Ginseng

Ginseng (Panax ginseng) is a slow growing perennial plant with fleshy roots, belonging to
family Araliaceae and genus Panax. It usually grows in cooler climates and belongs to north
America and eastern Asia. Panax quinquefolius cultivar of ginseng is found in Asia (Xiang and
Lowry, 2007) Ginseng is most widely used herbal drug. It has various therapeutic and
pharmacological activities (Bladt et al., 1990). Conventional breeding of ginseng requires long
period for maturity and growth, new plant may be different from parent plant. Ginseng is
characterized by the presence of ginsenoside and gintonin. Ginsenoside is a group of steroidal
saponins. About 40 ginsenosides have been isolated and characterized from ginseng plant (Tung
et al., 2009). Clonal propagation of ginseng can be achieved by the use of in vitro techniques. It
also promotes aseptic plant propagation. There are some problems associated with the in vitro
cultivation of ginseng are

 Excessive phenolics exudation

 Require rapid subculturing
 Contamimation problem

In vitro culture of Ginseng

From leaf-derived callus: (Nhut et al., 2011)

Explant sterilization and preparation
 Leaves will be surface sterilized with 70% (v/v) ethyl alcohol for 30 seconds and 0.1%
(w/v) mercuric chloride for 5 minutes.
 3-4 time, rinsing with sterile double distilled water.
 Leaves will be cut into small pieces of about 1mm.

Callus induction and proliferation

 Leave disks will be cultured on Murashige and Skoog (MS) medium supplemented with
0.1-0.5 mg L-1 TDZ (thidiazuron) and 1.0-5.0 mg L-1 2,4-D for callus induction.
 After callus induction it will be shifted in Schenk and Hildebrandt (SH) medium,
Murashige and Skoog (MS) medium and half strength MS medium supplemented with
0.2 mg L-1 TDZ (thidiazuron) and 1.0 mg L-1 2,4-D for callus proliferation.
Shoot formation
 After eight weeks the callus will be transferred to SH medium containing 0.5-4.0 mg L -1
6-benzylaminopurine (BA) for shoot regeneration from callus.

Root formation
 Regenerated shoots will be shifted in SH medium with 0.5-2.5 mg L -1 α-naphthalene
acetic acid (NAA) for rooting of shoots.
 pH of all the mediums will be adjusted at 5.7±0.1.
 Culture condition will be maintained at 25±2°C temperature, 70-80% relative humidity
and 10 hr photoperiod.

Acclimatization
 After 2 months plants will be acclimatized.

From root-derived callus: (Zhang et al., 2014)

Explant sterilization and preparation
 Roots will be washed with 70% alcohol for about 1 minute and will be blotted with
towel.
 Roots will be sterilized in 2.5% sodium hypochlorite along with a few drops of detergent.

 Adventitious roots will be sectioned into 10 mm in length.

Callus induction
 These sections will be placed in MS solid medium supplemented with 0.5-2 mg L -1 2,4-D
and 0.1-0.5 mg L-1 kinetin for callus induction.
 After 3 weeks callus will be transfered to MS medium along with different
concentrations of 2,4-D (0 mg/L, 0.5 mg/L, 1 mg/L) for callus proliferation and embryo
induction.

Shoot formation
 After somatic embryo induction it will be shifted in MS medium with 3-7 mg L -1
gibberellic acid for development and elongation of shoots.
Root formation
 At a hight of 3-4 cm the regenerated shoots will be transferred into 1/3 strength of Schenk
and Hildebrandt basal medium supplemented with 0.15-0.45 mg L-1 naphthalene acetic
acid for the development of well-developed taproots.
 pH of MS media will be maintained between 5.6-5.8.
 Growth conditions will be 23±2°C temperature with 16 hr light and 8hr dark period.

Acclimatization
 After this plants will be acclimatized by planting them in plastic pots containing a soil
mixture of peat moss, vermiculite and perlite (2:3:1 v/v) in growth room.

Excised Cotyledonary Explants : (Choi et al., 1999)

Explant selection and sterilization
 Seeds with immature embryos will be harvested in the month of july.
 Seeds will be stored at 10-15°C for 6 months to allow the embryos in the seeds to grew at
least 6mm in length.
 Seeds will be sterilized with 70% ethanol for 1 min, after that with 1% NaOCl for 1 hour
and then rinsed 3-4 times with sterile double distilled water.
 After dissection of seed, zygotic embryos will be cultured on hormone-free half strength
Murashige and Skoog (MS) basal medium (supplemented with vitamins and myo-
inositol) containing 3% sucrose and 0.5% agar for germination.
 Embryos at four different developmental stages will be selected at different time of
germination ie. mature zygotic embryos, 1, 2 and 4 week old seedlings.

Media for root and shoot induction

 Cotyledon bases will be transversely cut and will be cultured on MS medium containing
0.5 µM IBA (indol butyric acid) and 8.8 µM BA (N 6-benzyladenine), supplemented with
3% sucrose and 0.7% agar.
 Cultures will be transplanted into the same medium after one month.
  Epicotyl-like shoot primordia will be developed from cotyledon base after 2 weeks.
 Subsequently roots will develop near the base of the epicotyls-like shoots.
 pH will be maintained at 5.8.

Plant regeneration via adventitious bud formation: (Choi et al., 1998)

Preparation and sterilization of explant

 Seeds will be immersed in 70% ethanol for 1 mint.
 Sterilization will be done with 1% NaOCl for 1 hour, then rinsed with double distilled
water.
 Stratification of seed will be done at 10°C for 6 months.
 The embryo in the seed will grow 6mm in length
 Zygotic embryos will be dissected and transversely cut.

Adventitious bud formation

 Cotyledons will be placed on MS basal medium containing 3% sucrose and 0.7% agar.
 Cytokinins (BAP, Zeatin and Kinetin) will be used for adventitious bud formation with
different concentrations (0.1, 0.5, 1.0, 2.5, 5.0mg L-1)
 pH will be maintained at 5.8

Shoot formation
 Cotyledonary explants with adventitious buds will be shifted in MS medium
supplemented with GA3 (8, 10 and 12 mg L-1) for adventitious shoot formation.
Root formation
 Adventitious shoots will be transferred to half-strength MS medium with various
concentrations of IAA (0.1, 0.25, 0.5 mg L-1) for root induction.
Acclimatization
 After root and shoot formation plants will be acclimatized in square pots containing soil
and sand (1:1) in greenhouse.
REFERENCES

Bladt S, Wagner H, Woo WS (1990). HPLC fingerprint analysis and standardisation of

Eleutherococcus senticosus (Acanthopanax) extracts. Planta Med. 56: 78-79.

Choi, Y.E., D.C. Yang, E.S. Yoon and K.T. Choi. 1998. Plant regeneration via adventitious bud
formation from cotyledon explants of Panax ginseng C. A. Mayer. Plant cell reports 17:
731-736.

Choi, Y.E., D.C. Yang, E.S. Yoon and K.T. Choi. 1999. Plant regeneration via direct embryo
axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings.
In vitro Cell. Dev. Biol. 35: 210-213.VIA

Nhut, D.T., N.P. Huy, V.Q. Luan, N.V. Binh, N.B. Nam, L.N.M. Thuy, D.T.N. Ha, H.X. Chien,
T.T. Huong, H.V. Cuong, L.K. Cuong and V.T. Hien. 2011. Shoot regeneration and
micropropagation of Panax vietnamensis Ha et Grushv. from ex vitro leaf-derived callus.
Afr. J. Biotech. 10(84): 19499-19504.

Tung NH, Song GY, Park YJ, Kim YH (2009). Two new Dammaranetype saponins from the
leaves of Panax ginseng. Chem. Pharm. Bull. 57: 1412-1414.

Xiang, Q. and P.P. Lowry. 2007. Araliaceae. Flora of China. 13: 489-491.

Zhang, J.Y., H.J. Sun, I.J. Song, T.W. Bae, H.G.Kang, S.M. Ko, Y.I. Kwon, I.w.Kim, J.Lee,
S.Y.Park, P.o. Lim, Y.H. Kim and H.Y.Lee. 2014. Plant regeneration of Korean wild
ginseng (Panax ginseng Meyer) mutant lines induced by g-irradiation (60Co) of
adventitious roots. J. Ginseng Res. 38: 220-225.