Plant Biotechnology Student Oral Presentation Competition
The 2011 Plant Biotechnology Student Presentation Competition was moderated by Sukhpreet Sandhu. This session was a success with outstanding student presentations that were well-received by a large audience. The authors of the best five abstracts were invited to give a talk in this session. Abstracts from 11 student participants were evaluated by Jim Register (Pioneer Hi-bred Intl., USA), Prakash Lakshmanan (BSES Ltd., Australia), and Ian Curtis (Texas A&M, USA). Adjudged competition winners were presented with a certificate and a cash award of $250 for the first place winner, Sarah H Holt (Virginia Polytechnic Institute & State University, VA), $100 for the second place winner Man Zhou (Clemson University, SC) and $50 for the third place winner Jessica L. Rupp (Kansas State University, KS). Cash prizes were made possible through contributions from BASF. Participation certificates were awarded to Khanhvan T. Nyugen (Worcester Polytechnic Institute, MA) and Hyeong Je Jung (University of Florida, FL) for their excellent presentations.
Submitted by Sukhpreet Sandhu
Flower and Fruit Developmental Defects Caused by a Spontaneous DNA Deletion in a SEPALLATA3-like Gene in Fragaria vesca
Floral morphology and development are critical in the plant life cycle. Genetic control of floral patterning depends upon gene interactions, orchestrated by the MADS box transcription factors (TFs). These TFs are divided into five classes (ABCDE) of genes corresponding with the ABCDE model of floral morphology. These genes have overlapping function across the floral whorls and are necessary for proper organ development. Understanding how each TF impacts floral morphology has been greatly aided by the use of knockout mutants, either caused by random genetic mutation or intentional T-DNA insertional mutagenesis. Fragaria vesca L. is a rapidly developing model for Rosaceae and other fruit crops. This diploid strawberry possesses simple flowers and fleshy fruits that provide an excellent opportunity for studying MADS box TFs. Phenotypic screening of a T1 pCAMBIA insertional mutant population revealed a floral morphology mutation in F. vesca named Green Petal (GP). The phenotypic effects of the GP mutation include sepaloid petal morphology, pollen abortion, indehiscent anthers, spontaneous conversion of anthers to sepals, presence of trichomes on pistils, green carpels and ectopic fruit formation, thus affecting all of the three inner whorls. Southern analysis indicated that two T-DNA insertions were present in the GP mutant line. The flanking regions of the T-DNA insertions were sequenced by HiTAIL PCR. T1 Segregation analysis through T-DNA Zygosity PCR showed that neither T-DNA insertion associated with the GP phenotype. Based on the GP phenotype, we selected 30 MADS box TFs from strawberry as potential candidate genes responsible for this phenotype. RT-PCR of floral bud cDNA revealed a 37 bp deletion of the C-terminal domain in a putative SEPALLATA3 E-class MADS TF. Segregation analysis confirmed association of the deletion with the GP phenotype. The wide range of expression of the mutation suggests that this gene’s function may affect many downstream processes, offering new insight into flower development.
Sarah H. Holt, Department of Horticulture, Virginia Polytechnic Institute & State University, Blacksburg, VA. In Vitro Cellular and Developmental Biology, 47:S35-36, 2011.
Overexpression of a Rice MicroRNA319 Gene Enhances Drought and Salt Tolerance in Transgenic Creeping Bentgrass (Agrostis stolonifera L.)
miroRNAs (miRNAs) are short single-stranded molecules arising from primary miRNA transcripts (pri-miRNAs) encoded by miRNA genes. They regulate gene expression by negatively controlling both the stability and translation of target messenger RNAs. In plants, miRNAs mostly regulate other transcription factors and have critical functions in plant growth, development and stress responses. The miR319 family is one of the first characterized and conserved miRNA families in plants and it has been demonstrated to target TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) genes encoding plant-specific transcription factors known to be largely involved in leaf development. In rice, the miR319 comprises two members, OsmiR319a and OsmiR319b with the same mature sequence. We have investigated the role miR319 plays in plant development and plant response to abiotic stress in perennial grass species. Transgenic creeping bentgrass plants overexpressing the full-length cDNAs of OsmiR319a were produced. All transgenics exhibited dramatic morphological changes, including significantly decreased tiller numbers, wider leaves, and coarser stems. The five putative miR319 target genes in turfgrass, AsPCF5, AsPCF6, AsPCF7, AsPCF8 and AsTCP14 were all down-regulated in transgenic plants. Overexpression of miR319 also led to dramatically enhanced salt and drought tolerance in transgenics, which is associated with the down-regulation of at least one of the target genes, AsPCF5, indicating its direct involvement in plant response to salt stress. Transgenic analysis further elucidated the biochemical and physiological basis of the enhanced stress tolerance in transgenic plants. Data obtained in this research reveal the importance of miR319 in plant resistance to environmental adversities, and identify its direct target specifically involved in plant stress response. This will lead to development of novel molecular strategies to genetically engineer crop species for enhanced performance under unfavorable environmental conditions, contributing to agriculture production.
Man Zhou, Department of Genetics and Biochemistry, Clemson University, 110 Biosystems Research Complex, Clemson, SC 29634. In Vitro Cellular and Developmental Biology, 47:S37, 2011.
RNAi Mediated Viral Resistance in Transgenic Wheat
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV), are two viruses of the wheat mosaic complex affecting Triticum aestivum (L.) in the Great Plains of the United States. Although not fully effective, the current disease management strategy incorporates the deployment of resistant varieties, mite vector control and various cultural practices. As an alternative strategy, the use of interference RNA to generate resistance to these wheat viruses was evaluated. RNAi expression vectors were independently created from the sequences of the coat proteins (CP), the HC-Pro, 6K2-NIa and a portion of the VPG of both WSMV and TriMV. Immature embryos of the spring wheat cultivar ‘Bobwhite’ were independently co-transformed by biolistic particle delivery system with RNAi expression vectors and pAHC20, which contains the bar gene for glufosinate selection. After tissue culture, putative transformed plants were analyzed through PCR for the presence of the appropriate RNAi gene. Transgenic T1 seeds were collected and each line was tested for transgene expression via RT-PCR. To determine viral resistance, progeny were mechanically inoculated twice (day 1 and day 14) with the corresponding virus, and leaf samples were screened fourteen days after last inoculation by ELISA. Resistance was observed in the transgenic lines however the results did not segregate in a Mendelian fashion; there was evidence of transgene silencing. Analysis of T2 and T3 generation followed similar results. Regardless of these phenomena, consistent resistance response in two lines of WSMV CP-RNAi construct and one TriMV CP-RNAi transgenic line was found. T3 generations continue to exhibit a high level of resistance among these CP-RNAi constructs. Twenty-two crosses have been made with the commercially important winter wheat cultivar ‘Overly.’ In addition to the CP-RNAi events, plants expressing 6k2-NIa-RNAi transgene have been grown to the T2 generation and are currently undergoing bioassays and analysis. HC-Pro and VPG RNAi constructs for both viruses are also in the tissue culture process.
Jessica L. Rupp, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. In Vitro Cellular and Developmental Biology, 47:S36-37, 2011.
Plant Biotechnology Post-doctoral Presentation Competition
On Sunday, June 5, SIVB had an oral presentation competition for post-doctoral scientists. The top three contestants had been chosen to deliver talks based on the quality of their abstracts. There was a large turnout in a large conference room. All three talks were very good, and the contestants each fielded several challenging questions. Since the scientific topics were diverse it was difficult to choose one as better than the others. However, our judges Dr. Nancy Reichert, Dr. Prakash Kumar, and Dr. John Finer had to render a rank order. They selected M.O. Balogun for 3rd prize, Zhigang Li for 2nd prize, and M. Aydun Akbudak for 1st prize. The next day at our Plant Section Business Meeting, plaques and cash awards were made. We encourage qualified scientists to enroll for next year’s competition.
Submitted by Jeffrey Adelberg
FLPe-mediated Marker Gene Excision from Site-specific Gene Integration Locus in Rice
Site Specific Recombination (SSR) systems carry out precise recombination between target DNA sequence resulting in DNA integration or excision depending on the orientation and position of the target recombination sites. This principle has been applied to drive integration of transgenes into the engineered genomic sites and removal of marker genes in plant cells. These two major applications have been individually developed using different SSR systems such as Cre–lox, FLP–FRT, and R–RS. In the present work, a molecular strategy for combining the two applications was tested. This strategy was designed to accomplish Cre–lox-mediated gene integration followed by FLP–FRT-mediated marker excision to generate a marker-free site-specific integration locus. Two site-specific integration loci containing FRT sites to remove marker gene were developed to study the efficiency of FLP-FRT mediated marker excision. First, wild-type FLP (FLPwt) was introduced either transiently or constitutively to initiate FRT x FRT recombination. FLPwt-mediated marker excision on two integration lines resulted only in poor to undetectable excision of marker genes. Next, the enhanced (thermo-stable) version of FLP protein, FLPe, was introduced in the two lines. FLPe catalyzed efficient excision of marker genes that was detected by PCR and Southern analysis. The excision efficiency as calculated by real time PCR was found to be ~ 100% in majority of the cases. Thus, FLPe–FRT system is suitable for developing a robust marker-removal technology for plants.
M. Aydin Akbudak, Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR 72701. In Vitro Cellular and Developmental Biology, 47:S34-35, 2011.
Overexpression of a Cyanobacterial Flavodoxin in Transgenic Creeping Bentgrass (Agrostis stolonifera L) Leads to Enhanced Drought Tolerance
Flavodoxin (Fld) plays an important role in photosynthetic microorganisms as an electron carrier flavoprotein. It can be induced in response to undesirable environmental stress and has been demonstrated to be able to efficiently replace the stress-sensitive plant chloroplast ferredoxin (Fd) in most electron transfer processes. When introduced into tobacco, transgenic plants displayed enhanced tolerance to various sources of abiotic stress. We have studied the feasibility of using cyanobacterial Fld in perennial grasses for improving plant performance under environmental adversities. We generated transgenic creeping bentgrass plants, in which the overexpressed Fld was directed to chloroplast by a pea rbcS transit peptide. Twelve of the eighteen independent transgenic (TG) plants exhibited dramatically improved drought tolerance compared to non-transgenic wild type (WT) plants. The enhanced drought tolerance was associated with the higher relative water content and lower electrolyte leakage. Under water deficient conditions, the biomass increments in the TG plants were higher than in WT controls, and the TG plants had higher chlorophyll contents than WT controls under both normal and drought stress conditions. Moreover, transgenic creeping bentgrass plants also exhibited enhanced performance under other abiotic stresses including heat and salinity. Our results demonstrated that overexpression of cyanobacterialFld gene in transgenic turfgrass significantly impacted plant response to adverse environmental conditions, leading to enhanced performance under water, salinity and temperature stresses. This points to the promising potential of using similar strategy for genetic engineering of food crops and other environmentally and economically important perennial grasses, enhancing agriculture production.
Zhigang Li, Department of Genetics and Biochemistry, Clemson University, 110 Biosystems Research Complex, Clemson, SC 29634. In Vitro Cellular and Developmental Biology, 47:S35, 2011.
Effects of Plant Growth Regulators on In Vitro Tuber Initiation and Dormancy at Different Growth Phases in White Yam (Dioscorea rotundata)
Yam tuber production and dormancy are linked. They are also affected by plant growth regulators (PGRs). However, exogenous control of tuber dormancy is still inadequate. We investigated in vitro, microtuber production and dormancy at some PGR regimes applied at selected growth phases spanning the life cycle of yam. Three concentrations each of uniconazole-p (UP), gibberellic, jasmonic (JA) and naphthalene acetic (NAA) acids were applied at culturing, primary nodal complex (PNC) formation and to harvested microtubers in a split-plot design in two genotypes of white yam. Duration to 50% microtuber initiation (MI) and sprouting (SPR) were recorded. NAA applied at culturing initiated microtubers 28 days earlier than the control, but delayed it by 56 days when applied at PNC formation. GA (3µM) applied at culturing also initiated microtubers 28 days earlier than the control in TDr 93-23. JA (3µM) applied at PNC formation delayed microtuber initiation by 56 days in TDr 608 while 0.3µM applied at culturing induced earlier tuber initiation than the control in TDr93-23. The effects of JA and NAA on sprouting of microtubers were not significant. All regimes of UP inhibited microtuber production, except 1.7µM UP applied at PNC formation with MI of 126days in TDr 608. Microtubers produced from cultures treated with 1.7µM UP at PNC formation sprouted 90 days earlier than the control in TDr 608, while those produced when cultures were treated with 0.15µM GA at culturing or PNC formation did not sprout for 376 days post culturing. These results suggest an important role for GA and its inhibitor UP applied at different growth phases in in vitro microtuber initiation and dormancy.
M. O. Balogun, Department of Crop Protection and Environmental Biology, University of Ibadan, Nigeria. In Vitro Cellular and Developmental Biology, 47:S34, 2011.