Plant Science 148 (1999) 9–17 High-frequency transformation of oat via microprojectile bombardment of seed-derived highly regenerative cultures Myeong-Je Cho *, Wen Jiang 1 , Peggy G. Lemaux Department of Plant and Microbial Biology, Uniersity of California, Berkeley, CA 94720, USA Received 23 February 1999; received in revised form 3 May 1999; accepted 5 May 1999 Abstract A highly efficient and reproducible transformation system for oat (Aena satia L. cv. GAF/Park-1) was developed using microprojectile bombardment of highly regenerative tissues derived from mature seeds. Callus was induced under dim light conditions on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and high cupric sulfate. Highly regenerative tissues, generated from embryogenic callus, were used as a transformation target. From 327 individual explants bombarded with the -glucuronidase gene (uidA ; gus ) and a hygromycin phosphotransferase gene (hpt ), 84 independent transgenic events were obtained after an 8 – 12-week selection period on hygromycin. All events were regenerable, giving an effective transformation frequency of 26%; co-expression of GUS activity occurred in 70% of the independent events. Presence of the foreign genes in DNA from leaf samples of T 0 and T 1 plants was confirmed by polymerase chain reaction (PCR) amplification and/or DNA blot hybridization. Fertility of the plants from the transgenic lines was 63% (24/38) and the transgene(s) was stably transmitted to T 1 and T 2 progeny. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aena satia L.; Mature seed; Embryogenic callus; Highly regenerative tissue; Transformation www.elsevier.com/locate/plantsci 1. Introduction Introduction of genes using genetic engineering is dependent on efficient, reproducible in vitro culturing methods. Successful transformation of oat using microprojectile bombardment of cell sus- pension cultures or embryogenic calli developed from immature embryos was reported [1 – 4]. How- ever, the utility of these target tissues is limited because the regenerability decreases and so- maclonal variation increases during the prolonged culturing periods [5] necessary for transformant identification. Other in vitro-derived tissues have also been used in successful transformation experi- ments with oat. These include mature embryo- derived callus tissue [6,7], leaf base segments of young oat seedlings [8,9] and shoot meristem cul- tures initiated from the shoot apex of germinated seedlings [10 – 12]. We have recently established an efficient in vitro system to proliferate highly regenerative tissues from immature scutellar tissues of barley [13,14]. These tissues, which yield large numbers of shoots during regeneration and can be maintained for more than a year with minimal loss in regenerabil- ity, have been used for the successful transforma- tion of previously recalcitrant commercial barley cultivars [13,14]. Genomic DNA methylation analyses showed that barley plants regenerated from the highly regenerative tissues are less vari- able with regard to those regenerated from em- bryogenic callus tissues in methylation pattern polymorphism and agronomic performance [15]. In this report we describe the development from mature oat seed of similar highly regenerative tissues to be used as transformation targets. We Abbreiations: 2,4-D, 2,4-dichlorophenoxyacetic acid; BAP, 6-ben- zylaminopurine; CIM, callus-induction medium; GUS, -glu- curonidase; hpt, hygromycin phosphotransferase gene. * Corresponding author: Tel.: +1-510-6421347; fax: +1-510- 6427356. E-mail address: mjcho@nature.berkeley.edu (M.-J. Cho) 1 Present address: Calgene Inc., Davis, CA 95616, USA 0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(99)00082-5