Transgene copy number can be positively or negatively associated with transgene expression

Plant Molecular Biology 21: 17-26, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium. 17

Transgene copy number can be positively or negatively associated with transgene expression

Shaun L.A. Hobbs 1., Thomas D. Warkentin and Catherine M.O. DeLong National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Rd., Saskatoon, Saskatchewan, STN OW9, Canada; 1present address: CAB International, Walling)Cord, Oxon, OXIO 8DE, UK (* author for correspondence)

Received 19 March 1992; accepted in revised form 19 August 1992

Key words: additive gene action, antisense RNA, biolistics, gene suppression, methylation

Abstract

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and/3-glucuronidase activity; low- expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F 1 plants with GUS activity that equalled the parents and individual F 2 plants with 50~, 100%, 150~o and 200~o of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F1 and F2 generations with both types of insert had low GUS activity while F2 segregants that only had the H-type inserts had high GUS activity again. Sup- pression of the H-type gene was associated with increased methylation of the insert. Particle accelera- tion was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.

Introduction

Desirable alien genes can be stably introduced into the plant genome of many plants by manipulation of the naturally occurring transformation mechanism of the T-DNA in Agrobacterium tumefaciens [see 3 and 19 for reviews]. However, the T-DNA can be inserted more than once in the same plant genome during a transformation event

and there is conflicting evidence as to how such an increased copy number affects the expression of the introduced genes. The correlation between copy number and gene expression in transformants has been reported to be positive [4], inde- terminate [2, 9, 15, 16] or negative [7].

The situation in transgenic plants is further complicated by the fact that the introduction of additional copies of naturally occurring genes into

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plants can have a repressive effect on the endogenous homologues. The introduction of extra copies of genes coding for chalcone synthase (CHS) or dihydroflavonol-4-reductase into petunia resulted in a reduction rather than an enhancement of coloration [14, 18]. In addition, even the introduction of part of an endogenous gene, whether a naturally occurring gene [17] or one which has previously been successfully transformed into a plant in its entirety [5], can cause a reduction in the expression of the endogenous homologues. The underlying causes of such suppression have not yet been determined.

In a previous paper [7] we have reported variability in fl-glucuronidase (GUS) activity in individual transformed tobacco (Nicotiana tabacum L.) plants. After passage of each transformant through several generations and careful selection to ensure that only homozygous material was compared, there was a clear bimodal distribution of GUS activity, with material from individual transformants being differentiated into either high or low activity levels. It was shown that the differences in GUS activity were not due to epige- netic effects, but that Mendelian genetics could be used to explain the variability encountered. Sim- ilarly, the differences in GUS activity were shown not to be due to position effects or disruptions in a particular gene in the recipient genome caused by the T-DNA insertion, as independently gen- erated transformants, each with the T-DNA inserted at different positions in the genome, produced the same levels of GUS activity. Rather, those transformants with low GUS activity all had multiple copies of the T-DNA containing uidA, either at the same or different loci, whereas those transformants with high GUS activity all had single copies. Hence, the homozygous plants in the R2 population of transformants T5, T13, T14 and T19, which had single inserts, had the same levels of high GUS activity whereas all homozygous plants from transformants T4, T7 and T18, which had duplicated inserts at a single lo- cation had low activity. In addition, T6, which had many insertions, at least most of which were at different loci, had low activity. T3 had T-DNA inserts at two separate loci which segregated away

from each other. When both were present in the genome there was low activity, whereas when only the insert at locus 1 was present there was high activity. Unfortunately no segregants were ob- tained with only inserts at locus 2, which apparently itself contained multiple copies.

Hence it was clearly demonstrated that multiple copies of T-DNA inserts at the same or different loci were associated with reduced GUS activity. However, it was not determined whether this was a general effect, with all multiple insertions expected to result in reduced gene expression, or a specific effect, caused by the configu- ration of particular inserts due to rearrangement of the T-DNA during transformation, i.e. whether the multiple, often inverted, repeats at the same locus produced a repressive effect on GUS activity. In no case were plants available that had two individual inserts at different loci, where each locus could be shown to have only one T-DNA copy. It was therefore decided to perform cross- fertilization experiments between different transformants with high GU S activity to generate these plants, as well as to cross high and low transformants. The results of these experiments are reported here and clearly show that it is the type of insert that is important. Thus it is demonstrated that increasing the copy number of alien genes can both increase and decrease their expression in transgenic plants.

Materials and methods

The tobacco transformants have been fully de- scribed elsewhere as have conditions of plant growth, Southern and northern hybridisations and methods of determining DNA methylation and GU S activity [ 7 ]. Autoradiograms of northern blots were scanned with a Molecular Dynam- ics computing densitometer model 300A and intact versus degraded RNA hybridizing to the uidA probe was determined from scans which included only the bands at ca. 1.9 kb and those which included all signals below these bands. Parents for cross-fertilization were R2 progeny that had been selected to be homozygous for the T-DNA inser-

tion. Crossing was performed following the pro- cedure outlined by Wernsmann and Matzinger [20]. Leaf discs for determination of GUS activity were taken at various times during plant growth. Fully developed leaves at a similar stage of development were taken from all plants measured at the same time.

Tests for presence or absence of GUS activity in the F 3 generation were carried out by germi- nating seeds on 0.25 × Hoagland's solution. After 14 days, individual seedlings were transferred to wells in microtitre plates, covered with GUS ex- traction buffer [8] containing 4-methylumbelli- feryl-fl-D-glucuronide (MUG) and left overnight. It was found that wells containing seedlings with either high or low GU S activity fluoresced brightly when plates were put on an ultra-violet transillu- minator while those containing seedlings without uidA did not fluoresce at all. Hence, segregation of the T-DNA could be determined.

Tobacco leaf disc bombardment

Fully expanded leaves from post-flowering plants were surface sterilized by shaking in 70 ~o ethanol for 30 s, then for 15 min in a 8~o solution of com- mercial bleach containing a wetting agent (Tween 80, 0.025~ v/v). The leaves were subsequently rinsed four times in sterile distilled water.

Plasmid DNA was isolated using a QIAGEN- tip 500 column (Qiagen Inc., Chatsworth, CA) from constructs with different promoters driving the uidA structural gene with the nopaline synthase (nos) terminator. The promoters were: 35S (pBI-507); nos (pBI-509) and the duplicated 35S followed by AMV (the untranslated leader from RNA 4 of alfalfa mosaic virus) (pBI-505) [6]. pBI101, which contains a 'promoter-less' uidA- nos terminator cassette, was used as a control (Clontech Laboratories, Palo Alto, CA).

DNA was precipitated onto gold particles (1.0/~m diameter) according to the protocol de- scribed for the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, Rich- mond, CA). Tobacco leaf discs were cultured for one day, adaxial side up, on a medium contain-

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ing MS salts [ 13], myo-inositol (100 mg/l), thia- mine-HC1 (0.5 mg/1), pyridoxine-HC1 (1.0 mg/1), nicotinic acid (5.0mg/1), benzylamino purine (1.0mg/1), indole butyric acid (6.0mg/1), agar (7 g/l), and sucrose (30 g/l) at pH 5.9. Leaf discs (12 mm diameter) were bombarded under partial vacuum using a helium pressure of 10000 kPa. The distance between the stopping plate and target tissue was 6.5 cm. Each shot delivered 0.8/~g DNA on 500 #g of gold particles. Two days after bombardment leaf discs were removed from the medium and immersed for 24 h at 37 °C in a solution containing 1 mM 5-bromo-4-chloro- 3-indolyl-fl-glucuronic acid (X-gluc), 50raM sodium phosphate buffer (pH 7.0), 1 mM EDTA, 0.5 mM potassium ferricyanide, and 0 .5mM potassium ferrocyanide. Chlorophyll was removed by placing the leaf discs in 70~o ethanol for 48 h.

Results

GUS activity in populations from a high × low cross

Plants transformed with pBI121 [8], which contains a chimaeric gene, CaMV 35 S promoter-uidA structural gene-nos terminator, within its T-DNA, had been previously selected [7]. R2 progeny that were homozygous for the T-DNA insert were chosen from transformants that had either high (T5, T19) or low (T4, T7) GUS activity. These high and low plants were cross-fertilized and leaves from the F~ progeny were assayed several times during the course of development to determine their level of GUS activity and compare it to the parental levels. All F~ progeny resulting from such high × low crosses (T4 x T5, T19 x T4, T7 × T5 and T7 × T19) had GUS activity levels that differed significantly from the mid-parent value and were close to the levels of the low parent when expressed on either a per unit leaf area (Fig. 1) or per mg protein basis (data not presented). Similar results were found no matter which plants were used as male or female in a cross, indicating a lack of maternal effects. This showed that the presence of T-DNA inserts associated

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5

m =°2. _=

~ 0 . An T4 T5 T7 T19 T4xT5 T19xT4 T5xT19 T7xT5 T7xT19

POPULATION

Fig. 1. Levels of GUS activity in parental and F I populations from different crosses. Activity for each population is the average of six plants measured by the M U G assay at three different times during development. Bars show standard error.

with low GUS activity levels (L-type) in the same genome as those associated with high activity levels (H-type) resulted in the repression of the high activity.

This was confirmed by examination of F2 material. F 2 seed from the T7 x T19 cross were germinated and GUS activity was examined in 30 resulting plants. To avoid potential problems with variability from experiment to experiment, data are presented as a percentage of one of the high parents (T19). The GUS activities of the F2 population are definitely skewed towards the low end (Fig. 2). Southern blots showed that the T-DNA inserts from T19 and T7 could be differentiated and it was determined that all the highest expressers in the F 2 had only T19 inserts present. Indi- vidual F 2 plants containing T7 inserts only had low activity (ranging from 5 to 28 ~o) as did plants containing both T7 and T19 inserts (ranging from

14

I ' - 12 Z

a . u . 8

o ~ 6 u l

"1 2 Z

o i-lnl-ln

5O

GUS (as % of T19)

.I-'7. lOO

Fig. 2. Histogram of the GUS activity levels of 30 individual F 2 plants from a T7 x T19 cross. Activity for each plant was measured by the M U G assay twice just before flowering and the average was expressed as a percentage of the T19 parent.

4 to 15~). Seed from the highest (91 ~o) and two intermediate (50~o and 38~o) F2 individuals, which had only T19 inserts, were germinated and seedlings scored for presence or absence of GUS activity. All F 3 seedlings from the highest F2 individual were positive for GUS activity, whereas of the seedlings from each of the other two, 21 were positive and 9 negative which was not significantly different from 3:1 ratio, positive to negative. This demonstrated that the F 2 plant approaching 100~o of the T19 parent was homozygous for the T19 insert whereas those plants approaching 50~o were hemizygous for that insert and confirms that the uidA alleles on the T19 insert act in an additive manner when no T7 inserts are present in the plant genome. There was no apparent heritable effect of the interaction on the H-type insert which expressed normally again when it segregated away from the L-type.

G US activity in populations from a high × high cross

Cross-fertilization was made between two transformants (T5 and T19) using plants that were homozygous for the T-DNA inserts. These transformants both had high levels of GUS activity which Southern blots determined had resulted from different transformation events. The F 1

progeny had GUS activity levels that were not significantly different from the parents (Fig. 1) and again there were no maternal effects. This indicated that each of the individual H-type inserts had an additive effect on GUS activity. Seed from F 1 material was taken and 18 individual F 2 plants were grown and measured for GUS activity. This F 2 population showed transgressive segregation with levels of activity up to twice that of the parent and definite clusters of individuals around the 0, 50, 100, 150 and 200~o levels (Fig. 3).

To determine copy number in the F2 individuals, Southern blots were used to show which T-DNA inserts were present and seeds from each plant were germinated and the F3 seedlings tested for segregation. An F 3 population segregating for the presence or absence of GUS activity indicated that the F 2 parent was hemizygous at all the

o 5 ,

50 100 150 20O GUS (as % o f T 1 9 )

Fig. 3. Histogram of the GUS activity levels of 18 individual F 2 plants from a T5 x T19 cross. Activity for each plant was measured by the MUG assay twice just before flowering and the average was expressed as a percentage of the T19 parent.

T-DNA loci that were present in it. These data could be used to determine the exact copy number of individual inserts for 10 of the 18 F2 plants (Table 1). The genes acted in a completely additive manner, one copy of either giving half the expression of the parent and 2 copies, in any combination, giving the same expression as that of the parents. The remaining eight plants had either three or four copies, although the exact number could not be determined. Of these, seven had GUS activity levels between 123~o and 211~o. The remaining plant did not have the expected GUS activity (34~o) and it was not possible to determine if this was due to a physiolog- ical, environmental or genetic cause.

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RNA content

Northern blots of RNA extracted from four individuals of the parental types, four F~'s and 20 F2's from a T7 x T19 cross showed that the levels of RNA hybridizing to uidA were very much higher in the plants that had only the T19 inserts than in those which had the T7 inserts, either alone or in conjunction with the T19 ones (Fig. 4). In addition, densitometry scans of the autoradiograms showed significantly higher levels of degraded as opposed to intact RNA hybridizing to uidA in the T7 containing plants (79 + 6~o degraded) than in those with T 19 alone (21 + 13 ~) .

Transient expression in transformed plants

Leaf discs were taken from individual F2 plants from a T19 x T7 cross. These plants included those which Southerns indicated had only T19 inserts (high GUS activity), had only T7 inserts (low GUS activity), had both T7 and T19 inserts (low GUS activity) and those in which both inserts had segregated out (no activity). The discs were subjected to particle bombardment with DNA containing different promoters driving uidA and then stained with X-gluc. The average number of dark blue spots per leaf disc is presented in Table 2. Leaf discs from plants with no T-DNA

Table 1. Copy number, origin and GUS activity in F 2 plants from a T5 x T19 cross.

Plant Number of Origin of GUS activity number inserts inserts (yo of T19)

65 1 T5 58 68 2 T5 and T19 102 71 2 T5 103 73 1 T5 49 74 2 T5 102 75 2 T19 111 77 0 - 0 80 2 T5 and T19 111 81 0 - 0 82 1 T19 50

Fig. 4. Autoradiograms of northern blots of RNA from parents, F~ and representative F 2 individuals probed with uidA, main band is at 1.9 kb.

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Table 2. Number of dark blue spots appearing on leaf disks from plants with different types of T-DNA inserts present after bombardment with DNA containing different promoters driving the uidA gene. Numbers in parenthesis are number of leaf discs scored in two separate experiments.

Type of Promoter Average indigenous (with T-DNA insert none 35S nos 35S-35S-AMV promoter)

None 0 18.8 62.0 117.1 66.0 (24) (24) (24) (24) (72)

T7 only 0 1.2 0 7 2.7 (9) (8) (7) (7) (22)

T7 and T19 0 0.1 0.5 3.6 1.6 (13) (16) (5) (14) (35)

inserts showed a considerable number of dark blue-stained spots after bombardment with DNA that contained promoter-uidA fusions (Table 2), with 35 S- 35 S-AMV being the strongest promoter. The level of activity of the endogenous uidA genes in the discs from plants with low GUS activity was such that there was variable background staining even after bombardment with pBI101, the plasmid with no promoter in front of uidA. The dark blue spots associated with transient expression in discs with no inserts were apparently sufficiently intense that their presence would have been obvious over this light blue background. However, to avoid any possibility of background interference only those discs with no background blue staining were scored. As all of the discs from the plants without T-DNA showed spots it could be determined that this would not bias results due to bombardments having missed any leaf discs or staining reagent not having penetrated their tissues. Few, if any, dark blue spots were evident on discs from plants with the T7 insert (Table 1) indicating that the indigenous suppression was able to repress gene copies added in vitro. Leaf discs from the high activity plants (those with only T19 inserts) stained dark blue with X-gluc and no extra staining could be resolved with any of the bombarded DNA.

Methylation patterns

Ava I and Pst I do not cut at their recognition sites if the cytidine residues are methylated. Sites that

are recognized by these restriction enzymes exist in the introduced T-DNA in the 35S promoter and at the end of the uidA structural gene and these sites were used to provide information on the degree of methylation in the T-DNA. DNA samples from parental, F 1 and F2 individuals were digested with these enzymes in conjunction with Dra I and Eco I (which cut close to the right border and at about 700 bp from the left border of the T-DNA, respectively), Southern blots were probed with uidA or kan and the sizes of the re- suiting fragments were then used to indicate whether the internal Pst I and Ava I sites were methylated. Two crosses were examined in detail, T7 × T19 and T5 × T19. DNA was extracted from ten individual plants of each parental type and the F 1 for each cross, and from 30 F 2 plants from the T7×T19 cross and 18 F2 plants from the T5 × T19 cross. DNA was extracted from each plant sample and examined at least twice to ensure that any variability in cutting was not caused by contaminated DNA samples. Previous exper- imentation [7] determined that these combina- tions of enzymes and probes could give a clear indication of the state of methylation of the individual sites within the T-DNA inserts from the size of the individual bands produced.

Digestion with Pst I and Eco RI and hybridization with uidA produced a 3 kb band in all cases showing that the Pst I site at the beginning of the 35S promoter was not methylated in any of the plants examined. Digestion with Dra I and Ava I and hybridization with kan showed that the Ava I site in the 35S promoter was not methylated in

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Fig. 5. Autoradiograms of Southern blots of DNA from parents and representative F 2 individuals. A. DNA digested with Ava I and Dra I and probed with kan. B. DNA digested with Ava I and Eco RI and probed with uidA.

T19, only a 2.6 kb band being present (Fig. 5A) but at least partially methylated in T7, as an additional 3.4 kb band appeared. The F 1 of the T7 x T19 cross had both of these bands as did all F 2 individuals where both types of insert, or only the T7 insert, were present (Fig. 5A). Those individual F 2 plants which only had the insert from T19 only had the 2.6 kb band (Fig. 5A). Hence there was a correlation between the presence of partial methylation at this site, as evidenced by the appearance of the 3.4 kb band, and low levels of uidA expression. All parental, F1 and F 2 material from the T5 x T19 cross had only the 2.6 kb band.

Digestion with Eco RI and Ava I and hybridization with uidA also demonstrated a difference between parents. A 2.2 kb band in T7 and a 1.9 kb band in T19 (Fig. 5B) showed that the Ava I site between the uidA structural gene and the nos terminator was methylated in L-type but not in H-type inserts. DNA from the F1 of the T7 x T19 cross only had the 2.2 kb band present, as was the case in all individual F2 plants that had both types of T-DNA insert or the T7 insert alone (Fig. 5B). Both H and L types of insert were now methylated at the Ava I site at the end of the uidA gene. The 1.9 kb band, either alone or in conjunction with the 2.2 kb band, appeared in all those F2 individuals that had only the T19 inserts. Therefore, the appearance of the 1.9 kb band, and

hence the reduction in methylation at the Ava I site, was directly correlated to increased levels of expression of uidA. Once again, all parental, FI and F 2 plants from the T5 x T19 cross showed only the 1.9 kb band, indicating no methylation.

Discussion

The results show that the transformation of tobacco with pB1121, which introduced a chimaeric uidA gene into the plant genome, produced two different types of T-DNA insert with markedly different effects on the levels of expression of uidA (as yet, expression levels of kan, which was also introduced, have not been quantified). The H-type, found in T5 and T19, was associated with a high level of expression of uidA which resulted in high levels of uidA RNA and GUS activity in the transformants. There were no apparent interactions between alleles at the same or different loci and extra copies of the genes in the same genome acted additively, one copy of the insert producing half the level of GUS activity of two copies, a third that of three copies, etc. How- ever, the L-type of insert, found in T4 and T7, generally produced low expression of the introduced uidA and a resulting low level of uidA RNA and GUS activity. In addition, the L-type inserts interacted in trans with the H type, totally or par-

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tially suppressing the expression of its GUS genes. Hence the F1 population from a cross between two transformants with H-type inserts at different loci had GUS activity levels equal to the parents and individuals in the F2 population had levels up to twice parental levels. However, the F1 population from a cross between a transformant with H-type inserts and one with L-type inserts had GU S activity levels well below the mid-parent value as did all F2 individuals in which both types of insert were present. As all F2 plants with both types of insert had low GUS activity levels, a single L-type insert could apparently suppress the expression of at least two copies of the H-type. In the F2 population from a T7 x T19 cross, only in those individuals where the H-type inserts had segregated away from the L-type did the high GUS activity levels again appear. Although the number of F2 plants reported here is small, anal- ysis of further populations confirmed the results (data not presented). A very similar kind of allelic interaction has been reported associated with mutants at the nivea locus in Antirrhinum [ 1 ]. Null alleles of Niv ÷ were found that were semidomi- nant and, when heterozygous with the wild type allele, gave very pale flowers in contrast to the red of the wild type. Such null alleles acted in trans

to inhibit expression of Niv + 25- to 50-fold and mapping showed that there were inversions and multiple copies of niv gene sequences at the mu- tant loci.

The X-gluc assay for GUS activity is very much less sensitive than the M U G assay and as a result the plants with L-type inserts, i.e. with low endogenous GUS levels, often showed little or no histochemical staining. This meant that it could be determined whether or not further copies of uidA, introduced in vitro via particle bombardment, were being expressed in plants with L-type inserts. Unlike leaf discs from plants with no T-DNA inserts, where the uidA genes introduced via particle bombardment produced many dark blue staining spots indicative of high GUS activity, the leaf discs from plants with indigenous L-type inserts produced few, if any, blue spots following bombardment. Even when uidA was driven by the 35S-35S-AMV promoter, which

was very strong in its effect in tissue with no T-DNA inserts, little transient GUS activity was produced in the presence of the L-type inserts. These results indicate that the L-type inserts are able to repress not only the expression of genes on other T-DNA inserts in the same genome, but also that of still further uidA copies that are introduced in vitro.

One apparent physical difference between the two types of insert was that the L-types in T4 and T7 each had two copies of the T-DNA inserted at the same locus as inverted repeats. The T4 insert has the structure Dra I-kan-uidA-Eco RI: Eco RI-uidA-kan-Dra I and the T7 insert has the structure Eco RI-uidA-kan-Dra I: Dra I-kan-uidA-

Eco RI ([7], and confirmed here by further exper- imentation (data not presented)). However, it has been demonstrated that it is not just the presence of multiple T-DNA copies in a genome that re- suits in gene suppression as multiple copies of the H-type inserts act in a totally additive manner. The insertion of truncated genes into plants is known to reduce expression of endogenous, full- length homologues that expressed well in untrans- formed plants [5, 17]. Although in L-type inserts reported here, the transformation events apparently introduced double T-DNA copies at the same locus, Southerns showed that only full- length copies of both uidA and T-DNA were present [7]. The H-type inserts had only one full- length T-DNA insert present at each locus.

In many respects, therefore, the phenomenon reported here is very similar to co-suppression [see 10 for review], where the re-introduction of endogenous genes via plant transformation re- suited in the suppression and not enhancement of gene expression [ 14, 18]. This trans suppression of the wild-type gene was no longer apparent when it segregated away from the introduced gene during meiosis.

The causes of the various suppression effects of similar genes on each other have not as yet been established. Methylation, ectopic pairing and antisense production have been put forward as possible explanations [ 10] and the results presented here are also inconclusive in this respect. It is possible that the production of antisense RNA

could play a part as the two L-type inserts examined in detail are both composed of inverted repeats of the T-DNA. However, the production of the antisense RNA would have to be under different promoter control in T4 and T7 due to the different orientation of the inserts. In the T4 insert, antisense could be produced by the 35S promoter producing read-through transcription from one uidA copy straight into the reversed uidA copy. In the T7 insert, such read-through transcription would not be easy to envisage and any antisense would most likely be produced from plant genomic promoters close to the insert. Although in T4 and T7 the T-DNAs at the same locus are inverted repeats and antisense RNA could be an- ticipated to be a factor, another transformant, T18, which apparently also has an L-type insert, had two T-DNA copies in tandem [7]. The reduction in the amount of RNA that hybridized to uidA in the low expressing plants may also indicate an effect of antisense, although it could not be determined if this reduced uidA RNA level was due to a decrease in uidA transcription or a reduction in the stability of the RNA. The rapid and effective repression of the expression of uidA copies introduced via biolistics indicates the presence of some factor in the cell that recognises the DNA or its products and works rapidly to suppress their activity. This factor could be antisense RNA. It is difficult to reconcile such a dramatic reduction in transient expression of naked DNA with ectopic pairing and the exchange of chromatin structural components [ 10], especially as the particle bombardment presumably introduces many copies of the gene into cells where, at most, only two indigenous L-type inserts are present, albeit each with two uidA copies.

The transformants with high and low GUS activity have previously been found to vary in degree of methylation of the T-DNA [7]. This associa- tion of methylation and gene expression was also found in the segregants examined here and it is difficult to connect this to an antisense effect. In other segregating progeny, similar correlations have been found between the presence of one T-DNA insert and the methylation and suppression of an unlinked insert [7, 12].

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Copies of uidA introduced by biolistics were repressed in the presence of L-type inserts regardless of the promoter associated with them. The indigenous uidA genes were driven by the 35S promoter, but the 35S, nos and 35S-35S-AMV promoter-uidA fusions were all repressed. It is therefore the presence of the gene sequence itself that is important in the suppression of homolog expression in transgenic plants. This has also been found in other similar cases of suppression. The introduction into petunia of chimaeric genes where the 5'-flanking regions of CHS genes had been fused to the GUS gene resulted in GUS activity that was identical to the expression of the authentic CHS gene [ 11 ]. However, the reintro- duction into petunia of the CHS 5'-flanking re- gion still in conjunction with the CHS structural gene resulted, in some cases, in the suppression of both the introduced and the endogenous gene [181.

On the practical side, the production of transgenic plants with economic levels of expression of the alien genes is fundamental to the success of agricultural improvement through plant biotechnology. It is therefore important to determine whether increased activity of a gene product can be expected through manipulation of the copy number in a genome or whether the opposite would be expected. The data presented here showed that increasing the copy number can both increase and decrease the level of expression of alien genes in transgenic plants, the key criterion being the nature of the copy that is involved. With the correct type of T-DNA insert it is evident that increased expression of an introduced gene could be improved if initial transformants were not expressing at an economic level. By crossing two different transformants, each with H-type inserts, and selecting high expressers in the F2 population, a stably inherited increase of the expression of the introduced gene(s) should be very rapidly effected.

Acknowledgements

Seed from the original (Ro) transformants used in this and the previous paper [7] as well as pBI-

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505, pBI-507, and pBI-509 were kindly provided by Drs R.S.S. Datla and W.L. Crosby and Mr J. Hammerlindl, Plant Biotechnology Institute, National Research Council of Canada, Saska- toon, Canada.

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Transgene copy number can be positively or negatively associated with transgene expression