Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Effects of Colchicine Treatments on Physiological
Characteristics of Secondary Somatic Embryos of Oil Palm in
vitro.
Sainiya Samala1 and Sompong Te-chato1*
1
Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University, Hat Yai,
Songkhla, 90112, Thailand
*
Corresponding author: Sompong Te-chato, email: [email protected], phone: +66-8160-99034
Introduction
Oil palm (Elaeis guineensis Jacq) is a valuable economically important source of vegetable oil,
the most traded vegetable oil in the international market, and is increasingly used in the food industry. At
present, plant micropropagation is applied for plant breeding in order to overcome some limitations of the
conventional breeding, and clonal propagation of oil palm through tissue culture is common (Rabechault
et al., 1968; Teixeira et al., 1995; Aberlenc-Bertossi, 1999; Te-chato, S.2002 ; Rajesh et al., 2003).The
method for improving plant quality and quantity by inducing polyploids is of great important. Polyploidy
often generates variants that may possess useful characteristics and also provide a wider germplasm base
for breeding studies (Thao et al., 2003). Ploidy induction has been carried out for a variety of reasons. In
citrus, tetraploid (2n=4X) parents were produced to create seedless triploids by crossing between 4X and
2X parents (Wu and Mooney, 2002). In the medicinal plants, Scutellaria (Gao et al., 2002) and Artemisia
(De Jesus-Gonzalez and Weathers, 2003), tetraploidy was aimed to increase the amounts of the secondary
metabolites, baicalin and artemisinin. In azalea (De Schepper et al., 2004) and pomegranate (Shao et al.,
2003) chromosome doubling has been used to obtain new ornamental characteristics.
Chromosome duplication using colchicine has long been used in plant breeding program.
Colchicine, a compound that effectively arrests mitosis at the anaphase stage, has been found to have a
significant effect on polyploid induction. In the most plants, artificial polyploidy is often accompanied by
increased cell size, leading to larger reproductive and vegetative organs (Adaniya and Shira, 2001). In
breeding programs, it is important to determine the ploidy level in a quick and simple way in various
stages of plant development. In many of plant species, there are correlation between ploidy level and
physiological characteristics such as chloroplast number in guard cell, size of stomata cell, stomata
density and pollen grain diameter (Omidbaigi et al., 2010). The aims of this study were to use
physiological markers for verification of diploids and tetraploids plant from cholcicine-treated SSEs of oil
palm.
Materials and Methods
Plant material
SSEs of oil palm induced from young leaves of elite clone on MS medium supplemented with 1-5
mg/l dicamba, 3% sucrose, 0.2 M sorbitol and 200 mg/l ascorbic acid were used. The pH of medium was
adjusted to 5.7 and the cultures were maintained at 26±4
illumination (Te-chato, 2002) and subcultured every 4 weeks for more than 5 years.
Colchicine treatment
SSEs induced from haustorium embryo (HE) on Murashige and Skoog (MS) medium
supplemented with 0.2 M sorbitol for 90 days were treated with 0, 0.05, 0.10, 0.15 and 0.20% colchicine
for 12, 24, and 48 hours. Then, they were transferred to culture on MS medium without plant growth
hormones for 30 days.
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Physiological Characteristics analysis
Putative chromosome-duplicated plants such as thick leaves and dark-green leaves were collected
after one month of treating to analyse physiological characteristics such as size of stomata, chloroplast
number in guard cells and chlorophyll content as the following methods.
Size, density of stomata and chloroplast number in guard cell
Three samples of leaf from each treatment were collected. Epidermal layer were peeled from
lower surface (abaxial side), placed on a glass slide and observed under light microscope (Olympus
LABOMED model CXR II 110-260 V) in order to study chloroplast numbers in guard cell. For
investigation of size and density of stomata scanning electron microscopy (SEM) was used. For SEM
examination, leaves were fixed in 0.2 M Na2HPO4 –NaH2PO4 buffered solution (PB; pH 7.8) and
immersed in 2.5% glutaraldehyde (C5H8O2) in PB for 2 hours. After washing thrice in PB, leaves were
dehydrated through a graded ethanol series to the critical-point drying, mounted on stubs, and sputtercoated with gold in an ion sputtering device (Spi module). Leavess were examined using a SEM (Quanta
400, FEI) at an accelerating voltage of 1.0 kV (Hiroshi et al., 2008).
Chlorophyll content
Leaf tissues at approximately 0.1 gram fresh weight were homogenized in 4 ml of 80% acetone
solution and the crude extract was filtered with filter paper (Whatman no.1), then transferred to a 10 ml
volumetric cylinder and made up to 10 ml with distilled water. The contents of chlorophylls a, b and total
chlorophyll in the extracts were determined using spectrophotometer [MAPADA (v-1200/uv-1100/uv1200)] at OD 645-663. The content of chlorophyll were calculated from the value recorded at those
corresponding wavelengths of maximum absorption: 663 nm for chlorophyll a and 645 nm for
chlorophyll b. All studies were made in three replications and average results were calculated.by the
formula described by Witham et al. (1986).
chlorophyll a
= [12.7 (D663) – 2.69 (D645) ] x
V
1000 x W
chlorophyll b
= [22.9 (D645) – 4.68 (D663) ] x
V
1000 x W
total chlorophyll = [20.2 (D663) + 8.02 (D645) ] x
D645 =
D663 =
V =
W =
V
1000 x W
absorpbance at 645 nanometer
absorpbance at 663 nanometer
total volume of chlorophyll
weight of leaves
Results and Discussion
Size, density of stomata and chloroplast number in guard cell
Treating with 0.2% colchicine for 24 hours had the largest size of stomata at 19.11 x 29.23 µm
(width x length) However, density of stomata were decreased with the increment concentration of
colchicine (Table 1, Figure 1). The number of chloroplasts was increased up to 19.40 chloroplasts/
stomata from plant treated with 0.2% colchicine for 24 hours. However, size of chloroplast was decreased
when the concentration of colchicine was increased (Table 1, Figure 2).
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Table 1.Size and density of stomata revealed by SEM and chloroplast numbering guard cell of putative
chromosome duplicated-plants.
Characteristics
Stomata length (µm)
Stomata diameter (µm)
Stomata density (mm2)
chloroplast number/ stomata
Control
25.82
9.75
18.90
13.50
Concentration of colchicine (%)
0.10% (12h)
0.20% (24h)
26.61
29.23
14.39
19.11
12.00
7.10
13.70
19.40
Figure 1. Stomatal size and density of putative chromosome-duplicated plants as revealed by
SEM. (a) control, (b) treated with 0.10 % colchicine for 12 h and (c) treated with 20%
colchicine for 24 h.
Figure 2. Number of chloroplast in guard cell of putative chromosome-duplicated plants.
(a)control, (b) treated with 0.10 % colchicine for 12 h and (c) treated with 0.20%
colchicine for 24 h.
chlorophyll Content
Putative chromosome-duplicated plants had higher content of chlorophyll a, b and total
chlorophyll than control treatment (Table 2).
Table 2.Chlorophyll content of putative chromosome-duplicated plants.
Chlorophyll content (mg/gFW)
Chlorophyll a
Chlorophyll b
Total chlorophyll
control
0.270
0.181
0.434
Concentration of colchicine (%)
0.10% (12 h)
0.20% (24h)
0.329
0.362
0.198
0.199
0.518
0.603
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Proceedings of the 7th IMT-GT UNINET and the 3rd International PSU-UNS Conferences on Bioscience
Analysis of chlorophyll content revealed that SSEs treated with 0.2% colchicine for 24 hours
gave the highest content of chlorophyll a, b and total chlorophyll which was 0.362, 0.199 and 0.603
(mg/gFW), respectively.
Size and density of stomata, chloroplast number per stomata and content of chlorophyll between
control and treated plants were clearly different. Similar results of those researchers were also reported
(Madon et al.,2005; Gu et al., 2005; Nigel et al., 2007; Heping et al., 2008; Wen et al.,.2009).This
methods are suitable, proved to be an effective way to identify the tetraploid plant, however, confirmation
of ploidy level of treated plants, flow cytometric analysis and chromosome counting were required.
(Omidbaigi et al.,2010).
Conclusion
Present study indicates that estimation of morphological changes, size of stomata, stomata
counting, examination of chloroplast number in guard cells and contents of chlorophyll a, b and total of
chlorophyll is an effective method in primary screening of tetraploid plants in polyploidisation breeding
program and it is recommended flow cytometry to be used for accurate identification of ploidy level in oil
palm.
Acknowledgements
The financial support was provided by Graduate School and Center of Excellence in Agricuitural
and Natural Resources Biotechnology, Oil Palm Agronomical Research Center, Faculty of Natural
Resources, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
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