Micro-pR0PAgation of ACACIA
hybrid WK 004 AH Using Nodal
Explants
Budiyansah1,
Freddy Pangaribuan2, Sugito2,
Abriantomi3, J Sufitra4
1Tree Improvement PT
Wirakarya Sakti, SMF Group- Jambi
2Tissue Culture Laboratory
Unit- PT Wirakarya Sakti, SMF Group- Jambi
3Seed Orchard Management
Unit- PT Wirakarya Sakti, SMF Group- Jambi
4Tree Breeding Unit-PT
Wirakarya Sakti, SMF Group- Jambi
Email for corresponding
author : fpangari@gmail.com
Summary
Micro propagation protocols of Acacia hybrid been achieved
from nodal explants of adults trees (12
years old trees). Explants sterilization
technique using NaDCC 5000 ppm and Mercury Chloride (0.1%) is effective to control contamination from
explants and 63-70% sterile explants can be achieved . Using medium medium E6 ( Macro E6 + IAA 0.2
+ BAP 0.5) for shoot initiation gives good result in terms of number of shoot
response. As for multiplication, using
medium MS with BAP 1.0 gives good shoot growth and multiplication rate of 2.1.
Rooting rate using medium ½ MS+NAA 2 + IBA 1 gives 68% rooted shoot.
Keywords : Acacia hybrid, explants, sterilization,
contamination, multiplication, rooting
rate, shoot
INTRODUCTION
Acacia hybrid is a cross between Acacia mangium and Acacia auriculiformis which are
the two tropical acacias natural to Australia, Papua New Guinea and
Indonesia where both have the potential for timber and pulp production. A.
mangium, being one of the selected fast-growing species has become an
important choice of species in agro forestry, while A. auriculiformis has become a major source of firewood, its dense
wood and high energy (calorific value of 4 500 to 4 900 kcal/kg) contributes to
its popularity. It provides very good
charcoal that glows well with little smoke and does not spark (Asmah, H.N.
et.al, 2012)
Acacia
mangium Willd x A. auriculiformis A. Cunn. Ex Benth hybrids
possesses some of the outstanding intermediate characteristics of its parents
such as better stem form and longer clear bole height than A. auriculiformis and lighter branching, circular trunk, smoother
bark with whiter color and smaller phyllodes compared to A. mangium (Ahmad, D.H. and Ghani, R.A). Three seven years old tree of Acacia hybrid,
compared with both parents had longer fiber, and higher slenderness ratio,
fiber proportion and hellocellulose content (Yahya, R. et.al. 2010). In a study conducted in Sabah, Malaysia,
heart rot was reported to be present in five years old plantation of A magnum, but absent in similarly aged
plantation of A auriculiformis and A mangium hybrid (Ito and Nanis, 1994)
In 2012 naturally occurring acacia
hybrid Wk 004 Ah were selected as plus tree and sent to Tissue Culture Laboratory
for micro propagation and also sent leaf samples for testing of DNA
and result test confirmed its hybrid traits along with some other clones
(Sulistyono, 2013).
Due to increasing demand for
genetically superior plant materials by Sinarmas Forestry, micro propagation
studies have been carried out on A.
mangium x A. auriculiformis hybrid.
The main advantage of mass vegetative propagation compared to
propagation by seed is that higher yield planting materials with uniform
characteristics can be obtained within a minimum time period. In the case of A. mangium x A. auriculiformis hybrids, vegetative propagation
remains the only means of multiplication because of the very limited capacity
for producing interspecific hybrid seeds from bi-specific orchard. Beside, tissue culture technologies and
especially micro propagation are faster than horticultural vegetative
propagation methods to rejuvenate and mass multiply true to type adult selected
trees (Galiana et al., 2003; Beck et.al., )
Table 1. WKS –Jambi Acacia Hybrid Verification Using
Molecular Markers (Sulitstyono, 2012)
|
No
|
Sample ID
|
Source/site
|
Scar Marker-M33
|
Scar Marker-R01
|
Scar Marker-Ac27
|
|
1
|
Wk 004 Ah
|
Rawasari 1
|
A hybrid
|
A hybrid
|
A hybrid
|
|
2
|
Wk 007 Ah
|
Rawasari 4
|
A hybrid
|
A hybrid
|
A hybrid
|
|
3
|
Wk 017 Ah
|
Rantau kapas 6
|
A hybrid
|
A hybrid
|
A hybrid
|
|
4
|
Wk 062 Ah
|
Ture 17
|
A hybrid
|
A hybrid
|
A hybrid
|
|
5
|
Wk 056 Ah
|
Ture 11
|
A hybrid
|
A hybrid
|
A hybrid
|
|
6
|
Wk 060 Ah
|
Ture 15
|
A hybrid
|
A hybrid
|
A hybrid
|
|
7
|
Wk 061 Ah
|
Ture 16
|
A hybrid
|
A hybrid
|
A hybrid
|
|
8
|
Wk 063 Ah
|
Ture 17
|
A hybrid
|
A hybrid
|
A hybrid
|
|
9
|
Wk 068 Ah
|
Ture 23
|
A hybrid
|
A hybrid
|
A hybrid
|
|
10
|
Wk 103 Ah
|
Jambi 16
|
A hybrid
|
A hybrid
|
A hybrid
|
|
11
|
Wk 109 Ah
|
Jambi 22
|
A hybrid
|
A hybrid
|
A hybrid
|
|
12
|
Wk 115 Ah
|
Jambi 28
|
A hybrid
|
A hybrid
|
A hybrid
|
|
13
|
Wk 129 Ah
|
Jambi 42
|
A hybrid
|
A hybrid
|
A hybrid
|
|
14
|
Wk 130 Ah
|
Jambi 43
|
A hybrid
|
A hybrid
|
A hybrid
|
|
15
|
Wk 137 Ah
|
Jambi 50
|
A hybrid
|
A hybrid
|
A hybrid
|
|
16
|
Wk 002 Ah
|
Jl 170
|
A hybrid
|
A hybrid
|
A auriculiformis
|
|
17
|
Wk 003 Ah
|
Jl 170
|
A hybrid
|
A auriculiformis
|
A auriculiformis
|
|
18
|
Wk 009 Ah
|
Rawasari 6
|
A hybrid
|
A auriculiformis
|
A auriculiformis
|
|
19
|
Wk 048 Ah
|
Ture 4
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
|
20
|
Wk 049 Ah
|
Ture 5
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
|
21
|
Wk 055 Ah
|
Ture 11
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
|
22
|
Wk 064 Ah
|
Ture 12
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
|
23
|
Wk 065 Ah
|
Ture 13
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
|
24
|
Wk 069 Ah
|
Ture 17
|
A mangium
|
A auriculiformis
|
A auriculiformis
|
Figure 1. A
hybrid verification with scar marker SCAR-M33, SCAR-R)1, and SSR-Ac27
Objectives
Objectives of
this study are:
1. Micro
propagation of Acacia hybrid clone Wk 004 Ah and provides protocol for
mass propagation
2. To
study rooting properties of Clones Wk 004 Ah using several rooting medium
II METHODOLOGY
2.1. Place and Time
The Research was conducted in Tissue Culture Lab RDD PT Wirakarya Sakti
from March 2012 – March 2013.
2.2.
Material and Tools
Explants of Wk 004 Ah clone was
taken from PT Wirakarya Sakti R&D Nursery Centre in March 2012.
2.3 Design
2.3.1
Contamination Rate
Contamination rate only assessed in
sterilization stage by counting contaminated explants
2.3.2.
Multiplication Rate
Multiplication rate was assessed by
dividing the number of sub culturing result with number of bottles (original
bottles) using medium MS + BAP 1
2.3.3
Rooting Rate
Rooting rate was assessed by
counting the number of rooted shootlet
divided by number of shoot. Four rooting
medium were tested; control using medium B2RE + NAA 1 + IBA 0.5, R1;
1/2MS + NAA 2 + IBA 1, R2; 1/2MS + IBA
1, R3; 1/2MS + IBA 1.5
2.4. Measurement
Measurement was
conducted 20 days after sub culturing by
counting the clean explants left, number of
bottles after subculture and number of shoot rooted.
2.5. Procedures
Nodal segments of 3-5 cm was harvested from newly sprouted
branch of the stool plant (mother plant).
Leaves were trimmed and washed with detergent for 10 minutes. Explants were surface sterilized with
solution of Streptomycin sulfate 0.2% (w/v) and 0.2% (w/v) Fungicide (Dithane M
45) using shaker 250 rpm for 2 hours.
Explants rinsed using sterilized water five times and washed with NaDCC
5000 ppm for 20 minutes and rinsed with sterile water. Explants submerged in HgCl2 (0.1%)
for 3 minutes and rinsed with sterile water five times. Explants surfaced sterilized with NaDCC 5000
ppm for 20 minutes. All the aseptic manipulation was carried out in laminar
flow. Selection of medium for induction
medium using E6 (IAA 0.2 + BAP 0.5, Sucrose 30 gr/l and as gelling agent using
agar (5.5 gr/l). After inoculation culture were incubated in
cool white fluorescent light (1600-2000 lux) with 12-16 hours photoperiod at
temperature 25-32oC. The
explants were oriented in a vertical position.
The response of the explants was estimated on contamination rate.
Fig
2. WK 004 Eh explants after 2 weeks in
medium E6
Fig
3. Wk 004 Ah after 4 series of
subculture ( every 4 weeks) from induction stage (rejuvenation can be seen by
the leaf, which has phylloide)
III
RESULT AND DISCUSSION
3.1. Contamination Rate in Induction Stage
Contamination
control is one of the crucial step in clone development using micro
propagation. High contamination level
after sterilization procedures depends on several factors i.e; source and type
of explants, shoot harvesting time, chemicals being used and also the
sterilization protocols. Conditioning of
mother trees in the nursery with pest and disease control will significantly
reduced the number of species and colonies of microbe living in the plant
tissues.
Fig
4. Source/colonies of fungus and
bacterial contamination on plant tissue surfaces
Test of several shoot samples from
open area and stool plant shows that contamination rate from explants taken
from open area ranged from 63% to 100% are contaminated, while contamination
from stool plant ranged from 40% to 70%.
Most of the visible contamination comes from fungus, but bacteria also
visible longer after fungus contamination.
The fast growing fungus will soon occupies the medium within a week for
fast growing microbe, but slow growing microbe can be visible after one month
or may be new contamination from out side enter the medium accidently.
Our research also shows that fast
growing grows on the rich mineral and organic medium can be reduced using
systemic fungicide methyl thiophanate 100 ppm added prior to autoclaving and
without adverse effect on shoot initiation (Pangaribuan, unpublished
2012). Several research using Bavistin
as additives to medium within 10-100 ppm also being reported (Preethi et.al.
2011)
Table 2. Contamination of explants/shoot taken from
open standing trees (1) and from stool
plant (2) 4 months after cutting
|
No
|
Clone
|
Date
of Induction
|
No
of Explants
|
Contamination
Rate (%)
|
|
1
|
WK 004 ah
|
22/11/2011
|
27
|
63
|
|
2
|
Wk
004 Ah
|
6/3/2012
|
23
|
70
|
|
B
|
|
A
|
Figure 5. Shoot initiation, A. Clean explants amd
B. contaminated explants by fungus
(probably from the genus Aspergillus)
3.2.
Multiplication Rate and Rooting Rate
A high multiplication rate occurs
after the stage reach a propagules state, breaking down the propagules into
smaller unit multiply the number of shoot. Multiplication using medium MS + BAP 1 which
refer to Darus, 1989, on Acacia mangium which
shows BAP concentration 0.5 mg/l and 1.0 mg/l shows highest multiplication rate. In this experiment we use BAP 1.0 alone which
also shows good result on Acacia hybrid.
The variety of growth occurs between
clones as well as within clones in terms of emergence of phylloides. Phylloides is a s sign of juvenility, because
it is only developed during the first stage of germination. On the other hand some explants can not produce
phylloide immediately due to un known reason.
Table 3.
Multiplication Rate (MR) of Wk 004 Ah using medium MS + BAP 1
|
No
|
Subculture
|
Method
|
Original (vsl)
|
Result (vsl)
|
MR
|
|
1
|
12/9/2012
|
Break down propagules
|
36
|
109
|
3.0
|
|
2
|
27/9/2012
|
Break down propagules
|
25
|
25
|
1.0
|
|
3
|
11/12/2012
|
Break down propagules
|
32
|
75
|
2.3
|
|
4
|
13/12/2012
|
Break down propagules
|
22
|
50
|
2.3
|
|
5
|
30/1/2013
|
Break down propagules
|
23
|
50
|
2.2
|
|
6
|
31/1/2013
|
Break down propagules
|
35
|
86
|
2.4
|
|
7
|
1/2/2013
|
Break down propagules
|
53
|
93
|
1.7
|
|
8
|
2/2/2013
|
Break down propagules
|
41
|
50
|
1.2
|
|
9
|
7/3/2013
|
Break down propagules
|
22
|
73
|
3.3
|
|
10
|
21/4/2013
|
Break down propagules
|
37
|
60
|
1.6
|
|
|
Average
|
|
|
|
2.1
|
Rooting rate 44% can be achieved
using medium B2RE supplemented with NAA 1.0 and IBA 0.5.
Using NAA instead of IAA is preferred since IAA degraded with high
temperature (heat labile) while NAA more heat stable, further more, light
influences the physiological activity of IAA while synthetic auxin are not as
light sensitive (Klerk, D. 1999). The
rooting rate can be increased by selecting the quality shoot, or not to select
young shoot.
Table 3. Rooting rate of Wk 004 Ah 12 days after
planting in rooting medium
|
No
|
Treatment
|
Medium
|
Rooting Rate (%)
|
Average(%)
|
||||
|
1*
|
2
|
3
|
4
|
5
|
||||
|
1
|
Control
|
B2RE + NAA 1 +
IBA 0.5
|
40
|
30
|
60
|
50
|
40
|
44
|
|
2
|
R1
|
½ MS + NAA 2.0
+ IBA 1.0
|
60
|
60
|
60
|
60
|
100
|
68
|
|
3
|
R2
|
1/2MS + IBA 1.0
|
20
|
20
|
0
|
40
|
0
|
16
|
|
4
|
R3
|
½ MS + IBA 1.5
|
20
|
20
|
0
|
20
|
20
|
16
|
1One vessels consist of five shoot
Figure
6. Rooting result using R1 medium with ½
MS + NAA 2 + IBA 1.0
Figure
7. Rooting result using R2 medium with ½
MS + IBA 1.0
Figure
8. Rooting result using R2 medium with ½
MS + IBA 1.5
The
difference in root emergence was the result of the NAA presence in the medium,
since the medium contain NAA consistently shows better rooting compare to
medium without NAA. The
concentration of IBA in the medium (1.0 and 1.5 ppm) is probably sub optimum to
induce rooting in the early stage. Using
IAA also do not shows good result. The
poor result of rooting by IBA and IAA can be the result of sub optimum
concentration due to oxidation by plant tissues.
IV CONCLUSION
From the result, micropropagation of
Acacia hybrid can be accomplished using apical nodes as explants. Sterilization technique using NaDCC to
replace NaOCl can be done to produce sterile explants. Contamination during shoot initiation range
from 63-70%. Using medium MS + BAP 1 in
multiplication process gives good result, eith multiplication rate range from
1.0 to 3.3 times, with average 2.1, while rooting rate resulted from medium ½
MS + NAA 2+ IBA 1 gives best result with 68% rooted shoot.
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