INFORMATION SYSTEMS FOR BIOTECHNOLOGY


June 2006
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


.pdf version
IN THIS ISSUE:



A CONCEPT TO ENGINEER MALE REPRODUCTIVE STERILITY IN CONIFERS
Christian Walter

Conifers provide major plantation forest tree species, covering around 40 million hectares world wide. Wood from plantations is used for the production of paper, cardboard, structural timber, and furniture. Intensive conventional breeding programs have produced significant genetic gain in some species, and genotypes with improved growth and form characteristics, resistance to pathogens, and those lending themselves to improved forest management have been produced.

Biotechnology tools, such as gene expression analysis, genetic fingerprinting, marker assisted selection, and genetic engineering, contribute to a better understanding of the genetic basis for important commercial traits, and furthermore, these techniques provide the potential to introduce significant genetic gain. This in turn will ensure commercial viability and environmental sustainability of plantation forestry. It is envisaged that genetically enhanced forest tree plantation will, in the coming 20 – 40 years and probably starting in China and South America, significantly replace the use of timber resourced from natural forests, thereby providing the opportunity to leave native forests alone.

While modern biotechnology, including genetic engineering, has made an unprecedented impact on agricultural practice due to its efficiency and environmental sustainability, the uptake of this technology in forestry is lagging behind. This delay is due mainly to the long generation times of forest trees and the difficulties originally experienced with the development of tissue culture and genetic modification technology. In recent years, however, routine genetic transformation has successfully been developed for major plantation forest trees including conifers, and genetically modified conifers are at the stage of field testing and pre-commercial evaluation.

Some groups voice strong criticism and concern about the safety of genetically engineered forests, and significant protest has been mounted against the use of such technology. One of the main concerns is the potential spread of genetically modified material through pollen flow. Conifer pollen can transfer over huge distances, and while it is not viable for long periods of time, it has the potential to transfer engineered genes into wild interfertile populations.

Various risk mitigation strategies have been proposed and tested to address this problem, mainly focusing on male reproductive sterility, which can be induced by two strategies:

1. Cell ablation in which either the tapetum or pollen is directly influenced by the expression of a cytotoxic gene, causing abnormal pollen development, or ideally, complete absence of pollen formation. For example, the barnase gene codes for an RNAase which degrades RNA in cells targeted through expression from a cell specific promoter.1 Plants engineered with such a construct usually fail to produce fertile pollen. However, this kind of approach can potentially be problematic since leaky promoters could lead to damage of non-reproductive tissue, with potentially deleterious consequences for plant health and production. This is particularly serious when the long generation times of conifers are considered.

2. Suppression of genes specific to pollen development, which can be achieved through the targeted antisense- or RNAi downregulation of a pollen specific gene.2,3 A potential problem with this strategy is that full suppression of a target gene is often difficult to achieve, even when RNAi is used.

A cytotoxic approach, as published recently by Höfig et al. (2006)4, combines the use of a tissue specific promoter isolated from the target species (Pinus radiata7) with a cytotoxic gene that is predicted to be non-toxic to non-target cells. The grapevine stilbene synthase (STS) has been shown to compete with the enzyme chalcone synthase (CHS) for the substrates malonyl-CoA and coumaroyl-CoA. STS-induced sterility in tobacco is believed to result from a reduced or abolished flavonol biosynthesis. This has been confirmed by experiments where STS-sterile tobacco plants were regularly sprayed with flavonols5 and where fertility was partially restored.

STS, when expressed in non-tapetal cells, is not expected to have a toxic impact since there is no competing CHS present. Further, expression of STS in somatic cells of plants may have the additional benefit of providing resistance against fungal attack, through synthesis of the antifungal agent resveratrol. This makes STS an ideal sterility approach with inherent biological and operational safety.

Höfig et al. first isolated a P. radiata promoter expressed specifically in male reproductive tissues and fused it to the Vitis vinifera sts gene. Testing the sterility construct in radiata pine would be time consuming, since this species usually takes 7 – 8 years to reach sexual maturity, and also a field test with plants producing male reproductive structures would require considerable time to obtain regulatory approval. Consequently, the sterility construct was initially tested in the model system Nicotiana tabacum. It was expected that the P. radiata male cone-specific promoter would be conserved between species and hence express in a tapetum-specific manner in tobacco. This hypothesis was further supported by the fact that a P. radiata male cone specific promoter-uidA construct expressed specifically in male organs of transgenic Arabidopsis.6

Expression of the sterility construct in tobacco led to almost complete ablation of pollen formation. Ten independent transgenic lines were analysed for pollen fertility. Seven did not produce any pollen, and the remaining three lines produced only very small amounts, ranging from 0.03 to 2%. This pollen was not viable, as evidenced by germination experiments, and it was not capable of fertilizing the ovules of non-transgenic tobacco plants. Further cross-pollination experiments confirmed that transgenic, pollen-sterile tobacco plants produced fertile seed after fertilization of ovules with wildtype pollen grains. None of the transgenic plants showed pleiotropic effects of the transgene expression on plant growth or health.

Further studies were conducted to understand the sterility mechanism morphologically and biochemically. Microscopic examination (TEM) of transgenic tobacco plants revealed that anthers produced all tissue types found in non-transgenic control plants. However, during later stages of anther development, a failure to produce normal pollen grains became apparent. Most transgenic lines did not show any pollen development, and those few that did produced aberrant pollen. The results were further confirmed by confocal microscopy, which identified aberrant pollen as flat, flake-like structures compared to the rounded appearance of wildtype pollen. Also, as evidenced in these images, the normal sculpturing of the pollen wall, as seen with wildtype pollen, was missing in transgenic material.

To better understand the potential effect of the expression of the sts gene on flavonol production in transgenic pollen, wildtype and transgenic material was stained with diphenylborinic acid-ethanolamine ester, a flavonol specific stain. Fluorescence microscopy revealed that wildtype pollen contained significant amounts of flavonols only at very late stages of development, whereas at earlier stages, only small amounts of flavonols could be detected. Transgenic pollen also showed small amounts of flavonols at the beginning of development; however, elevated amounts could not be detected at later stages. In addition, the lack of flavonols as compared to wildtype pollen only became apparent long after other signs of aberrant development of transgenic pollen were confirmed. It was concluded that the reasons for aberrant pollen development did not appear to be related to lack of flavonol production.

TEM and pyrolysis GC-MS were applied to test the hypothesis that sterility may be a result of changes in the pollen exine, or in sporopollenin biochemistry. However, no major changes in exine morphology and no major biochemical changes were detected. From these experiments it was concluded that sterility may have arisen from a lack of malonyl-CoA or coumaroyl-CoA in the tapetum, inhibiting or slowing down other critical biochemical pathways required for fertile pollen production.

It is already known that the male cone-specific promoter used in the experiment is differentially expressed in P. radiata male reproductive tissue.7 It is expected that the sterility construct will express and produce stilbene synthase in transgenic radiata pine tapetum cells. Transgenic lines and young trees have been produced for further analysis. It has since also been demonstrated that the male cone-specific promoter expresses at low levels in embryogenic tissue of radiata pine. However, a negative effect of residual or leaky expression of the sts gene in transgenic embryogenic tissue and young plantlets has not been observed. This supports our original hypothesis that any expression of this gene in conifers may not harm, and may in fact benefit, the plant by providing some resistance against fungal attack.

STS-induced male sterility in conifers may provide a viable strategy to prevent the flow of transgenes from transgenic trees in forest plantations. This may enable both the field testing of other traits and the streamlined achievement of regulatory approval. These improvements will enable the commercial exploitation of new biotechnologies in forestry in a socially and environmentally acceptable way. In addition, the sterility strategy discussed here may provide a safeguard against potential promoter leakiness and against some decrease in gene expression levels over the rotation time. Further, it has been hypothesised that conifers that do not produce pollen may well redirect nutrient flow into growth, thereby providing a male sterile plant that is also more attractive from a forest grower's point of view. In species that have become problematic as a source of pollen-induced allergies in humans, such as experienced with the Japanese conifer Cryptomeria japonica, this strategy will also provide a mechanism to greatly reduce allergenic impacts.

Selected references

1. Burgess DG et al. (2002). A novel, two-component system for cell-lethality and its use in engineering nuclear male-sterility in plants. Plant Journal 31(1), 113-125

2. Mou Z et al. (2002). Silencing of phosphoethanolamine N-methyltransferase results in temperature-sensitive male sterility and salt hypersensitivity in Arabidopsis. Plant Cell 14, 2031-2043

3. Preston et al. (2004). AtMYB32 is required for normal pollen development in Arabidopsis thaliana. Plant Journal 40, 979-995

4. Höfig et al. (2006): Towards male sterility in Pinus radiata – a stilbene synthase approach to genetically engineer nuclear male sterility. Plant Biotechnology Journal 4, 333-343

5. Fischer R, Budde I & Hain R. (1997) Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco. Plant Journal 11, 489-498

6. Höfig K et al. (2003) Expression analysis of three Pine male cone promoters in the heterologous host Arabidopsis. Planta 217, 858-867

7. Walden AR, Walter C, & Gardner R. (1999) Genes expressed in Pinus radiata male cones include homologs to anther-specific and pathogenesis response genes. Plant Physiology 121, 1103-116

 

Christian Walter
Senior Scientist Genetic Engineering
Cellwall Biotechnology Center, SCION
Rotorua, New Zealand
www.scionresearch.com


MOST GROWERS AWARE OF, COMPLYING WITH BT CORN STANDARDS
Tracy Sayler

On-farm assessments and telephone surveys indicate that over 90 percent of growers are aware of and are adhering to Bt corn borer stewardship requirements. Bt corn hybrids are engineered to produce an insecticidal protein from the naturally occurring soil bacterium, Bacillus thuringiensis (Bt), that is toxic to European corn borers.

The National Corn Growers Association recently announced that more than nine out of 10 growers are complying with Insect Resistance Management (IRM) requirements as mandated by the Environmental Protection Agency (EPA). These findings are based on 2005 on-farm assessments along with an independent survey conducted on behalf of the Agriculture Biotechnology Stewardship Technical Committee (ABSTC).

Since 1999, Bt corn registrants – Dow AgroSciences, Monsanto, Pioneer Hi-Bred International, Inc., a subsidiary of DuPont, and Syngenta Seeds – have monitored adherence to the IRM requirements to help ensure that Bt corn borer technology remains effective against pests and is a tool readily available for all growers. This monitoring effort was expanded in 2002 with the development of the Compliance Assurance Program (CAP) which is designed to promote IRM awareness and assess implementation at the farm level.

The annual Bt corn borer IRM telephone survey, managed by an independent third party, indicated that 92 percent of U.S. Bt corn growers met or exceeded the minimum recommended refuge size in 2005. This level of adherence with the requirements is consistent with past results of 91 percent in 2004 and 86 percent in 2003. 

In a separate program of on-farm visits, more than 94 percent of producers assessed were found to be meeting the refuge requirements. Both large and small acreage growers are meeting refuge management requirements at similar levels.

An element of the on-farm assessment program that reinforces how seriously Bt technology providers regard resistance management is the potential penalties for non-compliance. Failure to properly plant and manage refuges in two crop seasons can lead to a grower's inability to access technology for use on their farm in the third year.

Farmers visited in 2004 and found to be out of compliance came back into compliance in 2005, and have maintained access to corn borer protected Bt corn for this growing season.

For the small set of growers found to be non-compliant in 2005, Bt providers responded in accordance with the uniform industry standards developed to bring growers back into compliance. These standards outlined in the CAP include letters and additional IRM educational materials sent to growers as well as on-farm compliance assessments. Growers who repeatedly fail to adhere to IRM requirements risk losing access to the technology.

The EPA-required Bt corn refuge obligates farmers to plant at least 20 percent refuge – that is, corn that does not contain a Bt gene for controlling corn borers. The rule is essentially a safeguard measure to help protect against resistance to the technology – the refuge supplies a source of Bt-susceptible European corn borer that could mate with resistant European corn borer potentially emerging from nearby Bt corn. In certain cotton areas of the South, growers are required to plant at least a 50 percent refuge. All areas of the country require that the refuge is planted within one-half mile of the Bt corn.

Growers reported that information from the National Corn Growers Association and affiliated state groups, along with seed companies marketing Bt hybrids, are the leading sources for IRM information. Most growers surveyed recall receiving an average of four pieces of IRM literature and more than three-fourths of those surveyed noted having had an individual conversation with a seed company representative or seed dealer regarding management requirements.

Tracy Sayler
Agricultural writer and ISB correspondent
Fargo, N.D.
tsayler@prairieacomm.com



GE TOMATO RESISTANT TO LEAF CURL DISEASE
Anupam Varma and Shelly Praveen

Geminiviruses cause devastating plant diseases, particularly in the tropics and semi-tropics. Some of these diseases have been adversely affecting crop production since the early 20th Century, but the present century faces severe disease epidemics caused by newly emerging and reemerging whitefly (Bemisia tabaci) transmitted geminiviruses (WTGs), which affect crops like cassava, cotton, cucurbits, grain, legumes, and tomato. The growers of these crops in various parts of the world suffer severe economic losses due to WTG infections.1 Despite management practices, menacing disease epidemics caused by WTGs are becoming frequent and are occurring even in new regions that were previously free from such diseases. The frequency with which WTGs are appearing shows that these viruses are still evolving. In recent years, some WTGs have also moved to temperate agriculture, causing concern in the production of vegetables in greenhouses.

Geminiviruses have unique twin particle morphology, and their genome is either a monopartite or bipartite covalently closed circular single stranded DNA. These viruses form the second largest plant virus family, Geminiviridae, which consists of three genera, Mastrevirus, Curtovirus and Begomovirus, distinguished by their vector, host range, and genomic characteristics. The WTGs belong to the genus Begomovirus, which represents more than 80% of the known geminiviruses. Some begomoviruses have a monopartite genome of about 2.7 kb designated as DNA-A, but mostly they have bipartite genomes of about 2.7 kb each, designated as DNA-A and DNA-B. In addition, satellite-like DNAβ molecules of covalently closed single stranded DNA of about 1.3 kb have also been found associated with most of the monopartite and some of the bipartite begomoviruses.

In the bipartite begomoviruses, both the genomic components are essential for infection. DNA-A encodes for replication associated protein (Rep), the replication enhancer (REn), transcriptional activator protein (TrAP), and the coat protein, whereas DNA-B codes for proteins required for symptom development and movement function. In DNA-A and DNA-B, the open reading frames (ORFs) are arranged in two divergent clusters (Fig. 1C), two viral sense and four complementary sense ORFs are located in DNA-A, whereas DNA-B has one viral and one complementary sense ORF, separated by an intergenic region. The single stranded DNA genome of the virus is replicated in the host nuclei via double stranded DNA intermediates using the rolling circle (RC) mechanism. Virus multiplication relies mostly on the host DNA replication apparatus. The product of the AC1 ORF, Rep, plays a key role by initiating RC replication by virtue of its nicking and ligation property. DNAβ is essential for the infectivity of some of the monopartite begomoviruses, but it is not an essential component of bipartite begomoviruses.

Tomato appears to be a favorite host for begomoviruses; over 50 species of begomoviruses naturally infect tomato. Most of these begomoviruses have come to light during the last two decades, particularly in the Americas where nearly 20 distinct begomoviruses have been isolated from tomato, and considering the limited information available, many more tomato infecting begomoviruses await detection. The most devastating begomoviruses affecting tomato are known by their generic names ‘tomato leaf curl virus' and ‘tomato yellow leaf curl virus'. So far, 18 distinct begomoviruses associated with tomato leaf curl disease (ToLCD) and 11 associated with tomato yellow leaf curl disease (TYLCD) have been identified.1 These viruses induce characteristic leaf curl diseases, which manifest as a severe reduction in leaf size, downward curling, crinkling of interveinal areas, interveinal and marginal chlorosis, occasional development of enations, purple discoloration of the abaxial surface of leaves, shortening of internodes, development of small branches, and reduction in fruit size and fruiting. The tomato diseases caused by begomoviruses are collectively known as either ‘leaf curl' or ‘yellow leaf curl' based on subtle differences in symptoms. However, the viruses causing these diseases are distinguished by their DNA-A nucleotide sequence analysis.

Efforts to develop tomato varieties resistant to ToLCV by traditional breeding have not been successful, as natural sources of resistance are not available. Control of the whitefly vector has not been effective in preventing epidemics and yield losses due to these viruses. Various cultural and chemical approaches for management of this disease have also not been found satisfactory.2

Several methods employing the use of genetic engineering have successfully demonstrated development of virus resistance through the production of transgenic plants. Pathogen-derived resistance (PDR)3 is leading to the development of virus resistant plants. PDR is mediated either by the transgene protein (protein-mediated) or by the transgene RNA (RNA-mediated)4, which has been interpreted as an example of homology-dependent gene silencing. The effectiveness of the rep gene for developing resistance was illustrated by the transgenic expression in antisense orientation against different begomoviruses. Two overlapping strategies have been used to generate novel resistance against begomoviruses in transgenic plants. The first is RNA-mediated resistance involving blocking the expression of the replicase (rep) gene by an antisense gene construct, and the second involves post-transcriptional gene silencing (PTGS) using a short sense RNA fragment based on homology dependent gene silencing.5

To obtain stable resistance against the ToLCD, an antisense gene strategy was adopted for development of transgenic tomato in our investigations. We have developed tomato transgenics resistant to ToLCD using replicase (rep) gene sequences of the Tomato leaf curl virus in antisense orientation, via Agrobacterium-mediated transformation. A binary vector carrying the antisense rep gene (untranslatable full length sequence 1086 bp) along with the npt II gene was used for transformation. A high level of resistance and inheritability of the transgene was observed up to the T2 stage following challenge inoculation with the virus. The mechanism of resistance appears to be RNA-mediated, since plants carry the untranslatable antisense rep gene, and have no detectable protein expression. Progeny analysis of these plants showed a classical Mendelian pattern of inheritance.6

It is important to note that two of the transgenic lines with a single transgene insertion have shown more than 80% resistance compared to the non-transformed control plants. These were selfed to produce progeny for resistance evaluation at the T2 stage, which followed the same pattern of resistance as the T1 stage. These lines have also been found promising in limited field trials to evaluate their performance under field conditions. Studies are in progress to determine the spectrum and mechanism of resistance. Patent protection of this construct has been initiated.

We have also recently shown the effectiveness of an antisense rep gene construct to induce silencing of the viral genome in transgenic plants derived from ToLCV-infected explants. The plants transformed with the ToLCV antisense rep gene construct fully recovered from the virus infection, while the non-transformed plants showed the typical leaf curl symptom (Fig. 2) and the presence of a large number of virus particles. The 'recovered' plants also showed faster growth compared to the non-transformed infected tomato plants at various stages in tissue culture. The indication of RNA-mediated virus resistance using an antisense gene construct came from studies on the presence/absence of viral genome using a coat protein gene of the virus as a marker.

These studies have opened up possibilities of developing transgenic tomato for resistance to begomoviruses. However, much more research is required before this technology is ready for practical use. A primary concern is the variability in the viral genomes of different viruses causing ToLCD worldwide and the emergence of new variants of these viruses through mutations and genomic recombinations. We plan to target conserved sequences for homology dependent gene silencing for deployment through RNAi to obtain broad spectrum resistance to begomoviruses in tomato. We also plan to develop tomato with resistance to multiple viruses as, apart from the begomoviruses, cucumo- and tospoviruses are also causing serious economic losses in tomato cultivation in India.

References

1.  Varma A & Malathi V G (2003) Emerging geminiviral problems: A serious threat to crop production. Ann. appl. Biol. 142, 145-164

2. Tripathi S & Varma A (2002) Eco-friendly management of leaf curl disease of tomato. Indian Phytopath. 55, 473-47811

3. Sanford JC & Johnston SA (1985) The concept of parasite-derived resistance – deriving resistance genes from the parasite's own genome. J. Theor. Biol. 113, 395-405

4. Baulcombe DC & English JJ (1996) Ectopic pairing of homologous DNA and post transcriptional gene silencing in transgenic plants.  Curr. Opinion in Biotech. 5,117-124

5. Praveen S, Dasgupta A & Varma A (2004) Phylogenetic Analysis and   Homologies of the Replicase of Tomato Leaf Curl Geminiviruses: Implications for Obtaining Pathogen Derived Resistance Virus Gene. 28(1),197-201

6. Praveen S, Mishra AK & Dasgupta A (2005) Antisense suppression of replicase gene expression recovers tomato plants from leaf curl infection. Plant Science 168, 1011-1014

Anupam Varma
Advanced Centre for Plant Virology
New Delhi, India
anupamvarma@vsnl.net



OMEGA-3 PIGS
Yifan Dai and Randall S. Prather

Polyunsaturated fatty acids (PUFAs) have 18 or more carbon atoms and two or more double bonds. They can be classified into two groups, omega-6 (n-6) and omega-3 (n-3), depending on the position of the double bond nearest the methyl end (the w carbon) of the fatty acid. The n-3 PUFAs include a-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The n-6 PUFAs include linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) and docosapentaenoic acid (DPA, 22:5n-6). PUFAs are generally synthesized from the modification of saturated fatty acid precursors by different desaturase enzymes. Mammals lack the desaturases necessary to synthesize 18:2n-6 (LA) and 18:3n-3 (ALA), which are the precursors for other PUFAs. Furthermore, the n-3 and n-6 PUFAs are not interconvertible in mammalian cells because mammals also lack the enzyme, omega-3 fatty acid desaturase, to convert n-6 PUFA to n-3 PUFA. Therefore, LA and ALA and their elongation and desaturation products are essential fatty acids (EFAs) to mammals and they must be taken from the diet.1

Human beings evolved consuming a diet that contained about equal amount of n-3 and n-6 EFAs. However, over the past 100-150 years, there has been an enormous increase in the consumption of n-6 PUFAs due to the increased intake of vegetable oils from corn, sunflower seeds, cottonseed, and soybeans. These all contain high amounts of n-6 PUFAs but none of n-3 PUFAs. Intake of n-3 PUFAs is much lower today because of the decrease in fish consumption and the industrial production of animal feeds rich in grains containing n-6 fatty acids, leading to production of meat and eggs rich in n-6 and poor in n-3 PUFAs. The same is true for cultured fish since fish can not make n-3 PUFAs by themselves. Today, in Western diets, the ratio of n-6 to n-3 fatty acids ranges from 20-30:1 instead of the traditional range of 1-2:1. Many studies in the last 20 years have shown the high n-6/n-3 PUFA ratio may contribute to the high prevalence of many modern diseases (e.g., heart disease, autoimmune disorders, and depression). Studies have also shown that n-3 PUFAs are essential for normal growth and development of the retina and brain and may play an important role in the prevention and treatment of coronary artery disease, hypertension, diabetes, arthritis, other inflammatory or autoimmune disorders, depression, and cancer. n-3 fatty acids affect coronary heart diseases beneficially not by changing serum lipid concentration, but by reducing blood clotting in vessel walls and ventricular arrhythmias.2

An n-3 fatty acid desaturase gene, fat-1, was cloned from Caenorhabditis elegans (roundworm) by Dr. Browse's group in Washington State University in 1997.3 Expression of the fat-1 gene in plants and mammalian cells showed FAT-1 protein converted n-6 PUFA to n-3 PUFA efficiently. The production of fat-1 transgenic mice was reported by Kang et al. in 2004. A humanized fat-1 gene with the optimized codons for mammals was used to increase the hfat-1 gene expression in mice. The results showed that the n-6/n-3 ratio of the hfat-1 transgenic mice was significantly reduced in all the tested tissues (e.g., muscle from 49:1 to 0.7:1; milk from 32.7:1 to 5.7:1), and the hfat-1 transgenic mice could produce all n-3 PUFAs (ALA, EPA, DPA and DHA).4

In 2004, we started our collaboration to generate fat-1 transgenic pigs by cloning. Our team also included Dr. Kang's group from Massachusetts General Hospital and Dr. Evans's group from the University of Pittsburgh. The hfat-1 cDNA driven by cytomegalovirus enhancer and chicken β-actin promoter was linked together with an expression cassette of pgk-neo as a selection marker. The construct, pST103, was transfected into primary male porcine fetal fibroblasts. The transfected cells were selected with G418, and the hfat-1 expression and the function of the FAT-1 enzyme in G418 resistant cells were confirmed by detection of high levels of n-3 PUFAs. These hfat-1 transgenic cells were then used to produce the hfat-1 transgenic pigs by cloning (somatic cells nuclear transfer). In total 14 hfat-1 transgenic piglets were produced, 11 of them showed a significantly higher amount of n-3 PUFAs in their body. The concentrations of total n-3 fatty acids in tail tissues of the transgenic piglets were 3-fold higher than in the wildtype piglets. Among them, EPA and DPA showed a 15-fold and 4-fold increase, respectively. On the other hand, the concentration of total n-6 fatty acids in the transgenic piglets was reduced by 23%. Consequently, there was a 5-fold reduction of the n-6/n-3 ratio in hfat-1 transgenic piglets compared with wildtype piglets (from 8.52 to 1.69, p<0.001). Examination of major tissues (muscle, liver, kidney, heart, spleen, tongue, brain, and skin) from the transgenic piglets showed a substantial reduction of n-6/n-3 ratios, indicating that hfat-1 was expressed in all of those tissues.5

Currently, the main source of the n-3 PUFAs for humans is from sea fish and shellfish, which accumulate n-3 PUFAs through the food chain. The bottom of the food chain is microalgae in the ocean, which are the original source of the n-3 PUFAs. However, while fish accumulate n-3 PUFAs through eating smaller fish, they also accumulate mercury, which usually stays with fat. Therefore, the fish at the higher position of the food chain have higher mercury contamination. The US Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) have constantly advised women who might become pregnant, women who are pregnant, nursing mothers and young children to avoid eating shark, swordfish, king mackerel, tilefish, and white tuna because they contain high levels of mercury. They have also advised those women and young children to reduce the intake of other sea fish. Ironically, it is the same group of people who need the n-3 PUFAs the most because n-3 PUFAs are essential to the development of the brain and retina of fetuses and young children. Because a pregnant woman has to provide her fetus with a large amount of n-3 PUFAs, it results in decreased n-3 PUFAs in her own body if she does not obtain enough n-3 PUFAs from her diet. It is the same for a breastfeeding mother. Studies have shown that a higher incidence of post-partum depression is co-related with lower n-3 PUFAs in those patients.

Today, in Western diets, the ratio of n-6 to n-3 has reached to an unhealthy 20-30:1 because people are eating fewer fish. If n-3 PUFA-rich pork is available to the general public (once approved by FDA) and consumed by people on a daily basis, it will likely have a huge impact on the people's health. It will significantly reduce the n-6/n-3 ratio from the current 20-30:1 to an ideal 1-2:1 in the western population. Feeding livestock and poultry a fish diet is not a good solution. First, mercury will accumulate in these animals; second, it will increase the cost of the meat and dairy product. In April 2004, a Canadian company introduced a line of n-3-high milk from cows fed a fish diet. The price was 20% higher than regular milk. And third, feeding livestock with flaxseeds or fish meal has a potential negative effect on meat quality. For example, the majority of n-3 PUFA in flaxseeds is ALA (18:3n-3), and it has been shown that an increase of ALA alone in pork by feeding pigs flaxseed could affect its sensory qualities (the bacon has a fishy taste). Pork quality can also be affected by a high intake of PUFAs in the diet because it will decrease the oleic acid content, increase the iodine value, and cause undesirable softening and yellowing of the carcass fat. However, it is unlikely to be an issue in the hfat-1 transgenic pigs since they only convert n-6 PUFAs to n-3 PUFAs and will not increase the total PUFAs (n-6 + n-3) percentage in pork. The transgenic pigs can produce all four n-3 PUFAs, but EPA and DPA are the major PUFAs.

The hfat-1 transgenic pigs have several other advantages. First, they will not increase production costs because they only need to be fed the regular n-6 rich diet; second, they will provide a mercury-free pork; and third, the significant reduction of the n-6/n-3 ratio in their bodies will improve their general health and reduce the early loss of piglets compared with non-transgenic farm pigs, which usually have high n-6/n-3 ratio due to their n-6 rich diet. The hfat-1 transgenic pig is also a good large animal model. It can be used to study the effect and the mechanism of n-3 PUFAs in prevention and treatment of coronary artery disease, hypertension, diabetes, arthritis, other inflammatory or autoimmune disorders, and cancer. Humans are more similar to pigs than mice, and the discoveries in the hfat-1 transgenic pigs will be more readily applied to humans.

Acknowledgements

The authors would like to acknowledge funding from the National Institutes of Health R01 RR13438 & U42 RR018877 (RSP) and R01 DK64207 (YD) and an unrestricted gift to the Thomas E. Starzl Transplantation Institute from the Eberly Family Fund for Transplant Innovation (YD).

References:

1. Simopoulos AP (1998) Overview of evolutionary aspects of omega-3 fatty acids in the diet. World Rev Nutr Diet., 83, 1-11

2. Simopoulos AP (2003) Importance of the ratio of omega-6/omega-3 essential fatty acids: evolutionary aspects. World Rev Nutr Diet. 92, 1-22

3. Spychalla JP, Kinney AJ, Browse J. (1997) Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis. Proc Natl Acad Sci U S A. 94(4),1142-7

4. Kang JX, Wang J, Wu L, Kang ZB. (2004) Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids. Nature 427(6974), 504

5. Lai L, Kang JX, Li R, Wang J, Witt WT, Yong HY, Hao Y, Wax DM, Murphy CN, Rieke A, Samuel M, Linville ML, Korte SW, Evans RW, Starzl TE, Prather RS, Dai Y. (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24(4), 435-436, Epub 2006 Mar 26.

Yifan Dai
Associate Professor of Surgery
Thomas Starzl Transplantation Institute
University of Pittsburgh School of Medicine
Pittsburgh, PA 15261
daiy@upmc.edu

Randall S. Prather
Distinguished Professor of Reproductive Biotechnology
Division of Animal Sciences
University of Missouri
Columbia, MO 65211
PratherR@missouri.edu



A NANOTECH REVOLUTION IN AGRICULTURE AND THE FOOD INDUSTRY
Phillip B C Jones

On March 20, 2006, U.S. Secretary of Commerce Carlos M. Gutierrez announced the launch of a state-of-the-art center for collaborative nanotechnology research at the National Institute of Standards and Technology. "The National Center for Nanoscale Science and Technology," Gutierrez said in a press release, "will help the private sector develop innovative products like more efficient batteries, lighter-weight and higher performing materials for aircraft and autos, and smaller computer chips to power digital devices."

Nanotechnology encompasses the ability to measure, model, and control matter at dimensions of about 1 to 100 nanometers. The groundbreaking potential of nanotech derives from the unusual physical, chemical, and biological properties of nanoscale-sized matter that differ from those of individual molecules and bulk matter. These unique properties allow the development of novel applications, noted by Gutierrez, in the fields of engineering and computer science. Nanotechnology will also bring innovations to the food industry and agriculture.

Effecting Big Changes with Small Alterations in the Food Industry

The Helmut Kaiser Consultancy (Tübingen, Germany) finds nothing small about the nanofood market, predicting that the market may reach over $20 billion dollars by 2010. Around the globe, over 400 companies research, develop, and produce nanofood-related products. The general aims of nanotechnology in this arena center on improving the quality of food.

Numerous food companies seek to use nanotechnology to create safer, more nutritious, and more flavorful products. Nanotech may provide improved functional properties, such as low sodium food products that taste salty due to nanotech-induced interactions with the tongue, and functional food components tailored to the individual consumer's preferences. Nanoparticles, nanoemulsions, and nanocapsules may be designed to enhance the availability and dispersion of nutrients, antioxidants, or nutraceuticals. These beneficial factors may even be delivered to targeted areas of the body at selected times.

Research and development efforts in the nanofood industry also focus on improved food packaging. Nanotech can enable two new types of food containers: active packaging and smart packaging.

An example of active packaging is a plastic film with dispersed clay nanoparticles that prevent oxygen, carbon dioxide, and moisture from reaching food. Other types of active packaging possess antimicrobial properties.

Smart packaging incorporates nanomaterials that respond to environmental conditions, engage in self-repair, or alert a consumer to the presence of chemical or pathogen contamination. For example, nanoparticle films and other packaging with embedded sensors will detect food pathogens. These nanosensors trigger a package color change to alert consumers that the food has become contaminated or has begun to spoil. Another type of packaging may incorporate a bio-switch that releases a preservative if the food within begins to spoil.

A Nanotech Transformation in Agriculture

Nanotechnology may support "precision farming," the application of information technologies applied to the management of commercial agriculture. Precision farming's enabling technologies include satellite-positioning systems, geographic information systems, and remote sensing devices. By connecting global positioning systems with satellite imaging of fields, farm managers could remotely detect crop pests or evidence of drought. Information about these conditions would trigger an automatic adjustment of pesticide applications or irrigation levels. Dispersed throughout fields, a network of sensors would relay detailed data about crops and the soil. These sensors would need to have nanoscale sensitivity to monitor conditions, such as the presence of plant viruses or the level of soil nutrients.

Other forms of nanotechnology may directly alter agricultural practices. Nanoparticles or nanocapsules could provide a more efficient means to distribute pesticides and fertilizers, reducing the quantities of these chemicals introduced into the environment. Livestock may be identified and tracked through commerce using implanted nanochips. Nanoparticles may deliver growth hormone or vaccines to livestock, or DNA for genetic engineering of plants.

Ultimately, nanotech innovations may enable the agricultural industry to precisely control and improve production. An ability to manipulate molecules may permit the food industry to design food with enhanced function at lower costs. The capability to introduce revolutionary changes in agriculture and food carries risks. Is the federal government prepared to oversee these new developments?

Ahead of the Curve in the Regulation of Agrifood Nanotech

The University of Minnesota's Jennifer Kuzma has emphasized the lack of a comprehensive U.S. oversight policy for nanotechnology, despite the federal government's annual investment of about one billion dollars in nanotech research. Kuzma sees parallels between the regulation of biotechnology and nanotechnology in food and agricultural industries: both technologies have raised debates about whether the government should regulate the process or the product, both technologies offer diverse applications that touch multiple regulatory agencies, and both technologies can be characterized by overlapping or missing regulatory jurisdiction.

As a step toward analyzing regulation of agrifood nanotechnology, Kuzma and Peter VerHage have created a database of nanotechnology food and agriculture-related research funded by the U.S. government. They also examined publicly available information from the U.S. Patent and Trademark Office.

Kuzma and VerHage presented analyses of their data on March 30, 2006, at a program hosted by the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for Scholars and via webcast. During the meeting, Kuzma suggested a bottom-up method for studying regulatory oversight of agrifood nanotech. The process would have three phases: (1) use the research and development database to assess applications of nanotechnology to food, agriculture, and agroecosystems; (2) select individual products to identify risks and benefits; and (3) after assessing particular products, extrapolate to analyze appropriate regulatory or non-regulatory governance systems for agrifood applications. Applying lessons from agbiotech, Kuzma suggested that independent research and safety studies should be performed and made available to the public, and that regulatory agencies should ensure a transparency in the product review and oversight process.

Most of the agrifood applications included in the database, Kuzma and VerHage predict, have a commercial timeframe of 5 to 15 years. David Rejeski, director of the Project on Emerging Nanotechnologies, noted that those concerned about nanotech and food issues enjoy a unique position. "We are ahead of the curve," he said, "and have time to prepare."

The Project on Emerging Nanotechnologies website provides copies of the database, which Kuzma and her colleagues will expand in the future (http://www.nanotechproject.org/index.php?id-11).

Selected Sources

Bello M (2006) Commerce Secretary Gutierrez Announces New Nanotechnology Center. March 20, 2006. Available at: http://www.nist.gov/public_affairs/news.htm.

Kuzma J (ed.) (2005) The Nanotechnology-Biology Interface: Exploring Models for Oversight. Hubert H. Humphrey Institute of Public Affairs (University of Minnesota). September 15, 2005. Available at: http://www.hhh.umn.edu/centers/stpp/nanotechnology.html.

Precision Agriculture - Nanotech Methods Used, Such as "Smart Dust," Smart Fields and Nanosensors. 2004. Available at: http://www.azonano.com.

Phill Jones
BiotechWriter.com
PhillJones@nasw.org


PLUM TREES GENETICALLY ENGINEERED FOR RESISTANCE TO PLUM POX

Environmental Assessment for Determination of Nonregulated Status

The USDA Animal and Plant Health Inspection Service seeks public comments by July 17, 2006, on a " ... petition from the U.S. Department of Agriculture's Agricultural Research Service seeking a determination of nonregulated status for plum designated as transformation event C5, which has been genetically engineered to resist infection by plum pox virus (PPV)."

Publicly available documents are to be posted in APHIS Docket Identification Number APHIS-2006-0084 at http://www.regulations.gov.

An electronic copy of the petition and an accompanying Environmental Assessment on it are posted at http://www.aphis.usda.gov/brs/aphisdocs/04_26401p.pdf and http://www.aphis.usda.gov/brs/aphisdocs/04_26401p.ea.pdf, respectively.

To obtain copies of the petition or the environmental assessment (EA), contact:

Ingrid Berlanger
Tel: 301-734-4885
Email: Ingrid.E.Berlanger@APHIS.USDA.gov

For further information from APHIS contact:

Dr. Michael Watson
Biotechnology Regulatory Services, APHIS
4700 River Road Unit 147
Riverdale, MD 20737-1236
301-734-0486

Source:
APHIS May 16 Federal Register: http://a257.g.akamaitech.net/7/257/2422/01jan20061800/
edocket.access.gpo.gov/2006/E6-7402.htm




ISB News Report
1900 Kraft Drive #103
Corporate Research Center
Virginia Tech
Blacksburg, VA 24061

The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture, or Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge.

ISB welcomes your comments and encourages article submissions. If you have a suitable article relevant to our coverage of the agricultural and environmental applications of genetic engineering, please e-mail it to the Editor for consideration.

Ruth Irwin, Editor (rirwin@vt.edu)

To have the News Report automatically e-mailed to you, send an e-mail message to isb@vt.edu with your request.
Connect to http://www.isb.vt.edu for internet access to ISB News Reports, textfiles, and databases.

Information Systems for Biotechnology, Virginia Tech, 1900 Kraft Drive, Suite 103, Blacksburg, VA 24061, tel: 540-231-3747, fax: 540-231-4434, e-mail: isb@vt.edu