Sudesh Chhikara, Advanced Centre for Biotechnology, M. D. University , Rohtak-124001, India Pawan K. Jaiwal, Advanced Centre for Biotechnology, M. D. University, Rohtak-124001, India, Email: jaiwalpawan@rediffmail.com

Indian mustard is a high biomass producing crop that accumulates heavy metals (Pb, Cd, Ni) in large amounts. The development of transgenic Brassica juncea will able to efficiently remediate heavy metal polluted soils of different origin and in a wide range of environments. In present study, a simple and efficient non-tissue culture approach for the generation of transgenic Indian mustard (Brassica juncea) plants using Agrobacterium tumefaciens has been developed for the first time. This procedure involves infiltration of inflorescence with flower buds in a suspension of Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301 that contained β-glucuronidase (GUS) gene (uidA) as a reporter gene  and neomycin phosphotransferase (nptII) gene for resistance to the antibiotic kanamycin as a selectable marker. The seeds of agro-infiltrated plants (T0) were germinated on 130 mgl-1 kanamycin containing medium and the seedlings which remained green produced T1 transgenic plants. The presence, integration and expression of transgenes in T1 plants were confirmed by PCR, genomic Southern and histochemical GUS assay. Southern analysis revealed independent stable transformants carrying single or two copies of transgenes. An overall transformation frequency of 0.8 % from 250 seeds was obtained.  The analysis of T2 generation indicates that transgenes inherited in Mendalian fashion.  This method may facilitate large-scale insertional mutagenesis for functional genomics of Brassica and the development of transgenic mustard with improved phytoremediation ability for toxic metals and other agronomic traits.

Plant Spacing for Optimal Arsenic Phytoremediation using Pteris cretica

Joshua Goldowitz, Environmental Management & Safety Department, Rochester Institute of Technology, 78 Lomb Memorial Drive , Rochester, NY 14623-5604, Tel: 585-475-7018 Fax 585-475-7560, Email: jxgctp@rit.edu
Sean O’Neil, Environmental Management & Safety Department, Rochester Institute of Technology, 78 Lomb Memorial Drive , Rochester, NY 14623-5604, Tel: 585-314-1175, Fax: 585-475-7560, Email: swo1834@rit.edu

The brake ferns Pteris vittata and Pteris cretica have been suggested for phytoremediation of arsenic contaminated soil. Edenspace Systems Corporation has demonstrated the practical application in an ongoing remediation project in the Spring Valley area of Washington DC . where Pteris species were grown on one foot centers. In that Pteris are tropical to subtropical (USDA plant hardiness zone 7-10), use in colder climates will be problematic. The plants inability to over winter will require annual replanting, and the radius of single-season root spread will determine minimum planting density to ensure root coverage of the remediation area. Researchers at Rochester Institute of Technology’s Environmental Management department determined the single season radial root spread of  P. cretica in a lab study using four soil types.  Three replicates of four soil types were prepared in cubic 1 ft³ planting containers. Seedlings of 4-6 fronds in 40 cm³ seed starting media were planted centrally and grown under light with abundant water. Plants were misted and fertilized and grown for seven months to simulate one growing season. Soil was then sectioned and excavated to determine root density Vs. depth and distance from the plant crown. Root density was determined using the standard measurement of dry root mass/soil volume and using a novel GIS pixel-based measurement of root length/soil volume. Preliminary results indicate significant root growth beyond the original starting media, but exponential decline in density with distance from the planting center. This indicates that most soil beyond 0.3 foot from the plant crown will not be within a root rhisosphere and will remain unremediated. An arsenic contaminated site planted with P. cretica on 1 ft centers likely contains nodes of remediated soil surrounded by unremediated soil after one growing season.

Analysis of Arsenic-Induced Transciptome of Crambe abyssinica to Isolate Genes for Phytoremediation of Arsenic  

Student Presenter

Bibin Paulose, Dept. of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, MA-01003, USA, Tel: 413-545-5231, Email: bpaulose@psis.umass.edu
Asma Zulfiqar , Dept. of Plant, Soil and Insect Sciences, University of Massachusetts , Amherst, MA-01003, USA, Tel: 413-545-5231, Email: asmazulfiqar@hotmail.com
Om Parkash Dhankher, Dept. of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, MA-01003, USA, Tel: 413-545-0062, Email: parkash@psis.umass.edu

Arsenic (As) has been known as a carcinogen as well as an acute poison for centuries. Well-known high As groundwater areas have been found all over the world, particularly in South and southeast Asia and central and south America.  Further, due to the repetitive use of As-contaminated water for irrigation, very high levels of As build up in the agricultural soils around the world. Millions of humans, livestock and vegetation face the increasing threat of As toxicity. Phytoremediation renders an eco-friendly and sustainable method to remediate the As polluted sites. Crambe (Crambe abyssinica)- a non-food high biomass crop, is reported to be able to tolerate and accumulate unusually high amount of As. Understanding the molecular mechanism of high As tolerance is essential to improve the efficiency of uptake thereby exploiting the plant for commercial phytoremediation.

The objective of the present study is to isolate and characterize the As responsive genes in Crambe. A PCR-Select Subtractive cDNA Hybridization approach was employed to isolate As-induced genes from Crambe seedlings. After differential screening, 105 positive cDNA clones from the subtracted library were sequenced. The sequences were categorized based on their similarity with reported sequences in the databases. Many novel sequences were present in the library, which were hitherto uncharacterized or not reported. A variety of reductases viz. peptide methionine sulfoxide reductase, oxophytodienoate reductase, dioxygenases, aldo-keto reductase and sulfite reductase, were found to be differentially expressed implying that the As alters the redox potential at cellular level.  Glutathione transferases, both the Tau and Phi subfamily that are involved in cellular detoxification were also represented in the library. Other important sequences included ATPases, drug transporter/antiporter, phosphosulfate kinases, adenylyl transferase, methionine synthetase and proteins involved in ubiquitin proteolytic pathway along with transcription factors and RNA binding proteins. Expression analysis and functional characterization is being carried out and expected to reveal the mechanism of plant arsenic tolerance. The candidate genes will be used to engineer non-food high biomass C. abyssinica plants for phytoremediation of As-contaminated soil and sediments.

Isolating Chromium-Induced Genes from Crambe abyssinica for Phytoremediation of Chromium Contamination  

Student Presenter

Asma Zulfiqar , Dept. of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, MA-01003, USA, Tel: 413-545-5231, Email: asmazulfiqar@hotmail.com
Bibin Paulose, Dept. of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, MA-01003, USA, Tel: 413-545-5231, Email: bpaulose@psis.umass.edu
Om Parkash Dhankher, Dept. of Plant, Soil and Insect Sciences, University of Massachusetts, Amherst, MA-01003, USA, Tel: 413-545-0062, Email: parkash@psis.umass.edu

Chromium (Cr) is a serious environmental pollutant due to its widespread use in industries such as tanning, corrosion, plating, pigment manufacturing and nuclear weapons production. Cr (VI) is generally considered to pose the greatest human health risk because of it being toxic, mutagenic and carcinogenic. There is no cost-effective environmental-friendly Cr remediation strategy available so far. Plants can be used to cleanup the Cr pollution by accumulating, stabilizing or transforming into less toxic form Cr(III). Previously, we have analyzed the uptake of Cr and other toxic metals in Crambe abyssinica and other Brassica species. Crambe accumulated high levels of Cr and As in the shoot tissues and thus has potential to be utilized as an ideal non-food crop for phytoremediation of heavy metals and metalloids. The present study was undertaken with an aim to isolate and characterize the genes induced in response to Cr stress in Crambe using a PCR-Select Subtractive cDNA Hybridization approach. After subtraction and differential screening, 71 positive cDNA clones from the subtracted library were sequenced. The sequences were categorized based on their similarity with reported sequences in the databases. Forty-five different types of genes were found to respond to Cr stress. Among these were Transcription factors, Chitinases, Thi-J like protein, Peroxidases, Glutathionases-S-Transferases , Aquaporins, oxidoreductases, harpins, zinc and iron binding proteins and many novel sequences with some unknown functions. Currently, we are analyzing these genes for expression analysis and functional characterization using both forward and reverse genetic approaches. The candidate genes will be used to engineer non-food high biomass C. abyssinica plants for phytoremediation of Cr contaminated soil and sediments.  

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