Abstract
The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l−1 thidiazuron (TDZ) and subjected to bombardment after 5–7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding β-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 μm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l−1 BA and hygromycin (20, 40 and 60 mg l−1) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T0 and T1 plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.
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Abbreviations
- BA:
-
N 6-Benzyladenine
- DMRT:
-
Duncan’s multiple range test
- GUS:
-
β-Glucuronidase
- HPT:
-
Hygromycin-phosphotransferase
- MS:
-
Murashige and Skoog basal salt media
- NAA:
-
∝-Naphthaleneacetic acid
- NPT:
-
Neomycin phosphotransferase
- PCR:
-
Polymerase chain reaction
- TDZ:
-
1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)
- X-Gluc :
-
5-Bromo, 4-chloro, 3-indolyl, β-d-glucuronide
References
Ahn YJ, Vang L, McKeon TA, Chen GQ (2007) High-frequency plant regeneration through adventitious shoot formation in castor (Ricinus communis L.). In Vitro Cell Dev Biol Plant 43:9–15
Anonymous (1988) MSTATC Microcomputer Statistical Programme. Michigan State University, Michigan, East Lansing, USA
Athma P, Reddy TP (1983) Efficiency of callus initiation and direct regeneration from different explants of castor (Ricinus communis L.). Curr Sci 52:256–257
Atsmon D (1989) Castor. In: Robbelen G, Downey RK, Ashri A (eds) Oil crops of the world their breeding and utilization. Mc Graw-Hill, New York, pp 438–447
Auld DL, Rolfe RD, McKeon TA (2001) Development of castor with reduced toxicity. J New Seeds 3:61–69
Bilang R, Zhang S, Leduc N, Iglesias VA, Gisel A, Simmonds J, Potrykus I, Stutter C (1993) Transient GUS expression in vegetative shoot apical meristems of wheat after ballistic microtargeting. Plant J 4:735–744
Brar GS, Cohen BA, Vick CL, Johnson GW (1994) Recovery of transgenic peanut (Arachis hypogea L.) plants from elite cultivars utilizing ACCELL technology. Plant J 5:745–753
Christou P (1996) Particle bombardment for genetic engineering of plants. RG Landes Co., TX
Deng XY, Wei ZM, An HL (2001) Transgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system. Cell Res 11:156–160
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15
Gheysen G, Van Montagu M, Zambryski P (1987) Integration of Agrobacterium tumefaciens T-DNA involves rearrangements of target plant DNA sequences. Proc Natl Acad Sci USA 84:9006–9010
Hagio T (1998) Optimising the particle bombardment method for efficient genetic transformation. JARQ 32:239–247
Hunold R, Bronner R, Hahne G (1994) Early events in microprojectile bombardment: cell viability and particle location. Plant J 5:593–604
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Klein TM, Wolf ED, Wu R, Sanford JC (1987) High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70–73
Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. Bio/Technol 6:559–563
Lin X, Zhang W, Takechi K, Takio S, Ono K, Takano H (2005) Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos. Plant Cell Rep 24:418–425
Malathi B, Ramesh S, Venkateswara Rao K, Dashavantha Reddy V (2006) Agrobacterium-mediated genetic transformation and production of semilooper resistant transgenic castor (Ricinus communis L.). Euphytica 147:441–449
McCabe DE, Swain WF, Martinelli BJ, Christou P (1988) Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technol 6:923–926
McKeon TA, Chen GQ (2003) Transformation of Ricinus communis, the castor plant. US Patent No 6,620,986
Molina SM, Schobert C (1995) Micropropagation of Ricinus communis. J Plant Physiol 147:270–272
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Potrykus I (1991) Gene transfer to plants: assessment of published approaches and results. Annu Rev Plant Physiol Plant Mol Biol 42:205–225
Reddy KRK, Bahadur B (1989) Adventitious bud formation from leaf cultures of castor (Ricinus communis L.). Curr Sci 58:152–154
Reddy KRK, Rao GP, Bahadur B (1987) In vitro morphogenesis from seedling explants and callus cultures of castor (Ricinus communis L.). Phytomorphology 37:337–340
Russell JA, Roy MK, Sanford JC (1992) Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation. Plant Physiol 98:1050–1056
Saeed NA, Zafar Y, Malik KA (1997) A simple procedure of Gossypium meristem shoot tip culture. Plant Cell Tiss Org Cult 51:201–207
Sarvesh A, Ram Rao DM, Reddy TP (1992) Callus initiation and plantlet regeneration from epicotyl and cotyledonary explants of castor (Ricinus communis L.). Adv Plant Sci 5:124–128
Sharma KK, Bhatnagar P, Thorpe TA (2005) Genetic transformation technology: status and problems. In Vitro Cell Dev Biol Plant 41:102–112
Sticklen MB, Oraby HF (2005) Shoot apical meristem: a sustainable explant for genetic transformation of cereal crops. In Vitro Cell Dev Biol Plant 41:187–200
Sujatha M, Reddy TP (1998) Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.). Plant Cell Rep 17:561–566
Sujatha M, Sailaja M (2005) Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Rep 23:803–810
Vain P, Mc Mullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12:84–88
Vignolo R, Naughton F (1991) Castor: a new sense of direction. Inform 2:692–699
Weissinger A, Tomes D, Maddock S, Fromm M, Sanford J (1988) Maize transformation via microprojectile bombardment. Genetic improvements of agriculturally important crops—progress and issues. In: Fraley RT, Frey NM, Schell J (eds) Current communications in molecular biology. Cold Spring Harbor Laboratory, NY, pp 21–25
Yang J, Lee HJ, Shin DH, Oh SK, Seon JH, Paek KY, Han KH (1999) Genetic transformation of cymbidium orchid by particle bombardment. Plant Cell Rep 18:978–984
Acknowledgments
The authors thank The Project Director, Directorate of Oilseeds Research, Hyderabad for extending all facilities for carrying out the work and D. Lakshmi and P. Kanaka Mahalakshmi for their excellent technical help. The financial support from Andhra Pradesh-Netherlands Biotechnology Programme is gratefully acknowledged.
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Communicated by R. Rose.
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Sailaja, M., Tarakeswari, M. & Sujatha, M. Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds. Plant Cell Rep 27, 1509–1519 (2008). https://doi.org/10.1007/s00299-008-0580-3
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DOI: https://doi.org/10.1007/s00299-008-0580-3