BIOTROPIA Vol. 28 No. 1,2021: 21 - 28 DOI: 10.1 1598/btb.2021.28.1.903 

GROWTH A N D  DEVELOPMENT OF OIL PALM SHOOTS 
UNDER DIFFERENT LIGHT QUALITIES 

HELENA PATRICIA MANOH','*, YANTIl A N D  NURITA TORUAN-MATHIUS' 

'Faculiy ofBiotechnology, AtmaJgya Catholic Universiiy ofIndonesia, Jakarta 12930, Indonesia 
'Plant Production and Biotechnology Divisian, Sn/LART Biotecbnology Center, PT SMART Tbk., Bogor 16810, Indonesia 

Received 22 September 2017/Accepted 19 December 2019 

ABSTRACT 

Light quality is one important factor that affects the growth and development of in vitro plants. This study 
examines the influence of different light qualities on the in vitro growth and development of oil palm shoots 
which were cultured in Murashige & Skoog medium under white fluorescent lamp, white light-emitting diode 
(LED), red LED, blue LED, combination of red and blue LED, and in darkness. The results showed that the oil 
palm shoots grew and developed differently under different light qualities. Root initiation and shoot elongation 
progressed well under red light, while chlorophyll and sugar content were better produced under white and blue 
light than under the red light. Both white fluorescent lamp and the combination of red and blue LED resulted in 
higher growth parameter compared to other light qualities. However, the results were not significantly different. 

Keywords: chlorophyll, Elaeisguineensis, hormone, in vitro, LED light 

INTRODUCTION 

In the production of oil palm (Elaeis 
gzlineensis) clones, the use of tissue culture 
through somatic embryogenesis is widely 
applied. However, the efficiency of tissue culture 
in oil palm is very low (Rohani et al. 2000; 
Icushairi et al. 2010). Hence, improvement on 
the efficiency of this technology on oil palm is 
important for its mass production. 

Plant growth and development in tissue 
culture are regulated by various environmental 
factors, wherein light is one of the most 
important. Undoubtedly, light is necessary for 
photosynthesis and photomorphogenesis, 
with certain specific wavelengths playing 
important roles in plant tissue culture efficiency 
(Fujiwara & I<ozai 1995). 

The lighting system generally used for 
maintaining plant tissue culture is the tubular 
fluorescent lamps (TFL). However, TFL has a 
wide spectrum and contain unnecessary 

wavelengths for plant growth. The light-emitting 
diode (LED), an alternative light source for 
plant tissue culture, has several advantages in 
comparison to other lighting systems. LED has 
wavelength specificity, durabhty, longer 
operating lifetime, has the ability to select 
spectral composition, has a smaller size, and 
emits less heat (Gupta & Jatothu 2013). The 
disadvantage of LED is the establishment cost. 
However, LED can still be profitable in the long 
run, even though it may require a high capital 
investment, because the long-term operating 
costs of LED are generally lower than other 
lighting systems. 

The LED lighting system using various light 
qualities in plant tissue culture has been applied 
to a variety of species, such as Cymbidizm 
(Tanaka et al. 1998), strawberry (Nhut e t  aL 
2003), grape (Poudel et al. 2008), Antbam'zm 
(Bucharto 2010), Gosgpizlm birsatam (Li et a/. 
2010), Onn'diam (Mengxl et al. 201 I), Brassica 
napas (Li et aL 2013), banana (Vieira et al. 2015), 

'Corresponding author, email: biotechnology@sinarmas- Vanilla planfoolid (Belle-Bell0 et al. 201 6 ) ,  and 
agri.com sugarcane (Ferreira et aL 2017). The effect of 



BIOTROPIA Vol. 28 No. 1,2021 

light quality varied depending on the plant 
species yet little is known about LED utilization 
with different light qualities in oil palm shoot. 
Hence, the influence of various light qualities on 
in vitro growth and development of oil palm 
shoot was evaluated in this study. 

MATERIALS AND METHODS 

Plant Materials and Light Quality 
Treatments 

The i n  v h o  oil palm shoots used in t h s  study 
were obtained through indirect somatic 
embryogenesis from young leaf explants 
following the Wong e t  aL (1997) method. The 
different lights used were white TFL (Phillips T5 
TCH086 EV 28W, white 6500 I<), white LED 
(Vanq VQ-GLT8020W-27, white 10000 I(), red 
LED (Vanq VQ-GLT8020W-27, wavelength: 
660 nm), blue LED (Vanq VQ-GLT8020W-27, 
wavelength: 460 nrn) and red blue LED (Vanq 
VQ-GLT8020W-27, wavelength: 660:460 nm 
(3:l)). 

Effect of Light Quality on Shoot 
Development 

The 4-6 cm shoots, which have more than 
two leaves were selected and transferred onto a 
modified Murashige & Skoog (1962) medium 
without plant growth regulator. All cultures were 
maintained in a culture room at a temperature of 
28 + 2 "C, relative humidity 50 f 10°/o, and with 
16/8 light/dark photoperiod under various light 
qualities. After two months of culture, the fresh 
weight, the number of new leaves, the shoot 
height, and its root formation were observed, 
and then whole shoot samples from each 
treatment were randomly collected for 
chlorophyll, sugar, and endogenous hormone 
analyses. The whole shoots were ground in 
liquid nitrogen and stored at -80 "C prior to 
analyses. 

Chlorophyll Content Analysis 

for chlorophyll a and at 645 nm for 
chlorophyll b. The chlorophyll content was 
calculated according to Lichtenthaler (1 987). 

Sugar Content Analysis 

The sugar content was measured, according 
to the method described by Dubois e t  al. (1951). 
The 2 g sample was homogenized in 10 mL of 
distilled water and kept in water bath at 95 "C 
for 5 min. The sample was then stored overnight 
at 70 "C. Subsequently, the sample was 
centrifuged at 5,000 rpm for 10 rnin and 2 mL 
of the supernatant was transferred into another 
tube then added with 0.02 mL of 80% phenol 
and 5 mL of sulfuric acid. The mixture was then 
incubated at 30 "C for 30 min. The optical 
density was measured using a 
spectrophotometer (BioTek Epoch microplate) 
at 490 nm. Fructose and glucose were used as 
the standard. 

Endogenous Hormone Content Analysis 

The endogenous hormone content was 
measured, according to I<elen e t  aL (2004), with 
some modifications using Ultra Performance 
Liquid Chromatography (YUater UPLC). 

Auxin, gibberellic acid (GA), and abscisic 
acid (ABA) were extracted from 5 g of sample 
and soaked overnight in 30 mL 80% methanol 
at 4 "C. The 5 g extracted sample was dissolved 
with 30 mL 0.1 M phosphate buffer (pH 8.5) 
and then partitioned with ethyl acetate two 
times. After removal of the ethyl acetate, the pH 
of the aqueous phase was adjusted to 2.5 with 1 
N HC1. The sample was partitioned again with 
ethyl acetate 2 times and passed through 
anhydrous sodium sulphate. The ethyl acetate 
was evaporated in rotary evaporator at 50 "C. 
The dry residue containing hormones was 
dissolved in 2 mL of methanol and filtered into 
a UPLC vial using a 0.2 pm Millipore syringe 
filter. An injection volume of 1 pL was used for 
each analysis. The column used was Water 
Acquity UPLC BEH C18 1.7 pm (2.1 x 50 mm). 
The mobile phases constituted of acetonitrile: 
water (26:74) p H  4 with a flow rate of 0.2 
mL/min and retention time of 3 minutes. The 

The chlorophyll content was extracted from signal of the compounds was monitored by a 
the sample using 10 mL of 100% acetone. The photodiode array (PDA) detector at 208 nm. 
optical density was measured using a Indole Acetic Acid (IAA), GA3, and ABA (100 
spectrophotometer (Hitachi U-2900) at 662 nm ppm) were used as the standard compounds. 



Growth and development of oil palm shoots under different light qualities - Patricia Manoh e t  al. 

For cytokinin isolation, 1 g of sample was 
extracted using 20 rnL 70% methanol for 4 
hours at 4 "C. The extracted sample was filtered 
and injected into a UPLC column as previously 
mentioned. The mobile phases constituted of 
water: methanol (2030) pH 2 with a flow rate of 
0.35 mL/min and retention time of 0.6 minutes. 
The signal of the compounds was monitored by 
a PDA detector at 210 nm. Zeatin (100 ppm) 
was used as the standard compound. 

Statistical Analysis 

This experiment used randomized complete 
block design with three block and six 
treatments. Data were analyzed statistically using 
analysis of variance (ANOVA), and the 
differences among the treatment means were 
tested using Duncan's multiple range test 
(DMRT) at P = 0.05. Statistical analyses were 
conducted using SAS version 9.1.3. 

RESULTS AND DISCUSSION 

Shoot Growth and Development 

The growth and development of oil palm 
shoots were influenced by the light quality 
(Table 1). Compared with other treatments, the 
no light (darkness) treatment gave the lowest 
growth. When under darkness, the number of 
new leaves and fresh weight were significantly 
lower than those under light treatments, while 
the results among different light treatments did 
not significantly differ from each other. 

However, the highest shoot height and fresh 
weight were observed in shoots growing under 
the combination of red and blue light-emitted 
diode (LED). Meanwhile, the highest number 
of new leaves was observed in shoots under 
white tubular fluorescent lamp (TFL) (Table 1). 
White TFL is the most widely used light in plant 
tissue culture. However, whte TFL produces a 
wide range of wavelengths that contain 
unnecessary wavelengths for plant growth and 
development, whde LED can emit light at 
specific wavelengths (Gupta & Jatothu 201 3). 

Different wavelengths generally bring about 
different responses on plant growth and 
development. In this study, the red blue LED 
gave the greatest growth compared to other 
LED colors (Table 1 & Fig. 1). Red and blue 
lights remarkably impacted plant growth, 
probably because these are the major energy 
sources for photosynthesis and photomor- 
phogenesis. Red and blue lights, either alone or 
in combination produce different responses in 
plant growth. Red light induces shoot elongation 
(Nhut e t  al. 2003; Cybularz-Urban et al. 2007; 
Poudel et al. 2008), while blue light induces 
chlorophyll and stomata formation (Poudel et al. 
2008; Muneer et al. 2014). The combination of 
these two lights may have produced higher 
growth rate, due to the combined advantages of 
red and blue lights for inducing plant growth 
and development. The combination of red and 
blue lights also enhance the growth of other 
plant species, such as strawberry (Nhut et al. 
2003), cotton (L et al. 2010), ginseng (Nhut etal. 
2015), and vanilla (Bello-Bello et al. 201 6). 

Table 1 Effect of different light qualities on growth and development of oil palm shoots 

Treatment Number of new leaves Fresh weight increase (mg) Shoot height increase (cm) 

Dark 0.51 f 0.03 
White TFL 0.78 + 0.03 a 
White LED 0.69 + 0.03 a 
Red blue LED 0.76 + 0.03 a 
Blue LED 0.76 f 0.04 a 
Red LED 0.70 + 0.03 a 

Notes: Values are mean + SE for n = 330. Different superscripts on the same column indicate significant differences at 
P = 0.05 by DMRT. 



BIOTROPIA Vol. 28 No. 1,2021 

Figure 1 Representative of oil palm shoots under different light qualities 
Notes: D = dark W ?m = %bite Tm;, W LED = white 

LED; RB LED = combination red blue LED; 
B LED = blue LED; R LED = red LED;, 

Light Quality and Endogenous Hormones 

Light quality affects plant gzowth and 
development by regulating the synthesis of 
endogenous hormones: c y t o k h ~ ~  gibberellic 
acid (GA), auxin and abscisic acid (AM). 

The endogenous hormone in the oil palm 
shoot showed varied responses to different light 
qualities (Table 2). Cytokinitl, GA and ABA 
contents differed significantly from a c h  other 
when exposed under the different hght quality 
treatments, indicating their different photo- 
responses to certain wavelengths. The highest 
cytokinin content was observed under the white 
LED and lowest in the darkness. Gibberellic 
Acid (GA] content was higher when the shoots 
were cultured under red, blue, or red and blue 
LED when compared with white light. ABA 
content was the highest under red blue LED, 
and the lowest in dark vestment. Auxin did not 
respond to any of the light quality treatments. 
Althaugh the auxin content did not differ 
signifimdy among treatments, the highest was 
observed in red LED treatment. 

Interaction berween Endogenous 
Hormones and Shoot Growth 

Synthesis of hormone in plant is regulated by 
plant photoreceptors, which include phpchromes, 
~ t o & , ; o m e ,  and phototropin (de Wit ~t .ti 
20 1 6). The interaction between hght quality and 
endogenous hormone plays an important role in 
plant growth and development. 

GA and auxin are key players in plant 
elongation. The stem increment and GA 
content of Norway spruce seedlings under red 
light were higher than under blue light, whereas 
U A  content under red light were lower 
(Ouyang t.t al', 2015). In contrast, GA eontent in 
petunia plants were higher under bhe light 
compared to red light (Fukuda e t  8 2016). Blue 
light enhances shoot elongation and red light 
inhibites shoot elongation in petunia plants. In 
this current study8 GA and auxin contents in oil 
palm shoots under LED light were not 
significantly different, but GA content in shoots 
under blue LED was higher than those under 
other lights. The. resulting differences may be 
attributed to plant species1 stage of plant 
growth, and other environmental factors. 

Table 2 Endogenous hormones cootent in oil palm shoots under different light qudties 

Treatment C p k i n i n  (mg/@ GA @/g) Auxin @g/@ kg/$$ 
Dark 0.48 f 0.04 3.39 f 0'33 3.51 f 0.68 8.13 f 5.63e 
White TFL 0.61 f 0.04 2.50 + 0.32 b 3.83 f 0.55 35.81 f 10,41 " 
Wlhite LED 0.70 f 0.03 2.24 -t 0,35 b 4.59 +. Q.65 a 29.15 f 1034 * 
Red blue LED 0.64 f 0.02 ab 2.89 f 0.09 ah 4.37 1: 0.60 a 42.08 f 12.1 1 
Blue LED 0.64 k 0.06 ab 3.06 f 9.26 3.99 f 0.78 a 19.77 f 9.70 bc 
Red LED 0.67 f 0.03 " 2.67 f 0.29 *b 4.79 + 0 9 3  a 30.40 f 8.51 * 

Nates: Values are mean f SE f ~ r  pr = 6. Different letters in the same column indicate significant difference at P = 0.05 
by D m  



Growth and development of oil palm shoots under different light qualities - Patricia Manoh e t  al: 

Shoot elongation seems to positively 
correlate with the interaction of endogenous 
hormones. In this study, shoot elongation under 
red LED was higher than those under other 
lights (Table 1) and this might be correlated with 
the higher auxin content under red LED (Table 
2). GA and auxin are known to stimulate plant 
elongation through different mechanisms. Auxin 
affects cell elongation by promoting the release 
of hydrogen ions from the plant cell and whch 
in turn reduces the stress on the cell wall. GA 
requires cell wall plasticity and cytoplasmic 
protein synthesis for cell elongation. To achieve 
this, cooperation with other hormones, such as 
auxin is required p a n g  & Irving 201 1). 

Cytokinin has a different effect as it inhibits 
plant elongation by inhibiting auxin-promoted 
extension growth (Cohen e t  al. 1991). Cytohnin 
and blue light induced inhibitory effects on 
hypocotyl growth of cucumber (Cohen e t  al. 
1991). In this study, cytokinin content and shoot 
elongation under light treatments were not 
significantly different. T h s  results showed that 
cytokinins might not be correlated with the 
elongation of oil palm shoots. 

Auxin also plays a key role in root initiation. 
In this study, auxin content in the oil palm 
shoots increased under the red LED (Table 2) 
and it induced the root initiation in the shoots 
(Fig. 2). The process of root formation is 

considerably complex and involves interaction 
with other hormones. Auxin regulates root 
growth by modulating the effect of GA-induced 
destabilization of DELLA protein growth 
repressor (Fu & Harberd 2003). In the presence 
of GA, DELLA is degraded and thereby 
modulates other plant hormones, such as auxin. 

Light Quality and Photosynthesis 

Light quality also influences photosynthesis 
activity. In this study, the effect of light quality 
on photosynthesis was determined by the 
accumulation of photosynthetic pigments 
(chlorophyll a and b) and primary photo- 
synthesis products (fructose and glucose). 
Chlorophyll and sugar content of the oil palm 
shoots were significantly lugher under light 
treatment than those under dark treatment (Figs. 
3 & 4). Light is an important factor that 
regulates chlorophyll biosynthesis. 

The shoots under white, red blue, and blue 
light accumulated higher chlorophyll, compared 
to those under the red or dark treatment (Fig. 3). 
Several studies showed that blue light is a good 
light for chlorophyll synthesis and that red light 
decreases chlorophyll content (Tanaka e t  al. 
1998; Li e t  al. 2010). Simdarly, in this study, the 
chlorophyll content in oil palm shoots decreased 
under red LED, and increased under white and 
blue LED (Fig. 3). 

Dark White White Red Blue Blue Red LED 
TFL LED LED LED 

Figure 2 Percentage of rooting in shoots under different light qualities 
Notes: Vertical bars indicate k SE of the means for n = 6. Different letters indicate significant differences at 

P = 0.05 by DMRT. 



BIOTROPIA Vol. 28 No. 1,2021 

Blue light is also important in chlorophyll 
synthesis. Blue light stimulates chlorophyll 
synthesis by inducing the expression of genes 
encoding the enzyme for two early step of 
chlorophyll biosynthesis, glutamate-1 - 
semialdehyde aminotransferase and 5- 
aminolevulinic acid dehydratase (Matters & 
Beale 1995). Chlorophyll content were also 
correlated to cytolilnin content in oil palm 
shoots. Chlorophyll content under light 
treatments was significantly higher than that in 
dark treatment (Fig. 3) and t h s  may be 
stimulated by high cytokinin content under light 
treatments (Table 2). Cytokinin affected 
chlorophyll biosynthesis by promoting the 
synthesis of 5-aminolevulinic acid, a precursor 

of chlorophyll synthesis, and enhancing activity 
of Protochlorophyllide Oxidoreductase, an 
enzyme in chlorophyll synthesis (Cortleven & 
Schmulling 201 5). 

Sugar content in the oil palm shoots under 
different light treatments did not significantly 
&ffer (Fig. 4). Apparently, different light 
qualities did not affect sugar content because the 
in vitro shoots are mainly heterotrophic, where 
the shoots were grown in culture media with 
sugar. Sugar is the main carbon source for 
heterotrophic growth (Yasseen e t  aL 2013). The 
presence of sugar in culture media might reduce 
the need for sugar production and limits 
photosynthetic activity. 

mg/g Chlorophyll a R Chlorophyll b 

Dark White White Red Blue Blue Red LED 
TFL LED LED LED 

Figure 3 Chlorophyll content in oil palm shoots under different light qualities 
Notes: Vertical bars indicate k SE of the means for n = 6. Different letters indicate significant differences at 

P = 0.05 by DMRT. 

mg/g Fructose F Glucose 

Dark White White Red Blue Blue Red LED 
TFL LED LED LED 

Figure 4 Sugar content in oil palm shoots under different light qualities 
Notes: Vertical bars indicate ? SE of the means for n = 6. Different letters indicate significant differences at 

P = 0.05 by DMRT. 



Growth and development of oil palm shoots under different light qualities - Patricia Manoh et al. 

CONCLUSIONS 

Light quality influenced the growth and 
development of the oil palm shoots. The 
different wavelengths produced different effects 
on the root and shoot activities. Root initiation 
and shoot elongation were well-produced under 
red light, while chlorophyll and sugar contents 
were better produced under the white and blue 
light. Although the results were not significantly 
different, the combination of red and blue LED 
resulted in higher growth than the other light 
treatments. Besides the white light, the red and 
blue light combination provides good lighting 
for promoting the growth and development of 
oil palm shoots. Hence, the red blue LED may 
be used as an alternative for the whte TFL used 
in oil palm tissue culture. However, further 
study may be needed to analyze the ratio of red 
and blue LED in order to optimize the 
protocols for oil palm tissue culture. 

ACKNOWLEDGEMENTS 

This study was fully funded by the Plant 
Production and Biotechnology Division of PT 
SMART Tbk. The authors would like to express 
their great appreciation to the Division Head of 
Plant Production and Biotechnology Division 
for permission to publish this article. The 
authors would also like to thank all technicians 
in the tissue culture and biotechnology 
laboratory at SMART Biotechnology Center, 
who provided their assistances in this study. 

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