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Romanian  Biotechnological  Letters 

Vol. 14, No. 4, 2009, pp. 4519-4523 

Copyright © 2008 Bucharest University  

Printed in Romania. All rights reserved 

Romanian Society of Biological Sciences 



ORIGINAL PAPER 

4519


The Effects of Different Culture Media on the Callus Production of Radish 

(Raphanus sativus L.)

Received for publication, February 22, 2009 

Accepted, June 20, 2009 

MOHAMMAD ALI AAZAMI

1

*, MOHAMMAD BAGHER HASSANPOURAGHDAM

1

*

1

Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh 

55181-83111, Iran. Email: Aazami58@gmail.com

Abstract

This study was conducted to investigate the effects of different genotypes, explants and culture 

media on the callogenesis, calli growth and regeneration of radish plants. To do this, four varieties of 

radish (‘Vermal’, ‘Chuhong’, ‘Caudatus’ and ‘Longipinnatus’), three culture media with different 

hormonal combinations based on MS medium as A (without hormone or control), B (2, 4-D 1 mgL

-1

and BAP 2 mgL

-1

) and C (2, 4-D 2 mgL

-1

 and BAP 1 mgL

-1

) as well as three explant types: hypocotyl, 

cotyledon and root were employed. After one month the callogenesis, diameter, fresh and dry weight of 

calli and the calli water content were evaluated. The results showed that, the effects of genotype, 

culture media and explants types were statistically significant for all of the traits. ‘Longipinnatus’ and 

‘Vermal’ cultivars represented the highest amount of calli growth. Meanwhile, ‘Chuhong’ and 

‘Caudatus’ had the greatest amount of calli percentage. A and B media had the highest values for calli 

related traits. These results are indicating the presence of the high internal hormonal concentration in 

the plants and probably it's inhibiting effects on calli production of the explants in high level hormone 

enriched media. 

Keywords: Raphanus sativus L., Callogenesis, Explants, Genotype, Culture media 



Introduction 

Radish (Raphanus sativus L., 2n=18) is a common vegetable in Asia and in most parts 

of the world. This plant produces a red colored edible root with different shapes [1]. Apart 

form culinary purposes, radish has diverse medicinal properties as well [2]. Major genetic 

improvement of radish has been achieved by conventional plant breeding methods, such as 

crossing [3]. However, these methods are time and labour consuming.  

In recent years, advances in plant genetic engineering have opened a new avenue for 

crop improvement and various transgenic plants with novel agronomic characteristics have 

been produced [2,4,5]. 

The success in plant genetic engineering is dependent upon several factors, from 

which an efficient tissue culture system, with high plant regeneration potential, is a crucial 

option.


However, to the best of our knowledge, there is limited information on the plant 

regeneration from cell and tissue cultures of radish [6,7]. Previous attempts to regenerate 

plants from leaf protoplasts of radish were not successful [1,6,7]. However, use of radish as 

one of the donors in the protoplast fusion studies has produced several novel fertile 

intergeneric somatic hybrids [8,9,10,11]. 

The aim of this research was to study for the first time the effects of explant types and 

culture media on the potential of calli production from four cultivars of radish. 


MOHAMMAD ALI AAZAMI,

MOHAMMAD BAGHER HASSANPOURAGHDAM

Rom. Biotechnol. Lett., Vol. 14, No. 4, 4519-4523 (2009) 

4520


Material and Method 

In the present study explants of hypocotyl, cotyledon and root were afforded from 6 to 

10 days old seedlings of radish cultivars: ‘Vermal’, ‘Chuhong’, ‘Caudatus’ and 

‘Longipinnatus’. For hypocotyl explants an 8-10mm sample of hypocotyl about 2mm beneath 

the cotyledonary leaves were used. About cotyledon explants, cotyledons were excised with 

about 2mm of their petiole and for root explant, a sample of about 1cm of root were used as 

propagules.

Culture media: Murashige and Skoog [12] medium enriched with L-arginin 

(0.2 mgL


-1

), nicotinic acid (0.05 mgL

-1

), pyridoxin (0.05 mgL



-1

), and thiamine (0.05 mgL

-1

)

was used for callus production. Three different media were used where, A was control 



(without hormone), B (2, 4-D 1 mgL

-1

 and BAP 2 mgL



-1

) and C (2, 4-D 2 mgL

-1

 and BAP 1 



mgL

-1

). Culture media were supplemented with 3% sucrose and 0.8% agar. The pH of the 



media was adjusted at 5.7 and the prepared media were autoclaved for 20 min at 121°C for 

sterilization. 



          Data  collection: Cultured explants were incubated in growth chamber with 

constant temperature of 25±2 °C and day/night period of 16:8. After one month, callogenesis, 

calli diameter, fresh and dry weight of calli and calli water content were evaluated. 

Callogenesis was estimated with the proporion of calli producing explants in relation to the 

total explants. The separated calli were weight, then wrapped in aluminum foil for dry weight 

measurement in an air forced oven at 70±2°C for 24 hrs. Calli water content was evaluated 

with this equation:  

                                    Calli fresh weight – calli dry weight 

Calli water content =   

                                                 Calli fresh weight



Statistical analysis: Variance analyses for traits were done with SAS 8.02 statistical 

software as 4×3×3 factorial design based on CRD with four replications. Duncan’s multiple 

range test at 5% probability level was employed for mean comparison of treatments. 

Results and Discussions 

Effects of genotype, culture medium and explant type were statistically significant 

(p 1%) for all traits. Interactive effects were significant as well for all traits except for calli 

dry weight.

Mean comparison for cultivars showed that ‘Chuhong’ had the minimum amount of 

callogenesis.

For calli diameter and fresh and dry weight of calli, ‘Longipinnatus’ and ‘Vermal’ 

cultivars had the greatest values (Table 1). 

The highest mean for callogenesis, calli diameter and fresh and dry weight of calli 

belonged to the B culture medium. On the contrary, B culture medium had the least amount of 

calli water content (Table 1).  

Hypocotyl explant was superior to the other explants in respect to the callogenesis but 

in contrast, it had the least amount of calli diameter.  

For all cultivars root explants had a relative advantage in comparison with other 

explants. Hypocotyl explants had the least amount of calli fresh and dry weight except for 

‘Longipinnatus’.



The Effects of Different Culture Media on the Callus Production of Radish (Raphanus sativus L.)

Rom. Biotechnol. Lett., Vol. 14, No. 4, 4519-4523 (2009) 

4521

Table1. Mean comparison for calli traits of  radish cultivars in different culture media 

Cultivar

Culture 

media 

Calli

percentage  (%) 

Calli

diameter (mm) 

Calli

fresh weight (g) 

Calli

dry weight (g) 

Calli

water content 

A 88.57ab  6.09ab  0.108b 0.0105b 

90.22a 

B 91.43a 



5.83b  0.098c 

0.0102 


81.69b 


‘Vermell’ 

C 82.66 


7.12a  0.168a 0.0202a 

87.93ab 

A 85.62b 

5.79b  0.092b 0.0097b 

88.91ab 


B 76.56c 

5.39c  0.073bc 

0.0085bc 

89.52a 


‘Chuhong’ 

C 89.75a 

6.83a  0.121a 0.0141a 

86.47b 


A 84.28b 

5.27b  0.097b 0.0101b 

92.22ab 

B 79.43bc 

4.31c  0.087bc 

0.0094bc 

91.48b 

‘Candutus’ 



C 91.22a 

6.87a  0.132a 0.0162a 

94.65a 

A 90.41ab 



7.77b  0.151b 0.0192b 

84.58ab 


B 86.15b 

6.69c  0.105c 

0.0112 



85.69a 



‘Longipinnatus

C 92.77a 



8.85a  0.212a 

0.0312 


82.35b 


Different letters in columns show significant difference based on Duncan s multiple range test at P 0.01 

A (without hormone or control), B (2, 4-D 1 mgL

-1

 and BAP 2 mgL



-1

) and C (2, 4-D 2 mgL

-1

 and BAP 1 mgL



-1

)

Root explant had the greatest values for mean fresh & dry weight of calli in both 



‘Chuhong’ and ‘Caudatus’ cultivars. About calli water content, hypocotyl explants had the 

greatest amounts except for ‘Longipinnatus’ cultivar (Table 2). 



Table2. Mean comparison for calli traits of radish cultivars with different explants 

Cultivar

Explant

Calli

percentage  (%)

Calli

diameter (mm)

Calli

fresh weight (g)

Calli

dry weight (g)

Calli

water content

Hypocotyl 80.3a  5.62b  0.09c 0.0083c 

91.33a 

Cotyledon 76.2b  6.51a  0.128b 



0.0131a 

90.01ab 


‘Vermell’ 

Root 72.3c 

5.98ab 

0.101ab 


0.0098bc 

91.12a 


Hypocotyl 76.7a  5.36b  0.07c 0.0051c 

94.48a 


Cotyledon 71.7b  6.14a 0.129ab 

0.0128b 


90.22ab 

‘Chuhong’ 

Root 96.5c 

6.31a 


0.149 

0.0161a 


88.91b 

Hypocotyl 

69.9a 5.53c 0.08c 

0.0077c 


92.86a 

Cotyledon 72.8ab  6.26b  0.141b 0.0151b 

89.41ab 

‘Candutus’ 

Root 70.4b 

6.93a 


0.194a 

0.0188a 


86.18b 

Hypocotyl 86.7a  7.48a  0.231a 

0.0241a 

84.36b 


Cotyledon 78.9ab  6.39b  0.172c 0.0169c 

88.56a 


‘Longipinnatus’ 

Root 76.6b 

7.33a 

0.214ab 


0.0221ab 

85.77ab 


Different letters in columns show significant difference based on Duncan s multiple range test at P 0.01 

Mean comparison for callogenesis between cultivars showed that, ‘Longipinnatus’ and 

‘Vermal’ had the highest and, ‘Chuhong’ had the lowest amount of callogenesis respectively. 

 

It seems that internal hormone concentrations of two former cultivars was higher than 



that of others, so that they had better growth in control medium or media with minimum 

hormonal concentrations. This trend shows that, in cultivars with high potential of internal 

hormones, calli growth is prohibited in hormone enriched media [13,14,15]. 


MOHAMMAD ALI AAZAMI,

MOHAMMAD BAGHER HASSANPOURAGHDAM

Rom. Biotechnol. Lett., Vol. 14, No. 4, 4519-4523 (2009) 

4522


Media with balanced hormonal combinations had the highest percentage of calli 

production, calli diameter and calli fresh and dry weight. There was root regeneration from 

callus in the B culture medium. In accordance with our results some plants of Brassica genus 

showed rooting potential from callus as well [13,16]. 

Sachiko and Hegazi [13] reported plant regeneration from radish callus for the first 

time. There are contradictory reports on the effects of phytohormones on the callus initiation 

of different plants [11,13,14]. 2, 4-D is a growth regulator essentially needed for callus 

initiation and growth in most of the in vitro culture studies but it limits regeneration of 

plantlets [13,17]. Furthermore, for regeneration purposes callus must be transferred to a 2, 4-

D free medium with balanced combinations of cytokinin and one another auxin [18]. In 



Raphanus sativus cultivars ‘Morignchi’ and ‘Nerimashiring’, plantlets were regenerated in the 

first subculture in a 2, 4-D free medium [13,16,18]. Same results were reported for 

cauliflower [19] and red cabbage [20]. Hypocotyl explants had a relative superiority for 

callogenesis in comparison with the other explants. There are several reports that diverse 

explants such as flower, leaf, petiole, stem, cotyledon, shoot-tip and cotyledon have been 

employed for callus initiation of different plant species [6,7,9]. However hypocotyl explants 

showed the premium callogenesis in radish. Furthermore, hypocotyl explants were reported as 

the most suitable explant for in vitro culture purposes especially for callus initiation in 

broccoli [2,15,21], red cabbage and mustard [20]. 

In some cases and for some genotypes of radish, concomitant production of callus and 

plantlets from hypocotyl explants were reported as well [15,22,23]. Shukla and sawny [24]  

reported that variations in shoot and root production of two rapeseed (Brassica napus)

cultivars were due to the differences in the internal levels of auxin and cytokinin. In fact 

differences in tissue culture responses and organogenesis would be dependant on the different 

potential of explants for internal hormones metabolism [25]. Findings of this experiment are 

initial steps toward in vitro culture multiplication as well as a trend into enhancing a genetic 

engineering potential of this plant. However, in the future there is need for novel and accurate 

protocols for optimization of plant regeneration from these explants. 



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The Effects of Different Culture Media on the Callus Production of Radish (Raphanus sativus L.)

Rom. Biotechnol. Lett., Vol. 14, No. 4, 4519-4523 (2009) 

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