Skip to main content

Advertisement

SpringerLink
Log in

Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shoot-derived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6- benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 µM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the β glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Awad R, Arnason T, Trudeau V, Bergeron C, Budzinski JW, Foster BC, Merali Z (2003) Phytochemical and biological analysis of skullcap (S. lateriflora L.): a medicinal plant with anxiolytic properties. Phytomed 10:640–649

    Article  CAS  Google Scholar 

  • Baylor NW, Fu T, Yan Y-D, Ruscetti FW (1992) Inhibition of human T cell leukemia virus by the plant flavonoid baicalin (7-glucuronic acid,5,6-dihydroxyflavone). J Infect Dis 165:433–437

    Article  CAS  PubMed  Google Scholar 

  • Behbahani M, Shanehsazzadeh M, Hessami MJ (2011) Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family) for lycopene production. Sci Agric (Piracicaba, Braz.) 68:69–76

    Article  CAS  Google Scholar 

  • Brearley TA, Vaidya BN, Joshee N (2014) Cytokinin, carbon source, and acclimatization requirements for in vitro propagation of Scutellaria barbata D. Don and Scutellaria racemosa Pers. Am J Plant Sci 5:3662–3672. doi:10.4236/ajps.2014.524382

    Article  Google Scholar 

  • Brock C, Whitehouse J, Tewfik I, Towell T (2014) American Skullcap (Scutellaria lateriflora): a randomised, double-blind placebo-controlled crossover study of its effects on mood in healthy volunteers. Phytother Res 28:692–698. doi:10.1002/ptr.5044 Epub 2013 Jul 22

    Article  PubMed  Google Scholar 

  • Chafin LG (2007) Field guide to the rare plants of georgia. State Botanical Garden of Georgia, Athens, pp 347–348

    Google Scholar 

  • Cole IB, Saxena PK, Murch SJ (2007) Medicinal biotechnology in the genus Scutellaria. In Vitro Cell Dev Biol-Plant 43:318–327

    Article  CAS  Google Scholar 

  • Da Silva Teixeira, Jaime A (2004) The effect of carbon source on in vitro organogenesis of chrysanthemum thin cell layers. Bragantia 63:165–177

    Article  Google Scholar 

  • Dandawate S, Williams L, Joshee N, Rimando AM, Mittal S, Thakur A, Lum L, Parajuli P (2012) Scutellaria extract and wogonin inhibit tumor-mediated induction of Treg cells via inhibition of TGF-beta1 activity. Cancer Immunol Immunother 61:701–711

    Article  CAS  PubMed  Google Scholar 

  • Ernst E (2006) Herbal remedies for anxiety: a systematic review of controlled clinical trials. Phytomed 13:205–208

    Article  CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Grzegorczyk-Karolak I, Kuźima L, Wysokińska H (2013) The use of long-term Scutellaria altissima callus cultures for shoot regeneration, production of bioactive metabolites and micropropagation. J Med Plants Res 7:3308–3313

    Google Scholar 

  • Grzegorczyk-Karolak I, Kuźma L, Wysokińska H (2015a) Study on chemical composition and antioxidant activity of extract from shoot culture and regenerated plants of Scutellaria altissima L. Acta Physiol Plant 37:1736

    Article  Google Scholar 

  • Grzegorczyk-Karolak I, Kuźma L, Wysokińska H (2015b) The effect of cytokinin on shoot proliferation, secondary metabolite production and anti-oxidant potential in shoot cultures of Scutellaria alpina. Plant Cell Tissue Org Cult. doi:10.1007/s11240-015-0804-5

    Google Scholar 

  • Huang WH, Lee AR, Yang CH (2006) Antioxidative and anti-inflammatory activities of Polyhydroxy flavonoids of Scutellaria baicalensis GEORGI. Biosci Biotechnol Biochem 70(10):2371–2380

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed  PubMed Central  Google Scholar 

  • Joshee N, Mentreddy SR, Yadav AK (2007) Mycorrhizal fungi and growth and development of micropropagated Scutellaria integrifolia plants. Ind Crops Prod 25:169–177

    Article  CAS  Google Scholar 

  • Joshee N, Parajuli P, Medina-Bolivar F, Rimando AM, Yadav AK (2010) Scutellaria biotechnology: achievements and future prospects. Bull UASVM Hortic 67(1):24–32

    Google Scholar 

  • Joshee N, Tascan A, Medina-Bolivar F, Parajuli P, Rimando AM, Shannon DA, Adelberg JW (2012) Scutellaria: biotechnology, phytochemistry and its potential as a commercial medicinal crop. In: Chandra S, Lata H, Varma A (eds) Biotechnology for medicinal plants: micropropagation and improvement. Springer, Heidelberg, pp 69–99

    Google Scholar 

  • Kim YS, Li X, Park WT, Uddin MR, Park NI, Kim YB, Lee MY, Park SU (2012) Influence of media and auxins on growth and falvone production in hairy root cultures of baikal skullcap, Scutellaria baicalensis. Plants Omics J 5:24–27

    CAS  Google Scholar 

  • Krakauer T, Li BQ, Young HA (2001) The flavonoid baicalin inhibits superantigen-induced inflammatory cytokines and chemokines. FEBS Lett 500:52–55

    Article  CAS  PubMed  Google Scholar 

  • Krzyżanowska D, Górecka K, Kiszczakand W, Kowalska U (2006) The effect of genotype and medium on plant regeneration from androgenic embryos. J Fruit Ornam Plant Res 14:121–127

    Google Scholar 

  • Li B-Q, Fu T, Yan Y-D, Baylor NW, Ruscetti FW, Kung HF (1993) Inhibition of HIV infection by baicalin: a flavonoid compound purified from Chinese herbal medicine. Cell Mol Biol Res 39:119–124

    CAS  PubMed  Google Scholar 

  • Li H, Murch SJ, Saxena PK (2000) Thidiazuron-induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin. Plant Cell Tissue Organ Cult 62:169–173

    Article  CAS  Google Scholar 

  • Li ZJ, Jayasankar S, Gray DJ (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res 13:143–154

    Article  CAS  PubMed  Google Scholar 

  • Lin MG, Liu LP, Li CY, Zhang M, Chen Y, Qin J, Gu YY, Li Z, Wu XL, Mo SL (2013) Scutellaria extract decreases the proportion of side population cells in a myeloma cell line by down-regulating the expression of ABCG2 protein. Asian Pac J Cancer Prev 14(12):7179–7186

    Article  PubMed  Google Scholar 

  • Marsh Z, Yang T, Nopo-Olazabal L, Wu S, Ingle T, Joshee N, Medina-Bolivar F (2014) Effect of light, methyl jasmonate and cyclodextrin on production of polyphenolic compounds in hairy root cultures of Scutellaria lateriflora. Phytochem 107:50–60

    Article  CAS  Google Scholar 

  • Middleton E, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751

    CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murch SJ, Vasantha Rupasinghe HP, Goodenowe D, Saxena PK (2004) A metabolic analysis of medicinal diversity in Huang-qin (Scutellaria baicalensis Georgi) genotypes: discovery of novel compounds. Plant Cell Rep 23:419–425

    Article  CAS  PubMed  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nhut DT, Le BV, Van KTT (2001) Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinins. In Vitro Cell Dev Biol Plant 37:44–49

    Article  Google Scholar 

  • Nhut DT, Da Silva JT, Aswath GR (2003) The importance of the explant on regeneration in thin cell layer technology. In Vitro Cell Dev Biol Plant 39:266–276

    Article  Google Scholar 

  • Nijveldt R, van Nood E, van Hoorn DEC, Boelens PG, van Norren K, van Leeuven PAM (2001) Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr 74:418–425

    CAS  PubMed  Google Scholar 

  • O’Brien TP, Feder N, McCully ME (1964) Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma 59:367–373

    Google Scholar 

  • Parajuli P, Joshee N, Rimando A, Mittal S, Yadav AK (2009) In vitro anti-tumor mechanisms of various Scutellaria extracts and constituent flavonoids. Planta Med 75:41–48

    Article  CAS  PubMed  Google Scholar 

  • Parajuli P, Joshee N, Chinni SR, Rimando AM, Mittal S, Sethi S, Yadav AK (2011) Delayed growth of glioma by Scutellaria flavonoids involve inhibition of Akt, GSK-3 and NF-kB signaling. J Neurooncol 101:15–24

    Article  PubMed  Google Scholar 

  • Patel PS, Joshee N, Rimando AM, Parajuli P (2013) Anti-cancer scopes and associated mechanisms of Scutellaria extract and flavonoid wogonin. Curr Cancer Ther Rev 9:34–42

    CAS  Google Scholar 

  • Paton A (1990) A global taxonomic investigation of Scutellaria. Kew Bull 45(3):399–450

    Article  Google Scholar 

  • Rahman MH, Islam R, Hossain M, Islam MS (2010) Role of sucrose, glucose and maltose on conventional potato micropropagation. J Agric Technol 6:733–739

    Google Scholar 

  • Shang X, He X, He X, Li M, Zhang R, Fan P, Zhang Q, Jia Z (2010) The genus Scutellaria an ethno: pharmacological and phytochemical review. J Ethnopharmacol 128:279–313

    Article  CAS  PubMed  Google Scholar 

  • Sinha S, Vaidya BN, Pokhrel S, Joshee N (1999) In vitro micropropagation and callus induction in Scutellaria discolor Colebr: a medically important plant of Nepal. Indian J Plant Genet Resour 12(2):219–223

    Google Scholar 

  • Stojakowska A, Malarz J, Kohlmuenzer S (1999) Micropropagation of Scutellaria baicalensis Georgi. Acta Soc Bot Pol 68:103–107

    Article  Google Scholar 

  • Stracke R, Ishihara H, Huep G, Mehrtens F, Niehaus K, Weisshaar B, Barsch A (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tascan A, Adelberg JW, Rimando AM, Tascan M, Joshee N, Yadav AK (2010) Hyperhydricity and flavonoid content of Scutellaria species in vitro on polyester-supported liquid culture systems. HortSci 45:1723–1728

    Google Scholar 

  • Tayarani-Najaran Z, Mousavi SH, Asili J, Emami SA (2010) Growth-inhibitory effect of Scutellaria lindbergii in human cancer cell lines. Food Chem Toxicol 48:599–604

    Article  CAS  PubMed  Google Scholar 

  • Vaidya BN, Brearley TA, Joshee N (2013) Antioxidant capacity of fresh and dry leaf extracts of sixteen Scutellaria species. J Med Act Plants 2:42–49

    Google Scholar 

  • Van Tran Thanh M (1973) In vitro control of de novo flower, bud, root and callus differentiation from excised epidermal tissues. Nature 246:44–45

    Article  Google Scholar 

  • Van Tran Thanh K (1980) Control of morphogenesis by inherent and exogenously applied factors in thin cell layers. Int Rev Cytol 32:291–311

    Google Scholar 

  • Zandi K, Lim T-H, Rahim N-A, Shu M-H, Teoh B-T, Sam S-S, Danlami M-B, Tan K-K, Abubaker S (2013) Extract of Scutellaria baicalensis inhibits dengue virus replication. BMC Complement Altern Med 13:91–100

    Article  PubMed  PubMed Central  Google Scholar 

  • Zarate R, Verpoorte R (2007) Strategies for the genetic modification of the medicinal plant Catharanthus roseus (L.) G. Don. Phytochem Rev 6:475–491

    Article  CAS  Google Scholar 

  • Zhang DY, Wu J, Ye F, Xue L, Jiang S, Yi J, Zhang W, Wei H, Sung M, Wang W, Li X (2003) Inhibition of cancer cell proliferation and prostaglandin E2 synthesis by Scutellaria baicalensis. Cancer Res 63:4037–4043

    CAS  PubMed  Google Scholar 

  • Zhang CG, Li W, Mao YF, Zhao DL, Dong W, Guo GQ (2005) Endogenous hormonal levels in Scutellaria baicalensis calli induced by thidiazuron. Russ J Plant Physiol 52(3):345–351

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful for the USDA-NIFA Capacity Building Grant (CSREES Award #2011-38821-30928) (PI: Dr. N. Joshee). The authors also gratefully acknowledge Dr. Dennis J. Gray, University of Florida, for the transformation vector pq35SGR. S.A. Dhekney holds the E.A. Whitney Endowed Professorship in the Department of Plant Sciences.

Author information

Authors and Affiliations

  1. Agricultural Research Station, Fort Valley State University, 1005 State University Drive, 113 Stallworth Biotechnology Building, Fort Valley, GA, 31030-4313, USA

    Brajesh N. Vaidya, Carissa L. Jackson, Zachary D. Perry & Nirmal Joshee

  2. Department of Plant Sciences, Sheridan Research and Extension Center, University of Wyoming, Sheridan, WY, 82801, USA

    Sadanand A. Dhekney

Authors
  1. Brajesh N. Vaidya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Carissa L. Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zachary D. Perry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sadanand A. Dhekney
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nirmal Joshee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nirmal Joshee.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vaidya, B.N., Jackson, C.L., Perry, Z.D. et al. Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties. Plant Cell Tiss Organ Cult 127, 57–69 (2016). https://doi.org/10.1007/s11240-016-1029-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-1029-y

Keywords

Search

Navigation