Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

Sri Lankan J. Agric. Sci. Vol. 42 - 2005, 20 - 33

ISOZYMIC AND MORPHOLOGICAL CHARACTERIZATION OF

BRINJAL (SOLANUM MELONGINA L) ACCESSIONS AND
CULTIVARS AVAILABLE IN SRI LANKA

K.A.B. Pathmarajah and J.P. Eeswara

Department of Crop Science, Faculty of Agriculture,

University of Peradeniya, Sri Lanka

and

H. Fonseka

Horticulture Research and Development Institute, Gannoruwa,

Peradeniya, Sri Lanka.

SUMMARY

The potential use of electrophoretically detected isozymic variability complement

to the morphological characteristics in brinjal cultivar identification was assessed
by studying six enzymes among 16 brinjal accessions and 3 cultivars available in
Sri Lanka. Starch gel electrophoresis was used to analyse extracts prepared from
young leaf tissues of seven-day-old seedlings grown under green house
conditions. Enzymes, diaphorase, esterase and glutamate oxaloacetate
transaminase exhibited three banding patterns while two banding patterns were
observed for malic enzyme. Acid-phosphotase and malate dehydrogenase were
highly polymorphic with six and eight banding patterns respectively. Cultivar
specific isozymic banding patterns were observed for all the enzymes except malic
enzyme and glutamate oxalo acetate transaminase. Unique combinations of
isozymic variants of all enzymes assayed were able to differentiate thirteen
accessions and two cultivars (79%). The remaining accessions and cultivars
could be separated into two groups.

Nine morphological characters (growth habit, leaf blade colour, leaf blade
lobing, fruit curvature, fruit colour distribution, % of type of flowers per plant,
number of fruits per infruitescence and days to first harvest) and four
agronomical characters (net photosyntheis, chlorophyll content, leaf area index
and bacterial wilt incidence) were examined. Three cultivars and six accessions

20
 K.A.B. Pathmarajah et.al.

(53%) could be differentiated by combining qualitative morphological characters

and rest could be categorized into four groups.

All the cultivars and accessions could uniquely be distinguished by combining the
morphological characteristics with isozyme finger prints. Results of the present
study suggest that sufficient variability is present in brinjal to allow the use of
isozyme analysis as a system for cultivar identification thus complementing the
traditional methods currently in use.

INTRODUCTION

Brinjal (Solanum melongina L), which belongs to family solanaceae is one of the
most common and popular vegetable crops grown in Sri Lanka. Production of
brinjal in 1999 was around 74,204 mt and the land area planted was 10,048 ha
(Anon., 2000). Average yield of brinjal in Sri Lanka under farmers’ condition is 8
mt/ha while the potential yield is 15 mt/ha. Thus, the enhancement of productivity
through breeding is essential to achieve the potential yield to increase total
production and profit of brinjal.

At present, three recommended brinjal varieties, SM 164, BW 11 and Thinnevelly

purple (Jaffna purple), are being cultivated in Sri Lanka. Although, number of
brinjal accessions are conserved at Plant Genetic Resource Center they have not
been evaluated or identified todate. Therefore, evaluation of performance and
characterization of these accessions are very important in future crop
improvement as well as in seed certification programs.

A technique useful for variety identification should have several characteristics. It

should be relatively simple, quick, consistent and inexpensive. The test should
also allow immediate observation of off types. At present, morphological and
agronomical features are commonly used to differentiate brinjal cultivars.
However, identification based on phenotypic characters is unreliable since they
can be affected by environmental conditions (Nittler, 1973; Ansary and Smith,
1976; Hamil and Camlin, 1984, Gottschalk, 1985). Moreover, a cultivar must be
judged by an individual who possesses a thorough knowledge of the
characteristics of the particular cultivar at precise growth stage (Wagner and
MacDonald, 1982). For morphological characterization the plant must be grown
either to flowering or fruiting stage, which is space and time consuming.
Therefore, it is desirable to develop cultivar identification tests based on
biochemical techniques or molecular techniques in addition to morphological
characterizations.

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Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

Isozymes have proven to provide simple and reliable markers since they do not
change with environmental conditions. In addition, electrophoresis of plant
enzymes is more rapid than field-testing and can be detected with a small amount
of plant tissue extracts (Wagner and McDonald, 1982). With several work done in
Sri Lanka, isozyme electrophoresis has shown promise in the identification of
field crops such as soybean (Glycine max (L.) Merill) (Eeswara and Peiris, 1998),
cowpea (Vigna unguiculata L. Walp) (Eeswara and Peiris, 1994) and Mungbean
(Vigna radiata (L) Wilcezk) (Eeswara and Peiris, 2001). However, studies have
not been done on brinjal. Therefore, the present study was conducted to
investigate the feasibility of separating brinjal cultivars by isozyme
electrophoresis.

MATERIALS AND METHODS

The experiment consisted of two parts, a field experiment and a laboratory

experiment. The field experiment was carried out at the Horticultural Crop
Research and Development Institute (HORDI) at Gannoruwa, Peradeniya and the
laboratory experiments were conducted at the Department of Crop Science of the
Faculty of Agriculture, University of Peradeniya.

Isozyme electrophoresis

Brinjal seeds from 16 accessions and 3 cultivars (Table 1) were obtained from the
germplasm maintained at the Plant Genetic Resources Center, Peradeniya, Sri
Lanka and grown under green house conditions in the Department of Crop
Science, Faculty of Agriculture, University of Peradeniya. Fresh leaf tissue
(approximately 0.5 g) from seven days old individual seedlings was extracted as
described by Weeden, (1982). The homogenate was centrifuged at 2300 g for 20
min at 4°C and the clear supernatant was stored at -10 °C prior to electrophoresis.

Horizontal starch gels were prepared using 25g of hydrolysed potato starch in 220
ml of buffer containing 55 ml electrode buffer (0.065 N L-histidine, pH adjusted
to 6.5 with 0.007 M citric acid) and 165 ml deionised water (Weeden, 1982). Gels
were cast in a rectangular gel mould (16.5 x 13.0 cm) and allowed to cool for 1 h.
After cooling, the gels were covered with a plastic wrap to prevent desiccation
and left to set overnight at room temperature (28+3ºC). Filter paper wicks (0.8 x
0.5 cm No. 470, Schleicher and Schuell) were used to absorb samples of thawed
supernatants for insertion into a slit cut 4.0 cm from the cathodal end of the gel.
Supernatants from all the brinjal varieties were placed into the slit spaced at 1.5

22
 K.A.B. Pathmarajah et.al.

cm intervals. A wick containing red colour dye marker was placed at one edge of
the gel as a marker to follow isozyme migration.

A fresh electrode buffer was used for each electrophoretic run. The gel was placed
in an electrophoretic tank kept in a refrigerator at 4.0°C and a 50 mA constant
current was supplied for 20 min. The wicks were removed and electrophoresis
resumed at 40 mA constant current and continued until the dye marker migrated
7.5 cm from the point of wick insertion. After electrophoresis, 1 mm thick slices
were cut from the gels and isozymic banding patterns were observed for 6
enzymes by immersing the gel slices in staining solutions. Thereafter, the gel
slices were incubated at 37 °C for 1 h with the exception of esterase and glutamate
oxaloacetate transaminase where they were incubated at room temperature to
reveal enzyme activity. Recipes for the staining solutions were identical to those
given in Weeden (1982).

After incubation the staining solution was poured off and the gels were fixed
either with 50% ethanol or 50% glycerol : 5% acetic acid mixture. The gel slices
stained for acid phosphatase (ACP), esterase (EST) and glutamate oxaloacetate
transaminase (GOT), were fixed with 50% ehanol while the slices stained for the
remaining enzymes were fixed with 50% glycerol : 5% acetic acid mixture. The
banding patterns were visually recorded and schematic diagrams (zymograms)
were prepared for clearer presentation. The relative migration (Rf values) of each
band was measured in each zymogram for every cultivar tested using the
following equation:

Distance migrated by the enzyme

Rf Value = band from the cathodal edge .
Distance migrated by the dye marker

After preparing zymograms, scoring was done based upon a qualitative

interpretation of the banding patterns (number of bands and their Rf values) and
arbitrarily assigned a letter designation.

Morphological characterization

Five qualitative (growth habit, leaf blade colour, leaf blade lobbing, fruit
curvature, fruit colour (purple) distribution), four quantitative morphological
characters (percentage of types of flowers, number of flowers per inflorescence,
number of fruits per infruitescence and days to first harvest) and four agronomic
characteristics (rate of photosynthesis, chlorophyll content, leaf area index and
bacterial wilt incidence) were obtained by observations on field plot at

23
Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

Horticultural Research and Development Institute. Descriptors for vegetables and

condiments (Anonymous, 1994) was used to characterize the qualitative and
quantitative morphological characteristics.

A cluster analysis was conducted using SAS computer package to relate character
variations within cultivars and accessions.

RESULTS AND DISCUSSION

Though there are practical advantages in the use of seeds, which are in
metabolically stable stage, the first true leaves of seven-day-old seedlings were
used for isozyme analysis since a greater number of banding patterns can be
achieved with leaf materials (Nehara, Kartha and Stushnoff, 1991). In addition,
leaf tissues are preferred for isozyme analysis of plants because of their
availability throughout the growing season. However, the presence of high
amounts of phenols in green leaf tissues interfere with enzyme extraction resulting
in poor resolution and low reproducibility of results (Loomis, 1974). The
problems with phenols could be minimized if the seeds were germinated in the
absence of light in a growth chamber. However, in the present study seeds were
germinated in the presence of light (12 h light/ 12 h dark, 28+3 °C) in a green
house. The problems caused by phenols in the analysis of leaf isozymes of brinjal
could be resolved by using young leaf tissues and addition of insoluble PVP into
the extraction medium as described by Nehara et al., (1991).

The extraction procedure followed in this study was successful for resolving
isozyme banding patterns of six enzymes, esterase (EST), malate dehydrogenase
(MDH), diaphorase (DIAP), malic enzyme (ME), glutamate oxaloacetate
transaminase (GOT) and acid phosphotase (ACP), by employing only one
extraction and separation buffer. The banding patterns of these enzymes are
shown in Fig. 1. Two enzymes, isocitrate dehydrogenase (IDH) and alcohol
dehydrogenase (ADH) did not produce clean band resolution. Therefore, these
two enzymes were not used for cultivar/accession identification. However, it may
be possible to reveal isozyme-banding patterns for these two enzymes by using a
combination of extraction and separation buffers as demonstrated by Ramirez et
al., 1987.

The highest enzyme polymorohism was exhibited by malate dehydrogenase for

brinjal accessions and cultivars examined (Fig. 1a). Malate dehydrogenase,
exhibited eight different banding patterns of which MD4, MD5, MD6, and MD7
were unique to accessions 07148, Arkekeshu, variety BW11 and the accession

24
 K.A.B. Pathmarajah et.al.

08534 respectively (Table 1). Other varieties and accessions shared the MDH
isozymic patterns and therefore, could be categorized into four groups.

+ a) + b)
RF RF
.18
.17

.14
.13
.12 .12

.08 .08
.05 .05
.04
.00 - .00
MD MD MD MD MD MD MD MD A A A A A A

+
+ + +
RF c) RF d) RF e) RF f)

.14 .14 .14

.12 .12 .12 .12
.09 .09
.06
.05

.00
- - -
E E E D1 D2 D3 G1 G2 G3 M1 M2

Fig. 1. Diagrammatic Representation of Electrophoretic Banding Patterns of

Brinjal Cultivars.
a) Malate Dehydrogenase,
b) Acid Phosphotase,
c) Esterase,
d) Diaphorase,
e) Glutamate Oxaloacetate Transaminast and
f) Malic Enzyme

25
Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

The sixteen accessions and three cultivars of brinjal tested for acid phosphotase
activity produced six banding patterns (Fig. 1b) and could be categorized into six
groups (Table 1). The accessions, wild type and 07153 produces accession
specific banding patterns and thus they, could be exclusively identified.

It has been reported that esterase, is an excellent enzyme for use in taxonomic
studies (Kuhns and Fretz, 1978). This is consistent with brinjal and based on the
location of the bands 16 accessions and 3 cultivars could be categorized into three
groups (Table 1, Fig. 1c). Five accessions (09632, 08534, 07157, wild type and
arkekeshv) and one cultivar (BW 11) belong to first group in which bands were
located at Rf values of 0.01, 0.12 and 0.14. The accession Thammanna which,
exhibited bands at Rf values of 0.01 and 0.06 belonged to third group and the
banding pattern was specific for Thammanna. Remaining two cultivars (SM 164
and Thinnewelly Purple) and 10 accessions belong to the second group in which
bands were observed at Rf values of 0.01 and 0.12.

Sixteen brinjal accessions and three cultivars tested for diaphorase (Fig. 1d), and
glutamate oxaloacetate transaminase (Fig. 1e) activity displayed three banding
patterns. Two variety specific isozymic banding patterns were observed for
diaphorase while glutamate oxaloacetate transaminase did not produce any
cultivar specific banding patterns (Table 1).

The least number of bands were observed for malic enzyme activity. This enzyme
produced only one band (Figure 1f). However, depending on the location of the
band, brinjal cultivars could be divided into two groups. The first group (M1)
contained all three cultivars and 14 accessions while the second (M2) group
contained two cultivars 07157 and Arkekeshv (Table 1).

The results of the present study suggest that all enzymes studied are not necessary
to separate the 19 brinjal cultivars/accessions. They could be separated by
combining the isozymic patterns of four enzymes, i.e. esterase, malate
dehydrogenase, malic enzyme and diaphorase. If more cultivars are to be
identified, all the enzyme systems may be required. Thus, the isozymic
fingerprints were prepared for 19 cultivars/accessions examined and presented in
Table 1.

26
 K.A.B. Pathmarajah et.al.

Table 1. Grouping of Sixteen Brinjal Accessions and Three Cultivars Based

on Isozymic Patterns
Isozyme Accession/ Cultivar
MDH ACP EST GOT DIAP ME
MD1 A2 E2 G2 D3 M1 08891
04994
MD2 A1 E3 G2 D3 M1 THAMMANNA
E1 G2 D3 M1 09362
A2 E2 G2 D3 M1 SM164
A2 E2 G2 D3 THINNEWELLY
PURPLE
A3 E1 G2 D1 WILD TYPE
A5 E2 G2 D3 M1 EG014
07151
MD3 A4 E2 G2 D3 M1 07153
A5 E2 G2 D3 M1 SA7 MTE-2
MD4 A2 E2 G2 D3 M1 07148
MD5 A5 E1 G1 D3 M2 ARKEKESHU
MD6 A1 E1 G2 D3 M1 BW11
MD7 A1 E1 G2 D3 M1 08534
MD8 A5 E2 G2 D3 M1 EG090
A6 E1 G3 D3 M2 07157
E2 G1 D2 M1 07145
E2 G3 D3 M1 08505
Variety specific banding patterns and differentiated cultivars and accessions are
shown in bold letters.

Furthermore, the results of the present study suggest that adequate isozymic
variability exists in brinjal for cultivar identification. Unique combination of
isozyme variants of all enzymes assayed, identified two cultivars and thirteen
accessions (79%). This could be increased up to 100% by combining with

27
Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

morphological characteristics as discussed below. Banding patterns produced by

the enzymes were very stable and reproducible. Beside the stability and
reproducibility, the presence of sufficient isozymic variability among the brinjal
cultivars is an advantage in cultivar identification. Furthermore, time and space
requirement is less for electrophoresis compared to morphological
characterizations via field evaluation. However, isozymic characters are largely
independent of the agronomically important traits. Therefore, it is necessary to
assess whether the accession or cultivar maintains its uniformity and stability of
agronomically important characters via field evaluation.

Morphological characterization

The classical methods used for identifying brinjal cultivars are mainly based on
the phenotypic expressions of different plant parts. Therefore, in the present study
twelve morphological and four agronomic characteristics were observed to
differentiate brinjal cultivars and varieties (Table 2 and 3). Thinnavelly Purple
was affected by bacterial wilt destroying all the plants. Therefore, morphological
and agronomic characters were not recorded for this cultivar. Combining five
qualitative morphological characters, two cultivars (BW 11 and SM 164) and
seven accessions could be identified (Table 2). Remaining brinjal accessions
could be divided into four groups containing two accessions in each (Table 2).

Quantitative morphological characteristics and agronomic characteristics recorded

are shown in the tables 3 and 4. With the help of cluster analysis (Fig 2 and Table
3) two brinjal accessions, 04994 and EG014, could be identified.

In the present study phenotypic expressions such as leaf blade colour and fruit
colour distribution were assessed by visually. However, visual detection of colour
is considered as unsuitable for variety identification owing to possible
inaccuracies involved in colour determination depending on the factors such as
light quality, coloured object, matching of the plant colour with the standard
colour charts and the observer. Furthermore, 14 morphological and agronomic
characteristics observed in the present study were not adequate to differentiate 19
brinjal accessions and cultivars examined. Thus, isozyme electrophoresis can be
used as a system for cultivar identification complementing the traditional methods
currently used.

28
Table 2. Differentiation of Brinjal Cultivars and Accessions Based on Qualitative Morphological Characters
Growth Habit Leaf Blade Leaf Blade Lobbing Fruit Colour Fruit Curvature Accession/
Colour (Purple) Cultivar
Distribution

Upright (3) Green (3) Indeterminate (5) Stripped (7) Curved (5) 04994
Strong (7) Stripped (7) Straight (1) 07153
Violet (9) Indeterminate (5) Uniform (1) Slightly Curved (3) EG 014
Indeterminate (5) Green (3) Indeterminate (5) Uniform (1) Straight (1) 07145
07151
Curved (5) Thammanna
Stripped (7) Curved (5) 07157
08505
Slightly Curved (3) BW11
Strong (7) Uniform (1) Straight (1) 08534
SA 7 MTE2
Snake Shaped (7) EG 090
Arkekeshu
Slightly Curved (3) SM164
Stripped (7) Curved (5) 07148
Sickle shaped (8) 08891
Light Green (1) Strong (7) Stripped (7) Sickle shaped (8) 09362

Differentiated cultivars and accessions are shown in bold letters.

29
Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

Table 3. Quantitative Morphological and Agronomic Characters of Brinjal

Accession / % of % of % of true Number of Number of Net Chlorophyll Leaf % Wilt
Cultivar long styled pseudo short fruits per days to Photo- content Area affected
flowers short styled styled infrutescence first synthesis Index
flowers flowers harvest
04994 41.3 27.5 31 1 77 22.5 45.94 3.11 10
07145 25.3 46.6 26 1 77 26.94 45.18 2.80 20
07148 16.4 24.1 59.3 1 65 20.1 45.98 2.72 20
07151 48.6 38.7 12.6 1 65 21.96 41.76 1.98 20
07153 21.3 46.2 32.4 1 65 24.4 43.94 2.26 25
07157 51.5 17.7 30.6 1 65 23.54 43.92 3.25 5
08505 - - - 1 77 26.12 44.86 100
08534 27.6 34.6 37.6 1.9 65 27.36 42.14 85
EG 090 31.3 3.7 64.8 1.76 65 27.8 45.58 3.81 25
09362 42.8 8.5 20 1 65 23.3 41.20 1.12 100
08891 20.5 34.4 45 1 65 20.68 42.54 2.24 0
EG 014 65.5 34.4 0.0 4.2 77 14.94 57.50 2.08 0
SA 7 MTE2 19.6 25.5 54.7 1.28 65 26.62 41.64 2.50 10
Thammanna 57.4 18.5 24 1 65 25.42 43.24 2.68 95
Arkekeshu 35.5 6.8 57.6 2.12 77 28.82 43.84 2.99 0
BW11 34.9 21.3 43.6 1 77 27.26 45.10 3.12 30
SM164 55.5 20.6 23.8 1 77 27.52 44.82 65

30
 Similarity

15.14

43.42

71.71

100.00

4 6 3 1 2 9 16 5 8 13 11 12 7 10 15 17 14
Observations

Fig. 2. Dendrogram for Cluster Anaysis of Brinjal Cultivars and Accessions

sions

Table 4. Differentiation of Brinjal Cultivars and Accessions Based on

Morpholohical and Agronomic Characters
Cluster Number Accession/ Cultivar
1 1 04994
2 12 EG 014
3 2 07151
4 07145
4 8 08354
13 SA 7 MTE-2
16 Arkekeshv
5 3 07148
5 07153
6 07157
7 08505
9 EG 090
10 09362
11 08891
14 Thammanna
16 BW 11
17 SM 164

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Isozymic and Morphological Characterization of Brinjal Available in Sri Lanka

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