Sujata Agarwal

Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens.

Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

BackgroundGrasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency.ResultsA high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the β-glucuronidase (GUS) gene). The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG) assays showed a peak of β-glucuronidase (GUS) enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1.ConclusionAgrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo) compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock. The addition of thiol compounds such as L-cysteine and dithiothreitol in combination with acetosyringone during cocultivation also increased transient expression. The combination of multiple wounding treatments along with the addition of thiol compounds during cocultivation increased transient expression levels from 6% to 54%. There were differences in temporal GUS expression induced by pCambia1305.1 and pCambia1305.2.

Floral Transformation of Wheat

A method is described for the floral transformation of wheat using a protocol similar to the floral dip of Arabidopsis. This method does not employ tissue culture of dissected embryos, but instead pre-anthesis spikes with clipped florets at the early, mid to late uninucleate microspore stage are dipped in Agrobacterium infiltration media harboring a vector carrying anthocyanin reporters and the NPTII selectable marker. T1 seeds are examined for color changes induced in the embryo by the anthocyanin reporters. Putatively transformed seeds are germinated and the seedlings are screened for the presence of the NPTII gene based on resistance to paromomycin spray and assayed with NPTII ELISAs. Genomic DNA of putative transformants is digested and analyzed on Southern blots for copy number to determine whether the T-DNA has integrated into the nucleus and to show the number of insertions. The nonoptimized transformation efficiencies range from 0.3 to 0.6% (number of transformants/number of florets dipped) but the efficiencies are higher in terms of the number of transformants produced/number of seeds set ranging from 0.9 to 10%. Research is underway to maximize seed set and optimize the protocol by testing different Agrobacterium strains, visual reporters, vectors, and surfactants.

Hematopoietic differentiation activity of a recombinant human interleukin-6 (IL-6) isoform resulting from alternatively spliced deletion of the second exon

We have previously identified and cloned an alternatively spliced form of human interleukin‐6 mRNA lacking exon II, which encodes amino acid residues known to be important in gp130‐mediated signal transduction pathways. We expressed and purified the recombinant protein (rIL6‐alt) resulting from this alternatively spliced mRNA and now report the initial characterization of its biologic activities with comparison to full length IL6 (rIL6‐full). rIL6‐alt was found to have 104 to 105 fold less activity in proliferation assays with 7TD1 murine plasmacytoma cells and did not competitively inhibit the stimulatory activity of rIL6‐full. In addition, like rIL6‐full, rIL6‐alt had antiproliferative activity toward M1 murine myeloblast cells and was 10–200‐fold less active than rIL6‐full. In contrast, in assays with human HL60 promyelocytic leukemia cells, rIL6‐alt had greater antiproliferative activity than rIL6‐full and more strongly upregulated phagocytosis as well as surface expression of the differentiation antigen CD11b. rIL6‐full and rIL6‐alt upregulated the level of lysozyme mRNA in HL60 cells approximately equally. These findings suggest that IL6‐alt, which lacks amino acid residues encoded by the second exon of the gene, is not a natural inhibitor of IL6‐full but may be relatively tissue specific and may play a role in modulation of hematopoietic cell growth and differentiation. Am. J. Hematol. 61:169–177, 1999.

High‐throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)

We apply CE for high‐throughput analysis of functional markers for marker‐assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence‐tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well‐differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 μL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC1F3 generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.

mRNA expression of canine ATP10C, a P4-type ATPase, is positively associated with body condition score

Mouse and human Atp10c genes are strong candidates for changes in bodyweight and glucose homeostasis. Using comparative genomic analysis, a novel canine P4-type ATPase, ATP10C, was identified. Expression of ATP10C was compared between sex-matched lean (body condition score, BCS<8; n=7) and obese (BCS⩾8, n=8) client-owned dogs of comparable ages. Canine ATP10C is highly expressed in visceral and subcutaneous fat at approximately 3-fold levels compared to the omental adipose depot. There was a 5-fold significant increase (P<0.0001) in mRNA expression of ATP10C in dogs with a BCS⩾8.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya ( Carica papaya L.)

BackgroundGenetically engineered (GE) ringspot virus-resistant papaya cultivars ‘Rainbow’ and ‘SunUp’ have been grown in Hawai’i for over 10 years. In Hawai’i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.ResultsWe assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.ConclusionsThis method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai’i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.