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Transgenic Plants of Mustard Czern and Coss: Brassica Juncea (L.)
Transgenic Plants of Mustard Czern and Coss: Brassica Juncea (L.)
245
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
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.
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.
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
248
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.
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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
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.
250
Table I.
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 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
Discussion
251
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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