Plant Molecular Biology and Genetic Engineering Laboratory

Group Leader: Dr.K.Venkateswar Rao,, Associate Professor

Dr.K.V.Rao's Ph.D Students

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KAMBAKAM  SEKHAR             

 

                            

E mail: kambakamsekhar@sify.com

M.Sc (Agricultural Biotechnology)  Acharya N.G.Ranga Agricultural University, Hyderabad. M.Sc dissertation (Isolation of full-length cDNA sequence from rice EST resources and construction of plant expression vectors for functional tests) at Prof. A.R.Reddy lab, school of Life Sciences, university of Hyderabad.  B.Sc (agriculture)  Acharya N.G.Ranga Agricultural University, Tirupati.

 

Topic of Research:

 

Functional validation of stress responsive genes in Arabidopsis.

 

     Drought stress is the major cause of yield instability in agricultural production across diverse crop ecosystems. Drought stress induces a plethora of biochemical and physiological responses in plants involving an array of different pathways associated with stress perception, signal transduction, gene expression, and synthesis of a number of proteins and other small molecular weight compounds. Given this complexity of the drought response, EST (Expressed Sequence Tag) approach would assist in gene discovery and genetic engineering of plants for drought tolerance.

     We isolated drought stress responsive genes from ESTs collection of drought stress pigeonpea. Functional validation of such drought stress responsive genes in vivo can be tested by developing transgenics in Arabidopsis. The present investigation is aimed at functional characterization of stress responsive genes with the following specific objectives:

 

Ø     Construction of plant expression vectors containing drought stress responsive genes.

Ø     Introduction of drought responsive genes into Arabidopsis through Agrobacterium-mediated transformation

Ø     Molecular characterization of transgenic plants

Ø     Functional analysis of those genes against drought stress.

 

  

P. SUBRAMANIAN               E.mail : paul_sub_2000@yahoo.com

 


 

Post M.Sc, certificate course in Plant Genetic engineering from Madurai Kamaraj University, 2001. M.Sc., Biochemistry from Sourashtra college affiliated to Madurai Kamaraj University, 1998-2000. B.Sc., Biochemistry from Sourashtra college affiliated to Madurai Kamaraj University, 1995-1998. Worked as a Junior Research Scientist for a collective period of 7 months in Avestha Gengraine Technologies Pvt. Ltd., and Metahelix Lifesciences Pvt. Ltd., Bangalore, 2002.

 

Topic of Research:                                          

                Development of transgenic cotton resistant to sap – sucking pests.

Introduction:

Cotton is an important commercial crop and is grown in more than eighty countries. The cotton cultivation is getting affected every year by major insect pests such as lepidopteran insects especially Helicoverpa armigera, and homopteran (sap-sucking) pest such as white fly (Bemisia tabaci). The white fly not only causes physical damage to the plant but also act as vector for cotton leaf curl virus (CLCV). Although, chemical pesticides have been used extensively to control these pests during cotton cultivation but met with little success. After the success of Bt cotton in controlling the major bollworms, the immediate need is to find out a suitable solution using biotechnological tools to control the sap sucking pest problem without further damaging the balance between prey and predators agro ecosystem of cotton cultivation zones of the country. Therefore the development of genetically modified cotton plant with resistance to these pests using desirable genes will add value to the integrated management of these pests.

OBJECTIVES:

 

1.     Standardization of transformation protocol for Cotton variety Coker 312 by using pTOK 233 plasmid, which has gus A reporter gene.

2.     Introduction of lectin genes into cotton variety Coker 312.

3.     Molecular characterization of transgenic cotton.

4.     Insect bioassays with T1 transgenic plants of Cotton.

5.     Selection and maintenance of T2 homozygous transgenic lines.

 

 

VIJAYA KUMAR SADUMPATI (  CSIR-JRF):

 


 

M.Sc.- Genetics from Osmania University, 2001-03, B.Sc. – (B.Z.C) from S.V University, 1997-2000, District merit scholar ship from VII to X, National Merit Scholar ship during Intermediate, Best out going student award after degree from S.V.Arts Degree College, M.Sc 1st Rank from Osmania University Genetics Department.

 E-mail : vijaycpmb@yahoo.com

Topic of Research:

Development of transgenic rice for Sheath blight resistance

 

Introduction:

Rice (Oryza sativa L) is one of the most important cereal crops, which serves as staple food for more than two thirds of the world’s population.  Rice productivity is adversely affected by both biotic and abiotic stress factors. It was estimated that annually, more than 200 million tons of rice is lost due to damages caused by pests and edaphic stress factors.  The worldwide losses due to pests and diseases have been estimated as 37% of the agricultural production, out of which 20-25% is due to fungal diseases.

 The insertion of specific chitinase gene into crops is showing value in integrated fungicidal management programs for controlling different fungal organisms.  Chitinase elicit a variety of responses that can defend plants from a range of factors including environmental stress, infections by bacteria, and fungi.  Many agricultural plants lack chitinase defenses, making them susceptible to yield losses from fungal infestations.

 

Objectives:

-         Establishment of genetic transformation system in popular indica rice varieties.

-         Construction of Ti- plasmid vector carrying the rice chitinase gene for Agrobacterium mediated transformation of rice.

-         Characterization of primary transformants by  molecular analyses

-         Fungal bioassays with T1 generation transgenic rice plants expressing rice chitinase gene.

-         Evaluation of transgenic indica rice variety containing snowdrop lectin gene (gna) against sheath blight.

-         Pyramiding of snowdrop lectin gene (gna) and chitinase gene for durable resistance to sheath blight.

 

Work carried out:

The E.coli plasmid pAHGII containing the expression unit of chitinase gene was isolated and cloned at Hind III site of PSB II intermediate vector of Agrobacterium. After conformation the recombinant clone containing chitinase gene was transferred into Agrobactirum strain LBA 4404 by triparental mating. Popular rice varieties like BPT 5204, MTU-1071 and Swarna were selected for production of transgenics with the chitinase gene.

 

 

 

 

Priyanka Bhyri   M.Sc Genetics (Osmania University, Hyderabad)

Research Topic:   

Isolation of stress inducible genes from Pigeon pea.

Email            :      priyasravan@rediffmail.com

 Abstract of the work:

Pigeonpea (Cajanus cajan L.) Mill sp. is a major grain legume of the semi-arid tropics of India and other developing countries of Asia, Africa and Latin America. Abiotic stresses, such as drought salinity and low temperature are major factors in limiting plant growth and productivity. It has been estimated that two thirds of the yield potential of major crops are routinely lost due to these stresses. In general pigeonpea is tolerant to drought and high temperature than most of the crops. No attempts were made to understand the drought tolerant mechanism of pigeonpea and its ability to tolerate drought conditions. The isolation and characterization of drought stress inducible genes from pigeonpea is highly desirable in order to overcome abiotic stress factors, especially for achieving sustainable yields from drought sensitive crops.

 OBJECTIVES:

·        Construction of subtractive cDNA libraries  from stressed and control tissues.

·        Screening of library for putative stress inducible genes

·        Molecular analysis and characterization of stress induced genes

·        Functional analysis of isolated stress tolerant genes.

 WORK CARRIED OUT

In order to isolate stress inducible genes, pigeonpea plants grown under control conditions were subjected to different levels of polyethylene glycol (PEG) and water stress. Total RNA was isolated from control and stressed plants and cDNA-subtracted libraries were constructed using PCR select cDNA subtraction and cDNA subtractive hybridization methods. More than hundred recombinant clones were obtained. Among these, more than fifty clones were subjected to DNA sequencing. One cDNA clone, obtained by subtractive hybridization, contained an ORF of 540 bp. The deduced amino acid sequence of the protein showed partial homology with the known protein encoding genes involved in stress tolerance.

 Five different PCR select cDNA subtraction libraries were constructed from the plants subjected to drought stress. The different recombinant clones obtained showed homology with stress induced ESTs of Glycine max, cDNAs of Medicago trancatulata, 14 kDa proline rich protein of Phaseolous vulgaris, dihydrofolate reductase, chlorophyll a/b binding protein, arabinogalactan protein, high mobility group proteins, protease inhibitors, peptide transporters, leucine aminopeptidases, fructokinases, and acetyl glutamate kinase. However, some of the cDNA clones are unique and did not show any homology with the known protein encoding genes. The Northern blot analysis confirmed the stress inducible nature of these genes. Southern blot analysis indicated the presence of these DNA sequences in the genome of pigeonpea. For the first time, we have deposited 22 cDNA clones of pigeonpea in the genbank with the accession numbers, CK394831 to CK394851.

 

   

RAGHU PEDAVALLI (B.Sc (Microbiology)- 1999-Nalanda Degree College - Nalgonda. M.Sc (Genetics)- 2002-Osmania University – Hyderabad,         Post Graduate Diploma in Computer Applications (P.G.D.C.A)

 

Topic of Research:

Transgene pyramiding for multiple insect resistance and their localization of integration in rice genome.

 E-mail             :  raghugenes@yahoo.com

 Objectives:

  •  Identification and localization of transgene using FISH and SSR markers in transformants.

  •  Development of transgenic hybrid rice resistant to lepidopteran pests.

  •  Pyramiding of different insect resistant genes for multiple resistance.

 I have identified the localization of integrated transgenes in two independent transgenic rice lines with the help of molecular markers, especially using SSRs. The position of integrated transgene in these two transformants was on Chromosomes 1p and 3q, respectively.

  I was also involved in a couple of projects entitled

1.      “Parentage analysis of Annona squamosa using RAPD”.

2.      “A tool for construction of protein Hydropathic curve.”

Bharathi Yerasi CSIR-JRF  (Bsc(Ag)- 2000-ANGRAU-Tirupati,     Msc(Ag)- 2002-ANGRAU-Tirupati)

 

Topic of Research:  

Genetic transformation and gene pyramiding for multiple resistance against major insect pests of rice.

 E-mail             :  Bharathiy2002@yahoo.co.in

Objectives:

  •  Isolation and characterization of garlic lectin gene

  •  Construction of Ti based plant expression vector of Agrobacterium tumefaciens 

  •  Production of transgenic rice plants

  •  Pyramiding of garlic lectin and snowdrop lectin genes for broad based resistance.   

Garlic lectin gene was isolated from garlic leaves and cloned in pET 21 vector of E. coli. After confirming the expression of garlic lectin in E.coli the same was cloned into pSB11 intermediate vector of Agrobacterium tumefaciens . Later, the recombinant clone was transferred into Agrobacterium  strain LBA-4404 by triparental mating. Popular rice varieties like Swarna and BPT 5204 were selected for production of transgenics with the garlic lectin gene.

 

Anumula Kavitha CSIR/JRF    ( M.Sc Botany Gold medalist,  Osmania University.,  PGDCA, SISI-CMTES).

 

Research Topic:      

Isolation of stress inducible promoters from pigeonpea.

 Email                :    akavi_99@yahoo.com.

Plant growth is greatly affected by environmental stress, in the form of abiotic and biotic stresses, such as drought, temperature, salinity, mineral nutrients, insects, bacterial and viral infections. Stress is usually defined as an external factor that exerts a disadvantageous influence on the plant. In general, plants respond to environmental stresses through a wide variety of biochemical and physiological adaptive changes, such as the accumulation of compatible solutes and synthesis of many regulatory proteins.

In recent years, different classes of genes encoding structural and regulatory proteins have been employed for the production of a range of abiotic stress-tolerant transgenic plants. However, only a few promoters are available for driving these stress tolerant genes. In order to isolate stress inducible promoters, we have selected the pigeonpea, which is known stress tolerant crop.

Objectives

·           Isolation of genomic DNA from pigeonpea plants.

·           Construction of genomic DNA library.

·           Screening of the library for the stress inducible genes.

·             Functional analysis of identified promoters.