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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.
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African Journal of Biotechnology Vol. 6 (7), pp. 861-867, 2 April 2007
Available online at
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
vitro organogenesis
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,
Gorgan, Iran.
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-
ases annually.
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:
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
Iron chelate
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
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 %).
Explant type
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 -
S. E.
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
shoot formation
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
explant type.
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
% level.
Influence of growth regulatores on vitrification of
canola explants
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
Mean of
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 -
Mean of
explant type (S.E.05=1.6) 60.9 27.1 5.7 - -
S. E.
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
= 2.7)
Mean of medium
= 1.9)
- 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
Mean of
explant type (S.E.05 = 1.6)
60.9 27.1 5.7 - -
S. E.
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 %).
Morphogenesis medium
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
= 0.47)
- 5.6
3 3 6.4 6.0
- 6.3
4 3 7.6 6.9
- 13.9
5 3 16.7 14.8
- 10.8
6 3 13.5 12.1
S. E.
for individual distinctions = 0.66. Precultivation of explants for 2 days on medium No. 2
followed by
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;
Tisserat, 2005).
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,
Moscow, Russia.
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.
... Although plant tissue culture is an attractive means for improving plants, its use requires an effective regeneration system from somatic tissues of cotton plants. Compared with many other crops, it is more difficult to obtain somatic embryogenesis and plant regeneration from cotton ;Ghasemi et al., 2007;;Ghaemi et al., 2011). Price and Smith (1979) were the first to report somatic embryogenesis in cotton, Journal of Applied Biosciences 47: 3256-3263 ISSN 1997-5902 Gossypium koltzchianum, although complete plants could not be regenerated. ...
... In vitro cultured cotton cells have been induced to undergo somatic embryogenesis in numerous laboratories using various strategies (Voo et al., 1991;Kumria et al., 2003;Ikram-ul-Haq, 2005;Wu et al., 2005;Hilarie et al., 2008;Ghaemi et al., 2011). Somatic embryogenesis and plant regeneration systems have been established from different tissues and regenerated plants have been obtained from explants such as hypocotyls, cotyledons and roots (Zhang, 2000;Ouma et al., 2004;Abdul Qayyum et al., 2006;Ghasemi et al., 2007;Ozyigit et al., 2007), anther (Zhang et al., 1996), embryo (Syed Sarfraz et al., 2004, 2005, protoplasts and ovules (Feng and Zhang, 1994), and from various cotton species and cultivars (Zhang, 1994b;Zhang et al., 2001;Tripathy and Reddy, 2002;Mishra et al., 2003;Jin et al., 2006;Han et al., 2009;Ghaemi et al., 2011). Regeneration procedures have been used to obtain genetically modified plants after Agrobacterium-mediated transformation of hypocotyls and cotyledons (Umbeck et al., 1987;Leelavathi et al., 2004;Mashayekhi et al., 2008) or by transformation of particle bombardment (Finer and McMullen, 1990;Rajasekaran et al., 2000). ...
... The ideal transformation scheme is that via somatic embryogenesis, because each transformed cell of callus has the potential to produce a plant. Somatic embryogenesis and subsequent plant regeneration has been reported in most of the major crop species (Abdol Qayyum et al., 2006;Ghasemi et al., 2007;Mashayekhi et al., 2008;Ghaemi et al., 2011). In this study different callus induction media with various concentrations of NAA and BAP hormones were tested using different explants (hypocotyls, cotyledons, and crowns with root) and different cotton cultivars. ...
Full-text available
Objective: The study aimed to optimize tissue culture conditions of cotton (Gossypium sp.). Methodology and results: The study was carried out at the Cotton Research Institute of Iran (CRII) in Gorgan with 4 cotton cultivars, i.e. Sahel, Sepid, Coker 312*349 and No.200. Cotyledonary leaves, hypocotyls and crown explants excised from 7 days-old seedlings grown under in vitro conditions were cultured on MS basal medium with B5 medium vitamins containing various hormonal combinations of Naphthalene Acetic Acid (NAA) and 6-Benzylamino purine (BAP). Analysis of results showed that percent of callus formation was significant at 1% level for hormonal combinations in medium, cultivar, explant type and all interactions of studied factors. The highest callus formation was observed in cv. Sahel and Sepid at 41.9%. The hypocotyl explants with 51.3% had the highest frequency of callus formation, followed by crown and cotyledonary explants. The highest callus formation (75%) was observed in the hypocotyl explants cultured on MS medium containing 2 mg/l NAA without cytokinin. For crown and cotyledonary explants the highest callus formation was observed in MS media containing 1 mg/l NAA without cytokinin. Conclusion and application of results: The results suggest that plant growth regulators are essential for callus formation. The higher levels of auxins can positively affect callus formation of different explants of cotton cultivars, but cytokinins have a negative effect. Therefore, low concentration of cytokinins with high concentration of auxins in nutrient medium increases callus formation frequency in cotton.
... Thus, cotyledons represent the best explant type for transformation experiments with rapid-cycling lines. Kamal et al. [41] also reported higher regeneration frequencies for cotyledons (31-100%) than hypocotyls (6-44%) in three commercial canola cultivars (Surigol ® , Quantum ® , Option 500 ® ). In contrast, Maheshwari et al. [22] found that plants of the B. napus cultivar Invigor 5020 ® displayed greater (~ 20% more) shoot induction with hypocotyls than with cotyledons and shoot induction was faster in hypocotyls than cotyledons. ...
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Background Rapid-cycling Brassica napus ( B. napus -RC) has potential as a rapid trait testing system for canola ( B. napus ) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus -RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus -RC plants. Results An in-vitro regeneration protocol was developed for B. napus -RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium -mediated transformation of B. napus -RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus -RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6 -overexpressing B. napus -RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6 -overexpressing plants showed less freezing injury than the wild type. Conclusions This study demonstrates that B. napus -RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.
... Maximum survival of seedlings was recorded in variety GSL-1 after four weeks in this treatment while lowest per cent of aseptic seed cultures and seedling survival was observed when seeds were surface sterilized in HgCl 2 (0.1%) for one minute duration after one and four weeks after treatment. These findings are in conformity with the findings of Bajaj and Nietesch (1975), Singh et al. (1981), Dieter (1982), Kamal et al. (2007) and Moghaieb et al. (2006) who reported that seed explants of Brassicas can be surface sterilised by using different sterilents such as 0.5% and 0.1% Hg cl 2 , 0.5% sodium hypochlorite or 70% ethanol. Dubey and Gupta (2014) also reported that Surface Sterilization -Combination of 70% Alcohol + 0.1% HgCl 2 for 5 minutes was recorded as best surface sterilization with lowest rate of contamination. ...
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The present study entitled "In vitro Regeneration Studies in Brassica napus, variety GSL-1 and DGS-1 was carried out in the Biotechnology Laboratory, Division of Genetics and Plant Breeding SKUAST-J. Brassica napus, variety GSL 1 and DGS-1 were cultured on MS Media supplemented with different concentrations of growth regulators. The sterilization treatment with HgCl 2 (0.1%) for 3 minutes resulted in highest per cent of aseptic seeds and survival of seedlings. Highest callus induction was observed in GSL-1. MS medium augmented with 2,4-D at 2-2.5 mg/l. Different concentrations of growth regulators in media showed a significant difference in the callus induction frequency (CIF) and regeneration frequency (RF) for both the genotypes tested. MS medium supplemented with BAP (5.0 mg/l) and 2, 4-D (0.5 mg/l) showed highest shoot regeneration frequency in GSL-1. Highlights • Seed Sterilisation with HgCl 2 (0.1%) for three minutes resulted (100%) aseptic callus. • Highest callus induction frequency was observed in GSL-1. • MS medium supplemented with BAP (5.0 mg/l) and 2, 4-D (0.5 mg/l) showed highest shoot regeneration frequency in GSL-1.
... In vitro regeneration offers a chance to grow attractive and virtually genetically identical plants quickly (Farooqet al.,2019).Many factors influence this in vitro regeneration including the composition of the culture medium, source of the explants,cultural environment and genotype (Bano et al., 2010). The in vitro tissue culture technique is an essential experimental approach in applied research (Bednarek and Orłowska, 2019).Organogenesis is the most frequently recorded method of adventitious shooting in Brassica crops and different explants were used such as hypocotyles (Gerszberg et al., 2015),cotyledons (Munshi et al. 2007;Kamal et al., 2007), leaves (Gambhir et al., 2017), shoot tips (Widiyanto and Erytrina, 2001), and protoplasts (Chikkalaet al., 2009). In various brassica species, for instance Brassica napus,various factors such as combinations of plant regulators of growth, explant type and age were investigated for finding an efficient approach for high frequency regeneration of Brassica napus (Dina et al., 2019). ...
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Broccoli, Brassica oleracea var.italica is an important plant due to its flavor and anticancer activity that received more attention in recent years. The leaf, stem and root explants of aseptic 15 day old seedlings, grown on MS medium were used for callus initiation.Theseexplants were cultured on agarsolidified MS medium containingBAP, NAA, TDZ plant growth regulators.Stem segments gave the highest rate of callusinduction that reached 98% on MS medium supplementedwith 0.2 mg L-1 NAA and 0.5 mg L-1 BAP. Also,callus had been initiated from root segments cultured on MS medium fortified with 1.5, 2.0 mg L-1 TDZ. The results indicated that shoot regeneration occurred after sub culturingthe callus in the same medium as for callus induction, in addition to MS medium supported with 4.0 mg L-1 BAP and 0.5 mg L-1 NAA. Moreover, different somatic embryos stages (globular, heart, torpedo and cotyledonary) were observed when stem callus wascultured on MS medium supplied with 0.5, 0.4 mg L-1 TDZ. In vitro regenerated shoots derived from callus and those that developed from somatic embryos were transferred to MS medium free from growth regulators as well as MS medium containing 1.0, 2.0 mg L-1 IBA for rooting. Rooted plantlets have beensuccessfully acclimatized. An efficient approach was used for inducing shoot regeneration in broccoli Brassica oleracea var.italica via organogenesis and indirect somatic embryogenesis. This research proved that TDZwas effective in inducing somatic embryos from stem and root callus.
... In this regard, Ghasemi et al. (2007) reported that, vitrification of regenerates was promoted by increasing the auxin NAA or cytokinin BAP, and ABA in the nutrient medium. Also, Lee et al. (2004) recorded that, the highest regeneration rate was obtained in most shoots from young leaves of Pyrus pyrifolia on a medium based on MS media supplemented with 0.1-1.0 ...
... The highest frequency of shoot regeneration from cotyledon explants (80%) was observed using 4 mg · L −1 6-BA along with 0.5 mg · L −1 NAA. A much higher concentration of 6-BA (8 mg · L −1 ) has been used in B. napus, resulting in 100% shoot regeneration frequency (Kamal et al., 2007). ...
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Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies. In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate on average. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5 to 4.0 months. Average transformation frequency is up to 10.83%. The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.
... In this regard, Ghasemi et al. (2007) reported that, vitrification of regenerates was promoted by increasing the auxin NAA or cytokinin BAP, and ABA in the nutrient medium. Also, Lee et al. (2004) recorded that, the highest regeneration rate was obtained in most shoots from young leaves of Pyrus pyrifolia on a medium based on MS media supplemented with 0.1-1.0 ...
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Methods to enhance sweetgum (Liquidambar styraciflua L.) in vitro axillary shoot formation and shoot establishment into soil are presented. Sweetgum shoots grown in an automated plant culture system (APCS) produced 400 to 500 shoots via axillary branching compared to only 40 shoots produced within Magenta vessels containing agar medium after 8 weeks of incubation. Vitrification was observed in as many as 80% of the axillary shoots produced in the APCS. A continuous carbon dioxide (CO2)-flow-through system was tested on both vitrified and non-vitrified sweetgum shoots transferred from the APCS to soil. One- and two-cm-long vitrified shoots were grown within CO 2-flow-through system chambers and subjected to 350, 1500, 3000, 10,000, or 30,000 μL·L-1 (ppm) CO2 for 4 weeks. Administering 10,000 μL·L-1 CO2 improved culture survival and enhanced overall shoot and root growth compared to shoots grown under ambient atmosphere (i.e., 350μL·L-1 CO2).
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The objective of the present study is to develop an efficient protocol for shoot and plant regeneration using five commercial canola cultivars grown under the Egyptian agricultural conditions. The regeneration efficiency from hypocotyl explants was examined. The data indicated that embryonic calli were formed within two weeks in the presence of 1 mgl-1 2,4-D. Adventitious shoots emerged from the embryonic callus in the presence of 4.5 mgl-1 BA. The cultivars showed a varied response to shoot regeneration. Regeneration frequency was high in the cultivar Sarow-4 (68%) followed by Masrri L-16 (64%) compared with the other cultivars tested. Hypocotyl explants from the cultivars Sarow-4 and Semu-249 were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing the neomycin phosphotransferase-II gene (NPT-II). The resulted putative transgenic plantlets were able to grow under knanamycin containing medium. The stable integration of the NPT-II gene into the plant genomes was tested by PCR using NPT-II -specific primers. The GUS gene expression can be detected only in the transgenic plants. The reported protocol in the present study is repeatable and can be used to regenerate transgenic canola plants expressing the genes present in A. tumifaciens binary vectors.
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A reproducible system to produce regenerated Brassica napus L., plants has been developed using hypocotyl explants. Different concentrations of Benzyladenine (1.5, 3.0 and 4.5 mg l -1 ) and Thidiazuron (0, 0.15 and 0.30 mg l -1 ) were evaluated for shoot regeneration using 7, 14 and 21 day old hypocotyl explants. Treatments were arranged in a factorial experiment as a randomized complete block design with three replications. Significant differences were found in the interactions of Benzyladenine and Thidiazuron concentrations as well as the ages of the explants. Maximum shoot regeneration was obtained using 4.5 mg l -1 Benzyladenine and 0.3 mg l -1 Thidiazuron. Shoot regeneration was highly affected by the age of the explants. It was found that 21 day old explants were more likely to undergo shoot development than the others. Under these culture conditions, the highest percentage of shoot regeneration was 174.0% for hypocotyl explants. Regenerated shoots rooted when cultured on a root induction medium supplemented with 2 mg l -1 of Indolebutyric acid. Rooted plantlets were successfully established in the soil which developed normal fertile flowers and viable seeds. In light of its efficiency, this hypocotyl regeneration method could be a suitable tool for genetic transformation using Agrobacterium.
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The effect of different durations of exposure to 2,4-D on hypocotyls and cotyledons cultured in vitro was studied in Brassica napus L. cv. Kana. Organogenesis or callogenesis depended on the duration of explant exposure to MS medium supplemented with 2 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid). Short treatment (1 and 3 days) with 2,4-D resulted in rhizogenesis on hypocotyls (100% and 98% of explants, respectively) and cotyledons (80% and 54% of explants, respectively). Adventitious shoots formed sporadically, with the highest frequency (14% of explants) on hypocotyls cultured 3 days on MS supplemented with 2,4-D and then transferred to hormone-free medium. Histological analysis clearly indicated that the basal part of hypocotyls is involved in root formation and callus production, and the apical part for shoots. Meristematic sites originated from groups of cells in the cortex layer (including cells of the endoderm), but the procambium, phloem and pericycle also showed meristematic activity. The present study indicated that the response of explants cultured on media containing 2,4-D at constant concentration depends on the duration of explant exposure to growth regulator.
Ten breeding lines including two CMS (Cytoplasmic Male Sterility) lines and one cultivar, Seoul, of Chinese cabbage {Brassica campestris ssp. pekinensis), known to be one of the most difficult vegetable crops to be regenerated from in vitro tissue culture, were used to establish the plant regeneration system. Two different types of explants, cotyledonary petiole and hypocotyl segments were cultured on MS basal medium supplemented with several combinations of plant growth regulators (PGRs). The optimum level of AgNC>3 for shoot regeneration ranged from 0.5 to 1.0 mg/1. An addition of AgNCb at higher than 1.0 mg/1 caused the high vitrification rate of regenerated shoots. There were great differences in the abilities in shoot regeneration among genotypes and media types. In all genotypes, MS basal medium supplemented with higher levels of PGRs (NAA 1.0 mg/1, BAP 3.0 mg/1, AgNC>3 0.5 mg/1) was far more effective in shoot regeneration than that containing lower level of PGRs(NAA 0.5 mg/1, BAP 2.0 mg/1, AgN03 0.5 mg/1). The best shoot regeneration (68%), however, took place when hypocotyl explants were cultured on MS basal medium in conjunction with NAA 1.0 mg/1, BAP 2.0 mg/1, zeatin 2.0 mg/1, AgNC>3 0.5 mg/1. RAPD (Randomly Amplified Polymorphic DNA) analysis of regenerants using several arbitrary decamer oligonuclotides showed that there were no major detectable somaclonal variations except for one regenerant, indicating that most of the regenerants were genetically stable. For genetic transformation experiments, hypocotyl segments of selected genotypes were precultured in the medium (NAA 1.0 mg/1, BAP 2.0 mg/1, zeatin 2.0 mg/1) for two days and cocultivated with Agrobacterium tumefaciens strains containing NPTII and bar (bialaphos resistance) genes for two days. Tobacco feeder cells were included in the medium from preculture to cocultivation procedure. Shoot regenerated directly from the explants in the selection medium (NAA 1.0 mg/1, BAP 2.0 mg/1, zeatin 2.0 mg/1, AgNCb 0.5 mg/1) containing kanamycin 10 mg/1 in a few weeks. Putatively transgenic plants were acclimatized to green house condition and applied to PCR analysis using bar primer. Ten out of 14 plants regenerated and survived from selection medium were found to be transgenic plants.
The aroA-M1 mutant consisting of an N-terminal portion of Es- cherichia coli 5-enolpyruvylshkimate-3-phosphate synthase (EPSPS) and a C-terminal portion of Salmonella typhimurium EPSPS confers high resistance to glyphosate because of a decrease in the Km(PEP) and an increase in the Ki(glyphosate). In this paper, we constructed a binary vector carrying the aroA-M1 mutant gene under the control of the CaMV 35S promoter for Agrobacterium-mediated gene trans- fer to Brassica napus L. Transgenic Brassica plants with increased resistance to glyphosate were obtained by cocultivation of hypocotyl explants with Agrobacterium tumefaciens and direct selection on media containing glyphosate. Southern blot and western blot analyses confirmed gene integration and expression in T0 transgenic Brassica plants. Transgenic plants exhibited increased resistance to glypho- sate compared with untransformed plants. Our results also indicated the aroA-M1 mutant gene may be a useful selectable marker for the transformation of plants and for the production of herbicide- resistant plants.
This study was made to clarify the effects of cytokinin type and their concentrations on shoot proliferation and hyperhydricity in in vitro Pyrus pyrifolia N. (`Hosui' and `Kosui') shoots. The shoots were subcultured in a woody plant medium supplemented with 0.5 M 3-indolyl-butyric acid, 3% (w/v) sorbitol, 0.8% (w/v) agar, and with cytokinins kinetin, 6-benzylaminopurine (BA), N-(2-chloro-4-pyridyl)-N9-phenylurea (CPPU), 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) added at concentrations 0.44, 4.40, 11.0 and 44.0 M. The highest number of shoots was induced by adding BA at concentration 11.0 M, then by 4.4 M BA, in both cultivars. TDZ and CPPU caused greater hyperhydricity in cultured explants than BA and kinetin. `Kosui' was more susceptible to hyperhydricity compared with `Hosui'.