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Studies on Ranunculus Population: Isozymic Pattern

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Species of Ranunculus is small herb grows at quite high altitudes, ranging from several hundreds to more than a thousand meter above sea level. They can occupy a variety of habitats such as moist soils or can even grow sub merged or floating in stream. A few numbers of species from different populations have been recorded to have morphological complexity, which could cause a problem for the work of taxonomists in making decisions. In order to support taxonomists in solving their problem, a new experimental method using SDS-PAGE will be used to explore the isozyme data. The main purpose of this research was to investigate whether or not isozyme data can be used to clarify the morphological complexity of Ranunculus species. In this study, nine species of Ranunculus from different populations were used. Five to twenty plants were sampled for electrophoresis studies. Four enzyme systems: peroxidase, esterase, malate dehydrogenase and acid phosphatase were chosen. The results showed that every enzyme gave its specific pattern in each species and common band always found in nine species tested. This experiment proved that genetic data (isozyme) quite promoting to be applied in higher plant taxonomy in solving the morphological complexity problem.
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BIODIVERSITAS ISSN: 1412-033X
Volume 2, Nomor 1 Januari 2001
Halaman: 85-91
Studies on Ranunculus Population: Isozymic Pattern
S U R A N T O
Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University Surakarta.
Received: December 24th 2000; Accepted: January 20th 2001
ABSTRACT
Species of Ranunculus is small herb grows at quite high altitudes, ranging from several hundreds to more than a
thousand meter above sea level. They can occupy a variety of habitats such as moist soils or can even grow sub
merged or floating in stream. A few numbers of species from different populations have been recorded to have
morphological complexity, which could cause a problem for the work of taxonomists in making decisions. In order to
support taxonomists in solving their problem, a new experimental method using SDS-PAGE will be used to explore the
isozyme data. The main purpose of this research was to investigate whether or not isozyme data can be used to
clarify the morphological complexity of Ranunculus species. In this study, nine species of Ranunculus from different
populations were used. Five to twenty plants were sampled for electrophoresis studies. Four enzyme systems:
peroxidase, esterase, malate dehydrogenase and acid phosphatase were chosen. The results showed that every
enzyme gave its specific pattern in each species and common band always found in nine species tested. This
experiment proved that genetic data (isozyme) quite promoting to be applied in higher plant taxonomy in solving the
morphological complexity problem. © 2001 Jurusan Biologi FMIPA UNS Surakarta
Key words: isozyme, PER, EST, MDH, ACP, Ranunculus.
INTRODUCTION
The genus Ranunculus consists of about
500 species, and is recorded as the large
genus within the family Ranunculaceae. The
Ranunculus species usually grow at quite high
altitudes, ranging from several hundred to
more than a thousand meters above sea level.
They can occupy a variety of habitats, in wet
or moist soils and muddy water, or can even
grow submerged or floating in steams.
Species are widely distributed around the
world and the number in different countries
varies. About 146 species have been recorded
in Russia (Komarov and Schischkin, 1963). On
the other hand, in Java (Indonesia) only 3
species so far have been recorded (Eichler in
Backer and Bakhuizen van den Brink, 1963).
Japan, India and Taiwan each have 20-30
species, and in New Zealand approximately 50
are recorded. This confirms they are typical
plants of cool climate regions. In tropical
countries such as Indonesia they can only
grow in the mountains. Table 1. shows the
distribution of Ranunculus species used in this
study.
Enzyme variations
The term "isozyme" has been used by
Brown (1990) for "the multiple zones of
enzyme activity that are observed when
electrophoresis gels are subject to
histochemical stains to detect the presence of
more or less specific enzymes". In addition, he
stated that isozymes might precisely or
correctly is applied for multiple bands coded
by more than one structural gene locus for
particular enzymes. Alternatively the allozyme
term is used if the occurrence of multiple
bands arises from the alleles which are
segregating at a single locus.
DOI: 10.13057/biodiv/d020101
BIODIVERSITAS Vol. 2, No. 1, Januari 2001, hal. 85-91
86
The use of isozyme in plant taxonomy
In recent years, uses of isozyme data for
plant taxonomic purposes have increased
quite rapidly. This method offers a very
powerful tool in studying lower hierarchies of
plant taxa such as the species, sub-species or
population level (Reisenberg et. al., 1988;
Burden et. al., 1980; Brown, 1990).
Isozyme data is particularly useful if the
morphological characters of species appear to
overlap. At the present time, there is no doubt
that isozyme data provides a powerful tool in
the era of molecular taxonomy. It is likely that
by the next decade the use of isozyme data in
the work of taxonomist will be intensively
adopted. Molecular biology techniques will use
data both from DNA sequence and isozyme
analysis for research purposes in plant
taxonomy.
DNA sequencing needs quite expensive
funds to run it, but it provides the best data,
and has wider applications for all levels of
taxa. On the other hand, "isozyme analysis" is
less expensive (even affordable) and relatively
easy and rapid to perform. It also has the
ability to handle quite a large numbers of
samples simultaneously (Brown, 1990). From
the undoubted advantages of both the above
approaches, financial considerations
frequently determine which experimental
taxonomic research will be adopted. In this
project, the second approach is the alternative
to be adopted.
Studies of enzyme patterns in higher plant
taxonomy have been carded on for more than
two decades (Mitra et. al., 1970; Conklin and
Smith, 1971; Reisenberg and Soltis, 1987;
Burden at al., 1980; Moran et. al., 1990;
Brown, 1990). These studies of plant enzymes
involved both alloenzyme variations (Crawford
et. al., 1988, 1985; James et. al., 1983), and
isozyme variations (Reisenberg, l987). The
principle methodology in studying isozyme and
allozymes in plant is electrophoresis.
Electrophoresis examines the movement of
the protein and enzymes in buffered gel when
they are subjected to an electric current.
Band forms on electrophoresis gels which
reveal the activity of enzymes using specific
enzymic detection methods, at least implies
functional similarity and analogy of the
proteins being compared (Mitra et. al., 1970).
Protein variations revealed by electrophoresis
have been considered as a powerful approach
in measuring the genetic diversity in
populations (Brown, 1978; Brown et. al.,
1978). In order to ascertain the level of generic
variation within and/or between populations,
isozymes and allozymes can be used as main
sources of data, because an enzyme marker
which is separated by electrophoresis
furnished a simple means for rapid partitioning
of the variability within and between
populations at the gene level.
Although studies of enzyme variations by
means of electrophoresis in Ranunculus
species is here being conducted for the first
time, this method has been extensively
employed for other species, for example;
Pisum sativum (Bowling and Crowden, 1970);
Datura species (Conklin and Smith, 1971);
Nicotiana species (Smith et. al. 1970;
Bredemeijer, l984); Dubautia agyroxiphium
and Wilkesia (Witter, 1988); Glycine
canescent and G. aryrea (Brown, 1990);
Glycine tomentella (Doyle et. al., 1986);
Chenovidium santae-clarae (Crawford et. al.,
1988); Eucalyptus and Acacia (Moran et. al.,
1990); Thuja plicata, Eremaea species
(Coates and Hnatiuk, 1990); Emex australis
(Panetta, l990).
Since multiform enzymes are presumably
the direct product of multiform (alleles) of
genes, they may serve as molecular markers
which are useful for analyzing genetic
dissimilarity among species (Conklin and
Smith, 1971). "Undoubtedly allozymes will
continue to be used in biosystematics and
phylogenetic applications, and the importance
of isozyme data will be strengthened if they
are treated carefully like any other group of
taxonomic characters, with criteria for category
assignments clearly stated and the possibility
of selective interactions considered" (Johnson,
1973). Although the use of isozyme data in the
studies of populations is increasing, it is better
that a group of enzymes be studied rather than
a single system.
In this present study four enzyme systems
were chosen, namely peroxidase, esterase,
malate dehydrogenase and acid phosphatase.
Peroxidase is an easy enzyme to detect in
gel electrophoresis, but interpretation of
isoperoxidase data requires can, as has been
reported in their studies of Shorea loprosula
and Xerospermum species. Pattern may
change during physiological development.
Conklin and Smith (1971) have reported an
increased number of bands in Datura species
as leaves approach maturity. In addition, they
SURANTO – Isozymic pattern of Ranunculus 87
recorded no difference in band patterns from
plants from the green house or from field
grown plants.
Like peroxidase, the esterase has
numerous isozymes, and more in herbaceous
plants than in woody plants. Smith et al.
(1970) reported band number of esterase in
Nicotiana species to range from 3-15. Even
though there are more esterase band present
than peroxidase, this enzyme system may not
be particularly useful in discriminating between
species, as Mitra et al. (1970) recorded in
Hordeum species.
Table 1. Ranunculus species and population sources
used for electrophoresis.
Population Species Plant
numbers
Liawenee R. triplodontus 16
Nive River R. triplodontus 20
Rat Castle R. triplodontus 15
Clarence Weir R. triplodontus 20
Ouse River R. triplodontus 13
Projection Bluff R. triplodontus 10
Wild Dog Plains R. triplodontus 12
Black Mary Plains R. pimpinellifolius 9
Pine Lake R. gunnianus 6
Lake Augusta R. gunnianus 5
Projection Bluff R. decurvus 10
Rat Castle R. decurvus 10
Projection Bluff R. collinus 10
Rat Castle R. collinus 10
Wild Dog Plains R. collinus 10
Liawenee R. pascuinus 10
Wild Dog Plains R. amphitricus 10
Green View R. lappaceus 9
Wild Dog Plains R. nanus 20
Camerons Lagoon R. nanus 20
Saint Patrict Plains R. nanus 20
Ouse River R. nanus 20
Clarence Weir R. nanus 20
Unlike esterase, malate dehydrogenase
isozymes are fewer in number, i.e. only about
3 or 4 as recorded by Gottlieb (1982). Moran
and Hooper (1983), again showed for malate
dehydrogenase that there are more isozymes
bands in herbaceous than in woody species.
Acid phosphatase isozymes are likely to
give many bands since this enzyme may be
dimeric. This enzymic marker gives good
resolution where polyploid species are
studied, for example in hexaploid bread wheat
(Gottileb, 1982). This enzyme system may
give significant results since several
Ranunculus have several polyploid species.
Recent studies of isozyme variations in plant
taxonomy have made substantial contributions
in examining inter relationships between
species or species differences, provided then
data are integrated with a proper analysis. The
value of using isozyme data in association
with morphological characters in resolving
problems species complexes is potentially
great.
To examine any possible effect of
environmental change on the isozyme patterns
in Ranunculus, both field and transplanted leaf
samples were examined Field harvested
leaves were of uncertain age, which may
account for some quantitative differences
observed within population sample. Leaves
examined after four months transplanting were
of uniform age and at an equivalent stage of
physiological development, and therefore
would be expected to give a uniform pattern
unless genetic variability existed within the
population.
MATERIALS AND METHODS
Gel Preparation
Acrylamide gel electrophoresis was
employed. The gel buffer was tris-citric buffer
pH 8.4 (Stock Solution A). Stock Solution A:
4.5 grams of TRI (Hydroxymethyl)
Methylamine (PURISS), 0.51 grams of citric
acid and 500 ml of deionized water. Stock
Solution B: 30 grams of acrylamide, 0.80
grams of N N'-methylene-bis-acrylamide and
100 ml of deionized water.
The gel was made by mixing 20 ml of
solution B and 40 ml of solution A. This
mixture was deaerated on a Buchi rotary
evaporator for 5 minutes after which 0.04 ml of
N, N, N', N'-tetramethyl-ethylenediamine was
added and with carefully mixed To polymerize
the gel, 0.06 grams of ammonium persulphate
was added and mixed carefully immediately
before pouring the solution into the gel mould
(BIO-RAD Model 360). Using this model, at
least 4 thin gels each with 10-14 slots can be
cast simultaneously.
Extraction and loading the samples
Laminas and petioles were examined
separately. Material from each plant was
BIODIVERSITAS Vol. 2, No. 1, Januari 2001, hal. 85-91
88
ground individually in a staining dish using
0.15-0.35 ml of protein extracting solution for
laminas and 0.1-0.15 ml for petioles. Despite
the voluminous literature on extraction
methodology which suggests the need to use
frozen plant material (liquid nitrogen), it was
found unnecessary for the systems studied in
this project to use other than an ice cool buffer
and to hold plant material and extracts in an
ice bath. The extracts were transferred to a
small glass vial, 2 mm diameter, 3 cm long,
and centrifuged at 3500 rpm for 15 minutes.
The supernatants were then applied in the gel
slots. The amount of sample loaded in each
slot was, for peroxidase about 10-15 ul, while
for the other enzymes about 15-24 ul.
The protein extracting solution consisted of
0.018 grams of cysteine, 0.021 grams of
ascorbic acid, 5 grams of sucrose, diluted in
20 ml of borax buffer pH 8.4 (tank buffer).
Electrophoresis
The electrophoresis chamber used in this
project was a mini vertical slab cell
manufactured by BIO-RAD, USA, model 360.
This model has advantages in allowing use of
very small amounts of samples, as well as
allowing a short running time.
Electrophoresis was conducted at a
constant current of 5 mA for peroxidase (PER)
and 7 mA for esterase (EST), malate
dehydrogenase (MDH), and acid phosphatase
(AP), at room temperature far about 60
minutes including a pre-electrophoresis time of
approximately 10 minutes. Electrophoresis
was stopped when the bromophenol blue
marker dye had traveled about 56 mm from
the slot toward the anode.
Staining Procedures
Four enzyme stains were used routinely.
Peroxidase
0.0125 grams of o-Dianisidine, dissolved in
2.5 ml of acetone, then add 50 ml of 0.2 id
acetate buffer pH 4.5 and 2 drops of H2O2.
Esterase
0.0125 grams of 1-naphthyl acetate
dissolved in 2.5 ml acetone, then add 50 ml of
0.2 M phosphate buffer pH 6.5 and 0.0125
grams of Fast Blue BE Salt
Malate Dehydrogenase
15 ml of 0.l M Tris-HCl pH 8 was mixed in
125 ml of deionized water. Then 10 ml of 0.2
M Sodium Malate pH 7.5, 0.020 grams of MTT
(2,5-Diphenyl tetrazolium Bromide) and 0.005
grams of PMS (Phenazine Methosulphate)
was added.
Incubate gel for 30-40 minutes in the dark,
and then transfer to a fresh solution containing
in addition 0.020 grams of NAD (Nicotilamide
Adenine Dinucleotide).
Acid Phosphatase
0.0125 grams of l-naphthyl phosphate
dissolved in 2.5 ml of acetone then add 75 ml
of 0.2 M acetate buffer pH 4.5, 0.025 grams of
Fast Black K Salt and 0.025 grams of Fast
Garnet GBC Salt.
A number of other enzyme systems, namely
Glucose-6-Phosphate Dehydrogenase. 6
Phospogluconate Dehydrogenase, Alcohol
Dehydrogenase, Isocitratase, and Tetrazolium
Oxidase were investigated and found
unsuitable.
All staining procedures in this experiment
were conducted at room temperature. For
Peroxidase and Esterase stains refer to Mills
and Crowden (1968), for Malate
Dehydrogenase stains refer to Brown et al.
(1978), and for Acid Phosphatase stains refer
to Adam and Jolly (1980).
RESULTS AND DISCUSSION
Figure 1-4 are interpretative drawings of
isozyme patterns for Peroxidase, Esterase,
Malate Dehydrogenase, and Acid Phosphatase
of the Ranunculus species examined. The
diagrams shown represent average band
patterns determined after examining 1 number
of populations far each species.
SURANTO – Isozymic pattern of Ranunculus 89
Figure 1. Peroxidase isozyme patterns of Ranunculus
species. A. R. triplodontus, B. R. collinus, C. R.
decurvus, D. R. pimpinellifolius, E. R. gunnianus, F. R.
pascuinus, G. R. amphitricus, H. R. lappaceus, I. R.
nanus.
Figure 2. Esterase isozyme patterns of Ranunculus
species. A. R. triplodontus, B. R. collinus, C. R.
decurvus, D. R. pimpinellifolius, E. R. gunianus, F. R.
pascuinus, G. R. amphitricus, H. R. lappaceus, I. R.
nanus.
Figure 3. Malate dehydrogenase isozyme patterns of
Ranunculus species. A. R. triplodontus, B. R. collinus, C.
R. decurvus, D. R. pimpinellifolius, E. R. gunnianus, F. R.
pascuinus, G. R. amphitricus, H. R. lappaceus, I. R.
nanus.
Figure 4. Acid phosphatase isozyme patterns of
Ranunculus species. A. R. triplodontus, B. R. collinus, C.
R. decurvus, D. R. pimpinellifolius, E. R. gunnianus, F. R.
pascuinus, G. R. amphitricus, H. R. lappaceus, I. R.
nanus.
BIODIVERSITAS Vol. 2, No. 1, Januari 2001, hal. 85-91
90
Peroxidase
Peroxidase showed most variations of the
four enzymes tested. The seven
isoperoxidases identified arranged in eight
distinct patters. Bands 1 and 2 occurred in all
species tested. The two species R.
triplodontus and R. amphitricus had the same
patterns (5 bands). These are aquatic or semi-
aquatic plants. Two other species R. collinus
and R. nanus also had identical peroxidase
patterns (6 bands). These species which often
co-occur in the same habitat sometimes show
overlapping leaf morphology, but they are
easily distinguished on floral characters. The
other species had distinctive peroxidase
patterns.
Esterase
Isozyme bands 1, 2, 4 and 6 appeared in all
species. Five species, i.e. R. collinus. R.
pimpinellifolius. R. gunnianus R. pascuinus
and R. amphitricus had the same banding
patterns (4 bands). The other 4 species R.
nanus, R. decurvus, R. lappaceus and R.
triplodontus had distinctive patterns. The main
variability was in the bands 7, 8 and 9.
Malate Dehydrogenase
Common isozyme bands for all species
were numbers 1, 2 and 4. For species R.
decurvus, R. pimpinellifolius, R. pascuinus and
R. amphitricus had the same bands (4 bands),
as did R. gunnianus (3 bands). R. lappaceus,
R. triplodontus and R. nanus had distinctive
patterns. R. nanus showed the most
complexity of isoenzyme patterns.
Acid Phosphatase
Common isozyme bands in all species tested
were bands 1, 2, 3, 5 and 7. Band 5 was
absent from R. amphitricus while band 7 was
very strong in this species. R. gunnianus
showed a very distinctive pattern, with the
unique bands 4 and 9. Six species R. nanus.
R. pascuinus, R. pimpinellifolius, R. gunnianus
and R. lappaceus were not resolved.
Each enzyme studied gave a different
result, and a different level of species
separation. Peroxidase gave the best
resolution of the species, compared to the
presently accepted taxonomy. However, one
would hardly expect all 11 species to be
separated on the basis of variation in 1
enzyme system. No sound taxonomic
classification will result from examination of a
single character, even though that character
may have a number of states.
The experimental methods used in this
study, of isozyme analysis using
polyacrylamide gel electrophoresis, applied to
9 species of Ranunculus have supported the
general view that such experimental data are
useful in delineating in a meaningful
(classification) sense. It is almost certainly that
the more extensive application of the
procedure(s) to include wider range of enzyme
tests, examination of more populations,
examination of plant organs other than leaves,
e.g. seeds, seedling, would broaden the data
base, and therefore would gave better result
for taxonomists in providing improved taxon
delineation
ACKNOWLEDGEMENTS
A great appreciation was given to AIDAB-
Australia for the funding during my study in
Tasmania. I would like to thank Dr R. Crowden
for his advice, patience and correction of the
manuscripts, while I was studying at the
Department of Plant Science (Botany),
Tasmania University-Hobart-Australia
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... Acrylamide gel electrophoresis and buffer solutions (extraction buffer, tank buffer, running buffer) were prepared and carried out as described by Suranto (2001), Setyawan et al. (2014) and Suratman et al. (2016). ...
... Loaded samples were electrophoresed at a constant current of 5 mA for peroxidase and 7 mA for esterase at room temperature for about 60 minutes. Electrophoresis was stopped when the bromophenol blue marker dye had traveled about 56 mm from the well toward the anode (Suranto 2001;Padmanaban et al. 2013, Setyawan et al. 2014. ...
... After electrophoresis, the gels were stained for the appropriate enzyme systems (esterase and peroxidase) as described by Suranto (2001), Setyawan et al. (2014) and Suratman et al. (2016) with some modifications. Gels were immersed in the staining solutions until bands appeared. ...
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... The methods used for extracting the leaf samples and electrophoretic isozymes was according to Suranto (2001) which was slightly modified especially the treatment of samples used. About 350 milligrams of infected rice leaf samples were picked up from the third leaves of the top plant during anthesis. ...
... Meanwhile, the total band number of five was recorded for the rice sample which was not infected by plant viruses (Siva, Kumar, & Rajasekaran, 2013). Using other plant samples, Suranto (2001;2002) also recorded a total of five bands in herbaceous plant of Ranunculus amphitricus. ...
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Tungro is one of the most common diseases of rice plants which caused by double infection of RTBV (Rice Tungro Bacilliform Virus) and RTSV (Rice Tungro Spherical Virus), and it gives a significant economic loss. These viruses are transmitted by green leaf hopper (Nephottetix virescens. Distant), and the interaction between host plant and the viruses were still quite difficult to be fully understood. In order to look at whether there are any differences between the healthy and tungro infected rice, this study was set to examine the character differences between the infected and the healthy plants based on isozyme banding pattern. The infected plants were collected from three districts around Surakarta (Sragen, Sukoharjo, Klaten) followed by Polyacrilamide Gel Electrophoresis (PAGE) to evaluate the isozyme banding patterns. There were peroxidase, esterase and acid phosphotase isozymes used. The results showed that the real differences of isozyme banding patterns of both healthy and tungro infected plants were discovered. In all cases, the Tungro infected rice had thicker and more band numbers compared to the healthy one. This evident suggested that Tungro infected rice could be detected early using PAGE method. © 2017, Agriculture Faculty Brawijaya University. All rights reserved.
... Anatomical characters are also valuable in taxonomy and identification of groups of plant (Rahayu et al. 2012; Chikmawati 2013). Isozyme as the classical biochemical marker can be used to determine genetic variation of cultivars, natural populations and accessions in germplasm collections, if the morphological characters appear to overlap due to strong influence of environment (Suranto 2001; Fernandez de Souza and Primo 2001; Padmanaban et al 2013). Isozymes have several advantages over traditional markers such as morphological or anatomical traits to study polymorphism because they are not influenced by environmental factors making identification possible in early stages of development (Torres 1990). ...
... Loaded samples were electrophoresed at a constant current of 5 mA for peroxidase and 7 mA for esterase at room temperature for about 60 minutes. Electrophoresis was stopped when the bromophenol blue marker dye had traveled about 56 mm from the well toward the anode (Suranto 2001; Padmanaban et al. 2013; Setyawan et al. 2014). ...
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Suratman, Pitoyo A, Kurniasari S, Suranto. 2016. Morphological, anatomical and isozyme variation among giant taro (Alocasia macrorrhizos) accessions from Central Java, Indonesia. Biodiversitas 17: 422-429. The objective of this study was to evaluate morphological, anatomical and isozyme variation among giant taro (Alocasia macrorrhizos (L.) G.Don) accessions from Central Java (Indonesia). A total of 20 giant taro accessions were collected from different collection sites in Central Java. Identification of morphological characters was done by direct observation of roots, leaves, stems, and corms. Anatomical characters were observed from both paradermal and transverse sections of leaf. Identification of biochemical markers was done by using peroxidase and esterase isozyme system. The genetic similarity among giant taro accessions was measured by using Group Average Clustering. The results of the analysis of variance revealed highly significant differences for majority of the tested morphological and anatomical characters suggesting that there was a high degree of diversity among the giant taro accessions. Isozyme polymorphism was observed in giant taro accessions using peroxidase (two banding patterns) and esterase (four banding patterns). Based on the dendogram, giant taro accessions were segregated into two major clusters. In Cluster I, the closest relationship were showed in KTN 2 and WNG 1 accessions from Klaten and Wonogiri that had 80.95% of similarity coefficient. The five accessions (SKA, SKH, WNG 4, KRA 3, KRA 4) from Surakarta, Sukoharjo, Wonogiri and some parts of Karanganyar were clustered separately as Cluster II with similarity coefficient of 50%.
... Tanaman sejenis akan bervariasi morfologinya apabila faktor lingkungan lebih dominan mempengaruhi tanaman dari pada faktor genetik. Tanaman tidak akan menunjukan variasi morfologi yang signifikan apabila faktor genetik lebih dominan mempengaruhi tanaman tersebut (Suranto, 2001). ...
... Faktor lingkungan berpengaruh terhadap morfologi tanaman, apabila faktor lingkungan lebih dominan dibanding faktor genetis maka tanaman akan mengalami perubahan morfologi (Suranto, 2001). Sehingga dalam jangka waktu lama dimungkinkan tanaman akan mengalami perubahan sifat genetiknya. ...
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Dioscorea alata L. adalah salah satu jenis tanaman lokal yang memiliki potensi untuk mendukung program ketahanan pangan di Indonesia. Wilayah Nganjuk memiliki beragam Dioscorea alata L., sehingga perlu dilakukan inventarisasi dan karakterisasinya. Penelitian ini bertujuan untuk:mengidentifikasi karakter morfologi umbi Dioscorea alata L. yang terdapat di Nganjuk dan untuk mengetahui kemiripan karakter umbi Dioscorea dengan membuat dendogram jarak kemiripan. Hasil karakterisasi dari pengamatan morfologi umbi dianalisis dengan fungsi similarity interval pada program NTSys berdasarkan koefisien DICE. Hasil penelitian menunjukan bahwa di wilayah Nganjuk terdapat 22 aksesi Dioscorea alata L. dengan 13 varian. Dioscorea alata L. dengan varian yang sama menunjukan jarak kemiripan yang dekat walaupun berasal dari wilayah yang berbeda.
... Isozymes were analyzed from young leaves taken from mature plants. Enzymes were extracted, electrophoresed, stained and characterized as described by Suranto (2001), Setyawan et al. (2014), Suratman et al. (2016) and Pitoyo et al. (2018). A total of 0.6 g young leaves of maderira vein were ground in mortar using 1800 µl of extraction buffer and then transferred to a 1.5 ml microtube. ...
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Restykania, Suratman, Pitoyo A, Suranto. 2019. Morphology and isozyme variation among madeira vine (Anredera cordifolia) accessions from southeastern part of Central Java, Indonesia. Biodiversitas 20: 3024-3032. The genetic diversity among madeira vine accessions from southeastern part of Central Java has not been investigated in detail. Information on genetic diversity and relationship among accessions of this plant will provide important input into determining resourceful management strategies and guiding the improvement of plants through plant breeding programs. The objective of this study was to evaluate variation of morphology and isozyme among madeira vine (Anredera cordifolia (Ten.) Steenis) accessions from southeastern part of Central Java. A total of 13 madeira vine accessions were used for morphological observations and isozyme analysis. Morphological diversity was characterized based on measurements and observations of vegetative structures such as stems, leaves, and aerial tubers. Identification of biochemical markers was done using peroxidase and esterase isozyme systems. A relationship dendrogram among accessions was constructed based on the genetic similarity matrix by applying a cluster analysis. The analysis of quantitative and qualitative morphological characters revealed that the majority of the tested traits showed variation, indicating that there was variability among the tested madeira vine accessions. Polymorphism was observed using isozyme of peroxidase (i.e. two banding patterns) and esterase (i.e. five banding patterns). Based on the dendrogram at a level of 63 % similarity, it showed distinct separation of 13 madeira vine accessions into two major clusters. Cluster I consisted of nine accessions and the closest relationship was shown between KLT 3 and KLT 1 accessions that had 94 % of similarity coefficient. The remaining four accessions were clustered separately as Cluster II with similarity coefficient of 73 %. Therefore, genetic characterization based on morphological and isozyme markers obtained in this study is valuable to understand genetic variability and relationship among madeira vine accessions in southeastern part of Central Java.
... This was evidenced from the results of research obtained, showing that pepper plants that were given a bio-fertilizer consortium of cellulolytic bacteria based on organic material have the same fruit length. According to [37], the appearance of variations can be influenced by genetic and environmental factors. If genetic factors had a stronger influence than environmental factors and the plant lives in any different environment it would not show morphological variations that were different from the place of origin. ...
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Red pepper production in 2016-2017 was increased. Although production has increased, demand for pepper will continue to increase, and efforts to increase soil fertility still need to be sought. To maintain and increase soil productivity, a combination of inorganic fertilizers with appropriate organic fertilizers is needed. One of the organic fertilizers is biofertilizers. This research aimed at finding out the formulation and dosage of biological fertilizers consortium of cellulolytic bacteria based on organic liquid waste that was appropriate for the growth and yield of red pepper plants. This research method used a Completely Randomized Design with 12 treatments, namely (5, 10, 15) ml of biological fertilizer of waste water washing rice, (5, 10, 15) ml of biological fertilizer of coconut water waste, (5, 10, 15) ml of biological fertilizer of tofu water waste, (5, 10, 15) ml of palm oil liquid fertilizer. The results showed that all biological fertilizer application based on organic liquid waste cellulolytic was able to give varied results on each parameter observed so that all wastes could be applied to pepper plants. Tofu wastewater biofertilizer with a dose of 10 ml give a good response to the production of pepper plants compared to other treatments.
... Total number of 45 leaves were collected from three different location (Figure 1). Procedures of running electrophoresis of isozymes which consisted of preparation of solution, acrylamide gel, extraction of sample, electrophoresis, and staining procedure were conducted according to Suranto (2001), with some modification. ...
Article
Aims of this research were to investigate the morphological character and isozyme banding patterns of Cucurbita moschata plants grown at three different altitudes. Samples in this study consisted of leaf, stem, and flowers. The morphological characters were conducted by direct observation in the field and analyzed descriptively as well as statically by one way ANOVA. The isozyme bands appearance of esterase and peroxidase of leaf samples were conducted using polyacrylamide gel electrophoresis (PAGE). Qualitative approach was used to analyze the presence and the absence of isozyme bands, while Retardation factor (Rf) was used to analyze quantitatively. The results showed that most plants grown at middle altitude (351-750 m asl.) were well-developed in terms of length of leaves, stems and flowers. Accordingly, the isozyme banding pattern of peroxidase was also found varied in plants grown at middle altitudes from which the presence of very unique bands was detected. Conversely, the band detected in plants grown at the lower and the highest altitudes was similar in term of band's number but it was different in the quality of the bands. Meanwhile, esterase isozyme banding pattern of plants grown at the lower and higher altitude had more bands than the middle altitude. Based on this result it is obvious that the isozyme data could be used to support in understanding the diversity morphological characters of plants grown in three different altitudes. This early result suggests that altitudes as a crucial factor in contributing the expression of isozyme appearance, which is useful for further pumpkin characterizations. © 2018, Society for Indonesian Biodiversity. All rights reserved.
... If the different varieties of taro belong to one group with a distance of close to 1 it is possible that the similarity comes from the older of taro. Environmental factors affect plant morphology, if the environmental factor is more dominant than genetic factors, the plant will experience a change in morphology (Suranto 1999(Suranto , 2001. In the long term it is possible crop genetic trait changes in her body. ...
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Abstract. Trimanto, Sajidan, Sugiyarto. 2011. Characterization of taro (Colocasia esculenta) based on morphological and isozymic patterns markers. Nusantara Bioscience: 7-14. The aims of this research were to find out: (i) the variety of Colocasia esculenta based on the morphological characteristics; (ii) the variety of C. esculenta based on the isozymic banding pattern; and (iii) the correlation of genetic distance based on the morphological characteristics and isozymic banding pattern. Survey research conducted in the Karanganyar district, which include high, medium and low altitude. The sample was taken using random purposive sampling technique, including 9 sampling points. The morphological data was elaborated descriptively and then made dendogram. The data on isozymic banding pattern was analyzed quantitatively based on the presence or absence of bands appeared on the gel, and then made dendogram. The correlation based on the morphological characteristics and isozymic banding pattern were analyzed based on the product-moment correlation coefficient with goodness of fit criterion. The result showed : (i) in Karanganyar was founded 10 variety of C. esculenta; (ii) morphological characteristics are not affected by altitude; (iii) isozymic banding pattern of peroxides forms 14 banding patterns, esterase forms 11 banding patterns and shikimic dehydrogenase forms 15 banding patterns; (iv) the correlation of morphological data and the isozymic banding pattern of peroxidase has good correlation (0.893542288) while esterase and shikimic dehydrogenase isozymes have very good correlation (0.917557716 and 0.9121985446); (v) isozymic banding pattern of data supports the morphological character data.
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The diversity of Dioscorea spp. both inter-species and intra-species are spreaded in several areas in Central Java, including Banjarnegara. Dioscorea is a plant that has the potentials to be developed and cultivated. The purposes of this reasearch are to identify the species, characterize the morphological characters (stem, leaf, and tuber) of eight varians Dioscorea spp from Banjarnegara. The sample used in this reasearch are the eight varians of Dioscorea spp from Banjarnegara. The Identification was done by using a identification keys. Characterization of morphological characters was done by using the guidebook of Descriptor for Yam. The results showed that eight cultivars of Dioscorea spp are grouped into two types: Dioscorea alata and Dioscorea esculenta. Dioscorea alata has a stem which rotate the to the right, rectangular shaped of stem, it’s has wing and doesn’t have a spin, elongated heart-shaped leaves, oval-oblong and round shaped tuber. Dioscorea esculenta has a stem which rotate to the left, rounded shaped of stem, it has not wing but spiked, dilated heart-shaped leaf, and oval shape tuber. Keywords: Dioscorea spp, identifikasi, characteristic, morphology
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Black rice is one of the functional foods due to its high anthocyanin content. Black rice grain was irradiated by gamma rays with a dose of 200 Gy and 300 Gy. The main purpose of this irradiation is to induce mutation to the black rice plant in order to achieve the improved organism. This study was undertaken to elucidate the morphological character and esterase isozyme pattern of black rice plant after irradiated by gamma rays. There were morphological differences on leaves, stems and grains between irradiated and non irradiated black rice plant. Gamma radiation dose of 200 Gy showed the significant influence of the length of the stem, number of internodes, and length of leaves. The radiation dose of 300 Gy showed the significant influence of the decrease value of diameter of 3rd internodes, number of branches and width of leaves. Flowering time is getting faster as increasing radiation dose. At the age of 74 days after planting there are 9.15% plants of 200 Gy radiation dose that have flowered faster than normal plants. This value increased into 11.45% at the dose of radiation 300 Gy. There were differences in the esterase banding pattern between radiation dose of 200 Gy and 300 Gy than the control plants, indicated that randomly mutation has occurred.
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The exploration, conservation and use of the genetic resources of plants is a contemporary issue which requires a multidisciplinary approach. Here the role of population genetic data, particularly those derived from electrophoretic analysis of protein variation, is reviewed. Measures of the geographic structure of genetic variation are used to check on sampling theory. Current estimates justify the contention that alleles which have a highly localised distribution, yet are in high frequency in some neighbourhoods, represent a substantial fraction of the variation. This class, which is the most important class in the framing of sampling strategies, accounts for about 20-30% of variants found in 12 plant species. The importance of documenting possible coadapted complexes and gene-environment relationships is discussed. Furthermore, the genetic structure of natural populations of crop relatives might suggest the best structure to use in the breeding of crops for reduced vulnerability to pest and disease attack, or for adaptation to inferior environments. The studies reported to date show that whilst monomorphic natural populations do occur, particularly in inbreeding colonisers, or at the extreme margins of the distribution, polymorphism seems to be the more common mode. It is stressed here that the genetic resources of the wild relatives of crop plants should be systematically evaluated. These sources will supplement, and might even rival, the primitive land races in their effectiveness in breeding programmes. We may look forward to a wider application of gel electrophoresis in the evaluation of plant genetic resources because this technique is currently the best available for detecting genetic differences close to the DNA level on samples of reasonable size.
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Leaf proteins of the 10 herbaceous species of the genus Datura were separated by electrophoresis in starch gels and stained to reveal peroxidases. A. total of 19 different sites of peroxidase bands was observed for the species as a whole. Developmental studies showed that the number of bands increased with age of the leaf during seedling growth as well as in more mature plants. Among species the number of band sites for one particular leaf (AL-4) varied from 4 to 9. Each species pattern was unique, and no single peroxidase band was common to all the species. Based on considerations of interspecific cross compatibility and chromosomal rearrangements the c assical taxonomic division of the genus into three sections was further subdivided to give five groups. The degree of phylogenetic relationship for species within vs. between these groups was assessed for the peroxidase patterns by use of statistical methods based on a hypergeometric distribution model. The probability that the observed degree of band matching was due to chance was less among species within a group than between groups, thus indicating a closer genetic association which is in agreement with the relationships arrived at by more conventional methods.
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Changes in soluble protein and in peroxidase, esterase, and amylase enzymes during development of the pea plant (P. sativum) have been investigated using a horizontal method of acrylamide gel electrophoresis. Both qualitative and quantitative variation between organs at different stages of physiological development are recorded.
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The majority of pistil peroxidases are involved in processes related to growth, development and senescence. Only the tissue specific peroxidases in the transmitting tissue of the style may play a direct role in the regulation of pollen tube growth. The pollen peroxidases may function mainly in growth regulation and tube wall formation and play a role in the interaction between pollen and pistil by metabolizing the phenolic compounds in the pistil.
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Data are concordant with the suggestion that L. maritima is relatively recently derived from L. minor. The switch from outcrossing to selfing and selecting of genotypes adapted to the chemically and physically unusual substrate on the seabird rocks are considered the critical steps in the evolution of L. maritima. - from Authors
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There are 15 populations of E. caesia Benth. on granite rocks in south-westem Australia which include a total of about 2120 plants. The level of genetic variation at 18 allozyme loci in 13 populations was estimated. Seven loci were monomorphic for all plants assayed. At a majority of the 11 polymorphic loci the level of polymorphism was very low in most populations. Within populations the mean number of alleles per locus was 1.31 and the genetic diversity 6.8%. However, populations differed markedly in allelic frequencies at a number of loci. The genetic diversity within populations was remarkably low for a tree species but the level of population differentiation was the highest reported for any tree species. The data suggest that genetic drift may in part have been responsible for the low overall genetic diversity and the extensive population differentiation. The optimal strategies for conservation of the genetic resources of this valuable ornamental are considered in the light of the results of this study.
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A simple, reliable and easily reproducible method, based on the use of isozyme polymorphisms, is described for distinguishing between the three morphological forms of Chondrilla juncea known to occur in Australia. The potential applications of these techniques are discussed.
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The plants of the Hawaiian silversword alliance area classic example of the process of adaptive radiation. The genetic basis of phenotypic variation among 23 taxa is proposed for the enzymes ADH, GA3PDH, GDH, G6PD, GOT, IDH, ME, PER, 6PGD, PGI, PGM and TPI. Dupiicate gene expression was observed for ADH, PGI, PGM and TPI, and is interpreted to be the result of polyploidy in the ancestor of the Hawaiian taxa.
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Gel electrophoresis was employed to assess genetic variation in progeny of plants from the one known population of Chenopodium sanctae-clarae Johow, a species endemic to the Juan Fernandez Islands. Gene duplications were detected for a number of the enzymes assayed. The chromosome number of 2n=36 for this species indicates that it is a tetraploid, and the extra isozymes reflect the polyploid nature of the plants. Minimal genetic variation was detected in the population, with only one allele per gene locus detected for all isozymes of seven of the 13 enzymes examined. For six enzymes, one or two alleles were detected for each locus. More than two alleles per gene were found for only one enzyme. The level of variation detected in this population is comparable to that found in many populations of widespread tetraploid species of Chenopodium occurring on mainland South America. It is, therefore, not possible to conclude that the lack of variation at isozyme loci in C. sanctae-clarae is the result of a founder event by its progenitor and/or small population size during the evolutionary history of the taxon.