show that a liquid culture system was superior. Growth and frequency of somatic embryogenesis were also enhanced when embryogenic tissues were cut into I - 2 mm in size.
Cut-up embryogenic tissues were cultured in 1/2 MS liquid medium with light (16 h photoperiod at 68 µEm·-2s-1) or in the dark. Small pieces of tissues (0.5 g) were inoculated into a 100 ml Erlenmeyer flask containing 50 ml liquid media and flasks were incubated at 25 ± 2℃ on a gyratory shaker at 100 rpm. Aner 6 weeks, growth (fresh weight) and somatic embryogenesis frequency were detennined. Growth of cut-up embryogenic tissues cultured in 1/2 MS liquid medium in the dark was 1.5 limes higher than the tissues grown under the light. In a period of 6 weeks, tissues in the dark grew approximately 15 times (7.7 g) the original inoculum of 0.5 g per flask. However, the number of somatic embryos formed per culture was the same, 236 in light-grown tissues and 231 in dark-grown tissues.|
|Rooting||Plantlet formation via shoot formation in germinating somatic embryo occurred on phytohormone-free medium. Highest number of shoots per culture was obtained from somatic embryos of petiole tissues (Pgb). P. ginseng plantlets could be transplanled inlo pots and acclimatized in a phytotron (25 ± 1 ℃, 35 µEm-2s-1).|
|Traints of regenerants||The ginsenoside contents of a 4-year old field-cultivated root were compared to ginsenoside contents in seedlings from zygotic and somatic embryos. Two-month old seedlings and somatic embryos cultured in 1/2 MS liquid medium for 2 months contained less ginscnosides than the 4-year old field-cultivated root. However, somatic embryos contained 1.7 times the amount of ginsenoside Rb1 and 2.3 times the amount of ginsenoside Re when compared to seedlings. It is of interest that ginsenoside Rd which was absent in seedlings, was detected in somatic embryos. It was found that the ginsenoside Re content in somatic embryos was comparable to that of a 4-year old field-cultivated root_ Embryogenic tissues grown on solid medium contained higher ginsenosides Rd and Rg1 levels than those in liquid medium. Levels of ginsenosides Rb1, Rc and Re were not much different between the liquid and solid culture system. The ginsenosides Rb1 and Rg1 levels in cut-up tissues were higher than levels in clump tissues. This was observed in both liquid and solid culture systems. The ginsenoside Rg1 content in cut-up embryogenic tissues grown on solid media was as high as that in a 4-year old field-cultivated root. Ginsenosidc yields were clearly higher in cut-up tissues than in clump tissues.|
|Ingredients analyzed||Ginsenosides ginsenoside Rb1, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1）|
|Extraction||Freeze dried tissues (50 mg) of a 4-year old field-cultivated root, seedlings from zygotic embryos (2 months aner germination), somatic embryos (2 months of culture in 1/2 MS liquid media) and embryogenic tissues (6 weeks of culture in 1/2 MS media) were extracted with methanol (7 ml) at 70 ℃ for 1 h. This procedure was repeated three times. The combined extract was centrifuged and the supernatant was evaporated to dryness.
The residue was dissolved in 2 ml water and adsorbed on a Sep-
Pak C18 cartridge (MILLIPORE®). The cartridge was washed with water (5 ml) and 30% methanol (5 ml) and eluted with 5 ml methanol. After evaporation to dryness, the residue was redissolved in 1 ml methanol and filtered.|
|Analitical methods||Ginsenosides Rb1, Rc and Rd in the resulting supernatant were analyzed by HPLC [column: TSKgel ODS 80TS, 4.6 mm i.d. x 150 mm (TOSOH); flow rate: 1.1 ml min-1; temperature: 40 ℃; solvent: acetonitrile / water (3/7); detection: UV 203 nm; retention time for ginsenosides: Rb1=13.01 min, Rc=16.73 min, Rd=35.01 min].
Ginsenosides Rg1 and Re were analyzed by HPLC conditions using either column: Wakosil II ODS 3C18 HG, 4.0 mm i.d. x 100 mm (Wako Chemicals, Japan); flow rate: 0.45 ml min-1, temperature: 40 ℃; solvent: acetonitrile / water (2/8); detection : UV 203 nm; retention time for ginsenosides: Rg1=33.34 min, Re=35.10 min, or column: Hibar Mightysi l RP-18, 4.6 mm i.d. x 150 mm (MERCK); flow rate: 0.75 ml min-1; temperature: 40 ℃; solvent: acetonitrile / water (2/8); detection: UV 203 nm; retention time for ginsenosides: Rg1=29.03 min, Re=30.40 min.|