Plant Science, 72 (1990) 245--252

Elsevier Scientific Publishers Ireland Ltd.

245

Transgenic plants of mustard Brassica juncea (L.)

Czern and Coss

Helena Mathews a,b, N. Bharathan a, R.E. Litz% K.R. Narayanan a, P.S. Rao b and
C.R. Bhatia b
University of Florida, Tropical Research and Education Center, Homestead, Florida 33031 (U.S.A.) and ~Bhabha Atomic Research
Centre, Bombay (India)
(Received May 9th, 1990; revision received July 16th, 1990; accepted July 19th, 1990)
The recovery of transgenic mustard plants from the R, progeny of regenerated plants using cotyledon explants treated with Agrobacterium tumefaciens (C58C1, pGV3850:: 1103) is described. Adventitious shoots were isolated from cotyledonary callus treated
with A. tumefaciens, and the shoot apices were sliced longitudinallyand grown on selection medium. Regenerants were also grown on
selection medium. After the second subculture, shoots with a higher ratio of green to white and/or purple were rooted. These were
transplanted into soil and selfed. Seeds from R0 generation were germinated in vitro and shoot tip and cotyledon explants were
screened for kanamycin resistance. Solid green shoots were obtained from cotyledon explants on selection medium containing 20 rag/
I kanamycin. These were transplanted into soil and grown to maturity. Southern blot analysis of R 1plants showed intact integration
of T-DNA into the Brassicajuncea genome. This was further confirmed by seed germination and seedling growth on medium containing 200 mg/l kanamycin.
Key words: Brassicajuncea; transformation; transgenic plants

Introduction
C o t y l e d o n s a r e excellent source e x p l a n t s f o r
g e n e r a t i n g t r a n s g e n i c p l a n t s [1,2]. In B r a s s i c a j u n cea, a d v e n t i t i o u s s h o o t s d e v e l o p r e a d i l y f r o m
c o t y l e d o n callus in vitro [ 3 1 5 ] ; b u t c o - c u l t i v a t i o n
o f c o t y l e d o n s with A . t u m e f a c i e n s d i d n o t p r o duce direct t r a n s f o r m a n t s . V e r y o f t e n , t h e cells
c o m p e t e n t for t r a n s f o r m a t i o n a r e n o t necessarily
c o m p e t e n t f o r r e g e n e r a t i o n [6]. I n r e p e a t e d experim e n t s a f t e r i n c u b a t i o n with A . tumefaciens, c o t y l e d o n e x p l a n t s have p r o d u c e d t r a n s f o r m e d callus
but not transformed shoots (our own observations). H e r e we d e s c r i b e a p r o t o c o l d e v e l o p e d for
the p r o d u c t i o n o f t r a n s f o r m e d m u s t a r d p l a n t s

Correspondence to: Helena Mathews, University of Florida,

Tropical Research and Education Center, Homestead, FL
33031, U.S.A.
Abbreviations: BA, benzylaminopurine; IBA, indole-3-butyric
acid; NAA, naphthalene acetic acid.

which involves j u d i c i o u s choice o f e x p l a n t s at v a r ious stages, m a n i p u l a t i o n o f t h e m i c r o e n v i r o n ment

for sequenced
promotion
of organ
d e v e l o p m e n t a n d p h a s e d e x p o s u r e to selection
pressure.

Materials and Methods

Explant
Seeds o f Brassica j u n c e a (L.) C z e r n a n d Coss
cv. Rai 5 ( m u s t a r d ) were surface-sterili_ed with
0.1070 ( w / v ) a q u e o u s H g C I 2 f o r 15 m i n f o l l o w e d b y
t h o r o u g h rinsing in sterile distilled water. Seeds
were g e r m i n a t e d o n M u r a s h i g e a n d S k o o g
m e d i u m (MS) [7] s u p p l e m e n t e d with 307o ( w / v )
sucrose, 550/~M i n o s i t o l , 2.4/~M p y f i d o x i n e H C I ,
0.3 buM t h i a m i n e H C I , 4.1 /~M nicotinic acid a n d
26.6 /~M glycine ( f u r t h e r r e f e r r e d t o as b a s a l
m e d i u m , BM). M e d i u m was s o l i d i f i e d with 0.807o
( w / v ) D i f c o B a c t o a g a r , a u t o c l a v e d at 121 C a n d
1.1. k g / c m 2 f o r 20 min. C u l t u r e s were m a i n t a i n e d

0168-9452/90/$03.50 1990 Elsevier Scientific Publishers Ireland Ltd.

Printed and Published in Ireland

246
in continuous fluorescent light of 25/aE. s-1. m -2
at 25 C.
Cotyledons from 6-day-old seedlings were cultured on BM with 0.54 ~ NAA and 4.4/~M BA
(regeneration medium). Cotyledons showing canlogenic callus were selected for A. tumefaciens
infection.

Preparation of bacterial culture

A single colony of Agrobacterium tumefaciens
C58C1, pGV3850::1103 was inoculated into 5 ml
of Luria Broth (LB) [8] with 50 mg/1 kanamycin
(pH 7.0) overnight. This was transferred to 50 ml
of LB at pH 5.6 with 30 ~M acetosyringone (AS)
(Aldrich) and kept on a shaker at 200 rev./min at
28 C for 16 h.

without kanamycin, and were transplanted to soil.

Mature plants were self-pollinated.

Recovery and selection of putatively transformed

shoots from seedling explants
Seeds from the first generation (R0) of regenerated plants were germinated on BM as described
above. Shoot tip (0.8--1.0 cm) and cotyledon
explants were cultured on regeneration medium
with 50 mg/1 and 20 mg/1 kanamycin, respectively, to suit the kanamycin tolerance of the particular explant [9].

Maintenance of putatively transformed shoots

Solid green shoots were cultured on regeneration medium with 50 mg/l kanamycin for multiplication.

Co-cultivation of bacteria and explant

Twenty cotyledons were cut transversely at the
region of caulogenic callus. A log phase culture of
A. tumefaciens activated with AS was smeared
onto the wounded surfaces of the caulogenic callus attached to the cotyledon explant. These were
recultured on regeneration medium for 3 days.

Tissue culture separation of transformed cell

layers
Cotyledon explants were rinsed with sterile distilled water after co-cultivation, blotted dry on
sterile filter paper and cultured on regeneration
medium supplemented with 10 mg/1 kanamycin
and 500 mg/l cefotaxime. After 25 days all the
shoot regenerants were excised from the cotyledon
explants and were subcultured.

Tissue culture separation of transformed cell

layers
Shoot tips (0.5--1.0 cm) excised from cotyledon-derived shoot regenerants after Agrobacterium co-cultivation were longitudinally cut into
equal haves. Each half was cultured separately on
regeneration medium with 300 mg/1 cefotaxime
and 20 mg/1 kanamycin. After 30 days, shoots
with a high ratio of green to white and/or purple
were segmented and subcultured.

Rooting and transplantation

Shoot regenerants that were at least 80% green
were rooted on BM with 100 mg/l cefotaxime and

Rooting of putative transformants

Completely green shoots derived from cotyledon explants on selection medium were cultured
on BM with 0, 50 and 100 mg/1 kanamycin for
root induction. The bases of unrooted shoots were
dusted with Hormex rooting powder No. 16
(Brooker Chemical, active ingredient, IBA). The
shoots were then transplanted to soil.

Southern blot hybridization

DNA was extracted from young leaves (1 g) of 7
putatively transformed plants and a non-transformed control plant by the method of Dellaporta
et al. [10]. Prior to endonuclease digestion, DNA
suspensions were treated with DNase-free pancreatic ribonuclease (RNase) to remove any contaminating RNA [11]. Fifty microliters of an
endonuclease reaction mixture contained 10/~1 of
miniprep DNA, 5/A of 10 reaction buffer, 2/al
of EcoRI (20 units), 2/~1 of bovine serum albumin
(BSA) and brought to volume with water.
Digestion was overnight at 37 C. Electrophoretic
separation was carried out on 0.8% agarose (w/v)
horizontal slab gels in TAE buffer (containing 40
mM Tris acetate and 1 mM ethylenediamine tetraacetic acid (EDTA), pH 8.0) at 30 V for 16 h, and
transferred to a nylon membrane (Amersham) by
capillary blotting [12]. The DNA was immobilized
on nylon membranes by baking at 80C for 2 h in
vacuum.
Hybridization probes were made from plasmid

247
p L G V n e o l l 0 3 , maintained in E. coli, HB101.
Plasmids were isolated by the alkaline extraction
procedure o f Birnboim and Doly [13] and digested
with EcoRI and ApaI restriction endonucleases.
This releases a 2 kb fragment containing the NPTII coding region fused on its 5' end with the promoter region o f the nopaline synthase gene and on
its 3' side to a fragment o f the octopine synthase
gene containing the polyadenylation signal (OCS
polyA) [14]. The 2-kb fragments were gel-purified
and labelled by a multiprime DNA labelling system (Amersham kit, 30 /~Ci [32p]dcTP. Probe
specific activities were 1 10~--1.5 109 c p m /
/~g DNA.
Prehybridization, hybridization and washes
were performed at 65C. Hybridization was in
6 SSC, (1 = 0.15 M NaC1/0.015 M sodium
citrate, pH 7.0), 5 Denhardt's solution (1 x =
0.02O7e polyvinylpyrrolidone, 0.027o ficoll, 0.027o
BSA), 10o70 sodium dodecylsulfate (SDS), 50o7o
(v/v) formamide containing 1.1--2.2 107 dpm
of 32p-labelled probe/ml. After overnight hybridization, filters were washed twice in 2 SSC at
65C for 15 min and once in 1X SSC at 68C for

Fig. 1.

10 min followed by exposure to Kodak-x-omat AR

X-ray film at - 7 0 C for 8 days using Cronex
intensifying screens.

Screening for surviving A. tumefaciens

Leaf and stem segments o f the putatively transformed plants (R~) were grown in liquid broth
medium specific for A. tumefaciens [15]. Ten
flasks, each containing five l-cm segments of stem
or leaves, with 50 ml of medium, were kept on a
shaker at 200 rev./min for 7 days. Aliquots o f
0.05 ml of this medium, after incubation with
plant material, were plated on broth agar plate. A
pure bacterial culture o f C58C1, pGV3850::1103
was also plated on similar broth medium.

Progeny test
Seeds from the putatively transformed plants
and a non-transformed control plant were cultured on BM with 200 mg/1 kanamycin and incubated in darkness. After 3 days the cultures were
exposed to continuous fluorescent light of 25
/~E" s-~ m -2 at 25 C. Seedlings which were green
and formed secondary and tertiary leaves in the

Cotyledonarycallus of R~generation producingsolid green regenerantson regenerationmeliumwith 20 mg/l kanamycin.

248

presence o f kanamycin were scored as resistant

and white seedlings which did not form true leaves
were scored as sensitive.
Results
After 3 days of co-cultivation and reculture on
regeneration medium, the wounded region of the
cotyledons continued to differentiate shoots.
Longitudinally sliced shoot tips on selection
medium with 20 mg/l kanamycin produced shoot
regenerants which were green, white, purple or a
combination o f the three colours. Shoots that
were either completely white or white and purple
were discarded, while shoots that were either
completely green or green with white a n d / o r purple were cut longitudinally and the shoot segments
were subcultured. Regenerants from the second
subculture were visually screened for a high ratio
o f green to white a n d / o r purple shoots. Eight

shoots that were at least 8070 green were rooted on

BM without kanamycin and were transplanted
into soil. All 8 plants of the R 0 generation were
selfed and seeds from 6 plants were available for
further analysis. An average of 20 seeds from each
of the 6 plants were germinated in vitro. Cotyledons and shoot tips, both from 7-day-old seedlings, were cultured on regeneration medium with
20 mg/l and 50 mg/l kanamycin respectively. The
shoot tip explants formed calli at the cut ends but
did not form multiple shoots. The cotyledon
explants formed caulogenic callus. From a total of
240 (20 x 2 x 6) cultured cotyledons, 15 explants
produced completely green shoots (Fig. 1). These
were separately subcultured on shoot multiplication medium with 50 mg/l kanamycin. Although
the majority of regenerants were green, occasionally purple or purple with white shoots were
observed during multiplication of these putatively
transformed shoots on selection medium with 50

Fig. 2.
A n autoradiogram of agarose gel showing Southern blot analysis of putative transformants of B. juncea probed with the
ApaI and EcoRI digest (2 kb) of pLGVneo1103. Lane 1 - - homologous hybridisation to 2.0 kb probe from pLGVneo1103. Lanes 2 - 5 and 7--9, 5 btg each o f digested D N A from 7 transformants. Lane 6 is 5 #g digested D N A from a control plant. Molecular weight
markers of HindIII digests are shown in (kb) to the left o f lane 1.

249
mg/l kanamycin. Green shoots were transferred to
BM with 0, 50 and 100 mg/l kanamycin for root
induction. Surprisingly,~the shoots did not form
roots even on non-selection medium (0 kanamycin). These shoots were then dusted with Hormex
rooting powder No. 16 (Brooker Chemical) at cut
ends and directly transferred to soil. Of 15 such
transplants of the R~ generation, 12 formed roots
and produced seeds after selfing.

Southern hybridization
Figure 2 shows the Southern blot results of
EcoRI cut DNA probed with a 2-kb fragment
from pLGVneoll03. Hybridization bands were
detected in all the lanes with transformed DNA
while the control lane did not show any signal.
Fragment sizes of approximately 6.5 kb and 5.2 kb
were observed in all the lanes except the control
lane.
Screening test for A. tumefaciens contamination
Samples of liquid medium inoculated with leaf

and stem explants of putative transformants (R~),

when plated on A. tumefaciens specific medium,
did not show any bacterial growth. Pure bacterial
cultures of C58C1, pGV3850:: 1103 readily produced large numbers of colonies on the plate.

Progeny test
Seeds from the 12 putative transformants of the
R~ regeneration and a non-transformed control
plant were cultured on BM with 200 mg/l kanamycin. During 3 days of incubation in darkness, all
control seeds germinated and hypocotyls were
approx. 2--4 cm in length. Seeds from putatively
transformed plants were of 3 types: (1) those
which germinated and grew like the control; (2)
seeds with barely sprouted embryos; (3) seeds
which did not germinate. Kanamycin-resistant
seedlings became green in the light, produced secondary and tertiary leaves and grew normally,
whereas the sensitive seedlings were white and did
not produce true leaves (Fig. 3 and Table I).
Resistant seedlings were transferred to soil for
future studies.

O
Fig. 3.

Kanamycinresistant(left)and sensitive(right)plants25 daysaftergerminationon BMwith200 mg/l kanamycin.

250
Table I.

Progeny analysis o f the R t generation.

Putatively
transformed
plants

No. o f
seeds sown

1
49
2
86
3
78
4
46
5
19
6
72
7
24
8
22
9
39
10
95
11
77
12
85
Control
51
(non-transformed)

No. o f seeds
not germinated

No. o f seeds showing

arrested growth of
sprouted embryo

No. o f seedlings
Resistant to
kanamycin

Sensitive to
kanamycin

10
ll
35
6
l
43
12
17
28
29
5
30
0

0
0
0
7
0
0
0
5
0
8
16
14
0

3
28
5
1
4
8
1
0
3
6
6
0
0

36
47
38
32
14
21
11
0
8
52
50
41
51

Medium BM + 200 mg/I kanamycin. Observations scored after 24 days.

Discussion

Although Brassica species in general are highly

susceptible to A. tumefaciens, attempts to obtain
transformed plants have been reported only in B.
napus [16--20]. Transformed callus lines have
been reported in B. campestris [21]. In B. juncea,
shoot regeneration from tumor callus and formation of rooty callus by wild and mutant strains of
A. tumefaciens have been reported [22,23]. In this
communication we report recovery of putatively
transformed plants from the R 1generation derived
from Agrobacterium-treated cotyledon explants.
The intact stable integration of foreign DNA into
the B. juncea genome was confirmed by Southern
blot analysis.
Plant material from putatively transformed
plants in A. tumefaciens specific medium was free
of bacterial growth. This confirmed that the presence of intact plasmid T-DNA in the plant genome
was not due to residual contamination from A.
tumefaciens.
Common fragments of 6.5 kb show the
expected hybridization pattern of EcoRI cut genomic DNA transformed with pGV3850::1103 [24].

The presence of the 5.2 kb fragment could be due

to the presence of additional homologous
sequences in the probe outside the NPT-II
sequences.
Extensive studies [9] carried out on the kanamycin tolerance of different explants of B. juncea
have clearly demonstrated that 200 mg/l kanamycin impairs chlorophyll formation and seedlings
do not form true leaves and that 20 mg/l kanamycin is suffient to suppress shoot regeneration from
cotyledonary explants. Accordingly, final screening for kanamycin resistance was carried out at
200 mg/l kanamycin for seed germination and at
20 mg/l kanamycin for cotyledonary explants.
Selection pressure was increased in a phased manner at the earlier stages as indicated in Materials
and Methods.
Since all the cotyledons from 20 seeds each of 6
R0 plants were pooled, independent transformation events were not determined and the frequency of transformation could not be precisely
determined. However, since at least one transformation event has taken place in the 20 initial
explants, the frequency of transformation is equal
to or better than 5 x l0 -2. Germination at 200

251

mg/l kanamycin confirmed inheritance of the

kanamycin resistance trait by the progeny.
Our earlier attempts to regenerate plants from
B. juncea callus transformed with A. tumefaciens
C58C1, PGV3850:: 1103 were unsuccessful. A similar situation has been reported in flax. Although
flax hypocotyl and cotyledon segments produce
shoots highly efficiently, incubation with C58C1,
pGV3850::1103 results in transformed callus but
no regeneration [25]. Jianping and Quiquan [26]
reported transformed B. oleraceae callus only
from hypocotyl segments, using C58C 1,
pGV3850::1103. Feldmann and Marks [27] treated
germinating seeds of Arabidopsis thaliana with A.
tumefaciens and recovered transgenic plants in the
second generation. A transformed plant was also
recovered from the progeny of a chimaeric regenerated plant that resulted from DNA introduction by the particle gun method into the meristems
of immature soybean seeds [28].
The procedure described in this report essentially involved: (1) formation of a chimaera consisting of transformed and non-transformed cells;
(2) separation of transformed and non-transformed cells by tissue culture; and (3) inclusion of
these cells in the germinal layer of the regenerated
plant enabling transmission of the transformed
cells into the seeds.
The frequency of seed germination and the
atypical segregation pattern of kanamycin resistant and sensitive seedlings in the progeny of putative transformants raise a few points. Are the
putative transformants of the R~ generation a
mosaic of transformed and non-transformed
cells? Is the higher-than-expected ratio of sensitive
to resistant seedlings due to lack of expression of
the KanR gene? Considerable variability of Kana
expression has been reported in many solanaceous
plants [29]. However, in the present study the high
number of sensitive seedlings could be due to the
presence of non-transformed cells, since the solid
green shoot regenerants recovered from cotyledon
explants occasionally produced purple and/or
white shoots in the selection medium with 50 mg/1
kanamycin. We feel that the kanamycin-resistant
plants obtained in the progeny of R 1 ought to be
considered as true transformants and one should

look at the progeny of the R 2 generation to study

the inheritance pattern of the Kan Rgene.
It is possible that the arrested growth of the
sprouted embryos and lack of seed germination
(Table I) are associated with the mutagenic role of
T-DNA insertion. The insertion of foreign DNA
could be functioning as a germline lethal. Embryo
lethal mutants were reported in the progeny of
transgenic tobacco plants [30]. The observation in
B. juncea that the putatively transformed shoots
were difficult to root, coupled with our earlier
finding that transformed callus never underwent
organogenesis indicates changes in the morphogenetic expression of the transformed genome. Control non-transformed B. juncea shoot explants
root easily in BM and cotyledons produce caulogenic calli. This suggests the possible role of TDNA as an insertional mutagen [31]. Transgenic
plants have been used to study the molecular
organization of plant genes [32].

Acknowledgements
We gratefully acknowledge Dr. An Depicker,
Rijkuniversiteit, Ghent for providing the Agrobacterium tumefaciens strain used in this study.
The assistance of Rose Hendrix, Sarah Wright and
W.R. Graves is greatly appreciated. Florida Agriculture Experiment Station Journal Series No. R0O659.

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