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F O R M A T T E D P R O O F
N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH, K.N. DHANRAJ AND R. GOWDA
* Author for correspondence
Nethra, N., Rajendra Prasad, S., Vishwanath, K., Dhanraj, K.N. and Gowda, R.
(2007), Seed Sci. & Technol., 35, 176-186
Identification of rice hybrids and their parental lines based on
seed, seedling characters, chemical tests and gel electrophoresis
of Total soluble proteins
N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH*, K.N. DHANRAJ AND R. GOWDA
Department of Seed Science and Technology, UAS, G.K.V.K, Bangalore-560065, India
(E-mail: vishwakoti@gmail.com)
(Accepted September 2006)
Summary
Identification of rice genotypes is of prime importance to ensure quality seed, which is required for achieving
global food demand. Three rice hybrids and their parental lines were identified on the basis of seed colour, seed
size and 1000 seed weight, response to different chemicals (Phenol, Modified phenol with Copper sulphate
or Ferrous sulphate, NaOH, GA3 and 2,4-D) and electrophoresis of soluble seed proteins (SDS-PAGE). All
the genotypes were identified by using key based on seed colour, 1000 seed weight and seed size except for
the female lines and their maintainers. No individual chemical test was able to distinguish all the genotypes.
However, the combinations of different rapid chemical tests were useful in identification of individual genotypes.
While all the cultivars were identified successfully by using electrophoresis of seed protein (SDS-PAGE) and
hence it could be used as a powerful tool to identify every genotype in a short period of time.
Introduction
Rice is the most important cereal grain of the world. It is estimated that half of the world’s
population depends on rice as its main source of food and Asia is considered as the
homeland of rice. India has the largest acreage under rice growing countries of world but
ranks second in production, owing to its lower productivity. To sustain its high production
and productivity, a number of high yielding varieties and hybrids have been developed
and notified in the recent past, out of which many varieties and hybrids are now in seed
production chain. The release of large number of rice hybrids has increased the task as
well as the responsibilities of seed technologists in order to ensure the quality of seed.
Seed technologists must be well equipped to identify different varieties and hybrids, both
at field and at seed level. Varietal descriptions given by the breeders most often relate to
field characters and not sufficient to identify genotypes or seed lot adequately. Frequently,
information is required rapidly, which can be only provided by identification at the seed or
seedling level or some chemical tests, which should be rapid, reliable and reproducible.

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Many researchers have used seed, seedling morphological characters and different
chemical tests for varietal identification. Electrophoresis is a relatively sophisticated reliable
and reproducible technique that has been used extensively by many workers for wheat,
soybean, barley, pearl millet, oat and other crop species for varietal identification (Grabe,
1957, Cooke, 1987; Vanangamudi et al., 1988; Varier, 1993; Cooke, 1993). The present
study was under taken to develop laboratory keys based on seed, seedling characters,
chemical test and electrophoretic analysis of total seed proteins for identification of some
rice hybrids and their parental lines.
Material and methods
Pure seeds of three elite rice hybrids, viz., KRH-2, APRH-2, and DRRH-1: their parental
lines (IR-58025A, IR-58025B, IR-62829A, IR-62829B, KMR-3R, MTU-9992R, and IR-
40750R) were obtained from Directorate of Rice Research, Hyderabad. Observations on
seed colour, seed size and 1000 seed weight were recorded. Fifty seeds of each genotype
were observed visually for seed colour with the aid of magnifying glass and Munsell soil
colour charts (Anon., 1954) were used for assigning seed colour classes. Seed length,
width and thickness were measured with the help of grain micrometer and seed size was
calculated by using the formula (l × w × t) 1/3
. Eight replications of 1000 seeds each were
used to calculate test weight.
Phenol test: The standard phenol test for varietal purity testing as suggested by Walls
(1965) was followed. Four replications of 100 seeds each were soaked in distilled water
for 24 hours. The seeds were then placed in petri dishes containing filter paper moistened
with 5 ml of 1% phenol solution and kept at room temperature (28°C) for 24 hours. After
that, the seeds were examined and grouped into different colour classes as no colour
change, light brown, brown and dark brown.
Modified phenol test with Copper sulphate or Ferrous sulphate: As described by Banerjee
and Chandra (1977), the procedure similar to the standard phenol test except that the
seeds were soaked in a solution of 0.5% CuSo4 or 1% FeSo4 instead of soaking the seeds
in distilled water. The seeds were examined and grouped into five distinct groups namely,
no colour change, light brown, brown, dark brown and black.
NaOH test: Four replications of fifty seeds each were soaked in 3% NaOH solution for 3
hours and thereafter the change in colour of the solution was observed. Based on intensity
of colour reaction, the genotypes were classified into three groups viz., no colour change,
light yellow and wine red.
GA3 test: Four hundred seeds were soaked in 25, 50 and 100 ppm GA3 and germinated
as per ISTA (1996). The shoot length was measured and the percentage increase in shoot
length over that of control was computed and different groups were made based on their
response viz., Low (<35%), Medium (51-75%), High (76-100%) and Very high (>100%).

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N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH, K.N. DHANRAJ AND R. GOWDA
2,4-D test: Four hundred seeds were soaked in 5 and 10 ppm 2,4-D and germinated as per
ISTA (1996). Observations were recorded on 7th
day in terms of decrease in shoot length
over that of control and genotypes were grouped as Tolerant (<50%), Susceptible (50-
60%) and Highly Susceptible (60%).
Electrophoretic technique of Total soluble seed proteins: SDS-PAGE of total soluble seed
proteins was carried out by using 15 per cent gels according to the methods prescribed by
Laemmli (1970) with slight modifications.
Protein was extracted from single seed by adding 0.2 ml Tris glysine extraction buffer (25
mM, pH 8.5). The suspension was centrifuged at 10000 rpm for 15 minutes. The extract
was dissolved in equal amount of working buffer (Tris-HCl 0.0625 M, pH 6.8, 2% SDS,
5% 2- mercaptoethanol, 15% glycerol and 0.001% bromophenol blue) and kept in boiling
water for 2 minutes, again centrifuged and the supernatant was used for loading on to the
gels. A current of 1.5 mA per well with a voltage of 80 V was applied until the tracking
dye crossed the stacking gel. Later the current was increased to 2 mA per well and voltage
up to 120 V. The electrophoresis was stopped when the tracking dye reached the bottom
of the resolving gel. Then the gel was stained using coomaasie brilliant blue solution
overnight and destained using a mixture of 227 ml of methanol, 46 ml of acetic acid and
227 ml of distilled water until the bands were clearly visible.
Results and discussion
The data for seed characteristics, presented in table 1, showed an ample amount of variation
for seed colour, seed size, and 1000 seed weight. Four types of seed colours were observed
in the genotypes studied viz., yellow (KMR-3R), yellowish white (IR-40750R), pale
yellow (MTU-9992R and IR-62829B) and very pale brown (IR-62829A, IR-58025A, IR-
58025B, KRH-2, APRH-2, DRRH-1). Pascual et al., (1983) and Ramaiah and Rao, (1953)
have also described different seed colour classes in rice seeds. Two groups were made
based on seed size namely medium (3.03-3.51; all the genotypes except MTU 9992R) and
large (>3.51; MTU 9992R). Based on the 1000 seed weight, the genotypes were grouped
into three different categories viz., light (<18.0g; IR-62829A) medium (18.1-23.0g; IR-
58025A, IR-58025B, IR-62829B, IR-40750R, KMR-3R, KRH-2, APRH-2 and DRRH-1)
and heavy (> 23.0g; MTU-9992R). A key based on the seed characters was constructed as
an aid for the identification of these hybrids and their parental lines (figure 4).
The genotypes were also grouped on the basis of the reaction of seeds to the various
chemical tests (table 2).
Standard Phenol test: The ten genotypes were grouped depending upon the intensity
of staining. Hybrid DRRH-1 stained dark brown, APRH-2 and IR-62829A stained light
brown, while rest of the genotypes stained brown. This test was unable to discriminate all
the cultivars.

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Table 1. Seed characters of rice hybrids and their parental lines.
Genotypes Seed Seed 1000 seed
colour Size (mm) weght (g)
Female lines IR 58025A Very pale brown 3.19 20.63
IR 62829A Very pale brown 3.32 17.61
Maintainers IR 58025B Very pale brown 3.36 19.45
IR 62829B Pale yellow 3.20 18.67
Restorer lines KMR-3R Yellow 3.51 19.00
MTU 9992R Pale yellow 3.67 23.90
IR 40750R Yellowish white 3.31 20.73
Hybrid KRH-2 Very pale brown 3.16 19.61
APRH-2 Very pale brown 3.04 18.59
DRRH-1 Very pale brown 3.22 18.65
S. Em ± 0.175 0.174
CD (0.05P) 0.524 0.519
Table 2. Changes in seed coat colour due to various chemicals in rice genotypes.
Genotype Standard Modified phenel test NaOH GA3 2,4-D
phenol test test test
test CuSo4 FeSo4
(0.5%) (1%)
IR 58025A B B B WR Low Susceptible
IR 62829A LB LB LB WR Medium Susceptible
IR 58025B B B DB LY Medium Susceptible
IR 62829B B B B LY Very high Tolerant
KMR-3R B B B WR High Susceptible
MTU 9992R B LB LB LY High Tolerant
IR 40750R B B BL LY Medium Susceptible
KRH-2 B BL B LY Medium Susceptible
APRH-2 LB B B LY Medium Susceptible
DRRH-1 DB BL BL LY Medium Susceptible
Note: NC – No colour change; BL – Black; LB – Light Brown; LY – Light Yellow; B – Brown;
WR – Wine Red; DB – Dark Brown

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N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH, K.N. DHANRAJ AND R. GOWDA
Modified Phenol test: The modified phenol test using Cu++
ions helped in further
subdividing the standard phenol groups (Jaiswal and Agarwal, 1995). Seeds of MTU992R
and APRH-2 with brown colour in standard phenol test were subdivided into light brown
and brown respectively by modified phenol test with Cu++
ions. Similarly, IR-58025A,
IR-58025B, KMR-3R and IR-40750R which were brown in standard phenol test were
further subcategorized into brown (IR-58025A and KMR-3R), dark brown (IR-58025B)
and black (IR-40750R) by using Fe++
ions.
NaOH test: Based on the NaOH test, genotypes could be divided into two groups: Reddish
brown (IR-62829B, MTU-9992R and APRH-2) and light yellow (rest of the genotypes
studied) (figure 1).
GA3 test: The present study, GA3 (25, 50 and 100ppm) was used and its effect on increase
in shoot growth over control was studied (Goyal and Baijal, 1980; Bansal et al., 1992).
Based on the response to 100ppm concentration it was possible to categorize the genotypes
into different groups; Low i.e. < 50% (IR-58025A), medium i.e. 51-75 % (IR-62829A,
IR-40750R, KRH-2, DRRH-1 and APRH-2), high i.e. 76-100 % (KMR-3R and MTU-
9992R) and very high i.e. > 100% (IR-62829B) (figure 2).
Figure 1. Response of rice genotypes to NAOH test.
Figure 2. Response of rice genotypes to GA3 (100ppm).

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Figure 3. Response of rice genotypes to 2, 4-D (10ppm).
2,4-D test: Based on the response to 2,4-D (5 and 10 ppm) shoot length inhibition, the
genotypes categorized into two groups; tolerant i.e. < 30% reduction over control (IR-
62829B, and MTU-9992R) and susceptible i.e. > 60% reduction over control (rest of the
other genotypes). The difference in reduction in seedling growth genotypes might be due
to differential ethylene production up on application of 2,4-D (Sundaru et al., 1983).
Based on seed colour genotypes KMR-3R (Yellow) and IR-40750 (Yellowish white)
could be distinguished from other genotypes. IR-62829B and MTU-992R showed pale
yellow seed colour and these two genotypes could be further distinguished by 1000 seed
weight (figure. 4). Most of the genotypes could be identified by using key based on seed
colour, 1000 seed weight and seed size except for female lines and their maintainers.
This showed that based on seed characteristics it is difficult to identify all the genotypes
unambiguously.
The genotypes IR-58025A and IR 58025B that showed same seed colour, 1000 seed
weight and seed size, could be distinguished clearly by their response to modified phenol
test with FeSO4 (figure 5). Further, all the genotypes could be grouped in to two different
categories based on response to NaOH. However, by standard and modified phenol tests
each cultivar was sub categorized and identified. Response of seeds to different chemicals
helped in grouping the genotypes rather than clear cut differentiation from each other.
No individual chemical test was efficient in distinguishing the genotypes individually.
However, they can use as supplement to each other.
The soluble seed proteins of rice inbreds/hybrids were extracted and separated by
an SDS-PAGE method. Based on the strongly staining bands in the central region of the
electrophorograms, it was possible to characterize all the genotypes individually (figure
6 and 7).

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N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH, K.N. DHANRAJ AND R. GOWDA
Total soluble seed proteins could be fractionated into 28 bands, which showed
heterogeneity among the genotypes. The highest number of bands (18) was observed in
APRH-2 (figure 7). Regions C (43 KD), D (29 KD) and E (20 KD) were found useful
to identify most of the genotypes studied, as the banding pattern was distinct for each
of the genotypes in these regions. The cultivars differed in the number of bands, their
mobility (position) and intensity. The results on the banding pattern of the protein profiles
suggested that the specific genotype could be differentiated by either based on either the
position or intensity of bands but not on number, as some of the genotypes expressed
similar number of bands (table 3). These results are inline with results of Chauhan and
Nanda (1984).
The results of the present study suggests that the genotypes of rice examined can be
characterized, distinguished and identified by combination of seed, seedling characters
and chemical tests. However, these need extended time to perform all these tests. In the
present study all the cultivars were identified successfully by using electrophoresis of seed
protein and hence it could be powerful tool to identify every genotype in a short period
of time.
Seed Colour
1000 Seed weight 1000 Seed weight
Seed size
Medium
IR-58025A&
IR-58025B
Light
IR-62829A
Medium
IR-58025A&
IR-58025B
Yellow
KMR-3R
Yellowish white
IR-40750R
Pale Yellow
IR-62829 B
MTU-9992R
Very pale brown
IR-62829A, APRH-2, KRH-2,
DRRH-1, IR-58025A, IR-58025B
Medium
IR-62829B
High
MTU-9992R
Figure 4. Identification of rice genotypes based on seed and seedling characters.

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NaOH test
Reddish brown Light Yellow
IR 62829B, MTU 9992R IR-58025A, IR-58025B, KMR-3R,
& APRH-2 IR-40750R, KRH-2 & DRRH-1
Standard Phenol test Standard Phenol test
Light Brown Brown Light Brown Brown Dark brown
IR 62829B MTU 9992R & APRH-2 IR-62829A IR 58025A, IR 58025B, DRRH-1
KMR-3R, IR-40750R & KRH-2
Modified Phenol test with CuSo4 Modified Phenol test with CuSo4
Light Brown Brown Brown Dark brown
MTU 9992R APRH-2 IR 58025A, IR 58025B, KRH-2
KMR-3R & IR 40750R
Modified Phenol test with FeSo4
Brown Dark brown Black
IR 58025A IR 58025B IR 40750R
& KMR-3R
GA3 test
Low High
IR 58025A KMR 3R
Lane 1: APRH-2, 2: IR-62829A, 3: IR-62829B, 4: MTU-9992R, 5: KRH-2, 6: IR-58025A, 7: IR-58025B,
8: KMR-3R, 9: DRRH-1, 10: IR-58025A, 11: IR-58025B, 12: IR-40750R, M: Standard Protein Marker.
1 2 3 4 5 6 7 8 9 10 11 12 M
Figure 5. Identification of rice genotypes based on chemical tests.
Figure 6. SDS-PAGE for rice genotypes.

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N. NETHRA, S. RAJENDRA PRASAD, K. VISHWANATH, K.N. DHANRAJ AND R. GOWDA
Table3.PresenceofseedproteinbandsatdifferentRelativemobility(Rm)inricehybridsandtheirparentallines.
BandNo.RmAPRH-2IR-62829AIR-62829BMTU-9992RKRH-2IR-58025AIR-58025BKMR-3RDRRH-1IR-40750R
280.039+++++++-++
270.059+++++++-++
260.084++-+------
250.107--------+-
240.119--------+-
230.238+++-------
220.259++++++++++++++++++++
210.283+++-+++-+-
200.298++++++++++++++++++++
190.333--------+-
180.358----------
170.388++++++++++++++++++++
160.411----------
150.435++++++++++++++++++++
140.456+++++++-+-
130.468+++-------
120.498+---------
110.522--+-------
100.552--+++-----
90.589----------
80.664----------
70.688++++++++++++++++++++
60.725++++++++++++++++++++
50.755+-------++
40.814----++----
30.871+++++++-+-
20.925++++++++++++++++++++++++++++++
10.97++++++++++
TotalNo.ofbands1816171415141381711
+:weak;++:medium;+++:highintensitybands

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Lane1:APRH-2,2:IR-62829A,3:IR-62829B,4:MTU-9992R,5:KRH-2,6:IR-58025A,7:IR-58025B,8:KMR-3R,9:DRRH-1,10:IR-58025A,
11:IR-58025B,12:IR-40750R,M:StandardProteinMarker.
Figure7.Electrophorogramsoftotalsolubleseedproteinofricegenotypes.
TotalNo.
ofbands18161714151413817141311
REGIONS
A97,000Daltons
(Phosphorylaseb)
B68,000Daltons
(Bovineresumalbumin)
C43,000Daltons
(Ovalbumin)
D29,000Daltons
(Carbonicanhydrase)
E20,000Daltons
(Soybeantrypsininhibit)
F14,300Daltons
(Lysozyme)
01
02
03
04
05
06
07
08
09
10
28––
27––
26––
25––
24––
23––
22––
21––
20––
19––
18––
17––
16––
15––
14––
13––
12––
11––
10––
9––
8––
7––
6––
5––
4––
3––
2––
1––
BANDNUMBERS
+123456789101112M

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Acknowledgements
Authors would like thank Indian Council of Agricultural Research (ICAR), New Delhi
for providing grants and Directorate of Rice Research (DRR), Hyderabad, for providing
seed material.
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