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Park S.-H.,Michigan State University | Ransom C.,Michigan State University | Mei C.,The institute for Advanced Learning and Research | Sabzikar R.,Michigan State University | And 4 more authors.
Journal of Chemical Technology and Biotechnology | Year: 2011

BACKGROUND: Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial cellulases. At least three cellulases including endo-cellulase, exo-cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these cellulases could be self-produced within cells of transgenic bio-energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo-cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars. RESULTS: Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4-β-cellobiohydrolases I (CBH I) exo-cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1:4:1 (E1:CBH I:cellobiase) of crude heterologous cellulases is ideal for converting ammonia fiber explosion (AFEX) pretreated corn stover into fermentable sugars. CONCLUSIONS: Corn plants that express all three biologically active heterologous cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the present microbial cellulase mix production for cellulosic biofuels. © 2011 Society of Chemical Industry.


Lowman S.,The Institute for Advanced Learning and Research | Lowman S.,Virginia Polytechnic Institute and State University | Kim-Dura S.,The Institute for Advanced Learning and Research | Mei C.,The Institute for Advanced Learning and Research | And 2 more authors.
Plant and Soil | Year: 2015

Background and aims: Sustainable agricultural production in the 21st century requires new approaches to reduce the use of synthetic nitrogen fertilizers. A newly recognized option is biological nitrogen fixation by commensal bacterial endophytes. The aim of this project was to explore strategies for supplying biologically fixed nitrogen to a bioenergy crop, switchgrass cv. Alamo. Methods: The tested strategies were: 1) harnessing the ability of horizontal gene transfer between a known N-fixing bacterium, Burkholderia phymatum STM 815, and a switchgrass growth promoting endophyte, Burkholderia phytofirmans strain PsJN, and 2) isolation and utilization of naturally occurring N-fixing endophytes from seeds of switchgrass cv. Alamo. Results: The ability to grow on nitrogen free medium was successfully transferred from B. phymatum STM 815 to B. phytofirmans strain PsJN. The resulting bacterium, PsJN+, outperformed PsJN in switchgrass growth promotion in vitro on a low nitrogen (75 mg/L) medium (69 % increase). An endophyte with FAME and 16S sequence most similar to Sphingomonas sp. was isolated from seedlings derived from surface sterilized seeds germinated and grown in nitrogen-free hydroponic medium, and was also able to promote switchgrass growth under low nitrogen conditions (27 % increase over control). Conclusions: A plant growth promoting endophyte Burkholderia phytofirmans strain PsJN transformed with genomic DNA containing the nif operon from Burkholderia phymatum STM 815, and Sphingomonas sp. strain NSL, a naturally occurring switchgrass seed endophyte capable of nitrogen fixation, were able to promote in vitro growth of switchgrass under low nitrogen conditions. © 2015 Springer International Publishing Switzerland


Veilleux R.E.,Virginia Polytechnic Institute and State University | Mills K.P.,Virginia Polytechnic Institute and State University | Baxter A.J.,Virginia Polytechnic Institute and State University | Upham K.T.,Virginia Polytechnic Institute and State University | And 14 more authors.
Plant Biotechnology Journal | Year: 2012

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T1 progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T0 launch pads, putative transposants in the T1 generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T1 plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T1 plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T0 generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3′ UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T2 transposon-tagged plants. The mutant collection has been catalogued in an on-line database. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.


Patent
The Institute For Advanced Learning And Research | Date: 2011-07-15

A system, method and media formulation for high-quality and large-scale micropropagation of graminaceous plants such as M.giganteus and switchgrass have been developed and include callus induction, callus propagation, plantlet regeneration, shoot multiplication, shoot quality improvement and rooting, resulting in high plant survival in the greenhouse and in the field. The systems and methods described herein are theoretically capable of producing more than 700 billion plants from one single shoot in one year.


Patent
The Institute For Advanced Learning And Research | Date: 2014-08-20

Systems, methods and media formulations for high-quality and large-scale micropropagation without callus phase and plant regeneration from callus of graminaceous monocot plants such as Arundo, corn and wheat involving composite meristem explants, leaf explants and other explants have been developed. Graminaceous plants and plantlets propagated and regenerated by these systems, methods, and medium formulations are also described herein.

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