isb news report - Information Systems for Biotechnology - Virginia Tech

ISB NEWS REPORT
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS
A UGUST 2004
In This Issue:
How Fit Are Genetically
Engineered Mosquitoes? ..........1
Enhanced Drought Tolerance in
Transgenic Rice .......................4
Biotechnological Improvements
of Tea ......................................5
Regulating Plant Genes and
Agbiotech Products –
International Style ....................8
India Produces Indigenous
‘GM Cotton’ .........................10
FAO Document - Molecular
Marker Assisted Selection .....10
I NSECT RESEARCH
HOW FIT ARE GENETICALLY ENGINEERED MOSQUITOES?
Mark S. Hoddle
Genetic engineering, the practice of using molecular tools to insert novel genetic
material of particular interest into an organism’s genome, is a research enterprise
spanning many laboratories globally. This precise form of genetic insertion and
manipulation has great potential for custom designing molecular-based solutions
to many pest and disease problems that currently appear intractable when
confronted with conventional management techniques, such as pesticides, or
cultural and biological control practices that aim to eradicate or reduce pest
populations to non-damaging levels.
One area of interest for molecular biologists is development of transgenic mosquitoes
refractory to transmission of human diseases such as malaria, dengue
fever, and yellow fever. Transformation of mosquitoes can be achieved in the
laboratory through transposable elements, units of DNA that move from one
DNA molecule to another where they insert at random. Transposable elements
(e.g., the hermes element from Musca domestica) can be designed to carry
marker (e.g., GFP from jellyfish, which glows green under UV light, facilitating
rapid identification of transgenic individuals) and strategic genes (i.e., genes that
make the transformant behave in the desired way, such as being unable to
transmit disease-causing organisms) that are expressed following successful
incorporation into the target’s genome.
While significant progress is being made in developing transgenic insect technologies
and effector genes to transform insect species of interest, very little work
has been done with laboratory-generated genotypes to determine the likelihood of
transgenic insect establishment, population growth, and persistence when competing
with non-transformed wild types in nature. Transgenes conferring fitness
advantages could act to promote the spread of particular genotypes while
transgenes resulting in fitness costs could be a significant impediment to the
establishment and competitiveness of transgenic organisms. Therefore, creating
insects with appropriate fitness will be critical to the field-level success of
transgenic-based control strategies. While many labs are routinely creating transgenic
mosquitoes expressing various novel characters, rigorous assessment of the
fitness of genetically engineered mosquitoes is lacking. Fitness of transgenic
mosquitoes can be affected by type of transgene inserted, placement of the novel
material within DNA and associated mutations or interruption of functional gene
sequences, and founder effects resulting from inbreeding between small numbers
of transformants when establishing newly transformed lines.

ISB News Report August 2004
THE ISB NEWS REPORT
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Fitness assessment of transgenic insects is an area of research
that needs greater attention immediately if shortcomings associated
with transgenesis are to be identified, and mechanisms underlying
fitness costs are to be understood and ultimately remedied during
the early stages of this emerging technology. University researchers
located largely in the United States, England, and Italy, using
various laboratory-based studies to investigate fitness of transgenic
mosquitoes, are paying closer attention to evaluating fitness of
transgenic mosquitoes. One study examined the fate of transgenes
in Anopheles stephensi, an important vector of Plasmodium that
causes malaria in humans 1 . In studies in which caged transgenic
mosquitoes could interbreed with non-transformed mosquitoes, the
frequency of transgenic alleles declined abruptly and in some
instances died out. The mechanisms underlying poor performance
were not elucidated but were assumed to be caused by insertion
and position effects of novel genes and inbreeding depression of
transgenic lines following initial creation.
Another research avenue to investigate fitness was to quantify
demographic parameters for three lines of transgenic Aedes
aegypti, the vector of yellow fever, and compare them with nontransformed
Ae. aegypti parameters. Several factors affect the
reproductive output of an insect and ultimately its population size
and stability. Important demographic factors are rates of sterility or
egg inviability (i.e., progeny production), sex ratio of offspring,
juvenile viability, development times, and adult longevity. Workers
at the University of California Riverside demonstrated that transgenic
Ae. aegypti had a significantly diminished capacity for
population increase in comparison to non-transformed Ae. aegypti,
and that significant differences existed between lines of transgenic
mosquitoes 2 . For example, fecundity was significantly impaired for
all transgenic lines in comparison to non-transformed mosquitoes,
and one transgenic line produced substantially fewer viable offspring
than its non-transgenic counterparts. In this study, the
authors demonstrated that negative effects of transgenesis on
fitness are not uniform, and a strain that performs poorly in one
area may outperform other strains when different characters are
measured. For example, impaired fecundity may be correlated with
a significantly shorter pre-oviposition period. Data collected from
this experiment were used to calculate the intrinsic rate of increase—the
rate at which a population can increase when resources
are unlimited and predators are not present. This value
was calculated for non-transformed and transformed Ae. Aegypti 2
and inserted into a simple logistic growth equation (Fig. 1). The
model began with a population of five individuals and continued for
100 generations with a maximum carrying capacity of 100 mosquitoes
in the water container.
In Fig. 1, non-transformed Ae. aegypti significantly outcompeted
all transgenic lines, reaching 50% and 100% of the environmental
carrying capacity 47% and 41% faster than transgenics, respectively.
Based on their intrinsic rate of increase, non-transformed
2

August 2004
ISB News Report
Another strategy for controlling pestiferous insects is to
use viruses engineered with specific activity towards
insects, especially caterpillars. Transgenic baculoviruses, a
group of entomopathogenic double-stranded DNA viruses
with genetically enhanced toxicity for insects, has been
assessed for fitness. These viruses were engineered to
express insect-selective toxins produced by spider and
scorpion genes and kill more rapidly than non-transformed
viruses. Laboratory and field experiments have demonstrated
that genetically engineered baculoviruses that
express insect-selective toxins have reduced reproductive
capacity and rates of transmission. Taken together, these
results suggest that engineered baculoviruses are less fit
than non-transformed parental wild types and will not likely
persist in the environment 4 . It is possible that fitness costs
incurred by genetically engineering insects and viruses will
have similar and fundamental underpinnings that could be
worth elucidating.
Figure 1. Logisitic growth curves for non-transformed and transformed
Ae. aegypti using intrinsic rate of increase values from Irvin et al. 2
mosquitoes would be predicted to outcompete transgenic
mosquitoes rapidly. The challenge facing molecular biologists
now is to create a “super-achiever”—a mosquito that
is competitively superior to wild types in nature. This would
require building a transgenic mosquito with superior fitness,
which would result in a shift of its logistic growth curve to
the left of non-transformed mosquitoes (Fig. 1). Development
of a “super-achiever” may be possible with use of
promoters that drive constructs (i.e., transposable element,
marker, and strategic genes) through the target population,
ultimately reducing or replacing non-transformed wild type
populations.
In contrast to the above study, workers in one laboratory
identified no fitness costs associated with mosquito
transgenesis 3 , and in some instances, important traits such
as longevity and fecundity were actually enhanced in
transgenics in comparison to non-transformed conspecifics.
Consequently, it is crucial to develop a comprehensive
understanding of the mechanisms driving these conflicting
outcomes and for the development of standardized protocols
for laboratories investigating fitness-related issues in
transgenic insects. Adoption of standardized evaluation
techniques would increase our ability to unambiguously
quantify and compare fitness of transgenic insects among
different laboratories.
Transgenesis is an exciting new technology that promises
to revolutionize pest and disease vector control. It is
expected to become an important tool in managing many
pest problems, most likely complimenting the fields of
biological, cultural, and pesticidal control. Like any new
emerging technology, there will be initial problems during
development, and fitness related issues is just one area
among many needing attention. With greater research
effort, many of the current problems associated with
reduced fitness may be solved once fundamental mechanisms
are better understood, thereby allowing the technology
to advance to field application.
References
1. Catteruccia F, Godfray HCJ, & Crisanti A. (2003) Impact of
genetic manipulation of the fitness of Anopheles stephensi
mosquitoes. Science 299, 1225-1227.
2. Irvin N, Hoddle MS, O’Brochta DA, Carey B, & Atkinson PW.
(2004) Assessing fitness costs for transgenic Aedes aegypti
expressing the GFP marker and transposase genes. PNAS 101,
891-896.
3. Moreira L, Wang J, Collins FH, & Jacobs-Lorena M. (2004)
Fitness of anopheline mosquitoes expressing transgenes that
inhibit plasmodium development. Genetics 166, 1337-1341.
4. Cory JS. (2000) Assessing the risks of releasing genetically
modified virus insecticides: Progress to date. Crop Protection
19, 779-785.
Mark S. Hoddle
Biological Control Specialist
University of California, Riverside
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ISB News Report August 2004
P LANT RESEARCH
ENHANCED DROUGHT TOLERANCE IN
TRANSGENIC RICE
Teresa Capell
Engineering the plant polyamine biosynthetic pathway
Manipulation of metabolic pathways in plants through
molecular genetics is now possible because of a significant
increase in our knowledge base of how such, often complex,
networks are controlled and regulated. In our ongoing
efforts to implement rational molecular approaches to
modulate plant metabolism, we chose the polyamine
pathway as a model to unravel key factors that still present
bottlenecks in engineering plant biosynthetic pathways.
The polyamine pathway is ubiquitous in living organisms.
Polyamines are low molecular weight polycationic molecules,
which are thought to play important roles in a
number of physiological and developmental processes 1 . In
animals and fungi, the diamine putrescine (the precursor of
the higher polyamines spermidine and spermine) is synthesized
directly from ornithine by the enzyme ornithine
decarboxylase (ODC). Plants have an alternative route to
the production of putrescine that is catalyzed by arginine
decarboxylase (ADC). Additional reactions convert
putrescine into spermidine and spermine. These steps are
catalyzed by spermidine and spermine synthases, which
add propylamino groups generated from S-adenosylmethionine
by S-adenosylmethionine decarboxylase (SAMDC).
Engineering of the plant polyamine biosynthetic pathway
has concentrated mostly on two species, tobacco and
rice 2,3 . We have generated a diverse rice germplasm with
altered polyamine content. Transgenic rice plants expressing
the Samdc cDNA accumulated spermidine and spermine
in seeds at two to three-fold higher levels compared
to wild type. In a different set of experiments, we were
able to measure a ten-fold putrescine accumulation in
transgenic rice plants harboring oat adc cDNA compared
to wild type. Reduction in endogenous adc transcript levels
in rice resulted in depletion of putrescine and spermidine
pools, with no concomitant changes in expression of
downstream genes in the pathway.
In general, studies focusing on spatial expression of these
transgenes demonstrate that more dramatic changes in
polyamine content occur in storage compared to vegetative
tissues, such as leaves and roots. Therefore, we believe
that the polyamine biosynthetic pathway in plants is regulated
strongly in a spatial manner 4 . In tomato, enhanced
fruit juice quality and prolonged vine life of fresh fruits with
increased lycopene was achieved by expression of yeast
Samdc driven by the ripening-inducible E8 promoter 5 .
Role of polyamines in stress response
In plants, polyamines accumulate under several abiotic
stress stimuli, including salt and drought. It has been
suggested that this increase in polyamine concentration
could be considered as an indicator of plant stress. The
first demonstration of involvement of polyamines in stress
responses in plants was documented by accumulation of
putrescine in response to sub-optimal K + levels 6 . Since
then, the link between increased putrescine levels and
several abiotic stresses was established. For example,
Krishnamurthy and Bhagwat 7 reported accumulation of
spermidine and spermine in salt tolerant rice cultivars and
accumulation of putrescine in rice sensitive cultivars in
response to salinity stress.
The physiological role of an increase in putrescine accumulation
following abiotic stresses is still unclear and is a
matter of considerable debate. It has been very difficult to
establish directly a cause and effect relationship between
increased polyamine levels in plants and abiotic stress. The
increase in putrescine levels in plants under stress might be
the cause of stress-induced injury or alternatively a means
of protection against stress. Earlier experiments by Roy
and Wu 8,9 expressing oat adc cDNA in rice under control
of an ABA-inducible promoter resulted in transgenic rice
plants with increased biomass when grown under salt
stress. The same authors expressed Tritordeum Samdc
cDNA in rice, under control of the same promoter. Under
salt stress these plants showed increased seedling growth
compared to wild type. Results from a number of studies
suggest that polyamines, particularly spermidine and
spermine, are involved in regulation of gene expression by
enhancing DNA-binding activities to particular transcription
factors. Polyamines are believed to have an osmoprotectant
function in plan cells under water deficit.
A threshold model linking polyamines and abiotic
stress response in plants
Osmoregulatory processes are important to all organisms
for stabilization of the intracellular milieu against environmental
fluctuations of water and ions. Despite divergence
of biochemical pathways, both prokaryotic and eukaryotic
organism share several physiological responses to
osmotic stress.
We put forward a threshold model that is consistent with
4

August 2004
high levels of adc gene expression leading to production of
putrescine. The production of putrescine needs to exceed
basal levels in order to generate a large enough metabolic
pool to trigger polyamine flux through the pathway leading
to increases in the levels of spermidine and spermine.
Transgenic rice plants expressing the Datura adc gene
accumulated up to two-fold putrescine in leaf tissues
compared to wild type. Such plants, when subjected to
drought stress induced by 20% polyethylene glycol, exhibit
a very significant divergent behavior compared to wild type
under the same conditions. Following 3 and 6 days of
drought stress, all wild type plants wilt and show droughtinduced
rolling of leaves. Such symptoms are completely
absent from Dadc-transgenic plants, which exhibit significant
putrescine accumulation during the same period. After
the 6-day drought stress period, the phenotype of transgenic
plants is indistinguishable from non-stressed wild
type. Transgenic plants with 2- to 4-fold higher levels of
putrescine develop and set seed normally.
Based on these observations, we put forward a model that
is consistent with a mechanism linking polyamine metabolism
to drought tolerance. Expression of the Dadc
transgene driven by the strong maize Ubi-1 promoter
would augment the putrescine pool to levels that extend
beyond the critical threshold required to initiate the conversion
of excess putrescine to spermidine and spermine 10 .
Spermidine and spermine de novo synthesis in transgenic
plants under drought stress is corroborated by the activation
of the rice samdc gene. Transcript levels for rice
samdc reach their maximum levels at 6 d after stress
induction. Such increases in the endogenous spermidine
and spermine pools of transgenic plants not only regulate
the putrescine response, but also exert an anti-senescence
effect at the whole plant level, resulting in phenotypically
normal plants. Wild type plants, however, are not able to
raise their spermidine and spermine levels after 6 d of
drought stress and consequently exhibit the classical
drought-stress response 11 .
Transgenic germplasm we have generated exhibiting
increased tolerance to drought stress is currently being
evaluated in field trials. We are very excited about the
prospects of this germplasm to make a positive contribution
towards sustainable rice production under stress conditions.
References
1. Malmberg RL et al. (1998) Critical Rev Plant Sci 17, 199-224.
2. Kumar A, Minocha SC (1998) Transgenic manipulation of
polyamine metabolism. In: Lindsey K (ed) Transgenic research in
plants. Harwood Academic Publishers UK, pp 187-199.
ISB News Report
3. Capell T, Christou P (2004) Current Opinion in Biotechnology
15, 148-154.
4. Trung-Nghia P et al. (2003) Planta 218, 125-134.
5. Mehta RA et al. (2002) Nature Biotech 20, 613-618.
6. Richards FJ, Coleman RG (1952) Nature 170, 460.
7. Krishnamurthy R, Bhagwat KA (1989) Plant Physiol. 91, 500-
504.
8. Roy M, Wu R (2001) Plant Science 160, 869-875.
9. Roy M, Wu R (2002) Plant Science 163, 987-992.
10. Bassie L et al. (2000) Trans. Res. 9, 33-42.
11. Capell T, Bassie L, Christou P (2004) Proc. Natl. Acad. Sci. of
USA 101, 9909-9914.
Teresa Capell
Department of Crop Genetics and Biotechnology
Schmallenberg, Germany
teresa.capell@ime.fraunhofer.de
BIOTECHNOLOGICAL IMPROVEMENTS OF TEA
Tapan Kumar Mondal
Tea (Camellia sinensis L.; family Theacea) is the oldest
non-alcoholic caffeine-containing beverage crop in the
world, and India is currently the foremost producer, consumer,
and exporter of commercial tea. The plant is a
woody perennial, traditionally propagated through either
seeds or stem cuttings, with a life span of more than 100
years. Tea is classified morphologically into two varieties:
Assam and China. The young leaves are processed into
different types of tea, such as black, green, and oolong.
Health benefits attributed to tea consumption are well
proven.
Background
Conventional tea breeding is well established, though timeconsuming
and labor intensive due to its perennial nature
and long gestation period (4 – 5 years). Vegetative propagation
is standard, yet limited by slow multiplication rate,
poor survivability of some clones, and need for copious
initial planting material. Seed-borne plants are heterogeneous
due to their highly allogamous nature; consequently,
it is difficult to maintain their superior character. Additionally,
tea breeding has been slowed by lack of reliable
selection criteria. Although few morpho-chemical markers
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ISB News Report August 2004
are available for identification of superior cultivars, these
markers are greatly influenced by environmental factors and
show a continuous variation with a high degree of plasticity.
To overcome these problems, a limited number of isozyme
markers have been used, resulting in less polymorphism.
With the advancements of molecular biology, however,
efforts have shifted to using various DNA markers.
Understanding genetic diversity at the molecular level of
tea germplasm will help to: (1) preserve the intellectual
property rights of the tea breeder; (2) identify individual tea
cultivars through use of a molecular passport; (3) prevent
duplicate entry of different genotypes into the tea gene
pool; (4) increase efficient selection of varieties for hybridization,
composite plant production, etc.; (5) classify tea
genotypes taxonomically using molecular markers; and (6)
improve tea varieties for agronomically important characteristics
through marker assisted selection. Consequently,
biotechnological tools appear to be the ideal choice to
circumvent problems of conventional tea breeding.
Micropropagation
The development of micropropagation, a rapid in vitro
multiplication method, of tea has passed through three
phases. Until the 1980s, emphasis was on standardizing
parameters of the in vitro protocol, such as using a suitable
explant, overcoming microbial contamination, and optimizing
media composition combined with growth regulation for
better proliferation. It is now accepted that nodal segments
(0.5-1 cm) cultured on MS medium with BAP (1-6 mg/l)
are best for multiplication of shoots, along with either a
high dose (500mg/l) pulse treatment or a low dose (1-2mg/
l) long duration treatment of auxin such as IBA for in vitro
rooting. Until the1990s, efforts turned toward hardening
micro-shoots to achieve a higher survival percentage.
Accordingly, several nonconventional approaches, such as
a CO 2
-enriched hardening chamber, biological hardening,
and micrografting, were developed. Presently, attention is
increasingly focused on evaluating field performance of the
micropropagated plant.
In our laboratory we developed a micropropagation protocol
by using a novel plant hormone, thidiazuron, which was
commercialized at the Research and Development Department
of Tata Tea Ltd., India. This protocol provides much
faster proliferation rates.
Somatic embryogenesis
One prerequisite for genetic transformation of tea is an
efficient system of regenerating the complete plant from a
single cell. Until today, somatic embryogenesis in tea was
considered the most efficient regeneration system. Unlike
micropropagation, tea somatic embryogenesis started in the
late 1980s. Thus, emphasis was focused on standardizing
parameters, such as genotypes, seed maturity, media
formulation, growth regulator, physical condition, etc. We
developed a complete pathway of tea somatic embryogenesis
in which somatic embryos were first induced within 6
– 8 weeks on the cotyledon segments of mature tea seed,
which were then further multiplied synchronously (Fig. 1).
A germination medium was formulated that yielded a 70%
conversion rate. Following this protocol, we transferred
3,000 plants to the field at the Research and Development
department, Tata Tea Ltd, India 1 .
Figure 1. Occurrence of somatic embryo on the tea cotyledon.
Bioreactor technology for secondary embryogenesis
Applications of bioreactor technology further ensure the
speedy, continuous, and large-scale supply of propagule. A
bioreactor system for repetitive embryogenesis in tea has
also been developed in Australia 2 in which uniform sizes of
globular somatic embryos were obtained for a bioreactor
technology called the temporary immersion system (TIS).
By controlling immersion cycles, synchronized multiplication
(24 fold) and embryo development were achieved with
greater consistency and with a high rate of plant recovery.
Plantlets recovered through this method were hardy, with a
well-formed taproot. Therefore, this technique was the first
significant step for commercial application of bioreactor
technology to produce large-scale tea somatic embryos.
Field performance of micropropagated raised plants
The ultimate success of any in vitro protocol depends upon
performance of plants in the field compared to vegetative
counterparts. For the last several years, researchers at the
Research and Development Department of Tata Tea Ltd,
India, have transferred more than 45,000 plants of eight tea
cultivars to the field, from which leaves are harvested
regularly to manufacture black tea.
6

August 2004
ISB News Report
Name of Technique
Objective
Status
Remark
Somaclonal and
Gamatoclonal variation
Develop a mutant resistant
to different stresses
Few plants of drought
tolerance observed in
the laboratory
No commercial success
Artificial seed
Storage of propagule
Nodal segment and somatic
embryos were encapsulated
Maximum 60 days storage
was possible without loss
of germination
Protoplast culture
Haploid and hybrid
plant production
Fusion of two protoplasts
was achieved
Regeneration was not possible.
Anther culture
Production of haploid plant
Development of micro-calli
Regeneration was not achieved.
Suspension culture
Secondary metabolite production
Many secondary
metabolites produced
Commercially exploited
Cryopreservation
Long term storage of propagule
Technique standardized for shoot
tip preservation in liquid nitrogen
Not exploited further for either
academic or commercial purposes
Table1: Summary of a sampling of tea cell culture techniques
A systematic study at 1.7, 4, and 8 year-old field-grown
micropropagated and vegetatively propagated tea plants in
our laboratory and elsewhere in India demonstrated that
overall yields and quality were comparable. Although
different physiological parameters such as photosynthetic
rate, chlorophyll content, etc. remained the same, two
morphological variations were noticed. First, the number of
lateral shoots produced after ‘centering’ were significantly
greater in micropropagated-raised plants compared to
vegetatively propagated plants. This is perhaps due to
effects of various growth regulator treatments applied
under in vitro conditions. Second, root volumes of tissue
culture plants were also greater than in vegetativelypropagated
plants. Micropropagated shoots were treated
with IBA to induce rooting, which may be responsible for
better root development in the field. Therefore, we concluded
that the micropropagation protocol should be used
only when required to produce a large number of plantings
from a limited source.
Other tissue culture techniques
Other techniques have been applied in tea with specific
objectives (Table 1). Efforts to improve these techniques
are ongoing at laboratories worldwide.
Genetic transformation
Transgenic technology has immense potential for genetic
improvement of tea; however, until 2000 there were no
reports on tea transgenesis. The initial challenge was to
develop a protocol for gene transfer. Recently, we reported
the optimization of transformation conditions and production
of transgenic tea via Agrobacterium tumefaciens 3 . In this
study, we produced transgenic tea using GUS reporter and
NPT-II marker genes under control of strong monocot
gene promoters, and stepwise antibiotic selection. Using
this protocol, further experiments are underway to transfer
the chitinase gene for production of a fungus-resistant
tea plant.
Biolistic-mediated genetic transformation is another
effective method to produce transgenics in a wide variety
of plant species. Although no transgenic tea plants have
been grown in the field using this technique, experimental
conditions have been standardized by Australian and
Chinese scientists.
Genetic characterization
Morphological markers such as leaf pose, dry matter
production, partitioning, flesh evenness, etc. and biochemical
markers such as total catechin/polyphenol content,
caffeine, etc. are used to identify the superior tea plant.
However, tea breeders are often unable to use markers
effectively because they are greatly influenced by environmental
factors and show a continuous variation with a high
degree of plasticity. Hence, to overcome these problems,
research has shifted to using more sensitive DNA markers.
Work on molecular markers in tea began in our laboratory
in 1994 and a significant amount of work is continuing
worldwide (discussed below).
Research in India:
We have used the random amplified polymorphic DNA
(RAPD) assay to characterize 25 important Indian tea
cultivars and two ornamental species (Fig. 2). In a separate
study, twenty-five diverse tea cultivars were analyzed
7

ISB News Report August 2004
and China tea cultivars were characterized through RAPD
or AFLP, and RAPD, respectively.
Figure 2. DNA fingerprinting pattern of different tea cultivars. Lane 1-
25 denotes different tea cultivar.
using the simple sequence repeat anchored polymerase
chain reaction (SSR-anchored PCR) or Inter SSR-PCR
(ISSR). In both cases, cultivars were analyzed using
Shannon’s diversity index, which revealed that the China
type tea group is more diverse than the Assam group.
Additionally, we noted that molecular classification
matches conventional classification of tea. A speciesspecific
primer was also developed for distinguishing
between the Assam and China type tea cultivars.
Amplified fragment length polymorphism (AFLP) markers
were also studied in depth to detect diversity and genetic
differentiation of several important tea clones, including the
famous ‘Darjeeling tea’, mainly to protect cultivars for
intellectual property rights purposes. Interestingly, the
RFLP technique was also used to detect adulteration with
cashew husk in 10 different tea samples 4 .
Research abroad:
Researchers in different countries have made fingerprints
of tea cultivars in their countries of origin. In Kenya,
fingerprints of popular tea cultivars were made through
RAPD and AFLP analysis at the Tea Research Institute,
Kenya. The same group also initiated a genetic linkage
map of tea. Work is ongoing to develop a complete tea
database with chemical as well as molecular data, which
will assist with easy identification of the different cultivars.
In Japan, a wide range of markers has been used with
various applications. The markers used for genetic characterization
of different green tea cultivars are RAPD,
AFLP, SSR, CAPS, and RFLP. Importantly, the RFLP
technique was also applied in Japan to prevent adulteration
of higher grade with lower grade tea. Several other minor
tea-producing countries have used different molecular
markers to characterize the tea gene pool of introduced tea
cultivars available to that country. Such efforts were made
using RAPD in Portugal, ISSR in Taiwan, and RAPD in
South Africa. All work focused on the genetic characterization
and molecular taxonomy of the introduced variety
available in the respective countries. Similarly, South Korea
Simple sequence repeats (SSR) were derived from C.
japonica, a closely related species of tea in Japan. Using
these primer pairs, 53 C. japonica ecotypes were
genotyped and population genetic parameters calculated.
Later, the same group investigated the spatial genetic
structure of C. japonica using four of these microsatellite
primers. Spatial distribution of individuals was also assessed
to obtain an insight into spatial relationships between
individuals and alleles.
Gene cloning and expression
Japanese researchers have isolated the cDNA chalcone
synthase (CHS) gene as well as ß-tubulin gene from the
Japanese green tea cultivar ‘Yabukuta’. More recently, a
few important genes such as phenyl ammonia lyase
(PAL), caffeine synthetase, and primeverosidase have
been isolated.
References
1. Mondal TK et al. (2004) Plant Cell Tissue Org Cult, Netherlands
76, 195-254.
2. Akula A, Akula C (1999) In: Jain SM, Gupta PK, & Newton RJ
(eds.), Somatic embryogenesis in Woody plants, Vol. 5, pp 239-
259. Kluwer Academic Publishers: The Netherlands.
3. Mondal TK et al. (2001) Plant Cell Reports 20,712-720.
4. Dhiman & Singh (2003) Planta Med 69(9), 882-4.
Tapan Kumar Mondal
Centre for Advance Study in Tea Science and Technology
Uttar Banga Krishi Viswavidalaya, India
mondaltk@rediffmail.com
R EGULATORY NEWS
REGULATING PLANT GENES AND AGBIOTECH
PRODUCTS – INTERNATIONAL STYLE
Phillip B.C. Jones
Challenges from pathogens, pests, and the climate fuel
perpetual efforts to develop new crops with mixed traits.
When a fungus decimated maize grown in the southern
United States, for example, plant breeders devised a way
to defeat the pathogen with a natural resistance trait
harbored within a variety of African maize. To combat
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August 2004
yellow dwarf disease in U.S. barley varieties, researchers
harvested resistance genes from Ethiopian plants, while
genes from plants originating in the Caucasus and Spain
provided the keys for resisting rust disease in U.S. commercial
wheat varieties.
Plant breeders can perform these feats of agricultural
innovation because they can select traits from a wealth of
genetic resources. And since no single country has the full
range of naturally occurring genetic resources, the collection
and exchange of germplasm requires international
cooperation. Yet not every nation considers this exchange
equitable. Accusations of biopiracy often arise from
countries in tropical and subtropical regions, which possess
the majority of the world’s agricultural genetic diversity. At
the same time, changing land use practices within these
countries lead to the loss of uncollected genetic resources.
The Food and Agriculture Organization (FAO) of the
United Nations estimates that about three quarters of the
genetic diversity found in agricultural crops has vanished
over the last century.
These concerns inspired the creation of the UN’s International
Treaty on Plant Genetic Resources for Food and
Agriculture, an agreement adopted after seven years of
negotiations by delegates from 116 nations. Although
drafted in November 2001, the Treaty only came into force
on June 29, ninety days after forty governments ratified it.
José Esquinas-Alcázar, secretary of FAO’s Commission on
Genetic Resources for Food and Agriculture, told BBC
News Online that the “treaty will ensure the conservation
and availability of raw material for agriculture.”
The agreement requires each contracting party to explore
and conserve its plant genetic resources for food and agriculture.
Parties can work toward this objective by surveying
their genetic resources and assessing any threats, and
by promoting both in situ conservation and the compilation
of genetic resources for preservation in public collections.
The treaty also mandates that contracting parties develop
and maintain measures to advance the sustainable use of
plant genetic resources. Examples of such measures
include extending the genetic base of crops available to
farmers and supporting plant breeding efforts that
strengthen the capacity to develop varieties adapted to
particular ecological conditions.
Under the Treaty, countries agree to establish a Multilateral
System to facilitate access to plant genetic material and to
share the benefits. The Multilateral System applies to plant
genetic resources for food and agriculture listed in the first
ISB News Report
annex of the Treaty that are under the control of the
contracting parties and in the public domain. The 35
itemized food crops and 29 forage crops “represent most of
the important food crops on which countries rely,” says
Esquinas-Alcázar. According to one estimate, the Treaty’s
annex lists crops representing 80 percent of the world’s
calorie intake. The 600,000 sample gene bank collection
held by the Consultative Group on International Agricultural
Research will also be administered under the Treaty.
Restrictions apply to the use of these genetic resources.
The material must be used to promote conservation,
research, breeding, and training for food and agriculture.
Any use of genetic material for chemical, pharmaceutical,
and other industrial applications falls outside the scope of
the Treaty. If an entity incorporates material accessed from
the Multilateral System into a commercial food or agricultural
product and does not permit others to use the product
without restriction for research and breeding, then the
Treaty requires payment of an equitable share of any
resulting monetary benefits.
The FAO asserts that the Treaty benefits agricultural
research because the Multilateral System will reduce
transaction costs for the exchange of plant genetic material
between countries. That is, researchers will no longer have
to negotiate bilateral agreements with each donor country
to obtain germplasm.
But before the Treaty can facilitate any transfer of plant
genetic material from its germplasm clearinghouse, the
countries that ratified the agreement must decide about
conditions for access and benefit-sharing, details that will
be embodied in a standard material transfer agreement.
Conditions recited in this key document will also determine
whether the United States will ratify the Treaty.
At least one mystery resides within the Treaty. Article 12
states that “recipients shall not claim any intellectual
property or other rights that limit the facilitated access to
the plant genetic resources for food and agriculture, or their
genetic parts or components, in the form received from the
Multilateral System.” Even delegates involved in the
drafting of the document could not agree about the meaning
of this statement.
It might have been the eruption of biopiracy complaints that
motivated the inclusion of the ambiguous passage. During
the drafting of the Treaty, protests from India provoked the
revocation of a U.S. patent covering a use of turmeric and
a European patent on a compound derived from the Neem
tree, while South American activists prompted the with-
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ISB News Report August 2004
drawal of a U.S. patent on an ayahuasca vine variety. In
2001 alone, India, Pakistan, and Thailand voiced grievances
about a U.S. patent on a type of basmati rice, and Mexico
protested a U.S. patent on yellow beans.
A copy of the Treaty is available from the FAO website
(http://www.fao.org/ag/cgrfa/itpgr.htm).
Labeling Food as Genetically Modified and Living
Modified Organisms as Pests
In June the Codex Alimentarius Commission released its
report of the 32nd Session of the Codex Committee on
Food Labeling. The delegates generally supported the
establishment of guidelines for end-use labeling—the
labeling of foods derived from genetic engineering that
have a significant change in composition, nutrient content,
or intended use.
Production labeling was met with less enthusiasm. Certain
delegates, including the U.S. delegation, asserted that
consumers would perceive labeling based solely on the
method of production as a safety warning. The Canadian
delegation argued that method of production labeling fails
to comply with Codex’s own rule: only factors accepted
worldwide should be taken into account as a basis for
decision-making. A copy of the report can be obtained
from the website of the Codex Alimentarius Commission
(http://www.codexalimentarius.net/).
On June 1, the FAO published new guidelines for determining
if a living modified organism (LMO) poses a hazard to
plants. About 130 countries have adopted this assessment
standard, which focuses on the risk that a foreign gene
may transform a plant into a weed. The document also
offers advice for determining whether genetically modified
insects, fungi, or bacteria could harm plant ecosystems. If
authorities deem an LMO a threat, then they can decide
whether to prohibit or restrict its import and domestic use.
The website of the International Plant Protection Convention
offers a copy of the guidelines, entitled “Pest Risk
Analysis for Quarantine Pests, Including Analysis of
Environmental Risks and Living Modified Organisms”
(http://www.ippc.int/).
Selected References
Anonymous. (2004) Plant gene treaty becomes law. BBC News.
June 29, 2004. Available at: http://news.bbc.co.uk/2/hi/science/
nature/3849489.stm.
Fowler C. (2003) The status of public and proprietary germplasm
and information: An assessment of recent developments at FAO.
IP Strategy Today, No. 7-2003. Available at: http://
www.biodevelopments.org/.
Sullivan SN. (2004) Plant genetic resources and the law. Past,
present, and future. Plant Physiology, 135: 10-15.
Phillip B.C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org
INDIA PRODUCES INDIGENOUS ‘GM COTTON’
P Janaki Krishna
Insects, disease, and drought present the greatest impediments
to realizing expected yields in major crops. In
addressing these problems, development of transgenic
varieties has assumed significance, primarily through use of
durable resistance genes. However, a few multinational
companies in developed countries own and patent many of
these genes. In developing countries, these novel genes are
sometimes available to scientists as ‘gifts’ through personal
contacts. Though initial transgenic crop development is
dependent on these borrowed genes, the varieties developed
from them would not be available for commercial
cultivation because of contractual obligations generally
underlying the ‘gift’ to investigators (as these genes
generally are available for academic and experimental
purposes only). At best, GM plants thus developed could be
tested only for their efficacy in solving designated problems
and not developed for commercial cultivation. Deployment
of borrowed genes in transgenic crops might also attract
patent problems under new IPR regimes.
Scientists, institutes, and seed companies have therefore
decided to convene and begin searching and licensing
indigenous genes and technologies for endogenous developments.
Apparently this exercise is having an effect, as
Monsanto’s monopoly on genetically modified cotton in
India will soon be broken by Swarna Bharat Biotechnics
Private Ltd (SBBPL), Hyderabad, India, a consortium of
seven Indian seed companies. SBBPL received licenses
for two genes derived from Bacillus thuringiensis (Bt),
which protect cotton against bollworm (Helicoverpa
armigera) and tobacco caterpillar (Spodoptera litura).
The genes are licensed from the National Botanical
Research Institute (NBRI), Lucknow, India, for Rs. 7.5 ($
0.16) million over a three year period and a royalty of 3%.
SBBPL is soon likely to get license for a third gene
(LecGNA 2) that directs production of lectin, a protein
lethal to sucking pests such as aphids, from the publicly
funded Centre for Plant Molecular Biology (CPMB),
Osmania University, Hyderabad, India.
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August 2004
According to NBRI’s Deputy Director, Dr Rakesh Tuli,
SBBPL has licensed two genes—Cry1Ac and another
killer gene called Cry1Ec. Of these two genes, Cry1Ec,
used against a tobacco caterpillar, is designed and synthesized
at NBRI. Because Cry1Ac, which confers resistance
against bollworm, is not protected in India, NBRI’s team
altered Cry1Ac promoters to create a version with greater
expression and stability. According to some experts,
although NBRI has modified the gene, there could be legal
implications: GM plants derived from this gene might come
under the category of ‘essentially derived varieties’
(EDVs) and cannot be registered under India’s Plant
Variety Protection Act - 2001. Even more, Monsanto might
contest the commercialization of Indian indigenous Bt
cotton after January 2005, the date when India has to
comply with patents covered under the WTO rules. Tuli
admits that only time might solve the problem. Meanwhile,
SBBPL is seeking regulatory approval in India for both
Cry1Ac and Cry1Ec, with a view toward introducing the
new Bt cotton by 2006.
The consortium’s aim is to enter an era of self-sustaining
agribiotech development. Satish Kumar, Managing Director
of SBBPL, reiterated that they are ready to source beneficial
genes from any publicly funded laboratory where they
are available. The consortium opines that the advantages of
sourcing indigenous technology are economic and strategic.
The profit generated by public sector institutes through
licensing helps support reinvestment in developing more
agribiotech products to serve local needs. The main benefit
for consortium members is economic, as the technology
access fee is shared by members of the consortium. In
addition, Indian partners help with the regulatory process to
obtain product approval. Kumar expects that the price of
SBBPL seeds would be two-thirds of Monsanto’s.
Through licensing these genes, Rs 10 ($ 0.2) billion spent
on chemical pesticides can be saved by SBBPL, as 90% of
cotton damage is from bollworm and sap sucking pests.
Indian farmers spend about Rs 16 ($ 0.35) billion on
chemical pesticides. With the introduction of novel Bt
cotton varieties, SBBPL, which has a 30% share of the
total Indian cottonseed market, expects to claim some of
the Rs 30 ($0.66) billion market per year that is presently
monopolized by the joint-venture company Monsanto-
Mahyco Biotech, Mumbai, India.
ISB News Report
Since scientific knowledge is indigenous in India, and
smaller players can access costly technology monopolized
by big multinational companies, many eminent scientists,
activists, scholars, and institutes welcomed this initiative of
SBBPL. The activist groups critical of Monsanto’s monopoly
are happy, as 42% of India’s transgenic research
has been based on Monsanto’s gene. “Finally, we seem to
be getting our act together,” said Suman Sahai, Convener
of Gene Campaign, New Delhi, India.
However, Monsanto is not daunted by the competition—
they have already co-licensed the Cry 1Ac gene to nine
more Indian companies whose products are at different
stages. Ranjana Smetacek, the company’s spokesperson in
India, says Monsanto welcomes the widespread usage of
Bt cotton. Meanwhile the Council for Scientific and
Industrial Research (CSIR), New Delhi, India, the parent
institute of NBRI, is finalizing the list of countries soon to
file patents for novel genes. Experts suggest that the
consortium may try to exploit those GM technologies on
crops for which patents held by multinational companies
are now expiring. Prabhakar Rao, Managing Director of
Nujiveedu seeds (Hyderabad, India), the largest company
in the consortium, said that membership would soon reach
19, as many other countries are willing to partner with
them. Hence, it appears that a few multinational seed
companies can no longer monopolize agribiotechnologies,
and local stakeholders can play a crucial role in technology
development and commercialization.
References
1. Nat. Biotechnol. 22, 255-256, 2004.
2. Nat. Biotechnol. 21, 590-591, 2003.
3. Nat. Biotechnol. 19, 895-896, 2001,
P Janaki Krishna
Biotechnology Unit, Institute of Public Enterprise
Hyderabad, India
jankrisp@yahoo.com
C ONFERENCE NEWS
Molecular Marker Assisted Selection Document
FAO has published a biotechnology-related summary of
an e-mail conference on “Molecular marker assisted
selection as a potential tool for genetic improvement
of crops, forest trees, livestock and fish in developing
countries,” which ran from 17 November to 14 December
2003. The document summarizes the main arguments
and concerns raised during the moderated e-mail conference.
The full summary is available at http://www.fao.org/
biotech/logs/C10/summary.htm.
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