Manjul Dutt

OPTIMIZING AGROBACTERIUM-MEDIATED TRANSFORMATION OF GRAPEVINE

SummaryA translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.

Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening)

Commercial sweet orange cultivars lack resistance to Huanglongbing (HLB), a serious phloem limited bacterial disease that is usually fatal. In order to develop sustained disease resistance to HLB, transgenic sweet orange cultivars ‘Hamlin’ and ‘Valencia’ expressing an Arabidopsis thaliana NPR1 gene under the control of a constitutive CaMV 35S promoter or a phloem specific Arabidopsis SUC2 (AtSUC2) promoter were produced. Overexpression of AtNPR1 resulted in trees with normal phenotypes that exhibited enhanced resistance to HLB. Phloem specific expression of NPR1 was equally effective for enhancing disease resistance. Transgenic trees exhibited reduced diseased severity and a few lines remained disease-free even after 36 months of planting in a high-disease pressure field site. Expression of the NPR1 gene induced expression of several native genes involved in the plant defense signaling pathways. The AtNPR1 gene being plant derived can serve as a component for the development of an all plant T-DNA derived consumer friendly GM tree.

Temporal and spatial control of gene expression in horticultural crops

Biotechnology provides plant breeders an additional tool to improve various traits desired by growers and consumers of horticultural crops. It also provides genetic solutions to major problems affecting horticultural crops and can be a means for rapid improvement of a cultivar. With the availability of a number of horticultural genome sequences, it has become relatively easier to utilize these resources to identify DNA sequences for both basic and applied research. Promoters play a key role in plant gene expression and the regulation of gene expression. In recent years, rapid progress has been made on the isolation and evaluation of plant-derived promoters and their use in horticultural crops, as more and more species become amenable to genetic transformation. Our understanding of the tools and techniques of horticultural plant biotechnology has now evolved from a discovery phase to an implementation phase. The availability of a large number of promoters derived from horticultural plants opens up the field for utilization of native sequences and improving crops using precision breeding. In this review, we look at the temporal and spatial control of gene expression in horticultural crops and the usage of a variety of promoters either isolated from horticultural crops or used in horticultural crop improvement.

Transgenic citrus expressing the antimicrobial gene Attacin E (attE) reduces the susceptibility of 'Duncan' grapefruit to the citrus scab caused by Elsinoë fawcettii

Citrus scab, caused by Elsinoë fawcettii (anamorph Sphaceloma fawcettii), is a common foliar fungal disease affecting many citrus cultivars, including grapefruit. No commercial grapefruit cultivar is resistant to scab, and the disease results in severely blemished fruit which reduces its marketability. Transgenic ‘Duncan’ grapefruit trees expressing the antimicrobial attE gene were produced via Agrobacterium-mediated transformation. In in vitro leaf and greenhouse assays, several transgenic-lines had significantly lower susceptibility to E. fawcettii compared to the non-transformed control (P < 0.0001). In the greenhouse studies, sporulation on all transgenic lines except 1 was significantly reduced (P < 0.0001) but the level of sporulation over time did not correspond to disease severity ratings. Lesion size was also significantly reduced on transgenic lines compared to the non-transformed control (P < 0.0001) and the least susceptible line A-23 had the smallest lesions, but in general there was no correlation between lesion size and disease susceptibility. The level of attE mRNA was inversely related to the number of copies detected by Southern blot. The least susceptible line had a single inserted copy of the attE transgene whereas more susceptible lines had multiple copies. Since the attacin mode of action was thought to be specific to Gram-negative bacteria, it was unexpected to find that there was a significant activity against E. fawcettii.

Comparative expression analysis of five caulimovirus promoters in citrus

Four caulimovirus-derived constitutive promoters were evaluated for gene expression in citrus and their expression levels were compared with a 35S promoter. Chimeric promoters made with duplicated enhancer sequences from the cauliflower mosaic virus (D35S), the cassava vein mosaic virus (DCsVMV), the figwort mosaic virus (DFMV), the mirabilis mosaic virus (DMMV) and the peanut chlorotic streak virus (DPCLSV) were inserted into a transformation vector fused to the gus reporter gene. Gene expression patterns driven by these promoters were analyzed in the transgenic citrus cultivar Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.). The histochemical and fluorometric measurement of GUS activity and the gene expression quantification by RT-qPCR analysis demonstrate that the DMMV promoter is able to direct gene expression in citrus as strongly as the D35S promoter and represents great application potential in citrus biotechnology. The DFMV, DCsVMV and DPCLSV constitutive promoters were weaker compared to the D35S promoter but can be considered for use in gene stacking strategies for the development of transgenic citrus. Our results also reveal the importance of the evaluation of specific promoter fragments for a particular crop cultivar due to the availability of species-specific transcription factors that can define the strength and tissue specificity of a determinate promoter.

Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide. New cultivars are needed to battle industry threatening diseases and to create new marketing opportunities. Citrus improvement by conventional methods alone has many limitations that can be overcome by applications of emerging biotechnologies, generally requiring cell to plant regeneration. Many citrus genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches to cultivar improvement. This chapter provides a brief history of plant somatic embryogenesis with focus on citrus, followed by a discussion of proven applications in biotechnology-facilitated citrus improvement techniques, such as somatic hybridization, somatic cybridization, genetic transformation, and the exploitation of somaclonal variation. Finally, two important new protocols that feature plant regeneration via somatic embryogenesis are provided: protoplast transformation and Agrobacterium-mediated transformation of embryogenic cell suspension cultures.

Development of a SCAR Marker Linked to Male Fertility Traits in 'Jinkyool' (Citrus sunki)

In Citrus, an F1 segregation population of 150 plants was constructed from a cross between ‘Kiyomi’ (C. unshiu × C. sinensis) carrying the male sterility trait and ‘Jinkyool’ (C. sunki). Sequence-related amplification polymorphism (SRAP) combined with bulked segregant analysis was used to develop markers linked to male fertility. In the F1 population, 66 out of 150 seedlings had aborted anthers and the ratio of male sterile plants to fertile plants in the progenies matched the expected Mendelian segregation ratio of 1:1 (χ² =2.16 at p=0.05). From the profiling of the 197 SRAP primer sets, three SRAP primer sets (F4/R27, F39/R60, and F15/R37) that were closely linked to the target trait were identified and successfully converted into a sequence characterized amplified region (SCAR) marker for selection of male fertility in citrus. The SCAR marker, using the pMS 33U/pMS 1462L primer set specifically, produced a single 1.4-Kb fragment that was linked to male fertility. Our results suggested that this SCAR marker can be useful for marker-assisted selection of male sterile individuals in breeding F₁ progenies in Citrus.

The Role of Calmodulin and Related Proteins in Plant Cell Function: An Ever-Thickening Plot

Stimuli-induced fluctuations in intracellular free calcium (Ca2+) serve as secondary messenger signals that regulate diverse biochemical processes in eukaryotic cells, such as developmental transitions and responses to biotic and abiotic stresses. Stimuli-specific Ca2+ signals are manifested as spatially and temporally defined differential Ca2+ signatures that are sensed, decoded, and transduced to elicit distal responses via an array of Ca2+ binding proteins (CBPs) that function as intracellular Ca2+ sensors. Calmodulin (CaM), the most important eukaryotic CBP, senses and responds to fluctuations in intracellular Ca2+ levels by binding to this ubiquitous second messenger, and transducing given Ca2+ signatures that differentially activate distal effector (target) proteins regulating a broad range of biochemical responses. Ca2+/CaM targets include an increasing number of proteins whose functions continue to be elucidated. Hundreds of reports have highlighted the importance of CaM, and other CBPs, in the transduction of Ca2+-mediated signals involved in transcriptional regulation, protein phosphorylation/dephosphorylation, and metabolic shifts. Other Ca2+-binding proteins are known to play significant functional roles in plant cells as well. This review is primarily focused on the role of CaM in some key plant processes, and discusses recent advances in understanding the pivotal role of CaM in an ever-increasing number of plant cell functions and biochemical responses. We also discuss recent work highlighting the emerging importance of CaM in nuclear and organellar signaling.

Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.

Initiation and transformation of grapevine embryogenic cultures.

Protocols for the production and transformation of grapevine embryogenic cultures are described. Embryogenic cultures are initiated from leaves or stamens and pistils and transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Cultures are transferred to induction medium in the dark for callus formation and proliferation. Resulting cultures are transferred to somatic embryo development medium to induce secondary embryogenesis and formation of transgenic somatic embryos. Transgenic embryos identified on the basis on GFP fluorescence and kanamycin resistance are transferred to germination medium to regenerate transgenic plants. The presence of transgenes in independent plant lines is confirmed by PCR.