E. Report of the Committee on Genetic Engineering

(A survey of representative cloning vehicles for rice)


A. Caplan and M. Van Montagu, Convenor

Laboratorium voor Genetica, Rijksuniversiteit Gent B-9000 Gent, Belgium


Intensive investigations into tissue culture conditions have recently yielded several protocols for the efficient regeneration of rice plants from isolated protoplasts (1; Thompson and Cocking, in press). Using these techniques in combination with at least one of the methods currently employed to transform other species should produce genetically modified rice plants within the next few years.

There are two proven methods of introducing DNA into plants stably. One employs the DNA transfer machinery of Agrobacterium tumefaciens to insert a predictably defined piece of DNA into the nuclear genome (2). The other method depends on DNA uptake facilitated by either polyethylene glycol (3) or electrical fields (4), and on the wholly unpredictable recombination between random sequences on this DNA and the host genome. Although only the latter method has been used successfully with rice (5), studies reported on maize (6) indicate Agrobacterium may yet prove valuable for transforming grasses. It is this very promise that makes it difficult to recommend one class of vectors over another. For this reason, we have compiled a partial list of representative vectors that might prove useful in the near future as tools for introducing or expressing genes in rice (Table 1-Properties of representative cloning vehicles). Most of these vectors were originally designed for use in conjunction with Agrobacterium. Although the T-DNA transfer sequences are not employed when purified DNA is absorbed by plant protoplasts (7), these vectors are of importance for direct DNA transfer since they offer the broades choice of cloning sites and screenable markers. The only remaining consideration for choosing a vector is that the smaller ones, or those with high copy numbers in E. coli, are easiest to purify when preparing DNA for DNA-uptake experiments. The first five vectors in Table 1-(Properties of representative cloning vehicles) are noteworthy for this reason; however only pCTIT3 was specifically designed for these uptake experiments.



Table 1. Properties of representative cloning vehicles

=============================================================================
Vector  Size  Cloning   Selectable Screen- cos  Bac-   Bac-   Ti        Ref.
site          sites     marker1    able    site terial terial helper-   
                                   marker       repli- sel-   plasmid
                                                con    ection
                                                       marker
=============================================================================
DNA  uptake vector
pCTIT3  16.3  HindIII,   pCAMV-VI:   nos  --  pBR322,  Str/  none           5
              SalI       NPT:CAMV-VI          pSa      Spc    
Multipurpose vectors
pEN4K   16.0  BamHI,     pNos:NPT:   --   +   pUC,pRK2 Cam;  A             25
              KpnI,SalI, none                          Kan
              XbaI
pAS2034  7.7  ApaI,EcoRI pTR2':NPT:  --   --  pBR322   Str/  pGV3850       26
         (BamHI,SalI)2   Ocs                           Spc;
                                                       Cbn
pGV831   8.9  BamHI      pNos:NPT:   --   --  pBR322   str/  pGV2260       12
                         none                          Spc;
                                                       Cbn
pMON200  9.5  BglII,     pNos:NPT:   nos  --  pBR322   Str/  pTiB6S3-Se    27
              ClaI,      Nos                           Spc
              EcoRI,HIndIII
              HpaI,StuI,
              XbaI,XhoI
pBin6   16.0  EcoRI,SalI pNos:NPT:   nos  --  pRK2     Kan   A             28
                         Nos
pGA471  15.6  BglII,     pNos:NPT:   --   +   pRK2     Tet   A             29
              EcoRI,     Nos        
              HindIII
pPC310  14.2  BamHI,     pNos:NPT:   ocs  +   pRK2     Cbn;  pMP90RK       13
              HindIII,   Ocs                           Tet  
              SalI
pC22    17.5  BamHI,     pNos:NPT:   --   +   pBR322,  Str/  A             30
              XbaI       none                 pRi      Spc;
                                                       Cbn
=============================================================================


General considerations

Regardless of the manner of introducing DNA, gene transfer vectors should provide (i) an origin of replication and selectable marker to allow DNA to be prepared conveniently from E. coli, (ii) an eukaryotic selectable marker to allow one to identify transformed plants, and (iii) unique restriction enzyme recognition sites in which to clone additional eukaryotic genes. If large pieces of DNA are to be introduced into the vector, it may be desirable to provide a cos site in order to exploit the in vitro packaging system of bacteriophage lambda (8). Finally, it is advisable to have an additional gene on the vector that can be used conveniently to verify that calli or plants selected with one marker are indeed transformed with the entire construct.


Bacterial markers

If DNA uptake is employed to introduce genes into plants, the vectors can be relatively small and use the prokaryotic origins and markers found on pBR322 (9) and its high copy number derivatives (10). In many cases, it is possible to select for recombinants between these plasmids and the Ti helper plasmid, pGV3850 (11), so that clones can be transferred to plants using Agrobacterium. In addition, a number of more specialized vectors have been made that can replicate in both E. coli and Agrobacterium, and there function as T-DNA plasmids when provided with the DNA transfer functions present on plasmids such as pGV2260 (12), pMP90RK, and LBA4404 (14).


Selectable and screenable markers

Most vectors in common use confer kanamycin resistance to transformed cells by means of a modified neomycin phosphotransferase II (npt-II) gene. Kanamycin (100 ug/ml) appears to be effective against untransformed rice during the first few divisions after protoplast regeneration, but larger calli can grow on 500 ug/ml of the antibiotic (R. Dekeyser and A. Caplan, unpublished results). For this reason, it is advisable to have an easily screened marker on the vector. Among these are the enzymes octopine synthase (15) and suitably modified forms of chloramphenicol acetyltransferase (16,17). The NPT-II enzyme activity may also be used as a sensitive assay for transformed cells (18,19). Thus, it may prove possible to select for transformants using modified hygromycin resistance genes (20,21), and NPT-II as a means of verifying transformation or comparing the activities of different promoters.


Cloning sites and gene expression

Most vectors have at least one or two sites that can be used to insert additional DNA sequences. These can be used to introduce genomic sequences harboring intact plant genes. Since there is evidence that some monocot regulation or processing signals do not function efficiently in dicots (22), one must anticipate cases where the reverse is true. For this reason, it may be necessary to employ "universal" promoters, such as those obtained from nopaline synthase (15), cauliflower mosaic virus (23), the T-DNA genes 1' and 2' (24), or maize sucrose synthase (19). Transient expression assays have shown the first three (and a variety of others) are functional in rice protoplasts (17; R. Dekeyser and A. Caplan, unpublished results), although to varying degrees. Polyadenylation and transcription termination signals have been obtained from a variety of T-DNA genes as noted in references 13, 17, 24, and elsewhere. The properties of some of these genes and promoters are noted in Table 2 (Additional components for gene expression). As techniques for the transformation of rice improve, one can expect to construct better expression systems using components derived from rice itself.



Table 2. Additional components for gene expression

=============================================================================
Markers
=============================================================================
Gene           Use                                          Reference
=============================================================================
pOcs:HPT:Nos      Selection of transformed cells               20
pNos:HPT:Nos      Selection of transformed cells               21
none:NPT:Ocs      Assay system for plant promoters             19
none:CAT:rbcS     Assay system for plant promoters            17,22

Additional promoters functional in grasses
=============================================================================
Source      Expression    Tested in            Reference
=============================================================================
pNos         moderate      wheat              31
                           maize              16
                           rice                5; R. Dekeyser and A. Caplan
                                                  (unpublished results)
pCOPIA       high          rice               17
pCAMV-35s    high          maize              16
                           rice               17
pCAMV-VI     high          ryegrass           32
pTR1Õ,2Õ     high          rice                R. Dekeyser and A. Caplan
                                                  (unpublished results)
p(sucrose synthetase)  
             high          wheat              19
=============================================================================
HPT, hygromycin phosphotransferase; NPT, neomycin phosphotransferase; CAT, chloramphenicol acetyltransferase; rbcS, ribulose-1, 5-bisphosphate carboxylase; CAMV, cauliflower mosaic virus.



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Acknowledgment

This research was supported by a grant from the Rockefeller Foundation (R.F. 84066).