All content in this area was uploaded by Asadollah Ahmadikhah on May 03, 2016
Content may be subject to copyright.
All content in this area was uploaded by Asadollah Ahmadikhah
Content may be subject to copyright.
Content uploaded by Gennady Karlov
All content in this area was uploaded by Gennady Karlov
Content may be subject to copyright.
African Journal of Biotechnology Vol. 6 (7), pp. 861-867, 2 April 2007
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2007 Academic Journals
Full Length Research Paper
Effects of genotype, explant type and nutrient medium
components on canola (Brassica napus L.) shoot in
Ghasemi Bezdi Kamal 1, 2*, Karlov Gennady Illich2 and Ahmadikhah Asadollah3
1Cotton Research Institute of Iran, Beheshti St, P. O. Box 49175-483, Gorgan, Iran.
2Russian State Agrarian University – MTAA named after K. A. Timiriazev, Department of Agricultural Biotechnology,
Timiriazevskaya St., No. 49, Moscow 127550, Russia.
3Agricultural Sciences and Natural Resources University of Gorgan, Department of Agronomy and Plant Breeding,
Accepted 5 March, 2007
The objective of the study was to develop an efficient method for shoot regeneration of canola
(Brassica napus L.) and to compare the regeneration capacity of different explants on MS medium with
several combinations of plant growth regulators. The experiments showed that the morphogenetical
potential of canola depends on genotype, primary explant, hormonal structure and concentration of
nutrient medium. Cotyledons possessed higher regeneration ability in comparison to hypocotyls and
roots. The best regeneration capability was exhibited by the cultivar 'Quantum'. Its frequency with
cotyledonary explants reached 68.8% on all used media. Addition of 3 mg/l ABA in nutrient medium
considerably increased the regeneration frequency. The highest shoot regeneration (100%), however,
took place when cotyledonary explants were cultivated on medium, containing 1.0 mg/l NAA, 8.0 mg/l
BAP and 3.0 mg/l ABA. Precultivation of explants on callus induction medium did not affect the shoot
regeneration frequency. Vitrification of regenerants was promoted by increasing the auxin NAA or
cytokinin BAP, and ABA in the nutrient medium.
Key words: Brassica napus, shoot regeneration, cotyledonary explants, nutrient medium, seedlings,
Canola (Brassica napus L.) is an important oil crop
grown in Canada, India, China, Europe and other
regions of the world, and is ranked third in global produc-
tion of oil crops (Kazan et al., 1999; Cardoza et al.,
2003). In Iran, the area under cultivation of canola incre-
The increase in the transformation efficiencies is desira-
ble in order to decrease the amount of resources needed to
produce transgenic plants, and to potentially provide a hig-
her baseline for subsequent transformation of other canola
*Corresponding author. E-mail: firstname.lastname@example.org.
Tel: (+98171) 2254960. Fax: (+98171) 2227781.
varieties. Two important factors governing the efficiency of
transgenic plant recovery are obtaining healthy shoots that
are not hyperhydrated and having a good rooting efficiency
(Cardoza et al., 2003).
In the development of new forms of transgenic plants
by genetic transformation methods, shoot regeneration
frequency has a great value. Considerable progress has
been accomplished in the cellular and molecular biology
of Brassica species in the recent years. Plant regenera-
tion has been increasingly optimized via organogenesis
and somatic embryogenesis using various explants and
by tissue culture improvements focusing on factors such
as age of explant, genotype and media additives. In this
study, we report an increase in the regeneration efficiency
862 Afr. J. Biotechnol.
Table 1. Components of different nutrient media for organogenesis of canola.
Components (mg/l) No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10
½ MS Based on MS ½ MS
Vitamins - Based on B5
BAP - - 4 4 4 8 4 0.5 6 -
NAA - 2 - 1 2 1 2 - 0.6 -
Kinetin - 4 - - - - - - - -
2,4-D - 0.1 - - - - - - - -
AgNO3 - - - - - - 10 5 -
IBA - - - - - - - - - 0.5
Sucrose (g/l) 5 30 10 10 10 10 10 10 10 20
pH 5.7 – 5.8
Agar (g/l) 7 6 6 6 6 6 7 7 6 7
of canola. Improved efficiency was achieved through
altering the shoot regeneration ability of 5-day-old cotyle-
donary leaves, hypocotyls and roots of Iranian canola
cultivars of 'Sarigol', 'Quantum' and 'Option 500' by
manipulating the nutrient media components required for
MATERIALS AND METHODS
All experiments with the plant tissues were carried out in vitro on
media based on MS (Murashige and Skoog, 1962) and vitamins
based on B5 medium (Gamborg et al., 1968), plus sucrose and
plant growth regulators in different concentrations (Table 1). The
media were solidified with 6 - 7 g/l agar (Bacto-agar "DIFCO".
USA). For regeneration of canola, four variants of phytohormones
BAP and NAA were used, with and without addition of abscisic
acid (ABA). Canola seeds of cultivars 'Sarigol', 'Quantum' and
'Option 500' were rinsed 1 min with 96% ethanol, then surface
sterilization was followed for 15 min with 0.5% sodium hypo-
chlorite, and 0.2% Tween 20 was added as a surfactant. The
seeds were thoroughly washed with sterile distilled water (3 - 5
times for 15 min). The disinfected seeds were germinated on MS
medium No. 1 (Table 1). The cultures were grown for 5 days in
growth chamber at 20 - 22°, under a 16/8-h (light/dark) photo-
period with light supplied by cool-white daylight fluorescent lights.
Cotyledonary leaves with a small petiole, whose size did not ex-
ceed 2 - 5 mm, hypocotyl (5 - 10 mm) and root (2 - 5 mm) segments
that were used as explant, were excised from 5-day-old seedlings.
The explants were precultivated at 24° in darkness on the MS
medium No. 2. After 2 days, they were transferred into media No. 3
- 6 and were cultivated in growth chamber within 3 weeks at 20 -
22° under a 16/8-h (light/dark) photoperiod.
The regenerated explants transferred to medium containing
silver nitrate (AgNO3) for shoot development and shoot elongation.
The elongated shoots were cut out and transferred into rooting
medium No. 10. The rooted plantlets were washed and transferred
to the autoclaved soil in pots. The pots were covered with clear
bags to provide 100% relative humidity. They were placed in an
acclimatization room under a 16/8 h photoperiod at 20 - 23°. After
2 weeks, acclimatizied plants were transferred to greenhouse and
allowed to grow to maturity. They normally passed generative
phase and produced seeds.
Regeneration frequency was calculated 21 days after precultiva-
tion as percentage of explants capable to shoot regeneration on
the media No. 3 - 6. The data were analyzed by ANOVA (analysis
of variance). In the tables, the means with a standard error were
shown. The means were compared using the Duncan multiple
comparison test at P < 0.05.
Estimation of in vitro morphogenesis
The morphogenesis potential of cotyledon, hypocotyl
and root explants of canola cultivars was estimated.
Occurrence of the first regenerated shoots was obser-
ved 13 - 18 days after precultivation, irrespective of the
studied genotypes. The result established that for shoot
regeneration of canola, cotyledons isolated from 5-day-
old seedlings were the best explants (Table 2). Depen-
ding on genotype, the average morphogenesis frequency
ranges from 27.1 to 35.9%. As 'Quantum' and 'Option
500' cultivars had the higher levels of morphogenesis,
they were selected for transformation studies.
Kamal et al. 863
Table 2. Dependence of shoot regeneration frequency on genotype and primary explant (in %).
Genotype Cotyledon Hypocotyl Root Mean of genotype (S.E.05 = 1.6 )
Sarigol 51.6 23.4 6.3 27.1
Quantum 68.8 31.3 7.8 35.9
Option 500 62.5 26.6 3.1 30.7
Mean of explant type (S.E.05 = 1.6 ) 60.9 27.1 5.7 -
for individual differences = 2.9. Precultivation of explants for 2 days on medium No. 2 with subsequently followed by
shoot regeneration. Values are average on all nutrient media.
Influence of nutrient medium components on in vitro
The change of cytokinin to auxin ratio in a nutrient
medium allows the induction of morphogenesis and ob-
taining the regeneration of shoots or roots, depended
on the object of the experiment. The influence of vari-
ous concentrations of phytohormones on the organoge-
nesis was studied with the purpose of increasing its
efficiency. Cotyledon, hypocotyl and root explants were
used on media No. 3 - 6 (Table 3). The investigated
parameters affected the phytohormonal component of
nutrient medium, genotype, type of primary explant and
the interaction of nutrient media, plant genotype and
For all explants and genotypes, the greatest frequen-
cy of shoot regeneration (39.6%) was observed on the
medium No. 5, while the least (17.3%) was obtained on
the medium No. 3. Media No. 5 and 6 had similar freq-
uencies of shoot regeneration (37.5 and 39.6%, res-
pectively). It is necessary to note that cotyledonary expl-
ants possessed high morphogenesis potential in compari-
son with hypocotyl and root segments on all used nutri-
ent media. Regeneration efficiency of cotyledons was
60.9%, whereas that of hypocotyls was 27.1%, and
that of roots was 5.7% (Table 3). Therefore, it is
suggested that in the further experiments, cotyledons must
be selected as primary explant.
Also from Table 3, cotyledons on the medium No. 6 con-
taining the highest concentration of cytokinins (8 mg/l BAP)
in combination with 1 mg/l NAA possessed the greatest
regeneration frequency of shoots (85.4%). However, med-
ium No 5 was the best nutrient medium for hypocotyls and
roots (41.7 and 12.5%, respectively).
Influence of ABA on shoot formation
Addition of ABA to morphogenesis induction medium
caused significant effects (at 5% level) on the shoot
formation, and considerably increased the regenera-
tion frequency in all investigated types of explants on
all nutrient media. The highest shoot formation freq-
uency (100%) was observed in the cultivation of coty-
ledons on the medium containing 8 mg/l BAP, 1 mg/l
NAA and 3 mg/l ABA (Table 4).
Effect of precultivation
The influence of precultivation of canola explants was
investigated for 2 days on media No. 2 and No. 5. From
the given results in Table 5, it follows that although
shoot regeneration frequency on the callus induction,
medium No. 2 (50.4%) was better than the control on
morphogenesis medium No. 5 (47.1%), in this parame-
ter were not observed significantly differences at the 5
Influence of growth regulatores on vitrification of
In our research, we also observed vitrificant shoots (Table
6). Depending on hormonal concentration of medium, the
average vitrification frequency ranges from 5.6 to 16.7%. The
greatest frequency of vitrificant shoots was observed on the
medium No. 5 containing the highest concentration of NAA (2
mg/l) in combination with 3 mg/l ABA.
Hardening of the plantlets and transfer to soil
The shoots obtained from cotyledonary explants on
root formation medium (No. 10) formed powerful root
system. The plantlets were potted up and hardened off
by gradually decreasing the humidity. Plants were
grown in the vegetative vessels in greenhouse. They
were fertilized, passed generative phase normally and
produced seeds (Figure 1).
Efficiency of plant regeneration is one of the main limiting
conditions influencing frequency of genetic transformation.
864 Afr. J. Biotechnol.
Table 3. Dependence of shoot regeneration frequency of canola cultivars on phytohormonal components of nutrient medium, primary
explant and genotype (in %).
Medium Cultivar Cotyledon Hypocotyl Root
Mean of medium
Sarigol 31.2 6.3 - 12.5
Quantum 43.7 18.8 - 20.8
Option 500 43.7 12.5 - 18.7
Mean of explant type and
medium (S.E.05=3.3) 39.5 12.5 0.00 -
Sarigol 43.7 25.0 - 22.9
Quantum 56.3 43.7 12.5 37.5
Option 500 62.5 31.2 - 31.2
Mean of explant type and
medium (S.E.05=3.3) 54.2 33.3 4.2 -
Sarigol 56.2 43.7 12.5 37.5
Quantum 75.0 43.8 18.7 45.8
Option 500 62.5 37.5 6.3 35.4
5 Mean of explant type and
medium (S.E.05=3.3) 64.6 41.7 12.5 -
Sarigol 75.0 18.7 12.5 35.4
Quantum 100 18.7 0.1 39.6
Option 500 81.3 25.0 6.2 37.5
6 Mean of explant type and
medium (S.E.05=3.3) 85.4 20.8 6.3 -
explant type (S.E.05=1.6) 60.9 27.1 5.7 - -
for individual differences = 5.7. Precultivation of explants for 2 days on medium No. 2 followed by shoot regeneration.
Table 4. Influence of ABA on shoot regeneration frequency of canola cultivars (in %).
Medium ABA (mg/l) Cotyledon Hypocotyl Root
Mean of medium
Mean of medium
- 25.0 8.3 - 11.1
3 3 54.2 16.7 - 23.6 17.3
- 41.7 29.2 - 23.6
4 3 66.7 37.5 8.3 37.5 30.5
- 58.3 29.2 12.5 33.3
5 3 70.8 54.2 12.5 45.8 39.6
- 70.8 16.7 - 29.2
6 3 100 25.0 12.5 45.8 37.5
explant type (S.E.05 = 1.6)
60.9 27.1 5.7 - -
for individual differences = 4.7. Precultivation of explants for 2 days on medium No. 2 followed by shoot regeneration. Average values
of the 3 cultivars are shown in the table.
The numerous factors influencing canola in vitro organoge-
nesis includes the plant genotype, age of plant (donor of explant), explant type and nutrient medium components
(Raldugina and Sobolkova, 1995; Cardoza et al., 2003;
Kamal et al. 865
Figure 1. Development of regeneration and obtaining normal plants of canola
(cultivar 'Quantum') from cotyledon isolated from 5-day-old seedlings: a)
rooting of shoots on nutrient medium No. 10; b) potting up and hardening off the
plantlets; and c) cultivation of canola plants in greenhouse and seed formation.
Table 5. Precultivation and comparison of shoot regeneration frequency in canola cultivar of 'Quantum' (in %).
Precultivation medium No. 5 No. 9
Mean of media for
No. 2 54.2 46.6 50.4
No. 5 52.8 41.4 47.1
Mean of media for
53.5 44.0 -
for individual differences = 2.5. Precultivation of explants for 2 days followed by
Malishenko et al., 2003; Jonoubi et al., 2004, 2005; Halina
et al., 2005; Reda et al., 2006; Wang et al., 2006).
Therefore, the first step to develop an effective technique
for plant regen-eration is to find an optimum combination of
four factors above.
To choose the primary explant, it is necessary to
study the organogenesis process. In the experiments,
explants capable of regenerating only roots were not
taken into account, because the roots possess low
frequency of stem regeneration.
From comparison of the results obtained on media
No 3 - 6, it is possible to conclude that the presence of
1 mg/l NAA in nutrient medium essentially increases
the organogenesis efficiency. Besides, the results
obtained on media No. 5 and 6 testify that high con-
centration of both BAP and NAA simultaneously leads
to increase in the quantity of regenerated explants.
These results indicate that canola organogenesis
depends on the ratio and concentration of cytokinins
and auxins in the nutrient medium as have been obse-
866 Afr. J. Biotechnol.
Table 6. Frequency of vitrificant shoots in cotyledonary explants of 'Quantum' cultivar (in %).
Medium ABA (mg/l) Vitrifiation (%)
Mean of medium (S.E
3 3 6.4 6.0
4 3 7.6 6.9
5 3 16.7 14.8
6 3 13.5 12.1
for individual distinctions = 0.66. Precultivation of explants for 2 days on medium No. 2
rved in other studies (Raldugina and Sobolkova,
1995; Maisurian et al., 2005; Jonoubi et al., 2005;
Reda et al., 2006). For example, medium No. 3 con-
taining 4 mg/l BAP in the absence of auxins results in
the least relative shoot regeneration frequency
(17.3%), while presence of NAA together with 4 or 8
mg/l BAP in media No. 4 - 6 results in greater relative
frequency of shoot regeneration.
As observed in Table 3, cotyledons on the medium No. 6
containing 1 mg/l NAA possessed the greatest regene-
ration frequency of shoots, while medium No. 5 containing
2 mg/l NAA was the best nutrient medium for hypocotyls
and roots regeneration. It is known that cotyledons are
capable of independently synthesizing auxins, but the
content of this phytohormone decreases in plants from the
top towards the stem base (Sparrow et al., 2004).
As mentioned, preliminary cultivation of explants on
the callus induction medium considerably increases the
ability of explants for stem formation (Halina et al.,
2005). In addition, the study of precultivation was
necessary for further joint cultivation of explants with
Agrobacterium in transformation studies, which is
usually carried out on the induction medium for
callusogenesis. Based on the studies of Raldugina and
Sobolkova (1995) on canola, the increase of preculti-
vation time of cotyledonary explants increased shoot
regeneration frequency. However, our results on pre-
cultivation of canola explants for 2 days on media No. 2
and No. 5 before proceeding to the morphoge-nesis
media show-ed that although shoot regeneration
frequency on the callus formation medium was more
than the control on morphogenesis medium, this
parameter was not significantly differences at the 5%
level. The precultivation was limited to 2 days, since
longer contact time of explants with Agrobacterium
complicates their release from bacteria at the
subsequent regeneration stages.
In the cultivation of plants in vitro, the vitrification pheno-
menon is often observed, which is found in the strong
hyperhydrated leaves and stems. Thus, in plants, are
formed leaves with abnormal morphology, with expanded
basis of stalk. As a rule, such plants gradually perish. There
is no common opinion about the reasons of this phenom-
enon. The factors responsible for vitrificant shoots forma-
tion includes high humidity in cultivation vessels due to
enveloping them by parafilm or foil that entails sharp
deterioration of gas exchange. Other factors are accumu-
lation of ethylene and carbonic gas-rich nutrient media
containing significant amounts of ammonium salts, sucrose
and vitamins, reduced gel concentration and increase tem-
perature, high doses of exogenous cytokinin and AgNO3
and high CO2 concentration (Curtis and Shetty, 1996; Lim
et al., 1998; Popadin, 2002; Kadota and Niimi, 2003;
From comparison of the data with nutrient medium
components, it is possible to conclude that the high
content of auxin NAA or cytokinin BAP in the nutrient
medium increases vitrification. By analyzing the influen-
ce of nutrient medium components on Brassica olera-
cea, Popadin (2002) reported that high content of NAA
in the nutrient medium promoted vitrification. Kadota and
Niimi (2003) also observed the stimulation of stem
vitrification of pear due to high concentration of cytoki-
nins in the nutrient medium. The same tendency was
observed with addition of ABA in the medium. In the full
absence of ABA, the frequency of vitrificant shoots was
lower than on the medium with 3 mg/l ABA.
Finally, genotype, type of explant, components of
nutrient medium (ratio of various concentrations of cyt-
okinins and auxins in nutrient media, influence of ABA
on regeneration), and the interaction of these
factors affected morphogenesis of canola.
We gratefully acknowledge the financial support of the
Agricultural Research and Education Organisation of the
Islamic Republic of Iran and Department of Agricultural
Biotechnology and the Molecular Biotechnology Center
of the Russian State Agrarian University - MTAA after K.
A.Timiriazev, Russia. We also thank Professor V. S.
Sheveloukha, Professor E. A. Kalashnikova and Dr. G.
N. Raldugina for assisting in this project as well as our
laboratory colleagues for their support.
Cardoza V, Stewart CN (2003). Increased Agrobacterium-mediated
transformation and rooting efficiencies in canola (Brassica napus L.)
from hypocotyl segment explants. Plant Cell Rep. 21: 599-604.
Curtis OF, Shetty K (1996). Growth medium effects on vitrification, total
phenolics, chlorophyll, and water content of in vitro propagated
oregano clones. Acta Horticul. 426: 498-503.
Gamborg OL, Miller RA, Ojima K (1968). Nutrition requirements of
suspension cultures of soybean root cells.
Expt. Cell Res. 50:151-
Halina H, Marzena P, Grzegorz G (2005). Morphological and
Histological Aspects of 2,4-D Effects on Rape Explants (Brassica
napus L. cv. Kana) Cultured In vitro. Acta Biol. Cracoviensia Series
Bot. 47(1): 219-226.
Jonoubi P, Mousavi A, Majd A, Daneshian J (2004). Improved Brassica
napus L. regeneration from hypocotyls using thidiazuron and
benzyladenine as cytokinin sources. Pak. J. Bot. 36(2): 321-329.
Jonoubi P, Mousavi A, Majd A, Jalali Javaran M, Salmanian AH,
Daneshian J (2005). Efficient Regeneration of Brassica napus L.
Hypocotyls and Genetic Transformation by Agrobacterium
tumefaciens. Biol. Plantarum 49(2): 175-180.
Kadota M, Niimi Y (2003). Effects of cytokinin types and their
concentration on shoot proliferation and hyperhydricity in in vitro pear
cultivar shoots. Plant Cell, Tiss. and Org. Cult. 72: 261-265.
Kazan K, Marcus JP, Goulter KC, Manners JM (1999). Application of
genes encoding antimicrobial peptides for the control of fungal
pathogens of canola. Proceeding of the 10th International Rapeseed
congress. Canbera , Australia . p. 508.
Lim HT, You YS, Park EJ, Song YN, Park HK (1998). High plant
regeneration, genetic stability of regenerants, and genetic
transformation of herbicide resistance gene (bar) in chinese cabbage
(Brassica campestris ssp. pekinensis). Acta Hort. (ISHS). 459: 199-
Kamal et al. 867
Maisurian AN, Kalizhenkova MD, Mazin VV, Liapkova NS, Dridze IL,
Kharchenko PN (2005). Development of spring resistant canola
against herbicide "BASTA" (Glyphosate). Methodical recomendation.
Russian State Agrarian University – MTAA named after KA
Timiriazev, Moscow, Russia.
Malishenko SI, Tulkina LG, Zvereva SD, Raldugina GN (2003).
Development of transgenic plants of Brassica campestris, expressing
gfp gene. Russian J. Plant Physiol. 50(2): 309-315.
Murashige T, Skoog F (1962). A revised medium for rapid growth and
bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
Popadin PV (2002). Application of Agrobacterium transformation in
selection of cabbage for desease resistance. PhD Dissertation, Russian
State Agrarian University–MTAA named after K. A. Timiriazev,
Raldugina GN, Sobolkova GI (1995). Factors, influencing on
organogenesis of cotyledonary explants of canola. Russian J. Plant
Physiol. 42: 916-922.
Reda EA, Moghaieb MA, El-Awady RG, El-Mergawy SSY, El-Sharkawy
AM (2006). A reproducible protocol for regeneration and
transformation in canola (Brassica napus L.). African J. Biotechnol.
Sparrow PAC, Townsend T, Arthur AE, Morgan CL, Dale PJ, Irwin JA
(2004). Genetic analysis of in vitro shoot regeneration from
cotyledonary leaves of Brassica oleracea. Theor. Appl. Genet. 108:
Tisserat B (2005). Establishing tissue-cultured sweetgum plants in
soil. Hort. Tech. 15:308-312.
Wang JX, Zhao FY, Xu P (2006). Use of aroA-M1 as a Selectable
Marker for Brassica napus Transformation. Crop Sci. 46: 706-711.