RESULT AND DISCUSSION 1 Bud culture
Bogor, 21-22 October 2015
806 at 22º C - 26º C, 70 humidity, in the dark condition. Medium were used for embryo
development and maturation MS medium Murashige et al. 1962 supplemented with different concentration of BAP benzyl-amino-purine : Bo = BAP 0 mgl; B1 = BAP 1 mgl;
B2 = BAP 2 mgl, B3 = BAP 3 mgl and 0,01 mgl NAA napthalene-acetic-acid, 40 gl Sucrose, and 2,5 gl Gelrite. The pH of all media is adjusted to 5.8 with 1N NaOH before
autoclaving. Embryo cultures are maintained in a growth chamber at 22º C - 26º C, 70 humidity, and 2000
– 3000 light intencity.
2.4 Design experiment The completely random design had only one independent variable or treatment. Therefore,
there was no other variable beside treatment influencing response of research dependent variable.Repetition consisted of 3 clones with different test sample. In rooting research,
factorial pattern was used, which was composed in completely randomized design consisting of 2 factors: first factor is type and concentration of base media with three levels: ½ MS, ½
GD, ½ WPM and the second factor is concentration of ZPT Kinetin with 5 level: 0 ppml, 0.25 ppml, 0.5 ppml, 0.75ppml and 1 ppml. Therefore, there were 3 x5 = 15 treatments,
with 20 samples in each treatment. Total sample was 300. Acclimatization research used factorial pattern composed in completely randomized design
consisting of 2 factors. First factor was planting media with 3 levels: M1 3 portions of volcanic ash; 1 portion of Purwobinangun topsoil in Pakem, Sleman; 1 portion of manure,
M2 3 portions of volcanic ash; 1 portion of Playen topsoil in Gunung Kidul; 1 portion of manure; M3 3 portions of volcanic ash, 1 portion of Kaliurang topsoil in Sleman; 1 portion
of manure. Second factor is primary parent species consisting of 3 levels: T1 calliandra; T2 purslane; and T3 chili. Therefore total treatment are3x3=9, with 3 repetition and 4 test
samples. Total sample were 9 x3x4 =108. Parameter measured in induction and multiplication stages are: observation of shoot length is measured from growing point to end of shoot at
centimeter cm unit and the number of shoot observation was done on shoot forming. Grown shoot is calculated their shoots. Parameter observed in rooting stage consisted of fixed
percentage and plantlet growth cm. Parameters observed in acclimatization consist of contamination percentage, mortality percentage, rooted plant percentage, and sandalwood
growth and rooting. Result of sandalwood tissue culture was analyzed using Varian analysis Anova at 5 and 1 level and Confidence Interval CI with Standard error and means in
multiplication stage. When result of variant analysis indicated significant difference, further test with Duncan Multiple range test DMRT was done at 5 test level.
3. RESULT AND DISCUSSION 3.1 Bud culture
Main objective of initiation is to make culture from explants that are free from microorganism and initiation of new growth. Axillary shoot culture is tissue culture using explants obtained
from plant organ of axillary end. Use of axillary shoot because the part is juvenile and part of tissue where its cells are still active dividing, so explants is expected to induce easier. Result of
induction indicated that of three clones indicted, clone A.III.4.14 gave highest response with 85 induction, while average rate was 74. Various responses in induction percentage is
cause due to variation on clone cultured. Then, response of media on induction is great, indicated that all clone can be induced using media Murashige and Skoog MS. Result of its
induction can be seen in Figure 1. Multiplication is activity of adding explants. It was done by changing shoot result of initiation
into media bottles containing ZPT to avoid contamination causing failure of explants growth.
Bogor, 21-22 October 2015
807 Bottles contained media planted with explants is put on culture shelf and placed in sterile
place at 24 C±2
C and 60-70 humidity and 2000-3000 lux illumination.
Figure 1: Shoot Induction of Santalum album on MS Medium
Figure 2: Multiple Shoots Regeneration from Internodal Explants of Santalum album
Figure 3: Root Induction of Santalum album Figure 4: Acclimatization of Santalum albumin Green house
Result of multiplication indicated that Clone A.III.4.14 from Seabela, Rote island, East Nusa Tenggara,gave best response where result of confidential interval CI obtain development of
highest shoot number 22.79±3.77 and highest shoot length growth 4.5± 0.35.
Bogor, 21-22 October 2015
808 Table 1: Shoot Multiplication Santalum album on MS Medium
Clone Provenans
Experimental unit
Mean number of
shoots Mean shoots
length cm Information
A.III.4.14 Seabela P. Rote
33 22.79 ±3.77
4.5 ±0.35 Genetic conservation area
2005 Group A WS6
Pailelang P. Alor 35
15±0,95 2±0,18
Genetic conservation area 20022003 Watusipat
WS28 Pailelang P. Alor
19 17,89±2,63
3±0,32 s.d.a
Rooting is phase in which explants will indicate root growth indicating tissue culture process done operated well. Observation was done every day to see growth and development of root
and to see contamination by bacteria or fungi. Contaminated explants will indicate symptom of white or blue caused by fungi or rotten odor bacteria. Objective of rooting is strong
shaping root and plantlet and plant end, so it can survive until moved from in-vitro environment to green house environment. After rooted for three months, from 15 treatments
combination studied give various responses on rooting induction. Highest response reached 60 on treatment ½ GD with addition of 1 mgl Kinetin.
Plant resulted from tissue culture is still difficult to keep according to green house condition because it is very sensitive to environment outside culture environment. Therefore, it requires
acclimatization to deal with the extreme condition, particularly transition from agar media to soil. Its objective is to have stronger and former rooting. Requirement of acclimatization are
cleanliness, high illumination, stable aeration, high and stable humidity and temperature, and acclimatization media having sufficient nutrition required by plant. After acclimated for three
months, of 108 plantlets acclimated, highest percentage was obtained from sandalwood seedling from acclimatization with treatment of chili host plant 67.
3.2 Somatic embryogenesis Somatic embryogenesis is a process somatic cells both haploid and diploid developed new
plants through specific stages of embryonic development without gamet fusion. The results of the study the effect of various clones and concentration of plant growth regulator PGR 2,4-
D is used to sandalwood callus induction after incubation for 8 weeks are presented in Table 1.
Media culture is one of the important factors of plant propagation in vitro culture. In this study medium were used Murashige and Skoog, 1962 because MS is a privilege of the media
content of nitrate, ammonium and potassium high, it contains a decent amount of inorganic nutrients to meet the needs of different types of plant cells in culture Wetter dan Constabel,
1982. In somatic embryo proliferation phase showed that the use of MS medium containing PGR 2,4-D 1- 5 mgl 2,4-D + 0,15 mgl kinetin provide good response, this is accordant
with research conducted by Revathy and Arumugam, 2011
described that high number direct somatic embryo proliferation from leaves explant was observed in MS medium containing 2,4-
D 3 mgl. When explants calluses with embryogenic structures were transferred to medium supplemented with different concentrations of BAP, they all survived.
Bogor, 21-22 October 2015
809 Figure 5: Embryo development maturation of Sandalwood
Figure 6: Sandalwood plantlet via somatic embryogenesis Table 2: The effect of clones and concentration 2,4-D to sandalwood callus Induction after
incubation for 8 weeks
Clones Percentage callus induction
Average embryo induction
CND1 CND3
CND5 A.II.4.5
75 75
Average Embryo induction 60
40 20
40 A.II.2.3
15 Average Embryo induction
20 6,7
A.IV.Ii.12 100
Average Embryo induction 20
40 20
B.VI.7.14 10
25 Average Embryo induction
T.A.12 5
5 Average Embryo induction
20 20
20 20
A.II.4.16 Average Embryo induction
60 60
40 B.VI.Ii.12
50 Average Embryo induction
20 40
30 TA.1
100 80
Average Embryo induction 60
40 60
54 K
60 70
50 Average Embryo induction
80 80
20 60
Average Embryo induction 30
Torpedo Plantlet
Stem Root
Leave
Bogor, 21-22 October 2015
810 However, the concentrations of BAP had a significant P0.05 effect on the percentage of
matured somatic embryos and number of matured embryos per explant Table 2. The basal medium supplemented with 1.2 mgl BAP had the highest percent 37.4 of matured
primary somatic embryos; however, the medium that contained 1.0 mgl recorded the highest number of matured embryos per explant. The embryos of media that were supplemented with
0.2-0.8 mgl BAP emerged from the base of explants and had yellow coloration, while the embryos in media with 1.0-1.6 mgl BAP were located on the top of the explant and were
whitish-yellow in color.