Plant Cell Reports
Plant Cell Reports (1988) 7:504-507
© Springer-Verlag 1988
Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens Vibha Srivastava, A. S. Reddy, and Sipra Guha-Mukherjee Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Received May 25, 1988/Revised version received October 12, 1988 - Communicated by I. K. Vasil
ABSTRACT
MATERIALS
A chimaeric neomycin p h o s p h o t r a n s f e r a s e II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its TDNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in r e g e n e r a t i o n of shoots and roots without any exogenous r e q u i r e m e n t of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of k a n a m y c i n r e s i s t a n t regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll d e f i c i e n t m u t a n t plant was also obtained.
Plant Material: Brassica oleracea botrytis cv. synthetica (cauliflower) grown in a peat-sand (1:3) mixture freshly emerged leaves were taken experiments.
INTRODUCTION Genetic t r a n s f o r m a t i o n of higher plants has been most e f f i c i e n t l y carried out by A g r o b a c t e r i u m tumefaciens, a natural gene vector of plants (for r e v i e w see Zambryski et al., 1984). For the improvement of the transformation vectors, chimaeric dominant selectable marker genes conferring r e s i s t a n c e to a m i n o g l y c o s i d e drugs have been constructed (Bevan et al., 1983). The c h i m a e r i c genes have been inserted into both oncogenic and non-oncogenic Ti plasmid vectors. The plant cells transformed by non-oncogenic Ti plasmid carrying chimaeric gene in T-DNA, have been r e g e n e r a t e d by hormonal manipulation in tissue culture media (De Block et al., 1984; Hain et al., 1985). The t r a n s f o r m a t i o n of higher plants with c h i m a e r i c gene inserted in o n c o g e n i c Ti plasmid has also been performed (HerreraEetrella et al., 1983), but no regeneration has so far been reported. Here, we are reporting the t r a n s f o r m a t i o n and r e g e n e r a t i o n of Brassica oleracea system by an o n c o g e n i c Ti plasmid vector containing a chimaeric neomycin phosphotransferase II gene in its T-DNA. Several r e g e n e r a t e d plants were obtained and a few of them were d i s t i n c t l y d i f f e r e n t from the normal (untransformed) plants.
Offprint requests to." V. Srivastava
AND
METHODS
var. were and for
B a c t e r i a l strain: An o n c o g e n i c ~grobacter--~m/m~ f ~ strain C ~ C~ Rif harbouring ~ ~ pLGVTi23~o lwas used for transformation of plants. The plasmid contains chimaeric neomycin phosphotransferase II (Nos/NPT II) gene inserted in its TDNA. The original strain was o b t a i n e d from Dr. M. Van M o n t a g u (Belgium) and has been described as oncogenic by Herrera-Estrella et al. (]983). Plant T r a n s f o r m a t i o n and Regeneration: Transformation of B. oleracea was performed by leaf disc t r a n s f o r m a t i o n m e t h o d (Horsch et al., 1985), with few modifications. Discs punched from surface sterlized leaves, a v o i d i n g mid-rib, were p r e - c o n d i t i o n e d on MS m e d i u m (Murashige and Skoog, 1962) containing BAP (0.5 ppm) and NAA (I ppm). After 24 h of p r e - c o n d i t i o n i n g , the leaf discs were mixed with a d i l u t e d culture of 48 h grown Agrobacterium (1:20 with MS liquid media} for 5-10 minutes. The discs were blotted dry and placed on hormone-free MS basal m e d i u m c o n t a i n i n g c a r b e n i c i l l i n or c e f o t a x i m (500 ~ g / m l ) . After about two weeks, the leaf discs were t r a n s f e r r e d to MS basal medium containing k a n a m y c i n (100 ~ g / m l ) . The leaf and petiole explants of regenerated t r a n s f o r m e d B. oleracea plants were placed on MS basal m e d i u m c o n t a i n i n g kanamycin (100 ~ g / m l ) f o r induction of callus and r e g e n e r a t i o n of shoots and roots. DNA
Isolation
and
Southern
Hybridization
:
High molecular weight total DNA was isolated from shoots of t r a n s f o r m e d and nont r a n s f o r m e d (control) B. oleracea as described by Cullis (1981). Total DNA was digested c o m p l e t e l y with Hind III, electrophoresed on 0.8% agarose gel and t r a n s f e r r e d to a n i t r o c e l l u l o s e filter (Southern, 1975). Southern h y b r i d i z a t i o n was carried out using
505 which were not infected with A. tumefaciens did not grow on the hormone-free medium. Most of the r e g e n e r a t e d t r a n s f o r m e d plants d e v e l o p e d roots also. F o r m a t i o n of callus and r e g e n e r a t i o n of shoot and root was o b s e r v e d from transformed explants of B. oleracea on a kanamycin s u p p l e m e n t e d h o r m o n e - f r e e media. The regenerated plants thus formed, b e h a v e d similar to their t r a n s f o r m e d "mother plants" ~Fig. Id). N P T II A s s a y : The dot assay was performed using r e g e n e r a t e d shoots of t r a n s f o r m e d and non-transformed {control) Brassica. The reaction was set up with crude protein extract of shoots. Dot signals were obtained in t r a n s f o r m e d Brassica shoots, whereas no signal a p p e a r e d in control Brassica shoots. To check the s p e c i f i c i t y of dot signal, a reaction was set up with crude protein extract of t r a n s f o r m e d shoots w i t h o u t kanamycin in mixture, no dot signal was obtained with such r e a c t i o n mixture. The dot signal of m a r k e r NPT II was also o b s e r v e d (Fig. 2).
a
b
c
d
Fig. I. T r a n s f o r m a t i o n and r e g e n e r a t i o n of B. oleracea a) T r a n s f o r m e d callus; b) Shoot regeneration from transformed ce!is~ c] Three months old regenerated plant~ d} Formation of callus, shoot and root from a t r a n s f o r m e d p e t i o l e explant. a 3.3 Kb Hind III fragment of Tn5, coding for NPT II gene. Plant DNA was b l o t t e d on n i t r o c e l l u l o s e filter with Dot blot apparatus (Hybriblot, BRL, USA) also and probed with p L G V T i 2 3 n e o DNA. Hybridizations were c a r r i e d out as d e s c r i b e d by M a n i a t i s et al. (1982). Ti p l a s m i d DNA was isolated from Agrobacterium using the m e t h o d of Kado and Liu (1981). NPT II Assay. Dot assay for d e t e r m i n i n g NPT II activity of r e g e n e r a t e d shoots of transformed and non-transformed {control) Brassica, was performed as described by Platt and Yang, (1987). ~he cell extract of A. t u m e f a c i e n s C~. CA Rif used in tranform a t i o n experimen{~, ~as take92as the marker of NPT II. We used 5 ~ C i ~ P ATP in each r e a c t i o n mixture. The P81 paper was e x p o s e d for 7 h. RESULTS
Transformation
and R e g e n e r a t i o n
The response from the cut edges of leaf discs was o b t a i n e d as early as 2 weeks after infection with A g r o b a c t e r i u m . Often, the leaf edges gave rise to compact green structures that formed shoots directly. Some of the leaf discs formed callus (Fig. la} and shoot r e g e n e r a t i o n was o b s e r v e d from them (Fig ]b). The r e g e n e r a t e d plants sustained s a t i s f a c t o r y growth in k a n a m y c i n containing medium (Fig. Ic). The control leaf discs
Fig. 2: Dot assay for NPT II activity. Dots in lane (a) t r a n s f o r m e d clones, lane (b) n o n t r a n s f o r m e d (control} clones, lane {c) t r a n s f o r m e d clones w i t h o u t k a n a m y c i n in r e a c t i o n mixture and lane (d) NPTII marker d e r i v e d from A g r o b a c t e r i u m used in transform a t i o n experiments.
Hybridization
Analysis
Southern b l o t t i n g of DNA isolated from a kanamycin resistant plant obtained by transformation mediated by Agrobacterium tumefaciens, r e v e a l e d two d i f f e r e n t bands of 2.3 Kb and 1.5 Kb, when h y b r i d i z e d with a Hind III fragment of Tn5 coding for NPT II gene. This probe is specific to the NPT II coding region of pLGVTi23neo, which has been shown to h y b r i d i z e with the probe also {Fig. 3a). In the dot blot analysis of plant DHA, Ti plasmid (pLGVTi23 neo) was used as a probe to confirm the p r e s e n c e of T-DNA in the t r a n s f o r m e d clones {Fig. 3b). M o r p h o l o g i c a l Variations: A wide morphological v a r i a t i o n in r e g e n e r a t e d plants of B. oleracea was observed. Several normal shoots were obtained° A few of the regenerated shoots showed m a r k e d d i f f e r e n c e from normal morphology. One such variant was a short plant, with small leaves having serrated margins (Fig. 4a) and the other variant was tall h a v i n g bigger leaves with smooth m a r q i n s (Fiq. 4b}. These two variants were
506 obtained as a result of transformation mediated by the oncogenic strain of Agrob a c t e r i u m tumefaciens. A mutant deficient in c h l o r o p h y l l (albino plant) was obtained, which could not be rooted (data not shown).
Fig. 3a: Southern blot analysis of kanamycin resistant plant. 10 ~ g of plant DNA was digested with Hind III, a n a l y s e d on 0.8% agarose gel and t r a n s f e r r e d to nitrocellulose filter. The filter was h y b r i d i z e d With a 3.3 Kb fragment of Tn5 cooing for NPT II gene. Lanes a: transformed~ b: non-transformed; c: Ti plasmid; Values are Hind III cut DNA size standards in kb. Arrows indicate the regions of NPT II gene. Fig. 3b: Dot blot analysis of total DNA of transformed plant. High mole cular weight DNA (l~g} was dot blotted and probed with Ti plasmid DNA. Dots a: transformed~ b: non-transformed; c: Ti plasmid.
Fig. 4a,b: Morphological variants of B. oleracea
DISCUSSION
Phytohormone autotrophic growth of transformed tissue is conferred by T-DNA encoded enzymes of auxin and c y t o k i n i n pathway (Garfinkel et al., 1981~ Nakajima et al., 1981). Usually, when both oncogenes
(tms and tmr) are present in t r a n s f o r m e d system, it leads to formation of undifferentiating mass of tissue called crown gall tumor (Chilton et al., 1980~. In the disarmed Ti vectors, one of the oncogenes are switched off, which make endogenous auxin/ c y t o k i n i n ratio favourable for shoot or root i n d u c t i o n (Amasino and Miller, 1982}. It has been shown that pathogenic Agrobacterium tumefaciens mediates teratomatous-like shoot f o r m a t i o n in S t y l o s a n t h e s spp., which failed to be rooted (Manners, 1987). In the present study, the regeneration of B r a s s i c a shoots a n d roots mediated by oncogenic Ti plasmid presents an interesting phenomenon. It is a significant event that oncogenes of Ti plasmids have c o n f e r r e d such endogenous ratio of auxin and cytokinin~ which favours r e g e n e r a t i o n of the system. The e x p r e s s i o n of foreign genes, NPT II and oncogenes is e v i d e n c e d by the sustained growth of t r a n s f o r m e d tissue in h o r m o n e - f r e e medium c o n t a i n i n g kanamycin, where as nont r a n s f o r m e d leaf discs did not grow on such medium. The dot assay for NPT II a c t i v i t y further confirms the e x p r e s s i o n of NPT iT gene in t r a n s f o r m e d plants. Differentiated shoots had not only r e t a i n e d the T-DN~, as is evident by the callus formation from their explant on h o r m o n e - f r e e m e d i u m supplemented with kanamycin, they also showed unaltered h o r m o n e - b a l a n c e as indicated by shoot and root regeneration from the explant. To confirm the presence of T-DNA and [{PT II gene in t r a n s f o r m e d regenerated plant, dot blot and southern blot analysis were performed using Ti plasmid and Tn5 encoded NPT II as probes respectively. The signals obtained in dot blot confirm the presence of T-DNA in the r e g e n e r a t e d plants. Southern analysis reconfirms the presence of foreign gene (NPT II) in t r a n s f o r m e d plants. Hormone independence and kanamycin resistance traits had been found to be tightly linked, as d e s c r i b e d by HerreraEstrella et al. {1983). Often, the regenerated plants of Brassica had altered morphology. Alterations at m o r p h o l o g i c a l level are expected due to the insertion of foreign DNA in plant genome. The genomic alterations include the events of chromosomal rearrangements and insertion mutagenesis. It is significant that such v a r i a t i o n s o c c u r r e d at high frequency. The events of genomic changes after the insertion of T-DNA in plant cells lead to the o c c u r r e n c e of m o r p h o l o g i c a l v a r i a t i o n and f o r m a t i o n of mutants. A slow growing clone of Brassica which regenerates into a short shoot having round leaves and serrated margins and another clone of a tall plant having bigger leaves with smooth margins are the examples of m o r p h o l o g i c a l variants obtained in the e x p e r i m e n t s described in this report. A Ti plasmid mediated transformation may result in the insertion of T-DNA in r e g u l a t o r y or coding regions of functional genes, thus giving rise to insertional mutants. We obtained a c h l o r o p h y l l d e f i c i e n t mutant {alibno plant) of B. o l e r a c e a which suggests the o c c u r r e n c e of insertion mutagenesis.
507 The aspect of endogenous hormonal changes in B r a s s i c a induced by i n s e r t i o n of T-DNA is being investigated.
Herrera-Estrella L, DeBlock M, Messens E, Harlensteens JP, Van Montagu M, Schell J ~1983) EMBO J 2: 987-995.
ACKNOWLEDGEMENTS:
Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG, Fraley RT 1985) Science 227: 1229-1231
The work was supported by a CSIR f e l l o w s h i p to V.S. and financial assistance from D e p a r t m e n t of Science and Technology.
REFERENCES
A m a s i n o RM, Miller CO (1982) 69: 389-392
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Chilton M-D, Saiki RK, Yadav N, Gordon MP, Quetier F (1980) Proc. Natl. Acad. Sci. {USA) 7 7 : 4 0 6 0 - 4 0 6 4 Cullis CA
{1979}
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42: 237-246.
DeBlock M, H e r r e r a - E s t r e l l a L, Van Montagu M, Schell J, Zambryski P (1984) EMBO J 3: 1681-1689. G a r f i n k e l DI, Simpson RB, Ream LW, White FF, Gordon MP~ Nester EW (1981) Cell 27: 143153. Hain R, Stable P, C z e r m i l o f s k y AP, Steinbib HH, Herrera-Estrella L, Schell J (1985) ~oI. Gen. Genet 199: 161-168.
Kado Cl, Lui ST 1365-1373.
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Maniatis T, Fritsch EF, 8ambrook J (1982) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Manners 207.
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N a k a j i m a H, Yokota T, Takahashi N, M a t s u m o t o T, Noguchi N {1981) Plant Cell Physiol. 22: 1405-1410. Platt SG, Yang N-S 162: 529-535. Southern 517.
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Plant Cell Reports (1988) 7:504-507
© Springer-Verlag 1988
Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens Vibha Srivastava, A. S. Reddy, and Sipra Guha-Mukherjee Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Received May 25, 1988/Revised version received October 12, 1988 - Communicated by I. K. Vasil
ABSTRACT
MATERIALS
A chimaeric neomycin p h o s p h o t r a n s f e r a s e II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its TDNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in r e g e n e r a t i o n of shoots and roots without any exogenous r e q u i r e m e n t of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of k a n a m y c i n r e s i s t a n t regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll d e f i c i e n t m u t a n t plant was also obtained.
Plant Material: Brassica oleracea botrytis cv. synthetica (cauliflower) grown in a peat-sand (1:3) mixture freshly emerged leaves were taken experiments.
INTRODUCTION Genetic t r a n s f o r m a t i o n of higher plants has been most e f f i c i e n t l y carried out by A g r o b a c t e r i u m tumefaciens, a natural gene vector of plants (for r e v i e w see Zambryski et al., 1984). For the improvement of the transformation vectors, chimaeric dominant selectable marker genes conferring r e s i s t a n c e to a m i n o g l y c o s i d e drugs have been constructed (Bevan et al., 1983). The c h i m a e r i c genes have been inserted into both oncogenic and non-oncogenic Ti plasmid vectors. The plant cells transformed by non-oncogenic Ti plasmid carrying chimaeric gene in T-DNA, have been r e g e n e r a t e d by hormonal manipulation in tissue culture media (De Block et al., 1984; Hain et al., 1985). The t r a n s f o r m a t i o n of higher plants with c h i m a e r i c gene inserted in o n c o g e n i c Ti plasmid has also been performed (HerreraEetrella et al., 1983), but no regeneration has so far been reported. Here, we are reporting the t r a n s f o r m a t i o n and r e g e n e r a t i o n of Brassica oleracea system by an o n c o g e n i c Ti plasmid vector containing a chimaeric neomycin phosphotransferase II gene in its T-DNA. Several r e g e n e r a t e d plants were obtained and a few of them were d i s t i n c t l y d i f f e r e n t from the normal (untransformed) plants.
Offprint requests to." V. Srivastava
AND
METHODS
var. were and for
B a c t e r i a l strain: An o n c o g e n i c ~grobacter--~m/m~ f ~ strain C ~ C~ Rif harbouring ~ ~ pLGVTi23~o lwas used for transformation of plants. The plasmid contains chimaeric neomycin phosphotransferase II (Nos/NPT II) gene inserted in its TDNA. The original strain was o b t a i n e d from Dr. M. Van M o n t a g u (Belgium) and has been described as oncogenic by Herrera-Estrella et al. (]983). Plant T r a n s f o r m a t i o n and Regeneration: Transformation of B. oleracea was performed by leaf disc t r a n s f o r m a t i o n m e t h o d (Horsch et al., 1985), with few modifications. Discs punched from surface sterlized leaves, a v o i d i n g mid-rib, were p r e - c o n d i t i o n e d on MS m e d i u m (Murashige and Skoog, 1962) containing BAP (0.5 ppm) and NAA (I ppm). After 24 h of p r e - c o n d i t i o n i n g , the leaf discs were mixed with a d i l u t e d culture of 48 h grown Agrobacterium (1:20 with MS liquid media} for 5-10 minutes. The discs were blotted dry and placed on hormone-free MS basal m e d i u m c o n t a i n i n g c a r b e n i c i l l i n or c e f o t a x i m (500 ~ g / m l ) . After about two weeks, the leaf discs were t r a n s f e r r e d to MS basal medium containing k a n a m y c i n (100 ~ g / m l ) . The leaf and petiole explants of regenerated t r a n s f o r m e d B. oleracea plants were placed on MS basal m e d i u m c o n t a i n i n g kanamycin (100 ~ g / m l ) f o r induction of callus and r e g e n e r a t i o n of shoots and roots. DNA
Isolation
and
Southern
Hybridization
:
High molecular weight total DNA was isolated from shoots of t r a n s f o r m e d and nont r a n s f o r m e d (control) B. oleracea as described by Cullis (1981). Total DNA was digested c o m p l e t e l y with Hind III, electrophoresed on 0.8% agarose gel and t r a n s f e r r e d to a n i t r o c e l l u l o s e filter (Southern, 1975). Southern h y b r i d i z a t i o n was carried out using
505 which were not infected with A. tumefaciens did not grow on the hormone-free medium. Most of the r e g e n e r a t e d t r a n s f o r m e d plants d e v e l o p e d roots also. F o r m a t i o n of callus and r e g e n e r a t i o n of shoot and root was o b s e r v e d from transformed explants of B. oleracea on a kanamycin s u p p l e m e n t e d h o r m o n e - f r e e media. The regenerated plants thus formed, b e h a v e d similar to their t r a n s f o r m e d "mother plants" ~Fig. Id). N P T II A s s a y : The dot assay was performed using r e g e n e r a t e d shoots of t r a n s f o r m e d and non-transformed {control) Brassica. The reaction was set up with crude protein extract of shoots. Dot signals were obtained in t r a n s f o r m e d Brassica shoots, whereas no signal a p p e a r e d in control Brassica shoots. To check the s p e c i f i c i t y of dot signal, a reaction was set up with crude protein extract of t r a n s f o r m e d shoots w i t h o u t kanamycin in mixture, no dot signal was obtained with such r e a c t i o n mixture. The dot signal of m a r k e r NPT II was also o b s e r v e d (Fig. 2).
a
b
c
d
Fig. I. T r a n s f o r m a t i o n and r e g e n e r a t i o n of B. oleracea a) T r a n s f o r m e d callus; b) Shoot regeneration from transformed ce!is~ c] Three months old regenerated plant~ d} Formation of callus, shoot and root from a t r a n s f o r m e d p e t i o l e explant. a 3.3 Kb Hind III fragment of Tn5, coding for NPT II gene. Plant DNA was b l o t t e d on n i t r o c e l l u l o s e filter with Dot blot apparatus (Hybriblot, BRL, USA) also and probed with p L G V T i 2 3 n e o DNA. Hybridizations were c a r r i e d out as d e s c r i b e d by M a n i a t i s et al. (1982). Ti p l a s m i d DNA was isolated from Agrobacterium using the m e t h o d of Kado and Liu (1981). NPT II Assay. Dot assay for d e t e r m i n i n g NPT II activity of r e g e n e r a t e d shoots of transformed and non-transformed {control) Brassica, was performed as described by Platt and Yang, (1987). ~he cell extract of A. t u m e f a c i e n s C~. CA Rif used in tranform a t i o n experimen{~, ~as take92as the marker of NPT II. We used 5 ~ C i ~ P ATP in each r e a c t i o n mixture. The P81 paper was e x p o s e d for 7 h. RESULTS
Transformation
and R e g e n e r a t i o n
The response from the cut edges of leaf discs was o b t a i n e d as early as 2 weeks after infection with A g r o b a c t e r i u m . Often, the leaf edges gave rise to compact green structures that formed shoots directly. Some of the leaf discs formed callus (Fig. la} and shoot r e g e n e r a t i o n was o b s e r v e d from them (Fig ]b). The r e g e n e r a t e d plants sustained s a t i s f a c t o r y growth in k a n a m y c i n containing medium (Fig. Ic). The control leaf discs
Fig. 2: Dot assay for NPT II activity. Dots in lane (a) t r a n s f o r m e d clones, lane (b) n o n t r a n s f o r m e d (control} clones, lane {c) t r a n s f o r m e d clones w i t h o u t k a n a m y c i n in r e a c t i o n mixture and lane (d) NPTII marker d e r i v e d from A g r o b a c t e r i u m used in transform a t i o n experiments.
Hybridization
Analysis
Southern b l o t t i n g of DNA isolated from a kanamycin resistant plant obtained by transformation mediated by Agrobacterium tumefaciens, r e v e a l e d two d i f f e r e n t bands of 2.3 Kb and 1.5 Kb, when h y b r i d i z e d with a Hind III fragment of Tn5 coding for NPT II gene. This probe is specific to the NPT II coding region of pLGVTi23neo, which has been shown to h y b r i d i z e with the probe also {Fig. 3a). In the dot blot analysis of plant DHA, Ti plasmid (pLGVTi23 neo) was used as a probe to confirm the p r e s e n c e of T-DNA in the t r a n s f o r m e d clones {Fig. 3b). M o r p h o l o g i c a l Variations: A wide morphological v a r i a t i o n in r e g e n e r a t e d plants of B. oleracea was observed. Several normal shoots were obtained° A few of the regenerated shoots showed m a r k e d d i f f e r e n c e from normal morphology. One such variant was a short plant, with small leaves having serrated margins (Fig. 4a) and the other variant was tall h a v i n g bigger leaves with smooth m a r q i n s (Fiq. 4b}. These two variants were
506 obtained as a result of transformation mediated by the oncogenic strain of Agrob a c t e r i u m tumefaciens. A mutant deficient in c h l o r o p h y l l (albino plant) was obtained, which could not be rooted (data not shown).
Fig. 3a: Southern blot analysis of kanamycin resistant plant. 10 ~ g of plant DNA was digested with Hind III, a n a l y s e d on 0.8% agarose gel and t r a n s f e r r e d to nitrocellulose filter. The filter was h y b r i d i z e d With a 3.3 Kb fragment of Tn5 cooing for NPT II gene. Lanes a: transformed~ b: non-transformed; c: Ti plasmid; Values are Hind III cut DNA size standards in kb. Arrows indicate the regions of NPT II gene. Fig. 3b: Dot blot analysis of total DNA of transformed plant. High mole cular weight DNA (l~g} was dot blotted and probed with Ti plasmid DNA. Dots a: transformed~ b: non-transformed; c: Ti plasmid.
Fig. 4a,b: Morphological variants of B. oleracea
DISCUSSION
Phytohormone autotrophic growth of transformed tissue is conferred by T-DNA encoded enzymes of auxin and c y t o k i n i n pathway (Garfinkel et al., 1981~ Nakajima et al., 1981). Usually, when both oncogenes
(tms and tmr) are present in t r a n s f o r m e d system, it leads to formation of undifferentiating mass of tissue called crown gall tumor (Chilton et al., 1980~. In the disarmed Ti vectors, one of the oncogenes are switched off, which make endogenous auxin/ c y t o k i n i n ratio favourable for shoot or root i n d u c t i o n (Amasino and Miller, 1982}. It has been shown that pathogenic Agrobacterium tumefaciens mediates teratomatous-like shoot f o r m a t i o n in S t y l o s a n t h e s spp., which failed to be rooted (Manners, 1987). In the present study, the regeneration of B r a s s i c a shoots a n d roots mediated by oncogenic Ti plasmid presents an interesting phenomenon. It is a significant event that oncogenes of Ti plasmids have c o n f e r r e d such endogenous ratio of auxin and cytokinin~ which favours r e g e n e r a t i o n of the system. The e x p r e s s i o n of foreign genes, NPT II and oncogenes is e v i d e n c e d by the sustained growth of t r a n s f o r m e d tissue in h o r m o n e - f r e e medium c o n t a i n i n g kanamycin, where as nont r a n s f o r m e d leaf discs did not grow on such medium. The dot assay for NPT II a c t i v i t y further confirms the e x p r e s s i o n of NPT iT gene in t r a n s f o r m e d plants. Differentiated shoots had not only r e t a i n e d the T-DN~, as is evident by the callus formation from their explant on h o r m o n e - f r e e m e d i u m supplemented with kanamycin, they also showed unaltered h o r m o n e - b a l a n c e as indicated by shoot and root regeneration from the explant. To confirm the presence of T-DNA and [{PT II gene in t r a n s f o r m e d regenerated plant, dot blot and southern blot analysis were performed using Ti plasmid and Tn5 encoded NPT II as probes respectively. The signals obtained in dot blot confirm the presence of T-DNA in the r e g e n e r a t e d plants. Southern analysis reconfirms the presence of foreign gene (NPT II) in t r a n s f o r m e d plants. Hormone independence and kanamycin resistance traits had been found to be tightly linked, as d e s c r i b e d by HerreraEstrella et al. {1983). Often, the regenerated plants of Brassica had altered morphology. Alterations at m o r p h o l o g i c a l level are expected due to the insertion of foreign DNA in plant genome. The genomic alterations include the events of chromosomal rearrangements and insertion mutagenesis. It is significant that such v a r i a t i o n s o c c u r r e d at high frequency. The events of genomic changes after the insertion of T-DNA in plant cells lead to the o c c u r r e n c e of m o r p h o l o g i c a l v a r i a t i o n and f o r m a t i o n of mutants. A slow growing clone of Brassica which regenerates into a short shoot having round leaves and serrated margins and another clone of a tall plant having bigger leaves with smooth margins are the examples of m o r p h o l o g i c a l variants obtained in the e x p e r i m e n t s described in this report. A Ti plasmid mediated transformation may result in the insertion of T-DNA in r e g u l a t o r y or coding regions of functional genes, thus giving rise to insertional mutants. We obtained a c h l o r o p h y l l d e f i c i e n t mutant {alibno plant) of B. o l e r a c e a which suggests the o c c u r r e n c e of insertion mutagenesis.
507 The aspect of endogenous hormonal changes in B r a s s i c a induced by i n s e r t i o n of T-DNA is being investigated.
Herrera-Estrella L, DeBlock M, Messens E, Harlensteens JP, Van Montagu M, Schell J ~1983) EMBO J 2: 987-995.
ACKNOWLEDGEMENTS:
Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG, Fraley RT 1985) Science 227: 1229-1231
The work was supported by a CSIR f e l l o w s h i p to V.S. and financial assistance from D e p a r t m e n t of Science and Technology.
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