Home Page Link AgBioWorld Home Page
About AgBioWorld Donations Ag-Biotech News Declaration Supporting Agricultural Biotechnology Ag-biotech Info Experts on Agricultural Biotechnology Contact Links Subscribe to AgBioView Home Page

AgBioView Archives

A daily collection of news and commentaries on
ag-biotech.


Subscribe AgBioView Subscribe

Search AgBioWorld Search

AgBioView Archives

Subscribe

 


SEARCH:     

Date:

November 25, 2000

Subject:

The "Golden Rice" Tale: Potrykus

 

The "Golden Rice" Tale

INGO POTRYKUS

Turning Point Article

Professor Emeritus, Institute of Plant Sciences, Swiss Federal Institute
of Technology, ETH Centre, LFW 53.1, CH-8092 Zürich, Switzerland
(potrykus@active.ch)

(received October 23, 2000; editor I.K. Vasil)

"Golden Rice" is, to date, a popular case – supported by the scientific
community, the agbiotech industry, the media, the public, the Consultative
Group on International Agricultural Research (CGIAR), the Food and
Agriculture Organization of the United Nations (FAO), the World Health
Organization (WHO), official developmental aid institutions, etc., but
equally strongly opposed by the opponents of genetically modified
organisms (GMOs). The first group likes "Golden Rice" because it is an
excellent example of how genetic engineering of plants can be of direct
benefit to the consumer, especially the poor and the disadvantaged in
developing countries, where GMOs offer many more opportunities for the
improvement of livelihood than for those living in well-fed developed
nations.

The GMO opposition, however, is concerned that "Golden Rice" will be a
kind of "Trojan Horse", opening the developing countries to other
applications of the GMO technology, and for improving acceptance of GMO
food. Indra Vasil persuaded me to write the Golden Rice Tale because the
background behind this success, which is embedded in numerous failures and
obstacles, and which covers the entire history of the development of plant
genetic engineering, might be of interest to those who are faced with the
numerous specific problems of strategic research, where the target is set
at the outset, where no attractive alternatives to existing academic
questions are available, where success is measured in relation to the
original target, and not in relation to possible attractive academic
solutions.

Motivation and technique development (1972 to 1987)

My scientific career and my interest in "genetic engineering" began in
1970 with the first protoplast experiments with Petunia in the laboratory
of Professor D. Hess in Stuttgart-Hohenheim, Germany. We regenerated
fertile plants from mesophyll protoplasts (Durand et al. 1973), introduced
isolated nuclei (Potrykus and Hoffmann 1973) and chloroplasts (Potrykus
1973) into protoplasts, and treated protoplasts with naked DNA in an
attempt to transfer genes (Hess et al. 1973). In one exciting experiment
we used DNA from a dominant red-flowering pure line of Petunia to
transform protoplasts of a recessive white flowering pure line. We
expected pink-flowering plants in case of success. When we finally
recovered a greenhouse full of pink-flowering plants, we realized that
something had gone wrong. As far as we could reconstruct, we had taken
leaves for protoplast isolation from a population of young heterozygous
plants that were grown in the same greenhouse to take advantage of the
heterozygous state for anther culture experiments. We fortunately had not
published, but on the basis of this experience I was very skeptical when
Peter Carlson reported about his famous N. glauca x N. langsdorfii
"somatic hybrids". Already at that time (1972) there were claims (from
those working with tobacco and petunia) that the new technology would
contribute to food security in developing countries. Obviously, to
contribute, one would have to work with important crop plants and not only
talk about them.

Even at the peak of success of the Green Revolution it was clear that
feeding the exploding population in developing countries would require
intensive new scientific research. I therefore began in 1973 to work
towards the development of the new technology for cereals (beginning with
barley) and tried to repeat what had been so easy with Petunia. Our
efforts gained the attention of the late Professor G. Melchers who
arranged for the opportunity to establish a small research group at the
Max-Planck-Institute for Plant Genetics in Ladenburg/Heidelberg. With
Emrys Thomas and Gerd Wenzel I had enthusiastic colleagues and with Horst
Lörz and Christian Harms very motivated graduate students. We all were
focusing on cereal tissue and cell culture, and could regenerate plants
from various tissues of wheat, maize, barley, and rye (Potrykus et al.
1976, 1977). However, a mesophyll protoplast regeneration system could
not be developed despite the fact that I challenged, with a most
sophisticated microdrop array cell culture protocol, more than 120,000
protoplast culture conditions including up to 7-factor gradient mixtures
of all known growth substances in a wide range of concentrations (Potrykus
et al. 1978). The best I could achieve with wheat mesophyll protoplasts
was the formation of ca. 60-celled "globular proembryos", which, however,
refused to develop further. This cereal work found the attention of the
Agrodivision of Ciba-Geigy which intended to complement the
Pharma-oriented research in the recently established Friedrich
Miescher-Institute (FMI) in Basel, Switzerland (a foundation for basic
research) with agrobiotechnology-oriented research (yes, already in
1975!).

The institute offered me in 1976 the task of establishing three Plant
Biology groups, which I tried to coordinate around genetic engineering,
mutagenesis, and haploids of cereals with my colleagues Patrick King and
Emrys Thomas (Potrykus et al. 1979). As the mesophyll protoplast approach
remained very recalcitrant, we studied the loss of competence during the
course of leaf differentiation. We found that beyond the basal 3 mm of a
young cereal leaf cells are terminally differentiated. We thus looked for
alternatives such as somatic embryos inducible from the basal leaf
segments, especially effective in Sorghum (Wernicke et al. 1981, 1982),
and the re-meristematizing response of maize tissue to Ustilago maydis
infection.

At the beginning of the 1980s it became evident that the crown gall tumor
was based on a natural transformation process. Not surprisingly, many
laboratories focused on the development of a transformation protocol based
on T-DNA transfer. As this was based on a wound-response leading to a
wound-meristem to allow for the proliferation of T-DNA transformed cells,
and as we knew that gramineous species have a wound response leading to
death of wound-adjacent cells, we could not believe in a future for
Agrobacterium and cereals (Potrykus 1990, 1991). Consequently, we were
focusing on the development of a vector-independent transformation system:
"direct gene transfer" via incubating protoplasts in DNA. This was
strongly supported through two hard-core molecular biologists joining the
FMI. Barbara and Thomas Hohn were attracted by the scientific potential of
the new research area in the institute. This close collaboration between
the tissue culture specialists and the molecular biologists soon produced
the Agrobacterium-independent transformation technique (Brisson et al.
1984, Paszkowski et al. 1984, Schocher et al. 1986). Instrumental in this
was Jerszy (Jurek) Paszkowski who joined my group as a fresh Ph.D. from
Warzawa (Poland) and who was the perfect link between the two groups. Only
half a year after the first Agrobacterium-mediated tobacco was reported we
could publish the first "direct gene transfer"-derived tobacco. However,
this was tobacco and not any cereal. It took two more years to produce the
first transgenic maize cell culture, but this then was a non-morphogenic
cell line (Potrykus et al. 1985).

The next breakthrough came from Indra Vasil’s concept of preventing
differentiation in cereals by establishing "embryogenic suspensions". This
eventually led to the development of the embryogenic callus-suspension
culture-protoplast systems for cereals, which as shown later, played a
critical role in the production of transgenic cereals (Vasil 1999).
Attempts to transform embryogenic cultures with Agrobacterium did not
yield convincing results. However, this was no longer necessary because by
then John Sanford and Ted Klein had invented the "crazy" biolistic
transformation method (Sanford 2000), which was used successfully for the
regeneration of transgenic plants in tobacco, cotton, etc. Embryogenic
suspensions were the ideal material for biolistic treatment and it was to
be expected that, with the necessary effort, it would produce transgenic
cereals. Embryogenic suspensions were, however, also the only source of
totipotent protoplasts of cereals. We chose this approach for our work
(Vasil and Vasil 1992).

At the end of 1985, I was offered a full professorship at the Swiss
Federal Institute of Technology (ETH) in Zürich. I was responsible for
building, together with the professor in crop physiology (Josef
Nösberger), a new institute, combining both basic and applied research.
This Institute of Plant Sciences was the ideal setting for my intentions
(which increasingly focused on the development and use of genetic
engineering technology to contribute to food security in developing
countries), and it provided longer range independent and stable financing
for continued approaches to genetic engineering of cereals. At the same
time Swapan Datta joined my group and this was the beginning of our work
with rice and an important turning point.

Focusing on rice as the outstanding food security crop (1987-1999)

Swapan Datta was accompanied by his talented wife Karabi. Swapan had
joined me to learn from our protoplast experience but I was following at
that time another idea. As the embryogenic response was rather
genotype-dependent and as I wanted a generally applicable technique, I
decided to challenge early sexual embryos down to isolated zygotes. For
transformation I wanted to optimize the micro-injection technique (Kost et
al. 1995, Lusardi et al. 1994). I convinced two scientists with exciting
experience in microinjection (Gunther Neuhaus and German Spangenberg) to
join me in Zürich for this purpose, and I invested the entire group,
except for Jerszy Paszkowski who was free to work with his group on
homologous recombination (Paszkowski et al. 1988, Baur et al. 1990) on the
isolation, culture and microinjection of sexual proembryos from wheat,
maize, rice, and later Arabidopsis. After many years of enormous effort,
we found much to our disappointment that the microinjection system worked
fine with protoplasts, but was extremely bad with cells surrounded by a
cell wall. With Christof Sautter we also focused on the development of
mirotargeting as a further genotype-independent transformation technique
(Sautter et al. 1991, Leduc et al. 1994a, 1994b, Sautter et al. 1995),
which too did proved to be ineffective for routine transformation.
Swapan Datta asked for permission to work part-time (over the weekends) on
the embryogenic protoplast transformation approach. And the Dattas made
it. There came hygromycin-resistant rice, the first transgenic Indica rice
(Datta et al. 1990, 1992, Peterhans et al. 1990, Linn et al. 1995), all
from embryogenic protoplasts and direct gene transfer.

Swapan introduced me to Gary Toenniessen and the Rockefeller Foundation,
which by that time had already spent considerable funds in the Rice
Biotechnology Program on colleagues many of whom were not really using
these funds for rice work. By 1990 we also were receiving Rockefeller
funding (for development of Indica rice transformation protocol) and we
were producing the first insect-resistant Indica rice (Wünn 1996). We had
been unsuccessfully using wild type Bt and it took us one year of
bargaining until we were allowed to use the synthetic Ciba-Geigy gene. At
the beginning of 1990 I had also learned that "food security for
developing countries" not only had a quantity aspect, but also a quality
component. The major malnutritions were identified with "iron > iodine >
vitamin A" and this was the beginning of the "Golden Rice" adventure, and
another major turning point.

The problem of iron- and vitamin A-deficiency and traditional solutions

IDA (iron deficiency anemia), the most common nutritional disorder in the
world, impairs immunity and reduces the physical and mental capacities of
people of all ages. In infants and young children even mild anemia can
impair intellectual development. Anemia in pregnancy is an important cause
of maternal mortality, increasing the risk of hemorrhage and sepsis during
childbirth. Infants born to anemic mothers often suffer from low birth
weight and anemia themselves. An inadequate dietary iron intake is the
main cause of IDA. According to UNICEF, nearly two billion people are
estimated to be anemic and about double that number, or 3.7 billion are
iron deficient, the vast majority of them women. In Africa and Asia UNICEF
estimates that IDA contributes to approximately 20 per cent of all
maternal deaths.

Each year more than one million VAD (vitamin A-deficiency) associated
childhood deaths occur. And, according to the World Health Organization,
as many as 230 million children are at risk of clinical or subclinical
VAD, a condition which is largely preventable. VAD makes children
especially vulnerable to infections and worsens the course of many
infections. Supplementation with vitamin A is estimated by UNICEF to lower
a child’s risk of dying by approximately 23 percent. VAD is also the
single most important cause of blindness among children in developing
countries, about 500,000 per year.

Rice plants do not produce carotenoid compounds in the grain consumed by
humans. Consequently VAD often occurs where rice is the major staple food.
The amount of bioavailable iron is dependent both on the level of dietary
iron consumption and on iron absorption during the digestive process.
Dietary iron in developing countries consists primarily of non-heme iron
of vegetable origin, whose poor absorption is considered a major factor in
the etiology of iron deficiency anemia. Also legume staples and grains,
including rice, are high in phytic acid, which is a potent inhibitor of
iron absorption. Foods that enhance non-heme iron absorption such as
fruits and vegetables rich in ascorbic acid are often limited in
developing countries. Heme iron, which is relatively well absorbed by the
human intestine, is found primarily in foods containing blood and muscle.
Due to their expense and lack of availability, heme iron-rich foods are
often a negligible part of a typical developing country diet.

Interventions applied, so far, to reduce both IDA and VAD are (a)
supplementation (e.g. distribution of vitamin A capsules), (b) food
fortification (e.g. adding iron to wheat flower), and (c) dietary
education and diversification. In a FAO/WHO World Declaration on Nutrition
(1992) the following strategy has been advocated: "Ensure that sustainable
food-based strategies are given first priority particularly for
populations deficient in vitamin A and iron, favoring locally available
foods and taking into account local food habits." "Supplementation should
be progressively phased out as soon as micronutrient-rich food-based
strategies enable adequate consumption of micronutrients." And Per
Pinstrup-Andersen, Director General of the International Food Policy
Research Institute has pointed out that a sustainable solution of the
problem will come only when it will be possible to improve the content of
the missing micronutrients in the major staple crops. This was exactly
what we were trying to achieve. As the necessary genes for such an
improvement were not available in the rice gene pool, genetic engineering
was the only technical possibility. As rice endosperm did not contain any
provitamin A, the task was to introduce the entire biochemical pathway. As
rice endosperm contains very little iron and considerable amounts of a
potent inhibitor of iron resorption, and as resorption from a vegetarian
diet is generally poor, the task was to increase the iron content, reduce
the inhibitor content, and add a resorption-enhancing factor.

Solving the scientific problems: "Golden Rice" (1992 to 1999)

Peter Burkhardt joined me in 1991 for a Ph.D. thesis work and it was not
difficult to motivate him for the provitamin A-project. I approached
Nestle, the world’s biggest food company, for funding but Nestle was
(fortunately) not interested. This was "fortunate" in retrospect because
it kept the project open for public funding with its important
consequences for later free distribution to developing countries. With
Peter Beyer at the nearby University of Freiburg, Peter Burkhardt found
the ideal scientific supervisor, and I found a perfect partner. Peter
Beyer was studying the regulation of the terpenoid pathway in daffodil and
was working on the isolation of those genes we would need to establish the
pathway in rice endosperm. We approached the Rockefeller Foundation for
funding and Gary Toenniessen responded with the organization of a
brainstorming session in New York (1992). Many of the participants thought
that such a project did not have much chance of success, but because of
its potential importance it was worth trying. Peter Burkhardt found out
that the last precursor of the pathway in endosperm was
geranlygeranyl-pyrrophosphate and consequently, theoretically, it should
be possible to reach b-carotene via four enzymes: phytoene synthase,
phytoene desaturase, z-carotene desaturase, and lycopene cyclase. There
were hundreds of scientific reasons why the introduction and coordinated
function of these enzymes would not be expected to work, and that it may
cause many problematic side effects.

Those with the necessary scientific knowledge were right in not believing
in the experiment. When we finally had "Golden Rice" I learned that even
my partner, Peter Beyer and the scientific advisory board of The
Rockefeller Foundation, except for Ralph Quatrano, had not believed that
it could work. This exemplifies the advantage of my ignorance and naivete:
with my simple engineering mind I was throughout optimistic, and
therefore, carried the project through, even when Rockefeller stopped
funding of Peter Beyer’s group. Altogether it took eight years but the
first breakthrough came when Peter Burkhardt recovered phenotypically
normal, fertile, phytoene synthase-transgenic rice plants, which produced
good quantities of phytoene in their endosperm (Burkhardt et al. 1997).
This demonstrated two important facts: it was possible to specifically
deviate the pathway towards b-carotene, and channeling a lot of GGPP away
from the other important pathways had no severe consequences on the
physiology and development. This success encouraged me to motivate a
further Ph.D. student, Paola Lucca, with an MS in pharmacy, to work on the
problem of iron deficiency. More on this later, when the provitamin A
story has been completed. The next gene to follow was phytoene desaturase,
and this caused problems for more than a year. Peter Burkhardt could
obtain only heavily distorted transgenic plants. As he left the lab I
transferred the continuation of the project to my postdoc Andreas Klöti,
who had done excellent work towards engineering RTBV tungro disease
resistance (Fütterer et al. 1997, Klöti et al. 1999) and gene silencing,
and was happy about an "easier" task. Andreas continued with single gene
transformations and the concept was to combine the genes via crossing. We
had used biolistic transformation of embryogenic suspensions and
precultured immature embryos and had the typical complex integration
pattern. And this caused the longer the more, problems with gene stability
and fertility.

When we finally had transgenic plants for all genes separately, we could
combine genes pairwise but all this did not look too promising. By that
time Andreas left the lab and the project was transferred to Xudong Ye,
who had done a Ph.D. in forage grass biotechnology in my group, under the
supervision of German Spangenberg (Takamizo et al. 1992, Wang et al.
1992). Xudong had survived a tough training and he had learned that
success in strategic research may require hard work. Xudong wanted to
invest only one year because he had plans to go to the U.S. He analyzed
the situation and decided, after discussions with Salim Al-Babili from
Peter Beyer’s group and our man behind many constructs, and Andreas, to
try a radical change in the approach: (a) change from biolistic to
Agrobacterium-mediated transformation, (b) use the Erwinia double
desaturase (crtI), and (c) introduce all genes together in a single
co-transformation experiment. Xudong recovered ca. 500 independent
transgenic lines. As our glasshouse had space only for 50 of them he
discarded 450 and grew the 50 best looking ones to maturity. Peter Beyer
polished the seeds, analysed them with HPLC, took beautiful photographs
and presented them to me at the farewell symposium I had organized on 31
March 1999, the date I had to retire because I had passed the age limit.
At this symposium Xudong Ye presented the results for the first time to
the public: the endosperm contained good quantities of provitamin A,
beautifully visible as "golden" color of different intensity in different
lines. The best provitamin A line had 85% of its carotenoids being
b-carotene. Other lines had less b-carotene, but interesting levels of
lutein and zeaxanthin, both substances of nutritional importance because
they have positive effects with regards to macula degeneration (Ye et al.
2000).

Development of "high iron rice" (1995-2000)

At the same farewell symposium Paola Lucca reported about her "high iron
rice". Iron deficiency is the biggest medical problem. This malnutrition
affects more than two billion humans, predominantly women and children.
Consequences are millions of birth-related deaths of mothers and children.
It impairs physical and intellectual development, the immune system, and
fitness. Concerning rice as the major staple there are three key problems:
(a) no other crop contains as little iron, (b) phytate, the phosphate
storage for seed germination is an extremely efficient inhibitor of iron
resorption (up to 98% of available iron can be blocked), and (c)
resorption from a vegetarian diet is rather poor. Our scientific advisor
for the project was Richard Hurrell, ETH professor for human nutrition,
with specialization in iron nutrition. Paola approached an improvement on
all three lines. Knowing that only 5% of the iron in the rice plant is in
the seed she created a sink for iron storage in the endosperm by
expressing a ferritin gene from Phaseolus (our request for funding was
turned down with the argument that we better study iron uptake into the
rice plant!) This led to a 2.5-fold increase in endosperm iron content. As
feeding studies with peptides from muscle tissue had shown that
cystein-rich polypeptides enhance iron resorption, Paola expressed an
appropriate gene, a metallothionin-like gene from Oryza and achieved a
7-fold increase in endosperm cystein. As it appeared unwise to interfere
with the phosphate storage (the inhibitor phytate) prior to germination,
Paola decided to approach inhibitor degradation after cooking. Thanks to
the permission from Hoffmann LaRoche, Basel, we could use a thermotolerant
mutant of a phytase from Aspergillus fumigatus, which refolded to 80%
activity after 20 minutes at 100ºC. To prevent activity which could
interfere with germination, the enzyme was excreted into the extracellular
space. One transgenic line expressed the phytase to levels 700-fold higher
than endogenous phytase. In small intestine simulation experiments the
phytase degraded phytate to zero levels within one hour at 37ºC. However,
much to everybody’s surprise, in the transgenic situation the enzyme did
not refold properly after cooking and had lost it’s thermotolerance. New
transgenic plants are meanwhile maturing, where the enzyme has been
targeted to the phytase storage vesicles to reduce the phytate content
directly. With the experience meanwhile available from low phytase
mutants, we hope that this will not too much affect germination. The three
"iron genes" are combined with the "provitamin A genes" by crossing.
Vitamin A supply is strategy No. 4 against iron deficiency, as it has been
shown that vitamin A-deficiency indirectly interferes with iron resorption
(Lucca et al. 2000a).

Attaining public recognition (2000)

The vitamin A rice project was considered a scientific breakthrough
because it was the first case of pathway engineering, and it was
representing also a considerable technical advancement. We felt it also
was a timely and important demonstration of positive achievements of the
GMO technology. GMO technology had been used to solve an urgent need and
to provide a clear benefit to the consumer, and especially to the poor and
disadvantaged. To make the information available to a wider audience for a
more balanced GMO discussion, we submitted the manuscript to Nature with a
covering letter explaining its importance in the present GMO debate. The
Nature editor did not even consider it worth showing the manuscript to a
referee and sent it back immediately. Even supportive letters from famous
European scientists did not help. From other publications in Nature at
that time we got the impression that Nature was more interested in cases
which would rather question instead of support the value of genetic
engineering technology.

Fortunately, Peter Raven (Missouri Botanical Garden, St. Louis, MO, USA),
had heard about the "Golden Rice", and asked for more information, and
invited me in the last minute to present the work to the XVI International
Botanical Congress, August 1999 at St. Louis. He also took care of a press
conference and encouraged Science to look at the manuscript. Science was
interested in publishing both the pro-vitamin A case as well as the
iron-case in one publication, but the space it could provide was too
narrow for both (Ye et al. 2000). The iron-rice publication is soon coming
in Theoretical Applied Genetics (Lucca et al. 2000a). The press conference
in St. Louis, the presentation at the Nature Biotechnology Conference in
London, the Science publication with the commentary (Guerrinot 2000) the
feature story in TIME Magazine all led to an overwhelming coverage of the
"Golden Rice" story on TV, radio, and in the international press. A simple
example illustrates the difference in attitude between Europe and the rest
of the world. When the feature story came out in TIME Magazine (31 July
2000) it was planned that it would appear in the European edition the
following week. It has not shown up until now (12 November 2000).

The challenge of donating a GMO to poor countries (1999-open)

"Golden Rice" was developed for the vitamin A-deficient and iron-deficient
poor and disadvantaged in developing countries. To fulfill this goal it
has to reach the subsistence farmers free of charge and restrictions.
Peter Beyer had written up a patent application and the inventors, Peter
and myself, were determined to make the technology freely available. As
only public funding was involved this was not considered too difficult.
The Rockefeller Foundation had the same concept, the Swiss Federal
Institute of Technology supported it, but the European Commission had a
clause in it’s financial support to Peter Beyer, stating that industrial
partners of the "Carotene plus" project, of which our rice project was a
small part, would have rights on project results (The IVth and Vth
framework of EU funding forces public research into coalitions with
industry and thus is responsible for two very questionable consequences:
Public research is oriented towards problems of interest to industry, and
public research is loosing it’s independence). We did not consider this
too big a problem because the EU funding was only a small contribution at
the end of the project. But we realized soon that the task of technology
transfer to developing countries, the international patent application,
and the numerous Intellectual Property Rights (IPRs) and Technical
Property Rigths (TPRs) we had used in our experiments, were too much for
two private persons to be handled properly. We urgently (because of the
deadline of the international patent application) needed a powerful
partner. In discussions with industry the definition of "subsistence
farmer" and "humanitarian use" was the most difficult problem to be
solved. We wanted a definition as generous as possible, because we not
only wanted the technology free for small-scale farmers, we also wanted to
contribute to poverty alleviation via local commercial development. Very
fortunately the company which agreed to the most generous definition was
also the company which had legal rights because of its involvement in the
EU-project. This facilitated the agreement, via a small licensing company
(Greenovation), with Zeneca. Zeneca received an exclusive license for
commercial use and in return supports the humanitarian use via the
inventors for developing countries. The cut-off line between humanitarian
and commercial is $ 10,000 - income from "Golden Rice". This agreement
also applies for all subsequent applications of this technology to other
crop plants. It turned out that our agreement with Zeneca and the
involvement of our partner in Zeneca, Adrian Dubock, was a real asset to
the development of the humanitarian project. He was very helpful in
reducing the frightening number of IPRs and TPRs and he organized most of
the free licenses for the relevant IPRs and TPRs such that we are now in
the position of having reached "freedom-to-operate" for public research
institutions in developing countries to go ahead with breeding and de-novo
transformation into best adapted local varieties. Publicity sometimes can
be helpful: only few days after the cover of "Golden Rice" had appeared on
TIME Magazine, I had a phone call from Monsanto offering free licenses for
the company’s IPR involved. A really amazing quick reaction of the PR
department to make best use of this opportunity.

Making best use, not fighting patens helps the poor and underprivileged

At this point it is appropriate to add a more general comment on patents
and the heavy opposition against patenting in life sciences. As we did
not know how many and which intellectual property rights we had used in
developing the "Golden Rice", and as further development for the
humanitarian purpose required "freedom-to-operate" for the institutions
involved, The Rockefeller Foundation commissioned an IPR audit through
ISAAA. The outcome was shocking (ISAAA briefs No. 20-2000). There were 70
IPRs and TPRs belonging to 32 different companies and universities, which
we had used in our experiments and for which we would need free licenses
to be able to establish a "freedom-to-operate" situation for our partners,
who were keen to begin further variety development. As I was in addition
blocked by an unfair use of a material transfer agreement, which had no
causal relation to "Golden Rice" development, I was rather upset. It
seemed to me unacceptable, even immoral, that an achievement based on
research in a public institution and with exclusively public funding, and
designed for a humanitarian purpose, was in the hands of those who had
patented enabling technology early enough or had sneaked in a MTA in
context of an earlier experiment. It turned out that whatever public
research one was doing, it was all in the hands of industry (and some
universities).

At that time I was much tempted to join those who radically fight
patenting. Fortunately I did a bit further thinking and became aware that
"Golden Rice" development was only possible because there was patenting.
Much of the technology I had been using was publicly known because the
inventors could protect their right. Much of it would have remained secret
if this had been the case. If we are interested to use all the knowledge
to the benefit of the poor, it does not make sense to fight against
patenting. It makes far more sense to fight for a sensible use of
intellectual property rights. Thanks to the public pressure there is a lot
of goodwill in the leading companies to come to an agreement on the use of
IPR/TPR for humanitarian use which does not interfere with commercial
interests of the companies.

There was a recent satellite meeting in context with the World Food Prize
Symposium 2000 at Des Moines, Iowa, which surfaced agreements on this line
between all participants, including major agboitech companies (for more
information contact C.S.Prakash; e-mail: prakash@acd.tusk.edu).

The challenge of safe technology transfer (2000-open)

Having solved the scientific problems, and having achieved freedom to
operate, leaves technology transfer as the next hurdle. This is a far
bigger task that anyone having no personal experience should assume.
"Golden Rice" is, of course GMO and this fact is sufficient to cause a
series of further problems. All care has to be taken that it is handled
according to established rules and regulations (where these do not exist,
they have to be established). And, of course, GMO is faced with emotional
and irrational opposition. Rational concerns and questions are taken care
of by the established regulations. Let us focus first on safe technology
transfer. Again we realized that we needed help, because this task is
beyond the capabilities of a retired professor (a private person) and an
already overworked associate professor with no infrastructure and heavy
teaching load. We established a "Golden Rice Humanitarian Board" to help
make the right decisions, and to have secretarial support. Again our
decision to work with Zeneca was extremely helpful. Adrian Dubock was
willing to care for the task of the secretary. We have additional
invaluable help from Katharina Jenny from ISCB (Indo-Swiss Collaboration
in Biotechnology), an institution jointly financed by the Indian
Department of Biotechnology (DBT) and the Swiss Development Corporation.
Golden Rice will be introduced into India in the established
organizational framework of ISCB, which has ten years of experience in
technology transfer. Thanks to this situation and thanks to the strong
commitment of the DBT and the Indian Council for Agricultural Research
(ICAR), India will take a leading role and can serve as a model for other
countries. The project starts with a careful needs assessment, analyzing
and comparing pros and cons of alternative measures and setting a
framework for optimal and complementary use of "Golden Rice". Of course,
there will be bioavailability, substantial equivalence, toxicology, and
allergenicity assessments and we are grateful for offers from specialists
to help. Careful socio-economic and environmental impact studies will help
to avoid any possible risk and make sure that the technology indeed
reaches the poor. Care will be taken that the material is given only to
institutions, which ensure proper handling according to rules and
regulations. Traditional breeding will transfer the trait into locally
best adapted lines, and again will make sure that varieties important to
the poor will be used and not fashionable varieties for the urban middle
class. There will be also direct de-novo transformation into important
varieties, and this will be done with mannose selection (Lucca et al.
2000b). "Golden Rice" so far has a hygromycin resistance gene, as it has
been introduced via co-transformation breeding has a chance to separate it
from the pro-vitamin A trait. All this costs a lot of money, which should
not affect the free distribution to subsistence farmers. Fortunately,
probably the World Bank, ICAR and DBT will share the costs for this
development in India. Agreements have been established with several
institutions in Southeast Asia, China, Africa, and Latin America and as
soon as the written confirmation of the "freedom-to-operate" is in the
hands of the "Humanitarian Board", material will be transferred.

The challenge of the GMO opposition (2000-open)

"Golden Rice" fulfills all the wishes the GMO opposition had earlier
expressed in their criticism of the use of the technology, and it thus
nullifies all the arguments against genetic engineering with plants in
this specific example.
* Golden Rice has not been developed by and for industry.
* It fulfills an urgent need by complementing traditional interventions.
* It presents a sustainable, cost-free solution, not requiring other
resources.
* It avoids the unfortunate negative side effects of the Green Revolution.
* Industry does not benefit from it.
* Those who benefit are the poor and disadvantaged.
* It is given free of charge and restrictions to subsistence farmers.
* It does not create any new dependencies.
* It will be grown without any additional inputs.
* It does not create advantages to rich landowners.
* It can be resown every year from the saved harvest.
* It does not reduce agricultural biodiversity.
* It does not affect natural biodiversity.
* There is, so far, no conceptual negative effect on the environment.
* There is, so far, no conceivable risk to consumer health.
* It was not possible to develop the trait with traditional methods, etc.
Optimists might, therefore, have expected that the GMO opposition would
welcome this case. As the contrary is the case, and GMO opposition is
doing everything to prevent "Golden Rice" reaching the subsistence farmer,
we have learned that GMO opposition has a hidden, political agenda. It is
not so much the concern about the environment, or the health of the
consumer, or the help for the poor and disadvantaged. It is a radical
fight against a technology and for political success. This could be
tolerated in rich countries where people have a luxurious life also
without the technology. It can, however, not be tolerated in poor
countries, where the technology can make the difference between life and
death, and health or severe illness. In fighting against "Golden Rice"
reaching the poor in developing countries, GMO opposition has to be held
responsible for the foreseeable unnecessary death and blindness of
millions of poor every year.

Opportunities from remaining scientific challenges (future)

My retirement came too early. We have been working on challenges which
might be worth taking up by other institutions. We have been working on
projects to rescue lost harvests and have been successful with insect pest
resistance, had success with wheat (Clausen et al. 2000) with fungal
resitance, but not with rice despite thousands of transgenic rice lines
with genes for most peptides with antifungal effects. We had also no rice
lines resistant to RTBV tungro disease despite excellent research of the
group of Johannes Fütterer over more than eight years. We were also
interested in better exploitation of natural resources and here are two
projects I would continue, if the necessary resources were available: We
convinced ourselves that engineering of C4 photosynthesis is feasible. We
do not think that simple transfer of one or two genes from maize is going
to do it. However, we know now that rice (and wheat) have the necessary
Krantz anatomy with bundle sheath cells surrounded by mesophyll cells, and
we have bundle sheath-specific and mesophyll-specific promoters that work
in rice as a basis to engineer the appropriate enzymes cell-specifically
to hopefully establish the crucial CO2 gradient. We also have been working
towards N2 fixation and our approach was to engineer the nif-regulon into
the chloroplast genome, maintaining it’s operon structures and regulating
it cell-specifically in such a way that it is activated from its natural
regulator gene placed separately into the nuclear genome. Expression would
be in the tissue with the lowest possible oxygen tension, the cortex, and
the signal to start expression would come from the signal which starts
degradation of the photosynthetic apparatus to provide nitrogen for the
protein requirement during the grain-filling period. The idea would not be
to make rice totally independent of external supply of nitrogen, but to
provide additional nitrogen during grain filling to rescue the
photosynthetic apparatus for a longer periode of time. To be able to do so
we need chloroplast transformation in rice and I had, therefore,
established an entire group with Roland Bilang, which did excellent work
over five years, but could not establish a functional protocol. And
despite an existing publication in Nature Biotechnology, I have the
impression that nobody has, so far, such a protocol. As plastid
transformation in meristematic or embryogenic cells poses far bigger
problems than with chloroplasts in mesophyll cells, I was still interested
in the old probem of cereal mesophyll protoplast culture. And,
surprisingly, R.V.Sairam convinced me that this is possible in principle
by repeating his work from ICRISAT with Sorghum mesophyll protoplasts in
my laboratory (Sairam et al. 2000); he could, however, not establish a
system efficient enough to be used for chloroplast transformation.

Epilogue

"Golden Rice" was possible because
* I had stable, public funding over a long period of time which I could
use independent of the opinion of others.
* I had with Peter Beyer the perfect partner, who understood the
underlying science and provided the necessary genes and analytical
expertise.
* The Rockefelller Foundation was willing to add substantial financial
support over a long time period (special thanks to Gary Toenniessen and
Ralph Quatrano).
* The Swiss Federal Institute of Technology supported the concept of
strategic research for developing countries.
* The project was embedded in an enthusiastic group of coworkers (over
60), all motivated to contribute to food security with their work.
* I was naive enough to believe in it’s success.
"Golden Rice" hopefully helps
* to achieve better acceptance of GMO technology,
* to encourage scientists and granting agencies to invest also into
projects with no a priori guarranteed success,
* to motivate public research to care more for the problem of food
security and less for additional funds from industry,
* to encourage those who have rights in key enabling technology to make
free licences available for humanitarian projects,
* for some scientists to consider that there can be more in a scientific
carreer than the chace for impact factor points, and
* to have some GMO opponents consider whether a differentiated discussion
of the GMO technology might not be the better strategy in the long run.

References

Baur, M.; Potrykus, I.; Paszkowski, J. Intermolecular homologous
recombination in plants. Mol.Cell. Biol. 10:492-500; 1990.

Brisson, N.; Paszkowski, J.; Penswick, J.; Gronenborn, B.; Potrykus, I;
Hohn, T. Expression of a bacterial gene in plants using a viral vector.
Nature 310:511-514; 1984.

Burkhardt, P.K.; Beyer, P.; Wünn, J.; Klöti, A.; Armstrong, G.; Schledz,
M.; von Lintig, J.; Potrykus, I. Transgenic rice (Oryza sativa) endosperm
expressing daffodil (Narcissus pseudonarcissus) phytoene syntahse
accumulates phytoene, a key intermediate of provitamin A biosynthesis.
Plant J. 11:1071-1078; 1997.

Clausen, M.; Krauter, R.; Schachermeyer, G.; Potrykus, I.; Sautter, C.
(2000) Antifungal activity of a virally encoded gene in transgenic wheat.
Nature Biotechnology 18:446-449; 2000.

Datta, S.K.; Peterhans, A.; Datta, K.; Potrykus, I. Genetically engineered
fertile Indica-rice plants recovered from protoplasts. Bio/Technology
8:736-740; 1990.

Datta, S.K.; Datta, K.; Soltanifar, N.; Donn, G.; Potrykus, I. Herbicide
resistant Indica rice plants from Indica breeding line IR72 after
PEG-mediated transformation of protoplasts. Plant Mol. Biol. 20:619-629;
1992.

Durand,J.; Potrykus,I.; Donn,G. Plantes issues de protoplasts de Petunia.
Z. Pflanzenphysiol. 69:24-32; 1973.

Fütterer J.; Rothnie, H.M.; Hohn, T.; Potrykus, I. Rice tungro bacilliform
virus open reading frames II and III are translated from polycistronic
pregenomic RNA by leaky scanning. J. Virol. 71:7984-89; 1997.

Guerinot, M.L. The Green Revolution strikes Gold. Science 287:241-43; 2000.

Hess, D.; Potrykus, I.; Donn, G.; Durand, J.; Hoffmann, F. Transformation
experiments in higher plants: prerequisites for the use of isolated
protoplasts.
Colloques Internationales CNRS 212:343-351; 1973.

Klöti, A.; Henrich, C.; Bieri, S.; He. X.; Chen, G.; Burkhardt, P.K.;
Wünn, J.; Lucca, P.; Hohn, T.; Potrykus, I.; Fütterer, J. Upstream and
downstream sequence elements determine the specifity of the rice tungro
bacilliform virus promoter and influence RNA production after
transcription. Plant Mol. Biol. 40:249-266; 1999.

Kost, B.; Galli, A.; Potrykus, I.; Neuhaus, G. High efficiency transient
and stable transformation by optimized DNA microinjection into N. tabacum
protoplasts. J. Exp. Bot. 46:1157-67; 1995.

Leduc,N.; Iglesias,V.A.; Bilang,R.; Gisel, A.; Potrykus,I; Sautter, C.
Gene transfer to inflorescence and flower meristems using ballistic
microtargeting.Sex. Plant Reprod. 7:135-143; 1994.

Li, H.Q.; Sautter, C.; Potrykus, I.; Puonti-Kaerlas, J. Genetic
transformation of cassava (Manihot esculenta Crantz). Nature
Biotechnology. 14:736-740; 1996.

Linn, W.; Datta, K.; Potrykus, I.; Muthukrishnan, S.; Datta, S.K. Genetic
enginering of rice for resistance to sheath blight. Bio/Technology
13:686-691; 1995.

Lucca, P.; Hurrell, R.; Potrykus, I. Genetic engineering approaches to
improve the bioavailability and the level of iron in rice grains. Theor.
Appl. Genet. (in press); 2000a.

Lucca, P.; Ye, X.; Potrykus, I. (2000) Effective selection and
regeneration of transgenic rice plants with mannose as selective agent.
Mol. Breed. (in press); 2000b.

Lusardi, M.C.; Neuhaus-Url,G.; Potrykus, I.; Neuhaus, G. An approach
towards genetically engineered cell fate mapping in maize using the Lc
gene as visible marker: Transactivation capacity of the Lc vectors in
differentiated maize cells and microinjection of Lc vectors into somatic
embryos and shoot apical meristems.
Plant J. 5:571-582; 1994.

Paszkowski, J.; Baur,M.; Bogucki,A.; Potrykus, I. Gene targeting in plants.
EMBO J. 7:4021-4026; 1988.


Paszkowski, J.; Shillito, R.D.; Saul, M.W.; Mandak, V.; Hohn, T.; Hohn,
B.; Potrykus, I. Direct gene transfer to plants. EMBO J. 3:2717-2722;
1984.

Peterhans, A.; Datta, S.K.; Datta, K.; Goodall, G.; Potrykus, I.;
Paszkowski, J. Recognition efficiency of Dicotyledoneae -specific promoter
and RNA processing signals in rice. Mol.Gen.Genet. 222:361-368; 1990.

Potrykus, I. Intra and interspecific fusion of protoplasts from petals of
Torrenia baillioni and Torrenia fournierii. Nature 231:57-58; 1971.

Potrykus, I. (1973) Transplantation of chloroplasts into protoplasts of
Petunia.
Z.Pflanzenphysiol. 70:364-366; 1973.

Potrykus, I. Gene transfer to cereals: an assessment. Bio/Technology
8:535-542; 1990.

Potrykus, I. Gene transfer to plants: Assessment of published approaches
and results. Annu.Rev.Plant Physiol.Plant Mol.Biol. 42:205-225; 1991.

Potrykus, I.; Burkhardt, P.; Datta, S.K.; Fütterer, J.; Ghosh-Biswas,
G.C.; Klöti, A.; Spangenberg, G.; Wünn, J. Genetic engineering of Indica
rice in support of sustained production of affordable and high quality
food in developing countries. Euphytica 85:441-449, 1995.

Potrykus, I.; Harms, C.T.; Lörz,H. Problems in culturing cereal
protoplasts. In: D. Dudits et al. (eds). Cell Genetics in Higher Plants.
Akademiai Kiado, Budapest, 1976:129-140; 1976.

Potrykus, I.; Harms, C.T.; Lörz, H.; Thomas, E. Callus formation from stem
protoplasts of corn (Zea mays L.). Mol.Gen. Genet. 156:347-350; 1977.

Potrykus, I.; Harms, C.T.; Lörz, H. Multiple-drop-array (MDA) technique
for the largescale testing of culture media variations in hanging
microdrop cultures of single cell systems. I. The technique. Plant Sci.
Lett. 14:231-235; 1978.

Potrykus, I.; Harms, C.T.; Lörz, H. Callus formation from cell culture
protoplasts of corn (Zea mays). Theor.Appl.Genet. 54:209-214; 1979.

Potrykus, I.; Hoffmann, F. Transplantation of nuclei into protoplasts of
higher plants. Z.Pflanzenphysiol. 69:287-289; 1973.

Potrykus, I.; Saul, M.W.; Petruska, J.; Paszkowski, J.; Shillito, R.D.
Direct gene transfer to cells of a graminaceous monocot. Mol.Gen.Genet.
199:183-188; 1985.

Potrykus, I.; Spangenberg, G. Gene transfer to plants. A Laboratory
Manual. Springer, Heidelberg, pp. 361; 1995.

Sanford, J.C. The development of the biolistic process. In Vitro Cell.
Dev. Biol. Plants 37: (in press); 2000.

Sautter, C.; Leduc, N.; Bilang, R.; Iglesias, V.A.; Gisel, A.; Wen, X.;
Potrykus, I. Shoot apical meristems as target for gene transfer by
microbiolistcs. Euphytica 85:45-51 (1995).

Sautter, C.; Waldner, H.; Neuhaus-Url, G.; Galli, A.; Neuhaus, G.;
Potrykus, I. Micro-Targeting: High efficiency gene transfer using a novel
approach for the acceleration of miroprojectiles. Bio/Technology
9:1080-1085; 1991.

Schocher, R.J.; Shillito, R.D.; Saul, M.W.; Paszkowski, J.; Potrykus, I.
Co-transformation of unlinked foreign genes into plants by direct gene
transfer.
Bio/Technology 4:1093-1096 (1986).

Takamizo,T.; Spangenberg, G.; Suginobu, K.; Potrykus, I. Intergeneric
somatic hybridization in Gramineae: Somatic hybrid plants between tall
fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium
multiflorum Lam.). Mol.Gen.Genet. 231:1-6; 1992.

Thro, A.M.; Taylor, N.; Raemakers, C.C.J.M.; Puonti-Kaerlas, J.; Schöpke,
C.; Visser, R.; Iglesias, C.; Sampio, M.J.; Fauquet, C.; Roca, W.;
Potrykus, I. Maintaining the cassava biotechnology network. Nature
Biotechnology 16:428-430 (1998).

Vasil, I.K. (ed.). Advances in Cellular and Molecular Biology of Plants,
Volume 4, Molecular Improvement of Cereal Crops. Kluwer Academic
Publishers, Dordrecht; 1998.

Vasil, I.K.; Vasil, V. Advances in cereal protoplast research. Physiol.
Plant. 85:279-283; 1992.

Wang, Z.Y.; Takamizo, T.; Iglesias, V.A.; Osusky, M.; Nagel, J.; Potrykus,
I.; Spangenberg, G. Transgenic plants of tall fescue (Festucua arundinaea
Scheb.) obtained by direct gene transfer to protoplasts. Bio/Technology
10:691-696; 1992.

Wernicke, W.; Brettel, R.; Wakizuka, T.; Potrykus, I. Adventitious embryo
and root formation from rice leaves. Z.Pflanzenphysiol. 103:361-366; 1981.

Wernicke, W.; Potrykus, I.; Thomas, E. Morphogenesis from cultured tissue
of Sorghum bicolor - the morphogenic pathway. Protoplasma 111:53-62; 1982.


Wünn, J.; Klöti, A.; Burkhardt, P.; Ghosh-Biswas, G.C.; Launis, K.;
Iglesias, V.A.; Potrykus, I. Transgenic Indica rice breeding line IR58
expressing a synthetic CryA(b) gene from Bacillus thuringiensis provies
effective insect pest control. Bio/Technology 14:171-176; 1996.

Ye, X.; Al-Babili, S.; Klöti, A.; Zhang, J.; Lucca, P.; Beyer, P.;
Potrykus, I. Engineering provitamin A (b-carotene) biosynthetic pathway
into (carotenoid-free) rice endosperm. Science 287:303-305; 2000.
------

Background information

Anonymous. UNICEF - The State of the World’s Children. Oxford University
Press; 1998.

Anonymous. World Health Organization. Nutrition for Health and
Development. Progress and Prospects on the Eve of the 21st Century. WHO
Geneva; 1999.

Anonymous. Transgenic Plants and World Agriculture. Report of seven
academies from developing and developed countries. The Royal Society
London, document 08/00; 2000.

Anonymous. Nuffield Council on Bioethics. Genetically Modified Crops: The
Ethical and Social Issues. Nuffield Foundation, London; 1999.

Conway, G. The Doubly Green Revolution. Penguin Books, London; 1997.

Evans, L.T. Feeding the Ten Billion. Cambridge University Press; 1998.

Lipton, M. Reviving global poverty reduction: What role for Genetically
Modified Plants. CGIAR Secretariat, The World Bank; 1999.

Persley, G.J.; Lantin, M.M. (eds.). Agricultural Biotechnology and the
Poor. CGIAR, Washington; 2000.

Qaim, M.; Krattiger, A.F., von Braun, J. (eds.) Agricultural Biotechnology
in Developing Countries: Towards Optimizing the Benefits for the Poor.
Kluwer Acad. Publ., Boston, Dordrecht, London; 2000.