0 Y-J cells carrying this hybrid plasmid are allowed specific sites on the cell walls. A circle of to insert the chimeric T-DNA into tobacco, DNA in the pathogen known as the Ti plas- petunia, cowpea, and sunflower cells by nat- mid then mobilizes the transfer of a piece of ural infection. Although such genes have DNA into the plant cell, where it becomes at- been transferred to plants, these genes are not tached to the plant’s nuclear DNA. The sub- stably maintained, particularly during meio- sequent expression of this implanted DNA, sis (chromosome reduction, division, and the T-DNA, results in proliferation of a segregation); however, the genes seem to be tumor and production of unusual com- more stable in crop and ornamental plants pounds known as opines, which are used by that are vegetatively propagated. A. tumefaciens as food sources. As with cauliflower mosaic virus, there are Scientists have already demonstrated that several limiting factors. The Ti plasmid car- this natural mechanism of DNA transfer can ries genes that cause tumors in plants, and it be harnessed as a vector for foreign genes: Ti will need to be “disarmed.” Once disarmed, plasmids can be genetically engineered to an efficient means of selecting transformed mobilize genes other than those normally The bacterium that causes crown gall in cells will have to be developed. Also, most transferred during the disease process. potato and other plants has potential plants susceptible to crown gall have not been Unfortunately, no one has yet obtained clear- as a vector for transfer of desirable regenerated successfully from cell culture, an cut evidence for expression of these foreign traits from one plant to another. essential step in the development of useful genes in crown gall cells. For this reason, the plants. Finally, stability of the T-DNA in next step will be to develop methods for gene can be spliced to the virus vector. Also, transformed plants needs to be enhanced. regeneration of crown gall cells into normal- the added gene affects the cell-to-cell move- Other potential gene vectors being studied appearing plants that retain and express ben- ment (invasiveness) of the virus, since the are transposition elements and organelle eficial foreign genes. virus can no longer mature. Moreover, the DNA. Well-defined segments of DNA that In our research, we have used crown galls virus is mainly limited to Cruciferae as host can insert themselves in random locations of tobacco plants to answer fundamental plants and therefore the prospects of infec- along a chromosome are known as transposi- questions about the physiology of the di- ting other major crops are narrow. Neverthe- tion elements (or “transposons”). Organelles sease. Two questions pertaining to crown gall less, in-depth studies on this virus will help such as chloroplasts and mitochondria carry genetics have been of particular concern: (1) provide important information on means to DNA with characteristics much like plas- Can T-DNA genes be retained and expressed overcome these natural obstacles. mids. The most promising aspect of these in structurally normal tissues? (2) What is the Another promising gene vector is the Ti studies is that of harnessing existing plant hormonal basis for the abnormal growth as- plasmid harbored in the crown gall bacterium DNAs as vehicles of genes that can be at- sociated with crown gall? Agrobacterium tumefaciens. Ti plasmids are tached to them. Experiments related to these questions have very large, autonomously replicating, circu- In all of these examples of gene vectors, the utilized crown gall tissue cultures maintained lar DNA molecules about 30 times larger than pressing problem has been to find a suitable on a semisolid nutrient medium consisting of the DNA of cauliflower mosaic virus. This gene capable of confemng the desirable trait(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 than 90 plant families. Although the mechan- plant cells. Two of the promising gene vectors plants representing seven strains of A. ism by which these tumors occur is not well are reported in the articles that follow. tumefaciens. Because we conduct individual 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- during infection. A portion of this plasmid, clusions about the physiology of the disease. known as the T-DNA, which carries genes Under appropriate conditions of tempera- for synthesis of unusual amino acids (opines) ture, lighting, and hormone concentrations, and phosphorylated sugars, is incorporated unorganized crown galls can be regenerated into the nuclear DNA of the plant cell. Thus, The Ti plasmid into normal tobacco plantlets with discern- during infection of a wound, the crown gall ible leaves, stems, and roots. In most cases, bacterium “genetically colonizes” the plant John W. Einset 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 phosphorylated sugars, which are assimilated crown gall, a bacterial disease of dicots A. tumefaciens, one can regenerate plantlets by the infecting bacterium. and gymnosperms, is characterized by tu- retaining crown gall characteristics, such as With the knowledge that A. tumefaciens morous overgrowths on infected plants. Be- the nonrequirement for hormones (auxin and inserts a piece of its genetic material into cause the disease involves gene transfer from 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 workers have isolated the portion of the Ti pression of new characteristics, crown gall from radioactively labeled arginine fed to the plasmid that is transferred into plant cells. has tremendous potential as a vector for ge- tissues. Foreign genes that confer resistance to certain netic manipulation of important agricultural These observations demonstrate that the antibiotics, such as methotrixate and chlor- crops. abnormal growth of crown galls can be over- amphenicol, and that direct the synthesis of Under natural conditions, Agrobacterium come without affecting other tumor-borne seed proteins have now been inserted in the tumefaciens cells in the soil enter plant tissues characteristics. They also give us confidence T-DNA on the Ti plasmid. A. tumefaciens through wounds and attach themselves to that future attempts to introduce foreign

CALIFORNIA AGRICULTURE, AUGUST 1982 15 genes via crown gall will lead to plants that re- Leaves from turnip plants infected tain and express new characteristics. In fact, with cauliflower mosaic virus. Leaf on left is infected with native, similar demonstrations of T-DNA expression wild-type virus. Those on right are in normal plant tissues have now been accom- from plants infected with mutants plished in at least five other laboratories produced by insertion of 12 worldwide. additional nucleotide pairs into Because the abnormal growth of crown the chromosome of the same virus 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 DNA plant viruses tion, enclothement in protein, or cell-to-cell 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 cytokinins in 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 tissues generally overproduce cytokinins at Richard C. Gardner chromosome provide sites for insertion of levels ranging from 8- to 1,600-fold greater foreign DNA, which is carried into the plant than normal. The predominant cytokinins in and replicated along with the DNA of the in- crown galls have been purified and identified A remarkably simple genetic system for fecting virus. as zeatin and ribosylzeatin, which are study of DNA multiplication and gene ex- Another region of the cauliflower mosaic N6-substituted derivatives of adenine and pression in plants is provided by DNA plant virus chromosome has been identified as adenosine, respectively. In addition, crown viruses. These viruses have only a half-dozen being responsible for the severity of disease. galls with extremely high total cytokinin con- or so genes that are believed to be regulated in This region is gene VI on the physical map. tents contain glucose derivatives of both of the same way as other plant genes. The DNA The other five genes appear to have little, if these cytokinins. replicates in nuclei and may be associated any, effect on symptom induction. A single Presumably, the hormone imbalance re- with nuclear proteins (histones) in the same change in gene VI can have a profound effect sulting from T-DNA expression in crown gall way as plant genetic material. Thus, the virus on disease expression: in one case, insertion cells underlies the abnormal growth we ob- provides a small-scale, readily manipulated of 12 base pairs at a particular location in serve. We have detected genes on the Ti plas- model for gene expression. gene VI almost abolished disease. Plants in- mid affecting cytokinin biosynthesis by the Viruses reproduce in living tissue by sup- fected with this mutant of the virus show very pathogen, and C. I. Kado has shown that plying a few of their own functions, while fill- mild symptoms and grow at the same rate as plasmid genes for auxin biosynthesis also ex- ing most of their needs by parasitizing the healthy plants. Information of this sort may ist. Identifying these genes and their products host. Each viral particle contains a small loop enable the investigator to control disease ex- is a first step toward controlling them. Ulti- of nucleic acid, the genetic component, en- pression, if portions of the virus chromosome mately, we would like to have effective Ti clothed in an outer shell of coat protein. The are eventually used as a recombinant DNA plasmid vectors that introduce desirable protein is shed soon after the particle enters vector for plants. genes into plants without also making tumors. the cell, and the DNA sets about reprogram- Cauliflower mosaic virus has been used as ming the cell to manufacture virus. In carry- a vehicle to reproduce foreign DNA in plant John W Einset, Assistant Professor, Botany and Plant Sciences, Cell Interaction Group, U.C., ing out these changes, the virus interferes cells and to carry this foreign DNA from cell Riverside. with normal cellular functions so that the cell to cell throughout the entire plant. However, becomes less well coordinated. The organism not enough foreign DNA 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 limitation ap- leaf puckering and yellowing are common ef- pears to be related to a low capacity of the fects of the DNA viruses. virus particle to accommodate additional One DNA plant virus, the cauliflower mo- DNA. Assembly of DNA and coat protein to 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- Small ,ntergen,c ple, an isolate has been found to have 8,031 rus will probably play an important role in reg>on pairs of nucleotides in its circular chromo- defining the biological activity of those se- Physical map of circular chromosome of some. Nucleotides make up the language quences involved in replication and expres- cauliflower mosaic virus. Genes defined by with which genetic information is expressed. sion of DNA in plants. nucleotide sequence are indicated by This sequence of nucleotides and other infor- arrowed lines I to VI. Box indicates naturally mation have been used to construct a physical Robert J. Shepherd, Professor, and Stephen D. occurring deletion of most of region I1 Dau bert, Postdoctoral Research Associate, Plant (strain CM4-184). Open triangles indicate map of the virus chromosome (see diagram). Pathology, U.C., Davis; and Richard C. Gard- three single-stranded interruptions. Of the half-dozen genes of this simple virus, ner, Principal Scientist, Calgene, Inc., Davis.

16 CALIFORNIA AGRICULTURE, AUGUST 1982