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Title:	Somatic embryogenesis and in vitro mutagenesis of limau madu (citrus reticulata Blanco)
Authors:	Dita Agisimanto (P41936)
Keywords:	Mandarin orange Mandarin orange-Somatic embryogenesis.
Issue Date:	7-Mar-2012
Description:	An efficient system for cell proliferation and somatic embryo induction is necessary for limau madu (Citrus reticulataBlanco) propagation and improvement using in vitro mutagenesis. The main factors affecting cell proliferation and subsequently somatic embryo induction in liquid medium using flasks and bioreactors were studied. For inducing callus from nucellus, 2,4-D, Dicamba, Picloram, benzylaminopurine (BAP) and Kinetin at 3 mg L-1 (13.3 μM) in Murashige and Skoog (MS) supplemented with 500 mg L-1 of malt extract, 146 mM sucrose and 2.5% gelrite were tested. BAP at 13.3 μM produced 261.13 mg FW of callus in 28 days, higher than 2.4-D, Dicamba, Picloram and Kinetin which produced 257.40, 220.95, 200.28 and 172.87 mg FW, respectively. BAP at 13.3 μM was then compared with BAP at other concentrations of 4.4, 8.9, 17.8 and 22.2 μM to confirm its effectiveness on callus induction. BAP at 13.3 and 17.8 μM induced 70 and 72% callus at growth rates of 0.46 and 0.45 mg day-1, respectively, while at other concentrations, lower quantity of embryogenic callus was produced. Further studies using flask and bioreactor cultures were conducted to obtain the best in vitro conditions for multiplying cells and for somatic embryo induction in liquid medium. For cell proliferation either in flask or bioreactor culture, MS with BAP at 6.7 μM and sucrose at 146 mM were selected. Initial cell density of 2 mg mL-1 supported the highest rate of cell multiplication of 11.5-folds at day 30 of culture in flask cultures. In similar medium, the cells at initial density of 5.5 mg mL-1 supported the highest rate of cell growth at 2567.60 mg day-1 at day 21 of culture in an airlift bioreactor supplied with air atmosphere at rate of 1.5 vvm while the maximum yield of 556,004.25 mg of FW was harvested at day 28. Somatic embryos were effectively induced when sucrose used as a carbon source in proliferating medium was replaced with 110 mM sorbitol and 36 mM galactose in medium without BAP. Initial cells of 100 g of FW were converted into 2,031.55 globular, 2,867.85 hearts, 4,078.73 torpedo and 5,077.54 cotyledonery-shaped embryos. The genetic fidelity of cells, embryos and plantlets cultured in the optimal condition for cell multiplication and somatic embryogenesis was tested.To increase genetic variability of limau madu, nucelli were exposed to gamma rays at 10, 20, 40, 60, 80, 100 and 120 Gy and cultured on MS medium with 13.3 μM BAP. Untreated explants survived and produced friable embryogenic callus tissue in the 2nd-3rd week of culture, whereas the irradiated nucelli (10, 20, 40 Gy) showed delayed responses (3-4th week). The doses of 100 and 120 Gy completely suppressed callus formation. After 35 days of culture, 697 and 660 mg FW of callus were harvested from the nucelli irradiated at 10 and 20 Gy, respectively; which were higher than those at 40 Gy (441 mg), 60 Gy (436 mg) and 80 Gy (380 mg). Based on callus formation as survival indicator, the effective dose was 20 Gy which sustained 45.45% of nucelli survival, while the calculated lethal dose of 50% was 30 Gy. DNA of expected mutants from the dosage of 20, 40 and 60 Gy produced from air-lift bioreactor were individually amplified using retrotransposon, inter simple sequence repeat and markers related to seedlessness. Results confirm genetic fidelity in 14-months-cycle of somatic embryogenesis from callus induction on semisolid medium, cell proliferation on semisolid, flask and bioreactor cultures. The seedlessness characteristic was not found in any of the callus or cells from irradiated nucelli.,Certification of Master's/Doctoral Thesis" is not available
Pages:	130
Call Number:	SB370.M34A364 2012 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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