American Journal of Plant Sciences, 2011, 2, 27-34
doi:10.4236/ajps.2011.21003 Published Online March 2011 (http://www.SciRP.org/journal/ajps)
27
Characterization of a Collection of Brassica
carinata Genotypes for in vitro Culture Response
and Mode of Shoot Regeneration
Javier Gil-Humanes, Antonio Martín, Francisco Barro
Institute for Sustainable Agriculture, CSIC. E-14080, Córdoba, Spain.
Received August 5th, 2010; revised September 21st, 2010; accepted September 28th, 2010.
ABSTRACT
Brassica carinata, a natural alloploid formed between B. oleracea and B. nigra, is a potential oil crop for th e Mediter-
ranean area in which genetic transformation could help to breed. In vitro culture and shoot regeneration are key fac-
tors in developing an efficient transformation method in the genus Brassica. However, the studies for in vitro culture
and shoot regeneratio n in B. carinata are limited to only a few g enotypes. The aim of this study was to evaluate the in
vitro culture respon se and shoot regeneration in a collection of B. carinata accessions to identify promising genotypes
with high shoot regeneration for genetic transformation experiments. Cotyledonary explants from 51 genotypes were
cultured in vitro and callus formation and swelling as well as the mode of shoot regeneration evaluated. A highly posi-
tive response to in vitro culture, i.e. callus formation or swelling, was observed in all the genotypes tested. Tissue
blackening occurred only in eleven genotypes. Parameters like callus formation and swelling and number of shoots per
explant were highly variable among genotypes. Fourteen genotypes regenerated only via callus formation, whereas
only one regenera ted only via swelling. Most genotyp es showed a higher p ercentage of callus formation than swelling.
The average number of shoots regenerating per explant among genotypes was the most variable factor measured. Six
genotypes regenerated more than 6 shoots per explant via callus phase. These genotyp es have been identified as having
a high regeneration potential and can be used in genetic transformation via Agrobacterium.
Keywords: Mustard, Tissue Culture, Genotype, Cotyledons, Swelling, Callu s
1. Introduction
Rapeseed (Brassica napus L.) is the third most important
source of vegetable oil in the world with 31 million cul-
tivated hectares in the year 2009 (FAOSTAT, 2011).
Modification of the fatty acid composition is currently an
important objective of plant breeding of this crop [1] and
there is considerable commercial interest in the devel-
opment of high erucic acid and/or low glucosinolates
lines targeted toward industrial end-use and in the de-
velopment of low (or zero) erucic acid, low linoleic and
high oleic lines for food industries.
Ethiopian mustard, B. carinata A. Braun (BBCC, 2n =
4x = 34), is a natural alloploid from B. oleracea L. (CC,
2n = 2x = 18) and B. nigra (L.) W.D.J. Koch, (BB, 2n =
2x = 16) which has several agronomical important traits
such as nondehiscent siliques and a much more devel-
oped and aggressive root system than B. napus. It is re-
sistant to a wide range of diseases and pests and is toler-
ant to many abiotic stresses [2-4], which makes it a suit-
able candidate as a winter crop in the Mediterranean
countries.
Plant transformation systems have been developed for
many economically important species of the genus
Brassica such as B. napu s [5], B. oleracea [6], B. juncea
[7], B. rapa [8], B. nigra [9] and B. carinata [10]. This
technology enables us to obtain transgenic plants with
modified agronomic traits. Many genetic improvements,
such as herbicide tolerance, improved oil quality and
production of pharmacological and industrial products,
have been achieved by genetic transformation in the
Brassica species. For example, B. napus seeds containing
high levels of gamma-linoleic acid were obtained by the
introduction of δ12-desaturase genes from the fungus
Mortierella alpine [11]. In addition, B. napus genotypes
were developed for sulfonylurea resistance and bro-

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and
Mode of Shoot Regeneration
28
moxynil resistance by Blackshaw et al. [12] and Zhong
[13] respectively. In B. carinata, Jadhav et al. [14] in-
creased the level of erucic acid in the seeds by co-sup-
pression and antisense repression of the endogenous FAD2
gene encoding the oleate desaturase FAD2.
In vitro regeneration is a key factor in developing an
efficient transformation method in plants. In vitro regen-
eration in Brassica ssp. is highly genotype-dependent as
reported in previous studies for B. napus [15,16], B.
juncea [17], B. rapa [18] and B. oleracea [19]. In addi-
tion, Dietert et al. [20] compared 6 species of the genus
Brassica for callus growth and plant regeneration and
reported a high influence of the genotype, with as much
inter-cultivar as inter-species differences in the response
to the in vitro culture. However, the available informa-
tion for the genotype variability for in vitro culture and
shoot regeneration in B. carinata is limited to a small
number of genotypes. This genotype-dependence of the
in vitro culture is a limiting factor for the application of
genetic engineering to a wide number of genotypes. For
that reason, it is important to identify highly regenerant
genotypes that can be used in transformation via Agro-
bacterium tumefaciens.
This study aims to evaluate the in vitro culture re-
sponse, i.e. callus formation and swelling, and the mode
of shoot regeneration of a collection of B. carinata ac-
cessions to identify promising genotypes with high re-
generation potential.
2. Materials and Methods
2.1. Plant Material
Fifty-one lines of B. carinata supplied by the Regional
Plant Introduction Station (Iowa State University, Iowa,
U.S.) (Table 1) were evaluated for in vitro culture and
shoot regeneration response. For comparison, two lines
were used as controls; a B. carinata DH line (BC71),
which showed a good response to microspore culture [21]
and a B. oleracea DH line (AG1012) provided by the
Department of Crop Genetics (John Innes Centre, Nor-
wich, UK) which was selected for its high regeneration
and transformation potential [22].
2.2. In vitro Culture
Seeds were surface sterilized with 100% ethanol for 2
min and with a 15% sodium hypochlorite solution con-
taining 0.1% Tween-20 for 15 min. Then, seeds were
washed 3 times with sterile distilled water and air-dried
in flow hood chamber. Seeds were germinated in 15 x 90
mm Petri dishes containing 25 ml of germination me-
dium, which consisted of 4.3 g/l MS salts [23] plus 3%
sucrose and 0.8% phytoagar at pH 5.8. After autoclaving,
Table 1. List of Brassica. carinata genotypes used in the
experiment supplied by the Regional Plant Introduction
Station (Iowa State University, Iowa, U.S.). Genotypes are
ordered by accession number.
Origin
Code
number
Accession
number Country State Plant name
1 193459 Ethiopia Shewa Unknown
2 193460 Ethiopia Harer NU 51639
3 193467 Ethiopia Shewa NU 51640
4 193759 Ethiopia Shewa Unknown
5 193760 Ethiopia Shewa NU 51642
6 193959 Ethiopia Shewa NU 51643
7 194251 Ethiopia Kefa NU 51645
8 194252 Ethiopia Kefa NU 51646
9 194253 Ethiopia Kefa NU 51647
10 194254 Ethiopia Kefa NU 51648
11 194255 Ethiopia Kefa NU 51649
12 194900 Ethiopia Gonder NU 51651
13 194901 Ethiopia Gonder Unknown
14 194903 Ethiopia Gonder NU 51653
15 194904 Ethiopia Gonder Unknown
16 195552 Ethiopia Welo NU 40525
17 195923 Ethiopia Welo NU 51657
18 196836 Ethiopia Welega Unknown
19 197402 Ethiopia Unknown Unknown
20 197403 Ethiopia Unknown NU 51660
21 199947 Ethiopia Unknown NU 51661
22 199949 Ethiopia Gonder NU 16543
23 199950 Ethiopia Kefa NU 51663
24 209023 Puerto Rico Unknown Unknown
25 226545 Ethiopia Shewa NU 51664
26 231046 Ethiopia Shewa Unknown
27 273636 Ethiopia Harer NU 51666
28 273640 Ethiopia Shewa Gomenzer
29 274283 Ethiopia Unknown NU 51668
30 280230 Ethiopia Unknown NU 51669
31 331377 Ethiopia Unknown Unknown
32 331378 Ethiopia Unknown Unknown
33 360879 Sweden Unknown 68-5702-1
34 360880 Sweden Unknown 68-5702-4
35 360881 Sweden Unknown 68-5702-6
36 360882 Sweden Unknown 68-5702-10
37 360883 Sweden Unknown 68-5702-16
38 360885 Sweden Unknown 68-5702-12 PLT 1
39 360886 Sweden Unknown 68-5702-15 PLT 9 S
40 360887 Sweden Unknown 68-5702-16 PLT 5 S
41 390133 Pakistan Unknown P-1
42 390134 Pakistan Unknown P-58
43 596535 Spain Cordoba BC-815-2
44 596536 Spain Cordoba BC-876-2
45 596537 Spain Cordoba BC-738-5
46 596538 Spain Cordoba BC-834-2
47 596539 Spain Cordoba BC-831-2
48 597822 Sweden Unknown 85-0572-69
49 633075 Tanzania Unknown Swahili
50 633076 Kenya Unknown WIR 4325
53 633080 Zambia Unknown BRA 1028/79

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and
Mode of Shoot Regeneration
29
filtered vitamins myo-inositol (100 mg/l), thiamine-HCl
(10 mg/l), pyridoxine (1 mg/l) and nicotinic acid (1 mg/l)
were added to the medium. Seeds were sown at a density
of 20-25 seeds per plate, incubated overnight at 15ºC in
the dark and then transferred to a culture room at 23ºC
under a 16-h photoperiod of 70 µmol m-2 sec-1 for 72h.
Cotyledons containing petioles of 1-2 mm in length
were excised from 4-day-old seedlings and placed at a
density of 10-11 cotyledons per plate into 20 x 90 mm
Falcon dishes containing 50 ml of regeneration medium.
This medium was the same germination medium de-
scribed above plus 2 mg/l of 6-benzylaminopurine and
500 mg/l of carbenicillin. Explants were cultured in this
regeneration medium for three weeks at 23ºC under 16-h
photoperiod and then transferred onto fresh regeneration
medium and subcultured for one week. Then, they were
evaluated for blackening in the petiole, formation of cal-
lus or swelling in the base of the petiole and finally for
the number of shoots produced via callus phase and via
direct regeneration.
2.3. Statistics
Data was analyzed using the SPSS version 11.0 statistical
software package. Arcsine√x transformation was carried
out on blackening, callus, swelling and no response fre-
quencies before analysis. The General Analysis of Vari-
ance and the LSD pairwise comparisons of means were
used to determine significant differences.
3. Results
Shoot regeneration from cotyledons in the genus Bras-
sica can be produced via an indirect callus phase (Figure
1(a)). However, not all genotypes produce callus and, in
some genotypes, direct shoot regeneration is observed
via swelling at the petiole base (Figure 1(a)). The oc-
currence of blackening is thought to result from an inter-
action between the cut surface of the cotyledon petiole
and the medium, and is highly genotype dependent (Fig-
ure 1(a)). The absence of tissue culture blackening is an
important factor for transformation success in B. ol-
eracea and B. napus [22,24].
In this work we have evaluated the in vitro culture re-
sponse, i.e. callus formation, swelling and blackening,
and shoot regeneration in 51 genotypes of B. carinata
and in two DH lines used as controls. Table 2 shows the
results for all genotypes, indicating the number of culti-
vated explants, the explants with no response to in vitro
culture, the explants with blackening, and the explants
regenerating either via callus or via swelling at the base
of the petiole.
Positive response to in vitro culture, as determined by
callus formation or swelling of explants, was observed in
(a)
(b)
Figure 1. (a) Mode of shoot regeneration: callus formation
(left), swelling at the base of the petiole (center) and tissue
blackening (right). (b) Variability in number of shoots per
explant regenerated via callus in three genotypes of Bras-
sica carinata: 273636 (high number of shoots), 273640 (av-
erage number of shoots) and 209023 (low number of
shoots).
all the genotypes tested (Table 2). In genotypes 4, 29, 31
and 1012, all the cultivated explants responded to in vitro
culture, whereas genotypes 7, 18 and 41 showed the
highest percentage of non-responding explants. Although
genotypes showed regeneration both via callus and via
swelling, most of the genotypes regenerated better via
callus phase than via swelling, showing higher percent-
ages of explants regenerating via callus than via swelling
(Table 2). Only 7 genotypes (7, 8, 9, 10, 12, 14 and 17)
showed higher percentage of swelling than callus forma-
tion. The maximum percentage of explants producing
swelling at the base of the petiole occurred in genotype 9
with 76.9%, while the maximum percentage of callus
formation occurred in genotype 29 with 100% of the ex-
plants responding via callus formation (Table 2). Tissue
culture blackening occurred in 2.3% of the 2795 culti-
vated explants and affected 11 of the 53 genotypes tested.
The maximum percentage of blackened explants was
34.6% for genotype 7. This genotype also showed the
highest percentage of non-responding explants (57.7%)
and all explants regenerated via swelling (Table 2).
Figure 2 shows the number of shoots per explant re-
generated either via callus phase or via swelling phase
(direct regeneration). The mean number of shoots per
explant regenerated via callus was 2.9, and the maximum
value was observed in genotypes BC71 and 31 with 8.7
shoots per explant each. Six genotypes with more than 6
shoots per explant produced via callus were identified
(21, 27, 31, 32, 46 and 71) while twenty-five genotypes
produced less than 2 shoots per explant. Figure 1B shows
three genotypes with maximum, average and minimum
shoot regeneration via callus phase.

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and
Mode of Shoot Regeneration
30
Table 2. In vitro culture response of cotyledonary explants from a collection of Brassica carinata and two DH lines: BC71
(Brassica carinata) and AG1012 (Brassica oleracea).
No response Blackening Callus Swelling
Code number Accession numberNumber of explantsNo. (%) No. (%) No. (%) No. (%)
1 193459 66 2 3.0 0 0 55 83.3 9 13.6
2 193460 47 5 10.6 0 0 36 76.6 6 12.8
3 193467 63 10 15.9 0 0 35 55.6 18 28.6
4 193759 50 0 0 0 0 39 78.0 11 22.0
5 193760 64 3 4.7 12 18.8 61 95.3 0 0
6 193959 46 3 6.5 0 0 38 82.6 5 10.9
7 194251 26 15 57.7 9 34.6 0 0 11 42.3
8 194252 28 4 14.3 5 17.9 11 39.3 13 46.4
9 194253 39 7 17.9 7 17.9 2 5.1 30 76.9
10 194254 49 10 20.4 0 0 13 26.5 26 53.1
11 194255 40 1 2.5 0 0 37 92.5 2 5.0
12 194900 38 4 10.5 0 0 7 18.4 27 71.1
13 194901 52 3 5.8 0 0 46 88.5 3 5.8
14 194903 41 7 17.1 0 0 13 31.7 21 51.2
15 194904 47 15 31.9 1 2.1 22 46.8 10 21.3
16 195552 57 12 21.1 0 0 42 73.7 3 5.3
17 195923 27 5 18.5 0 0 5 18.5 17 63.0
18 196836 45 25 55.6 0 0 20 44.4 0 0
19 197402 50 3 6.0 0 0 44 88.0 3 6.0
20 197403 39 2 5.1 0 0 36 92.3 1 2.6
21 199947 48 6 12.5 0 0 42 87.5 0 0
22 199949 29 8 27.6 0 0 19 65.5 2 6.9
23 199950 38 1 2.6 1 2.6 29 76.3 8 21.1
24 209023 65 11 16.9 0 0 48 73.8 6 9.2
25 226545 29 3 10.3 4 13.8 24 82.8 2 6.9
26 231046 33 5 15.2 0 0 28 84.8 0 0
27 273636 55 2 3.6 0 0 53 96.4 0 0
28 273640 51 5 9.8 0 0 46 90.2 0 0
29 274283 61 0 0 0 0 61 100.0 0 0
30 280230 60 2 3.3 4 6.7 58 96.7 0 0
31 331377 58 0 0 0 0 53 91.4 5 8.6
32 331378 63 5 7.9 0 0 49 77.8 9 14.3
33 360879 56 7 12.5 0 0 47 83.9 2 3.6
34 360880 59 6 10.2 0 0 49 83.1 4 6.8
35 360881 50 12 24.0 0 0 36 72.0 2 4.0
36 360882 65 6 9.2 0 0 55 84.6 4 6.2
37 360883 58 17 29.3 0 0 41 70.7 0 0
38 360885 54 2 3.7 4 7.4 46 85.2 6 11.1
39 360886 63 12 19.0 0 0 50 79.4 1 1.6
40 360887 66 6 9.1 0 0 60 90.9 0 0
41 390133 65 23 35.4 0 0 42 64.6 0 0
42 390134 66 2 3.0 0 0 52 78.8 12 18.2
43 596535 33 5 15.2 0 0 25 75.8 3 9.1
44 596536 39 3 7.7 9 23.1 36 92.3 0 0
45 596537 48 15 31.3 0 0 33 68.8 0 0
46 596538 66 5 7.6 0 0 59 89.4 2 3.0
47 596539 64 8 12.5 0 0 53 82.8 3 4.7
48 597822 76 6 7.9 0 0 63 82.9 7 9.2
49 633075 53 5 9.4 0 0 42 79.2 6 11.3
50 633076 44 4 9.1 0 0 36 81.8 4 9.1
53 633080 95 15 15.8 0 0 74 77.9 6 6.3
71 BC71 44 5 11.4 0 0 39 88.6 0 0
1012 AG1012 127 0 0 3 2.4 100 78.7 27 21.3

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and
Mode of Shoot Regeneration
31
Figure 2 Number of shoots per explant regenerated via cal-
lus or swelling from cotyledonary explants collected from
51 lines of Brassica carinata. Two DH lines, BC71 (Brassicca
carinata) and AG1012 (Brassica oleracea) were included for
comparison.
The mean number of shoots regenerated via swelling
was 1.7, and the genotype producing the maximum
number of shoots per explant was also genotype 31 with
10.4 shoots per explant. Only three genotypes (20, 13
and 31) produced more than 6 shoots per explant by di-
rect regeneration while thirty-seven genotypes produced
less than 2 shoots per explant.
In thirty-two genotypes, shoots were produced both by
callus and swelling phase, whereas seventeen genotypes
regenerated shoots only by callus phase and in genotypes
7, 9 and 17 all the shoots were regenerated by direct re-
generation.
General analysis of variance (Table 3) revealed that
there were highly significant differences among geno-
types for the absence of response to the in vitro culture,
the mode of response, i.e. percentage of callus or per-
centage of swelling, the occurrence of blackening, and
the number of shoots produced either via callus or via
direct regeneration.
4. Discussion
Shoot regeneration in B. carinata can occur directly from
explant tissue as well as indirectly from callus that pro-
liferate from the cut edge of the explants. In many geno-
types, direct shoot regeneration has been described as
less genotype dependent [25] and regenerants show more
genetic stability [26], whereas callus phase is more asso-
ciated with somaclonal variation. However, direct shoot
regeneration has limitations in being used for DNA
transfer as totipotent cells are less accessible to Agro-
bacterium during cocultivation [27]. In fact, dedifferen-
tiation of explant cells into callus was necessary for the
efficient transformation of B. carinata [10]. Therefore,
the identification of genotypes with high regeneration
potential that regenerate mostly via callus formation can
largely influence the recovery of transgenic plants. Spar-
row et al. [22] found three phenotypic markers highly
influencing the transformation efficiency in B. oleracea.
These are: susceptibility to A. tumefaciens [28], shoot
regeneration potential and the mode of shoot regenera
Table 3. Analysis of variance for in vitro culture response
and shoot regeneration in a collection of Brassica carinata
accessions. Values expressed as a percentage were trans-
formed using the arcsine√x function for the analysis of va-
riance.
Parameter MS df F-test
No response (%) 0.1298 52 3.48***
Blackening (%) 0.0476 52 3.14***
Callus (%) 0.1865 52 4.61***
Swelling (%) 0.1617 52 4.90***
Shoots per explant from callus 19.742 51 10.00***
Shoots per explant from swelling15.596 38 2.16**
Significance probability levels: **1%, ***0.1%. MS; Mean Square, df;
degree of freedom.

Characterization of a Collection of Brassica carinata Genotypes for in vitro Culture Response and
Mode of Shoot Regeneration
32
tion. They also found that the absence of blackening tis-
sue associated with callus formation is critical for trans-
formation success. We have applied these criteria to
evaluate a collection of 51 genotypes of B. carinata for
in vitro culture and shoot regeneration potential, includ-
ing the mode of shoot regeneration. We have found a
highly positive response to in vitro culture, i.e. callus
formation and swelling, in all the genotypes tested in this
study. Other studies have previously confirmed the re-
sponsiveness of B. carinata seedling explants to tissue
culture [29-31]. For both, callus formation and swelling,
there were significant differences among genotypes. In
our study, the number of genotypes that proliferate via
callus formation was much higher than those via swelling.
In addition, the percentage of callus formation was
higher than swelling in all the genotypes, except in 7
genotypes (7, 8, 9, 10, 12, 14 and 17) in which the per-
centage of swelling formation was higher.
Callus formation at the base of the petiole has been
demonstrated to be an interesting trait in obtaining a
great number of shoots per explant. Sparrow et al. [22]
observed that shoot regeneration via the callus phase
resulted in well established and morphologically defined
shoots that were easy to isolate and propagate. Results
reported here showed that shoot production was very
variable among genotypes. This result agreed with stud-
ies in Brassica spp., which also reported that shoot pro-
duction was highly variable among different genotypes,
indicating that the regeneration ability is strongly influ-
enced by genotype [15-20]. Zhang et al. [18] reported a
maximum of 10.7 shoots per explant in a cultivar of
Chinese cabbage. In our study the maximum number of
shoots per explant was 8.7 and 10.4, regenerated respec-
tively from callus and direct regeneration, both by geno-
type 31. We have found six genotypes (21, 27, 31, 32, 46
and 71) producing more than 6 shoots per explant via
callus formation, although three of them (31, 32 and 46)
also produced shoots via swelling. These six genotypes
had a percentage of callus formation between 77.8% and
96.4% and no blackening tissue at the base of the petiole
was observed. These six genotypes produced an average
of 7.1 shoots per explant via callus. This average is 2.4
times higher than the 2.9 shoots of average produced via
callus by all genotypes. This high-frequency of regenera-
tion from callus might be of great importance in regen-
eration of transgenic plants. Mukhopadyay et al. [27]
reported that the transformation frequency of B. campes-
tris is strongly influenced by the mode of shoot regenera-
tion. In addition, Babic et al. [10] reported that efficient
regeneration from callus was responsible for the high
frequency of transformation in B. carinata.
The occurrence of tissue blackening resulted from an
interaction between the cut surface of the cotyledon peti-
ole and the medium. Although blackening does not pre-
vent the formation of shoots, we have observed a nega-
tive correlation between the percentage of blackening
and the number of shoots per explant. In addition, geno-
types with no blackening produce more shoots than those
presenting blackening. Therefore, the absence of tissue
blackening influences the successful regeneration of
shoots and this could be a critical factor to regenerate
transgenic plants.
In conclusion, in this work we have characterized the
tissue culture response and the mode of regeneration of a
collection of B. carinata. The mode of shoot regeneration
is important in the selection of genotypes with a high
regeneration potential and that regenerate mainly from
callus. Six genotypes (21, 27, 31, 32, 46 and 71) have
been identified as having a high regeneration potential.
They produce the highest number of shoots per explant
via callus formation and do not present tissue blackening
at the base of the petiole. We are currently incorporating
these 6 genotypes in transformation experiments to de-
termine their susceptibility to A. tu mefac iens. The results
of these experiments should allow us to develop a trans-
formation program using genotypes highly susceptible to
A. tumefaciens and with a high level of regeneration via
callus.
5. Acknowledgments
The authors acknowledge funding by the Spanish Comi-
sión Interministerial de Ciencia y Tecnología (C.I.C.Y.T.),
project AGL2004-03361-C02-2 and to the Regional Plant
Introduction Station (Iowa State University, Iowa, U.S.)
for supplying seeds. Javier Gil-Humanes acknowledges
the CSIC for a pre-doctoral I3P fellowship and to Dr.
Penelope Sparrow from the Crop Genetics Department of
John Innes Center (UK) for the critical reading of the
manuscript. Technical assistance by Azahara Vida is also
acknowledged.
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Mode of Shoot Regeneration
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