Genes for Africa

Front matter

Genes

Genes for Africa Genetically modified crops in the developing world Jennifer A Thomson

Genes for Africa: Genetically modified crops in the developing world First published 2002 c UCT Press, 2002 PO Box 24309, Landsdowne 7779 This book is copyright under the Berne Convention. ln terms of the Copyright Act, No. 98 of 1978, no part of this book may be reproduced or transmitted in any form or by any means (including photocopying, recording, or by any information storage and retrieval system) without permission in writing from the publisher. lSBN 1 919 713573 Copy editing by Ally Ashwell Proofreading by Helena Reid Design and typesetting by Catherine Crookes Cover design by Catherine Crookes Printed and bound in South Africa by Mills Litho, Maitland, Cape Town Cover photographs c Keith Young/Cape Photo Library, cc Eric Miller/iAfrika The author and publisher have made every effort to obtain permission for and acknowledge the use of copyright material. Should any infringement of copyright have occurred, please contact the publisher and every effort will be made to rectify omissions or errors in the event of a reprint or new edition.

Chapter 1: Plant Breeding And Jumping Genes

Nematode: a round worm; a common plant parasite, found in huge numbers in soil.

These genetic improvements depend on cross-po1lination between plants with different characteristics. However, soybean flowers carry both the male and female organs. When the pollen is shed, it drops onto the female part of the same flower, resulting in self-pollination. Fortunately for plant breeders, the stigma (part of the female organ) is receptive to pollen at least one day before the anthers (the -ma le organs) are sufficiently developed to shed pollen. This lag time permits the breeder to introduce pollen from another soybean plant in order to obtain hybrid seed. Breeding soybeans is a tedious process. The flowers are only ab?ut 6 mm long and the plant breeder must use tweezers to transfer pollen from the anthers of the donor plant to the stigma of the recipient flower. One person can cross-pollinate about 50 to 150 flowers per day. Less than half of these are likely to set seed because many flowers abort after the crosses are made.

The role of back-crossing ln any plant breeding process, after the first hybrid offspring has been produced, it is crossed with the parent plant that is being improved. This technique is called back-crossing. The parent used in the back-cross is called the recurrent parent because it recurs, or is used repeatedly for crossing. The other parent is the donor parent because it contributes the desirable gene(s).

During hybridisation, the genes of the donor parent mix with those of the recurrent parent in a random fashion. The purpose of back-crossing is to recover any desirable genes from the recurrent parent that may have been lost in the formation of the original hybrid plant. Each back-cross recovers an additional 500/o of the genes of the recurrent parent, amounting to 750/o after the first back-cross and 870/o after the second. Breeders continue back-crossing until they have recovered the desirable level of genes from the recurrent parent. 1t usually takes three back-crosses to recover virtually all of the original genes. 8 - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Back-crossing: crossing hybrid offspring with the parent plant that is being improved.

Food for thought? l shall discuss questions around food safety of products derived through conventional breeding in Chapter 6 but they are already worth mentioning here. ln an attempt to breed a 'super-broccoli' with high concentrations of a compound known to lower the risk of cancer, plant breeders in the United Kingdom recently crossed normal broccoli with wild Sicilian broccoli (24 May 2000; http://www.agbioworld.org). ln this process many hundreds of genes from the wild were introduced into the new variety. We do not know what these genes are or whether they code for any allergenic or toxic compounds. Many plant traits required for surviva 1 in the wild may not necessarily be good for human health or agriculture. What are the long­ term health and environmental impacts of this broccoli? No one has ever addressed this question and no regulatory body has ever tested the effects of broccoli on food safety or on the environment. lf the same broccoli had been developed using biotechnology, a whole battery of tests would have been required. ln fact, it has often been claimed that, had many of our current food products been developed through biotechnology, they would never have been accepted as they would not have made it over the regulatory hurdles. This applies to potatoes, which contain a class of toxins called solanine that can be toxic to humans if consumed at high levels. Soybeans and other legumes contain proteins called protease inhibitors, which inhibit enzymes that degrade other proteins. Prior to human consumption, these products ,, must be subjected to processing at high temperatures to inactivate these proteins. Would such food products and food processing requirements have been acceptable had they been introduced by biotechnology? This is indeed food for thought! biotechnology: the exploitation of biological processes to produce commercially valuable products and processes.

CHAPTER 1: PLANT BREEDING AND JUMPING GENES

Mutations - speeding up the process What 1 have described above is the crossing of two plants that, by chance, had developed the characteristic traits that the breeder was looking for. However, breeders can speed up the process by treating plants with agents that can introduce changes (mutations) into the DNA. These mutations occur randomly but at a greater rate than normal changes in the DNA, which happen by chance.

The breeder subjects the plant or seeds to agents that cause mutations (mutagens) and then selects the plants that express the desired traits, such as increased yield. Mutagens include chemicals and radiation, such as ultra­ violet light a.nd X-rays. Thus, the reason why you should avoid excessive exposure to sunlight is that the ultraviolet rays in sunlight can mutate your DNA and this may result in skin cancer.

Good examples of the use of mutagenesis in plant breeding have been the development of plants for improved oil qualities. Plant breeders mutated sunflowers to produce high levels of the nutritionally desirable oleic acid, and flax and canola to have low levels of the nutritionally undesirable linolenic acid (Downey and Robbelen, 1989; Green and Marshall, 1984). There are many other examples of the successful use of mutagenesis in plant breeding: ■ A mutation introduced herbicide resistance into a single soybean cultivar. Traditional crop breeding methods were then used to increase the number of cultivars resistant to herbicides (lAEA, 1995). ■ ln the Netherlands, irradiation of chrysanthemums introduced a range of new floral colours that quickly replaced previous cultivars (Van Harten, 1998}. ■ Mutant peanuts containing relatively high levels of monounsaturated oils that promote cardiovascular health are now being grown on a large scale. These peanuts contain 900/o of healthy oleic acid and only 50/o of linoleic acid, a type of fat associated with cardiovascular disease. Naturally occurring peanuts contain about 500/o oleic acid and 400/o linoleic acid (23 April 2000; http://www.agbioworld.org). A 1995 database of the international Food and Agricultural Organisation lists 1 790 varieties of 158 plant species developed through the use of purposely induced mutations. These have been officially released in 52 countries. None of these crops has been subjected to the intense levels of scrutiny that GM crops in commercial use have undergone. When breeders induce mutations they are likely to discover useful traits that do not exist in natural populations. However, even with the help of mutagens, mutation remains a random process that is limited in terms of the type of traits that can be created. Muta genesis thus rarely produces the particular desired characteristics.

DNA: deoxyribonucleic acid, the complex molecule that makes up genes and chromosomes and stores genetic information. Mutation: an inheritable change in DNA; in other words, the change gets passed on from one generation to the next.

Mutagen: an agent that causes mutations.

Conventional breeding can produce toxins Many wild plants contain toxic compounds that have evolved, for example, as defence mechanisms against predators. Through the ages, a major goal of plant breeding has been to reduce or eliminate these toxins. The wild potato (Solanum acaule) is the progenitor of cultivated varieties. lt contains a potentially toxic compound at concentrations about three times that of cultivated strains. Similarly, the wild cabbage (Brassica oleracea) is the progenitor of cabbage, broccoli, and cauliflower. lts leaves contain about twice the level of potential toxins compared to cultivated cabbage. Similar reductions in toxicity through plant breeding have been reported in lettuce, lima bean, mango and cassava. ln contrast, there are several well-documented examples where plant breeding has inadvertently introduced higher levels of toxins. ln order to reduce the use of synthetic chemical pesticides, plant breeders have developed plants that are more resistant to insects. However, these plants often contain high levels of natural toxins, some of which they use to repel insects. A new variety of highly insect-resistant celery was introduced in CHAPTER 1: PLANT BREEDING AND JUMPING GENES .11

America. Centers for Disease Control from a 11 over the country received a flurry of complaints because people who handled the celery developed rashes and burns when their skin was subsequently exposed to sunlight. Some detective work found that the pest-resistant celery contained 6 200 parts per billion {ppb) of carcinogenic and mutagenic substances called psoralens, instead of the 800 ppb present in normal celery (Ames et al., 1990). ln another case, a new potato, developed at a cost of millions of dollars, had to be withdrawn from the market because of its acute toxicity to humans. This was due to two natural toxins, solanin and chaconine, that block nerve transmission (ibid.). Many such toxic plant varieties never come to market as they are detected by the quality control procedures of the seed companies. As mentioned above, stricter approval procedures for crops produced by genetic modification significantly reduce the likelihood of such mishaps (Butler and Reichhardt, 1999).

Carcinogen: a compound that can cause cancer.

Chaconine: a plant compound that blocks nerve transmission. Psoralen: a compound found in many plants that can make humans and animals more sensitive to exposure to light. Solanin: a plant compound that blocks nerve transmission. Jumping genes One characteristic that has assisted plant breeders, although they did not know it until fairly recently, is the fact that DNA is inherently 'plastic'; in other words, DNA is not a rigid molecule but it can undergo considerable rearrangements. Barbara McClintock, a maize geneticist, first described such rearrangements in the 1950s (McClintock, 1951). She noticed something odd going on with her maize plants. Sometimes the patterns of pigmented spots in the seeds of the offspring differed from those in either of their - - - - - - - - - - - - - - - - - - - - - - - - - -- - parents. These changes could not be explained by classical Mendelian genetics. Moreover, these patterns could change back again in subsequent generations.

The cause of these changes are elements of DNA that can 'jump' around the chromosomes. McClintock called them 'transposable elements' or 'transposons' but we can simply call them 'jumping genes' . . Gregor Mendel (1822-1884) developed the principles of heredity based v: . 'On his research or:i peas. He used his findings to explain the behaviour of , factors1, now called genes, from generation to generation. ln today's errns, Mendel's first law states that cells, except for eggs and sperm, contain two copies of each gene. Eggs and sperm have only one copy an<;i ·therefore need to fuse during fertilisation to produce an offspring. enoel'.� second law states that pairs of genes' are located on pairs of _ rornosomes that occur independently in the cell. Mendelian genetics: laws of heredity proposed by the Austrian monk, Gregor Mendel, in the nineteenth century Chromosome: a structure composed of a very long molecule of DNA that carries hereditary information. Humans have 23 pairs of chromosomes.

Transposon Oun1ping gene): a mobile genetic element, consisting of a gene or series of genes, that can insert itself at random into chromosomes.

Unfortunately, McClintock's theory of transposons was rejected by scientists of the day. Eveylen Fox Keller (1983) explains the reasons for this in a spellbinding biography entitled A feeling for the organism. That 'feeling' was McClintock's greatest strength, but unfortunately for the acceptance of her theory, also her greatest weakness. Other scientists just did not share McClintock's feeling for her maize. Because they could not understand the complexity of the system she described, they chose to reject CHAPTER 1: PLANT BREEDING AND JUMPING GENES her theory out of hand. 1t was only when jumping genes were discovered in bacteria, a much simpler system, that scientists realised that she had been correct all along. Barbara Mcclintock received the Nobel Prize for this discovery in 1983.

Jumping genes certainly increase the variability of DNA in an organism. We call this the plasticity of an organism's genome. 1t provides breeders increased diversity with which to work. lt was this unfettered jumping around of genes that caused another Nobel Laureate, Werner Arber, to wonder aloud whether the bacteria we work with today are the same as the ones·we worked with yesterday (W. Arber, personal communication, 1978). Fortunately, nature has evolved mechanisms of 'damping down' jumping genes so that they do not create havoc in the genome. Readers should be aware that the random jumping around of pieces of DNA is a natural process. People opposed to the use of GM crops should note this before they condemn scientists for randomly inserting DNA into plants. The same process is happening constantly all around us in nature. Genome: the genetic composition of an organism.

References and further reading Ames, B. N., Profet, M. and Gold, L. S. (1990) Nature's chemicals and synthetic chemicals: comparative toxicology. Proceedings of the National Academy of Sciences, USA. Vol. 87, pp. 7782-7786.

Butler, D. and Reichhardt, T. (1999) Long-term effect of GM crops serves up food for thought. Nature. Vol. 398, pp. 651-656.

Downey, R. K. and Robbelen, G. (1989) Brassica species. In: Robbelen, G., Downey, R. K. and Ashri, A. (eds) Oil crops of the world. McGraw-Hill, New York, pp. 339-362.

Gianessi, L. P. and Carpenter, J. E. (2000) Agricultural biotechnology: benefits of transgenic soybeans. Report for National Center for Food and Agricultural Policy (ncfap@ncfap.org).

Green, A. G. and Marshall, D. R. (1984) Isolation of induced mutants in linseed (Unum usitatissimum) having reduced linolenic acid content. Euphytica. Vol. 33, pp. 321-328.

IAEA. (1995) Induced mutations and molecular techniques for crop improvement: proceedings of a symposium, Vienna, 19-23 June 1995. International Atomic Energy Agency, Vienna.

Keller, E. F. (1983) A feeling for the organism. W. H. Freeman and Company, New York.

McClintock, B. (1951) Chromosome organization and genie expression. Cold Spring Harbor Symposium on Quantitative Biology. Vol. 16, p. 40. Paabo, S. (1999) Neolithic genetic engineering. Nature. Vol. 398, pp. 194-195. Van Harten, A. M. (1998) Mutation breeding: theory and practical applications. Cambridge University Press, Cambridge.

Van Rensburg, J. B. J. (undated) The history of maize. Summer Grain Centre, Potchefstroom.

CHAPTER 1: PLANT BREEDING AND JUMPING GENES

Chapter 2 What is genetic mod,ification of plants?

Genetic engineering - the basics As discussed in the previous chapter, conventional plant breeding has been practis�{?r centuries and has succeeded in producing a wide variety of commerci<1f pfants and crops with a range of important agricultural traits. lt has succeeded in converting a- Mexican grass into maize and a Middle Eastern grass into wheat. However, it is to a large extent a hit-or-miss process, combining large parts of plant genomes in a rather uncontrolled fashion. Furthermore, the rate of increase in crop yields due to conventional breeding is slowing down. We need additional options to keep pace with growing demands for food. Genetic engineering allows scientists to transfer well-characterised and specific genes into plants, resulting in the intro­ duction of one or more defined traits into a particular genetic background. This process is called 'plant transformation' and the genes involved are expressed to produce proteins responsible for the particular trait. An added advantage is that the transferred gene(s), or transgene(s), can come from Gene: the biologjcal unit of inheritance that transmits hereditary information and controls the appearance of a physical, behavioural or biochemical trait It is composed of DNA and consists of a series of bases comprising its genetic code. %. :�'<:. 1'91nism: an individual plant, animal or micro-organism (e.g. a bacterium) that can independently carry out life functions. 16 - � - - - - - - - - - - - - - - - - - - - - - - - - - -

Protein: a molecule consisting of amino acids. The sequence of the amino acids is encoded in the gene for that particular protein. Proteins can be enzymes (biological catalysts) or structural proteins (keratin that forms the basis of hair and feathers is a structural protein).

Transgene: a gene transferred from one organism to another by genetic engineering.

Input and output traits Most traits transformed into plants to date involve herbicide tolerance and resistance to viral, bacterial and fungal diseases, as well as resistance to herbivorous insects. These so-called input traits are characteristics that improve the productivity of a crop and decrease dependency on chemical pesticides and herbicides. They are therefore mainly of benefit to seed companies and farmers. However, the environment also benefits because of the decrease in the use of pesticides. These traits will be discussed further in Chapter 3. The second generation or output traits include improvements to the quality of foods, delayed ripening of fruit and vegetables, and the production of vitamin A in rice. They have a more direct benefit to consumers and will be discussed in greater detail in Chapter 4. This chapter explains how these input and output traits are introduced into plants. 1t will cover the basic details of how transgenic plants (plants carrying introduced genes) are made.

Input trait: a characteristic that improves the productivity of a crop and decreases dependency on inputs like chemical pesticides and herbicides.

Output trait: an improvement in the quality of the food or crop produced that is of benefit to consumers.

Transgenic plant: a plant into which a gene from another species of plant, or another organism (e.g. a bacterium), has been transferred. CHAPTER 2: WHAT IS GENETIC MODIFICATION OF PLANTS?

At present most transgenic plants contain only one or a few transgenes as the traits involved usually depend on the expression of a single gene. However, in future, genetic engineering of plants is likely to involve traits encoded by a number of different genes. This will require more complicated genetic engineering techniques but the basic principles outlined in this chapter will still hold.

DNA, genes and their functions Before explaining how new genes are inserted into plants, it is probably a good idea to cover some basic facts about DNA, genes and how they function in an organism. James Watson and Francis Crick discovered the structure of DNA in 1953. The story is told in a fascinating book by James Watson called The double helix (Watson, 1968). The structure of the DNA molecule looks like a double helix: two chains wrapped around each other rather like a spiral staircase (see Figure 2 in the colour section on page 117). The two strands of DNA are held together by base pairing. The steps of the DNA staircase consist of four chemical bases: adenine, thymine, guanine and cytosine. These bases are arranged in various permu­ tations that constitute the genetic code. This is an alphabet which, despite relying on only four letters, delivers messages to the cell that enable it to behave in different ways.

Base: DNA has four chemicals called bases that form the 'rungs' of the ladder of the double helix: adenine, thymine, cytosine and guanine. The genetic code is determined by the sequence of the bases.

Base pairing: the links between the bases on the matching 'rungs' of the double helix. In DNA, adenine pairs with thymine and cytosine with guanine. It is this base pairing that holds the two strands of DNA together, forming the double helix. Genetic code: the sequences of bases on a DNA molecule. - - - - - - - - - - - - - - - - - - - - - - - - - - - -

A gene is a sequence of DNA that has a start, a middle and an end. Genes carry the coded information that enables the cell to make proteins. Proteins include both structural proteins like the keratin in hair and skin, and enzymes that control the biochemical processes in the cell. Protein synthesis is the process by which a cell manufactures the proteins it requires. During protein synthesis, an enzyme first recognises the start of the gene coding for the required protein. The enzyme then makes a copy of one strand of the DNA in that gene. This copy is in the form of a very similar molecule called RNA, which, unlike DNA, is a single strand. This process of rewriting the DNA into a different form is called transcription. The RNA molecule is called messenger RNA (mRNA) as it carries a message from the DNA to the rest of the cell. ln the next stage, the cell 'reads' the genetic code in the mRNA and produces a protein made up of a sequence of units called amino acids. This process is called translation as it translates the code into its final form. The term gene expression is used to describe the overall process whereby information present in DNA is converted into a protein. The processes of transcription and translation are shown diagrammatically in Figure 3 on the next page.

Amino acid: the basic unit of a protein. There are 20 different kinds of amino acids. Proteins consist of chains of amino acids in the order laid down by the gene that codes for that protein. Enzyme: a protein that acts as a catalyst to enable a biochemical reaction to take place. The enzyme itself is not changed in the process.

Gene expression: the process whereby a gene is transcribed into mRNA and then translated into a protein.

RNA: ribonucleic acid. It is a molecule synthesised from DNA in a process called transcription.

Transcription: the synthesis of RNA from DNA. ___ __ ____________ - - - - - - - - - - - · 19 CHAPTER 2: WHAT IS GENETIC MODIFICATION OF PLANTS?

Translation: the process by which a particular sequence of bases in messenger RNA (mRNA) determines the sequence of amino acids in a protein.

Information transfer Start J,C" Gene Stop �..;.--�--�J - - - ,.

- --\__/-::. _- . - - -- - - -- - - -- --- - Transcription mRNA (messenger) ·-· 'f" Translation Unfolded protein .___ _____________ __, Functional membrane enzyme (protein) Protein folding FIGURE 3: Gene expression is the process whereby information in DNA is transcribed into messenger RNA. from which it is translated into a protein. The gene is said to code for the protein.

DNA is generally found in the nuclei of plant and animal ce1ls, where it forms strands known as chromosomes. A gene is a segment of DNA on a chromosome. ln the laboratory, enzymes can be used to cut particular genes out of chromosomes. The excised gene can then be spliced into another piece of DNA. ln this way genes can be transferred into plants in a process ca11ed plant transformation.

Plant transformation: the process whereby any gene can be introduced into a plant. Such a plant is then known as a transgenic plant.

Plant transformation Plant transformation is possible because plant cells are ·totipotent. This means that a single, isolated plant cell can grow into an entire.Jy new plant; for example, if a single cell of a carrot is put into tissue culture, a whole new carrot plant can regenerate, or grow out of it. Thus if a gene is trans­ ferred into this isolated plant cell, every cell of the regenerated plant will contain that gene.

This is, however, not true of animals or humans. You cannot take a finger cell and grow a whole person from it! This is because animals are not genetically capable of being totipotent. One way to think about this is to imagine that a cell from an adult plant has a 'genetic memory' of when it was first formed at fertilisation. Such a cell, when placed in tissue culture, 'remembers' what it was like to develop into an adult plant and does so. Figure 4 (see page 118 in the colour section) shows shoots and roots devel­ oping from single cells in tissue culture. ln practice, before the required gene is introduced into the plant DNA, it is spliced next to a 'marker gene' that codes for resistance to a herbicide or to an antibiotic. The plant receiving the genes is naturally sensitive to the particular herbicide or antibiotic. Thus only transformed cells that contain and express the genes for herbicide or antibiotic resistance will be able to grow on the tissue culture medium. The use of antibiotic resistance genes in plant genetic engineering has been the subject of considerable controversy and will be discussed in Chapter 5.

Totipotent: the ability of a single cell to form a complete organism. A single plant cell can develop into an adult plant because its cells are totipotent.

Herbicide: a chemical compound that kills weeds. Antibiotic: a substance isolated from a micro-organism that destroys or inhibits the growth of other micro-organisms, especially disease-causing bacteria and fungi. Many antibiotics are made synthetically.

CHAPTER 2: WHAT IS GENETIC MODIFICATION OF PLANTS?

Transformed plant cells are placed on a gel-like medium containing the selected herbicide or antibiotic. They divide into an undifferentiated mass of cells called callus, but only callus expressing the genes for resistance to the herbicide or antibiotic will grow. Plant hormones such as auxin and cytokynin are used to trigger plants to develop from the transformed callus. Once plants are fully developed they are taken out of the medium and planted in soil for 'hardening off'. The process is shown in Figure 5 below. Antibiotic resistance gene: a gene coding for resistance to an antibiotic. Such genes are often used in the production of transgenic plants as transformed plants will grow in the presence of the antibiotic and non-transformed ones will be killed. Callus: a mass of relatively unspecialised tissue used in plant tissue -culture as the starting material for the propagation of plant clones. A clone is a group of genetically identical cells or organisms. Selection i Hormones Transform t withDNA--1 Plant cells Transgenic plant Mature plant FIGURE 5: The process of plant transformation.

Plant material is transformed with DNA carrying the gene of interest and a selectable marker. Callus tissue develops on selective tissue culture media. Shoots develop following the addition of plant growth hormones.

Withdrawal of these hormones promotes root development.

Monocot and dicot plants Flowering plants can be divided into two groups: dicot yledonous plants (dicots} and monocotyledonous plants (monocots}. Dicots are plants that develop from two (di} cotyledons in the seed. They can be recognised by the branching veins in their leaves. Commercially valuable dicots include many horticultural plants such as petunias, and crops like tobacco, tomatoes, cotton, soybeans and potatoes. Monocots develop from a single (mono} cotyledon in the seed and can be recognised by the parallel veins in their leaves. Most of the world's crops are grains, which are all monocots, including maize, wheat, rye and sorghum. Monocots and dicots are trans­ formed by different processes.

Dicotyledonous plants (dicots): plants that develop from two (di) cotyledons in the seed. They can be recognised by the branching veins in their leaves. Examples are tomatoes and petunias. Monocotyledonous plants (monocots): plants that develop from a single (mono) cotyledon in the seed. They can be recognised by the parallel veins in their leaves. Examples are maize and wheat.

Transformation of dicotyledonous plants Agrobacterium tumefaciens is a soil bacterium that may be harmful to dicots. 1t makes a tumour on plants it infects (tumefaciens is derived from the Latin for 'swollen'). Because these tumours are often found in the crown region of the root, the disease is called Crown Gall. Scientists were amazed to discover that when these bacteria infect a plant, they transfer part of their DNA into the plant cells where it becomes integrated into the plant's genetic material. Not long after this discovery, scientists realised that the introduction of a foreign gene into A. tumefaciens DNA would enable its transfer to the plant cell nucleus. This led to the development of dicot plant transformation.

Bacterium: a small, single-celled organism lacking a nucleus. - - - - - - - - - - - - - - - - - - - - - - - - - - - - 23 CHAPTER 2: WHAT IS GENETIC MODIFICATION OF PLANTS?

The process of dicot plant transformation takes three to four months and involves the following steps: ■ Cut small discs from the leaves of the plant and place them in dishes containing a liquid medium. ■ lnoculate the surface of the leaf discs with A. tumefaciens bacteria containing the gene to be transferred to the plant and incubate for two to three days. ■ Transfer the leaf discs to selection medium containing the herbicide or antibiotic of choice. ■ Transformation occurs along the cut edges of the discs where a mass of undifferentiated cells called callus tissue forms. ■ Tra_nsfer the callus tissue to a different medium, also containing the antibiotic or herbicide, which allows only resistant transgenic plants to develop. ■ Add growth hormones to promote the growth of a plant from the callus tissue. ■ Withdraw the hormones to promote root development. We know little about how these bacterial genes are integrated into the DNA of plant cells. lt appears to be a random process and much research is being done to understand how it works. Scientists are also investigating ways of integrating genes at specific target sites in the plant DNA. Examples of transformed dicot plants include: ■ petunias producing a range of attractive flower colors and patterns; ■ tomatoes with delayed ripening; ■ cotton resistant to insect pests and herbicides; ■ soybeans with improved oil quality and tolerance towards herbicides; and ■ potatoes resistant to viruses and insects.