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Title:	In vitro polyploid induction in Acacia
Authors:	Yap Jing Wei (P41938)
Supervisor:	Mahani Mansor Clyde
Keywords:	Acacia Universiti Kebangsaan Malaysia -- Dissertations Dissertations, Academic -- Malaysia
Issue Date:	24-May-2011
Description:	The development of seedless triploid planting material represents a possible strategy for overcoming the inherent weediness of introduced Acacia in Malaysia. In addition, triploid Acacia may have superior traits than diploids, as observed in other commercial crops. Triploids may be developed by first inducing tetraploids through the use of mitotic inhibitors such as colchicine. Tetraploids can later be crossed with diploids to produce seedless triploid progeny. In this study, colchicine immersion treatments and flow cytometry (FCM) nuclei isolation buffers were tested on in vitro cultures of an F1 Acacia hybrid (A. mangium × A. auriculiformis) clone for the purpose of developing a general colchicine treatment and FCM screening protocol for Acacia. The efficacy of five nuclei isolation buffers, [general purpose buffer (GPB), woody plant buffer (WPB) Otto's, LBO1, and Partec CyStain® PI absolute P buffer (PCPIP)], and two incubation periods (10 and 60 min), were evaluated on Acacia hybrid in vitro leaf nuclei suspensions. The resulting FCM histograms were evaluated according to four parameters namely, the relative fluorescence intensity of the propidium iodide (PI)-stained nuclei (FL), half peak coefficient of variation (CV) of the 2C DNA peaks as a measure of nuclear integrity and variation in DNA staining, a debris background factor (BF) as a measure of sample quality, and a nuclear yield factor (YF) to compare the amount of nuclei in suspension independently of the amount of leaf tissue used. Although minor variation existed in terms of performance, the results indicate that, with the exception of WPB which showed an undesirable combination of overall low YF and high CV, Acacia are generally amenable to a variety of nuclei isolation buffers. Ultimately, GPB was selected for Acacia hybrid FCM analysis as it demonstrated a good all round performance (overall low CV, low BF, and most importantly, a stable YF with respect to incubation time). Using GPB with PI as a fluorochrome and Glycine max cv. 'Polanka' as the internal reference standard, the 2C DNA amounts was estimated for diploid A. mangium (2n = 2x = 26) as 1.48 pg (1440 Mbp), for diploid A. auriculiformis (2n = 2x = 26) as 1.49 pg (1460 Mbp) and for the diploid F1 Acacia hybrid as 1.46 pg (1420 Mbp). In vitro Acacia hybrid shoot tips were subjected to a range of colchicine concentrations (0, 1.25, 2.5, 5 and 10 mM) for a range of exposure times (0, 3, 6, 12, 24, 48 and 96 h). FCM ploidy analysis of in vitro leaves from regenerating shoot tips demonstrated that putative DNA mixoploids (cytochimeras composed of diploid and tetraploid cells) of Acacia may be generated by exposures to 1.25 mM of colchicine or higher for as little as 3 h with the highest mean frequency of mixoploids (25.8%) occurring at 5 mM colchicine for 6 h. However, no conclusion could be drawn due to the large standard deviations in the data set. This research has therefore established colchicine treatment and FCM parameters for the production and evaluation of polyploid Acacia.,“Certification of Master's/Doctoral Thesis” is not available,Master Science
Pages:	79
Call Number:	SD397.A2Y346 2011 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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