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In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant

Abstract

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog’s medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog’s medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.

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References

  1. Bhalothia P, Alok A, Mehrotra S, Mehrotra R (2013) AACA element negatively regulates expression of protein phosphatase 2C (PP2C) like promoter in Arabidopsis thaliana. Am J Plant Sci 04:549–554. doi:10.4236/ajps.2013.43071

    Article  Google Scholar 

  2. Devi KD, Punyarani K, Singh NS, Devi HS (2013) An efficient protocol for total DNA extraction from the members of order Zingiberales- suitable for diverse PCR based downstream applications. Springerplus 2:669. doi:10.1186/2193-1801-2-669

    Article  PubMed  PubMed Central  Google Scholar 

  3. Elfahmi SS, Chahyadi A (2014) Optimization of genetic transformation of Artemisia annua L. Using Agrobacterium for artemisinin production. Pharmacogn Mag 10:S176–S180. doi:10.4103/0973-1296.127372

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Haider SZ, Mohan M, Andola HC (2014) Constituents of Artemisia indica Willd. From Uttarakhand Himalaya: a source of davanone. Pharmacognosy Res 6:257–259. doi:10.4103/0974-8490.132607

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Han J, Wang H, Ye H, Li G (2005) High efficiency of genetic transformation and regeneration of Artemisia annua L. Via Agrobacterium tumefaciens-mediated procedure. Plant Sci 168:73–80. doi:10.1016/j.plantsci.2004.07.020

    CAS  Article  Google Scholar 

  6. Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet MGG 163:181–187. doi:10.1007/BF00267408

    CAS  Article  PubMed  Google Scholar 

  7. 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 

  8. Li J-F, Park E, von Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6. doi:10.1186/1746-4811-5-6

    Article  PubMed  PubMed Central  Google Scholar 

  9. Mallavarapu GR, Kulkarni RN, Baskaran K, Rao L, Ramesh S (1999) Influence of plant growth stage on the essential oil content and composition in Davana (Artemisia pallens Wall.). J Agric Food Chem 47:254–258. doi:10.1021/jf980624c

    CAS  Article  PubMed  Google Scholar 

  10. Nathar VN, Yatoo GM (2014) Micropropagation of an antidiabetic medicinal plant. Artemisia pallens Turk J Botany 38:491–498

  11. Rech EL, Vianna GR, Aragao FJL (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418

    CAS  Article  PubMed  Google Scholar 

  12. Ruikar AD, Khatiwora E, Ghayal NA, Misar AV, Mujumdar AM, Puranik VG, Deshpande NR (2011) Studies on aerial parts of Artemisia pallens wall for phenol, flavonoid and evaluation of antioxidant activity. J Pharm Bioallied Sci 3:302–305. doi:10.4103/0975-7406.80768

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Sharafi A, Sohi H, Mirzaee H, Azadi P (2014a) In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss. Physiol Mol Biol Plants 20:487–494. doi:10.1007/s12298-014-0248-0

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Sharafi A, Sohi H, Sharafi AA, Azadi P, Mausavi A (2014b) Tissue culture and regeneration of an antima larial plant, Artemisia sieberi Besser. Res J Pharmacogn 1:15–20

    CAS  Google Scholar 

  15. Shukla V, Pala Z, Alok A, Desai N (2015) Screening of different Artemisia spp. from Western Ghats of Maharashtra for an anti-malarial compound-artemisinin. Am J Plant Sci 06:1619–1632. doi:10.4236/ajps.2015.69162

    CAS  Article  Google Scholar 

  16. Subramoniam A, Pushpangadan P, Rajasekharan S, Evans DA, Latha PG, Valsaraj R (1996) Effects of Artemisia pallens Wall. On blood glucose levels in normal and alloxan-induced diabetic rats. J Ethnopharmacol 50:13–17. doi:10.1016/0378-8741(95)01329-6

    CAS  Article  PubMed  Google Scholar 

  17. Sujatha G, Ranjitha Kumari BD (2007) High-frequency shoot multiplication in Artemisia vulgaris L. Using thidiazuron. Plant Biotechnol Rep 1:149–154. doi:10.1007/s11816-007-0028-1

    Article  Google Scholar 

  18. Sujatha G, Zdravković-Korać S, Ćalić D, Flamini G, Kumari Ranjita BD (2013) High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: hairy root production and essential oil analysis. Ind Crop Prod 44:643–652. doi:10.1016/j.indcrop.2012.09.007

    CAS  Article  Google Scholar 

  19. Tiwari S, Tuli R (2012) Optimization of factors for efficient recovery of transgenic peanut (Arachis hypogaea L.). Plant cell. Tissue Organ Cult 109:111–121. doi:10.1007/s11240-011-0079-4

    CAS  Article  Google Scholar 

  20. Upadhyay SK, Kumar J, Alok A, Tuli R (2013) RNA-guided genome editing for target gene mutations in wheat. G3 (Bethesda) 3:2233–2238

    CAS  Article  Google Scholar 

  21. Vergauwe A, Van Geldre E, Inzé D, Van Montagu M, Van den Eeckhout E (1998) Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. Plant Cell Rep 18:105–110. doi:10.1007/s002990050540

    CAS  Article  Google Scholar 

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Acknowledgment

The authors express their gratitude for providing research facilities to School of Biotechnology and Bioinformatics, D. Y. Patil University, Navi Mumbai, India.

Author contribution

Conceived and designed the experiments: AA and ND. Performed the experiments: AA, VS, ZP, SS and JK. Analyzed the data: AA, VS and ND. Wrote the paper: AA, JK and ND.

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Correspondence to Neetin Desai.

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Alok, A., Shukla, V., Pala, Z. et al. In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant. Physiol Mol Biol Plants 22, 261–269 (2016). https://doi.org/10.1007/s12298-016-0353-3

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Keywords

  • A. pallens
  • Agrobacterium tumefaciens
  • Attached cotyledons
  • Transient transformation
  • GUS