Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization,fruit characters,and nutrients were analyzed for T4 generation of transgenic papaya.All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field.The RP transgene can be steadily inherited to,and expressed at RNA level,the progenies.The growth characteristics of transgenic papaya were much better than nontransgenic papaya in the field.The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV.They died quickly and never grew to produce fruit.The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time,while most oftransgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV,and produced more,bigger,and high quality fruits.Compared with non-transgenic plants,the fresh fruit length of T4 generation of transgenic papaya increased 2.6%-5%,and the diameter decreased 0.6%-1.5%.The flesh thickness of fresh fruit increased 12%-15%,which made it fitter for eating.Although the fresh fruit quality changed,there was no significant difference between transgenic and non-transgenic papaya.The quality characteristics of dry fruit including the contents of water,lipid,N,protein,reduced sugar,vitamin A,vitamin C,and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents.This means that transgenic plants and non-transgenic plants are substantially equivalent,and the transgene has no effect on dry fruit quality.In this study,we found that vitamin A and vitamin C in red-fleshed papaya were 1.4-1.8 and 1.78-2.07 times more than the yellow-fleshed ones,respectively,while N and protein were only 84.2%-92.1% and 82.1%-98.9% of the yellow-fleshed ones.     

T4转基因番木瓜遗传性和果实品质分析

对T 4代转基因番木瓜进行了分子生物学和果实品质分析,结果表明,筛选获得的转基因番木瓜均为转番木瓜环斑病毒(PRV)复制酶突变体基因(RP),且对PRV抗性达到了高抗或免疫,RP基因在转基因植物中能稳定遗传至后代并在RNA水平上表达。在田间种植时,转基因木瓜的生长状况普遍好于普通番木瓜,尤其在生长后期(10月以后),普通番木瓜100%发病(大规模种植时),而大部分(约91.8%)转基因植株生长良好,果实较多且表面光洁、基本上无环斑。与非转基因亲本相比,T 4代转基因番木瓜的果实长度增加2.6%~5%,果实直径变小0.6%~1.5%,果肉厚度增加了12%~15%,因而果实形状与亲本相近或更好,且信用价值更高。转基因番木瓜果实中水分、蛋白质、氮、脂肪、还原性糖、维生素A、维生素C和类胡萝卜素的含量与对照都无显著性差异,即转基因番木瓜与亲本具有实质等同性,这表明转入的外源基因对番木瓜果实品质没有不良影响。     

Assaying for Pollen Drift from Transgenic ‘Rainbow’ to Nontransgenic ‘Kapoho’ Papaya under Commercial and Experimental Field Conditions in Hawaii

In 1992, papaya ringspot virus (PRSV) was discovered in the Puna district of Hawaii island where 95% of the state of Hawaii’s papaya was being grown. By 1998 production in Puna had decreased 50% from 1992 levels. A PRSV-resistant transgenic papaya ‘Rainbow’ containing the coat protein gene of PRSV was released commercially in Hawaii in 1998, and saved the papaya industry from further devastation. In the ensuing years since the release of the transgenic papaya, a number of farmers grew hermaphrodite nontransgenic ‘Kapoho’ papaya in close proximity to plantings of hermaphrodite transgenic ‘Rainbow’ papaya. These plantings provided a unique opportunity to assay for transgenic-pollen drift under commercial conditions. Between 2004 and 2010, assays for the GUS (beta-glucuronidase) transgene in embryos were done to study transgenic-pollen drift in commercial ‘Kapoho’ plantings and in replicated field plots. Very low pollen drift (0.8%) was detected in fruit of ‘Kapoho’ trees in the border row of one plantation when 90 embryos were assayed per fruit, while no pollen drift was detected in four other commercial plantings in which eight embryos were tested per fruit. Pollen drift averaged 1.3% of tested embryos in field plots where individual hermaphrodite ‘Kapoho’ trees were adjacent to two or four ‘Rainbow’ trees. In contrast, 67.4% of tested embryos were GUS positive in similarly located female ‘Kapoho’ trees. The very low transgene flow to close-by ‘Kapoho’ plantings is likely due to the fact that hermaphrodite trees are used commercially in Hawaii and that these trees are largely self-pollinated before the stigma is exposed to external pollen.     

转基因番木瓜研究进展

番木瓜环斑病毒 (PRSV)使热带亚热带的重要水果番木瓜的生产受到严重影响 ,在众多方法防效不佳的情况下 ,利用病原获得抗性防治PRSV给番木瓜的生产带来了光明。综述了近年来转PRSVCP基因番木瓜中影响番木瓜转化因素和转基因番木瓜的抗性因素。转PRSV外壳蛋白 (CP)基因的番木瓜中多以胚性组织为转化材料 ,被转化材料的生理状态和基因型 ,是影响转化效率和转基因植株质量的主要因素。所获得的转基因番木瓜对PRSV的抗性在很大程度上依赖于接种PRSV与所转化PRSVCP基因的序列同源性、转基因拷贝数和所转基因的位置等。     

Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.     

A protocol for efficient transformation and regeneration of Carica papaya L.

Summary A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya. Based in part on a presentation at the 1997 SIVB Congress on In Vitro Biology held in Washington, DC, June 14–18, 1997.     

Field Released Transgenic Papaya Affects Microbial Communities and Enzyme Activities in Soil

Soil properties, microbial communities, and enzyme activities were studied in soil planted with transgenic or nontransgenic papaya under field conditions. The transgenic papaya contained a replicase (RP) mutant gene of the papaya ringspot virus (PRSV), which conferred resistance to the virus, the neomycin phosphotransferase II (NPT II) marker gene, which conferred Km resistance, and a cauliflower mosaic virus 35S promoter (CaMV 35S). There were significant differences (P < 0.05) in the total number of colony forming units (CFUs) of bacteria, actinomycetes, and fungi between soils planted with RP-transgenic and nontransgenic plants; total CFUs of bacteria, actinomycetes, and fungi in soil planted with transgenic papaya were significantly higher by 0.43, 0.80, and 0.46 times, respectively. Significantly higher (P < 0.05) CFUs of bacteria, actinomycetes, and fungi resistant to kanamycin (Km) were present in soils planted with the transgenic papaya than in those planted with nontransgenic papaya. Resistance quotients (CFU in the presence of a chemical relative to that without) of Km-resistant bacteria, actinomycetes and fungi were higher in soil planted with transgenic papaya, and the resistance quotients of Km-resistant bacteria, actinomycetes, and fungi in soils planted with transgenic papaya increased statistically significantly (P<0.05) from 1.5 to 2.5, from 1.2 to 2.6, and from 0.9 to 2.8 times, respectively. Soils planted with transgenic papaya had significantly higher enzyme activities of arylsulfatases (+5.4 times), alkaline phosphatases (+0.5 time), invertase (+0.5 time) and phosphodiesterases (+0.2 time), but lower enzyme activities of proteases (−2.1 times), polyphenol oxidases (−1.4 times), urease (−0.2 time) than the soils planted with nontransgenic papaya. Our results suggest that transgenic papaya could alter chemical properties, enzyme activities, and microbial communities in soil.     

Risk assessment of transgenic apomictic tetraploid bahiagrass,cytogenetics, breeding behavior and performance of intra-specific hybrids

Pollen-mediated gene transfer from stress tolerant or herbicide-resistant transgenic plants may cause environmental or agronomic problems. Apomictic seed production found in some bahiagrass cultivars may serve as a natural transgene containment system. Under greenhouse conditions, the average gene transfer frequency from an herbicide-resistant apomictic tetraploid to a population of sexual diploid bahiagrass genotypes or apomictic tetraploid bahiagrass was 0.16% when the transgenic pollen donor was placed at 0.5–1.5 m distance from the non-transgenic pollen receptors. The herbicide-resistant hybrids were characterized for transgene integration, expression and ploidy, by Southern blot analysis, immuno-chromatography and flow cytometry, respectively. Hybrids resulting from open pollination of non-transgenic diploid female plants with transgenic tetraploid male plants were triploids or near-triploids, with 2n = 26–34. These hybrids displayed a wide range of phenotypic variability, including some non-persistent or non-flowering dwarf-type hybrids with good vigor, or hybrids with vegetative growth similar to non-transgenic plants, but with significantly reduced seed set. Non-flowering aneu-triploids with good vigor/field performance will provide the highest level of transgene containment. Embryo sac analysis of pollinated spikelets confirmed a high proportion of aborted ovules. An apospory-linked RFLP marker was detected in 13 of the 15 near-triploid hybrids. All flowering aneuploid hybrids displayed significantly reduced seed set, and none of the sexual near-triploid hybrids produced any seeds. All tetraploid gene transfer events carried the apospory-linked RFLP marker, suggesting that despite the presence of the aposporus locus, a low degree of sexuality co-exists in apomictic tetraploid cultivars. Thus, tetraploid apomictic bahiagrass does not provide complete transgene containment, although intra-specific gene transfer is drastically reduced compared to sexually reproducing perennial grasses.     

Transgene-specific and event-specific molecular markers for characterization of transgenic papaya lines resistant to Papaya ringspot virus

The commercially valuable transgenic papaya lines carrying the coat protein (CP) gene of Papaya ringspot virus (PRSV) and conferring virus resistance have been developed in Hawaii and Taiwan in the past decade. Prompt and sensitive protocols for transgene-specific and event-specific detections are essential for traceability of these lines to fulfill regulatory requirement in EU and some Asian countries. Here, based on polymerase chain reaction (PCR) approaches, we demonstrated different detection protocols for characterization of PRSV CP-transgenic papaya lines. Transgene-specific products were amplified using different specific primer pairs targeting the sequences of the promoter, the terminator, the selection marker, and the transgene, and the region across the promoter and transgene. Moreover, after cloning and sequencing the DNA fragments amplified by adaptor ligation-PCR, the junctions between plant genomic DNA and the T-DNA insert were elucidated. The event-specific method targeting the flanking sequences and the transgene was developed for identification of a specific transgenic line. The PCR patterns using primers designed from the left or the right flanking DNA sequence of the transgene insert in three selected transgenic papaya lines were specific and reproducible. Our results also verified that PRSV CP transgene is integrated into transgenic papaya genome in different loci. The copy number of inserted T-DNA was further confirmed by real-time PCR. The event-specific molecular markers developed in this investigation are crucial for regulatory requirement in some countries and intellectual protection. Also, these markers are helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.     

Analysis and stability of the Respiratory Syncytial Virus antigen in a T<Subscript>3</Subscript> generation of transgenic tomato plants

The integration, expression, and stability of the Respiratory Syncytial Virus (RSV)-F protein was analyzed in a T₃ generation of transgenic cherry tomato, Solanum lycopersicum L. cv. Swifty Belle, plants. Expression of the RSV-F antigen, under the control of the fruit-specific promoter E-8, was investigated in T₃ plants derived from a transgenic line, identified as #120. Transgene integration of the RSV-F gene in the T₃ generation was initially determined by polymerase chain reaction (PCR). PCR analysis from line 120-7-2 revealed that all T₃ plants were homozygous for the transgene; whereas, line 120-6-4 showed segregation for the transgene. Enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of RSV-F protein in these plants, and protein levels ranged from 0–22 μg/g of fresh weight, with an average of ~3 μg/g fresh weight. Southern blot analysis of the highest expressing plants revealed presence of a single copy of the RSV-F transgene in these plants.     

Papaya Ring Spot Virus: An Understanding of a Severe Positive-Sense Single Stranded RNA Viral Disease and Its Management

Viral diseases have been studied in-depth for reducing quality, yield, health and longevity of the fruit, to highlight the economic losses. Positive-sense single-stranded RNA viruses are more devastating among all viruses that infect fruit trees. One of the best examples is papaya ringspot virus (PRSV). It belongs to the genus Potyvirus and it is limited to cause diseases on the family Chenopodiaceae, Cucurbitaceae and Caricaceae. This virus has a serious threat to the production of papaya, which is famous for its high nutritional and pharmaceutical values. The plant parts such as leaves, latex, seeds, fruits, bark, peel and roots may contain the biological compound that can be isolated and used in pharmaceutical industries as a disease control. Viral disease symptoms consist of vein clearing and yellowing of young leaves. Distinctive ring spot patterns with concentric rings and spots on fruit reduce its quality and taste. The virus has two major strains P and W. The former cause disease in papaya while the later one in papaya. Virion comprises 94.4% protein, including a 36 kDa coat protein which is a component responsible for a non-persistent transmission through aphids, and 5.5% nucleic acid. Cross protection, development of transgenic crops, exploring the resistant sources and induction of pathogen derived resistance have been recorded as effective management of PRSV. Along with these practices reduced aphid population through insecticides and plant extracts have been found ecofriendly approaches to minimize the disease incidence. Adoption of transgenic crops is a big challenge for the success of disease resistant papaya crops. The aim of this review is to understand the genomic nature of PRSV, detection methods and the different advanced control methods. This review article will be helpful in developing the best management strategies for controlling PRSV.     

An assessment of gene transfer by pollen from field-grown transgenic potatoes to non-transgenic potatoes and related species

Information on the extent of transgene dispersal by pollen to adjacent potato plots and to related weed species is an important requisite for risk assessment; a procedure followed before novel transgenic plants are evaluated under field conditions. The purpose of the investigation was to determine the frequency of cross-pollination between potato (Solanum tuberosum) plants at different distances, using a kanamycin resistnace transgene (nptII) as a selectable marker. All potato plants were from the variety Désirée. Non-transgenic potato plants, used as potential recipients of transgene-containing pollen, were planted in 12 sub-plots, at distances of 0–20 m from the nearest transgenic potato plants. Seeds harvested from the non-transgenic plants were screened for resistance to kanamycin, and molecular methods were used to confirm that resistant progeny contained thenptII gene. Where transgenic and non-transgenic potato plants were in alternate rows (leaves touching), 24% of seedlings from the non-transgenic parent plants were kanamycin-resistant. Comparable seedlings from plants at up to 3 m distance had a resistance frequency of 2%, at 10 m the frequency was 0.017% and at 20 m no resistant progeny were observed. Plants of the weed speciesS. dulcamara andS. nigrum were also planted close to the transgenic potatoes to test for evidence of hybridization, and no kanamycin-resistant seedlings were observed among progeny fromS. dulcamara andS. nigrum. This investigation provided evidence that the extent of gene dispersal from transgenic potatoes to non-transgenic potatoes falls markedly with increasing distance, and is negligible at 10 m. There was, also, no evidence of transgene movement from potato toS. dulcamara andS. nigrum under field conditions. These data will be valuable in defining genetic isolation procedures for the early field evaluation and the use of novel transgenic potato genotypes.     

Enhancement of Growth and Fruit Maturity in 2-year-old Grapevines cv. Delaware by 5-Aminolevulinic Acid

The influence of 5-aminolevulinic acid (ALA) on the growth, photosynthesis, and fruit quality of gibberellin-induced seedless 2-year-old “Delaware“ grapevine was examined. The result of soil treatment (0.1–10 mg l⁻¹) and foliar spray treatment (30–300 mg l⁻¹) with ALA after flowering was significant growth improvement, up to 55% in the lateral shoot, and up to 38% in leaf area. Optimal doses were 1 mg l⁻¹ soil treatment and 100 mg l⁻¹ foliar treatment. The photosynthetic rate of plants treated with ALA increased by a significant 9.2%–22.5%. Relative to the control, based on measurements of the 5th leaf of each shoot in the ripening period, the optimum concentrations for growth and photosynthesis enhancement in grapevines were 100 mg l⁻¹ (foliar treatment) and 1 mg l⁻¹ (soil treatment). Total plant weight at harvest increased a significant 13% at the optimal treatment doses. In terms of fruit quality, the cluster fresh weight increased a significant 44.9%–53% and fruit colour showed a tendency to become darker in all plants treated with ALA. The °Brix value in the plant treated with 100 mg l⁻¹ ALA was a significant 2.7% higher than that of the control. We consider that leaf area and photosynthetic rates during cultivation are important for the acquisition of photoassimilates and that these are likely to be causally associated with improvement of total dry weight and advance of fruit quality. In addition, a possibility of advancing the harvest time of grapes by ALA treatment was shown.     

dsRNA介导的番木瓜环斑病毒（PRSV）的抗病性研究

以模式植物拟南芥（Arabidopsis thaliana）和烟草（Nicotiana tabacum）及PRSV寄主植物番木瓜（CaricapapayaL.）作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究。利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR（简称PHG12-CPIR）分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中。对转基因植株进行攻毒试验并分析了其抗病性。在接种3~7d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同程度的黄化、皱缩和枯斑等症状。在接种PRSV后,番木瓜和拟南芥转化植株表现症状的叶片的比例与对照相比,结果显著低于对照,而在烟草植株上症状表现的差异不明显。在3种植物上RT-PCR检测结果显示,在接种番木瓜环斑病毒PRSV后,野生型植株中有高浓度的病毒积累,而转pHG12-CPIR基因植株中几乎没有病毒积累,推测转pHG12-CPIR基因植株中瞬时表达系统已启动RNAi机制抑制了CP基因的表达。     

Transgenic yellow lupin (Lupinus luteus)

 Transgenic yellow lupin (Lupinus luteus L.) plants have been generated by meristem co-cultivation with Agrobacterium tumefaciens. The binary plasmid pPZBNIa contains the bar gene under the control of a CaMV 35 S promoter. The transformation method involves inoculation of embryonic axis explants with A. tumefaciens, flooding the meristem with glufosinate, and initial culture on non-selective medium. Shoots were transferred to culture medium containing 20 mg/l glufosinate. Following subculture, shoots were grafted onto non-transgenic narrow-leafed lupin (L. angustifolius L.) seedling rootstocks, or rooted in vitro. The overall transformation efficiency, as determined at the T₁ generation, was 0.05%–0.75%. The transgenic nature of plants grown to the T₆ generation was confirmed by phosphinothricin acetyl transferase, PCR and Southern analyses. Received: 20 March 1999 / Revision received: 17 July 1999 / Accepted: 17 August 1999     

Utilization of the bar gene to develop an efficient method for detection of the pollen-mediated gene flow in Chinese cabbage (Brassica rapa spp. pekinensis)

To develop an efficient screening method for detection of the transgene in Chinese cabbage (Brassica rapa spp. pekinensis) utilizing Basta spray, optimal conditions for Basta application were examined in this study. Two transgenic Chinese cabbage lines were obtained through Agrobacterium-mediated transformation and used as transgenic positive controls in the Basta screening experiment. Differential concentrations of glufosinate-ammonium were sprayed into three different growth stages of 12 commercial Chinese cabbage cultivars. The results showed that no plants could survive higher than 0.05% glufosinate-ammonium, and plants at the 2–3 leaf stage were most vulnerable to glufosinate-ammonium. On the other hand, no damage was observed in the transgenic control plants. Reliability of the Basta spray method was proven by showing perfect co-segregation of the tolerance to glufosinate-ammonium and the presence of the bar gene in T₁ segregating populations of the transgenic lines, as revealed by both PCR and Southern blot analyses. Using the developed Basta screening method, we tried to investigate the transgene flow through pollen dispersal, but failed to detect any transgene-containing non-transgenic Chinese cabbages whose parents had been planted adjacent to transgenic Chinese cabbages in field conditions. However, the transgene was successfully detected using Basta spray from the non-transgenic plants bearing the transgene introduced by hand-pollination. Since the Basta spray method developed in this study is easy to apply and economical, it will be a valuable tool for understanding the mechanism of gene flow through pollen transfer and for establishing a biosafety test protocol for genetically modified (GM) Chinese cabbage cultivars.     

Physiological responses to salt stress of T2 alfalfa progenies carrying a transgene for betaine aldehyde dehydrogenase

Glycinebetaine is an important quaternary ammonium compound generated in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by a Betaine Aldehyde Dehydrogenase (BADH) gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. In this study, a BADH gene was over expressed in transgenic alfalfa (Medicago sativa L) plants using Agrobacterium-mediated transformation. Transgenic alfalfa plants grown under 9‰ NaCl grew well; while non-transgenic control plants turned yellowish in color, wilted, and eventually died. Polymerase chain reaction (PCR) and Northern blot hybridization analyses demonstrated that the BADH gene was transferred into the T2 generation and segregated in a Mendelian fashion. Transgenic alfalfa plants expressing BADH showed significantly higher BADH enzyme activity and betaine contents when grown under 6‰ NaCl. Moreover, proline content in T2 lines were higher while electrolyte leakage and malonaldehyde content were lower in T2 lines compared with non-transgenic plants. These findings indicated that transgenic plants expressing BADH transgene exhibited higher salt tolerance than non-transgenic plants.     

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T₅) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6–21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.     

Apomixis and ploidy barrier suppress pollen-mediated gene flow in field grown transgenic turf and forage grass (Paspalum notatum Flüggé)

Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar ‘Argentine’ is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5–3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.