Embryo Transformation, A Practical Approach for Realizing Transgenic Plants of Safflower (Carthamus tinctorius L.)

A gene transfer system that ensured recovery of whole plant transformants was developed for safflower ( Carthamus tinctorius L.). Embryo axes of germinating seeds with one of the cotyledons removed were pricked with a sterile sewing needle at the cotyledonary node and infected by gentle agitation for 10 min in a suspension of Agrobacterium tumefaciens . Following a 24 h co-cultivation and decontamination with cefotaxime for 1 h, they were placed on soilrite moistened with water to allow germination to progress. Later, the seedlings were transferred to soil in pots where they grew into normal healthy plants in the greenhouse. The histochemical assay of an uid A gene that expresses only in plant tissues and PCR amplification of uid A and npt II marker genes were used for early determination of putative transformants, whereas Southern analysis of T 0 and T 1 plant DNA was used to confirm integration of the transgenes. The combined results indicated that the frequency of transformation was 5.3% in safflower ‘A-1’ and 1.3% in ‘A-300’. Four T 0 plants of ‘A-1’ yielded transformed T 1 progeny. The strategy, in principle, should be applicable to all cultivars and genotypes of safflower which are susceptible to Agrobacterium tumefaciens infection. Thus far, this is the only procedure available for safflower that could successfully be used to generate whole plant transformants. Keywords Safflower ( Carthamus tinctorius L.), transformation, non-tissue culture method, embryo axes, Agrobacterium , transgene expression References LITERATURE CITED 1 RG Atkinson RC Gardner Agrobacterium -mediated transformation of pepino and regeneration of transgenic plants Plant Cell Reports 10 1991 208 212 2 RG Atkinson RC Gardner Regeneration of tamarillo plants Plant Cell Reports 12 1993 347 351 3 CM Baker WE Dyer Improvements in rooting regenerated safflower ( Carthamus tinctorius L.) shoots Plant Cell Reports 16 1996 106 110 4 M Cheng RL Jarret Z Li A Xing JW Demski Production of fertile transgenic peanut ( Arachis hypogaea L.) plants using Agrobacterium tumefaciens Plant Cell Reports 15 1996 653 657 5 SL Dellaporta J Wood JB Hicks A plant DNA minipreparation: version II Plant Molecular Biology Reporter 1 1983 19 21 6 K Eimert C Schroder F Siegemund Expression of the NPT II-sequence in cauliflower after injection of Agrobacterium into seeds Journal of Plant Physiology 140 1992 37 40 7 KA Feldmann MD Marks Agrobacterium -mediated transformation of germinating seeds of Arabidopsis thaliana : a non-tissue culture approach Molecular and General Genetics 208 1987 1 9 8 BJ Janssen RC Gardner Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium Plant Molecular Biology 14 1989 61 72 9 RA Jefferson Assaying chimeric genes in plants, the GUS gene fusion system Plant Molecular Biology Reporter 5 1987 387 405 10 DA Johansen Plant microtechniques 1940 McGraw-Hill Book Co New York 11 Y Junhi J Guhung DNA uptake by imbibition and expression of a foreign gene in rice Physiologia Plantarum 94 1995 453 459 12 V Katavic GW Haughman D Reed M Martin L Kunst In planta transformation of Arabidopsis thaliana Molecular and General Genetics 245 1994 363 370 13 UK Laemmli Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 1970 680 685 14 V Lev'ee MA Lelu L Jouanin D Cornu G Pilate Agrobacterium tumefaciens -mediated transformation of hybrid larch ( Larix kaempferi × L. decidua ) and transgenic plant regeneration Plant Cell Reports 16 1997 680 685 15 A KA Mandal AK Chatterji S Dutta Gupta Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of safflower Plant Cell, Tissue and Organ Culture 14 1995 197 206 16 T Murashige F Skoog A revised medium for rapid growth and bioassays with tobacco tissue cultures Physiologia Plantarum 15 1962 473 497 17 TK Orlikowska WE Dyer In vitro regeneration and multiplication of safflower ( Carthamus tinctorius L.) 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