MATERIAL AND METHOD 1 Material and Location

Bogor, 21-22 October 2015 172 In line with the strategy, selected populations Gunung Kidul with the highest CCO Crude Calophyllum Oil content 50.00-50.12 among six nyamplung populations in Java, was established in Wonogiri Central Java as provenance seeds stand. Provenance seed stand is an area where the potential provenance or land race are established and managed intensively and entirely for seed production Zobel Talbert, 1984. The objective of the establishment above was to verify the potency of biofuel content in a different site. Besides the biofuel content, the growth, flowering, and fruiting characteristic were also investigated to determine its adaptability and to select individual tree with high productivity in fruits. Sites can affect performance in growth parameters of nyamplung, inappropriate environment and seed sources provenances land races are factors to affect in optimum plant productivity Putri et al., 2013; Hasnah Windyarini, 2014. This results conveyed that different site characteristic showed different performance of nyamplung from the same sources Hasnah Windyarini, 2015; Windyarini Hasnah, 2015. The ability to flower, bear fruits and produce seeds is key parameters to support the successful of breeding strategy program of nyamplung, since flowering characteristic and seeds productivity are important information. In the next step, individual having high productivity in fruits and biofuel content will be verified using DNA technology to assess their GCA General Combining Ability. Planting stocks of nyamplung with high seed productivity, biofuel content and GCA will then be reproduced through vegetative propagation in order to shorten its reproductive age. Production population to support mass plantation of nyamplung will be fulfilled by tissue culture to produce uniform growth and seeds in a large quantities. This paper presents the growth, flowering, fruiting and biofuel content of nyamplung observed during the first three years after planting in the provenance seed stand in Wonogiri, Central Java. 2. MATERIAL AND METHOD 2.1 Material and Location Data used in this study were obtained from provenance seed stand of nyamplung in Wonogiri research station, during the first three years after planting 2011-2014. The Provenance Seed Stand was established in December 2011 with spacing 5 x 5 m of 5 hectares using genetic material from selected population Gunung Kidul, Yogyakarta with the highest CCO Crude Calophyllum Oil content 50.00 –50.12 among six nyamplung populations provenance land race in Java. Wonogiri research station is located in Wonogiri district under the province of Central Java with vertisol soil type and loamy soil texture, slope 2 –3, pH 5.5–6.5, altitude 141 m asl, rainfall 1,878 mmyear with 6 –7 dry months and temperature 20–38 o C. Biofuel analyses was undertaken using nyamplung seeds collected from trees in the first fruit season on provenance seed stand. The seed pressing process and analyses of nyamplung biofuel were carried out at Bioenergy Laboratory, Centre for Forest Biotechnology and Tree Improvement CFBTI, Yogyakarta. Materials for biofuel analyses included lint, H3PO4, aquadest, methanol, NaOH, H2SO4. Tools and equipments used for carrying out the research included analytical balance, wooden hammer, fruit and seed cracker, grinder, screw press expeller, funnel, separating funnel, erlenmeyer, measuring glass, heater and magnetic stirrer. 2.2 Method Height and diameter breast height dbh growth were measured annually in the permanent plots until the trees were 3 years old, while tree branching was assessed using a subjective score of 1 –3 1 = poor branching, 2 = average branching, 3 = good branching. The Bogor, 21-22 October 2015 173 observation of flower and fruit were conducted when the trees flowering and fruiting. In the provenance seed stand, the first fruit season in the third year was observed and this were conducted every two weeks from January to December 2014. Biofuel analyses was conducted with the following stages: 1 Seed pressing using screw press expeller after sun-dried for three days to obtain dry seed weight with water content of 8 –12. 2 Dried seeds were transferred into screw press expeller to release the dark green raw oil. The raw oil was then set for 1 full day before squeezing to separate its residu and then it contained into mesuring glass as Crude Oil CCO. 3 Degumming is a process of releasing CCO gum. The method was used by diluting 1 of phosphoric acid H 3 PO 4 into CCO, followed with heating up at 6 C and 30 minute stirring. After heating, CCO was set for 4 –6 hours, so that the gum will be separated down at the bottom while the degumming oil will set on the top. The oil was taken as Refined Oil Refined Crude Calophyllum Oil RCCO. 4 Estherification is a reaction process of changing Free Fatty AcidFFA into FAME Fatty Acid Methyl Eter compounds, a generic name of biodiesel. In this estherification process, oil contented from degumming RCCO process was reacted with 20 of methanol by using 2 of H 2 SO 4 catalist. The sulfic acid H 2 SO 4 was mixed into methanol and stirred to perfectly dissolve before pouring it into RCCO. It was then heated up to 60 –65 C with 2 hour stirring. In this estherification process, the oil will release its acid grease that stays at the bottom separated from ester oil at the top. 5 Transestherification is a reaction to change triglyceride into FAME compounds. In this process, esther oil was reacted to 20 methanol by using 1 NaOH catalyst. NaOH was mixed into the methanol and it needs to be perfectly dissolved before pouring it into Esther oil. It was then heated up to 60 –65 C for 2 hours. Tranestherification content was set to make separation between oil crude biodiesel and glyserol. 6 In producing biodiesel, crude biodiesel transestherification content needs to be washed and dried. Washing was carried out by mixing 30 warm water at 50 C into Crude biodiesel, before 3 minute stirring. It was then set for 1 hour to separate water, at the bottom and wet biodiesel, at the top. Washing was conducted until the water is clear. Wet biodiesel that posses high water content looks blurry, therefore for producing clear biodiesel, it needs heating up to 90 –100 C to make water evaporate. 2.3 Data analyses Growth traits height and diameter were performed as average and ranges for each measurements, while flowering and fruiting were calculated as percentage of the trees when flower bloom and fruit ripe, periodically. Biofuel analyses of nyamplung was carried out to verify the content of the CCO, RCCO, and biodiesel from provenance seed stand. Formula used to calculate those three parameters are as follow: Weight of Biofuel extraction Biofuel content = × 100 Seed weight Where: Biofuel content = content of CCORCCOBiodiesel Weight of Biofuel extraction = weight of CCO RCCO Biodiesel kg Seed weight = weight of dry nyamplung seeds kg Bogor, 21-22 October 2015 174 3. RESULT AND DISCUSSION 3.1 Growth