Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

Authors: Ha, Chi; Lemaux, Peggy; Cho, Myeong-Je

Source: In Vitro Cellular and Development Biology - Plant, Volume 37, Number 1, January 2001 , pp. 6-11(6)

Publisher: Springer

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Abstract:

An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10-30 μE m⁻²s⁻¹, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3-4-month selection period for hygromycin resistance using 30-100 mg l⁻¹ hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30-40%.

Keywords: Poa pratensis L; Kenblue; recalcitrant cultivar; transformation; highly regenerative tissue; transgene expression

Document Type: Research article

DOI: http://dx.doi.org/10.1007/s11627-001-0002-5

Affiliations: 1: Email: mjcho@nature.berkeley.edu

Publication date: 2001-01-01