Surveying of pollen-mediated crop-to-crop gene flow from a wheat field trial as a biosafety measure

Outcrosses from genetically modified (GM) to conventional crops by pollen-mediated gene flow (PMGF) are a concern when growing GM crops close to non-GM fields. This also applies to the experimental releases of GM plants in field trials. Therefore, biosafety measures such as isolation distances and surveying of PMGF are required by the regulatory authorities in Switzerland. For two and three years, respectively, we monitored crop-to-crop PMGF from GM wheat field trials in two locations in Switzerland. The pollen donors were two GM spring wheat lines with enhanced fungal resistance and a herbicide tolerance as a selection marker. Seeds from the experimental plots were sampled to test the detection method for outcrosses. Two outcrosses were found adjacent to a transgenic plot within the experimental area. For the survey of PMGF, pollen receptor plots of the conventional wheat variety Frisal used for transformation were planted in the border crop and around the experimental field up to a distance of 200 m. Although the environmental conditions were favorable and the donor and receptor plots flowered at the same time, only three outcrosses were found in approximately 185,000 tested seedlings from seeds collected outside the experimental area. All three hybrids were found in the border crop surrounding the experimental area, but none outside the field. We conclude that a pollen barrier (border crop) and an additional isolation distance of 5 m is a sufficient measure to reduce PMGF from a GM wheat field trial to cleistogamous varieties in commercial fields below a level that can be detected.     

Ecological impacts of trees with modified lignin

Few experiments have yet been performed to explore the potential ecological impacts of genetic modification in long-lifespan species such as trees. In this paper, we review the available data on GM trees with modified lignin focussing on the results of the first long-term field trials of such trees. These trials evaluated poplars expressing antisense transgenes to reduce the expression of the lignin biosynthesis genes cinnamyl alcohol dehydrogenase (CAD) or caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) with the aim of producing trees with improved pulping characteristics. The trees were grown for 4 years at two sites in France and England, and their ecological impacts and agronomic performance were assessed. Modifications to lignin in the poplars were maintained over the 4 years of the trial. The trees remained healthy throughout and growth was normal. The lignin modifications had no unexpected biological or ecological impacts. Interactions with leaf-feeding insects, microbial pathogens and soil organisms were unaltered although the short-term decomposition of transgenic roots was slightly enhanced. Investigation of the ecological impacts of the GM trees was curtailed by the early termination of the field trial when it was attacked and largely destroyed by anti-GM protestors. To supplement our work on the decomposition of GM plant materials with modified lignin, we have therefore turned to the study of transgenic tobacco lines where we can perform more comprehensive and controlled analyses of the biological and ecological effects of lignin-gene suppression.     

Transgene stability and dispersal in forest trees

Transgenics from several forest tree species, carrying a number of commercially important recombinant genes, have been produced, and are undergoing confined field trials in a number of countries. However, there are questions and issues regarding stability of transgene expression and transgene dispersal that need to be addressed in long-lived forest trees. Variation in transgene expression is not uncommon in the primary transformants in plants, and is undesirable as it requires screening a large number of transformants in order to select transgenic lines with acceptable levels of transgene expression. Therefore, the current focus of plant transformation is toward fine tuning of transgene expression and stability in the transgenic forest trees. Although a number of studies have reported a relatively stable transgene expression for several target traits, including herbicide resistance, insect resistance, and lignin modification, there was also some unintended transgene instability in the genetically modified (GM) forest trees. Transgene dispersal from GM trees to feral forest populations and their containment remain important biological and regulatory issues facing commercial release of GM trees. Containment of transgenes must be in place to effectively prevent escape of transgenic pollen, seed, and vegetative propagules in economically important GM forest trees before their commercialization. Therefore, it is important to devise innovative technologies in genetic engineering that lead to genetically stable transgenic trees not only for qualitative traits (herbicide resistance, insect resistance), but also for quantitative traits (accelerated growth, increased height, increased wood density), and also prevent escape of transgenes in the forest trees.     

Vegetative and generative dispersal capacity of field released transgenic aspen trees

Transfer of genes by pollen or wind-dispersed seed is considered a main potential risk when field release experiments with transgenic trees are initiated. In Germany, the first release experiment with genetically transformed trees was initiated in 1996. To ensure that the transgenic trees remained in the vegetative phase, the duration of the experiment was limited to 5 years. In total, 457 1-year-old trees including eight transgenic aspen lines carrying either the 35S- rolC or the rbcS- rolC gene construct, and three control clones were transferred to the field. In 1998 and 2000, 12 plants of transgenic lines all carrying the 35S- rolC gene construct formed female flower buds. Furthermore, one young aspen plant identified as a root sucker was observed in 1999 followed by an increasing number of root suckers derived from transgenic and non-transgenic trees in 2000 and 2001. In 2001, the last year of the field trial, 15 root suckers were detected outside the field. In total, 234 root suckers were harvested in 2000 and 2001 and analysed for their transgenic status. More than half of the roots suckers investigated showed the presence of the rbcS- rolC gene construct. We concluded that in addition to the widely accepted generative propagation, vegetative dispersal capacity of transgenic perennial plants is also important and must be included in risk assessment studies.     

转基因林木潜在生态风险研究进展

近20年来,转基因技术在林木上的应用已使林木遗传育种研究取得了可喜的成绩,部分转基因林木已经进行了田间试验,还有一些获得了商品化许可。但与此同时,转基因林木的生态安全问题也逐渐引起了人们的关注,这些问题主要体现在外源基因的水平转移,垂直流动以及对昆虫、土壤生态系统、病毒等的潜在影响上。本文简要介绍了转基因林木田间试验、商品化生产情况及其潜在的生态风险,并对转基因林木的进一步发展进行了展望。     

Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple

Although cultivation of genetic modified (GM) annual crops has been steadily increasing in the recent 10 years, the commercial cultivation of GM fruit tree is still very limited and reports of field trials on GM fruit trees are rare. This is probably because development and evaluation of GM fruit trees require a long period of time due to long life cycles of trees. In this study, we report results from a field trial on three rolB transgenic dwarfing apple rootstocks of M26 and M9 together with non-transgenic controls grafted with five non-transgenic scion cultivars. We intended to investigate the effects of transgenic rootstock on non-transgenic scion cultivars under natural conditions as well as to evaluate the potential value of using the rolB gene to modify difficult-to-root rootstocks of fruit trees. The results showed that all rolB transgenic rootstocks significantly reduced vegetative growth including tree height regardless of scion cultivar, compared with the non-transgenic rootstocks. Flowering and fruiting were also decreased for cultivars grown on the transgenic rootstocks in most cases, but the fruit quality was not clearly affected by the transgenic rootstocks. Cutting experiment and RT-PCR analysis showed that the rolB gene was stably expressed under field conditions. PCR and RT-PCR analyses displayed that the rolB gene or its mRNA were not detectable in the scion cultivars, indicating no translocation of the transgene or its mRNA from rootstock to scion. Our results suggest that rolB modified rootstocks should be used in combination with vigorous scion cultivars in order to obtain sufficient vegetative growth and good yield. Alternatively, the rolB gene could be used to dwarf vigorous rootstocks of fruit trees or produce bonzai plants as it can significantly reduce the vegetative growth of plants.     

A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

The field performance of genetic containment technologies–considered important for certain uses of transgenic trees in forestry–is poorly known. We tested the efficiency of a barnase gene driven by the TA29 tapetum-dominant promoter for influencing growth rate and inducing male sterility in a field trial of transgenic hybrid poplar (Populus tremula?×?Populus tremuloides). When the growth of 18 transgenic insertion events with the sterility transgene were compared to non-transgenic controls after two growing seasons, they grew 40 % more slowly in stem volume, and all but one transgenic event grew significantly more slowly than the control. In contrast, when we compared the growth of transgenic trees containing four kinds of β-glucuronidase (GUS) reporter gene constructs to non-transgenic trees—all of which had been produced using the same transformation method and poplar clone and grown at the same field site—there were no statistically significant differences in growth after three growing seasons. In 2 years where gross pollen release from catkins was monitored and found to be abundant in the control, no pollen was visible in the transgenic trees; microscopy suggested the cause was tapetal collapse and revealed the presence of a very few normal-sized pollen grains of unknown viability. In two additional years when viable, well-formed pollen was microscopically documented in controls, and no pollen could be observed in any transgenic trees. We conclude that this construct resulted in robust and possibly complete male sterility that was stable over 4 years in the field.     

Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant

We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

     

Control of pollen-mediated gene flow in transgenic trees

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.     

Fate of transgenes in the forest tree genome

During the last two decades, genetic engineering (GE) has been progressing at a steady pace in the forest trees. Transgenic trees carrying a variety of different transgenes have been produced, and are undergoing confined field trials in the world. However, there are questions regarding stability of transgene expression, and transgene escape that need to be addressed in the long-lived forest trees. Although relatively stable transgene expression has been reported for several target traits, including herbicide resistance, insect resistance, and lignin reduction in the vegetative propagules of several forest tree species, there were still unintended unstable events in transgenic plants. Long-term stability of transgene expression involved in these traits and others affecting yield (impacting growth) would be desirable in the vegetative propagules, and also in the generative progeny of the forest trees. Transgene escape through pollen, seed, and vegetative propagules from GE trees to native forest populations, although inevitable, remains an important regulatory issue. However, it may be possible to manage/minimize the risk of transgene spread via isolation in confined areas, and use of incompatible genotypes of feral tree populations in the vicinity of transgenic forest trees. Therefore, it is desirable to produce genetically stable transgenic trees, and have biocontainment measures in place for testing and deployment of the GE forest trees. Toward these goals (transgene stability and containment), innovative biotech strategies are being actively pursued, with reasonable success, in forest trees.     

The role of psyllids (Hemiptera: Psyllidae) and bell miners (Manorina melanophrys) in canopy dieback of Sydney blue gum (Eucalyptus saligna Sm.)

Abstract The presence of the aggressive, colony-forming honeyeater, Manorina melanophrys (bell miner), in the canopies of unhealthy eucalypts has been well reported. There is, however, some debate as to the actual mechanisms producing these unhealthy trees. To investigate further some of the processes that may be contributing to this form of canopy dieback, two field trials were carried out in Olney State Forest, near Wyong, New South Wales. The study site contained Eucalyptus saligna (Sydney blue gum) with canopy dieback and was occupied by a large colony of bell miners. Close examination of the foliage revealed a large and diverse suite of phytophagous insects, including at least 16 species of psyllid (Hemiptera: Psyllidae). In the first trial, the use of bird exclusion cages over selected branches significantly improved leaf survival compared to leaves exposed to a relatively high density of bell miners. It is proposed that colonization by bell miners may interfere with the efficacy of both other insectivorous birds (through aggressive interspecific territoriality) and the invertebrate predators and parasitoids. Interference with such regulatory factors may enable some phytophagous insect populations to rise to sustained damaging levels. In the second trial, an insecticide application combined with reduced competition from the dense understorey and neighbouring trees was required to significantly improve trunk diameter and crown condition scores. After 12 months, neither treatment, by itself, significantly improved both growth measures. Possibly both treatments were required because the E. saligna trees were suffering from another source of stress (e.g. drought) in addition to the relatively high level of insect attack.     

Evaluation of transgenic cotton varieties and a glyphosate application on seedling disease incidence

A study was conducted to determine whether stand densities of transgenic cotton (Gossypium hirsutum) varieties, with or without glyphosate, were similar to conventional varieties of the same lineage group in Georgia and Mississippi. Transgenic and conventional cotton varieties were placed into five lineage groups of related varieties and seedling disease was evaluated in three greenhouse tests and a field trial using Rhizoctonia solani AG-4. Seed vigor was determined by standard germination studies were conducted evaluating conventional and transgenic varieties of similar lineage. Results showed that no interactions occurred for the heights and dry weight data across treatments within the lineage groups in any of the experiments. No interactions were shown between stand densities at different inoculum rates and inoculated versus uninoculated pots (plots). Across all greenhouse studies, stand counts of PM 1220 were similar to the transgenic varieties PM 1220 RR and PM 1220 B/RR with or without a glyphosate application. In the field trial, PM 1220 B/RR + glyphosate had significantly lower stands than all other treatments expect PM 1220 RR (no glyphosate treatment) prior to and after glyphosate application. Stand densities for varieties within the lineage group DPL 5415 were also inconsistent when compared between the greenhouse and field trials with no apparent trends occurring. However, the Coker 312 varieties containing glyphosate tolerance had consistently lower stand counts compared to the conventional variety of Coker 312 but only during the greenhouse studies. Seed germination of Coker 312 could not be correlated with either the greenhouse or field trial data. In general, the commercially available varieties such as PM 1220, DPL 5690, DPL 5415, and DPL 50 with glyphosate tolerance had similar stand count, height, and dry weight data when compared to the conventional varieties from the same lineage group regardless of a glyphosate application. When differences did occur, no consistent trends could be determined within these four lineage groups. An erratum to this article is available at .     

A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass,precinct vegetation and the microbial community

A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.     

Evaluation of transgenic cotton varieties and a glyphosate application on seedling disease incidence

A study was conducted to determine whether stand densities of transgenic cotton (Gossypium hirsutum) varieties, with or without glyphosate, were similar to conventional varieties of the same lineage group in Georgia and Mississippi. Transgenic and conventional cotton varieties were placed into five lineage groups of related varieties and seedling disease was evaluated in three greenhouse tests and a field trial using Rhizoctonia solani AG-4. Seed vigor was determined by standard germination studies were conducted evaluating conventional and transgenic varieties of similar lineage. Results showed that no interactions occurred for the heights and dry weight data across treatments within the lineage groups in any of the experiments. No interactions were shown between stand densities at different inoculum rates and inoculated versus uninoculated pots (plots). Across all greenhouse studies, stand counts of PM 1220 were similar to the transgenic varieties PM 1220 RR and PM 1220 B/RR with or without a glyphosate application. In the field trial, PM 1220 B/RR + glyphosate had significantly lower stands than all other treatments expect PM 1220 RR (no glyphosate treatment) prior to and after glyphosate application. Stand densities for varieties within the lineage group DPL 5415 were also inconsistent when compared between the greenhouse and field trials with no apparent trends occurring. However, the Coker 312 varieties containing glyphosate tolerance had consistently lower stand counts compared to the conventional variety of Coker 312 but only during the greenhouse studies. Seed germination of Coker 312 could not be correlated with either the greenhouse or field trial data. In general, the commercially available varieties such as PM 1220, DPL 5690, DPL 5415, and DPL 50 with glyphosate tolerance had similar stand count, height, and dry weight data when compared to the conventional varieties from the same lineage group regardless of a glyphosate application. When differences did occur, no consistent trends could be determined within these four lineage groups.     

Field and pulping performances of transgenic trees with altered lignification

The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.     

跨国公司生物技术知识产权保护策略分析——以棉花转化体MON531为例

近年来,转基因棉花种植面积在多个国家得到了快速增长。为深入研究转基因抗虫棉花的技术体系和知识产权保护状况,现以棉花转化体MON531为例研究跨国生物公司的知识产权保护策略。结果显示,目前MON531转化体涉及3件处于有效保护期限内的专利,并在多个国家受到保护,实现了从功能基因及调控元件到最终商业化品种的全方位有效保护。申请人还通过申请植物品种权对下游的品种进行保护,美国有19件品种获得授权,在中国仅有1件相关申请处于审查中。对棉花转化体MON531技术体系的知识产权保护策略的研究,可以为我国研发转基因作物并采取知识产权保护提供有利的借鉴。     

Expression of endogenous proteins in maize hybrids in a multi-location field trial in India

Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2–26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12–64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.     

Problems in monitoring horizontal gene transfer in field trials of transgenic plants

Transgenic crops are approved for release in some countries, while many more countries are wrestling with the issue of how to conduct risk assessments. Controls on field trials often include monitoring of horizontal gene transfer (HGT) from crops to surrounding soil microorganisms. Our analysis of antibiotic-resistant bacteria and of the sensitivity of current techniques for monitoring HGT from transgenic plants to soil microorganisms has two major implications for field trial assessments of transgenic crops: first, HGT from transgenic plants to microbes could still have an environmental impact at a frequency approximately a trillion times lower than the current risk assessment literature estimates the frequency to be; and second, current methods of environmental sampling to capture genes or traits in a recombinant are too insensitive for monitoring evolution by HGT. A model for HGT involving iterative short-patch events explains how HGT can occur at high frequencies but be detected at extremely low frequencies.     

No unintended impacts of transgenic pine (Pinus radiata) trees on above ground invertebrate communities

As part of an investigation into the potential unintended ecological impacts of transgenic trees, invertebrates were sampled from a field trial of transgenic Pinus radiata D. Don carrying the expressed antibiotic resistance marker gene neomycin phosphotransferase II (nptII) along with other genes known to affect reproductive development in plants and from nontransformed control trees. Invertebrate species abundance, richness, diversity, and composition were measured and compared among trees of five different transclones and nontransformed isogenic control trees. Invertebrates were sampled at six-monthly intervals over a period of 2 yr. In total, 19,162 individuals were collected comprising 279 invertebrate recognizable taxonomic units. Total invertebrate populations as well as populations of herbivorous lepidopteran larvae and Hemiptera were compared among transgenic and control trees. The results show that the transclones had no significant unintended influence on species abundance, richness, diversity, or composition for all populations investigated.     

Expression of multiple resistance genes enhances tolerance to environmental stressors in transgenic poplar (Populus × euramericana 'Guariento')

Commercial and non-commercial plants face a variety of environmental stressors that often cannot be controlled. In this study, transgenic hybrid poplar (Populus × euramericana 'Guariento') harboring five effector genes (vgb, SacB, JERF36, BtCry3A and OC-I) were subjected to drought, salinity, waterlogging and insect stressors in greenhouse or laboratory conditions. Field trials were also conducted to investigate long-term effects of transgenic trees on insects and salt tolerance in the transformants. In greenhouse studies, two transgenic lines D5-20 and D5-21 showed improved growth, as evidenced by greater height and basal diameter increments and total biomass relative to the control plants after drought or salt stress treatments. The improved tolerance to drought and salt was primarily attributed to greater instantaneous water use efficiency (WUEi) in the transgenic trees. The chlorophyll concentrations tended to be higher in the transgenic lines under drought or saline conditions. Transformed trees in drought conditions accumulated more fructan and proline and had increased Fv/Fm ratios (maximum quantum yield of photosystem II) under waterlogging stress. Insect-feeding assays in the laboratory revealed a higher total mortality rate and lower exuviation index of leaf beetle [Plagiodera versicolora (Laicharting)] larvae fed with D5-21 leaves, suggesting enhanced insect resistance in the transgenic poplar. In field trials, the dominance of targeted insects on 2-year-old D5-21 transgenic trees was substantially lower than that of the controls, indicating enhanced resistance to Coleoptera. The average height and DBH (diameter at breast height) of 2.5-year-old transgenic trees growing in naturally saline soil were 3.80% and 4.12% greater than those of the control trees, but these increases were not significant. These results suggested that multiple stress-resistance properties in important crop tree species could be simultaneously improved, although additional research is needed to fully understand the relationships between the altered phenotypes and the function of each transgene in multigene transformants.