Genotypic and phenotypic changes in new plant varieties

~‘ifiii‘en:s a?c,ut g~e:k‘ii.4li! enginecirrd organ!sms have CW~I. ~-cc: around (I) 1.i~ f.:ffect of tjle ~ri:r(i duced kxeigx genie trait(s) on !Ix normal physica!ogy of an engineerec organism, (2) genera&ion of phenotype change other than that predicted from the transferred foreign gene(s) 1and (31 the effect and regulation of releasing engineered organisms into ecological environments. I till address the frst two of these CQnCernsin this article. Transferring forei genes into8 target Cells or tissues is 5ne way of altering the genotype and phenotype of plants. Another is to regenerate plants (somaclonal variants) from celis grown in tissue culture. Both methods produce genetically stable alterations in the plants. But while somacional variants of many pkmt species are commercially available and widely consumed, genetically ered crops are being subto intensive and lengthy small-scale field testing. Among those involved with the planned reiease of genetiCal!y engli-ieered organisms? it is widely accepted that it is the product, and not the means of producing it, that should b$ regulatedZJ. it becomes appropriate, therefore, to Compare the changes t0 plants from genetic engineering tith those wrought by tissue culture techniqiies .

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;trrtiSic!tx w-ii;: az;e). For plant celIs ii) tCq?rt’sS~a foreign gene consistently. the I!SA must be integrated intxt Into ,;ipp!,opriate chromosomai iocations of the: hf3st cellslr~l~. SIX-cessful integration of foreign gene5 is most often identified by selection of transf5irmed cells on culture media containing an antibiotic to which the reporter gene confers resistance. Transformed plant cells are commonly selected at frequencies between 1O-3 and 1O-4 (one in 103--IO4 of the treated cells expresses the gene sufficiently well to become resistant to the antibiotic)aJ2J”.

to divide continuously and to rege into piants, inserted forei A must apparently disturb proper functioning of the plant genome as little as possible. Mukip,le insertion, for instance, is mQre likely to destroy important endogenous genes. If genes which are responsible for housekeeping metabolism, cell and tissue differentiation, specific organ development and sexual reproduction are destroyed, the resulting plant will be neither healthy nor fertile. However, before a foreign gene can be expressed, at least one copy must be integrated intact, and it must be inserted into a chromosomal site where gene expression occurs Gene Wansferand i~t~gr~ti~~ consistently10-12. Most of the DNA Gene transfer techniques are on plant chromosomes is not exrapidly progressing, atid some have pressed Ontrons, intragenic regions, become routine in many Labora- ‘pseudogenes, etc.) and other sites tories. Methods developed for omosome may be only &ch tasks in&de: A&obactehzm: ~transiently active at certain stages of imediated ’tr&sformatiQn3, ,,electro- z: #plant development. Insertion of inporation4, ‘miiKroinjection5,and par-: 1!$act genes into inactive sites may ‘ticlk bomba&nent6 For piant ,,ah result in the lack of cell L transfor&tion, “hundreds of ; EiexpressiorPJ2. With ‘reporter’ thousands N.of’icbpiesof one’ or two ”$ene selection, many of these plants spe’cific fo$ei,&i~genesof interest are ‘with such inappropriate insertions Aelivered’to each target ccl!. Desir‘willnot be recovered. &!e foreign~genes need to be strin,gently de$n$d~ both biochemically Altered genetic content ‘~and’genet&A!$;;,,sothat they can be ‘I’ransgenic plants that exhibit &ffectivel# ~;m&itored in’en&eered normal phenotypes are then further .$ants. Thbn$& the cellular mechansubjected to small-scale plant‘isms are not yet fully ‘understood, a breeding programs for selection of small ‘1fi-ii&x$ df .tlie ~$iI3_tt, ‘foreign best individual lies for high and DNA molec&s (one ito I.several stable expression of target foreign copies) stiEC+fulIy .irjiteg+$es into gene traits, with a minimum of disthe host cel3,I;chromosomes., bappar- ruption of the plant’s physiology. entlq in ,a r&&k&nfasMbn7-‘1. To monitor the pattern of integration of foreign ‘genes genomic Southern blot anaiyses are commonly performed. Transformed plants often contain one to a few copies of the

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broken or rearranged foreign genes may also be present in the same Restriction organism. ma.pping analysesH,Y,14 determine wh.ether the multiple copies are integrated individually in.to separate sites of the chromosomes, or are present as a cluster of tandem genes (as observed for some gene-transfer methodsra). Genomic blots can also be used to show that inserted foreign genes are stable during development of engineered plants and are inherited stably by tiear progeny in B mendelian fashion7s. One study showed that an introduced foreign gene had high meiotic stabifityl”. Copy number and integration pattern of foreign genes Can be stably maintained through sexual reproduction. Therefore, foreign DNA stably integrated into host chromosomes will be bred as normal chromosomal genes in Conventional bree programs.

The production of most genetically engineered plants requires that cells are grown in tissue culture. Tissue culture without genetic engineering is also used for producing novel plant varieties. So, how much variation is due to genetic engineering and how mu& to tissue Culture? There is a high incidence of phenotypic changes in plants regenerated horn tissue culture procedures. Most culture-derived variations are physiological, caused by the conditions in the culture. These physiological changes are not seen in the sexually derived Fr progenies I However, some phenotypic variants, the somaclonal variants, are gentically stable. Somaclonal variants have been obtained by various different techniques of plant tissue culture, including protoplast regeneration, protoplast fusion, somatic embryogenesis and organogenesis. Many have been selected and bred to generate useful cultivars for potato, tomato, and other CrQpS15. Genetic explanations of the origin of somaclonal variants have been notable by their absence. Recently, however, ‘cryptic’ transposons (transposable genetic elements) on plant chromosomes which become activated in tissue culture have been foundr7. Excision and reintegration, (transposition) of chromosomal genes from one genomic site to

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A dkecl: comparison of the number and type of phenotgipic changes using large numbers of regenerated pkm.ts derived km genetic engineering versus non-engineered, conventiona ti+e ctilteure methods has not b’een repor+xL But reotaks

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obtained WOKboth engineered and non-engineered pl.ants from tissue cultures (Ref. 16 and N-S. Yang, unpublished). Therefore, it is not surptising m plants from tksue culture to f?nd pheec&ypic changes nol caused by the foreign gene or ita insertion in en Foreign DNA in engine constitutes only a smali the host cell genome (often at a ratic of 1: I million). The inserfionai ar,d

Gelzetic engineers generally want to maintain all of the dlesirable properties of the tar,get p!ant and include a:m additional phenotype predicteldi from insertion of a foreign gene. in plant genetic &omplications engineering experiments incluck several factors that now cannot be controiied. These includ.e the waIsted insertion of the gene into a. ‘silent’ portion of tbe chromosome, the deleterious insertion into a gene that is important for factors such as, disease or drought resistance, and the generation of variants by somaclonal variation. Hopefh is learned about plant systems, ii: will become possibie to manage such interfering phenotypes. This should increase the chance of producing useful plants t,hr5ugb genetic engineering.

NING-SUN

YANG

Agracetus, 8520 Univerfity Gl;een, Middleton, WI 53562, USA.

Refkenceo

I l$ecdmbinant DNA Safe8 Coksideratiijns: Safe& Considerations for In&striab. kzgricultural (alid 1Entir5nme$tal &$&cations of Organisms Derived by Recombinant DNA Techniques

,'?K! 1iw111121'171 11-c:

.Issues t 1987) 1J:s I‘l;lticjl;al AZczth'-111.' (:. %%nct:s, ‘WashingtwL ’ 2 Frale y, Ei.( Horsch, K. , ?‘tl
Ymg, N-S. (1987)PlantPhysiol. 8!j, 1803-1109 F Ursic, D ~,Slightom, J.L. and Kemp, J.D. (1983) Mol. Gen. Genet. 190, 494-503 SODe BEoch, M., Herrera-Estrella, L..,

Van Kontzgu, M., Shell, J. and Zambryski, P. (1984) Elw30/. 3, 16811-1689 I I Potrykus, I., P&kowski, J . , Saul, M.W., Petmska, J. and Shito, RD. (1985) i&L Gen. Genet ‘699, 169-177 ia Fiihr, R., Kuhlemeier, C., Nagy, F. and Chua, N-M. (1986) Science 232, X06-1112 83 Riggs, C. and Bates, G. (1986) BYK NatLlAcad. Sci. USA 83,560~~5606 I4 Muiler, AJ., Mendei, RR., Schiemann, J., Simoens , C. and Inze, XI~ (198’7)Mol. Gen. &net. 207, 171-175; 15 Evans, C.A. and Sharp, W.R. (1986) ir Handbook ofPlant Cell Culture, Vol. 4 (Evans, D.A., Sharp, W.R. and

Ammirato, P.A., eds), pp. 97-132, Macmillan IB Barbier, M. and Dulieu, H. (1983) PlaantSci. Lett. 29,2Ol-206 97 Peschke, V.M.? Phillips, Gengenbach, B.G. (1987) Science 238, 804-807,