Einset, John W. : John W. Einset, Assistant Professor, Botany and Plant Sciences, Cell Interaction Group, U.C., Riverside.

0 specific sites on the cell walls . A circle of cells carryingthis hybrid plasmid are allowed J - Y DNA in the pathogen known as the Ti plas - to insert the chimeric T - DNA into tobacco , mid then mobilizes the transfer of a piece of petunia , cowpea , and sunflower cells by nat - DNA into the plant cell , where it becomes at - ural infection . Although such genes have tached to the plantâ??s nuclear DNA . The sub - been transferred to plants , these genes are not sequent expression of this implanted DNA , stably maintained , particularly during meio - the T - DNA , results in proliferation of a sis ( chromosome reduction , division , and tumor and production of unusual com - segregation ) ; however , the genes seem to be pounds known as opines , which are used by more stable in crop and ornamental plants A . tumefaciens as food sources . that are vegetatively propagated . Scientists have already demonstrated that As with cauliflower mosaic virus , there are this natural mechanism of DNA transfer can several limiting factors . The Ti plasmid car - as a vector for foreign genes : Ti be harnessed ries genes that cause tumors in plants , and it plasmids can be genetically engineered to will need to be â?? disarmed . â?쳌 Once disarmed , mobilize genes other than those normally an efficient means of selecting transformed transferred during the disease process . cells will have to be developed . Also , most The bacterium that causes crown gall in Unfortunately , no one has yet obtained clear - plants susceptibleto crown gall have not been potato and other plants has potential of desirable as a vector for transfer cut evidence for expression of these foreign regenerated successfullyfrom cell culture , an traits from one plant to another . genes in crown gall cells . For this reason , the of useful essential step in the development will be to develop methods for next step plants . Finally , stability of the T - DNA in gene can be spliced to the virus vector . Also , regeneration of crown gall cells into normal - transformed plants needs to be enhanced . the added gene affects the cell - to - cell move - appearing plants that retain and expressben - Other potential gene vectors being studied ment ( invasiveness ) of the virus , since the eficial foreign genes . are transposition elements and organelle virus can no longer mature . Moreover , the In our research , we have used crown galls DNA . Well - defined segments of DNA that virus is mainly limited to Cruciferae as host of tobacco plants to answer fundamental can insert themselves in random locations of infec - plants and therefore the prospects of the di - questions about the physiology along a chromosome are known as transposi - ting other major crops are narrow . Neverthe - sease . Two questions pertaining to crown gall tion elements ( or â?? transposons â?쳌 ) . Organelles less , in - depth studies on this virus will help of particular concern : ( 1 ) genetics have been such as chloroplasts and mitochondria carry provide important information on means to DNA with characteristics much like plas - Can T - DNA genes be retained and expressed overcome these natural obstacles . ( 2 ) What is the in structurallynormal tissues ? mids . The most promising aspect of these Another promising gene vector is the Ti hormonal basis for the abnormal growth as - studies is that of harnessing existing plant plasmid harbored in the crown gall bacterium sociated with crown gall ? DNAs as vehicles of genes that can be at - Agrobacteriumtumefaciens . Ti plasmids are tached to them . Experimentsrelated to thesequestionshave very large , autonomously replicating , circu - In all of these examplesof genevectors , the utilized crown gall tissue cultures maintained 30 timeslarger than lar DNA molecules about pressing problem has been to find a suitable on a semisolidnutrient medium consisting of the DNA of cauliflower mosaic virus . This gene capableof confemng the desirabletrait ( s ) mineral salts , vitamins , and sucrose . We plasmid carries genes that cause crown gall on a plant . To solve this problem further derived these bacteria - free tissue cultures tumors in many plants , representing more studies are required on gene maintenance in about four years ago from tumors on tobacco 90plant families.Although the mechan - than plants representing seven strains of the promising genevectors of A . plant cells . Two ism by which these tumors occur is not well tumefaciens . Because we conduct individual are reported in the articles that follow . understood , A . tumefaciens naturally intro - experiments on a variety of types of crown duces the Ti plasmid DNA into plant cells galls , we feel justified in making general con - of this plasmid , during infection . A portion clusions about the physiology of the disease . known as the T - DNA , which carries genes of tempera - Under appropriate conditions for synthesisof unusual amino acids ( opines ) ture , lighting , and hormone concentrations , and phosphorylated sugars , is incorporated unorganized crown galls can be regenerated The Ti plasmid into the nuclear DNA of the plant cell . Thus , into normal tobacco plantlets with discern - during infection of a wound , the crown gall ible leaves , stems , and roots . In most cases , John W . Einset bacterium â?? genetically colonizes â?쳌 the plant unfortunately , these plants seem to be free of by converting normal cells into tumor cells all tumor characteristics.If however , one uses that are directed to produce the opines and crown galls produced by a specific strain of c r o w n gall , a bacterial disease of dicots phosphorylated sugars , which are assimilated A . tumefaciens , one can regenerate plantlets and gymnosperms , is characterized by tu - by the infecting bacterium . as retaining crown gall characteristics , such A . tumefaciens morous overgrowths on infected plants . Be - With the knowledge that the nonrequirement for hormones ( auxinand cause the disease involves gene transfer from inserts a piece of its genetic material into cytokinin ) by explants in tissue cultures and plants , Davis plant pathologists and other a bacterium to a plant cell and subsequent ex - the ability to produce radioactive opines of the Ti of new characteristics , crown gall workers have isolated the portion pression from radioactively labeled arginine fed to the plasmid that is transferred into plant cells . has tremendous potential as a vector for ge - tissues . These observations demonstrate that the Foreign genes that confer resistanceto certain netic manipulation of important agricultural antibiotics , such as methotrixate and chlor - crops . abnormal growth of crown gallscan be over - come without affecting other tumor - borne amphenicol , and that direct the synthesis of Under natural conditions , Agrobacterium tumefacienscells in the soil enter plant tissues characteristics . They also give us confidence seed proteins have now been inserted in the T - DNA on the Ti plasmid . A . tumefaciens to introduce foreign that future attempts through wounds and attach themselves to CALIFORNIA AGRICULTURE , AUGUST 1982 15 Leaves from turnip plants infected genes via crown gallwill lead to plants that re - with cauliflower mosaic virus . Leaf tain and express new characteristics . In fact , on left is infected with native , similardemonstrations of T - DNA expression virus . Those on right are wild - type in normal plant tissueshave now been accom - from plants infected with mutants plished in at least five other laboratories by insertion of 12s produced additional nucleotide pair into worldwide . the chromosome of the same virus Because the abnormal growth of crown strain . Plants infected with the gall tissues is a major obstacle to crown - gall - mutant on the right grow almost mediated plant genetics , we have also investi - as rapidly as healthy plants . gated the unusual hormone metabolism associated with the disease to determine whether it is the cause of the abnormal growth . The fact that crown gall tissues differ from nontransformed plant tissues in being one ( gene 11 ) is not essential for reproduc - able to grow on a simple nutrient medium tion , enclothement in protein , or cell - to - cell DNA plant viruses lacking auxin and cytokinin suggests that movement . Recent work at Davis indicates crown galls produce these hormones at ele - that this nonessential gene is probably in - Robert J . Shepherd vated rates . Our own studies on cytokininsin volved in insect transmission of the virus in Stephen D . Daubert a variety of crown galls indicate that these nature . These dispensible regions of the virus C . Gardner Richard tissues generally overproduce cytokinins at chromosome provide sites for insertion of 8 - to 1,600 - fold greater levels ranging from foreign DNA , which is carried into the plant than normal . The predominant cytokinins in and replicated along with the DNA of the in - A remarkably simple genetic system for crown galls have been purified and identified fecting virus . study of DNA multiplication and gene ex - as zeatin and ribosylzeatin , which are Another region of the cauliflower mosaic pression in plants is provided by DNA plant N6 - substituted derivatives of adenine and virus chromosome has been identified as viruses . These viruseshave only a half - dozen adenosine , respectively . In addition , crown being responsible for the severity of disease . or sogenesthat are believed to be regulatedin galls with extremelyhigh total cytokinin con - This region is gene VI on the physical map . the same way as other plant genes . The DNA tents contain glucose derivatives of both of The other five genes appear to have little , if replicates in nuclei and may be associated these cytokinins . any , effect on symptom induction . A single with nuclear proteins ( histones ) in the same Presumably , the hormone imbalance re - a profound effect change in gene VI can have as plant genetic material . Thus , the virus way sultingfrom T - DNA expressionin crown gall on disease expression : in one case , insertion provides a small - scale , readily manipulated cells underlies the abnormal growth we ob - of 12 base pairs at a particular location in model for gene expression . serve . We have detected genes on the Ti plas - in - gene VI almost abolished disease . Plants Viruses reproduce in living tissue by sup - mid affecting cytokinin biosynthesis by the fected with this mutant of the virus show very plying a few of their own functions , while fill - C . I . Kado has shown that pathogen , and as mild symptoms and grow at the same rate ing most of their needs by parasitizing the plasmid genes for auxin biosynthesis also ex - healthy plants . Information of this sort may a smallloop host . Each viral particle contains ist . Identifyingthese genesand their products enable the investigator to control disease ex - of nucleic acid , the genetic component , en - is a first step toward controlling them . Ulti - of the viruschromosome pression , if portions clothed in an outer shell of coat protein . The mately , we would like to have effective Ti are eventually used as a recombinant DNA protein is shed soon after the particle enters plasmid vectors that introduce desirable vector for plants . the cell , and the DNA sets about reprogram - genes into plants without also making tumors . as Cauliflower mosaic virus has been used ming the cell to manufacture virus . In carry - a vehicle to reproduce foreign DNA in plant John W Einset , Assistant Professor , Botany and ing out these changes , the virus interferes cells and to carry this foreign DNA from cell Plant Sciences , Cell Interaction Group , U.C . , so that the cell with normal cellular functions Riverside . to cell throughout the entire plant . However , becomes less well coordinated . The organism not enough foreignDNA can be inserted into as a whole is affected and shows disease the virus chromosome to bring about useful symptoms . Reductions in growth rate and transformations of plants . This limitationap - leaf puckering and yellowing are common ef - pears to be related to a low capacity of the of the DNA viruses . fects virus particle to accommodate additional One DNA plant virus , the cauliflower mo - to DNA . Assemblyof DNA and coat protein saic virus , has received more attention than form virus particles seems to be necessary the others . Its biology has been intensively before the DNA will move from cell to cell in studied during the last few years , and the the plant . It does not appear , therefore , that DNA from two strains of this virus has been the virus in its present form will be useful as a completely sequenced . At Davis , for exam - recombinant DNA vector . However , this vi - ple , an isolate has been found to have 8,031 Small , ntergen , c rus will probably play an important role in reg > on pairs of nucleotides in its circular chromo - defining the biological activity of those se - some . Nucleotides make up the language Physical map of circular chromosome of quences involved in replication and expres - cauliflowermosaic virus . Genes defined by with which genetic information is expressed . sion of DNA in plants . by nucleotide sequence are indicated of nucleotides and other infor - This sequence arrowed lines I to VI . Box indicates naturally Robert J . Shepherd , Professor , and Stephen D . mation have been used to construct a physical occurring deletion of most of region I1 Daubert , Postdoctoral Research Associate , Plant map of the virus chromosome ( see diagram ) . ( strain CM4 - 184 ) . Open triangles indicate Pathology , U.C . , Davis ; and Richard C . Gard - ner , Principal Scientist , Calgene , Inc . , Davis . three single - stranded interruptions . Of the half - dozen genes of this simple virus , 16 CALIFORNIA AGRICULTURE , AUGUST 1982