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CALLUS INDUCTION OF BRINJAL BY GENOTYPE AND GROWTH

REGULATORS

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J. Expt. Biosci. 5(2):35-42, July 2014 ISSN 223-9626 (Online). ISSN 2077-3358 (Print)

CALLUS INDUCTION OF BRINJAL BY GENOTYPE AND GROWTH REGULATORS

M. A. Alim1, B. K. Biswas2, M. Hasanuzzaman2, Pronay Bala3*and U. K. Roy4
*Corresponding Author; E-mail:kbdpronay@yahoo.com

Abstract

The investigation was carried out in Tissue Culture Laboratory of the Department of Genetics
and Plant Breeding, Hajee Mohammad Danesh Science and Technology University, Dinajpur
during the period from September, 2011 to January, 2012 with a view to study the effect of
genotypes and growth regulators on callus induction of Brinjal (Solanum sp.). Three genotypes
of Solanum viz. Protab, Green Ball and Ghemma Begun (Local) were used to assess their
regeneration ability. Leaf segments of three genotypes of Solanum were cultured on MS
medium with different concentrations and combinations of hormones. Callus induction was
ranged from 66.00 to 82.67% among the genotypes, Protab showed early callusing (7.533 days)
with maximum rate of callus induction (82.67%). The highest percentage of callusing appeared
in MS + 3 mg L-1 2,4-D + 0.05 mg L-1 BAP considering all genotypes. Based on the overall
performance, the genotype Protab appeared as the best for number of callus initiation (16.53).

Key words: Callus induction, Brinjal genotype and Growth regulators.

Introduction

Brinjal (Solanum melongena L.), belongs to the family Solanaceae, is one of the most popular, palatable
and nutritious vegetable crop in Bangladesh. It is thought to be originated in Indian sub-continent with
the secondary centre of origin in China (Zeven and Zhukovsky, 1975). It is known as eggplant,
aubergine, melongena and Gunia squats in different countries of the world. Eggplant is native to India
and China and was probably introduced to Europe by Arabic traders and then brought to North America
by early European settlers .The area of brinjal cultivation in Bangladesh is 45.57 thousand hectare and
production is 3.40 lac metric tons (BBS, 2011). It also plays a vital role in the national economy as a
cash crop. Brinjal is an economically important vegetable comprising an imperative supply of dietary
protein, carbohydrate, vitamin and mineral particularly for the vegetarian population of developing
countries. It can be cultivated as year round but the productivity and quality of this crop suffer due to its
susceptibility to a number of diseases and insect pests (Sadilova et al, 2006).
This problem has been addressed by hybridizing eggplant with wild resistant Solanum species, which
present a wide genetic diversity and are source of useful agronomic traits. The application of in vitro
methodologies to eggplant has resulted in considerable success. Eggplant tissue present a high
morphogenetic potential that is useful for developmental studies as well as for establishing
biotechnological approaches to produce improved varieties, such as embryo rescue, in vitro selection,
somatic hybridization and genetic transformation. The ability of eggplant regeneration in tissue culture
has allowed of somaclonal variation, haploidy, somatic hybridization and genetic transformation of
eggplant (Collonier et al., 2001).
Therefore, the present study was undertaken to investigate the in vitro regeneration of brinjal for
probable genetic manipulation of the crop by plant biotechnological approaches.

1
Assistant Production Manager, ACI (Seed) 2Professor 3Ph. D Fellow, Dept. of Crop Physiology & Ecology, Hajee
Mohammad Danesh Science & Technology University (HSTU), Dinajpur, 4Deputy Production Manager, Supreme
Seed Company Ltd., Bangladesh.

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Alim et al.

Materials and Methods

The experiment was carried out between September, 2011 and January, 2012 in the Biotechnology
Laboratory at the Department of Biotechnology of Hajee Mohammad Danesh Science and Technology
University, Dinajpur. The seeds of Solanum genotypes were collected from A.R. Mallik Seeds Co.
Metal Agro Ltd. and Localy from Dinajpur. The following Culture media were used in the present
investigation depending on specific purposes as mentioned below-

A. For seed germination

MS (Murashige & Skoog, 1962) medium
B. For callus induction
T1 = MS medium containing 3 mg L-1 2, 4-D + 0.05 mg L-1 BAP
T2 = MS medium containing 2 mg L-1 2, 4-D + 0.01 mg L-1 BAP
T3 = MS medium containing 1.5 mg L-1 2, 4-D + 1 mg L-1 BAP
T4 = MS medium containing 1mg L-1 2, 4-D + 0.01 mg L-1 BAP
T5 = MS medium containing 0.5 mg L-1 2, 4-D + 0.01 mg L-1 BAP
C. For shoot regeneration
T1 = MS medium containing 1 mg L-1 NAA + 0.1 mg L-1 BAP
T2 = MS medium containing 1.5 mg L-1 NAA + 0.5 mg L-1 BAP
T3 = MS medium containing 1.5 mg L-1 NAA + 1 mg L-1 BAP
T4 = MS medium containing 1 mg L-1 NAA + 1.5 mg L-1 BAP
T5 = MS medium containing 0.5 mg L-1 NAA + 2 mg L-1 BAP

Preparation of culture media

For the induction of callus and regeneration of plantlet in brinjal, a number of culture media have been
advocated by different scientists MS (Murashige & Skoog, 1962) medium was used for investigating
the present piece of work. A nutrient medium consists of organic and inorganic salts, irons, a carbon
source, some vitamins and growth regulators. Based on the types of explants, different media along
with different concentrations of hormone were used.

Preparation of stock solutions

The first step in the preparation of the medium was the preparation of stock solutions. The various
constituents of the medium were prepared into stock solutions for ready use to expedite the preparation
of the medium. Separate stock solutions for macro-nutrients, micro-nutrients, iron, vitamins, growth
regulators etc. were prepared and stored appropriately for use.
i) Stock solution A (Macro-nutrients)
For preparation, of this stock solution 10 folds (10 x) of the particular salt required per liter of the
medium was weighed accurately and dissolved in 750 ml of distilled water. The final volume was made
up to 1000 ml by further addition of distilled water. The stock solution was filtered and poured into a
sterilized bottle and stored in a refrigerator at 4ºC for later use.
ii) Stock solution B (Micro-nutrients)
The preparation of micro-nutrients stock solution 100 folds (100x) of the particular salt required per
liter of the medium was weighed accurately and dissolved in 750 ml distilled water. The stock solution
was made up to the mark (1000 ml) by further addition of distilled water. The stock solution was
filtered, labeled and stored in a refrigerator at 4ºC for later use.
iii) Stock solution C (Iron source)
This was prepared at 10 folds (10 x) the final strength of FeSO4 and Na2EDTA in distilled water and
dissolved by heating on a heater cum magnetic stirrer. Then the volume was made up to 1000 ml by
further addition of distilled water. Finally, the stock solution was filtered and stored in a refrigerator at
4º C for further use.

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J. Expt. Biosci. 5(2):35-42, July 2014 ISSN 223-9626 (Online). ISSN 2077-3358 (Print)

iv) Stock solution D (Vitamins)

Each of the desired ingredients except myo-inositol was taken at 100 folds (100x) of their final strength
in a measuring cylinder and dissolved in 400ml of distilled water.Then the final volume was made up to
1000 ml by further addition of distilled water. The solution was dispensed into 10 ml aliquots and
stored at -20º C. Myo-inositol was used directly at the time of media preparation.

Hormonal stock solution for callus induction media

Stock solution of hormones were prepared separately at 100 ppm by dissolving the 100 mg of
ingredients in appropriate solvent and made the final volume of 1 litre with distilled water and stored in
a refrigerator at 4ºC for later use. The following growth regulators were used in the present
investigation.
Auxins:
2,4-Diclorophenoxy acetic Acid (2,4-D)
-Naphthalene acetic acid (NAA)
Cytokinins :
6-Benzyl amino purine (BAP)
IBA (Indole butyric acid)
For the preparation of stock solution of the above mentioned hormones, 25 mg (concentrated) of each of
the hormones was taken on a watch glass with 1 ml of the particular solvent. The mixture was then
collected in a 250 ml measuring cylinder and the volume was made up to the mark by further addition
of distilled water. The solution was then stored at 4ºC for use up to two weeks.

Preparation of culture media

MS medium was used as cultural media. To prepare 1L of MS (Murashige and Skoog, 1962) medium,
the following steps were followed:
About 400 ml distilled water was taken in a flask.100 ml of macro-nutrients, 10 ml of micro-nutrients,
100 ml of irons and 10 ml of vitamins were taken from each of these stock solutions into a 2-litre
Erlenmeyer flask on a heater cum magnetic stirrer.100 (One hundred) mg of myo-inositol was added
directly to the solution and dissolved. Thirty g of sucrose was added to this solution and gently agitated
to dissolve completely. The solution was poured into a 1000 ml measuring cylinder and the volume was
make up to 1000 ml with addition of distilled water. This solution was poured into 8 beakers containing
125 ml of the solution in each beaker. Required volume of hormones solution was directly added to the
solutions in the beakers. pH of the medium was adjusted to 5.8 with the addition of 0.1 N NaOH or 0.1
N HCl. After adjusting the pH, 9 g of agar was divided into 8 groups and in each solution, 1.12 g of agar
was added. Each mixture was then gently heated in a hot plate magnetic stirrer till complete dissolution
of agar took place. Care was taken so that the solution did not get boil while melting agar. Required
volume of hot medium was dispensed into culture vessels. After dispensing and proper cooling of the
medium, the culture vessels were plugged with cork and non-absorbent cotton and marked with
different codes with the help of a glass marker to indicate specific hormonal combinations and then
autoclaved at 1210C and 15 psi for 15 minutes.

Sterilization of culture media

To ensure aseptic condition under in vitro, all instruments, glassware and culture media were sterilized
by autoclaving.
Sterilization of culture medium
The glass vials contained the medium were autoclaved with 22 g cm-2 of pressure at 121ºC for 20
minutes. After autoclaving the culture vessels were allowed to cool under normal condition.
Sterilization of glassware and instruments
Beakers, test-tubes, conical flasks, pipettes, metallic instruments, like forceps, scalpels, needles and
spatula were sterilized in an autoclave at a temperature of 121ºC of 40 minutes at 22 g cm-2 pressure.

37
Alim et al.

Sterilization of culture room and transfer area

The culture room was initially cleaned by gentle washing with a detergent followed by wiping with
70% ethyl alcohol; the process of sterilization was repeated at regular intervals. Generally, laminar air
flow cabinet was sterilized by wiping the working surface with absolute ethyl alcohol and with the help
of UV light.

Precaution of aseptic condition

All inoculation and aseptic manipulations were carried out in a Laminar Air Flow Cabinet. The cabinet
was exposed on the UV light for 30 minutes before use and cleaned with 90% ethyl alcohol to minimize
the chance of contamination. The instruments like scalpels, forceps, needles etc. were sterilized by an
alcoholic dipping and flaming method inside the Laminar Air Flow Cabinet, Others requirements like
petridishes, distilled water and glassware were sterilized by an autoclave. Aseptic conditions were
maintained during each and every operation to minimize the chance of contamination.

Sterilization of seeds
Healthy seeds were washed thoroughly in running tap water. The floated seeds were discarded and
others were rinsed in 70% ethyl alcohol for one minute, and then thoroughly washed with sterilized
water. The alcohol treated seeds were immersed into 0.1% HgCl 2 solution for 8-10 minutes, few drops
Tween-20 per 100 ml was also added at that time. The seeds were then washed 5-6 times aseptically
with sterilized distilled water.

Culture method
The following culture methods were employed in the present investigation.
i) Axenic culture
ii) Callus induction and
iii) Subculture
i) Axenic culture
Sterilized seeds were placed into seed germination medium in vials. Six seeds were placed in each vial.
The culture was then incubated in dark till the germination of seeds. These were then transferred to 16
hours light for normal seedling growth. 21 days old seedlings were used as source of contamination free
explants.
Explant culture
Leaf segment from each germinated seedling were cut into small pieces using sterilized scalpel under a
Laminar Air Flow cabinet. Four pieces of leaf segments were arranged on each vials and gently pressed
into the surface of the sterilized culture medium with various combinations and concentrations of
growth regulators.
The culture vials containing explants were placed under fluorescent light in a room with controlled
temperature (22ºC  2) using 16 hours photoperiod.
ii) Subculture
a) Subculture of the callus for shoot regeneration
After 20-25 days of incubation of explants, the calli about 20-25 mm in diameter, were removed
aseptically on a sterilized vials and cultures on freshly prepared medium containing the different
hormonal combinations of BAP and NAA for shoot induction.
b) Subculture of the regenerated shoot for root induction
The sub cultured calli continued to proliferate and differentiated into shoots. When these shoots grew
about 2-3 cm in length, they were rescued aseptically from the culture vials and separated from each
other and again cultured on freshly prepared root induction medium to induce root.

Data collection:
The effect of different treatments and response of different varieties to callus induction and plant
regeneration, data were recorded under the following parameters.

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J. Expt. Biosci. 5(2):35-42, July 2014 ISSN 223-9626 (Online). ISSN 2077-3358 (Print)

Callus induction
a) Days to callus initiation
Generally callus initiation started seven days of inoculation of explant. The number of callus initiated
over a number of days was recorded.
b) Percent callus induction
Percent callus induction was calculated on the basis of the number of explant placed and the number of
callus induced.
Number of explants induced calli
Percent callus induction = ×100
Number of explants incubated

c) Nature of callus: After twenty days of incubation, nature of callus was recorded and graded as 3 for
compact texture 2 for friable texture and 1 for loose texture.
d) Abundance of callus:
After twenty days of incubation, abundance of callus was recorded and graded as 3 for plenty, 2 for
moderate and 1 for poor.

Statistical analysis of data

The data for characters of the present study were statistically analyzed wherever applicable. The
experiments were arranged in Completely Randomized Design. The analysis of variance for different
parameters was performed and means were compared by Least Signification Difference (LSD) at 5%
level of significance.

Results and Discussion

In vitro regeneration of plant from induced calli offers the unique facilities of reproducible protocol as
well as recovery the somaclonal variants that can be utilized for the future crop improvement programs.
The ultimate goal of this experiment was plant regeneration via unrecognized calli derived from
explants. Callus initiation was the first step for successful plantlet regeneration of Solanum sp. The
three genotypes of Solanum sp. were taken under consideration and leaves from the fifteen days old
seedlings were used a explants and implanted on MS medium supplemented with different
concentrations of auxin (2,4-D) and different concentration of (BAP) to assess their callus inducing
potentiality. No. of explants showing callus showed significant mean square values for both variety and
treatment and their interaction also showed significant at 1% level of probability (Table 1).
Days required for callus initiation showed a highly significant difference against variety and treatment
(Table 1). Percentage of callus induction showed significant differences in variety, level of hormonal
treatment and their interaction also.

Table 1: Effect of varieties on callus induction

Total no. of % callus Days required Colour of Nature of Abundance

Variety Callus induction for callus callus callus of callus
induction initiation
Protab 16.53 82.67 7.533 Creamy Friable Moderate
Green Ball 13.20 66.00 9.600 Creamy Friable Moderate
Ghemma Begun
(Local) 14.87 74.33 8.267 Creamy Compact Plenty
LSD(0.01) 0.7620 3.811 0.5984 - - -
CV (%) 6.87% 6.87% 9.12% - - -

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Alim et al.

Effects of genotypes on callus induction

Mean values of genotypes on callus inducing characters like total no. of callus induction, % callus
induction and days required for callus initiation, colour of callus, nature of callus, and abundance of
callus were found statistically significant. The results of the different genotypes observed in the present
study are presented in Table 2. Leaf explants started callus initiation by changing their shape after six
days of incubation, and callus formation was completed within thirty days of incubation. The highest
percentage (82.67%) of callus induction was found in of Protab followed by local Ghemma Begun
(74.33%) and Green Ball (66.00%). Protab started callus initiation early (7.533 days) in comparison to
other genotypes such as local Ghemma Begun (9.267 days) and Green Ball (9.600 days). Callus
initiation was late in Green Ball (9.600 days). From the above results, it may be concluded that Protab
showed the best performance on callus induction.

Effects of phytohormones on callus induction

Total no. of callus induction, percent callus induction and days required to callus initiation, colour of
callus, nature of callus, abundance of callus showed significant differences among the hormonal
treatments (Table 2). Callus induction was highest (87.22%) in MS +3 mg L-1 2,4-D+0.05 mg L-1 BAP
(T1)treatment (Figure1) and lowest (65.56%) in MS+0.5mg L-1 2,4-D + 0.01 mg L-1 BAP(T5 ) Ms
medium supplemented with 2,4-D (0.5 mg L-1 and BAP (0.01 mg L-1 retarded callus initiation (10.22
days) among the treatment under study. From the above results, it may be concluded that MS +3 mg L-
1
2, 4-D+0.05 mg L-1 BAP ( T1) showed the best performance on callus induction. This result was in
agreement with those obtained by Jayasree et al (2001).

Table 2: Effect of treatment on callus induction

Days
Total no. of
% callus required for colour of Nature of Abundance of
Treatment Callus
induction callus callus callus callus
induction
initiation
T1 17.44 87.22 7.556 Creamy Compact Moderate
T2 14.89 74.44 7.889 Creamy Friable Moderate
T3 15.67 78.33 8.222 Creamy Friable Moderate
T4 13.22 66.11 10.11 Creamy Compact Moderate
T5 13.11 65.56 10.22 Yellow Loose Poor
LSD (0.05) 0.9837 4.919 0.7726 - - -
CV(%) 6.87 9.12 6.87 - - -
Here, T1=MS medium containing 1 mg L-1 NAA + 0.1mg L-1 BAP,T2 = MS medium containing 1.5 mg L-1 NAA
+ 0.5 mg L-1 BAP,T3 = MS medium containing 1.5 mg L-1 NAA + 1 mg L-1 BAP, T4 = MS medium containing 1
mg L-1 NAA + 1.5 mg L-1 BAP,T5 = MS medium containing 0.5 mg L-1 NAA + 2 mg L-1 BAP

Hormone × genotype interactions on callus induction

The maximum no. of explants showing callus was found in Protab genotype with (T1) MS +3 mg L-1
2,4-D+0.05 mg L-1 BAP. Protab with the highest percentage (93.33%) of callus (Table 3). The
genotypic differences for hormone levels can be evidenced from the callus quality also (Figure 2).
Above results showed that callus induction was highest in Protab (82.67%) and lowest in Green Ball
genotype (66.00%). Protab took minimum days (7.53) for callus initiation, while Green Ball took
maximum days (9.60). MS medium supplemented with 3 mg L-1 2, 4-D +0.05 mg L-1 BAP was the
best medium for both callus induction and initiation at shortest time. Callus induction gradually
increased with increasing 2, 4-D concentrations with different concentrations of BAP. The interaction
between Protab and MS medium supplemented with 3 mg L-1 2,4-D+0.05 mg L-1 BAP showed best
performance for callus induction with shortest initiation days (7.533 days).
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J. Expt. Biosci. 5(2):35-42, July 2014 ISSN 223-9626 (Online). ISSN 2077-3358 (Print)

Table 3: Combined effect of variety and hormone concentrations on callus induction

Number %callus Days Colour Nature of Abundance of

of formation required of callus callus callus
Hormone × genotype explants for callus
showing initiation
Callus
Protab 18.67 93.33 6.000 Creamy Compact Plenty
T1 Green Ball 18.00 90.00 8.000 Creamy Compact Moderate
Ghemma
15.67 78.33 8.000 Yellow Friable Moderate
Begun (Local)
Protab 14.67 73.33 9.67 Creamy Compact Moderate
T2 Green Ball 14.33 71.67 8.33 Yellow Loose Poor
Ghemma
15.00 75.00 8.67 Creamy Compact Moderate
Begun (Local)
Protab 16.00 80.00 9.00 Creamy Compact Moderate
T3 Green Ball 17.33 86.67 6.67 Yellow Friable Moderate
Ghemma
13.67 68.33 9.67 Creamy Friable Poor
Begun (Local)
Protab 14.67 73.33 9.67 Creamy Compact Plenty
Green Ball 11.33 56.67 11.00 Creamy Compact Plenty
T4
Ghemma
11.67 58.33 10.67 Creamy Compact Moderate
Begun (Local)
Light
Protab 14.67 73.33 9.67 Compact Plenty
green
T5 Green Ball 14.67 73.33 10.00 Creamy Compact Moderate
Ghemma
10.00 50.00 11.00 Yellow Friable Moderate
Begun (Local)
LSD (0.05) 1.70 8.52 1.34 - - -
CV (%) 6.87 6.87 9.12 - - -
Here, T1=MS medium containing 1 mg L-1 NAA + 0.1mg L-1 BAP,T2 = MS medium containing 1.5 mg L-1 NAA
+ 0.5 mg L-1 BAP,T3 = MS medium containing 1.5 mg L-1 NAA + 1 mg L-1 BAP, T4 = MS medium containing 1
mg L-1 NAA + 1.5 mg L-1 BAP,T5 = MS medium containing 0.5 mg L-1 NAA + 2 mg L-1 BAP

Fig.1 Development of callus from Fig.2 Development of callus from

Protab Variety using MS + 3 mg Ghemma Begun (Local) Variety
L-1 2,4-D + 0.05 mg L-1 BAP -1
using MS + 1.5 mg L 2,4-D + 1
-1
mg L BAP

41
Alim et al.

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