The document summarizes research on micropropagation and genetic transformation of Scutellaria ocmulgee, a threatened medicinal plant. Leaf and stem explants from S. ocmulgee were used in thin cell layer culture (tTCL) on different growth media. Murashige and Skoog medium with benzylaminopurine and naphthaleneacetic acid produced the highest number of shoot buds. Shoots were rooted and hardened into plants. An Agrobacterium-mediated transformation protocol was also developed using tTCL to insert marker genes. Results showed tTCL is effective for micropropagation and provides a system for genetic transformation of S. ocmulgee.

1. Micropropagation and Genetic Transformation of Scutellaria ocmulgee
Brajesh N. Vaidya and Nirmal Joshee
Agricultural Research Station, Fort Valley State University, Fort Valley, GA 31030. E-mail: josheen@fvsu.edu
3rd Annual Conference, American Council for Medicinally Active Plants, May 22 – 25, 2012. ASU, Jonesboro, AR
ABSTRACT
S. ocmulgee (Ocmulgee skullcap) is a medicinal plant with anti-tumor property and requires immediate conservation efforts due to its threatened status,
confined to a few counties in the state of Georgia. We present successful use of leaf and stem explants for rapid induction of shoot buds, elongation and
generation of complete hardened plants using transverse Thin Cell Layer Culture (tTCL) technology. Murashige and Skoog (1962) medium supplemented
with 0.5 or 2.5µM benzyl amino purine (BAP) with 0.5µM α-naphthalene acetic acid (NAA) induced highest number of shoot buds in comparison to other
treatments. Thidiazuron (TDZ) (25.0µM) with 0.5 µM indole-3-acetic acid (IAA) exhibited similar patterns but less number of shoot buds were produced in
comparison to BAP. Elongated microshoots rooted easily in MS basal or MS media containing 5.0µM indole butyric acid (IBA). Rooted plants were hardened
in a mist chamber and then potted in containers. An Agrobacterium mediated genetic transformation protocol using tTCL is also being developed.
Agrobacterium tumefaciens (EHA105) harboring binary vector pq35SGR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS)
fusion gene, and an enhanced green fluorescent protein gene (reporter) was used to optimize transformation process.
INTRODUCTION
Scutellaria is a member of Lamiaceae (mint family) spread throughout Northern Hemisphere in Europe, Africa, Asia and North America with 360 to 400
species (Paton, 1990). There are 90 species reported from N. America and about 20 species are growing in and around the state of Georgia (Joshee et al.,
2002). S. ocmulgee leaves have shown remarkable anti-tumor properties (Parajuli et al., 2009, 2011; Dandwate et al., 2012).
Status: Scutellaria ocmulgee is listed as threatened species in Georgia with 19 known populations. Habitat loss by logging, quarrying, commercial
development, trampling, mechanical clearing as well as herbivory are the major threats to this species (Chafin, 2007). The skullcap refers to the shape of the
calyx which has anatomical resemblance to medieval armored helmet worn by knights and warriors.
Micropropagation and tTCL: Micropropagation of S. ocmulgee was successfully conducted using MS and Gamborg’s B5 supplemented with various
levels cytokinins and auxins (unpublished, Richardson 2011). The transverse tTCL culture, first reported in 1973 (Tran Than Van, 1974), has been used in
some plants to regenerate new shoots. As tTCL contains different tissue types (epidermal cortical, cambium, perivascular, medullar tissue and parenchyma),
it has given positive results in in vitro morphogenic pathways. This is also the first report of tTCL of S. ocmulgee being used for genetic transformation.
MATERIALS AND METHODS
tTCL: Leaves and shoots were sliced transversely into tTCL sections 0.5 – 2.5 mm (Fig. 1. A-C). The tTCLs were inoculated on MS and B5 medium with
3% sucrose (w/v) supplemented with plant growth regulators (PGRs) either alone or in combination at various concentrations: 0.5 or 2.5µM BAP with
0.5µM NAA (0.5, 2.5, 5.0, and 10.0µM) and TDZ (0.5, 2.5, 5.0, and 10.0µM) with 0.5µM IAA. The medium was solidified with 0.8% agar (w/v)
(Phytotechnology Lab). The pH was adjusted to 5.8 before autoclaving at 121 oC for 15 min. Cultures were placed at 28 oC under 16h photoperiod. After 21
days, the percentage of explants giving shoot induction and shoot regeneration were recorded. Three replicates per treatment were maintained and the data
was pooled for statistical analysis. The adventitious shoots were transferred to MS medium supplemented with IBA for rooting of the shoots.
Agrobacterium mediated Transformation: Agrobacterium tumefaciens (EHA105) mediated genetic transformation of S. ocmulgee tTCLs was carried out.
Putative transgenic shoots were visualized under fluorescent Olympus SZX12 microscope (Fig. 1 Q-S).
RESULTS
• Leaf and stem tTCL of S. ocmulgee can be successfully integrated into micropropagation and genetic transformation programs.
REFERENCES
ACKNOWLEDGEMENT: NJ is thankful for the USDA-NIFA Capacity Building Grant that he received as PI (CSREES Award # 2008-38814-04737). BNV is thankful to Dr. Will R. Getz and Niraj K. Yadav for their help in
statistical analysis. We thank Vicki Owen, Research Assistant, for taking care of Scutellaria germplasm.
A C
D
B
E
GF
H I
J K
L
M
N RQP
O
S
Fig.1. Steps in the tTCL culture of Scutellaria ocmulgee. A and B are the tTCL explants using stem and leaf.
C. Experimental set up. D. and E. Early and late stages of leaf explants. F-K. Various stages of
organogenesis in stem tTCL explants. L and M. Leaf and stem tTCL explants with multiple shoots covering
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shoots. O. A clump has shoots of varying length, and P. Stage of microshoot for transfer to rooting medium.
Q-S. Stages in Agrobacterium tumefaciens mediated genetic transformation.
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B5BN: Gamborg’s B5 basal medium with BAP and NAA; B5-2: Gamborg’s B5 basal medium with TDZ and IAA;
MSBN: Murashige and Skoog’s basal medium with BAP and NAA