Plant Molecular Biology 5: 213-222, 1985 © 1985 Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Expression of shoot-inducing Ti TL-DNA in differentiated tissues of potato (Solanum

tuberosum cv Maris Bard) M. M. Burrell, D. Twell, A. Karp & G. Ooms Biochemistry Department, Rotharnsted Experimental Station, Harpenden, Herts. A L5 2JQ, U.K.

Keywords: Agrobacterium tumefaciens, gene expression, potato, T-DNA

Summary In potato line Mbl501B one or possibly two normal size Ti TL-DNA copies per tetraploid genome were detected by Southern blot analysis, but no TR-DNA. The TL-DNA probably contained the entire transposon Tn 1831 inserted at the T-DNA auxin gene for transcript 2. Northern blot analyses of the steady-state RNA in different Mb 1501B tissues isolated from (i) shoots cultured in vitro (ii) grafted plants and (iii) tubers, showed that that TL-DNA transcripts 3, 4, 6a and 7 were expressed most abundantly in the cultured shoots. They formed approximately 0.0023 to 0.0007% of the total poly(A) RNA. Transcripts 1,5 and 6b were not detected in any of the tissues analysed. This indicated even lower levels of expression (below approximately 0.0001% of the total poly(A) RNA or, making certain assumptions, an abundance of less than one T-DNA derived RNA molecule per cell). As expected, transcript 2 was not detected in any of the Mb 1501B tissues. The abundance of the transcripts was reduced in grafted plants and tubers compared with cultured shoots with the greatest decrease (5 ×) for transcripts 4, 6a and 7. Transcript 4, the one most responsible for the changed growth and development of Mb 1501 B, formed approximately 0.0003% of the poly(A) RNA from both grafted plants and tubers.

Introduction Octopine-type crown galls are induced by infection of wounded dicotyledonous plants with octopine-type Agrobacterium tumefaciens strains (7, 13, 23). Crown gall formation occurs when one or severalcopies of one or sometimes two distinct DNA segments from the same bacterial tumour-inducing (Ti-) plasmid become integrated in the plant nuclear genome (4, 8, 9, 32). Usually these transferred DNA segments or T-DNA's are 13 kbp for the left hand segment (TL-DNA) and 8 kbp for the right hand segment (TR-DNA) and each are bordered by unique imperfect direct repeat 25 bp integration sequences (4). In octopine-type crown gall cells there is always TL-DNA which contains eight genes (see for reviews, 7, 13, 23). One gene, ocs produces transcripts (tr3) coding for octopine synthase which is the en-

zyme responsible for the synthesis of a group of tumour cell-specific opines (6, 16, 20), represented by octopine. A second TL-DNA gene, ons coding for transcript 6a, is responsible for the active excretion of opines from transformed cells (21). T-DNA mutagenesis studies and complementary biochemical analyses (10, 18, 24, 38, 39) have shown that two T-DNA genes, T-auxl and T-aux2 (for trl and 2) concertedly enhance auxin biosynthesis in tumour cells (14, 29, 33). The T-DNA cytokinin gene T-cyt (for tr4) stimulates the first step in cytokinin biosynthesis (2, 5). The function of the three remaining TL-DNA genes (for transcripts 5, 6b and 7) is less clear, but at least two (for tr5 and 6b) appear to be involved in plant hormonal effects (10, 18, 28). From the T-DNA mutagenesis studies it became clear that A. tumefaciens strains inactivated for either of the auxin genes induced tobacco tumours from which shoots developed. The tumours con-

214 tained highly increased concentrations of endogenous cytokinins compared with untransformed cells (1) probably as a result of the expression of T-cyt alone. Interestingly, steady state RNA analysis of undifferentiated tumour tissues and transformed shoots developing directly from the undifferentiated tissues showed that T-cyt is similarly expressed in both (3, 36). Grafting of primary tobacco tumour shoots onto rootstocks of normal tobacco plants caused the phenotype to become more normal (19, 35, 41,42). The plants ultimately flowered and T-DNA was transmitted through meiosis. Some transformed tobacco plants formed roots and did not need grafting to reach maturity. In general, the lines analysed showed considerable differences in T-DNA structure, copy number and expression despite the fact that similar or identical Agrobacterium strains were used in the initial transformation events. Recently we have regenerated similarly transformed potato shoots (25). In this and the preceeding paper (26) one line, Mbl501 B, was investigated in greater detail. MbI501B shoots, like the tobacco shoots, became phenotypically more normal after grafting onto untransformed rootstocks. In the process, the endogenous cytokinin concentration decreased but stayed higher than in untransformed control plants. In addition grafted Mb 1501B plants tuberised precociously. In this report we present the results of the molecular analysis of the T-DNA structure, the chromosome number and differences in expression of TDNA genes between grafted and ungrafted Mb 1501B tissues. Transmission of T-DN A induced traits through tuber propagation is demonstrated and the reduced expression of at least some T-DNA genes, including T-cyt in grafted plants vs. cultured shoots is discussed. Prospects to study potato development and tuberisation in a novel way are indicated.

Materials and methods

Plants and culture conditions Potato line Mbl501B is an octopine producing cell line that originated from a single shoot tip from a stem tumour induced by infection with Agrobacterium tumefaciens strain LBA 1501 (25) on an in

vitro cultured shoot of Solanum tuberosum L cv. Maris Bard. Mb 1501B and untransformed cv. Maris Bard were micropropagated on Murashige and Skoog medium (22) without hormones but with 2% sucrose at 25°C 8 h dark; 16 h light. Plants in soil were grown under a 16 h light (20°C) and 8 h dark (18°C) photoperiod. Cytological analysis The chromosome number of Mbl501B was determined in Feulgen stained root tip squashes of occasionally developing roots in vitro, as described by Karp et al. (15). Plant DNA isolation Plant DNA was isolated according to Kreis et al., (17). In short, 5-10 g potato leaf and stem tissue was powdered in liquid nitrogen, thawed in 25 ml DNA extraction buffer (17.1% sucrose; 50 mM Tris HCI pH 8.0; 20 mM EDTA: 25 mM KC1; 1% SKS; plus CsCI to a final concentration of 1.05 g/ml), debris was removed using muslin or cheese cloth, 2.5 mg (from a stock solution of 10 mg/ml) of ethidium bromide added and centrifuged to equilibrium at 37 k rpm for 42 h. The DNA band was removed from the CsCI-EtBr gradient, extracted repeatedly with butan-l-ol saturated with 20 X SSC and the DNA was purified further using standard methods. Plant R N A isolation The plant tissues analysed were grown as described in the accompanying paper (26). For RNA analyses from shoot culture material only shoots without the callus tissue at their base was used. From the grafted plants RNA preparations were isolated from soil-borne transformed tubers, aerial transformed tubers and from the green tissues from the transformed scions (leaves and stems without tubers). Plant tissue was frozen in liquid nitrogen and powdered with a pestle and mortar. It was added to 2 volumes (ml:g) of RNA extraction buffer (200 mM Tris-HCl pH 8.5; 400 mM NaCI, 60 mM Mg Acetate, 50 mM EGTA, 1% SDS, 5 mM DTT and 0.25 M sucrose), and homogenised for 1 min at 4°C in a Kenwood liquidizer. Following centrifugation (21 000 g; 10 min; 4°C) the supernatant was added

215 to 1/2 vol cold phenol saturated with four times diluted RNA extraction buffer. An additional 1/2 vol of chloroform:butanol (50:1) was added after 10 min, stirred and the layers separated by centrifugation at 2 500 g for 10 min. Phenol-chloroform extractions were repeated (room temperature) until no protein layer remained visible and then followed by a final chloroform extraction. Nucleic acids were precipitated at -20°C with 2.5 vol'absolute' ethanol (96%) and 1/40 vol. 4 M NH4 Acetate. The precipitate was collected by centrifugation at 17 000 g for 25 min, resuspended in sterile distilled water and reprecipitated at 4°C with an equal volume 5 M NaCl. The poly A containing RNA (approx 1 #g/g fr wt starting material) was obtained by oligo dT cellulose chromatography (2 X). The eluate was precipitated twice in 70% cold ethanol and finally dissolved in sterile distilled water. ttybridisation

Plant DNA and RNA were analysed by Southern and Northern blots respectively. For DNA analysis 10 #g potato DNA treated with a restriction endonuclease was used per experiment. To provide one copy reconstruction experiments, 15.6 ng total A. tumefaciens strain LBAI501 DNA or 30.5 pg plasmid pRAL3252 or 75.8 pg plasmid pRAL3076 DNA was mixed with 10 #g herring sperm DNA. DNA was separated in 0.7% agarose gels in Trisacetate (80 mM Tris pH 8.0, 4 mM EDTA, 40 mM Na acetate) buffer and after fragmentation under UV light and denaturation, transferred onto biodyne A (PAL) membrane filters using standard techniques and following manufacturers instructions. Using similar standard procedures, Northern blots were prepared from usually 40 or 50 #g denatured (glyoxylated) poly(A) RNA separated on 1.1% agarose gels in 10 mM phosphate pH 7.0. For comparison, denatured DNA reconstruction samples were routinely included in the RNA analysis. Probes were obtained by nick translation using 32p d A T P (SA3000 Ci/mmol; Amersham) with cloned Ti plasmid fragments (Fig. 2). These plasmids were isolated by standard procedures and 0.15 #g DNA was used per hybridisation. Routinely the SA of the labelled DNA was 8-9 108 dpm/#g. Hybridisations were at 42°C for 24 48 h, depending on the complexity of the probes in hybridisation solution of 50% formamide; 5 X Denhardt (0.1% Ficoll, 0.1%

PVP, 0.1% BSA); 5 x SSPE (0.9 M NaCI, 50 mM Na phosphate pH 8.3, 5 mM EDTA); 0.2% SDS and 250 #g/ml denatured herring sperm DNA. The blots were washed (3 X) in 2 X SSC (0.3 M NaCI, 30 mM Na citrate), 0.1% SDS at 55°C and (3 X) in 0.1 X SSC, 0.1% SDS at 55°C and exposed to Fuji RX X-ray film. Opines

Octopine assays were done essentially as described by Otten and Schilperoort (27) except that enzyme incubations usually were overnight at 29°C. R N A abundance estimates

Estimates on the actual amounts of T-DNA derived RNA molecules in total poly(A) RNA preparations were obtained from comparative hybridisations of32p-labelled T-DNA fragments with poly(A) RNA and reconstruction DNA. On one gel, denatured plant RNA and denatured restriction endonuclease fragments of Ti plasmid DNA were resolved according to size and blotted onto Biodyne A filters. Such blots were probed and bands appearing on the autoradiograms were compared for intensity. To estimate the percentages of each of the T-DNA derived RNA's an individual transcript was compared with a reconstruction fragment hybridising to as near similar intensity as possible. The weight of DNA in the appropriate reconstruction fragment was known since it was a known proportion of the weight of reconstruction plasmid DNA loaded in that particular lane. After adjusting the weight of fragment for any difference in intensity between it and the specific RNA band the % abundance was calculated from the weight of RNA loaded. The number of RNA molecules per cell for each T-DNA derived RNA was calculated from the % abundance figures using the assumptions of Goldberg et al. (11) for poly(A) RNA from tobacco (one average size, 1240b, RNA molecule per cell had an abundance of approximately 0.0002%).

Results

Chromosome number in Mbl501 B

Potato plants regenerated from untransformed

216 explants (leaves, stems etc.) or protoplasts often have chromosome numbers that differ, sometimes grossly, from the normal karyotype (2n = 4x = 48) (15, 30, 3 I, 37). Plants that are aneuploid for one or few chromosomes can be phenotypically indistinguishable from the parental line probably because potato is tetraploid and buffered against the imbalance caused by aneuploidy. Aneuploids with considerably higher chromosome numbers, however, of sometimes up to 96 or more, invariably have a grossly abnormal morphology. Such plants are frequently found among protoplast regenerants but are less frequent among explant regenerants. As these genetic changes are introduced by the regeneration procedures used, although as yet by unknown mechanisms, it is therefore desirable to verify the chromosome number in regenerated transformed plants isolated via similar procedures. Mbl501B in culture occasionally formed roots which allowed a chromosome number of 49 per cell to be determined by root tip squashes (Fig. 1).

F(e. 1. The chromosome number was determined in root tip squashes prepared from the roots which occasionally developed on in vitro cultured M b 1501B. It was 49 instead of the normal 48 chromosomes per cell.

Ti-plasmid fragments that cover crown gall specific TL-DNA and TR-DNA (Fig. 2). Fig. 3A shows an autoradiogram representing the hybridisation patterns for EcoRl, BamHl and Smal digests of MbI501B DNA (lanes 3, 6 and 10) and Maris Bard DNA (lanes 4, 5 and 11) probed with 32p-labelled plasmid pRAL3252, specific for TL-DNA. Further hybridisation patterns were obtained when the

T-DNA structure o f M b l 5 0 1 B

The T-DNA structure in line MbI501B shoot cultures was analysed using Southern blots that were probed with 32p-labelled plasmids containing

5kb

Sma I ] 17 I BamH] I Hindm ] 18c EcoRI 3

16a ]

Tn 1831

i

I 8 22c ~ ~36~ [32g|

1Oc I ]281

3b

1

17a

I 11 I

7

19a

I

Tn 1831 ~"JffJ/~

T -DNA =RAL 3076 pRAL 3916

pRAL 3252 pRAL 3905 5

4= 7

¢=~= '2"

•

•

~.. 1

~ 4

~ 6a

¢== 6b

4,

, 3

Fig. 2. Map of T-DNA structure and possible transcripts in Mbl501B. A: Restriction map of T-DNA region in octopine Ti plasmid pRAL 1501. B: Probable structure of inserted T-DNA in Mb 1501B. The hatched areas at the ends of the broad bar refer to uncertainties in the localisation of the M b 1501 B T-DN A. The exact position of the usually preferred integration sites are indicated within the hatched areas by a marked transition. C: T-DNA region fragments used as 32p-labelled probes, either as part of a bacterial plasmid or as inserts purified from the plasmids. D: localisation and polarity of T-DN A coded transcripts. Transcript 2 is absent because of the integration of Transposon Tn 183 I.

217 same Southern blot was probed with 32p-labelled plasmid pRAL3076, specific for TR-DNA although it overlaps with the right part of the TL-DNA (Fig. 3B). In these experiments we also included 1 × and 5 × reconstruction experiments (lanes 1, 2, 7, 8 and 9).

Fig,. 3. Southern blot analysis of T - D N A in potato line M b 1501B. Lanes in which DNA was analysed from either Maris Bard, M b 1501 B or from reconstruction mixtures (see materials and methods), plus the restriction endonucleases used, are specified at the top of the figure. The blot was either probed with A) plasmid pRAL3252 (see Fig. 2) or B) plasmid pRAL3076 labelled with 32p.

The autoradiograms show that MbI501B contained T-DNA with sequences homologous both to pRAL3252 and pRAL3076 and that MbI501B probably contained only one or two copies of TDNA per tetraploid genome; based on the similar intensity of a number of bands representing 'internal' T-DNA restriction fragments (see also below). The borders defining T-DNA in MbI501B are probably at the specific 25 bp integration sequences of TL-DNA. These sequences are located within the Ti-plasmid restriction endonuclease fragments Smal 17 and EcoRl 19 (Fig. 2). In general, identical restriction endonuclease fragments are excised from transformed plant DNA and Ti-plasmid DNA when both the restriction enzyme recognition sites of a particular fragment are located between the integration sequences ('internal' restriction endonuclease fragments). As would be predicted, internal fragments, like BamHl fragments 8 jl and 17a, Smal 16a 1, 16a ~j and 10c, were detected in digested Mbl501B DNA. The smaller fragments such as BamH1 28 were only faintly visible in the autoradiographs. Also as expected, no restriction endonuclease fragments of hybridising plant DNA comigrated with any of the reconstruction Ti-plasmid fragments that carried the integration sequences. This is consistent with the generation of restriction endonuclease'junction' fragments comprising some q-i-plasmid derived T-DNA (of predictable length) and some plant DNA (of unpredictable length) with a 25 bp integration sequence combining the two. It is noted that hybridisation with pRAL3076 (Fig. 3B) did not indicate the presence of any TR-DNA sequences. The hybridisation patterns obtained also provide evidence for the integration of the bacterial transposon Tn1831 into plant DNA as part of Mbl501 B T-DNA (Fig. 3 A lane 10). Transposon Tn 1831 is 17 kbp in size (24) and Smal digests of either the Ti-plasmid DNA from LBAI501 or of MbI501B T-DNA itself show two characteristic large size plasmid-transposon junction fragments (Fig. 3A lane 9). Each of these fragments comprise some Ti plasmid DNA (from SmaI fragment 16a; 2.6 kbp) and a substantial part of transposon Tn 1831 DNA. The detection in DNA preparations from transformed as well as untransformed tissues of restriction endonuclease fragments that hybridised faintly to both of the 32P-labelled plasmids was unexpected (Fig. 3). Because similar background hybridization

218 patterns were found when plasmid pBR322 (the vector plasmid used to isolate pRAL3252 and pRAL3076) was used as 32p-labelled probe, it is unlikely that these bands represent homology of potato D N A with T - D N A specific sequences although the exact origin of the bands in plant D N A remains to be traced. The smear of radioactivity at the bottom of Maris Bard lanes (particularly Fig. 3A) was probably due to similar hybridisation to plant R N A that was co-isolated with the D N A in that particular DNA preparation. T - D N A transcription in M b I 5 O I B

Expression of T - D N A was analysed by isolating steady-state RNA from in vitro grown MbI501B shoots and from morphologically normalised plant material (grafted shoots and tubers). It is noted that for all tissues except aerial tubers a minimum of two extractions were examined with consistent results. Isolated RNA molecules were separated according to size by gel electrophoresis and Northern blots were prepared. These were hybridised with the 32p_ labelled recombinant DNA plasmids, pRAL3076 and pRAL3252 mentioned above and the smaller plasmids pRAL3916 and pRAL3905 which are specific for certain T - D N A derived transcripts (Fig. 2). The Northern blots (Figs. 4 and 5) showed that transcripts 1, 5 and 6b remained undetected in any of the RNA preparations, despite using 32p-labelled probes with SA in excess of 10 9 d p m / # g and 50 #g RNA per lane on the blots. Long exposure times also failed to reveal these transcripts. Transcript 2 was also not detected, but this was expected because of the insertional inactivation by transposon Tn 1831. In cultured MbI501B shoots transcript 3, 4, 6a and 7 were expressed at approximately 0.0023, 0.0014, 0.0007 and 0.0009% respectively of the total poly(A) RNA (see Materials and Methods for calculations). It is noted that repeated use of the same nylon blot, led to an apparent greater loss of signal from hybridisations to blotted RNA than to blotted DNA. Therefore quantitative estimates of abundance have been taken from Fig. 4A and B. When the specific probes plasmid pRAL3916 and pRAL3905 were used, strong background bands were observed (Fig. 4D, 5A). Therefore the experiments were repeated with just the insert fragments showing that background bands were

Fig. 4. Northern blot analysis of T-DNA expression. 50/~g RNA was used for each poly A RNA preparation (lanes 1, 2, 3, 4) and the reconstructions(lanes 5, 6, 7) wereprepared as for Fig. 3. The same blot was hybridized 4 times; A with pRAL3252, B pRAL3076, C-pRAL3916and D pRAL3905.

not due to T - D N A encoded transcripts (compare Fig. 5A and 5B). As with the Southern, the background bands were also observed when just pBR322 was used as a probe (not shown). In Mbl501B grafted plants and tubers the relative abundance of the detected T - D N A transcripts was reduced although to markedly different extents. This brought their abundance closer to or sometimes below our limits of detection (less than 0.0001%). Transcript 3 showed about no more than a two fold reduction in relative abundance in grafted plants (to 0.001%) and about three to five fold (to ca. 0.0005%) in Mb 1501B tubers (below soil) which could only be determined by using the highly specific sub probe pRAL3916 (Fig. 5A, 5B). The abun-

219 these transcripts to assess more accurately their reduced abundance. It should be noted that tr6 seen in Fig. 4B is mainly if not entirely tr6a since pRAL3916 is specific for tr6b as well as tr3 but did not detect tr6b (Fig. 5B).

Octopine synthesis in Mbl501B A typical result for the in vitro stimulation of octopine synthesis by extracts from shoot cultures, grafted plants and tuber derived plants is shown in Fig. 6. Extracts from plants grown from transformed M b 1501B tubers produced similar amounts of octopine as extracts from grafted plants. Extracts from tubers showed little or no octopine synthesis but extracts from roots that sporadically developed from cultured M bl501 B shoots (25) did give positive results (not shown). It is noted that octopine synthesis in cultured Mbl501B shoots has remained constant in numerous tests over a culture period of almost three years suggesting little variation in its expression.

Fig. 5. Differences in expression ofTi T-DNA transcripts 3 and 4 in differentiated Mbl501B tissues, A. Northern blot with 40 #g RNA from the tissues shown was hybridised with plasmid p RAL3916 specific for transcript 3. B. Same as A except that an insert fragment from pRAL3916 was used as a 32P-labelled probe. C. Same as A except that an insert from plasmid pRAL3905, specific for transcript 4, was used. To aid in the quantitative estimates of the level of T-DNA expression in M b 1501 B, shoot culture R NA was applied at 1.6 #g ( 1/ 25), 8 ~tg (1 ,,5) and 40 ~tg (1): lanes 6, 7 and 8 respectively.

dance of the Tcyt transcript 4 was reduced by approximately five fold (to ca 0.0003% both in RNA from grafted Mbl501B plants and in RNA from Mb 1501B tubers irrespective of whether the tubers developed above or below soil (Fig. 4B, 5C). This could only convincingly be demonstrated by using the specific sub probe pRAL3905 (Fig. 5C). Although transcripts 6a and 7 were also less abundant in RNA from grafted plants (ca 3-5 fold) and tubers (not detected; Fig. 4) no sub probes were used for

Fig. 6. Electropherogram of the products in octopine assays on various potato tissues. Stimulation of octopine synthesis from arginine and pyruvate in the presence of NADH was measured with extracts from 10 mg fresh weight of tissue. Lanes a and b refer to reaction mixtures at t = 0 and t - 18 h of incubation respectively. 1: Marls Bard 2: MbI5OIB shoot culture 3: M b 1501B grafted plant 4: tuber-derived plant.

220 Discussion

Potato line MbI501B was examined for its TDNA structure, T-DNA expression and its chromosome complement in order to provide'some insight into the molecular events that underly its difference in growth and development compared with the untransformed parental line cv. Maris Bard. It is likely that the introduced T-DNA was largely if not exclusively responsible for the changes in growth and development. Mb 1501B contained 49 chromosomes per cell and previous observations on plants regenerated from untransformed potato cells suggested that this degree of aneuploidy is unlikely to influence significantly the phenotype of regenerated potato plants (15). Furthermore, independently isolated potato lines, also transformed with TDNA from LBA 1501, all had the same morphology as Mb 150 i B (25; unpublished) with characters such as a lack of root formation and reduced apical dominance. Similarly transformed tobacco plants (36) and tobacco plants transformed with only the T-DNA cytokinin gene (2) also had this changed phenotype. Thus it is.likely that Tcyt was the major if not the only genetic factor causing the changed morphology and development of Mbl501B. Mb 1501B contained sequences homologous to the TL-DNA regions of the transforming A. tumefaciens strain LBA 1501 but no sequences homologous to the TR-DNA region (Fig. 3). The integrated TL-DNA consisting of one or possibly two copies per tetraploid potato genome, had not been rearranged and included the bacterial transposon Tn 1831 that had been inserted at the position of the T-DNA position of the T - D N A auxin gene for transcript 2 (Fig. 3). The T-DNA boundaries were within those restriction endonuclease fragments in which the 25 bp direct repeats that are of importance to T-DNA integration, are located (4). This makes it likely that T-DNA in Mb 1501B is confined exactly by these preferred integration sequences. It is noted that we did not determine the T-DNA structure in tuber-derived plants but did find TDNA coded traits in such plants. However, one would expect T-DNA to behave as any other normal DNA segment in vegetatively propagated cells and therefore that its structure would have remained unchanged during propagation via tubers. It is also noted that in certain tuber cells the ploidy

level may vary substantially (34) although plants with normal ploidy develop from tubers. Since it is not known how variation in ploidy relates to variation in the relative abundance of specific chromosomes in tuber cells it is not known whether T-DNA copy number per cell varies within the tuber. Examination of the steady-state RNA levels in different Mb 1501B tissues showed that in all tissues analysed (shoots, grafted plants, tubers) all T-DNA transcripts were very low in abundance (Fig. 4, 5). Transcript l, 5 and 6 b remained below our detection limit and therefore probably formed less than approximately 0.0001% of the total poly(A) RNA. Transcripts 3, 4, 6a and 7 in RNA from cultured shoots were estimated to represent respectively 0.0023, 0.0014, 0.0007 and 0.0009% of the total poly(A) RNA. In grafted plants and tubers they were, to varying degrees less abundant suggesting that their reduced expression was brought about by different mechanisms. Transcript 4, the most important for the present study, showed a similar, about five fold, decrease in abundance (to ca. 0.0003% of the total poly(A) RNA) in grafted plants and tubers, irrespective of whether the tubers developed above or below soil. Transcript 3 however, was perhaps slightly less abundant in grafted plants, three-five fold less abundant in tubers below soil and was barely detectable in tubers above soil. It is noted that in general the levels of abundance for T-DNA transcripts in Mbl501B shoots were similar to those found previously in tobacco crown gall tissues (38, 39, 40). Taking such percentages it can be calculated from experiments and assumptions described by Goldberg et al. (l l) for polysomal RNA in tobacco leaves that Tcyt was expressed on average at five to ten transcript 4 molecules per cell for Mb 1501B shoots and only on average at one to two Tcyt RNA molecules per cell for grafted plants and tubers. Note that this approximate fivefold reduction in the abundance of transcript 4 in transformed shoots following grafting is correlated with an average twenty-fold reduction in cytokinin level (26). Although the molecular mode(s) of action by which T-DNA expression was reduced is unknown the initial cause was the change in growth conditions. Grafting Mbl501B shoots and growth in soil in a growth room always restored morphology and therefore it was positively directed and not random. The mere presence of roots was probably not direct-

221 ly r e l a t e d to the r e d u c e d a b u n d a n c e o f t r a n s c r i p t s , p a r t i c u l a r l y o f t r a n s c r i p t 4 since g r a f t e d M b 1501B s h o o t s k e p t u n d e r n o r m a l tissue c u l t u r e c o n d i t i o n s c o n t i n u e d to g r o w like u n g r a f t e d M b 1 5 0 1 B s h o o t s ( u n p u b l i s h e d o b s e r v a t i o n ) . T h i s i m p l i e d t h a t the e x p r e s s i o n o f at least T~Tt was s i m i l a r in these g r a f t e d a n d u n g r a f t e d shoots. T h e fact t h a t T - D N A e x p r e s s i o n a p p e a r e d directed, i r r e s p e c t i v e of the m e c h a n i s m of r e d u c t i o n a n d i r r e s p e c t i v e o f w h e t h e r it was c o m m o n f o r diff e r e n t t r a n s c r i p t s , suggests t h a t it s h o u l d be possible to i d e n t i f y specific r e g u l a t o r y s e q u e n c e s t h a t are i m p o r t a n t f o r e x p r e s s i o n a n d r e d u c t i o n in e x p r e s sion. S e q u e n c i n g a n d SI m a p p i n g e x p e r i m e n t s have already revealed several sequences with putative c o n t r o l l i n g i n f l u e n c e on T c y t e x p r e s s i o n (4, 12), S p e c i f i c m u t a g e n e s i s o f T c y t f l a n k i n g r e g i o n s together with biological analyses after introduction of the m u t a t e d g e n e i n t o p l a n t s s h o u l d give m o r e insight i n t o the r e l a t i v e i m p o r t a n c e o f these r e g u l a t o r y s e q u e n c e s . This a p p r o a c h e x t e n d e d by r e g e n e r a t i n g the c o r r e s p o n d i n g t r a n s f o r m e d ~ p o t a t o p l a n t s s h o u l d l e a d to f u r t h e r c h a n g e s in p o t a t o d e v e l o p m e n t w h i c h are d i r e c t l y r e l a t e d to a l t e r e d T c y t e x p r e s s i o n . P e r h a p s s u c h e x p e r i m e n t s will lead to a b e t t e r u n d e r s t a n d i n g of c o n t r o l of t u b e r i s a t i o n by l i n k i n g specific g e n e t i c f a c t o r s w i t h h o r m o n a l c h a n g e s a n d e n v i r o n m e n t a l f a c t o r s s u c h as photoperiod.

Acknowledgements W e t h a n k the M o l b a s R e s e a r c h G r o u p , L e i d e n , T h e N e t h e r l a n d s a n d in p a r t i c u l a r D r P. J. J. H o o y k a a s , f o r p r o v i d i n g us w i t h s u b c l o n e s o f Tip l a s m i d f r a g m e n t s . P a r t o f this w o r k was s u p p o r t e d by r e s e a r c h c o n t r a c t no. 471 o f the B i o m o l e c u l a r E n g i n e e r i n g P r o g r a m m e o f the C o m m i s s i o n o f the European Communities.

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Received 21 February 1985; in revised form 5 August 1985; accepted 19 August 1985.