Ploidy duplication in Acacia hybrids with colchicine

1. Ploidy duplication in Acacia hybrids with colchicine Son, L. 1, Price, A.J.2, Harbard, J.L.2, Griffin, A.R.2 Koutoulis, A. 2 1. Research Centre for Forest Tree Improvement (RCFTI) - Forest Science Institute of Vietnam (FSIV) 2. School of Plant Science, University of Tasmania (UTAS) Introduction: Acacia mangium and A.auriculiformis are native to northern Australia but are popular plantation species in Malaysia , Indonesia and Vietnam. Their naturally occurring hybrids were observed in Vietnam in the early 1990s due to their increased vigour when compared to their parent species. Research was started by Prof. Le Dinh Kha [1], former director of FSIV, identifying elite clones. As a fast growing and multi-purpose species, these hybrids now account for more than 300,000 ha of plantations in Vietnam increasing by 46,000 ha/year. It was proposed to develop putative tetraploids to: (i) compare growth rates and wood properties of tetraploid (4x) and diploid (2x) clones; and (ii) through interploidy crossing produce sterile triploids (3x) to control the Acacia hybrids invasiveness. Seven in vitro clones were sent to the School of Plant Science, UTAS, in 2009 with putative 4x induced in four clones with the genome-doubling agent, colchicine (CAS 64-86-8). Results: Only treatments that produced putative tetraploids are shown. Flow cytometry results for three clones [number (%)] Methodology: Elite Acacia hybrid clones, BV 10, BV 16 and BV 33, were grown in vitro on modified Murashige and Skoog (MS) [2] media. Explants were treated with 0.01, 0.05 and 0.1% colchicine in liquid ½ MS medium for 24 and 48h, 29°C at 100 rpm [3] [4]. For flow cytometry analysis nuclei were collected by chopping 1 cm2 of leaf material in Galbraith’s buffer [5] + 3% PVP-10 [6] on ice, stained with 1 mg/l propidium iodide and analysed using a Coulter Elite ESP flow cytometer equipped with a 100 W high-pressure mercury arc lamp (excitation using a 488 nm air cooled argon laser and fluorescence collected using a 600 nm dichroic LP and 575 nm BP). Pea (Pisum sativum) cv. Torstag nuclei were used as an internal standard [7] [8]. Histograms of BV 33 treated material: L-R mixoploid, diploid and tetraploid RH peak: internal standard, P. sativum Outcomes: Putative 4x lines of Acacia hybrid clones have their ploidy status confirmed twice, by flow cytometry, before sending to Vietnam, in culture. To date, 8, 4 and 13 putative 4x lines of BV 10, BV 16 and BV 33 respectively, have been sent. Rooted plants will require a further confirmation of their 4x status by flow cytometry before interploidy crossings can occur. Hybrid clone BV 10: Comparison of growth of the 4x Line 575 and 2x stock. Hybrid clone BV 16: 36 days after 0.1%/24h treatment References: [1] Kha L D (2001) Studies on the use of natural hybrids between Acacia mangium and Acacia auriculiformis in Vietnam. Agricultural Publishing House, Hanoi. [2] Murashige T & Skoog F (1962) Physiol. Plant 15(3) 473-497. [3] Roy A T et al.(2001) Plant Cell Rep. 20(6) 489-495. [4] Dhooghe E et al. (2010) Plant Cell, Tiss. Org. 1-15. [5] Galbraith D W et al. (1983) Science 220(4601) 1049-1051. [6] Beatson R A et al. (2003) Euphytica 134(2) 189-194. [7] Bennett M D et al. (2000) Annals of Botany 85(3) 351-357. [8] Praça-Fontes M et al. (2011) Plant Cell Rep. 30(7) 1183-1191. Acknowledgments: We thank Mark Cozens (UTAS) for technical assistance and the Australian Centre for International Agricultural Research (ACIAR) for research funding.