Centre for Plant Molecular Biology and Biotechnology   CPMB&B Home | TNAU Home
    Estalished in 1987    
         
 
 
 
Department of Plant Biotechnology
 

 

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Research activities
 

I. Genetic engineering
II. Genomics
III. Marker-assisted breeding
IV. Micropropagation

I. Genetic engineering:
I. a. Genetic engineering of insect resistance
I. a. i. Cry1Ac Bt Brinjal


One of the major constraints in brinjal is the damage caused by the fruit and shoot borer (FSB) of brinjal. The loss due to the insect alone may range up to 60 to 70%. Conventional methods of pest management are less effective as the insect thrives inside the fruit. Development of pest resistant lines is one of the major strategies for effective management of this pest. FSB resistant brinjal plants have been developed in four regional specific GM brinjal lines exploiting well proven Bt technology. In this programme four elite genotypes were chosen as target genotypes in back-cross breeding programme to introgress cry1Ac gene from Mahyco’s elite event. These genotypes (Co2, MDU1, PLR1 and KKM1) were chosen keeping regional preference in mind. The advanced breeding materials were field tested under multi-location research trials conducted at Madurai and Coimbatore.
The Bt open pollinated varieties (Co2-Bt, MDU1-Bt, PLR1-Bt and KKM1-Bt) were tested under multi-location field trial during 2007-08. Bt brinjal OPVs showed a significantly lower damage resulting from BFSB feeding in comparison to non-Bt counterparts. Bt brinjal OPVs did not have any effects on non-target insects and beneficial insects. Results of these multi-location research trials indicate that the Bt brinjal OPVs offer adequate level of resistance to BFSB and show potential for significantly higher marketable yield. Since the Bt technology is introduced in varietal background, the farmers can save their own seeds as the Bt brinjal lines being developed are in varietal background. These lines are expected to be commercialized in 2009.

I.a.ii. Indigenous Bt constructs
About 500 isolates of B .thuringiensis were collected from different environmental samples (soil, grain, leaves etc.) of diverse regions of Tamil Nadu, especially from the Western Ghats. Potent isolates of native Bt were selected by insect bioassay against lepidopteran pests of rice, brinjal and cotton. Indigenous Bt genes were cloned from potent isolates of Bt and plasmid constructs were made to express codon optimized synthetic Cry2A genes in rice and brinjal plants. In addition, a chimeric Cry2A gene has been made using DNA sequences of two indigenous cry2A genes of B. thuringiensis. The chimeric Cry2A protein isolated from recombinant bacterium showed significantly higher toxicity to H. armigera (cotton bollworm) than the Cry2Ab protein which is currently used in the Bt-hybrids of private companies. Therefore, arrangements have been made to get IPR for the chimeric Bt gene of TNAU. To hasten the delivery of the chimeric Cry2A gene technology of TNAU to the farming community of India, TNAU has joined hands with other leading public and private sectors through a project funded by the 'New Millennium Indian Technology Leadership Initiative' (NMITLI) of CSIR, GOI.

I. b. Genetic engineering of disease resistance
I. b. i. Sheath blight resistant rice

A rice line (ASD16) expressing rice chitinase generated in the transformation laboratory has been evaluated for disease resistance under transgenic greenhouse conditions over generations. Based on its performance, the line has been advanced to multi location research trials (MLRT). This line was evaluated under MLRT to assess the efficacy under field conditions during 2006. The line showed moderate level of resistance to sheath blight under field conditions.

I. b. ii. Bunchy top virus (BBTV) resistant Banana
Hill bananas (two ecotypes (AAB) namely, Virupakshi and Sirumalai), known for their special flavour and long shelf life, are unique to the state of Tamil Nadu, India. Like other bananas, hill bananas are also susceptible to banana bunchy top virus (BBTV). BBTV has been the sole cause for reduction in hill banana cultivation from 18,000 ha in 1970s to a mere 2,000 ha at present. None of the strategies now available are able to completely protect hill bananas against the virus. Efforts have been made at the Centre for Plant Molecular Biology, TNAU with a view to engineering resistance in hill banana cultivar, Virupakshi through RNAi technology. A suitable regeneration protocol has been standardized for the hill banana using immature male flower bud as explant. At present standardization of hill banana transformation using the reporter gene is in progress.

Isolating Replicase gene from BBTV
A full length, 850bp replicase gene of BBTV was isolated from infected hill banana. The isolated sequence showed 100% identity to the already reported BBTV replicase gene sequences from India available in NCBI databases. At present construction of RNAi vector utilizing the partial replicase gene is in progress, which will be used for the genetic transformation of hill banana to impart BBTV resistance.

I. b. iii. PRSV resistant Papaya
In the recent time, Papaya Ring Spot Virus (PRSV) has become a serious disease in papaya that greatly affected the productivity in India. Conventional breeding is not successful due to the lack of resistant germplasm. All the popular papaya cultivar grown in India is susceptible to PRSV. Transgenic papaya resistant to PRSV is commercially grown in Hawaii State of USA and Taiwan to overcome PRSV disease problem. PRSV resistance in transgenic papaya was obtained using the pathogen derived genes like coat protein and replicase gene. In this centre, a transformation protocol is established for the papaya cultivar, Co7. The coat protein and replicase gene were isolated from papaya samples infected with PRSV collected from different districts of Tamil Nadu. High level of sequence identity is observed among the isolated coat protein and replicase gene of PRSV from Tamil Nadu. These genes will be utilized for the construction of RNAi vector, which will be used for the transformation of papaya to overcome PRSV problem.

I. c. Genetic engineering for nutritional improvement
I. c. i. Golden Rice

Elite locally adapted genotypes, ADT43 and ASD16 are being converted into golden rice lines with a view to improving pro-vitmain A content of rice grains. The pro-vitamin A biosynthetic pathway genes (phytoene synthase and phytoene desaturase) were introgressed from an elite golden rice event of Syngenta (GR1 event) into the target genotypes through molecular marker assisted back-cross breeding. The breeding lines are in advanced stages of development. The lines are expected to be field tested during 2010.
I. c. ii. Low phytate maize
Maize is one of the major source of poultry feed. Maize is the richest source for phosphorus and other minerals to the birds. A significant portion of the phosphorous (P) and other mineral such as iron in mature cereal grains is present in phytate form. Phytate molecule and nutrients bound to it cannot be absorbed in the digestive tract without enzymatic degradation by phytases. Since the birds have insufficient phytase to effectively digest phytate, inorganic phosphorous is added to poultry feed. Phytase produced by microorganisms in the digestive tract can be very efficient in degrading phytate. Microbial phytase is today widely used in diets for monogastric farm animals including birds. With a view to make bio-available the nutrients, attempts are made to express phytase in maize. In this direction, a phytase gene from Aspergillus niger has been isolated. This will be expressed in maize to make nutrient rich maize suitable for poultry industry. As a first step towards expressing the phytase in maize, transformation protocols are being standardized. The tissue culture protocols have already been standardized in transformation laboratory.

II. Genomics
II. a. Isolating genes conferring drought tolerance in rice
Despite of our concerted efforts, conventional breeding approaches are resulting in slow progress in developing drought/salinity tolerant rice genotypes. Under these circumstances, intervention through biotechnological tools such as Genetic Engineering will be an opt strategy to target/manipulate specific pathways associated with drought/salinity tolerance. Genetic Engineering provide us the powerful way of improving/incorporating stress tolerance in crop plants by introducing foreign genes conferring stress tolerance. In this context, availability of suitable candidate gene(s) is highly necessary before adapting this strategy. We plan to isolate novel genes responsive to drought/salinity stress from a drought tolerant upland rice genotype called "Apo" which can be used for engineering drought/salinity tolerance in rice. We are in the process of isolating drought tolerance related genes namely, Osmotion, Dehydrin, DREB1A and DREB1B. Genes have been isolated and cloned. After confirmation by sequencing they will be used for genetic engineering of drought tolerance in rice.

II. b. Functional genomics of abiotic stress tolerance in rice
To understand the molecular basis of drought and salinity tolerance in rice, a functional genomic approach has been initiated through a DBT funded network project. CPMB is working in collaboration with scientists of PBS, CPBG in identifying the loss of function and gain of function mutants differing in their degree of drought tolerance and salinity tolerance. Few putative salinity tolerant mutants have been identified through preliminary screening of N22 mutants which will be taken for further functional genomic studies.

III. Marker-Assisted Breeding
III. a. Marker-Assisted Breeding for Biotic Stress Resistance
III. a. i. Yellow stem borer resistance in rice

A recombinant inbred population (250 RILs) and a doubled haploid population (250 DH lines) were developed using the parents viz., CO43 and W1263 as susceptible and moderately resistant to yellow stem borer respectively in rice. Mapping population was phenotyped for YSB resistance both under green house and field conditions. Microsatellite markers associated with yellow stem borer resistance at vegetative and reproductive stages were identified by deploying single marker analysis (SMA). Twenty RILs were selected out of 72 based on the SSR markers associated with both YSB resistance and yield components. Of which two RILs viz., YSB RIL # 143 and YSB RIL # 479 were found to be superior in yield and YSB resistance. They recorded an average grain yield of 6.5 t/ha with moderate resistant to YSB. These superior RILs are being tested under AICRIP trails during 2008-09. Efforts have been made to initiate collaborative research programs with a private firm M/s. Advanta India Pvt. Ltd., Hyderabad.

III. a. ii. Leaf folder resistance in rice

A recombinant inbred population of 250 lines was developed from the cross of IR36 (susceptible to leaf folder) and TNAU831311 (resistant to leaf folder) by single seed decent (SSD) method to map the genes associated with leaf folder resistance in rice. Marker phenotype association resulted in the detection of QTLs for leaf folder resistance on the linkage groups viz., 7 (RM5499, RM432 and RM11), 9 (RM257, RM242 and RM3909) and 10 (RM271and RM244).



IV. Micropropagation
IV. i. Tamarind
Nodal segments are found to be best explants for tamarind micropropagation. Multiple shoots are induced on MS medium supplemented with thidiazuran (TDZ) (0.1 mg/L), benzyl aminopurine (BAP) (0.5 mg/L) and calcium pantothenate, biotin each 0.1 mg/L The induced shoots are elongated on MS media with indole acetic acid (IAA) (2 mg/L) and BAP (1.5 mg/L). The shoots are rooted in half strength MS medium with naphthaleic acetic acid (NAA) (0.5 mg/L) and indole butryic acid (IBA) (2 mg/L).
IV. ii. Soft seeded pomegranate
Auxillary buds from nodal segments are induced to produce multiple shoots on MS medium with BAP (1.5 mg/L) and NAA (0.5 mg/L).
IV. iii. Banana
Explants collected from healthy suckers of Dwarf Cavendish on MS medium supplemented with 5 mg/L BAP with 150 mg/L Adenine sulphate produce multiple shoot induction. The shoots are elongated on MS medium with BAP 2 mg/L + IAA 0.5 mg/L. In vitro rooting is best achieved in half strength MS medium with IBA (5 gm/L), NAA (0.5 mg/L) and 0.05 per cent activated charcoal. The well rooted plantlets are hardened for 15-20 days in the mist chamber. The rooted plantlets are hardened in polybags in the mist chamber using 1:1 ratio coarse sand and red soil.

 
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