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Abstracts
Inaugural Speech
Transgenic Potato Cultivars with Combined Resistance to Viral, Bacterial and
Fungal Pathogens
Alejandro Mentaberry
INGEBI-CONICET & FCEyN-UBA. Vuelta de Obligado 2490, 1428, Buenos Aires, Argentina
Developing countries produce one-third of world’s potatoes, surpassing 300 billion tons a year.
Nevertheless, per capita consumption is relatively low as compared with that of developed countries,
and there is a considerable potential to expand the use of this crop for alimentary purposes. A major
impediment to increase yields is the high incidence of phyto-sanitary problems, which introduce
heavy increases in production costs.
A project to genetically engineer various potato cultivars by introducing resistance genes to viral,
bacterial and fungal pathogens is presently in advance. To this aim, several two-gene constructions
carrying different combinations of resistance genes were introduced in already adopted cultivars. A
consortium composed by thirteen Latin American and European laboratories is contributing to
different aspects of this project.
A set of sixteen genetic constructions carrying different two-gene combinations was developed.
Transformation vectors were designed in such a way that a maximum of six genes can be introduced
in the same plant using different markers. At present, a large number of transgenic lines belonging to
nine potato cultivars that are commonly grown in Chile, Argentina, Uruguay, Cuba, Brazil and Spain
have been obtained. Transformants expressing combinations of two and four genes have been
obtained and several transgenic lines exhibiting resistance to different pathogens have been isolated
in greenhouse assays. Some of these plants are presently being tested in field trials located in the
participating countries.
Plant Biotechnology at CIAT: Powerful Tools for the Characterization, Conservation, and
Broadening the Genetic Base of Breeding Germplasm
Zaida Lentini.
CIAT (International Center of Tropical Agricultura). Biotechnology Research Unit/ Rice Genetics. A.A.
6713. Cali, Colombia. [email protected]
The assessment of diversity at the DNA level is providing information on potential new sources of
variability for broadening crop genetic base, and for linking diversity in-situ with ex-situ. Molecular
mapping of crop genomic regions associated with traits of interest, jointly with genetic engineering are
used to direct the modification of crop plant genomes. The usefulness of these powerful tools for
breeding germplasm can be illustrated by the following examples on the development of rice
germplasm adapted to the Latin American agroecosystems.
One of the major tasks of rice breeding nowadays is to increase by 70% rice production in the year
2025 in order to satisfy the future demand for this staple crop. Relying only on phenotype selection
has jeopardized the introgression of new alleles from wild species towards this goal. Currently, the
use of RFLPs and microsatellites allowed to map chromosomal regions associated with higher yields
(15-25% increase) and yield components in interspecific backcross progenies between rice (O.
sativa) and the wild species Oryza barthii or O. rufipogon, aiding the follow-up introgression of these
regions in the O. sativa genome.
Incorporation of resistance to major diseases, is one of the main targets when developing control
strategies less dependent on the use of agrochemical. Breeding for resistance to rice blast and rice
white leaf virus (RHBV, acronym for rice hoja blanca virus in Spanish) is the main tasks for the
Americas. Rice blast (Pyricularia oryzae) is particularly severe in Latin America. The great variability
in virulence exhibited by the blast pathogen is associated with the quick breakdown of resistance in
rice varieties. Analysis on population dynamics of the blast population by classical approaches is now
complemented with the characterization of the genetic structure of the pathogen population using
DNA-fingerprinting. This new approach has broaden the understanding of the resistance - virulence
interactions, and this knowledge is currently key for the design and implementation of novel breeding
strategies for durable resistance to this disease.
Rice hoja blanca virus (RHBV), is a tenuivirus endemic of tropical America. This viral disease causes
cyclic epidemics with up to 100% yield losses. Immunity for this virus has not been identified yet, and
the natural resistance source does not protect plants when younger than 25-day-old. We have cloned
the complete RNA 3 sequence, which encodes for the nucleocapsid (N) protein. Transgenic Latin
American indica rice carrying the N- gene (N-pro rice) showed 60-90% reduction in the development
and severity of the disease, and 46-64% increase in yield respect to the non-transgenic susceptible
rice. The most resistant N-pro lines show low RNA expression of the RHBV-N gene, do no
accumulate the N-protein, and some of them have truncated versions of the transgene showing the
5'end and 3'end regions but missing about 500 bp of the central part of the RHBV-N gene. Crosses
between N-pro rice and non-transgenic genotypes indicate that the protection conferred by the
RHBV-N transgene is inherited and expressed independently of the genotype background, and the
RHBV-N transgene complements the natural breeding resistance source by increasing the resistance
of 10-day-old from 30% to 70%. The first field trials of N-pro rice will be conducted in 1999. Current
international collaborative effort between Rutgers University, IDEA, and CIAT is underway to add new
transgenes towards the immunity to RHBV.
Crop genetic engineering for disease resistance – an African perspective
Dr. David Berger
ARC-Roodeplaat Vegetable and Ornamental Plant Institute
Three issues dominate this technology on a global scale. These are (i) huge investments in the
"North" by large university and multinational laboratories; (ii) aggressive patent protection of the
technology; and (iii) biosafety. In this context, how can countries in Africa develop and/or access this
technology to try to address pressing issues such as food security and the international
competitiveness of our nations? I will attempt to answer this question in part by giving some examples
from the crop genetic engineering programme at ARC-Roodeplaat. Development of a transgenic crop
requires both good science and a multidisciplinary approach. A project team should include at least a
plant physiologist, a molecular biologist and a plant breeder.
ARC-Roodeplaat has experience of the complete technology cycle - from gene isolation to transgenic
field tests. Genetic engineering can target local diseases, which may not necessarily be important
outside the region. For example, important South African potato cultivars were transformed for
resistance to potato leafroll virus, and tested in the first field trial of transgenic potato in Africa.
Polygalacturonase inhibitor proteins (PGIP) are attractive candidates for engineering fungal
resistance, since they have a different mode of action from other antifungal proteins. In collaboration
with a local partner, maize was transformed with a pgip gene, and tested for resistance to
Stenocarpella maydis. This was the first field trial of locally transformed maize. A biolistic
transformation protocol was developed for an indigenous flower, Ornithogalum. The aim is transgenic
virus resistance, since a virus limits production of this valuable export flower.
The way forward includes capacity development in Africa, for example through the UNESCO
Biotechnology Education and Training Centre at ARC-Roodeplaat that has already trained 163
scientists from 22 African countries in Plant Biotechnology. Biosafety structures are required in each
country. The South African Committee on Genetic Experimentation (SAGENE) and the new GMO
Law serve as good models. Scientists have a responsibility to participate in public education to inform
consumers that government approved GMO products are safe. Currently, only a few genes (eg. Bt,
Herbicide resistance) dominate the global industry. Novel gene discovery from indigenous sources
using some of the latest molecular techniques should be used to tap local genetic wealth.
Mechanism of Engineered Disease Resistance in Higher Plants by Transgenic Expression of a
Proton Channel
Eric Lam
Biotech Center, 59 Dudley Road, Rutgers University, New Brunswick, NJ 08903, USA.
The hypersensitive response (HR), characterized by a rapid and localized cell death, is frequently
implicated in disease resistance of plants against pathogens. The mechanism of induction and
execution of this programmed cell death remains unknown. In the past few years, we have shown
that one can mimic the HR-pcd process by the ectopic expression of a bacterial proton pump called
bO (Mittler and Lam, Plant Cell 7: 29). By all the criteria that we have examined, which included in
situ detection of nuclear DNA fragementation and resistance to pathogen challenge, we found that
these transgenic tobacco plants exhibit all the classical HR and systemic acquired resistance (SAR)
characteristics. Recently, we showed that similar phenomena are observed in transgenic
Arabidopsis, potato and eggplants as well. Thus, spontaneous HR-pcd lesions are observed in these
plants and like in tobacco, the severity of these lesions is temperature sensitive. Expression of bO in
these plant species has been correlated with heightened resistance to particular viral (TMV, TNV),
bacterial (Pseudomonas syringae) and fungal (Phytophthora infestans) pathogens. In order to
elucidate the molecular basis for the induction of this HR-like response and provide some information
about early events that may trigger the HR, specific amino acid residues have been mutated in the
bO protein and the corresponding mutants have been expressed in transgenic tobacco. The
phenotype of these transgenic plants indicate that proton channel function is required for bO to
induce HR and disease resistance phenotypes, supporting the hypothesis that ion fluxes could play a
causal role in triggering this process.
In addition to triggering cell death, the expression of bO induces the production of PR proteins and
salicylic acid. Using plants expressing various levels of bO protein, a quantitative analysis of the
resistance against Pseudomonas syringae pv. tabaci in such plants showed that resistance activation
and cell death induction may have different thresholds and that PR induction is not quantitatively
correlated with resistance against this bacterium. In contrast, resistance against viral pathogens such
as TMV is tightly dependent on expression levels of bO. These results suggest that a pathway distinct
from SAR may be activated by bO to inhibit the proliferation of certain bacterial pathogens.
Genetic Engineering for Maize Improvement in China
Guo-ying Wang.
National Lab for Agrobiotechnology, China Agricultural University, Beijing 100094, China
Maize is one of the most important cereal crops in China. Its planting acreage is more than 20 millions
of hectares and total yield exceeds 100 million tons every year. With the increase of population and
living standard in China, more and more demand for maize will be expected in the future. Genetic
engineering techniques have been used to improve maize. (1) Genetic engineering for maize
resistance to corn borer. Bt insecticidal protein gene (Cry IA), proteinase inhibitor II gene from potato
(Pin II), cowpea trypsin inhibitor gene (CpTI) have been transferred into maize and some of the
transgenic plants are highly resistant to Asian corn borer. (2) Genetic engineering for maize tolerence
to salt. Glucital-6-phosphate dehydrogenase (gut D) gene from E. coli were introduced into maize
embryogenic calli by using microprojectile bombardments and fertile transgenic plants were
recovered. A preliminary results showed that transgenic plants maintained a higher water potential,
delayed development of salt damage symptoms under salinized conditions (150 mM and 200 mM
NaCl). (3) Genetic engineering for resistance to maize dwarf mosaic virus (MDMV). MDMV coat
protein gene has been cloned and transferred into maize. The progeny of one transgenic plant were
innoculated with MDMV and attained a disease rating of 2.4 compared with a rating of 6 on
untransgenic plants in greenhouse.
Applied Genomic Research in Rice Genetic Improvement
Qifa Zhang
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan
430070, China
Genome research will make profound impacts on crop genetic improvements. In this presentation, I
will report the progresses made in our group in the applications of genome research to important
problems related to rice genetic improvements.
A major progress has been made in the study of the genetic basis of heterosis. Analyses of the
relationships between molecular marker heterozygosity and hybrid performance in a number of diallel
crosses using molecular markers produced variable results suggesting the complexity of the genetic
basis of heterosis. Characterization of the genetic components underlying the strong heterosis of
"Shanyou 63", the best hybrid in China, showed that epistasis played an important role as the genetic
basis of heterosis.
Progresses have also been made in identification, molecular marker-based mapping and genetic
analyses of important genes for rice improvements. The list includes the genes for fertility restoration
of wild-abortive cytoplasmic male sterility, photoperiod sensitive male sterility, wide-compatibility,
grain quality, and resistance to blast and bacterial blight. The analyses greatly enhanced the
understanding of the genetic basis underlying the expression of these important traits. Tightly linked
markers were obtained for most of the genes. We are now isolating several of the genes following the
map-based cloning approach. Physical maps have been completed for genomic regions surrounding
the pms1 locus for photoperiod sensitive male sterility and the S5 locus for wide-compatibility.
Another important effort we have made is improving hybrid rice using molecular techniques. Using
back crosses and marker-assisted selection, we have transferred Xa21, a gene for bacterial blight
resistance, into Minghui 63 as an attempt to improve the resistance of the hybrid "Shanyou 63". The
improved Minghui 63 is genotypically the same as the original variety, except the genomic segment of
less than 3.8 cM in length that carries the Xa21 locus. We have also nearly completed
marker-assisted selection for improving grain quality of the same hybrid, which is a major problem in
rice production in China. It appears promising that genome research has made molecular breeding in
rice a reality.
Broad spectrum pathogen resistance by expression of nontoxic forms of pokeweed antiviral
protein
Nilgun E. Tumer, Rong Di, Katalin Hudak and Oleg Zoubenko
Biotech Center for Agriculture and the Environment, Cook College, Rutgers
University, New Brunswick, NJ 08901-8520
Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana removes an
adenine residue from the a-sarcin/ricin loop of the large rRNA of the eukaryotic and prokaryotic
ribosomes, thus inhibiting translation. Transgenic tobacco plants expressing PAP confer resistance to
a broad spectrum of pathogens including plant viruses and a soil born
pathogenic fungus Rhizoctonia solani. Both acidic and basic PR proteins were induced in transgenic
plants expressing a nontoxic C-terminal deletion mutant, PAPc, and they were resistant to viral and
fungal infection. Another mutant of pokeweed antiviral protein, PAPn, was also nontoxic and
conferred strong resistance to viral and fungal infection. In contrast to wild type PAP and PAPc,
transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis related
proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic
plants. These results indicated that expression of PAPn in
transgenic plants activates a particular SA-independent stress-associated signal transduction
pathway that may be responsible for the broad spectrum disease resistance. Since the a-sarcin loop
has been placed near the peptidyl transferase center in E. coli ribosomes, we investigated the
effects of alterations at the peptidyl transferase center on the activity of PAP. We demonstrated that a
chromosomal mutant of yeast, harboring the mak8-1 allele of peptidyl transferase-linked ribosomal
protein L3 (RPL3), is resistant to the cytostatic effects of PAP. Unlike wild-type yeast,
ribosomes from mak8-1 cells were not depurinated when PAP expression is induced in vivo,
indicating that wild-type L3 is required for ribosome depurination. Co-immunoprecipitation studies
showed that PAP binds directly to L3 or Mak8-1p in vitro, but does not physically interact with
ribosome-associated Mak8-1p. L3 is required for PAP to bind to ribosomes and depurinate the 25S
rRNA, suggesting that it is located in close proximity to the a-sarcin loop. These results demonstrated
for the first time that a ribosomal protein provides a receptor site for an RIP and allows depurination of
the target adenine.
Development of Resistance Management Strategies for Commercial Cultivation
of Bt Cotton in China
Shi-Rong Jia*
Biotechnology Research Center, Chinese Academy of Agricultural Sciences, Beijing 100081
Large scale field trials of Bt cotton, developed by Chinese scientists, have been conducted in different
locations since 1996. In 1997, the Biosafety Committee, Ministry of Agriculture approved
commercialization of Bt cotton in 5 provinces in China. Field performances show it has strong
insecticidal activity to the cotton bollworm. In this article, the development of resistance management
strategies for Bt cotton is discussed in detail. Based on experimental data, we predict Bt cotton may
at least be used for 8 to 9 years. Data also showed transgenic tobacco with Bt/CpTI gene may delay
resistance development in cotton bollworm population.
Homology-based Cloning of R-Gene Candidates from Rice
Zhang-Liang Chen, Li-Jia Qu, Chonglin Yang, Guofeng Zhu, and Hongya Gu
College of Life Sciences, Peking University, Beijing 100871, China
Fax: 10-62752497 E-mail:[email protected]
Several pairs of degenerate primers were designed based on the conserved leucine rich repeat
(LRR) domains and nucleotide binding site (NBS) domains of cloned R genes' products. DNA
fragments were amplified from the genomic DNA of rice cultivar Zhonghua 8 (Oryza sativa var.
japonica), then cloned and sequenced. For LRR primers, one fragment (designated RL3.2) is found
to have a typical LRR structure. For NBS primers, another two fragments (designated RN15 and
RN18) are found sharing significant homology with NBS domain. A rice BAC library was screened
using RL3.2, RN15 and RN18 as probes and several positive clones were obtained respectively.
For LRR clones, one BAC clone was selected. After a series of subcloning and hybridization, a
fragment which is 4.5 kb in length was found to give hybridization signal to the probe. Sequence
analysis reveals that it contains an open reading frame encoding for a receptor- kinase protein of
1089 amino acid residues This deduced peptide, which shares certain sequence homology with that
of rice bacterial blight disease resistant gene Xa21, contains 24 perfect LRRs and 3 partial LRRs in
its N-terminus and three typical ser/thr kinase conserved domains in its C-terminus, i.e. Vib region
(DLKPSN), VII region (DFGLAKE) and VIII (GSVGYIAP). Comparing with Xa21, the product of this
gene has a longer receptor domain but shorter ser/thr kinase domain. The transformation and
challenging of pathogens to transgenic plants are being carried out. For NBS clones, seven clones
were obtained, among which three were found to have different restriction pattern. Partial sequence
analysis reveals that these three clones share significant homology with NBS domains of cloned
R-genes' products. Several conserved domains have also been found in one of the three BAC clones
(RNP5) even outside of the NBS domains sharing homology with those of other R genes including N,
L6, Rps2 and Rpm1. These three genes are being sequenced.
Functional Genomic Study of Rice Against Rice Dwarf Virus
LI Yi, WEI Chunhong, and CHEN Zhangliang
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life
Sciences, Peking University, 100871,Beijing, China
Rice dwarf virus (RDV) causes serious disease on rice production in many Asian countries,
developing control methods for this disease would be of great importance. In the past few years full
genome with 12 dsRNA segments of RDV Chinese isolate was cloned and sequenced. E. coli,
baculo- and rice expression systems have been established. Based on the analysis of expressed
products and amino acid sequence, functions for each gene segment are outlined. S1 is suggested to
encode the viral polymerase. RDV S2-encoded protein is essential in virus attachment and/or
penetration of virus into insect cells. S3 encoded-protein P3 shares significant homology to P3 of rice
gall dwarf virus (RGDV), VP4 of rotavirus at the amino acid level. It is suggested that RDV S3
encoded P3 protein is a major core protein and may involved in virus replication. S4 encoded-protein
contains a putative zinc-finger and GTP-binding motif. S5 encoded-protein has GTP binding activity
and is assumed to be the mRNA guanyltransferase. S6 encoded protein contains the viral
NTP-binding like motif. S7 encoded P7 has been found in viral particle and are suggested that is a
minor core protein. Immunoelectromicroscopy with antibody raised against S8 product showed that
S8 encode protein is viral outer coat protein with a molecular weight of 46KDa. Outer capsid protein
(P8) heterogeneity exists in purified virus particles, RDV infected rice and transgeneic rice expressing
P8 and E. coli expression P8 product. N-terminal amino acid sequencing revealed that P8’ (42KDa) is
a post cleavage product of P8. The cleavage occurs specifically at the residues of Asp362 and
Pro363 (Zheng et al., 1997, Theor Appl Genet 94:522-527 and Mao et al., Archives of
Virology,143:1-8, 1998). Amino acid sequence of RDV S11 encoded-protein contains a putative zinc
finger and five flanking basic regions at the C-terminus. The protein shares homology with WTV S12
coded protein, sea urchin histone H1 and VP6 of blue tongue virus (BTV). It was suggested that RDV
S11 encodes a nonstructural and nuclei acid binding protein. We found that S11 encoded P11 is a ssand ds- RNA and DNA binding protein by South- and North-western. The binding ability is sequence
-nonspecific. The binding-ability of different truncation mutations of Pns11 showed that the binding
ability needs both the zinc-finger and the C-terminal basic regions (Xu et al., Virology,
240:267-272,1998). RDV gene segment S2, S6, S7, S8 and S9 have been transferred into rice
genome. The functions of these genes in transgenic rice plants are in identification.
The Chinese Rice Genome Program
Hong Guofan
Center of Biotechnology in Shanghai under the Chinese Academy of Sciences
Professor Hong Guofan was coordinator of the Chinese Rice Genome Program during the period of
1992 -1998, and he has completed contig maps of the rice genome using the Bac library and finger
printing strategies. He has constructed a number of contigs of various lengths covering 12
chromosomes with 92% coverage of the full genome. Based on the physical maps he has achieved
he does large scale sequencing of the genes of Chromosome 4 and further identification of genes
that may have agricultural significance.
Managing emergence of resistance in insects against insecticidal toxins of Bacillus
thuringiensis
Raj K. Bhatnagar and A. Selvapandiyan,
International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi-110067
Insecticidal toxins produced by the strains of gram positive bacterium, Bacillus thuringiensis are
highly specific to the target pest and are environmental friendly. Genes coding for these toxins have
been incorporated into the genome of several crop plants. Expression of these toxin coding genes in
plants have protected them from insect predation. Generally the expression levels of native
insecticidal toxin coding genes in plants is not adequate, necessitating several molecular
modifications. Some of the modifications which have resulted in an enhanced expression of bacillar
genes in plants include the choice of promoter elements, optimisation of codon bias in favor of plants,
removal of potential polyadenylation signal sequences and truncation of the insecticidal toxin coding
gene etc. Thus, modification of toxin coding gene keeping in view optimisation parameters for
expression in plants have led to the development of transgenic crops resistant to predation by
insects.
Over the past couple of years since the release of Bt-transgenics in field, emergence of resistance by
pest to the toxins has been observed. The resistance is monogenic and dominant and is
simultaneous to structurally related toxins. Several pest management practices are being devised to
delay the emergence of resistance in the pest. From the structural aspect of the insecticidal toxins it is
obvious that the structurally dissimilar toxins will interact at different receptors at the midgut of the
susceptible insect larvae. Thus if structurally unrelated toxins that are active against the same pest
are expressed in the plant the emergence of resistance will be delayed since two simultaneous
mutational events will be required for insect to become resistant. During screening of several strains
of B. thuringiensis we have identified, cloned, sequenced and expressed two different insecticidal
toxins genes, cry1Ia5 and vip (vegetative insecticidal protein). A structural comparison of the two
proteins revealed near lack of homology. Analysis of toxicity spectrum revealed that E. coli expressed
cry1Ia5 is toxic to pest - Spodoptera litura, Plutella xylostella, Chilo partellus, Anomis flava,
Cnaphalocrocis medinalis and Phthorimea opercullela. Vegetative insecticidal protein is active
against, Chilo partellus, Spodoptera litura Earias vitella and Plutella xylostella.
In the pest management programme it will be pertinent to exploit structural uniqueness of the vip
toxin and use it in combination with other relevant toxins already characterised. We have analysed
the minimum toxicity domain of vip against a few pests and also assessed its suitability for
expression in plants. Results of these experiments will be presented.
Genetic engineering of plants for abiotic stress tolerance
S. K. Sopory,
International Centre for Genetic Engineering & Biotechnology (ICGEB), P. O. Box 10504, Aruna Asaf
Ali Marg, New Delhi - 110 067
One of the areas for future research in Agriculture Biotechnology will greatly focus on developing
novel plants, which will help in stabilizing agriculture production irrespective of variations in
environment & soil conditions. There are many soil-oriented stress factors, which affect crop
development, survival & yield. While on one hand there is a need to better understand the
correlations in the soil-plant-water system, yet at the same time improvement programs should be
taken up to impart defence mechanisms in important crop plants to enhance their ability to cope up
with stress environment. It has been emphasized repeatedly in many fora that abiotic environmental
stress conditions are among the most important factors limiting world agriculture productivity. Their
impact has both economical & social relevance, especially for the developing world.
Salt affected soils occur throughout the world and salination process is a continuous phenomenon.
Conventional breeding, it is felt, cannot make a breakthrough in achieving success in developing
drought and salinity tolerance. The present trend of research does indicate that molecular biology
techniques are a powerful tool to enable a basic understanding of abiotic stress tolerant mechanisms
and in manipulating various pathways to confer tolerance to crop plants. In view of this we have
cloned the gene encoding glyoxalase - I protein from Brassica juncea and shown the potential of
upregulating glyoxalase level in conferring salt and metal stress tolerance in transgenic tobacco. We
have also made transgenic plants with a gene coding for a calcium binding protein. The seeds of
transgenic plants expressing the protein germinated & grew better than wild type plants under salt
stress. The various aspects of role of glyoxalase enzyme and glutathione and of calcium binding
proteins in stress-tolerance, and attempts to genetically modify the enzymes of these pathway(s) in
crop plants will be discussed.
Chloroplast transformation for enhanced expression of foreign genes
V. Siva Reddy
Plant Transformation, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali
Marg, New Delhi - 110 067, India
The higher plant mesophyll cells contain 50-100 chloroplasts with each plastid containing 50-100
copies of its genome. Introduction of foreign gene into plastid genome, therefore, can result in the
amplification of introduced gene ranging from 5000 - 10,000 copies per cell. As a result, the
expression of foreign gene in plastid transformed plants is expected to be very high when compared
to nuclear transformed plants. In addition, the possibility of site specific integration of genes into
plastid genome eliminates the variations in the expression of foreign gene due to positional effects
among different transgenic plants is eliminated, a common problem in the nuclear transformation.
Also, as the plastid encoded traits are inherited maternally, the chances of introduced genes
escaping into wild relatives through pollen is remote in plastid transformed plants, thus making it
environmentally a safe approach. Therefore, we have focused our efforts to introduce unmodified Bt
cryV gene into chloroplasts of tobacco in order to obtain highly insect resistant transgenic plants.
Although various formulations containing Bt products have been used for the control of insects for
more than three decades, production of transgenic plants is an elegant and effective delivery system
for Bt Toxin. The Bacillus thuringiensis insecticidal gene, cryV, is toxic against colepteran and
lepidopteran pests. Previously, we have shown that an unmodified cryV gene expressed in tobacco
plants offered complete protection from Heliothis armigera neonate larvae (Mol Breed. 4: 473-4778,
1998). To increase the level of resistance through enhanced expression levels of the toxin, we
transformed tobacco chloroplasts with the same unmodified cryV gene. Western blot analysis
revealed that 2-2.5% of the total protein was CRYV, a 50 to 100 fold increase when compared to
nuclear transformed plants. The bioassays revealed that the plastid transformed plants offered
protection against 2nd and even 3rd instar larvae. The progeny of transgenic plants were grown to
maturity in a green house and evaluated for various growth parameters. The results indicated that the
plants expressing cryV in chloroplasts are morphologically indistinguishable from the untransformed
wild-type plants.
Plant biotechnology for crop improvement in Bulgaria
Atanas Atanassov
Institute of Genetic Engineering, Bulgaria
Agriculture is the oldest and still the most efficient industry in Bulgaria. For the last 20 years 95 % of
the arable land in Bulgaria for the most important agriculture crops was planted with cultivars
developed by local breeding Institutes. A National Program for plant biotechnology was launched in
1985 when the Institute of Genetic Engineering (IGE) was established into the frame of the
Agricultural Academy as National Center for Plant Cell and Molecular Genetics Research in order to
strengthen the crop breeding in Bulgaria. Such goal was possible to achieve because of the well
coordinated activities within the country and the well established cooperation with the leading foreign
research center, universities and private companies. An important issue was the development of
National guidelines for biosafety of genetically modified higher plants. Bulgaria was the first country
and commercialization of plant biotech varieties establish inside and outside of the country.
The cellular and molecular methods, widely used at IGE, complement the routine tissue culture
techniques performed by the breeding institutes. Both of the above modern techniques have been
implemented into the breeding programs and have given the first expected results.
Many valuable breeding lines have been already developed for wheat, barley, rice, sugar beet,
sunflower, alfalfa, tobacco et. Tobacco variety BIOPRESLAVNA, Datura innoxia-INKA, rice
MARIANA and wheat ZDRAVKO have been created on the base of the anther cultures. Gene cloning
and transfer methods are expected in the next few years to lead to the creation of the first tobacco
cultivars, resistant to economically important diseases, herbicides, parasites , Orobanche, drought,
cold and heavy metals, based on unique CMS developed by somatic fusion with the wild species of
tobacco.
As a second phase similar varieties are going to be developed in tomato (resistant to TSWV and
unique CMS lines); alfalfa (improving yield and digestibility, drought and Fusarium resistance), grape
(resistance to viral diseases (GFLP) and low temperature), carnation (new color changes ) etc. New
promoters related with the male fertility have been also isolated and characterized.
The economical and political changes in Bulgaria have also affected plant biotechnology. IGE started
to realize six years ago a restructuring program, aimed to overcome the financial restraints by
intensifying its international cooperation and diversification of the financial sources. This is achieved
by involvement into various international (such as ICGEB, EU’s, COPERNICUS and FACE,
FAO/IAEA, etc.) and bilateral programs and performing contract research for major multinational
corporations. A four years ago IGE was invited and joined the newly formed Norman Borlaug Institute
of Plant Science Research, founded by the De Monfort University, Leicester, UK which we consider
as a most valuable outcome of our strategy for internationalization of science.
We expect that this meeting could lead to the development of new type of effective international
collaboration between various countries of member and member of ICGEB from oneside but also with
the private business in the field of the agricultural biotechnology