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Agrobacterium-mediated genetic transformation of safflower (Carthamus tinctorius L.)

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Abstract

Reproducible and highly efficient protocols for shoot regeneration and genetic transformation mediated by Agrobacterium have been established for safflower (Carthamus tinctorius L.). Agrobacterium tumefaciens strain LBA 4404 with gus reporter gene and hygromycin (hpt gene) as plant selection marker was used as the plant transformation vector. Genetic transformation experiments were carried out to evaluate the efficacy of various parameters such as genotype, seedling age, co-cultivation period, bacterial titer, enzymatic pre-treatment of target tissues, use of compounds that induce vir-gene enhancer, acetosyringone (AS), explant type and explant injury to enhance transformation efficiency. Transformation frequency was high when root and hypocotyl explants of 8-day-old seedlings of safflower cv. HUS-305 were co-cultivated with bacterial cell density of 0.5 OD600 during a period of 2 days followed by selection regime of 10–15–15 mg/l hygromycin. The frequency of rooting of the primary transformants was low (18.0%) when compared with the regenerated shoots (70.0%), and seven shoots survived on transfer to soil. The putative transformants were confirmed by β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR), reverse-transcriptase PCR (RT-PCR) and Southern blot analysis. With the optimized transformation protocol, putative transformed shoots were obtained with frequency of 51.0% within 8–10 weeks of culture initiation.

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Abbreviations

2,4,5-Cl3POP:

2,4,5-Trichlorophenoxypropionic acid

2,4-D:

2,4-Dichlorophenoxyacetic acid

2iP:

N6-[2-Isopentenyl]adenine

AS:

Acetosyringone

AgNO3 :

Silver nitrate

BA:

6-Benzyladenine

GUS:

β-Glucuronidase

hpt :

Hygromycin phosphotransferase gene

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

KN:

6-Furfurylaminopurine/kinetin

MS:

Murashige and Skoog medium

NAA:

α-Naphthaleneacetic acid

PCR:

Polymerase chain reaction

RT-PCR:

Reverse-transcriptase polymerase chain reaction

PGA:

Phloroglucinol

TDZ:

Thidiazuron

X-GlcA:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronic acid

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Acknowledgments

We thank Dr. D. M. Hegde, Project Director, Directorate of Oilseeds Research, Hyderabad, India for extending all the research facilities for carrying out the study. The guidance and support of Dr. S. M. Balachandran, Directorate of Rice Research, Hyderabad, India in carrying out Southern blot hybridization work is acknowledged. The first author is grateful to Jawaharlal Nehru Memorial Fund, New Delhi, India for providing a fellowship for the work.

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  1. Crop Improvement Section, Directorate of Oilseeds Research, Rajendranagar, Hyderabad, 500030, India

    K. Sri Shilpa, V. Dinesh Kumar & M. Sujatha

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  1. K. Sri Shilpa
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Correspondence to K. Sri Shilpa.

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Sri Shilpa, K., Dinesh Kumar, V. & Sujatha, M. Agrobacterium-mediated genetic transformation of safflower (Carthamus tinctorius L.). Plant Cell Tiss Organ Cult 103, 387–401 (2010). https://doi.org/10.1007/s11240-010-9792-7

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