22 MATERIALS AND METHODS Location and Time This work was carried out in the Laboratory of Plant Biotechnology and Tissue Culture, Faculty of Agriculture, Bogor Agricultural University. It was started from August 2010 until September 2011. Plant Materials and Equipments Three kinds of plant materials have been collected to follow this study. The starting materials for callus induction include immature female flower, immature leaves and mature zygotic embryos isolated from Tenera variety donor palm. The selected trees were about 9 and 13 years old. The donor palm was selected according to some phenotypical characters such as fresh bunches borne, height, vigor, and health conditions. The vegetative material samples have been collected from the IPB Experimental Station of Cikabayan oil palm plantation. The three types of explants used in this study are shown in Figure 6. A B C Figure 6. The type of oil palm explants used in these experiments. A. Leaf explant, B. Zygotic embryos explant, and C. Female flower explant Media Preparation Two modified basal nutrient medium, Murashige and Skoog (1962) medium and Eeuwens and Blake (1976) medium, containing macro and micro salts (Table 2 & 3), were used for callus induction and embryo proliferation. These medium were supplemented with NAA, glutamine, asparagine, 2,4-D and Picloram.
23 Table 2. Murashige and Skoog (MS) medium composition used in these experiments Stock Compound Stock solution concentration (g/l) Pipet volume (ml/l media) Media concentration (mg/l) A NH4NO3 82.500 20 1 650 B KNO 3 95.000 20 1 900 C KH 2 PO 4 34.000 5 170 H 3 BO 3 1.240 6.2 Na 2 MoO 4.2H 2 O 0.050 0.250 CoCl 2.H 2 O 0.005 0.025 Kl 0.166 0.830 D CaCl 2.2H 2 O 88.000 5 440 E MgSO 4.7H 2 O 74.000 5 370 MnSO 4.4H 2 O 4.460 22.3 ZnSO 4.7H 2 O 1.720 8.6 CuSO 4.5H 2 O 0.005 0.025 F Na 2 EDTA.2H 2 O 3.730 10 37.3 FeSO 4.7H 2 O 2.780 27.8 VIT Thiamine 0.010 10 0.1 Niacin 0.050 0.5 Pyrotoxine 0.050 0.5 Glycin 0.200 2.0 Myo Myo inositol 10.000 10 100 Source: Murashige and Skoog (1962)
24 Table 3. Eeuwens and Blake medium (Y 3 ) composition Stock Compound Stock solution concentration (g/l) Pipet volume (ml/l media) Media concentration (mg/l) A NH 4 Cl KCl 26.750 74.600 20 535 1492 B KNO 3 40.400 20 2020 C NaH 2 PO 4.2H 2 O 55.200 5 276 H 3 BO 3 0.620 3.1 Na 2 MoO 4.2H 2 O 0.048 0.24 CoCl 2.6H 2 O 0.048 0.24 Kl 1.660 8.30 NiCl 2.6H 2 O 0.0048 0.024 D CaCl 2.2H 2 O 58.800 5 294 E MgSO 4.7H 2 O 49.400 5 247 MnSO 4.H 2 O 2.240 11.2 ZnSO 4.7H 2 O 1.440 7.2 CuSO 4.5H 2 O 0.050 0.25 F NaEDTA.2H 2 O 3.720 10 37.2 FeSO 4.7H 2 O 1.390 13.9 VIT Thiamine 0.050 10 0.5 Niacin 0.005 0.05 Pyrotoxine 0.005 0.05 CaP 0.005 0.05 Biotin 0.005 0.05 Myo Myo inositol 10.000 10 100 Source: Eeuwens and Blake (1976)
25 Sterilization Compounds Standard sterilizing agents such as alcohol (5%; 10% and 70%), Sodium hypochlorite (bleach) (5.25% NaClO), detergent,fungicide (Dithane M-45 2g/l, Agrept 2g/l) and antibiotic (Amoxicillin 1g/l), aquades steril were used in these experiments. Laboratory Equipments and Tools The equipments used in these experiments are pipet filler 1 ml and 10 ml, autoclave 2 l, wrapping plastic, glass bottles (baby food jar) 250 ml and 270 ml, magnetic stirrer, magnetic shaker, digital analytical balance scale, bunsen burners, erlenmeyer volumetric flask 250 ml; 500 ml and 1000 ml, graduate cylinder 100 and 500ml, hot plate, laminar air flow cabinet, spatula, pincet, sterilized blades, scalpel (knife), forceps, ph meter, petri dish, thermometer, pot holder, refrigerator, gas ring, labeler, cup holder 1000 and 2000 ml, towel, paper, sprayer, disposable mask, disposable hand gloves Methods Three independent experiments were applied in this study related to the three kinds of explant sources. Experiment 1. Callus and Somatic Embryos Induction from Young Leaf Explant Oil Palm Tree Sampling and Explant Processing The donor palm was a commercial Tenera variety with 9 years old. After the donor palm was identified, the spear was carefully removed by using a curved knife and with the help of a specially designed protocol. The procedure should ensure that the donor palm and the candidate spear are not damaged during the isolation process. As soon as the spear was isolated from the palm, it was sprayed with 96% alcohol, wrapped and placed inside a sampling bag for protection. The protected spear was then taken immediately to the laboratory for processing (Figure 7A.). The spear is a cluster of unopened young leaves or fronds which are different in terms of their age (Figure7B). Regarding their age, the leaves were carefully removed from the spear in laminar air flow cabinet of the dissecting or plant preparation room and grouped per cluster (Figure 7D). The leaves were then soaked in a 10 % NaOCl (household bleach) solution for 10 minutes of sterilization and rinsed three times in sterile distilled water. Each cluster of leaves was then soaked in 10% sterile sucrose solution. Leaves were sliced into 1 cm length and inoculated onto callus induction media using four explants per vessel. From one spear, around 3600 pieces of explants were extracted.
26 A B C Figure 7. Young leaves sample started material (spear) preparation process from the field to laboratory. A. Isolated spear, B. Young leaves clusters in the spear, C. Isolated cluster of young leaves, and D. Soaked Young leaves into sucrose solution after sterilization. D Callogenesis Mixing Medium (Combination) Ten treatments have been applied C1, C2, C3, C4, C5, C6 for Murashige and Skoog (1962) medium and four others M1, M2, M3, M4, for Eeuwens and Blake (1976) medium (Y 3 ). The tested medium compositions were as follow: C1 = MS + 0 µm 2,4-D + 107.41µM NAA C2 = MS + 22.62 µm 2,4-D + 107.41µM NAA C3 = MS + 45.24 µm 2,4-D + 107.41µM NAA C4 = MS + 67.86 µm 2,4- D + 107.41µM NAA C5 = MS + 450 µm picloram C6 = MS + 450 µm 2,4-D M1 = Y3 + 0 µm 2,4-D + 107.41µM NAA M2 = Y3 + 22.62 µm 2,4-D + 107.41µM NAA M3 = Y3 + 45.24 µm 2,4-D + 107.41µM NAA M4 = Y3 + 67.86 µm 2,4 D + 107.41µM NAA The ph of nutrient media (C1, C2, C3, C4, M1, M2, M3, and M4) was adjusted to 5.7 with 1N NaOH or HCl before the purified agar was added and autoclaved. For each media, 5% of commercial sugar and 0.9 % of purified agar to make solid medium were used.
27 According to C5 and C6 media, 3% of commercial sugar was used while the ph was adjusted to 5.8 before adding 0.3g/l charcoal and 0.9% of purified agar. The Media were dispensed into culture jar (250 ml) and covered by using heat resistant plastic. Then media were autoclaved for 20 minutes at 121 o C, 0.1 bar (17.5 psi). The experiment was designed into factorial randomized complete block design with 3 replications. Each treatment was applied into 30 jars so that a total of 960 explants have been used per replication. The first factor is the rank of leaf with 5 levels (L-5, L-6, L-7, L-8, L-9) and the second factor is the concentration of the tested growth hormone with 10 levels (C1, C2, C3, C4, C5, C6, M1, M2, M3, M4). However another factor as the type of basal media solution with two levels (MS media and Eeuwens media) was also taken in account. Environmental Culture Conditions Cultures were incubated under darkness to induce callus in a temperature-controlled room at 28 ± 1 o C. Observations were done every week for the percentage of contamination and every four weeks for the percentage of explant bearing callus, the percentage of embryogenic callus, and the percentage of embryoid. Qualitative variables observing explants color (browning), the color of callus, and the callus structures (nodular, root-like, and embryogenic) were recorded. As soon as calluses were initiated, they were transferred in the new same media composition. The subculture of the initial explant for callus initiation was done every 12 week intervals, while the initiated callus was subcultured every 8 week intervals. Culture Conditions of Embryos The embryoid stage marks the beginning of the light phase of the oil palm tissue culture process. The embryogenic cells and embryoid cultures were transferred from continuous dark culture to continuous light culture room for incubation with more light intensity of 1000 lux at 29 ± 1 o C. The number of embryo cells was recorded on the input sheet. Embryogenesis Mixing Medium Calluses are undifferentiated tissues which have the ability to give rise to unspecialized cells (embryogenic calluses) that are meristematic and can produce embryoids when good conditions are available. As soon as embryogenic calluses or embryoids were produced they were transferred to embryoid media for their proliferation or multiplication and maturation. This media was consisted of the same basal medium composition but with 25% of
28 gradually reduced concentration of 2,4-D and NAA from the 3 rd to 4 th subcultures. Each subculture was done after 6 week intervals. Subculture s Flow Chart of all the three Experiments Cultured explants Subculture every 3 months in dark Induced callus Embryogenic callus or embryoids Subculture every 2 months in dark Subculture every 45 days in light and 25% of gradually reduction of PGR after 2 subcultures in the same media Experiment 2. Induction of Somatic Embryo from Mature Zygotic Embryo Explant Explant Sampling and Sterilization Process Oil palm fresh mature fruit bunches were cut from 9 and 13 years old palms of Tenera variety in the farm. A total of 3600 fruits were isolated from bunches (Figure 8A) in the tissue culture laboratory and all their mesocarp was also removed (Figure 8B). 1800 seeds were treated for seed production process. The endocarp and endosperm of the obtained seeds from seed production process were then cracked and zygotic embryos were isolated for sterilization. The endocarp of the other 1800 fresh seeds was also cracked so that the kernel of seeds was isolated (Figure 8C) and stored in closed container to be sterilized. For both seeds, the sterilization have been done by mixing 4 g/l of fungicide (2g/l dithane and 2g/l benlate) for 1 hour; then soaked into 1 g/l of antibiotic for 1 hour, and 10 % commercial bleach for 20 minutes. In the laminar, isolated endosperm was then cracked to expose the fresh zygotic embryo (Figure 8D), so that these embryos were carefully isolated and placed into petridishes for sterilization by using 5 % commercial bleach for 5 minutes. The sterilized zygotic embryos have been cultured into treatment media for 10 explants per vessel (Figure. 8E).
29 A B C D E Figure 8. Mature zygotic embryo explants preparation process obtained from the laboratory. A. Isolated oil palm fresh fruits, B. Isolated seeds, C. Isolated kernels, D. Cracked endosperm seeds with zygotic embryo, and E. Isolated and cultured zygotic embryos Callogenesis Mixing Medium (Combination) Ten medium composition have been formulated as in the first experiment C1, C2, C3, C4, C5, C6 for Murashige and Skoog (1962) medium and four others M1, M2, M3, M4, for Eeuwens and Blake (1976) medium (Y 3 ). The experiment was designed into factorial randomized complete block design with 3 replications. Each treatment was applied into 12 jars so that a total of 1200 explants were used per replication. The first factor is the type of zygotic embryo explants with 3 levels (young embryo, mature embryo and embryo from seed production process) and the second factor is the concentration of the tested growth hormone with 10 levels (C1, C2, C3, C4, C5, C6, M1, M2, M3, M4). However another factor as the type of basal media solution with two levels (MS media and Eeuwens media) was also taken in account. Environmental Culture Conditions As in the first experiment, the same environmental culture conditions were also applied for callus induction and embryogenic callus obtained from mature zygotic embryo explant in this experiment.
30 Embyogenesis Mixing Medium Embryogenic calluses were transferred to embryogenic callus media for their proliferation and maturation. This media was consisted of the same basal medium composition with 25% of gradually reduced concentration of 2,4-D and NAA from the 3 rd to 4 th subcultures. Each subculture was done after 6 week intervals. Experiment 3. Induction of Somatic Embryo from Immature Female Flower Explant Explant Sampling and Sterilization Process Isolated immature female inflorescences from 9 and 13 years old oil palm trees of commercial Tenera variety were used as explant source. As soon as these inflorescences were cut, they were sprayed by using 96% alcohol and carefully plasticized (Figure 9A) in the field then brought to the laboratory. In the plant preparation room, the inflorescence spathes were carefully removed aseptically. Spikelets were isolated from inflorescence (Figure 9B) and sterilized by 10% of commercial bleach for 30 minutes. Immature flowers were isolated from spikelets and sterilized using 70 % alcohol for 10 minutes then soak into 10 % of commercial bleach for 20 minutes and excised. From these inflorescences 3 600 pieces of flower explants (Figure 9C) were extracted and sterilized using 5 % of commercial bleach for 5 minutes then cultured onto callus initiation medium. The entire, half and quarters sliced length flowers were used as explants. Firstly eight explants were inoculated into medium per vessel then after one month, the number of four explants was applied per bottles media. A B C Figure 9. Immature female flower explant preparation process in the laboratory. A. Isolated female inflorescence from selected donor palm, B. Isolated spikelets, and C. Isolated female flower explants
31 Callogenesis Mixing Medium (Combination) As in the experiments one and two, the same ten medium composition were formulated for this experiment. There are C1, C2, C3, C4, C5, C6 for Murashige and Skoog (1962) medium and four others M1, M2, M3, M4, for Eeuwens and Blake (1976) medium. The treatments have been arranged into factorial randomized completely block design with 3 replications. A total of 1200 explants have been used per replication. The first factor is the age of the oil palm explant (9 and 13 years old), and the second factor is the concentration of the tested growth hormone with 10 levels (C1, C2, C3, C4, C5, C6, M1, M2, M3, M4). However another factor as the type of basal media solution with two levels (MS media and Eeuwens media) was also taken in account. Environmental Culture Conditions The same environmental culture conditions and observations were also used for this experiment as in the first and second experiment, to induce callus and embryogenic callus. Data Analysis The obtained data was analyzed using Excel software and ANOVA. The means were separated with Student-Newman-Keuls tests at the 0.01 and 0.05 level of probability, respectively. The F-test was used to show significant differences among means.