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[The Importance of Biotechnology]
[Evolution of Biotechnology at
CIAT] [Molecular Markers]
[Gene-Transfer Technology] [Tissue
Culture] [Biosafety and Public
Information]
The Importance of Biotechnology
Biotechnology,
in its widest sense, covers a spectrum of ancient and
modern techniques for harnessing the biochemical processes
that take place within living things. End products can
be useful organic materials visible to the naked eye
like preserved foods, pharmaceutical drugs, liquid fuels,
plant tissues, and even whole plants. Or they can be
microscopic aids. These include enzymes for manipulating
DNA (deoxyribonucleic acid); DNA fragments for tagging
important variable genes in people, plants, and other
life forms; and cloned genes for creating transgenic
organisms. Information—in the form of molecular
maps, databases, and new techniques—is also an
important product of biotechnology and related research.
Modern
biotechnology, as practiced by CIAT and other advanced
research centers relies heavily on molecular biology
and is used mainly for genetic studies, diagnostics,
breeding, plant and microbial propagation, and genetic
conservation. Modern biotechnology is also now widely
applied in the otherwise traditional production-oriented
food industries, as well as in evolving areas like waste
management and renewable energy production.
Agricultural
biotechnology is based on an understanding of how organisms,
especially plants and disease-causing microorganisms,
work at various micro levels. To generate and exploit
that knowledge requires expertise from several disciplines:
genetics, molecular genetics, cell biology, molecular
pathology, breeding, physiology, entomology, botany,
agricultural geography, and others. The methodological
requirements of biotechnology are likewise diverse.
For example, to extract, synthesize, and analyze DNA
requires expertise in biochemical, electromechanical,
electronic, statistical, and computing techniques.
Several
facts and trends in today’s unfolding global food
production scene give biotechnology a prominent place
in agricultural science and in CIAT’s day-to-day
operations.
First,
most of the world’s crop-based food, whether for
people or livestock, is based on a shallow gene pool.
Just a handful of botanical genera and species—notably
rice, wheat, maize, potatoes, common beans, cassava,
soybeans, and a few forages—account for the bulk
of world food consumption. At the same time, on-farm
genetic diversity has shrunk in recent decades as the
proportion of arable land planted to commercial varieties
has gone up. Biotechnology can identify and help transfer
useful genes into food and forage crops. For many species,
the necessary material is readily available in the breeder’s
immediate gene pool. Alternatively, it may have to be
found in wild relatives of the target crop, more distantly
related species, or even in entirely different organisms.
A
second trend is the heavy and widespread environmental
pressure on two natural resources essential to world
agriculture—soil and water. Further stress will
be placed on natural resources, as the world population
swells by more than half, to about 9.4 billion people,
in the next 50 years. Feeding 80 million more people
each year over several decades will require the world
grain harvest to grow by some 26 million tons per year,
a gargantuan challenge to scientists and farmers. Biotechnology
can help make food crops more efficient in their use
of soil nutrients and water and less dependent on agrochemical
inputs like pesticides. And by contributing to greater
yield per unit area, it can ease production pressure
on vulnerable land.
Third,
there is the pervasive influence of world market liberalization.
While this creates viable new opportunities for many
farmers in developing countries, it also threatens traditional
livelihoods. Small producers of certain staple grains
are particularly vulnerable as they attempt to compete
against cheap, high-volume imports from efficient foreign
producers. Among the farmers who manage to adapt, the
need and demand for new technologies is on the rise.
Of special interest to them are nonstaple cash crops
like tropical fruits, garden vegetables, herbs, and
flowers. They are also looking for new varieties of
more traditional crops with value-added traits.
Hand
in hand with globalization is the economic influence
of agricultural biotechnology itself. Despite widespread
public controversy (especially in Europe) over the potential
health and environmental risks of transgenic plants,
biotechnology is in its own right a fast-moving trend
that appears to be widening the competitiveness gap
between North and South. For example, global sales of
transgenic crops went up nearly 30-fold between 1995
and 1999, from US$75 million to an estimated US$2.1
to US$2.3 billion. More than 80 percent of the crops
that generated this revenue were grown in industrialized
countries.
If
centers like CIAT are to help small farmers in developing
countries survive and thrive in the new world economy,
we must improve our understanding and use of the genetic
diversity of tropical crops, including alternative species
that have not so far received much attention. Molecular
mapping, easy and rapid plant propagation, and gene-transfer
technology are vital to achieving this aim. And given
the widening North-South scientific knowledge gap, it
is equally important that these tools and the products
they furnish be developed, deployed, and monitored in
partnership with national research programs and biosafety
authorities in developing countries.
Evolution
of Biotechnology at CIAT
CIAT’s
biotechnology work started in the late 1970s with efforts
to improve cassava germplasm conservation and prevent
the spread of disease. Research centered on cellular
physiology and tissue culture development within our
Genetic Resources Unit.
In
1985 we set up a separate Biotechnology Research Unit
in response to a review by an external panel of experts.
By 1992 the current lines of research had taken shape:
tissue culture, genetic transformation, molecular biochemistry,
and the application of molecular markers to conservation
and breeding.
Under
the CIAT project Assessing and Utilizing Agrobiodiversity
through Biotechnology, these major thrusts are now integrated
with our research on four commodities: rice, beans,
cassava, and forages. Biotechnology is also being applied
to selected alternative crops as demand and opportunities
arise.
To
pull together the necessary mix of disciplines and skills
for this work, CIAT has created what we sometimes refer
to as a “lab without walls.” In this open,
international learning environment, scientists can study
in increasingly fine detail genetic differences within
and between species and track the inheritance of physical
traits by their progeny. Through that research we are
enhancing the global plant-gene pool, helping conserve
it, and designing new products for the benefit of poor
farmers and consumers in tropical countries.
The
sections that follow give an overview of this work,
with selected examples showing the application of. three
broad categories of biotechnology tools used at CIAT:
(1) molecular markers, (2) gene-transfer technology,
and (3) tissue culture.
Molecular
Markers
Molecular
markers are segments of an organism’s DNA that
show genetic variability between individuals in the
same population or species. Known as polymorphisms,
these DNA sites reflect a simple but essential fact
of heredity: A gene on the chromosome donated by one
parent is not necessarily identical to the complementary
gene (the “allele”) on the corresponding chromosome
from the other parent.
In
sexual reproduction each parent organism contributes
one of its two alleles to the offspring. This happens
for each gene in each chromosome. Heredity plays out
like a perpetual game of coin tossing, with “heads”
and “tails” alleles each having an equal chance
of being passed on to the progeny. As in all structured
games of chance, though, combinations of outcomes can
be assigned a level of probability. And over many tosses
of the coin (i.e., generations), obedience to the laws
of probability emerges in an orderly, statistical fashion
that can be tracked and analyzed by geneticists and
breeders.
Plants
and higher organisms have multiple pairs of chromosomes
(23 pairs in people, for example), and each chromosome
contains many thousands of genes. The number of possible
gene combinations involved in producing unique individuals
is thus extraordinarily large. Alleles that are routinely
identical in a species account for the sameness of its
members—for example, the geometric pattern of leaves
on rice plants. The presence of unlike alleles (along
with environmental influences) accounts for observable,
though not always obvious, differences. These can be
minor, like skin and eye color in people, or major ones,
like predisposition to breast cancer.
This
allelic variation, or polymorphism, is crucial to the
work of specialists in agricultural biotechnology. When
recorded in the form of molecular markers on a genetic
map, it provides scientists with a powerful tool for
enhancing the genetic base of crops and designing better
germplasm for farmers.
Molecular
markers are the miniature workhorses of modern biotechnology.
They have three key applications in CIAT’s assessment
and use of biodiversity: (1) to build molecular maps
of plant and other genomes; (2) to analyze genetic variation
within and between specific populations, as in DNA fingerprinting;
and (3) to assist in otherwise conventional plant breeding.
This
last and most recent application is called MAS, for
marker-assisted or marker-aided selection. Breeders
use DNA markers to direct and verify the movement of
useful genes—and therefore the inheritance of important
traits like yield and disease resistance—from one
plant generation to the next. MAS, though still not
widely practiced, allows for precision in this molecular
juggling act. It also drastically cuts the time needed
to create new plant genotypes and, ultimately, new improved
varieties. A major reason is that large numbers of segregating
progeny do not have to be grown to maturity. By examining
DNA from very young plants or even cultured tissues,
breeders can determine in the lab whether the molecular
markers, and therefore the genes that code for desirable
traits, have been inherited.
Several
molecular marker methods are used at CIAT:
-
Restriction
fragment length polymorphisms (RFLPs)
-
Random
amplified polymorphic DNA (RAPDs)
-
Sequence
tagged sites (STSs)
-
Simple-sequence
repeats (SSRs), often called microsatellites
-
Amplified
fragment length polymorphisms (AFLPs)
-
Sequence
characterized amplified regions (SCARs)
-
Expressed
Sequences Tags (ESTs).
Marker
methods differ with respect to the type, specificity,
and volume of genetic data generated, lab time required,
and the cost of equipment and materials. RFLPs, while
highly specific, allow only very limited comparison
of individuals (two bands on an autoradiograph). RAPDs
(10-15 bands) and AFLPs (up to 100 bands) are much more
sensitive to genetic differences at the molecular level.
AFLPs are the preferred tool of genetic fingerprinting.
In
general, the choice of marker technology depends on
the type of analysis being conducted. With the exception
of RFLPs, all the marker methods listed above use polymerase
chain reaction (PCR) to amplify the DNA under study.
The use of molecular markers to analyze and compare
DNA samples consists of four main steps:
-
Preparing
target DNA for analysis
-
Combining
it with probes or primers (depending on the marker
method), which bind to the target DNA, thereby revealing
sites of genetic variation
-
Capturing
the results on autoradiographs (X-ray films) or
as chemical assay readouts
-
Reading
and analyzing data, often with the aid of statistical
tools.
Each
step includes substeps or protocols, such as chemical
processes to extract, fragment, sort, and stabilize
DNA samples. (An accompanying document, written in layman's
terms, explains this and other procedures as well as
underlying concepts.)
Rice
Blast, a Moving Target
Scientists
at CIAT and Purdue
University in the USA are jointly working on an
integrated approach to fighting rice blast, the most
widespread and damaging disease of rice. The strategy
combines conventional breeding and pathotyping (characterizing
individual strains of a pathogen) with two types of
molecular marking, RAPDs and RFLPs. The goal is to develop
broad, long-lasting resistance to this highly variable
fungus.
Pyricularia
grisea blast attacks both irrigated and rainfed
(upland) rice, although the latter is more susceptible
to epidemics. Yield loss due to outbreaks ranges from
about 5 to 20 percent. The fungus is hard to fight,
because there are many strains and they mutate rapidly.
New resistant rice varieties usually become susceptible
to new strains within 2 or 3 years of their official
release. Major instances of massive resistance breakdown
have been recorded in countries as environmentally diverse
as Egypt, South Korea, the Philippines, and the USA.
As a countermeasure, farmers often apply heavy doses
of fungicide. This not only is very costly but also
damages the environment.
Using
a conventional breeding approach, CIAT spent many years
developing two fully resistant rice cultivars in a Colombian
“hot spot” of rice blast. This was done in
collaboration with the Colombian Institute of Agricultural
Research (ICA)
and the country´s national Rice Growers Federation
(FEDEARROZ).
One of these varieties, Oryzica Llanos 5, has now been
grown for more than 5 years without the resistance breaking
down.
The
CIAT-Purdue strategy centers on exploiting this hard-won
genetic resistance to produce new rice cultivars resistant
to families of blast rather than single strains. For
Latin America, the potential benefits of exploiting
such resistance stability in rice are estimated at US$1.6
billion (net present value) over 15 years.
In
the early 1990s, CIAT used conventional pathotyping
assays (the only diagnostic tools then available) to
identify 56 strains or pathotypes of blast within a
collection of 151 Colombian isolates gathered in the
late 1980s. These were classified according to the level
of infection they cause in a standard set of rice cultivars
with known levels of resistance.
More
recently, DNA molecular marking techniques have allowed
for more in-depth analysis of these isolates. Using
RFLP fingerprinting based on the DNA probe MGR586 (found
in all types of rice blast), the CIAT-Purdue research
team discovered a chink in the armor of rice blast:
The various pathotypes fell into just six genetically
distinct families or lineages. On average, fingerprints
for different isolates within each lineage were 92 percent
similar.
Fortunately,
each blast lineage turned out to be strongly associated
with a specific subset of rice cultivars. RFLPs, based
on DNA probes from Cornell
University’s molecular map, as well as RAPDs,
are now being used to identify rice breeding lines with
specific resistance genes. This molecular marking work
recently identified genes associated with resistance
to blast strains belonging to one of the six lineages.
The results suggest that the strategy of identifying
gene combinations that confer resistance to lineages
rather than to individual strains is on the right track.
The approach could also serve as a model for tackling
similar fungal diseases in potato, wheat, sorghum and
millet.
Using
Wild Rice for Crop Improvement
Another
promising area of rice research that draws heavily on
molecular marker technology is the search for useful
genes in wild rice species to help break the rice yield
barrier. CIAT has successfully produced rice hybrids
by crossing improved rice cultivars with three wild
species. One cross in particular, between the wild species
Oryza rufipogon and the elite rice cultivar BG90-2,
shows good yield potential and resistance. Some families
of backcrosses (the result of mating a parent with its
offspring) had a significantly higher yield than BG90-2.
RFLPs
and microsatellites were used to evaluate families of
this cross for quantitative trait loci (QTLs), gene
combinations that control single phenotypic traits,
including important ones such as grain yield. Preliminary
results indicate yield-associated sites on six chromosomes.
A similar set of tests on another promising cross, between
the elite Lemont and the wild rice O. barthii,
showed an association between microsatellite markers
and yield on one chromosome.
Cassava
Mosaic Disease
A
major focus of CIAT’s cassava-related molecular
marking work is the mapping of genetic resistance to
cassava mosaic disease (CMD). This research is being
done in cooperation with CIAT’s sister organization,
the International Institute of Tropical Agriculture
(IITA),
headquartered in Nigeria.
CMD,
which at times causes total crop failure, is caused
by a geminivirus transmitted by the whitefly Bemisia
tabaci. At the moment the disease is restricted
to Africa. However, rapid and worldwide spread of whitefly
has caused concern that CMD could mutate and move elsewhere,
especially Latin America and Asia, which are also major
regions of cassava production.
Although
CMD-resistant varieties are available in Africa, the
pathogen is constantly changing and new strains are
highly virulent. Such a superstrain set off an epidemic
in Uganda in 1986, devastating production over several
years. Severe food shortages ensued and some people
died of starvation. So new sources of genetic resistance
urgently need to be found and harnessed.
Breeding
resistant cassava varieties is a slow process, in part
because of the crop’s long growth and reproduction
cycle. Researchers therefore consider it important to
develop varieties that carry as many different genes
for CMD resistance as possible. Such “gene pyramiding”
requires the use of molecular marker-assisted selection,
preceded by studies to tag the necessary genes.
The
groundwork for this has been laid under a CIAT-IITA
project funded by the Rockefeller
Foundation. Researchers have built a molecular map
of cassava containing 250 RFLPs, 186 SSRs, 100 RAPDs,
10 ESTs, and three isozyme markers. The map has allowed
researchers to analyze a collection of CMD-resistant
and susceptible cultivars, both landraces (farmers'
varieties) and improved varieties.
This
work has begun to pay off. Recently, researchers found
a region of the cassava molecular map that is linked
to CMD resistance. The region, flanked by one SSR marker
and one RFLP marker, accounts for half the phenotypic
variance of CMD resistance observed in the germplasm
under study. While more effort is needed to saturate
this part of the molecular map with markers, the success
to date paves the way for improving CMD resistance in
African and other cassava and preventing CMD from becoming
a menace in areas not yet affected.
In
a parallel project, CIAT scientists are using AFLPs
and microsatellites to locate the genetic basis of resistance
to the whitefly itself, a trait observed in some cassava
lines. In this instance, the work focuses on resistance
to Aleurotrachelus socialis, a direct pest of
cassava rather than a disease carrier. The insect sucks
sap from leaves, which leads to loss of plant vigor
and lower yield. Whitefly excretions on leaves, called
honeydew, make matters worse by serving as a substrate
for a leaf mold that reduces the cassava plant’s
photosynthesis capacity. In a comparison of resistant
cassava genotypes with susceptible ones, researchers
recently found 22 AFLPs and 90 microsatellites to be
polymorphic. That is, these markers were inherited only
by those cassava plants that were also resistant to
whitefly.
Visit the Web
site of the Tropical Whitefly Integrated Pest Management
(TWF-IPM) Project
Abiotic
and Biotic Stresses in Beans
Beans
are a major food crop in Latin America as well as central
and eastern Africa, especially for the poor. Beans are
remarkably diverse in yield, plant shape, and the range
of soil conditions and elevations where they can be
grown as well as in grain size, color, and texture.
Much of this phenotypic variation is genetically based,
and molecular markers now serve as valuable aids in
CIAT’s research on this protein-rich staple.
During
the 1990s, CIAT breeders screened thousands of specimens
(accessions) from the bean
germplasm collection held at the Center. This intensive
effort identified materials with superior yield potential,
including bean lines that do well under the low soil-phosphorus
conditions experienced by many small farmers. To aid
in the transfer of the promising genetic material into
new breeding lines, we recently used RAPDs to look for
multiple genes (QTLs) responsible for the observed high
yields. The target germplasm in these experiments was
a plant population resulting from the cross between
two promising bean lines: one with high biomass production
but with poor yield under low-phosphorus conditions
and the other with excellent yield despite the stress
of low phosphorus.
Our
researchers recently discovered several gene combinations
(QTLs) with a remarkably strong influence on yield.
For example, in the case of beans grown under low phosphorus,
they identified a relatively long segment of DNA that
accounted for more than 300 kilograms of yield per hectare.
That is roughly one-third of total yield. Interestingly,
the contributing QTLs differed according to the soil-phosphorus
level under which the beans were grown. This suggests
that MAS strategies should be tailored to soil phosphorus
conditions.
With
such strong links between genes and yield, MAS holds
out great promise for developing varieties for small
farmers who cannot afford to buy much fertilizer. And
there could be an added bonus: CIAT scientists suspect
that the genes responsible for tolerance to phosphorus
deficiency may also protect beans from drought.
In
the 1970s, bean golden mosaic virus (BGMV), a devastating
pathogen transmitted by whiteflies, began spreading
in Latin America, especially Central America. The virus
turns leaves a brilliant yellow and distorts both the
leaves and bean pods, resulting in undersized seeds.
CIAT responded quickly by developing and introducing
new resistant varieties in the late 1970s. National
research programs and farmers welcomed the solution.
By 1996 about 40 percent of Central America’s bean
area was planted to the improved varieties. And in some
areas badly hit by the virus, coverage has been as high
as 80 percent.
As
with the effort to tap the genetic basis of tolerance
to low-phosphorus stress, CIAT has been using molecular
markers to zero in on the bean genes responsible for
this valuable resistance to BGMV. One key source is
a recessive gene, dubbed bgm-1. Our research team has
developed a SCAR marker based on a RAPD marker identified
by USDA in Puerto Rico. The gene comes from a Mexican
landrace.
Going
a step further, the scientists converted the RAPD to
a SCAR. The latter is more efficient in marker-assisted
selection., because it can locate precisely on the plant
genome individual occurrences of a specific DNA sequence
linked to the gene being tracked by the breeder. The
biotechnology lab then screened 8,000 young plants for
the marker and gave the results to breeders just a little
over a month after planting. To make their crosses in
the second phase, the bean breeders used only plants
bearing the bgm-1 SCAR marker. The experiments
proved not only the effectiveness of SCAR-based MAS
strategy for bean but also its efficiency. In conventional
or classical breeding, a large number of plants have
to be grown to maturity and then examined to see which
are resistant. The research team found the MAS approach
cut breeding time and effort by about 60 percent.
Peppers,
Palms, and Passion Fruit
Besides
its research on staples like beans, rice and cassava,
CIAT also works with partner organizations to study
genetic diversity in several other tropical crops of
growing or potential economic importance. Among these
are peppers, palms. and passion fruit.
Peppers
(genus Capsicum) are a popular spice, condiment,
and vegetable in kitchens around the world. Hot chili
peppers are particularly good for adding zest to meats,
vegetables, sauces, and soups. Capsicum originated
in the equatorial region of the New World, and the Colombian
Amazon has been a major area of domestication over the
past six millennia or so. Although Colombia now produces
about 60 percent of the world’s green chili peppers,
all its production ironically comes from imported seed.
CIAT
is now working with the SINCHI
Institute in Bogotá and the University
Nacional de Palmira on a joint project to shed new
light on the biochemical, morphological, and genetic
traits of peppers. Our contribution is on the genetics
side. Preliminary AFLP analysis of wild species, collected
from the villages of 15 indigenous Amazonian peoples,
has revealed three major pepper groupings. Several cultivated
species were also included in the analysis for reference.
This classification work is a first step in the genetic
improvement of Capsicum—a way to help Colombia
tap its rich pepper heritage.
Similar
partnerships in biotechnology have also been struck
with other Colombian organizations. CIAT is working,
for example, with the Alexander
von Humboldt Institute for Research on Biological
Resources on a genetic study of endangered palms as
well as other plant and animal species. It is also collaborating
with the Colombian Corporation for Agricultural Research
(CORPOICA)
on the genetic characterization of passion fruit (Passiflora).
Gene-Transfer
Technology
In
gene-transfer technology, a potentially beneficial gene
from one organism is isolated, sequenced, cloned, modified,
and then inserted into the genome of the target plant.
The immediate destination of the new gene is not the
whole plant but one or several possible cells of regenerative
tissues extracted from the target plant (see the section
below on tissue culture). When cultured, the cells grow
into transgenic plants containing the foreign gene of
interest.
Candidate
genes for transfer to rice, beans, cassava, tropical
forages, and other crops can come from any of three
major sources: wild relatives, genetically more distant
plant species, or completely different organisms from
other taxa like viruses and bacteria. Transgenic rice
recently generated at CIAT, for example, harbors a gene
from the rice hoja blanca ("white leaf")
virus, which results in plant resistance to that virus.
We are also making progress toward the production of
transgenic cassava that will contain an insect-resistance
gene derived from Bacillus thuringiensis. This
bacterium, widely applied as an organic pesticide, has
also been used by other organizations to create transgenic
maize, cotton, and other crops.
The
actual insertion of a gene into the target tissue is
done either directly by mechanical, electrical, or chemical
means, or indirectly by a vector. The most commonly
used direct method is biolistics. A high-speed particle
gun, driven by compressed helium, bombards the tissue
with thousands of tiny gold "bullets," each
coated with the foreign DNA (the gene of interest).
Gold and helium are inert elements and thus do not interfere
in the biological process of gene incorporation.
The
second, more conventional method is called Agrobacterium-mediated
transformation. Agrobacterium is a soil-borne
bacterium, which naturally transfers some of its genes
into the plant genome (genetic transformation). In the
laboratory the transferable genes from the bacterium
plasmid (a circular chromosome found in several kinds
of bacteria, including the genus Agrobacterium)
are replaced by splicing in copies of the foreign gene
or genes of interest, called the "gene cassette."
The engineered bacteria carrying the gene cassette are
cultured with tissues from the target plant. There the
gene cassette incorporates itself into the tissue's
cellular DNA.
Two
other genes that serve as monitoring and screening tools
during the transgenic regeneration exercise usually
accompany the gene of interest in the gene cassette.
The monitor, called the "marker" gene, serves
as an indirect indicator of how well the beneficial
foreign gene has been incorporated into the plant tissue's
DNA. The most commonly used marker is the gus
gene, which encodes for an easily detected enzyme. The
level of gus expression in the early stages of
transformation, when the cultured plant tissue is growing,
can be observed as enzyme-related blue color differences
in the tissue. The second screener in the cassette is
called the "selectable" gene. It encodes for
resistance to a compound included in the culture medium.
The most commonly used selectable genes confer resistance
to a specific antibiotic. Once plantlets have regenerated
from cells in the tissue culture, they are treated with
that antibiotic. Those containing the gene of interest
will nearly always contain the selectable gene as well.
They survive the treatment because they are resistant
to the antibiotic. All other plantlets die. Thus, the
selectable gene serves to screen out nontransgenic plants.
The
aim of gene transfer is that the recipient organism
will express the trait for which the gene encodes and
transfer the trait on to future generations. The end
result, assuming the transgene successfully "segregates"
to the progeny, is referred to as a transgenic organism
or, more popularly, a genetically modified organism
(GMO). While plant biotechnologists are often successful
in the actual gene-insertion process, gene expression
and inheritability are not always a sure outcome. Number
of copies, changes, and position of the inserted gene
or genes in the plant genome may affect their stable
expression and inheritance.
The
term "genetically modified organism," while
often seen in the popular press, is something of a misnomer.
Confusing to scientists and the public alike, it has
made for difficult and often acrimonious public debate
over the safety issues surrounding the release of genetic
engineering products. (See the section
on biosafety and public information below.) Time-honored
methods of selecting and breeding plants and animals
through conventional means--by farmers, scientists,
and others--result, strictly speaking, in genetically
modified organisms. Even domesticated dogs and cats,
after centuries of manipulation by human beings, can
be considered GMOs. Random mutation in nature, interspecific
hybridization (crossing organisms that are members of
separate but similar species), and gene transfer-technology
also produce GMOs. A clearer definition of genetic modification
is needed if discussion of biosafety is to be put on
a sound footing. In the rest of this document, the term
GMO is avoided.
Gene
transfer technology at CIAT is still in the early stages
of development. This is in part due to its high costs
and complexity. Another reason is that until recently
it has not been possible to conduct, in Colombia (CIAT's
host country), the necessary follow-up field trials
of transgenic plants in the natural environment. In
late 1998, however, the Colombian government put in
place a biosafety review and regulatory structure (www.ica.gov.co/Normatividad/normas/Archivos/A002.pdf).
And CIAT has recently obtained approval of a request
for a permit to conduct outdoor field trials of transgenic
rice. (See the discussion of biosafety
issues below.)
While
very promising, gene-transfer methods are not a magic
bullet for eliminating the many constraints on food
production, whether they relate to rice or any other
crop. At CIAT we see this high-profile technology as
a single, though important, element in our overall research
strategy for enhancing and using genetic resources.
Turning
a Virus of Rice against Itself
Rice
is the world's most important crop. In Latin America,
where its consumption as a staple food by rich and poor
alike has grown in recent decades, production is regularly
threatened by outbreaks of a disease caused by the rice
hoja blanca virus (RHBV). Specific to Latin America,
the pathogen can wipe out an entire rice crop during
an epidemic. Desperate farmers often spray five or six
times with pesticide to control the leafhopper, Tagosodes
orizicolus, which transmits the virus.
Some
Latin American rice varieties do have a measure of resistance
to RHBV but not full immunity. Unfortunately, this protection
only kicks in when plants are about 20 days old; thus,
an epidemic early in the cropping season can be highly
damaging. New sources of resistance are needed to complement
the achievements of conventional breeding.
CIAT
biotechnology specialists identified a protein on the
surface of the virus (called a nucleocapsid), which
they thought would give rice better protection against
the virus if the plants could be induced to manufacture
the protein themselves. The resistance involved, akin
to the antibody-based immunity to disease that people
develop when vaccinated, is referred to as coat protein
gene-mediated protection.
With funding from the Rockefeller
Foundation, researchers successfully cloned and
sequenced the gene (in this case, a stretch of RNA)
for the viral nucleocapsid. They used a particle gun
to insert the gene into tissues from the rice variety
Cica 8, which they then cultured to recover whole plants.
Subsequent studies revealed the presence of the protective
gene in progeny.
The
transgenic rice was crossed with other commercial, nontransgenic
indica varieties. Seven generations of the transgenic
line have now been grown, and the plants show high levels
of resistance to RHBV. The resistant transgenic lines
and derived crosses with commercial varieties are currently
undergoing selection for other agronomic traits.
Simultaneously,
we are making good progress in using Agrobacterium-mediated
transformation for transferring other valuable genes
to rice.
Defeating
Cassava Stem Borer
On
Colombia's North Coast, many farmers grow a starch-rich
cassava variety for the industrial starch market. Unfortunately,
it is highly susceptible to the cassava stem borer Chilomina
clarkei. Other cassava growing areas in Colombia,
Cuba, Ecuador, and Venezuela also suffer infestations
by this insect pest.
The
stem borer is hard to get at with conventional insecticide
sprays. It spends two-thirds of its life inside the
cassava stem, a natural shield against insect-control
tactics. And when farmers cut cassava stakes as planting
material for the next crop, stow-away insects serve
as pioneers of the next infestation.
In
a survey conducted by CIAT and the Colombian Corporation
for Agricultural Research (CORPOICA),
Colombian farmers said they needed starch-rich cassava
varieties resistant to the insect pest. In response
CIAT recently began investigating transgenic methods
for endowing cassava with the necessary resistance.
Gene cassettes (engineered plasmids) that harbor the
insecticidal foreign gene are commercially available
for this purpose. In this case a gene coding for a natural
insecticide from the bacterium Bacillus thuringiensis
(Bt) is used. Our work in this area of pest control
focuses on perfecting tissue culture methods for regenerating
sufficient numbers of transgenic Bt cassava plants and
on evaluating how well the resulting toxin combats the
pest. One promising technology under investigation is
the so-called friable embryogenic callus (FEC) system,
which became available in 1996. CIAT scientists are
currently working to boost the proportion of transgenic
somatic embryos that mature, germinate, and convert
to transgenic plants under this system.
Bypassing
Recalcitrance to Genetic Transformation in Beans
CIAT
is now attempting to use its successful hybridization
work with common and tepary beans as a springboard to
deal with a long-standing problem in agricultural biotechnology:
Common beans do not respond well to gene-transfer technology.
If they did, a new door would be open to eliminating
a number of hurdles to bean production. The problem
does not lie not in the regeneration of common bean
plants from cultured tissues. Rather, foreign genes
transferred by Agrobacterium-mediated or biolistics
methods simply do not incorporate themselves into the
target bean tissues (meristematic calli) that have been
used efficiently for plant regeneration. Since tepary
beans have to date proven more responsive to genetic
transformation than common beans, CIAT is now screening
fertile hybrid populations to identify any lines that
may be more amenable to these techniques.
Tissue
Culture
Plant
tissue culture is the cultivation in vitro of
all plant parts, whether a single cell, tissue or organ,
under aseptic conditions, and then regenerating them
into whole plants. The main tissues used at CIAT are:
-
Meristems,
such as apical or axillary buds, where active cell
division takes place
-
Embryonic
leaves (cotyledons) extracted from seedlings
-
Young
leaves
-
Immature
or mature embryos
-
Anthers,
the ends of stamens containing pollen
Genetic
variation does not play a role in tissue culture, as
the only genetic material involved is the donor tissue.
Thus, plants regenerated from tissue culture are clones
of the original plant, except when the tissues are subjected
to mutation during the culture process or are used to
produce a transgenic plant, in which case the regenerated
plant may vary in one or more genes.
Recent
tissue culture research and applications at CIAT concentrate
on three areas:
-
Production
of disease-free planting materials for farmers and
researchers through better propagation techniques
-
Embryo
rescue for recovering wide crosses with wild species
-
Long-term,
large-scale storage of germplasm collections at
extremely low temperatures (cryopreservation)
Here
are a few examples of CIAT research in these areas.
Propagation
of Planting Materials
Many
crops, particularly perennials, are much easier to propagate
from vegetative cuttings than from seed, a fact well
known to ornamental plant lovers. A simple method for
many species is to take a clean cutting from a healthy
plant, put it in a glass of water, wait till roots grow,
then transplant it to a pot. A little growth hormone
is often a big help, particularly with woody species.
Such home remedies are examples of basic tissue culture.
CIAT
scientists, working with colleagues from CORPOICA, have
developed an inexpensive method for vegetative propagation
of cassava. A major advantage is that it cuts the disease-transmission
risk typically associated with the traditional method
of planting cuttings (called stakes). It is also an
easy-to-use technology suited for small farmers, who
cannot afford to buy more sophisticated propagation
kits. All it requires is a few items from the household
food pantry and some do-it-yourself biochemistry.
The
package is named RITA. It is a variation of the commercially
available Magenta Box, which contains mostly imported
components and requires commercial growth mediums and
grow lamps. Instead of the molded plastic containers
that house the Magenta propagation system, farmers can
use empty baby food jars. Another replaceable ingredient
is the Magenta growth medium, a solid substrate based
on an expensive derivative of seaweed, agar. Substituting
for this is a liquid medium composed of water and two
food products widely available at local supermarkets:
gelatin and cassava starch.
Not
only is RITA inexpensive, it also increases the cassava-cutting
propagation rate by three to five times over the Magenta
Box. As the latter is already much better than farmers'
traditional stake method, RITA represents a quantum
leap in propagation technology. Testing of the propagation
package is under way with cassava farmers in Colombia's
Cauca Department.
Another
producer-oriented technology developed by CIAT is a
micropropagation system for tropical fruits. This is
a joint effort with CORPOICA and the Corporación
Biotec, a consortium of Colombian public and private
organizations.
While
the system is intended for use with several species
of tropical fruits, research centers on guanabana
or soursop, a fruit of the Annonacae family (custard
apple). In Colombia demand for guanabana juice
is so high that the industry has to import 40 percent
of the fruit it processes. Other commercially important
fresh and juicing fruits are mora (blackberry)
and lulo.
Seed
is not the best way to propagate fruit species, since
open pollination makes it difficult to control paternity.
Grafting, a form of vegetative propagation, is needed
to control genetic makeup, thereby ensuring the best
progeny. However, this creates a serious risk of disease
being spread to producers' fruit orchards and fields.
Given strong Latin American demand for tropical fruits,
CIAT saw the marriage of grafting and tissue culture--called
micrografting--as a viable way to accelerate production
of disease-free planting material for commercial fruit
farmers. The new process starts with a soursop seedling
and an auxiliary shoot or "minibud," both
grown in vitro to eliminate pathogens.
The seedling becomes the rootstock onto which the minibud
(or "scion") is grafted. As the micrograft
grows into a new plant, it provides new buds that can
be harvested for further micrografting. This also eliminates
the need to establish mother plants as a source of new
scions, thus speeding up the process. It is possible
to get four buds from one micrograft in just 6 weeks.
These in turn are micrografted for further bud production.
With such exponential growth in a closed sanitary system,
as many as 20,000 healthy plants can be produced in
1 year from a single clean micrograft. Trees propagated
by CIAT using the new technique are now several meters
high and flowering correctly.
Transgenic
Plants and Wide Crosses
Another
tissue culture method for harnessing useful genes is
embryo rescue, a method used for decades. Genetic transformation
is not involved. Rather, two distantly related species
are crossed by conventional breeding. However, since
nature tends to erect reproductive barriers between
species, embryos from a wide cross often abort spontaneously.
Here, tissue culture can save embryos and regenerate
them into hybrid plants. An improved version of the
technique, combined with backcrossing, allowed CIAT
scientists to successfully hybridize common bean (P.
vulgaris) with a distant relative, the tepary bean
(P. acutifolius). Tepary beans contain genes
for resistance to common bacterial blight (CBB), leafhoppers,
and drought-stresses that also affect common beans.
The resulting breeding lines showed good resistance
to CBB and were distributed to national bean research
programs for further evaluation.
Cryopreservation
In
agriculture the cold is not always an enemy. When it
comes to conserving germplasm, the extreme environment
created by liquid nitrogen can be a long-term ally.
For the past few years, CIAT has been adapting a method,
known as cryopreservation, for treating and freezing
cassava shoot tips and then regenerating them into whole
plants.
The
technique has several clear advantages over the standard
in vitro method normally used by CIAT. With that
system some 6,000 cassava clones are maintained under
controlled slow-growth conditions within the gene bank
and grown out every 12 to 18 months to produce fresh
clones. Cryopreservation involves less work and is cheaper
than maintaining either an active in vitro collection
or outdoor conservation areas (in situ sites).
The prepared shoot tips take up very little space and
can be stored indefinitely without being disturbed.
The
cassava tissues are first treated with sodium alginate
to provide a thick protective coating. They are then
picked up by suction with pipettes and dropped into
a solution of calcium chloride, which makes them coagulate
into beads. In effect, the process creates artificial
cassava seeds.
Next,
the water content in the beads is reduced by treatment
with a sucrose solution, and then by immersion in silica
gel for 12 to 14 hours. These two steps mimic the natural
seed-drying process and prevent the formation of destructive
ice crystals while the beads are stored in liquid nitrogen.
CIAT
scientists subjected clones of 45 cassava cultivars
to the bead treatment and freezing in liquid nitrogen,
followed by regeneration procedures. They recorded a
plant-regeneration success rate of about 20 percent
but estimate this can be boosted 5 to 10 percent by
improving growing conditions. The next round of experiments
will more than double the number of clones tested.
Biosafety
and Public Information
As
the operator of a major biotechnology laboratory involved
in genetic transformation of plants, CIAT maintains
the highest standards of biosafety. Though Colombian
biosafety regulations for transgenic plants were established
only in late 1998, CIAT has followed international biosafety
regulations guidelines since the early 1990s, when our
genomic and genetic transformation work was begun. CIAT's
transgenic products were confined in containment biosafety
laboratory facilities and biosafety greenhouses. Once
the Colombian Biosafety Committee granted permission
for field testing of these products, field trials were
conducted under Colombian regulations and inspection.
Also, CIAT has an in-house Biosafety Committee that
consists of a multidisciplinary team representing expertise
in genetic resources, breeding, plant health, soil health,
economics, participatory research, and molecular biology.
We
are also committed to transparency and open communication
with other scientific organizations, national authorities,
and the public on this important issue. In late 1999
we organized an international workshop on biosafety
that brought together experts in breeding and biosafety
from eight Latin American countries, plus the USA and
the UK. The meeting explored issues surrounding the
release and eventual cultivation of transgenic crops
in Latin America and the Caribbean. The development
of CIAT's transgenic rice, resistant to RHBV, served
as the main case study for analysis and, we believe,
provides a model for biosafety in the region.
Meetings
such as these allow researchers and regulatory authorities
to examine biosafety risks and issues and propose solutions.
In the case of transgenic rice, two potential problems
include the movement of viral-resistance transgenes
into weeds genetically related to rice (so-called gene
flow) and the risk of transgenes combining with other
viruses to produce potent new plant pathogens.
Studying
the distribution of weed species and other wild relatives
of rice before a transgenic variety is tested in the
open environment or released for commercial use helps
quantify the risk of gene flow. Authorities charged
with deciding whether and where to restrict production
of the crop need such information. Among cautionary
measures under development or already in use are land
management techniques, such as selective weeding and
the use of buffer zones around transgenic rice crops
to prevent cross-pollination with weedy or wild species.
CIAT is currently conducting a project in which gene
flow from crop to wild/weedy relatives is being analyzed
in rice and beans, with the aim of evaluating potential
risks, developing management practices to reduce risks,
and developing monitoring tools for use in farmers'
fields and natural environments.
Download
the followingrelated Posters:
Gene
Flow Analysis from Rice into Wild/Weedy Relatives
in the Neo-Tropics: Morphological and Phenological
Characterization of Red Rice (220
kb)
Molecular
characterization of rice and wild/ weedy relatives
by microsatellites and their use to assess gene flow
in the Poster (236 kb)
As
for the potential for viral recombination, studies of
the genetic relatedness of tenuiviruses that attack
rice have led Latin American researchers to conclude
that the risk of new virulent strains emerging is very
low. In CIAT's transgenic rice, the low level of expression
of the RNA transgene minimizes the chance of this happening.
The
CIAT biosafety workshop recommended that national legislation
be flexible to allow rapid response to new developments
in transgenic research. It also underlined the need
for technical databases regarding the release of transgenic
organisms and for better communication of scientific
information to the media, both proactively and in reaction
to breaking news stories.
In
connection with this issue, CIAT organized a biotechnology
briefing and laboratory demonstrations for Colombian
journalists in 1999 and again in 2001. The event was
designed to help the media ensure that public perceptions
of transgenic technology are based on scientific fact.
Other such workshops have been custom designed to meet
the capacity building needs of Colombian biosafety policy
makers and the Ministry of Environment.
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