Characterization of cassava starch and detection of starch synthase gene under different micro nutrient fertilizer levels by using scanning electronic microscopy (SEM) and Real Time PCR

Abstract

The objectives of this study were to evaluate vegetative growth and root storage of cassava applied by micro nutrient fertilizer, to compare root storage of yield treated by micro nutrient fertilizer as 0, 20, and 40 kg/ha, to evaluate the structure of cassava starch by using Scanning Electron Microscopy (SEM), and to identify the activity of gene starch synthase with Real Time PCR. There were three different dosages of micro nutrient fertilizer as 0, 20, and 40 kg/ha. Variables observe in this study were leaf number, leaf dry weight, storage root number, storage root wet weight, diameter of starch granule, visual starch granule by SEM, and gene starch synthase activity by Real Time PCR. The result showed that leaf number of cassava applied by high dosages of micro nutrient fertilizer (20 & 40 kg/ha) tended to be lower compared to those without micro nutrient fertilizer application. Yet, the application of high dosages of micro nutrient fertilizer, tended to produce relatively high leaf dry weight. Such condition could support the production of high storage root number and weight. Additionally, starch structure applied by high dosages of micro nutrient fertilizer seemed to be denser due probably to high starch granule diameter. The increase in storage root number and density of cassava applied by 20 kg micro nutrient fertilizer/ha was concomitant with high gene starch synthase activity, approximately as many as 200 times compared to those without micro nutrient fertilizer application.

Full text

Characterization of cassava starch and detection of starch synthase gene under
different micro nutrient fertilizer levels by using scanning electronic microscopy
(SEM) and Real Time PCR
Kukuh Setiawan1), Paul B TImotiwu1), Agustansyah1), M. Syamsoel Hadi1),
Ardian1), and Wawan A Setiawan2)
1) Researchers of Department of Agriculture and Horticulture, College of Agriculture,
University of Lampung
2) Researchers of Department of Biology, College of Mathematics and Sciences,
University of Lampung
Abstract
The objectives of this study were to evaluate vegetative growth and root storage of cassava
applied by micro nutrient fertilizer, to compare root storage of yield treated by micro nutrient
fertilizer as 0, 20, and 40 kg/ha, to evaluate the structure of cassava starch by using Scanning
Electron Microscopy (SEM), and to identify the activity of gene starch synthase with Real
Time PCR. There were three different dosages of micro nutrient fertilizer as 0, 20, and 40
kg/ha. Variables observe in this study were leaf number, leaf dry weight, storage root
number, storage root wet weight, diameter of starch granule, visual starch granule by SEM,
and gene starch synthase activity by Real Time PCR. The result showed that leaf number of
cassava applied by high dosages of micro nutrient fertilizer (20 & 40 kg/ha) tended to be
lower compared to those without micro nutrient fertilizer application. Yet, the application of
high dosages of micro nutrient fertilizer, tended to produce relatively high leaf dry weight.
Such condition could support the production of high storage root number and weight.
Additionally, starch structure applied by high dosages of micro nutrient fertilizer seemed to
be denser due probably to high starch granule diameter. The increase in storage root number
and density of cassava applied by 20 kg micro nutrient fertilizer/ha was concomitant with
high gene starch synthase activity, approximately as many as 200 times compared to those
without micro nutrient fertilizer application.
Keywords: micro nutrient fertilizer, Real Time PCR, starch granule, starch synthase
Introduction
Cassava storage root was frequently harvested approximately less than 360 days after
planting (DAP) or 12 months after planting (MAP). Such condition happens due mainly to
the need for cash. Assuming the price of starch is highly stable, the increase in starch yield
would dramatically improve the economic social life. Lampung is the centre cassava in
Indonesia, in 2014 the harvest area for planting cassava is around 304,468 ha (BPS, 2015)
most (> 85%) of areal cassava belonged to small holder of farmers and the rest (around 15%)
belonged to private company (BPS, 2015). Young cassava storage root could absolutely

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reduce the quality of starch especially decrease in granule density as well as starch content.
Based on the habitual farmers, they usually harvest cassava before 7-8 MAP or less than 12
MAP. The other problem is farmers are very rare to fertilize cassava plant with micro
nutrient fertilizer. To increase storage root yield, Cock et.al. (1979) informed that population
of 20.000 plants/ha with optimum control of pest and disease, cassava storage root yield
would be up to 30 metric ton/ha at 12 MAP. Additionally, early planting and late planting
could influence cassava storage root yield. Agbaje dan Akinlosotu (2004) concluded that
cassava storage root yield was 44t /ha harvested at early planting and 31 t/ha at late planting.
Study on the application of micro nutrient combined with macro nutrient fertilizers to
increase cassava starch yield was conducted by Panitnok et al. (2013). They showed that in
Thailand, fresh weight of storage root and starch content would increase up to 29% when
cassava is applied by Zn. Moreover, in Indonesia Hadi (2010) gave information that
application of micro nutrient could increase fresh weight of storage root (yield increment of
0.47 kg) when early harvested at 210 DAP.
According to (Zhu, 2014), cassava starch has finer surface than potato starch and the more
cassava age to be harvested the more clearly granule distribution. It seems that starch cassava
would increase as cassava is harvested at 12 MAP. Consequently, the clear distribution
would increase starch content if harvested at 12 MAP. Miao et al. (2014) conducted study the
gene starch synthase (GSS) in banana. They concluded that activity of GSS would increase
gradually as the growth of banana fruit was developing.
Dos Santos et.al. (2014) concluded that absorption of micro nutrient would be influenced by
cassava age and N content in plant. Howeler (2001) stated that the application of fertilizer
for cassava in Thailand and Vietnam tended to be more P than N and K. Moreover, he also
reported that N was more in leave; K was more in storage root. However, absorption of Fe
showed high concentration in leaf and storage root, as 0.45dan 0.38 kg/ha, respectively in
cassava plant without fertilizer application (Howeler, 2001). Fageria (2009) reported that
absorption of Fe would be high in the condition of low pH. Panitnok et.al. (2013) reported
that combination of Zn, Mg and S did not show the effect on storage root production but
would be significant in the production of starch, starch content would be 28.5% when applied
by Zn and 24.,9% when no Zn (only Mg and S).

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You-Zhi et al. (2015) reported that there were four questions from scientist due mainly to
limited proof from research result as whether high starch accumulation in storage root related
to strong capacity of transport from stem part, whether high starch accumulation in storage
root as a result of low efficient of starch degradation, whether simple sugar move via
transportation far distance from stem, whether only simple sugar transport correlate with the
ability of starch accumulation in part of storage root.
The objectives of this study were to vegetative growth and root storage of cassava applied by
micro nutrient fertilizer, to compare root storage of yield treated by micro nutrient fertilizer
as 0, 20, and 40 kg/ha, to evaluate the structure of cassava starch by using Scanning Electron
Microscopy (SEM), and to identify the activity of gene starch synthase with Real Time PCR.
Material and Method
This study was conducted on dry land of Central Lampung from February to December 2016.
The planting date was in March 2016 using variety cassava of Thailand (UJ3). Fertilizers
used in this study were 200 kg urea/ha, 100 kg SP-36/ha, and 200 kg KCl/ha. These fertilizer
were applied at 30 days after planting (DAP), urea was split into two parts of application, first
was 30 DAP and second was 120 DAP. The application of micro nutrient fertilizer was at 120
days after planting (DAP) together with the application of second urea with the dosage of
0,20, and 40 kg/ha.
Treatment of micro nutrient fertilizer was arranged by randomized block design with 4 reps
used as a block. There were two plants of cassava used as destructive sample for each block.
Variables observed in this study were leaf number, leaf dry weight, storage root number,
storage root wet weight, structure of granule starch, and the expression of starch synthase
gene activity. Data was recorded at 190 DAP, the sample for structure of granule starch and
gene starch synthase activity were observed and measured by SEM and PCR Real Time,
respectively. The PCR Real Time was conducted and compared by using the sample of
storage root from cassava treated by 0 and 20 kg micro nutrient fertilizer/ha. Data were
analyzed with simple statistic as box plot with the mean value then the effect of treatments
was analyzed by MStat based on 5% probability.

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The activity of gene starch synthase of storage root sampled from 0 and 20 kg micro nutrient
fertilizer/ha based on quantity of transcription of gene starch synthase IV by using Real Time
PCR. Fresh storage root from cassava treated with 0 and 20 kg micro nutrient fertilizer/ha
was aseptically taken approximately 0,2 g then put in the solution of RNA-later to protect
RNA from contamination during the process of sampling until in laboratory. The extraction
of RNA was conducted by the method of trizol. After getting the RNA total, synthetic of
cDNA was analyzed by using RevertAidTM First-Strand cDNA Synthesis kit from
Fermentas Co. Moreover, the design of primer for gene starch synthase was based on gene
starch synthase of type IV with access code of starch synthase KT033500.1. cDNA. Then
each quantity of gene starch synthase type IV was determined by Real Time.
Result and Discussion
The leaf number and leaf dry weight tended to be influenced by micro nutrient fertilizer.
Leaf number tended to be decreased as the application dosage of micro nutrient fertilizer
increased (Fig 1) yet not significantly different (Table 1). Interestingly, leaf dry weight
would increase as the application dosage of micro nutrient fertilizer increased (Fig 2). It
seems that micro nutrient fertilizer induces and increases the photoassimilate as a source.
The average of leaf dry weight per plant of cassava without application of micro nutrient
fertilizer was 62.6 g and would reach up to 67.8 g at 20 kg/ha then to be 76.1 g at 40 kg/ha.
Two factors would affect starch yield of cassava as storage root wet weight and starch
content. Starch content could be influenced by quality of starch granule especially granule
size (diameter). The application of 40 kg micro nutrient fertilizer/ha would increase storage
root number from 4.25 to 6.63 (Table 2.
Storage root wet weight increased from the application of micro nutrient fertilizer in the
dosage of 0, 20, and 40 kg/ha (Fig 3) yet not significantly different (Table 2). The storage
root wet weight would increase up to 209 g from application of 20 to 40 kg micro nutrient
fertilizer/ha. It seems that the high leaf dry weight could increase the weight storage root.
Without application of micro nutrient fertilizer (0 kg/ha), diameter range of starch granule
was 8,357 - 16,49 µm. In this treatment, density of starch granule does not seem full visually
(Fig. 4a,b). The application of micro nutrient fertilizer (20 kg/ha), diameter range of starch
granule was 13.53 - 17.58 µm (Fig. 5a,b). It seems that diameter of starch granule of without

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micro nutrient fertilizer is less dense than those of 20 kg. Interestingly, application of 40 kg
micro nutrient fertilizer seems much denser than those of 20 kg/ha (Fig. 6a,b). The diameter
range of application of 40 kg micro nutrient fertilizer/ha was 20.30 – 26.79 µm. Between
starch granules under application of 40 kg micro nutrient fertilizer, there is “filler” and this
does not exist in both treatment of 0 and 20 kg micro nutrient fertilizer. Such condition might
cause the increase in starch content due mainly to high dense granule.
The expression of starch synthase gene activity on the storage root of cassava treated by 20
kg micro nutrient fertilizer at 190 DAP showed as many 200 times as that of 0 kg micro
nutrient fertilizer (Fig. 7). It proves that micro nutrient fertilizer application absolutely
affects the increase in starch content by increasing high dense of granule starch. This could
be proposed the mechanism of high quality cassava starch by application of micro nutrient
fertilizer (Fig 8). The application of micro nutrient fertilizer increased expression of starch
synthase gene resulting in the increase of photosynthate as a source (leaf dry weight). Such
source would transport to storage root as a sink part, resulted in heavier storage root. On the
other side, starch quality as granule enlarges the diameter lead to the much denser of granule
starch. Both of heavier storage root and high dense of granule could probably increase the
starch content.
Conclusion
Based on result and discussion, it could be concluded that:
a. The application of micro nutrient fertilizer tended to decrease leaf number however,
leaf dry weight tended to be increased by application of micro nutrient fertilizer at 190
DAP.
b. Storage root number would be increased by application of micro nutrient fertilizer.
However, the application of micro nutrient fertilizer did not signifantly increase
storage root wet weight.
c. Starch granule diameter of cassava generally bigger under application of high micro
nutrient fertilizer than that of no micro nutrient fertilizer. Especially, the application
of 40 kg micro nutrient fertilizer seems to induce “filler” resulted in high density of
starch granule.

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d. The application of 20 kg micro nutrient fertilizer could induce the expression of high
gene starch synthase as many as 200 times compared to no application of micro
nutrient fertilizer.
Acknowledgements
We would like to thank to LPPM, University of Lampung giving opportunity to conduct the
valuable research to characterize and identify cassava starch both physically (SEM) and
genetically (PCR Real Time). Respectful appreciation is also addressed to Mr. Subadi
(cassava farmer), students (University of Lampung and Sekolah Tinggi Pertanian Surya
Dharma Bandar Lampung for their help and support in maintaining, recording, and
harvesting. Collaboration and cooperation with Integrated Laboratory of University of
Lampung (chairman and technicians) are also highly appreciated for the success of Scanning
Electron Microscope (SEM) and PCR Real Time analysis.
References
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40200
80
70
60
50
40
30
Dosis
JD
41
41.25
49.25
Boxplot of JD
Fig 1. Leave number of cassava applied by different dosage of
micro nutrient fertilizer
40200
90
80
70
60
50
Dosis
BKDaun
76.125
67.75
62.625
Boxplot of BKDaun
Fig 2. Leave dry weight of cassava applied by different dosage of
micro nutrient fertilizer

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Table 1. Value of mean square, general means, and probability of observed variables
No
Variables
Mean square value
General
means
Probability
Dosage
Block
1.
Leave number (piece)
176,167
79,111
43,3
0,12
2.
Leave dry weight (g)
371,542
275,222
68,8
0,02
3.
Tuber number (no)
11,292
9,500
5,4
0,02
4.
Wet tuber weight (g)
89461,500
72493,944
774,3
0,29
Significantly different if P < 0,05 and not significantly different if P > 0,05.
Table 2. Leave number, leave dry weight, tuber number, and wet tuber weight of cassava
applied by different dosage of micro nutrient fertilizer as, 0, 20, dan 40 kg/ha.
No.
Variable
Dosage of micro nutrient fertilizer kg/ha
0
20
40
1.
Leave number (piece)
49,3a
41,3a
41,0a
2.
Leave dry weight (g)
62,6a
67,8ab
76,1b
3.
Tuber number (no)
4,25a
5,38ab
6,63b
4.
Wet tuber weight (g)
661a
792a
870a
Number in the same column followed by the same letter indicated not significantly difference
under Tukey’s procedure in the level of 5%
40200
1200
1000
800
600
400
200
Dosis
BSUmbiTanm2
870
792
660.75
Boxplot of BSUmbiTanm2
Fig 3. Wet weight of cassava tuber applied by different
dosage of micro nutrient fertilizer

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Fig 4a. Structure of cassava starch granule
applied by 0 kg micro nutrient fertilizer
/ha
Fig 4b. Diameter of cassava starch granule
applied by 0 kg micro nutrient
fertilizer /ha
Fig 5a. Structure of cassava starch granule
applied by 20 kg micro nutrient
fertilizer /ha
Fig 5b. Diameter of cassava starch granule
applied by 20 kg micro nutrient
fertilizer /ha

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Fig 6a. Diameter of cassava starch granule
applied by 40 kg micro nutrient
fertilizer /ha
Fig 6b. “Filler” between starch granule
applied by 40 kg micro nutrient
fertilizer per ha
Fig 7. Expression of gene starch synthase in the cassava tuber
harvested at 190 DAP.
Fig 8. Proposed mechanism of the micro nutrient fertilizer
effect on high cassava starch production via increase in
gene starch synthase activity