Genetic transformation of Indian bread (T. aestivum) and pasta (T. durum) wheat by particle bombardment of mature embryo-derived calli.
BACKGROUND
Particle bombardment has been successfully employed for obtaining transgenics in cereals in general and wheat in particular. Most of these procedures employ immature embryos which are not available throughout the year. The present investigation utilizes mature seeds as the starting material and the calli raised from the hexaploid Triticum aestivum and tetraploid Triticum durum display a high regeneration response and were therefore used as the target tissue for genetic transformation by the biolistic approach.


RESULTS
Mature embryo-derived calli of bread wheat (Triticum aestivum, cv. CPAN1676) and durum wheat (T. durum, cv. PDW215) were double bombarded with 1.1 gold microprojectiles coated with pDM302 and pAct1-F at a target distance of 6 cm. Southern analysis using the bar gene as a probe revealed the integration of transgenes in the T0 transformants. The bar gene was active in both T0 and T1 generations as evidenced by phosphinothricin leaf paint assay. Approximately 30% and 33% primary transformants of T. aestivum and T. durum, respectively, were fertile. The transmission of bar gene to T1 progeny was demonstrated by PCR analysis of germinated seedlings with primers specific to the bar gene.


CONCLUSIONS
The transformation frequency obtained was 8.56% with T. aestivum and 10% with T. durum. The optimized protocol was subsequently used for the introduction of the barley gene encoding a late embryogenesis abundant protein (HVA1) in T. aestivum and T. durum. The presence of the HVA1 transgene was confirmed by Southern analysis in the T0 generation in case of Triticum aestivum, and T0 and T1 generation in Triticum durum.
Patnaik
Debasis
D
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110 021, India. debasispatnaik@hotmail.com
Khurana
Paramjit
P
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000613:Aminobutyrates / Q000378:metabolism; D019470:Biolistics / Q000379:methods*; D046508:Culture Techniques; D005819:Genetic Markers / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D012038:Regeneration; D012639:Seeds / Q000235:genetics / Q000502:physiology; D013045:Species Specificity; D014170:Transformation, Genetic; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
D000613:Aminobutyrates; D005819:Genetic Markers; C003121:phosphinothricin; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase; D019868:Kanamycin Kinase; D005966:Glucuronidase