it is a long way to gm agriculture - univ- · pdf file · 2011-09-08it is a long...

PP62-FrontMatter ARI 20 April 2011 14:6

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PP62CH01-vanMontagu ARI 4 April 2011 16:10

It Is a Long Way toGM AgricultureMarc Van MontaguInstitute of Plant Biotechnology for Developing Countries, Department of PlantBiotechnology and Genetics, Ghent University, Ghent 9000, Belgium;email: [email protected]

Annu. Rev. Plant Biol. 2011. 62:1–23

First published online as a Review in Advance onFebruary 11, 2011

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev-arplant-042110-103906

Copyright c© 2011 by Annual Reviews.All rights reserved

1543-5008/11/0602-0001$20.00

Keywords

Agrobacterium, crown gall, Ti plasmid, T-DNA, opines, vir genes,T-DNA binary vector, plant genetic engineering, plant geneticmodification, GM plants, GMO

Abstract

When we discovered that crown gall induction on plants by Agrobac-terium tumefaciens is a natural event of genetic engineering, we were con-vinced that this was the dawn of a new era for plant science. Now, morethan 30 years later, I remain overawed by how far and how rapidly weprogressed with our knowledge of the molecular basis of plant growth,development, stress resistance, flowering, and ecological adaptation,thanks to the gene engineering technology. I am impressed, but alsofrustrated by the difficulties of applying this knowledge to improve cropsand globally develop a sustainable and improved high-yielding agricul-ture. Now that gene engineering has become so efficient, I had hopedthat thousands of teams, all over the world, would work on improvingour major food crops, help domesticate new ones, and succeed in dou-bling or tripling biomass yields in industrial crops. We live in a worldwhere more than a billion people are hungry or starving, while the lastareas of tropical forest and wild nature are disappearing. We urgentlyneed a better supply of raw material for our chemical industry becausepetroleum-based products pollute the environment and are limited insupply. Why could this new technology not bring the solutions to thesechallenges? Why has this not happened yet; what did we do wrong?

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Contents

THE EARLY YEARS . . . . . . . . . . . . . . . . 2PRIMARY AND HIGH SCHOOL . . . 3STATE UNIVERSITY GHENT . . . . . 4THE FASCINATION FOR

RESEARCH . . . . . . . . . . . . . . . . . . . . . . 5RNA PHAGES . . . . . . . . . . . . . . . . . . . . . . . 7PLANT TUMORS . . . . . . . . . . . . . . . . . . . 9THE GREAT SHIFT: DISCOVERY

OF TI PLASMID AND T-DNA . . . 9Functional Mapping of the

Ti Plasmid . . . . . . . . . . . . . . . . . . . . . 10THE WAY TO PLANT

TRANSFORMATION . . . . . . . . . . . . 11Plant Gene Vectors . . . . . . . . . . . . . . . . 11Selection and Regeneration

of Transformed Plants . . . . . . . . . . 12The First Genetically

Modified Plants . . . . . . . . . . . . . . . . . 12THE IMPACT OF

THE DISCOVERY . . . . . . . . . . . . . . . 12THE INNOVATION

CHALLENGE . . . . . . . . . . . . . . . . . . . . 14ENTREPRENEURSHIP. . . . . . . . . . . . . 14GENETICALLY MODIFIED

AGRICULTURE TODAYAND BEYOND . . . . . . . . . . . . . . . . . . . 15

GENETICALLY MODIFIEDORGANISM POLEMIC . . . . . . . . . . 16

BIOTECHNOLOGY FORDEVELOPMENT . . . . . . . . . . . . . . . . 18Institute of Plant Biotechnology for

Developing Countries (IPBO) . . . 18International Industrial

Biotechnology Network (IIBN) . . 18FINAL THOUGHTS . . . . . . . . . . . . . . . . 19

THE EARLY YEARS

Born in 1933 in Ghent, Belgium, in a periodof great economic recession and raised in aworking-class neighborhood, I was an unlikelycandidate ever to do higher studies, let alonemake contributions of importance to society.

My mother died during my delivery. Deathof either the mother or the newborn was verycommon in those days. My mother was the onlysurvivor of my grandmother’s nine pregnan-cies. For the first three years I was raised bymy maternal grandmother and her sisters, sur-rounded by lots of love and attention, being theonly child of my generation in the whole family.When my father remarried, I went to live withhim and my stepmother, who took care of melike a real mother. They had decided not to haveanother child for fear of treating us differently.The contact with my maternal grandmother re-mained close, and during the next ten years, Ispent several days a week with her and her sister.

The neighborhood I grew up in was typ-ical for a city relying on a flourishing textileindustry: large factories surrounded by a net-work of dead-end alleys with small working-class houses. Most houses did not have run-ning water; there was a central tap in the street.Some even had common toilets in the middleof the street. Light came from petroleum or gaslamps; few houses had electricity. Heating wasdone mostly with a coal stove, which also servedfor the cooking. Since the bedrooms were notheated, there were fascinating ice flowers on thewindows during winter.

The factories were dark and very noisy, andclouds of cotton dust would be floating aroundthe spinning machines. They were so frighten-ing and convincingly repulsive that I felt I neverwanted to be obliged to work there. At noon-time, when it was not raining, many workerswould sit in the street on the sidewalk, eat-ing their bread and drinking from a gourdethey had brought from home. They were com-pletely covered by the white dust, which madethem look like ghosts. I often made remarksabout these disturbing working conditions tomy grandmother. She insisted that this was somuch better than before, when she was a child.Now they were working only a good 8 hours;before it was 10, sometimes even up to 12 hoursa day. In my grandmother’s youth 50% ofthe workers were children, many younger than10 years of age. They were important for slid-ing under the machines and knotting the broken

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treads. Her father-in-law had been one of thefounders of the workers movement in Ghent inthe 1870s. My stepmother also came from a mil-itant socialist family, from Bruges. That meantthat during all of my youth I was solidly em-bedded in a politically conscious environment.May 1st (our labor day) was the most importantholiday of the year. Never would we miss theparade, and the whole day we would sing mili-tant songs. Of course that was before the Warand later again after 1945.

Political activities remained very central un-til my graduation, after which science took over,although the striving for more social justice andbetter living conditions remained a basic moti-vation. Did not the Enlightenment teach us thatknowledge would set us free?

PRIMARY AND HIGH SCHOOL

A brother-in-law of my grandmother was theonly family member with three years of sec-ondary education. He became a school teacherand retired as a director of a primary school.He insisted that I go to the best primary schoolwithin walking distance. That certainly gave mea good head start.

The daily school routine was disturbed inearly 1940. Part of the school building wasturned into army barracks. Family memberswere drafted into the army. As the Germantroops were approaching, many bridges of thecity were blown up, and the barges on the canalswere sunk with explosives. I was puzzled by theview of hundreds of Belgian soldiers wavingwhite handkerchiefs above their heads, march-ing in surrender toward two German soldiers.School continued, although during the follow-ing years the atmosphere in class changed.Learning French was now optional, and the les-son was moved to the late afternoon. Duringmusic lesson we had to learn songs expressingour devotion to Flanders. Nobody dared makeremarks. Intuitively we felt these were danger-ous times. Great excitement arose during thecloudy nights: The blackout made streets verydark; one could really bump into somebody.Also, starting from 1943, bomb alarms were fre-

quent at night. The sky would be lit by search-lights and exploding antiaircraft shells, a beau-tiful son et lumiere for a 10-year-old.

I had the good fortune that the teacher ofthe last year in primary school insisted, as I wasamong the top pupils of the class, that I shouldgo on to high school—preferably to the Latinsection of the Atheneum, a state school with anoutstanding reputation. In a city where 80% ofschools were run by the Catholic Church, theranking of the best went to some Jesuit colleges,but these were for children of the middle or up-per class, not of the working class. As nobodyin the family, not even friends or acquaintances,was religious, it was self-evident that if I con-tinued with the schooling system, I would goto the Atheneum. Going on with my educationwas a big decision, but the family concluded thatit was worth trying because everybody noticedthat the only thing I seemed able to do was toread books all day long.

The first years were not brilliant. Educatedwith the strong imprint never to disturb or tryto draw attention, I was isolated and lonely. Ialways managed to sit on a bench in the firstrow, but that was the most daring thing I everdid; I never went any further and did not attractthe attention of the teachers.

A complete change occurred in the thirdyear. The mathematics teacher was an out-standing pedagogue who really made an effortto have the whole class follow him. Also, thecourses of physics and chemistry fascinated me.This was the first year of a new section calledLatin/Science. The best teachers had made aneffort to obtain an assignment to this section,and the program was novel and experimental.

I became most captivated by chemistry anddecided to start a small laboratory in the atticof our house. In winter, as there was only a coalstove on the ground floor, conditions were a bitharsh. But the heat of the Bunsen burner wassufficient, and, yes, I could convince the parentsto extend the gas line to the attic.

From then on I made my first class friend,Hubert Sion; he too had installed a lab in hishouse. His parents were clearly financiallymuch better off than mine, but interactions

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between us went very well. At the end of thesixth year of Athenaeum I was again in thetop group of the class. The great uncle, theschoolmaster, suggested that I should try forUniversity. It was financially possible becausethe inscription fee for the State UniversityGhent, as it was then called, was minimal andI would have few other costs because I livedand ate at home. Walking for 40 minutes ortaking my bicycle cut the transportation costs.The minimal tuition fee was 5000 Belgianfrancs (today 120€ or $165) and representeda one-month salary for my father, which wasa lot of money. If I had started working as anoffice clerk, I might have earned that amountof money. But the parents were so proud withthe idea of their son entering university, a levelnever attained by anybody in the family, thatwithout much discussion they accepted that Ishould try a year. By then I was already resoluteto study the chemistry of living organisms,biochemistry as it seemed to be called.

I found out that there was indeed a Professorof Biochemistry at the University of Ghent,teaching in the Veterinary School and in theSchool of Agriculture. Upon asking him whatstudies to start, he answered that pharmacy wasthe best mix between chemistry and biology.

I did not feel like following his advice. I wasafraid that I would end up in a chemist shop. Ihad made good friends with a pharmacist in myneighborhood; it was the place where I boughtmy chemicals. I did not see myself being satis-fied with this profession. So I decided to choosechemistry, a choice I have never regretted.

STATE UNIVERSITY GHENT

In those days, the Ghent University was a smalluniversity (5000 students in the Fifties, now36,000) with a good tradition in chemistry.The university was founded in 1817, after theNapoleonic wars, when Belgium was unitedwith The Netherlands. The courses where firstgiven in Latin. Later, when Belgium becameindependent in 1830, the courses were given inFrench, but in the 1930s it switched to Flemish,the language of the region.

In the middle of the nineteenth century theorganic chemistry section of Ghent Universityattracted international reputation with AugustKekule (structure of benzene), Adolf von Bayer(the synthesis of Aspirin, barbiturates), and LeoBaekeland (the invention of Bakelite). From thefirst year of university onward, I was very activein the student movements and the political,philosophical, and even the natural sciencescircles. This was a great worry for my parents,because several days a week I came home after2AM. One rule was strict: Be ready for breakfastat 7AM and leave the house at 7:45 for theclasses. The second year was even worse: I hadalready become either president or a memberof the directory council of several studentorganizations, so all evenings were taken. Thecourses, however, became more interestingsince there was organic chemistry. A verypositive point was an introduction to biologyby a fascinating man, Lucien De Coninck. Hetalked about evolution but also about DNAand RNA, cell biology, and the compartmen-talization of biochemical reactions. That wasin 1952, a year before the Nature publicationon the work of Watson and Crick. He was inclose contact with the team of Jean Brachet(1909–1988), Raymond Jeener (1904–1995),and Hubert Chantrenne (1918–2007) at theFree University of Brussels (ULB). During thewar the ULB had been closed by the occupationforces because quite a fraction of the professorswere either Jewish, freemasons, or left-wingmilitants. Jean Brachet went undergroundin the forests of the Ardennes, where heinstalled a small lab in which he developed theUnna-Brachet staining for DNA and RNA. Hedemonstrated that DNA was concentrated all inthe nucleus and that RNA was present all overeach cell but highly concentrated in the areas ofintense protein synthesis, called ergastoplasma.This was also what we learned in De Coninck’scourse. Other professors still mentioned thatthe DNA was for animal cells (Thymo NucleicAcid) and the RNA (Zymo Nucleic Acid) wasfor bacteria, yeast, fungi, and plants. In thebotany course, it was mentioned that in a plantcell, besides the chloroplast, one could often

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observe strange dot-like structures. They hadreceived many different names, varying fromchondriokont to mitochondria. However, wewere told not to worry too much because it wasnot sure whether they really were important.This was my first fascination for what we nowcall cell biology and was further stimulatedseveral years later, when Jean Brachet came toGhent for a lecture series (Francqui chair) on“The Cell” and when I learned how ChristianDe Duve discovered the lysozomes.

Only in the fourth year did I again have con-tact with the life sciences, by choosing biochem-istry as an optional course and by convincing theorganic chemistry professor to be the mentor ofa masters (then called licentiate) thesis in thatdiscipline. My study results were not brilliant,as I had become the National President of thesocialist students. I often had to travel to Brus-sels and Liege. It was also too hard to refusean invitation to fly to Warsaw, my first flightever, and join a student group that was to visitAuschwitz. I managed to convince the physi-cal chemistry professor that this was importantand that I could skip his practical courses. Any-way, I passed each year and finished with “dis-tinction,” essential for being accepted for PhDresearch.

THE FASCINATIONFOR RESEARCH

My parents had probably hoped that after thesefour years I would start to work. However,during my research thesis in biochemistry, Iwas asked by the Professor of PhysiologicalChemistry of the Medical School, LaurentVandendriessche, to become research assistant.

As this position yielded a salary equivalent tothat of a high-school teacher, I did not hesitate.Above all, should not my parents be glad thatI enjoyed the prospect of doing a PhD? Theywere happy, but they remained convinced thatI would go into politics since I continued to goto so many meetings.

The research topic was immediately frontierresearch: resolve the structure of the phospho-diester bond in RNA by asking whether it is

a 2′- or a 3′-5′ diester bond. The experimen-tal approach was enzyme kinetics measured bydilatometry, when a (2′,3′) ribonucleotide esterwas digested by pancreatic RNase.

Walter Fiers had started this work sixmonths earlier. He taught me the dedicationto careful experimentation and the fascinationfor research. He was my real mentor. We hadfirst met in the Biochemistry lab, during mylast year as an undergraduate. He was an intro-vert who knew immediately what was impor-tant and relevant. We were the first to open thepackages with science journals that the mailmanbrought and to discuss what intrigued us. A newscience called molecular biology was emergingand there were so many topics to follow.

The real revelation came when, duringanother Francqui lecture, Raymond Jeenerexplained how, in the United States, moleculargenetics had developed thanks to SalvatoreLuria, Max Delbruck, and the Cold SpringHarbor Phage School. Both Walter and Idecided that this was the topic we wantedto study. That was also the opinion of anundergraduate zoology student attending theselectures, Jeff Schell (1935–2003) (Figure 1).However, there was no phage research inGhent, so we asked ourselves if we shouldmove to the ULB. This would be expensiveand would imply a change of language, so thephage research had to wait. Jeff started hisPhD in the laboratory of Microbiology. Theresearch topic, taxonomy of Acetobacter, did notenthuse him much, but stimulated him to applyfor summer grants in the United Kingdom.

Bill Hayes, with whom Jeff stayed with ashort-term fellowship at the HammersmithHospital in London, confirmed that Jeff ’scuriosity in bacterial genetics and phages wasclearly timely. So Jeff entered in the mysteriousworld of restriction and modification, a topicthat would keep him interested during all of theSixties. Meanwhile, Walter decided for a bigchange and left the Physiological Chemistry labfor CalTech and the lab of Robert Sinsheimer.His research topic was the genome of theEscherichia coli phage, ϕX-174. Sinsheimer hadshown that this phage was very UV sensitive


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Figure 1Marc Van Montagu, Walter Fiers, and Jeff Schell during the 1974 EMBO workshop in Ghent (Drongen),Belgium.

and had proposed that it had a single-strandedDNA genome. Walter could demonstrate thatit was a single-stranded circle. Previously,Bill Hayes had predicted from recombinationfrequencies that the genetic map of E. coliwould be a circle. This was the first proof thata circular genome existed as a physical entity.This result made the front page of the New YorkTimes, but remained unnoticed in Belgium.

In the meantime, I followed a completelydifferent path, and after one year of research,mixed with political activities, I received theremarkable proposition to become deputydirector of a novel institute for training tech-nicians and technical engineers for the nuclearindustry. My task would be to discuss thecontent of the future courses with the scientistsof the nuclear reactor center in Mol and toidentify the possible teaching staff. Three anda half days on the spot was sufficient during theweek, so I could still continue with my PhDresearch. An offer difficult to refuse, especiallywhen one is barely 23 years old. I was evenable to select as teaching staff well-qualifiedand appreciated colleagues from around my

very age group. This experience became aunique learning process that lasted severalyears of working out teaching programs andsetting up laboratories where all organic andanalytical chemistry was done on a microscaleand, with a dedicated team, where solidarityand mutual friendship were high. In those daysthe students in this area of northern Belgiumoften did not have the economic possibility tostart university studies, so the creation of thiskind of higher institute was a real opportunityfor them. For us it was a reward to have suchhigh-quality students. Later, several students,among whom Ivo Zaenen, the first authoron our paper describing the isolation of theTi-plasmid (54), joined our research team inGhent and made important contributions.

After some years and a change of govern-ment, however, it became clear that we wouldnever obtain the quality research laboratoriesthat were promised. In this context, a deputydirector who takes care of the science qual-ity of the courses made no sense. So I aban-doned the position, did my military service, stillcompulsory in those days, and again joined the

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laboratory of Physiological Chemistry of theGhent University, now on a full-time basis.Walter had just left for CalTech, and the re-search group was looking for novel nucleasesand RNases in plants. As an organic chemist Iwas supposed to synthesize original substratesto facilitate the identification of such nucleases.The early Sixties was the period when GobindKhorana was very active in the chemical synthe-sis of the ribo- and deoxyribo-oligonucleotides.The game of using protective groups that couldbe selectively removed was mesmerizing.

Some defeatists wondered what was theuse of synthesizing some milligrams of di-or trinucleotides or some microgram of alonger oligonucleotide. I admired Khorana’swork and followed his papers closely. I alsostarted to make new derivatives and protectivegroups. The head of the laboratory, LaurentVandendriessche, was appreciative whensomebody was dedicated to a subject and didnot interfere but instead tried to help. He wasalready very involved in the International Sci-ence Organisation and traveled regularly to theSoviet Union and other Eastern Europeancountries. One day he announced that a wholeteam in a lab of the Academy of Sciencesin Prague was specializing in the chemicalsynthesis of nucleotide derivatives and saidthat I should join it for a short term. Hetook care of a grant and the invitation by thedirector of the Institute of Organic Chemistryand Biochemistry, Frantisek Sorm. A superiorpersonality, and former Minister of Education,he tried to democratize the system. Whenthe situation became difficult, particularly forJewish scientists, he helped them escape to theWest. Later, in 1968, he was too openly activeagainst the Soviet intervention, which causedhis destitution and banishment into forcedlabor. He died a year later.

In the summer of 1963, I went to Prague forthree months. It was a definitive step towardmy decision to remain in fundamental research.Whatever the problems of the country were,whatever the shortage of equipment and chem-icals the Institute had, the unit was hard work-ing and enthusiastically dedicated to progress in

Figure 2Marc Van Montagu, 1965.

science. Uridylic and cytidylic acids were pre-pared in kilogram amounts and bartered forother chemicals with U.S. companies. As thepreparation started with an acid hydrolysis ofRNA, a method that destroyed the purineanalogs, I worked only with pyrimidine deriva-tives and synthesized the corresponding tripletsUCU, CUC, etc. The protecting group reagentwas rather original; it was unstable and had tobe synthesized as a condensation intermediatebetween HCN and ketene, a reaction I was notwelcome to use once back home. Upon my re-turn I learned that Khorana’s team had alreadysynthesized the 64 different triplets. But thatwas not a major problem for my PhD defense(Figure 2).

RNA PHAGES

Meanwhile Walter had returned from theUnited States and decided to work on an RNAphage that was capable of infecting E. colibacteria that carried an F plasmid. This phagehad just been described by David Baltimore inNorton Zinder’s lab at Rockefeller Universityand the only bacterial messenger easy topurify, because the viral particles could beprepared in gram amounts. In the Fifties we had


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tried to work with tobacco mosaic virus (TMV)RNA, a plant virus that we also prepared ingram amounts, but in vitro protein synthesiswas not yet ready to exploit the molecularbiology knowledge that this mRNA could gen-erate. Walter had decided to develop methodsto sequence MS2 RNA, quite a daring decision,which generated many smiles. Nevertheless,10 years later he succeeded and his team wasthe first to sequence a viral genome (19).

I was given the possibility to start my ownresearch unit in the department of Histologyof the Medical Faculty. I had access to an elec-tron microscope and could teach cell biology.For research I decided to join Walter, but fo-cused on the genetics of the phage. This wasthe period when the genetic code was unrav-eled, when Brenner and Crick identified theUAG and UAA stop codons with their sophisti-cated mutagenesis of the phage T4rII locus, andwhen the one gene–one enzyme theory flour-ished. As recombination with RNA phages wasunknown, it became clear that the only way tomap the mutants would be via the sequencingof the mutant proteins—a challenge, becauseamino acid sequenators existed but they wereprohibitively expensive for a beginning team.So I convinced a bright beginning PhD studentnamed Joel Vandekerckhove to join me andto use the Fred Sanger method of separatingpeptides by paper chromatography and elec-trophoresis to elute the spots, hydrolyze them,and determine their composition.

I had the good luck that this was really fron-tier research at the time and many labs likethose of Fred Sanger (Cambridge, U.K.) andJim Watson (Cambridge, U.S.) had taken RNAphages as model systems for establishing thediscipline of molecular genetics. As a conse-quence, I was accepted when I applied for thefirst European Molecular Biology Organization(EMBO) courses and for the first Spetsai meet-ing in August 1966.

This was a very essential step because itintroduced me to many young U.S. postdocs,who remained friends for life, and the topicspresented opened new horizons. Indeed, wenever had formal graduate schools where the

emerging science was presented; we wereexpected to acquire this knowledge throughappropriate reading.

Evolving knowledge about tRNAs and ribo-somes and the universality of the genetic codewere stimulating discussion points. The phagesλ T4 were well represented, and I realized theirimportance as model systems for moleculargenetics. Back home, I intensified my contactwith Rene Thomas at the ULB. His team wasalready the world leader in the genetics of phageλ. The whole molecular biology department ofthe ULB had just moved to a nice new suburbanlocation in St-Genesius-Rhode. Every weekleading scientists from all over the world gaveseminars, something entirely nonexistent inGhent. The fact that in 1963 I had moved fromGhent to Brussels helped me to develop bettersocial contact with the Brussels group andthe visiting seminar speakers. I became partof the world community of phage geneticists.For visualizing the phages and their anatomicstructure, electron microscopists had workedout nice enhancing methods (negative stains).An EMBO course in Geneva, organized by theKellenbergers and Werner Arber, was veryhelpful for getting acquainted with thesetechniques. I also learned the Kleinschmidttechniques for visualizing DNA. Spreadingheteroduplexes between wild-type and phageλ DNA containing insertions or deletionsallowed the physical localization of the al-teration in the phage genome. Later weused this method for localizing IS sequences,transposons, and phage Mu insertions inplasmids and for positioning cloned restrictionfragments on the Ti plasmid.

Meanwhile, Walter had become professorin molecular biology and had started his ownunit in the Faculty of Science. Jeff Schell wasstill located in the Veterinary School where bio-chemistry and microbiology were initiated. Atthe end of the Sixties he also moved to the Lede-ganckstraat and introduced us to the E. coli K andB restriction and modification system.

Our understanding of the RNA phages hadmeanwhile progressed well, but we rapidly real-ized that we should start considering eukaryotic

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model systems. Cancer research became moremolecular, and tumor viruses became fashion-able model systems. Walter decided that, in ad-dition to his efforts to sequence phage MS2, hewould start SV40 research, including sequenc-ing. Jeff and I, with our limited budgets, thoughtit could be worthwhile to start plant tumors.Plant cell culture had recently been developedthat did not need expensive media, CO2 in-cubators, or special deep freezers. Using liveanimals as test systems was also not appealingto either of us. The microbiology lab where Jeffdid his PhD kept a collection of 150 Agrobac-terium strains of which some were called tume-faciens because they were able to induce crowngalls on a large variety of plants, and others,seemingly very related, that could not, werecalled radiobacter.

PLANT TUMORS

Let me first give some milestones on plant tu-mors studies. In the late 1940s plant pathol-ogists, microbiologists, and chemists had de-veloped a keen interest in crown-gall-inducingstrains of the bacterium Agrobacterium tumefa-ciens (47) because the interaction between thebacteria and their hosts displayed many un-usual features. Unlike other pathogenic bac-teria that are known to cause plant tissues todie, wilt, or become discolored, A. tumefacienshad the unusual ability to cause infected plantcells to proliferate and form a tumor. The bac-terium could not be detected intracellularly,either in plant cells that had been transformedor in the cells of sterile crown-gall tumorsgrown in vitro even in the absence of growthhormones for many years. This observation ledBraun to introduce, in 1947, the concept of atumor-inducing principle (TIP), a postulationthat implied that this principle was transferredby the bacteria to the plant cell to induce trans-formation (5). Importantly, Braun proposedthat the TIP must be capable of replication, be-cause it was never lost by dilution. Much workensued to identify the TIP, but only in the late1960s the first indications were forthcomingthat bacterial DNA may somehow be involved

in tumor induction. DNA sequences from A.tumefaciens were claimed to be present in sterilecrown-gall tumor DNA preparations, suggest-ing that bacterial DNA had been transferred tothe plant cells (46), and later, expression of somebacterial genes was demonstrated by the detec-tion of bacterial antigens in sterile crown-galltissue. Ever since the discovery of lysogeny inA. tumefaciens (2), much attention has beenfocused on the possible relationship betweenlysogeny and pathogenicity. However, effortsto determine the causative role of temperateA. tumefaciens phages in tumorigenicity were in-conclusive and contradictory. One of the mostimportant indications of phage involvement intumor induction by A. tumefaciens was the de-tection of �-group temperate phage DNA se-quences in strain A6-induced sterile crown-galltissue (45). Nevertheless, no phage could be in-duced from strain A6, and no �-phage DNAsequence could be detected in DNA extractedfrom A6. Furthermore, presumably curedstrains derived from lysogenic strains were noless pathogenic than the parental strain (28, 29).These results still did not rule out the possibilitythat supposedly cured strains contain a defec-tive or cryptic prophage that may be present asa plasmid or may be part of a bacterial plasmid.

THE GREAT SHIFT: DISCOVERYOF TI PLASMID AND T-DNA

To explore the plasmid hypothesis, a systematicsearch was conducted for the presence of a plas-mid DNA in a number of pathogenic and non-pathogenic strains of agrobacteria. This workculminated in the landmark discovery of a largesupercoiled plasmid in all virulent strains, butinterestingly, this plasmid was not detected inany of eight avirulent strains tested (54).

Careful experimental design was crucialto the success of these experiments because itwas essential to distinguish between possiblesupercoiled plasmids and covalently closedcircular forms of phage DNA, which wasnot easy with such large (200,000 base pairs)plasmids. Also at that time, only 20 years afterLederberg’s discovery of plasmids, the plasmid


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isolation methodology was in its infancy.To avoid possible contamination with phageDNA, strain B6S3 was chosen because it couldnot be induced to produce phage particles. De-tection of a large 54.1-μm plasmid in alkalineand neutral sucrose sedimentation gradientsand in cesium chloride gradients of B6S3was confirmed by electron microscopy. Viacomparison of a large number of pathogenic,virulent, and avirulent strains using thismethodology, conclusive insights into the roleof the plasmid in tumorigenicity were obtained.All pathogenic Agrobacterium strains testedharbored a large plasmid, including all curedbut still pathogenic derivatives, but none of theavirulent strains tested. Furthermore, whethera tumor-inducing strain of Agrobacterium waslysogenic for an inducible prophage did notinterfere with the presence of a plasmid inthat strain. Conversely, an Agrobacterium straincould be lysogenic without harboring a plasmidbecause two of the avirulent strains includedin the study produced phage particles uponinduction but did not contain plasmid DNA.So, Braun’s TIP had been identified 27 yearsafter it had first been proposed. We designatedthese plasmids tumor-inducing (Ti) plasmids.This landmark discovery prompted a world-wide intensification of efforts to prove directlythat these plasmids were responsible for thetumor-inducing capacity of their host strains.

The evidence for association of an extrachromosomal plasmid with virulence was ir-refutable. Direct proof that the Ti plasmidswere responsible for the tumor-inducing capac-ity of their host strains came shortly thereafterthrough isolation of plasmid-free derivativesfrom tumor-inducing strains. For some time,strain C58 of A. tumefaciens had been known tobe possibly “cured” of its virulence by growthat 37◦C and not at the normal growth temper-ature of 28◦C, and, predictably, strain C58 wasfound to have lost its plasmid when grown at37◦C. A further landmark was achieved in 1975when nononcogenic A. tumefaciens strains weredemonstrated to acquire virulence as a result ofplasmid transfer (48), in confirmation of Kerr’sexperiments (37) that first pointed out the pos-

sibility of mobile genetic elements as the ba-sis for virulence. A year after the initial discov-ery of the Ti plasmid, the first genetic marker,Agrocin 84 resistance, was shown to be encodedon the Ti plasmid, thereby opening the wayfor more refined genetic analysis (17). With in-creased focus on the Ti plasmid, a worldwideeffort was launched to unravel the molecularbasis for tumorigenicity, and our group con-tinued to dominate the field. The finding thatoncogenicity and virulence were determined bya mobile, extrachromosomal element was rev-olutionary and opened up hitherto unforeseenscenarios for initiating the molecular geneticsof a plant-microbe interaction.

Functional Mapping of the Ti Plasmid

In the late 1960s Morel’s group in Francedemonstrated that crown galls generate copi-ous amounts of novel metabolites, octopine,and nopaline and that crown-gall cells thatwere free from bacteria were still able to pro-duce them (42). They showed also that theAgrobacterium strain, not the plant, determinesthe type of opine made by the tumor and thateach Agrobacterium strain can catabolize solelyits own particular type of opine. Morel pro-posed that Braun’s TIP must be the cause,or it must include a gene responsible foropine synthesis in the plant. To account forthe strain specificity of the opine catabolism,he proposed that a single enzyme catalyzesopine synthesis in the plant and opine break-down in the bacterium. At that time it was dif-ficult to accept the notion that a bacterial genecould enter a plant cell and function there. Fiveyears later, with the Ti plasmid in hand, we werein a position to test the idea and to prove itby demonstrating integration of T-DNA intothe plant nuclear genome. Building on exper-tise in bacterial genetics and the use of phagemutagenesis in functional mapping (20, 50), weset about dissecting the genetic structure ofthe Ti plasmid and the molecular basis of themetabolic interaction between pathogen andhost. Transposon-insertion mutagenesis of theTi plasmid revealed the functional organization

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of the nopaline Ti plasmid pTiC58, and a yearlater the functional organization of the octopinepTiB6S3 had been established (12, 30), therebyconfirming the strain dependency of the opinesynthesis observations of Morel. Transposonhits in T-DNA were found to eliminate opineproduction, to alter tumor morphology, or tohave no phenotypic effect at all. The morpho-logical mutations were used to demonstrate thatgenes encoding a pathway for the synthesisof the plant-growth regulators auxin and cy-tokinin mapped to the T-DNA. The same mu-tagenesis approach showed that another groupof mutations affecting tumor induction mappedto a sector of the Ti plasmid, separate from T-DNA, the virulence (vir) region (16, 23, 30).

As soon as the structure and function of theTi plasmid were understood, the concept thatthe Ti plasmid could be used to deliver novelgenes into plants was born, raising the possibil-ity that the molecular dissection of plant phys-iology through target manipulation of gene ex-pression was in sight. The further possibilityof introducing, at will, genes that could con-fer new desirable properties to a plant and thepotential for revolutionizing plant breeding andcrop production was obvious. Galvanized by therealization of this outstanding opportunity inplant science, research progressed rapidly, bothin analysis of the Ti plasmid and in understand-ing the process by which the plasmid DNA isintegrated into the plant genome. The race forthe development of a workable plant gene vec-tor was tight, particularly between Chilton’s labin St. Louis and our lab in Ghent. In 1977, bothlabs had access to the novel technique of South-ern blot and we could demonstrate that onlycertain parts of the Ti plasmid, the T-DNA,are integrated into the plant genome. Unfortu-nately, we lost the battle to publish first (6), andthe only record of our result is a talk given ata Cold Spring Harbor Symposium in 1978 andin Angers (8). The fact that between differentTi plasmids sequences are conserved pointedtoward their central role and essentiality foroncogenicity (15). Adding quickly to this dis-covery, at a time when little was known aboutDNA sequence determinants for heterologous

integration, a 25-basepair direct repeat on theTi plasmid was identified at the borders of whatis incorporated into the plant genome (39, 56).It was shown that these sequences define T-DNA on the plasmid. At the same time, ele-gant cell fractionation analyses revealed that theT-DNA integrated into the nuclear, and notthe chloroplastic or mitochondrial, genome ofplants (7, 53).

THE WAY TO PLANTTRANSFORMATION

Plant Gene Vectors

Conversion of the Ti plasmid of Agrobac-terium into a gene vector progressed in multiplestages. Unraveling the functional organizationof the Ti plasmid was central to our efforts touse the integrative properties of T-DNA forthe genetic modification of plants. Methodswere developed for site direct mutagenesis andfor insertion of DNA into any part of theTi plasmid by homologous recombination bymeans of a co-integrate plasmid, which is theproduct of homologous recombination througha single crossover between a small plasmid ofbacterial origin and an Agrobacterium Ti plas-mid. Integration of the two plasmids requiresa region of homology present in both plas-mids introduced into A. tumefaciens (41, 49). Wegained understanding that no mutation in theT-DNA could block T-DNA transfer and thatall the genes affecting the process of T-DNAexport to the plant cell mapped in the so-calledvir region, outside the T-DNA borders.

Knowing which sequences on the Tiplasmid were required for integration ofthe bacterial DNA and, importantly, whatneeded to be removed from the T-DNA toavoid tumor development and to producetransformed plants, we designed the firstnononcogenic Ti plasmid (pGV3850) capableof transforming plants without tumor forma-tion (55). Besides demonstrating the feasibilityof creating transgenic plants, this work was thefirst demonstration that the T-DNA borders isall you need as T-DNA elements to integrate


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foreign DNA into the plant genome. Such co-integrated vector system was used extensivelyas a gene vector for plant transformation (10).

The functional organization of the Tiplasmid opened the way for disarming theT-DNA completely and for the constructionof vir region-containing plasmids lacking eventhe T-DNA borders. A versatile vector systemwas developed, now known as binary vectors,in which the T-DNA element and the vir geneswere located on separate replicons (27). Thestrategy to clone transgenes in their T-DNAwas to take advantage of rare restrictionendonuclease sites that were introduced intoT-DNA sequences. More recently, the T-DNA binary vectors were redesigned to benefitfrom novel in vitro recombinational cloningschemes, mainly based on the site-specificGateway R© system (Invitrogen, Carlsbad, CA)(22, 36). Gateway-compatible binary destina-tion vectors have been developed for a widerange of gene-function analyses, includingoverexpression, promoter fusion, proteinfusion, and silencing, and they have beenoptimized for various plant species (35). Thistechnology even facilitates the recombinationof multiple segments of DNA contiguously orin independent expression units (18, 33, 34).

Selection and Regenerationof Transformed Plants

An ongoing problem was how to regenerateviable plants from cells that had been trans-formed with the T-DNA. Of great help wasthe availability of a large bank of T-DNA mu-tants. Thanks to this bank, gene vectors couldbe constructed in which the cytokinin syn-thesis genes had been disrupted. By screeningrooty tumor cells for nopaline production, re-searchers showed that these, unlike crown-galltumor cells, were able to regenerate into wholeplants that passed the T-DNA copies to theirprogeny as Mendelian traits (1, 38).

However, screening the transformantsfor their ability to synthesize nopaline ortheir inability to grow in the absence ofexogenous cytokinin was cumbersome and was

incompatible with the main goal of selectingfew transformants from a large backgroundof false positives. Sequencing the nopalinesynthase (NOS) and the octopine (OCS) genes(3, 13, 14) and fusing their promoter and ter-minator sequences to a kanamycin-resistanceencoding gene enabled the creation of aselectable marker for plant cells. The bacterialregulatory sequences that were known to func-tion in planta would drive the expression of agene that would allow survival of transformantsunder antibiotic-selection conditions.

The First Genetically Modified Plants

The way to obtain the first transgenic plants hadbeen paved, and almost simultaneously, our lab,Monsanto’s, and Chilton’s reported success inthe use of Ti-derived plant gene vectors, antibi-otic selection of transformants, and regenera-tion of fertile plants that passed on the chimericgene in a stable and Mendelian manner to theirprogeny (4, 21, 25).

Only nine years after the discovery of theTi plasmid, the “Golden Era” of plant molec-ular genetics had begun, and the developmentof plant transgenic technologies would expanddramatically in the 1980s and 1990s.

THE IMPACT OFTHE DISCOVERY

Funding agencies always make a difference be-tween fundamental and applied research whenin fact there is no dichotomy in science. Thediscovery of the Ti plasmid is a classic case ofcuriosity-driven research that ultimately led tomajor scientific breakthroughs both in the pro-liferation of new research tools and quantumleaps in fundamental and applied knowledgeabout control circuits in biological systems.Indeed, Agrobacterium is an extremely interest-ing biological system, and its study has led to afeast of fundamental insights (58).

For example, (a) the recognition that bacte-rial genes could integrate into the plant genomeand direct the synthesis of plant growth regu-lators, thereby creating the microenvironment

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(the tumor) for their growth, was revolutionaryand formed the basis for much of what we knowabout horizontal gene transfer. The discov-ery that the T-DNA intermediate is a single-stranded molecule (44) elicited the hypothesisthat T-strand transfer occurs by a conjugativemechanism, where the recipient is a eukaryoticplant cell (24, 52). This interkingdom DNAconjugation stimulated basic research in bac-terial conjugation. In the postgenomics era, wenow know that horizontal DNA transfer is re-sponsible for a great deal of adaptive responsesand invasive behavior and that the genomesof living organisms are much more fluid thanpreviously imagined. (b) The fact that T-DNAgenes trigger plant growth regulator synthe-sis not only explained why crown-gall calli canbe grown on minimal media without additionof exogenous cytokinin or auxin, but also, andmore importantly, paved the way for a morerational understanding of how plant growthregulators function in plants and for plant generegulation. It must be kept in mind that in thesedays “hormonal control of plant growth and de-velopment” theories were rampant in the ab-sence of hard gene function data, and researchwas largely in the grasp of “spray and pray”approaches that to a large extent obscured whatwas really going on. (c) The induction of virgenes by plant phenolics was one of the firstexamples of communication between microbesand plants in the soil environment (43). Today,the importance of many more developmentalregulatory circuits has been identified.

In contrast, the demonstration that Agrobac-terium could be used as a vehicle to trans-fer and integrate any foreign gene into theplant genome undoubtedly opened the doorto a complete new area of plant sciences andplant engineering. For the academic world,this technology allowed researchers to studyplant processes through gain of function andenabled a systematic and refined analysis ofthe impact of single genes on all aspects ofplant biology. In the early years, the technol-ogy was applied in model plants, such as tobacco(Nicotiana tabacum) and Arabidopsis thaliana andwas the basis to identify and study fundamental

principles, such as the cis-regulatory elementsin plant genes (e.g., promoters), the translo-cation of proteins in the plant cell (e.g., thesignal peptides to transport a protein to thechloroplast) (26), and transcriptional regulationin plants (e.g., the signals needed to induce ex-pression of genes by light). These initial suc-cesses convinced an ever-increasing number oflabs throughout the world to rapidly adopt thenovel genetic engineering technology. Much oftoday’s detailed knowledge of how plants growand develop as well as phenomena such as thereaction of plants toward pathogens or abioticstress (such as drought and salt) have been un-raveled by creating transgenic plants throughAgrobacterium-mediated transformation.

Some of the first efforts to understand thefunction of all genes in a genome have beenlaunched in plants, to a large extent, thanksto the availability of the efficient gene-transfertechnology offered by Agrobacterium. The pos-sibility of using T-DNA as an insertional muta-gen had been realized early on, and the publiclyavailable populations of T-DNA-tagged mu-tants in Arabidopsis are now so large that nearlyevery gene in the genome is tagged. In addition,reverse-genetic strategies on DNA from popu-lations of T-DNA mutants allow researchers tostart with one gene sequence and to identifya mutant line. The use of these mutant lines inlabs throughout the world has led to a thoroughunderstanding of the physiology, biochemistry,and developmental programs of plants.

Much of our current understanding of howplants grow and develop; how primary and sec-ondary metabolic pathways are controlled; aswell as how plants defend themselves againstpests, diseases, and harsh environments hasbeen obtained by using transgenic plants. Geneinactivation by T-DNA insertion, RNA inter-ference, and the introduction of new genes andectopically altered transgenes have been invalu-able tools for plant molecular biology.

Many interesting fundamental questions re-main regarding the information encoded by theT-DNA and the interaction of the T-DNAwith the chromatin. Techniques for an in-depthstudy of the functional genomics of different


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T-DNAs are available. The phenotype of thecrown-gall tissues induced by these differentT-DNAs can vary spectacularly, from shootyteratoma tissues to smooth, seemingly undif-ferentiated galls. The demonstrated differencesin the cytokinin/auxin ratio might not be theonly cause of these differences. Our presentknowledge of regulatory small RNAs and theemerging understanding of hundreds of plant-encoded peptides suggest that it can be reward-ing to analyze the T-DNAs for the possiblepresence of small RNA and peptide-encodingsequences.

THE INNOVATION CHALLENGE

We were quite aware that genetic engineeringof crop plants would transform the agriculturalworld. Until then, new plant varieties could beobtained only by classical breeding. But we hadno clue of the legal steps for seeking patent pro-tection, and there was no technology-transferofficer at the Ghent University. In those days,our universities were centers of knowledge withlittle or no contact with the industrial world.Luckily, in 1978, Jeff Schell had accepted a po-sition as director at the Max Planck Institutein Cologne where the expertise of filing patentapplications was available. This is the reasonwhy the patent to engineer plants via A. tume-faciens belonged to the Max Planck Institute,even though the research had been performedin Ghent. A few years later a new start-up com-pany in Ghent, Plant Genetic Systems (PGS)(see below), had the funds to buy the patent asan important asset. Today all transformationsof the major crops, including maize, soybean,cotton, rice, and cereals are done primarily withAgrobacterium.

ENTREPRENEURSHIP

With the first gene-cloning experiments, it be-came evident that this technology had enor-mous economic potential for producing novelpeptides and proteins for the pharmaceuticalindustry. Particularly in the United States, aclimate developed for start-up companies that

were often directed by a business person and aleading scientist and were established at a tech-nology campus close to a leading university.

When our Agrobacterium story started, en-trepreneurs decided to try out plant biotechnol-ogy start-ups. One of these, Advanced GeneticSciences (AGS), with Laurence Bogorad (1921–2003) as Chair of the Science Board, asked Jeffand me to join its scientific board.

AGS had raised an—for us—impressiveamount of money, had set up in a record timeexcellent laboratories, and had attracted out-standing scientists. Why not try and start a sim-ilar venture in Belgium? As a good civil servantI approached our Minister of Education andResearch. He was a law professor but had thegood sense to see that it was not the task of theuniversity to establish a start-up company. Hedirected us to a just-established regional invest-ment company (GIMV) and a board of industri-alists (Innovi). The latter appointed a CEO andcontacted some private investors, among whichwere the Belgian Sugar Factory of Tienenand a Swedish equivalent called Hilleshog.Laboratory space was rented from the Schoolof Engineers at the Ghent University. MarcZabeau, who did his PhD with us on restrictionalleviation of phage λ, was hired as laboratorydirector, and the research started in 1983. Manyscientists and technicians from my laboratorywanted to join this exciting novelty, PGS. Quitea number of the original team remained afterPGS was sold to Hoechst/Aventis in 1996 andare still there after the transfer to Bayer in 2001.

As cofounder, member of the Board, andinitial Scientific Director of PGS, I remaineda 100% state employee as professor at GhentUniversity and part time at the Vrije Univer-siteit Brussels (VUB) (Figure 3). PGS had thegood sense to focus on some early successes:the cloning of a Bacillus thuringiensis insecticidalprotein and the engineering of tobacco plantsthat expressed this gene at a level to convey re-sistance to Manduca sexta (tobacco hornworm)(51). The other breakthrough was the expres-sion in plants of a bacterial gene that detoxified aherbicide, produced by the companies Hoechstand Meiji, termed Basta (9) in cooperation with

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Biogen, then established in Geneva, and initi-ated by their laboratory director Julian Davies.We operated with the same Science Board asthat of AGS, with, in addition to LawrenceBogorad, Dick Flavell, Bob Goldberg, HowardGoodman, and Ingo Potrykus.

PGS grew rapidly. Jan Leemans replacedMarc Zabeau as lab director when Marc leftto start Keygene (Wageningen, The Nether-lands), and Walter De Logi became CEO afterJozef Bouckaert had joined AGS in California.We also realized that to demonstrate that ournovel plants had commercial value we had tointroduce plant breeding. Willy De Greef,who had started as an oil palm breeder withUnilever in Africa, immediately appreciatedthe opportunities offered by the tapetum-specific genes cloned in Bob Goldberg’s lab atUCLA. Expression of an RNase from Bacillusamyloliquefaciens, Barnase, under control of atapetum-specific promoter, was so tight thatit inhibited pollen production but did notinterfere with the normal development of thegenetically modified (GM) plant. Barnase waschosen because the gene was available thatencoded an inhibitor protein, Barstar, whichthe Bacillus produces to protect itself againstthe toxicity of Barnase. Expression of Barstarunder the same tapetum-specific promoterrestored the pollen production inhibited byBarnase. Meanwhile we had developed a nu-clear male sterility/restorer system that couldbe the basis of hybrid vigor in quite some cropplants (40). Around that time we convincedSuri Sehgal, a veteran of the seed industry, tojoin PGS as chief operating officer. He imme-diately streamlined the organization, stoppedseveral ongoing projects where value recoverywas questionable, brought crop-trait focus inselect crops, and convinced everybody to focuson a crop with high commercial value—hybridcanola (Brassica napus). These moves created theproducts and identified the real value of PGS.

While AGS was under the direction of JohnBedbrook, it was sold to Dupont. The takeoversbecame a trend in the 1990s. All the start-upsin plant biotechnology were acquired by bigmultinational firms because they were, and still

Figure 3Marc Van Montagu, 1984.

are, the only companies that can afford the hugecosts of the full product-development chain.

GENETICALLY MODIFIEDAGRICULTURE TODAYAND BEYOND

I feel warmly rewarded in that GM crops arehaving a durable and unprecedented effect onimproving the quality of life and on sustain-able food, feed, and fiber production. From thefirst commercial launch in 1996, the global GMcrop area had grown by 2009 to some 130 mil-lion hectares, representing more than 9% ofthe total agricultural land. GM crops are cul-tivated primarily in the Americas and in China.The global seed value of GM crops is over$10 billion. Notably, 90% (13 million) of thebeneficiary farmers are resource-poor farmersin developing countries (31).

The data clearly show that the currentGM crops achieve higher yields in a moresustainable way. At the same time, novelapplications that provide environmental ben-efits appear as technologies mature and aremore widely adopted. Remarkable progressin genomics and functional genomics has


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Figure 4Marc Van Montagu and Jeff Schell, 1993.

brought the first insights into the gene pooland transcriptional regulation of model plantsand of some important crop species.

The adaptation of plants to biotic and abi-otic stress conditions is now open to molecularanalysis and manipulation. Through these ap-proaches, the next wave of crops will be resis-tant to biotic and abiotic stresses and will beable to grow productively on more marginalland. Yield gains are important for food securityand land conservation, particularly in a chang-ing climate. Our planet requires the prompt andwidespread adoption of more efficient and sus-tainable agricultural practices to improve foodsecurity and to reduce the negative effects of in-tensive agriculture on the global environment.To close the yield gap between productivity inthe field and what can be achieved best will re-quire further innovation, be it in providing in-formation and infrastructure or in generatingnew crop varieties that are better adapted tospecific local environments.

In this context, the potentials of biotech-nology are manifold. GM crops will help tomaintain sufficient availability of food, but also

to “domesticate” crops for biomass and bioen-ergy production. Renewable raw materials offeran alternative to the chemical industry and canplay a role in rural income growth and povertyalleviation in developing countries. Nutrition-ally enhanced GM crops can improve peoples’health, and, last but not least, GM crops canbring about environmental benefits, for exam-ple, by decreasing pesticide use or by reducingsoil erosion.

GENETICALLY MODIFIEDORGANISM POLEMIC

In spite of the above-mentioned benefits, GMcrops have aroused passionate opposition. Thatone can engineer a gene from a species be-longing to a certain kingdom into a speciesfrom another kingdom has struck the imagi-nation of many and frightened the public atlarge. The fears for health and environmentalrisks have been disproportionate and could notbe reassured by science-based risk-assessmentanalysis. Anti-GMO (genetically modified or-ganism) activists bombarded the media with

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unfounded claims that GM foods are not safefor human and animal consumption, that GMcrops would become super weeds, and that theinvoluntary spread of transgenes into the envi-ronment would be irreversible and devastatingfor our habitat. On top of that, some groups areconcerned about adverse social implications ofGM crops because they are in the hands of pow-erful multinationals. Rather than tackling theseissues, they use the precautionary principle as afirm philosophical basis for saying “no” to GMcrops, arguing that there is remote chance ofirreparable damage.

Personally, I admit that it was a surprise tous, plant molecular geneticists, to realize thatsociety did not follow our scientific rational-ity, because we see our planet as one largenatural genetic laboratory where all the liv-ing organisms continuously activate and silencepart of their genomes in response to environ-mental changes. It is essential for adaptation,and hence for evolution, that plants can altertheir genomes through transposition of mov-able elements, accumulation of deletions, inser-tions, gene amplifications, and point mutations.Genomic studies during the past decade haveclearly documented that a genome is not a staticentity but a dynamic structure continuously re-fining its gene pool. So, for a geneticist, gen-erating a transgenic organism is a chirurgicalalteration compared with the genomic changesinduced during crossing and breeding eventsexploited in agriculture and animal husbandry.The tools of molecular biology offer precision,speed, and ability to reach this invaluable en-deavor of species domestication.

It is not simple to answer the following con-cern: “Give proof that GM crops are safe andpose no risk.” It seems a scientific question butit is not. Science can prove the presence ofdanger, but not its absolute absence. The ex-perts’ claim that a GM variety is not more orless a health or an environmental risk than thenon-GM parent crop does not answer the ques-tion. Scientists thought that it was sufficient toshow that intensive agriculture causes environ-mental damage, independent of whether it is

GM or not, and that, on the contrary, we canmake novel GM varieties that are more envi-ronmentally friendly. Society wanted step-by-step examples of studies to support such state-ments. Now, after 25 years of field releases and15 years of commercial use without evidence ofharm, fears continue to trigger the precaution-ary principle in Europe. It is important for thesake of humanity and our planet to abandon thisdeliberately one-sided position and determinethe advantages and disadvantages of this tech-nology on the basis of scientifically sound riskassessment.

The regulatory requirements to get a GMcrop into the market are costly and constitute amajor obstacle that adds to the chronic underin-vestment in science and technology. Scientistsfrom the public sector cannot afford such regu-latory compliance costs, which range from tensof thousands to millions of dollars (32). Theresult is that, although the present generationof GM crops can be traced back to discoveriesmade in the public sector, there is a mispercep-tion that biotechnology is the exclusive domainof a handful of multinationals.

We, scientists of the public sector in-volved in biotechnology for public good, havetaken steps to fight for a regulatory frame-work that is less counterproductive. In 2004we started a worldwide initiative, the Pub-lic Research and Regulation Initiative (PRRI)(http://www.pubresreg.org/) (11), to offerpublic researchers involved in modern biotech-nology a forum through which they can par-ticipate in and are informed about relevantinternational discussions and agreements thatinfluence national regulations. The goals areto advise negotiators about the objectives andprogress of public research in modern biotech-nology, to bring science to the negotiations, andto inform the negotiators about concerns pub-lic researchers may have. We hope in this wayto curb the prohibitive costs and length of theregulatory requirements and that technologiesthat are pro-poor, pro-environment, and pro-economy find their way to those who need themthe most.


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BIOTECHNOLOGY FORDEVELOPMENT

Institute of Plant Biotechnology forDeveloping Countries (IPBO)

The pro-poor biotechnology has always beena goal for me. While Director of the Lab ofGenetics at Ghent University I was keen to re-ceive trainees from all over the world. Hun-dreds of scientists were trained during the 1980sand 1990s, many from developing countries.Together we contributed to major advances inplant sciences, most notably in the fields of plantgrowth, development, and flowering as well asbiotic and abiotic stress. Most of them returnedto their home countries where they have con-tinued their plant biotechnology research.

When I reached the “emeritus age,” thechoice to dedicate myself full time to activi-ties to promote biotechnological applicationsthat meet the needs of poor farmers was obvi-ous. With the support of the S.M. Sehgal Foun-dation and the Flemish Government, I startedin 2000 at Ghent University the Institute ofPlant Biotechnology for Developing Countries(IPBO), dedicated to fostering plant biotech-nology in low- and middle-income countries.

The challenge of IPBO is to help findbiotechnological solutions to improve the lifeof the rural poor, who make up 80% of theworld’s 1.4 billon hungry people. No segmentof humanity depends more directly on envi-ronmental resources and services than the ruralpoor. Their lives are interwoven with the sur-rounding environment in ways that make themboth particularly valuable as custodians of en-vironmental resources and particularly vulnera-ble to the impact of environmental degradation.When population pressure grows and food isscarce, hunger can drive them to plough underor overgraze fragile rangelands and forest mar-gins, threatening the very resources upon whichthey depend.

I am well aware that science and technologyalone will not have the power to overcomethe challenges. Solutions must come fromconcerted actions of different segments ofsociety. It will require political will and strong

commitments of the nations, as it will lead to afull revision of the way we perceive our societyand our interaction with the environment. Imaintain, however, that in the near future, asthe technology matures further and the fullimpact of the postgenomics era is felt, we willbe able to tackle some of the most intractableproblems in plant productivity. Then the ben-efits will be more obvious, particularly in thedeveloping world, where economies are largelydependent on the health of the agriculturalworkforce and where much of the impact ofthe Green Revolution was not felt. How canIPBO contribute? The strategy I propose isto take advantage of the extensive network ofcooperation that I have established during myyears as director of the Lab of Genetics andto promote international actions to respondto the needs of developing countries. The aimis not only to transfer technology, but alsoto stimulate competitiveness and independentbiotechnological research for the developmentof locally relevant crop improvements. IPBOhelps partners in developing countries to iden-tify agricultural needs that cannot be workedout by conventional means and then contactresearch groups from the network to developstrategies for solving the problems. This planincludes providing basic training. The explicitgoal is to create a network of experts to helpestablish independent research capacities inthe least-developed countries and to stimulatethe interest of highly qualified institutes in thericher economies to investigate the constraintsimposed on poor farmers. In addition to its roleof facilitator, IPBO carries out its own researchaddressing the agricultural productivity needsof small-holder farmers and capturing thevalue of biodiversity in developing countries.

International Industrial BiotechnologyNetwork (IIBN)

Beyond the traditional agricultural products offood, feed, and fiber, the remarkable break-throughs in the fundamental plant sciences arefueling new opportunities in agriculture andtransforming the bio-based economy. Faced

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with a global energy crisis and concerns over cli-mate change, we may find a reasonable fractionof the energy demand to be met through theexploitation of plant-based resources. Modifi-cation of lignin biosynthesis, increased biomassproduction and yield, resistance to abioticstress, and metabolic engineering to improve oilcontent and composition for biodiesel as well assugar and starch for ethanol are examples of thebiotechnology solutions for bioenergy.

Metabolic engineering will also become animportant approach for increasing nonfuel bio-products. Plants are being used more and morein industrial approaches that are not depen-dent on petroleum, such as biodegradable plas-tic or intermediates for the chemical industries.Indeed, advanced bioproducts might be thegreatest long-term benefit of the current bio-fuels research race. There is significant roomfor growth of this sector given that 60% of thechemical industry is carbon based. It is highlylikely that a large number of presently under-utilized plant commodities will emerge in thecoming years as sources of raw material for thecarbon-based chemical industry.

Plants also are being manipulated for useas vehicles for development and manufactureof high-value pharmaceuticals. The productionof pharmaceutical proteins in plants has severalpotential advantages over current systems suchas mammalian and bacterial cell cultures, in-cluding the lower costs and scalability of agri-cultural production and the absence of humanpathogens. Another interesting aspect is that insome cases crops, e.g., fruit, leafy vegetables,or grains, can also serve as delivery systems ofthese high-value proteins to humans and ani-mals. Research and development in plant-madepharmaceuticals include a number of vaccinesalready progressing to clinical trials, antibodies,and nutraceuticals.

The sustainable use of plants as feedstockfor industry and energy has already attractedsignificant investments in the technologicallyproficient countries, but much needs to bedone to promote an enabling environment forthe development of a plant-based industry inthe least-developed countries. It is urgent to

develop mechanisms to empower developingcountries so that, for once, they will not bemarginalized and will be able to participatein—and contribute to—the emerging globalknowledge-based bioeconomy.

Aware of the importance of applying scienceand technology to agricultural production pro-cesses, and of the need to create strong linkagesbetween agriculture and industry, the UnitedNations Industrial Development Organization(UNIDO), together with IPBO, launched inMarch of 2010 the International IndustrialBiotech Net (IIBN) aimed at advocating,promoting, and facilitating access to industrialbiotechnology for the generation of value-added products from genetic resources of devel-oping countries. The IIBN will create a uniqueplatform for participatory, proactive, and pre-competitive sharing of new technologies,knowledge, and best practices for productdevelopment. Through this network we wantto seize and exploit the new opportunities thatbiotechnology offers to utilize biomass andbiodiversity for the production of bioenergyand added-value products derived from feedstocks that are renewable and environmentallyfriendly.

FINAL THOUGHTS

Like most scientific innovations that impactour society, the field of plant biotechnologydid not emerge from targeted research effortsto increase agronomic productivity. Rather, itis the by-product of curiosity and basic scien-tific questioning. Plant biotechnology is nowexpected to contribute to strategies for meet-ing the U.N. Millennium Development Goalsthe fact that it is even on the agenda for sucha noteworthy cause is the result of the scien-tific discovery of the Ti plasmid and the storythat spans the birth and growth of recombi-nant DNA technology. This discovery and therevolution that swept science in its wake aredramatic examples of how science works bestand how basic research can lead to practicalresults that were unimaginable when the re-search began. The story is also a significant


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example of how inquisitive scientific thoughtfreed from preconceptions can create an entirescientific culture and philosophical frameworkable to comprehend an increasingly complexlarger picture through attention to the minutestdetail.

Despite the fact that such enormousprogress has been made in fundamental science,the expected innovation boom did not happen.The molecular plant community is still smallcompared with other life science disciplines.Still, I believe that it will continue to grow oncethe value of the tremendously wide range ofpossible applications for humankind has beenrecognized.

Genetically engineered plants and plantbiotechnology have the potential to revolution-ize agriculture in a sustainable manner; improveenvironmental quality; yield new medicines; actas biofactories for the production of pharma-ceutical proteins (molecular pharming); gen-erate biofuels; contribute to a less-pollutingindustry; and profoundly improve the health,quality of life, and livelihood of mankind.

The economic and environmental benefitsof GM plants should, however, not be the soleprivilege of the developed world. Much workhas to be done to lessen and simplify the regu-latory burden and to make the technology freelyavailable for those who need it the most.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

I thank Dulce de Oliveira and Sylvia Burssens, who for the past ten years have helped IPBO bringplant biotechnology to developing and emerging countries and who both had a major input in thisarticle during this, for health reasons, difficult year. I am grateful to Patricia Zambryski, MarcelleHolsters, and Jan Leemans for controlling the history of the plant gene engineering; to MartineDe Cock, who for more than 30 years carefully and indefatigably helped prepare my manuscripts;and to Karel Spruyt for taking such good care of the photographic archives of our department. Iacknowledge the many hundreds of coworkers and students here in Ghent, who over four decenniashaped plant sciences. My deep gratitude goes to my spouse, Nora Podgaetzki, who from the firstday of our marriage in 1957 understood that science was a mission and accepted that I was activeall over the world, but seldom at home. This article is dedicated to the memory of Jeff Schell, whoinitiated the Agrobacterium research. Without his drive and sportive ambition I would not havehad the courage for these achievements.

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Annual Review ofPlant Biology

Volume 62, 2011

Contents

It Is a Long Way to GM AgricultureMarc Van Montagu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Anion Channels/Transporters in Plants: From Molecular Bases toRegulatory NetworksHelene Barbier-Brygoo, Alexis De Angeli, Sophie Filleur, Jean-Marie Frachisse,

Franco Gambale, Sebastien Thomine, and Stefanie Wege � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �25

Connecting the Plastid: Transporters of the Plastid Envelope andTheir Role in Linking Plastidial with Cytosolic MetabolismAndreas P.M. Weber and Nicole Linka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �53

Organization and Regulation of Mitochondrial Respiration in PlantsA. Harvey Millar, James Whelan, Kathleen L. Soole, and David A. Day � � � � � � � � � � � � � � � � �79

Folate Biosynthesis, Turnover, and Transport in PlantsAndrew D. Hanson and Jesse F. Gregory III � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 105

Plant Nucleotide Sugar Formation, Interconversion, and Salvageby Sugar RecyclingMaor Bar-Peled and Malcolm A. O’Neill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 127

Sulfur Assimilation in Photosynthetic Organisms: Molecular Functionsand Regulations of Transporters and Assimilatory EnzymesHideki Takahashi, Stanislav Kopriva, Mario Giordano, Kazuki Saito,

and Rudiger Hell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Signaling Network in Sensing Phosphate Availability in PlantsTzyy-Jen Chiou and Shu-I Lin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Integration of Nitrogen and Potassium SignalingYi-Fang Tsay, Cheng-Hsun Ho, Hui-Yu Chen, and Shan-Hua Lin � � � � � � � � � � � � � � � � � � � � 207

Roles of Arbuscular Mycorrhizas in Plant Nutrition and Growth:New Paradigms from Cellular to Ecosystem ScalesSally E. Smith and F. Andrew Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

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The BioCassava Plus Program: Biofortification of Cassava forSub-Saharan AfricaRichard Sayre, John R. Beeching, Edgar B. Cahoon, Chiedozie Egesi, Claude Fauquet,

John Fellman, Martin Fregene, Wilhelm Gruissem, Sally Mallowa, Mark Manary,Bussie Maziya-Dixon, Ada Mbanaso, Daniel P. Schachtman, Dimuth Siritunga,Nigel Taylor, Herve Vanderschuren, and Peng Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 251

In Vivo Imaging of Ca2+, pH, and Reactive Oxygen Species UsingFluorescent Probes in PlantsSarah J. Swanson, Won-Gyu Choi, Alexandra Chanoca, and Simon Gilroy � � � � � � � � � � � � 273

The Cullen-RING Ubiquitin-Protein LigasesZhihua Hua and Richard D. Vierstra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 299

The Cryptochromes: Blue Light Photoreceptors in Plants and AnimalsInes Chaves, Richard Pokorny, Martin Byrdin, Nathalie Hoang, Thorsten Ritz,

Klaus Brettel, Lars-Oliver Essen, Gijsbertus T.J. van der Horst,Alfred Batschauer, and Margaret Ahmad � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

The Role of Mechanical Forces in Plant MorphogenesisVincent Mirabet, Pradeep Das, Arezki Boudaoud, and Olivier Hamant � � � � � � � � � � � � � � � � 365

Determination of Symmetric and Asymmetric Division Planesin Plant CellsCarolyn G. Rasmussen, John A. Humphries, and Laurie G. Smith � � � � � � � � � � � � � � � � � � � � � � 387

The Epigenome and Plant DevelopmentGuangming He, Axel A. Elling, and Xing Wang Deng � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 411

Genetic Regulation of Sporopollenin Synthesis and PollenExine DevelopmentTohru Ariizumi and Kinya Toriyama � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 437

Germline Specification and Function in PlantsFrederic Berger and David Twell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 461

Sex Chromosomes in Land PlantsRay Ming, Abdelhafid Bendahmane, and Susanne S. Renner � � � � � � � � � � � � � � � � � � � � � � � � � � � � 485

Evolution of PhotosynthesisMartin F. Hohmann-Marriott and Robert E. Blankenship � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 515

Convergent Evolution in Plant Specialized MetabolismEran Pichersky and Efraim Lewinsohn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 549

Evolution and Diversity of Plant Cell Walls: From Algaeto Flowering PlantsZoe Popper, Gurvan Michel, Cecile Herve, David S. Domozych,

William G.T. Willats, Maria G. Tuohy, Bernard Kloareg,and Dagmar B. Stengel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 567

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