Transgenic Pineapple Research Articles

Overexpression of AcWRKY31 Increases Sensitivity to Salt and Drought and Improves Tolerance to Mealybugs in Pineapple.

Pineapple is a globally significant tropical fruit, but its cultivation faces numerous challenges due to abiotic and biotic stresses, affecting its quality and quantity. WRKY transcription factors are known regulators of stress responses, however, their specific functions in pineapple are not fully understood. This study investigates the role of AcWRKY31 by overexpressing it in pineapple and Arabidopsis. Transgenic pineapple lines were obtained using Agrobacterium-mediated transformation methods and abiotic and biotic stress treatments. Transgenic AcWRKY31-OE pineapple plants showed an increased sensitivity to salt and drought stress and an increased resistance to biotic stress from pineapple mealybugs compared to that of WT plants. Similar experiments in AcWRKY31-OE, AtWRKY53-OE, and the Arabidopsis Atwrky53 mutant were performed and consistently confirmed these findings. A comparative transcriptomic analysis revealed 5357 upregulated genes in AcWRKY31-OE pineapple, with 30 genes related to disease and pathogen response. Notably, 18 of these genes contained a W-box sequence in their promoter region. A KEGG analysis of RNA-Seq data showed that upregulated DEG genes are mostly involved in translation, protein kinases, peptidases and inhibitors, membrane trafficking, folding, sorting, and degradation, while the downregulated genes are involved in metabolism, protein families, signaling, and cellular processes. RT-qPCR assays of selected genes confirmed the transcriptomic results. In summary, the AcWRKY31 gene is promising for the improvement of stress responses in pineapple, and it could be a valuable tool for plant breeders to develop stress-tolerant crops in the future.

AcMYB266, a key regulator of the red coloration in pineapple peel: a case of subfunctionalization in tandem duplicated genes.

Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.

Chemical composition of roots of transgenic pineapple plants

Chemical composition of roots of transgenic pineapple plants

AcWRKY28 mediated activation of AcCPK genes confers salt tolerance in pineapple (Ananas comosus)

Unfavorable environmental cues severely affect crop productivity resulting in significant economic losses to farmers. In plants, multiple regulatory genes, such as the WRKY transcription factor (TF) family, modulate the expression of defense genes. However, the role of the pineapple WRKY genes is poorly understood. Here, we studied the pineapple WRKY gene, AcWRKY28, by generating AcWRKY28 over-expressing transgenic pineapple plants. Overexpression of AcWRKY28 enhanced the salt stress resistance in transgenic pineapple lines. Comparative transcriptome analysis of transgenic and wild-type pineapple plants showed that “plant-pathogen interaction” pathway genes, including 9 calcium-dependent protein kinases (CPKs), were up-regulated in AcWRKY28 over-expressing plants. Furthermore, chromatin immunoprecipitation and yeast one-hybrid assays revealed AcCPK12, AcCPK3, AcCPK8, AcCPK1, and AcCPK15 as direct targets of AcWRKY28. Consistently, the study of AcCPK12 over-expressing Arabidopsis lines showed that AcCPK12 enhances salt, drought, and disease resistance. This study shows that AcWRKY28 plays a crucial role in promoting salt stress resistance by activating the expression of AcCPK genes.

Histological evaluation of pineapple transgenic plants following 8 years of field growth

Pineapple is among the most important fruits worldwide. However, the narrow genetic base of current commercial cultivars renders the industry at risk from environmental and other biological threats. Hence, there is a need to develop improved cultivars with tolerance to biotic and abiotic stresses. Our prior research resulted in the establishment of a method for genetic transformation of pineapple. The genetically modified pineapple plants generated were characterized following acclimatization and during the subsequent 8 years under field conditions. The current work highlights the results obtained for histological characterization of roots and leaves of transgenic pineapple plants. The four treatments investigated were macropropagated control plants (untransformed), micropropagated control plants (untransformed), and micropropagated genetically transformed plants (clones 90 and 98). Twenty indicators were measured in plants: two of them did not show statistically significant differences among the four groups of plants (2/20 = 10.0%); six showed “Low” overall coefficients of variation (OCV) (6/20 = 30.0%); 12 showed “Medium” OCVs (12/20 = 60.0%); and only two indicators showed “High” OCVs (2/20 = 10.0%). Consideration of medium OCVs showed that the biomass of transgenic plants was slightly greater than those of the non-transgenic controls. Statistically significant differences between the lines were recorded except in the central cylinder diameter of roots and the D leaf adaxial cuticle thickness. “High” OCVs were observed for root exo- and endodermis thickness where transgenic clones showed statistically significantly higher values. In terms of the roots, five out of nine histological indicators showed “Medium” OCVs: cortex, rhyzodermis, and parenchyma thicknesses, pith diameter, and thickness of the transversal root ratio. Again, genetically transformed materials showed statistically significantly higher averages. Only three out of seven histological indicators showed “Medium” OCVs in the leaves: D leaf abaxial cuticle thickness, chlorophyll parenchyma thickness, and D leaf aquifer parenchyma thickness. As noted above, statistically significantly higher values were recorded in the transgenic pineapple plants. Since the differences observed did not ultimately affect fruit yield (as shown in our previous work), they do not appear to be consequences of genetic transformation that impact on reproductive yield.

An Efficient Agrobacterium Mediated Transformation of Pineapple with GFP-Tagged Protein Allows Easy, Non-Destructive Screening of Transgenic Pineapple Plants.

Quite a few studies have been conducted to improve the Agrobacterium-mediated transformation of pineapple, which is the second most important commercial tropical fruit crop worldwide. However, pineapple transformation remains challenging, due to technical difficulties, the lengthy regeneration process, and a high labor requirement. There have not been any studies specifically addressing the introduction of GFP-tagged genes into pineapples through Agrobacterium-mediated transformation, which would enable easy, non-destructive expression detection. It would also allow expression localization at the organelle level, which is not possible with GUS a reporter gene that encodes β-glucuronidase or a herbicide resistance reporter gene. Here, we report a method for the introduction of GFP-tagged genes into pineapples through Agrobacterium-mediated transformation. We used embryonic calli for transformation, and plants were regenerated through somatic embryogenesis. In this study, we optimized the incubation time for Agrobacterium infection, the co-cultivation time, the hygromycin concentration for selection, and the callus growth conditions after selection. Our strategy reduced the time required to obtain transgenic plants from 7.6 months to 6.1 months. The expression of GFP-tagged AcWRKY28 was observed in the nuclei of transgenic pineapple root cells. This method allows easy, non-destructive expression detection of transgenic constructs at the organelle level. These findings on pineapple transformation will help accelerate pineapple molecular breeding efforts to introduce new desirable traits.

Mineral composition of a transgenic pineapple clone grown in the field for 8 yr

Mineral composition of a transgenic pineapple clone grown in the field for 8 yr

Characteristics evaluation of transgenic delay-flowering pineapple (Ananas comosusL. Merr.)

Characteristics evaluation of transgenic delay-flowering pineapple (<i>Ananas comosus</i>L. Merr.)

The third vegetative generation of a field-grown transgenic pineapple clone shows minor side effects of transformation on plant physiological parameters

Genetic modifications of pineapple provide the potential to increase fungal resistance without the use of pesticides. Previously, we developed a procedure for pineapple callus transformation, introducing the antifungal chitinase and AP24 genes, and the herbicide-tolerance conferring bar gene as a selectable marker. The three recombinant genes were under the control of the constitutively expressing promoters OCS-35S CaMV-rice actin I, 35S CaMV, and maize Ubi1, respectively. The objective of this study was to analyze whether these genetic modifications caused unintentional side effects on growth-related properties under field conditions. We analyzed the third vegetative generation of a transgenic pineapple clone, approximately 7 years after the genetic modification occurred. Plant height and diameter, chlorophyll and phenolic contents, fruit mass and D leaf (middle-age) mass, water content, succulence, sap pH, transpiration, CO2 assimilation, and water use efficiency were recorded and compared to a micro-propagated cultivar. A macro-propagated variant was also cultivated at the field site in order to distinguish unintended effects of the propagation method from that of the genetic modification. While all parameters analyzed in this study were highly similar to each other for the three cultivars, statistically analyses revealed significant deviations of the genetically modified variant for the succulence index being higher at in the morning and evening, carbon dioxide assimilation being lower at noon and, at night, the water use efficiency was lower. The minor deviations were all within the range which can be expected between common cultivars grown together.

Field evaluation of transgenic pineapple (Ananas comosus (L.) Merr.) cv. ‘Smooth Cayenne’ for resistance to blackheart under subtropical conditions

A comparative analysis of transgenic pineapple lines transformed with a polyphenol oxidase (PPO) gene (ppo) and the untransformed cultivar ‘Smooth Cayenne’ was made from plants grown in a series of field trials under cool subtropical conditions in southeast Queensland. In the four field trials where blackheart was recorded, all of the control lines expressed blackheart on each occasion and exhibited the greatest incidence (50%) and severity (34%) of symptoms. Irrespective of the gene transfer method or the gene construct used, 38% of the lines produced were regarded as blackheart resistant, having no blackheart symptoms in two or more trials. Five blackheart resistant transgenic lines consistently performed as well as or better than control plants in terms of fruit characteristics and quality.

Characterization of a field-grown transgenic pineapple clone containing the genes chitinase, AP24, and bar

We previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. The present report focuses on the evaluation of the first vegetative generation of a transgenic clone containing these genes. Three materials were compared: macropropagated controls (non-transformed), micropropagated controls (non-transformed), and micropropagated transformed plants. From each group, 50% of the plants were sprayed with FINALE® 3 mo after the experiment initiation. The characterization was performed after 1 yr of field growth. FINALE® killed all non-transgenic plants. Plants that survived the herbicide application showed 2n = 50 chromosomes in their roots after 1 yr in the field. Micropropagated transformed plants sprayed with FINALE® did not show phenotype differences from micropropagated transformed plants not sprayed with the herbicide. Between the micropropagated transformed plants sprayed with FINALE® and the micropropagated control plants, the following differences were observed: modifications in levels of cell wall-linked, free and total phenolics, and total proteins. Moreover, changes of the fruit mass without crown were also recorded. Between the micropropagated transformed plants sprayed with FINALE® and the macropropagated control plants, levels of chlorophyll b, total chlorophyll pigments, and proteins were different. Furthermore, activities of phenylalanine ammonia-lyase, superoxide dismutase, and glutamine synthetase were dissimilar. The plant height and diameter, and the crown height were also different. Until now, we have evaluated transformed pineapple plants during hardening and field growth. Although some unexpected variations were recorded, we believe they are not relevant enough to justify rejection of transgenesis as an important tool for pineapple genetic improvement.

Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.

Biochemical side effects of the herbicide FINALE® on bar gene-containing transgenic pineapple plantlets

Pineapple is one of the most important tropical fruits and therefore intensive genetic improvement programs are being carried out in many countries, including Cuba. Our research team has previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. Herein, we report on the biochemical side effects of the herbicide FINALE® on these transgenic plantlets during hardening. Levels of aldehydes and chlorophylls, and peroxidase activity were recorded. The transformed clone studied here, not sprayed with FINALE®, showed the following side effects because of transgenesis only. Levels of malondialdehyde, other aldehydes, chlorophyll b, and total chlorophyll pigments decreased. The most remarkable biochemical differences between transgenic and non-transgenic plantlets after application of FINALE® follow. Levels of malondialdehyde and other aldehydes in transgenic material were not decreased by FINALE®, perhaps because these levels were already low as a result of transformation. FINALE® increased peroxidase activity in transgenic plantlets but such increase was higher in non-transgenic material. The herbicide increased contents of chlorophyll pigments (a, b, total) in transformed plantlets. However, as expected, non-transgenic plantlets decreased levels of chlorophylls (a, b, total) after application of FINALE®. The genetic transformation of pineapple with the bar gene not only conferred resistance to the herbicide FINALE®, but also promoted other biochemical changes.

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.

Transformation and regeneration of pineapple

We have developed efficient methods for plant regeneration, via both organogenesis and embryogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. A range of different types of embryogenic tissues has been developed with varying properties in terms of growth rate and state of development (Firoozabady and Moy, 2004). Two of the embryogenic systems, namely friable embryogenic cell clusters (ECCs) and chunky non-dispersible embryogenic tissues (ETs) have been used for transformation of pineapple. The tissues were cocultivated for 2???3 days with Agrobacterium tumefaciens disarmed strain C58 carrying a binary vector containing either surB gene conferring resistance to chlorsulfuron or the nptII gene conferring resistance to geneticin (G418). After cocultivation and a recovery period, tissues were selected on media containing chlorsulfuron or G418. On average, about 50 or 120 independent transgenic lines were obtained from each gram of ECCs or ETs, respectively, inoculated with Agrobacterium. Transformed embryogenic tissues were transferred to maturation media to form somatic embryos, which subsequently produced transgenic pineapple plants. Transformation has been confirmed by GUS assay, polymerase chain reaction, and by Southern hybridization. Thousands of plants from independently transformed lines were transferred to the greenhouse and to the field to evaluate clonal fidelity and somaclonal variation.

OPTIMISING TRANSIENT ASSAYS FOR STUDYING THE REGULATION OF PPO GENE EXPRESSION IN PINEAPPLE BLACKHEART

To study factors influencing the development of pineapple blackheart we are undertaking functional analyses of a polyphenol oxidase promoter whose expression is induced in blackheart affected tissues. Since transgenic pineapple fruit take three years to develop, such an analysis must be undertaken using transient assays. In this report we describe experiments aimed at optimising a transient assay system using biolistic DNA delivery in pineapple fruit. Two factors are shown to be critical, namely the use of unmethylated plasmid DNAs prepared using dam E. coli host strains, and the use of an internal standard to control for the inherent variability of transient expression assays.

Herbicide-tolerant Transgenic Pineapple ( Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple ( Ananas comosus‘Phuket’) were produced following microprojectile-mediated delivery of the plasmid AHC25, carrying the β-glucuronidase ( gus) reporter gene and the bialaphos resistance ( bar) gene for herbicide tolerance, into leaves of micropropagated shoots. Transformed plants were regenerated from bombarded leaf bases on Murashige and Skoog-based medium containing 0.5 mg l −12,4-dichlorophenoxyacetic acid, 2.0 mg l −16-benzylamino purine and 0.5 mg l −1phosphinothricin. Integration and expression of the bar gene in regenerated plants was confirmed by Southern analysis and RT-PCR, respectively. Regenerated plants were assessed in vitro and under glasshouse conditions for their tolerance to the commercial herbicide Basta ™, containing glufosinate ammonium as the active component. Plants sprayed with Basta ™containing concentrations of glufosinate ammonium up to 1400 mg l −1remained healthy and retained their pigmentation. The generation of herbicide-tolerant pineapple will facilitate more efficient weed control in this widely cultivated tropical crop.

TOWARDS THE PRODUCTION OF TRANSGENIC PINEAPPLE TO CONTROL FLOWERING AND RIPENING

In Queensland, pineapple plants are artificially induced to flower by spraying with chemical agents as a standard industry practice; nevertheless an important percentage of plants flower naturally with fruit maturing ahead of the normal summer harvest. This results in the need of extra harvest passes and significantly increases growing costs. In pineapple, ethylene is thought to be responsible for natural flowering due to low temperatures. ACC synthase is the key regulatory enzyme in the biosynthesis of ethylene. Our research team has isolated and characterised an ACC synthase gene induced by cold temperature treatment of pineapple plants followed by an extended warm period. We are producing transgenic pineapple plants carrying sense and antisense copies of the flowering-related ACC synthase gene in order to down-regulate the expression of the gene and therefore suppress natural flowering. Pineapples are not climacteric fruits but nevertheless ethylene is believed to play a significant role during ripening. Important quality traits such as skin texture and colour are linked to the production of ethylene by the fruit. To this aim we have studied pineapple fruit ripening as our model system. We have cloned and characterised several ACC synthase and ACC oxidase genes and have shown for the first time that the two key genes involved in ethylene biosynthesis are induced during pineapple fruit ripening. Transgenic plants containing sense and antisense copies of the ripening-related ACC synthase gene are being produced. We expect to study the effects of suppression of internal ethylene in the transgenic fruits.

THE DEVELOPMENT OF BLACKHEART RESISTANT PINEAPPLES THROUGH GENETIC ENGINEERING

We are attempting to control blackheart by inhibiting expression of PPO in genetically engineered pineapple plants. PPO genes have been isolated from various pineapple tissues using PCR with redundant oligonucleotides and a cDNA which is specifically induced in blackheart affected fruit has been cloned. Suitable promoters to drive gene expression in pineapple have been defined and gene constructs suitable for transformation of the crop have been prepared. To generate transgenic pineapple we have established an efficient plant regeneration system based on the production of highly regenerable callus from leaf bases of tissue cultured plantlets. A transformation system based on micro-projectile bombardment of this regenerable callus has been developed. In addition, we have also transformed pineapple using Agrobacterium-mediated gene delivery to leaf bases, from which we have generated stably transformed plantlets. For biolistics we have obtained over a dozen independent transformation events in at least four separate experiments, transformation in some instances has been confirmed by Southerns and shoots have been regenerated from this material. For Agrobacterium, transformed plants have been regenerated and multiple events have been observed from at least four separate experiments.