Plant Transformation

PLANT BIOLOGY AND BIOTECHNOLOGY / Crop Improvement

Research Interests

Molecular farming based on chloroplast genetic engineering, Regulation of plastid gene expression at translation level; Submergence tolerance in Northeast Indian rice cultivars. Plant transformation, Chloroplast genetic engineering, molecular farming, Genetic improvement of cotton and rice.

Description of Research

Genetic engineering of chloroplasts: Genetic engineering based on the introduction of foreign genes into nuclear genome to modify existing trait or introduction of new trait has become very common and many laboratories are now able to produce Genetically Modified Organisms (GMOs). However, introduction of foreign genes into chloroplast genome still remain a major challenge and only few laboratories in the world have developed and using this technology. Chloroplasts have their own genome, transcription and translation machinery that resemble prokaryotes.  At ICGEB, PTG group has developed transformation system for chloroplasts/plastids and expressed a number of foreign genes. Expression of transgenes in the chloroplasts lead to very high level expression of recombinant proteins and the foreign genes introduced are contained due to maternal inheritance of plastids. So far we have tested the expression of a number of cellulolytic enzymes, required in huge qualities in bioenergy programs and in technical industry. Besides the applied aspects, our group is also interested in understanding the regulation of chloroplast gene expression. In this regard, a systematic study involving expression of foreign genes under different non-start codons was tested to assess the expression levels under non-AUG codons. Our results suggest that non-AUG codons are efficiently used by chloroplasts to express proteins where turnover is very high. The expression levels achieved under some of the non-AUG codons are much more as compared to the expression level obtained under AUG start codon (Figure).

Molecular farming and Bioenergy: Molecular farming based on chloroplast expression of foreign genes is considered to be more sustainable for large-scale production of industrial enzymes when compared to other expression systems as it is highly cost-effective and easy to scale up the production levels. Collaborative research on bioenergy with the University of Pavia, Italy showed that plant biomass can be converted into simple sugars which in turn can be converted further into ethanol. Xylanase was taken as a model enzyme and over expressed for the first time using chloroplast genetic engineering. Following the successful demonstration of xylanase expression by our group, a large number of genes coding for cellulolytic enzymes from bacterial and fungal sources were tested.  Work is ongoing to study the depolymerisation of lignocellulolytic biomass using some the recombinant enzymes produced using chloroplast transformation system at a large scale.

Genetic improvement of cotton and rice: Previously, our Group developed efficient transformation systems for both cotton and rice. Using such transformation systems, agronomically useful genes such as Cry1Ac, Cry2ab and VIP were transformed into cotton. More recently, promoter of Annexin gene that over expressed in cotton boll/fiber tissue and unregulated in response to drought stress has been characterized.  Our results showed that GhANXA1 promoter is highly active in all types of trichomes  and the expression is upregulated in the presence of stress related signaling molecules. Utility of GhANXA promoter has been tested to express cry1Ac, a gene coding for insecticidal protein in trichomes.

Submergence tolerance in rice is very fascinating as the plants use multiple mechanisms to cope with stress. Rice cultivars grown in Northeast escape flooding/water logging by rapidly elongating internodes. Our lab initiated a major program supported by the Department of Biotechnology, Govt. of India, to understand the basic mechanisms involved in the submergence tolerance in these rice cultivars.  Over expression of sub1A1 gene in these cultivars showed that elongation due to SNORKEL pathway can be reduced to some extent. Efforts are on to see if the down regulation of SNORKEL pathway can make these plants tolerant and yet do not elongate that result in loss of yield.

Plant Transformation Figure
Comparison of GUS expression under psbC and psbA promoters with different start codons in tobacco cholorplasts. To study the significance of non-AUG codon in the expression of proteins in chloroplasts, uidA (GUS) gene has been expressed under both promoters. Expression data showed GUS expression is highest under GUG; GUS expression was highest under AUG; however, GUS expression was below the detection level under CUG.

Recent Publications

Pagliano, C., Bersanini, L., Cella, R., Longoni, P., Pantaleoni, L., Dass, A. Leelavathi, S., Reddy, V.S. 2017. Use of Nicotiana tabacum transplastomic plants engineered to express a His-tagged CP47 for the isolation of functional Photosystem II core complexes. Plant Physiol Biochem 111, 266-273 PubMed link

Dass, A., Zainul Abdin, M., Reddy, S.R., Leelavathi, S. 2017. Isolation and characterization of the dehydration stress-inducible GhRDL1 promoter from the cultivated upland cotton (Gossypium hirsutum). J Plant Biochem Biotech 26, 113-119

Faè, M., Accossato,S., Cella, R., Fontana, F., Goldschmidt-Clermont, M., Leelavathi, S., Reddy, V.S.,Longoni, P. 2017. Comparison of transplastomic Chlamydomonas reinhardtii and Nicotianatabacum expression system for the production of a bacterial endoglucanase, Appl Microbiol Biotechnol 101, 4085-4092 PubMed link 

Cuu, N.V., Khiem, N.V., Henary, S.C., Guruaribam, S.D., Reddy V.S., Sadhu, L. 2016. Fast recovery of transgenic submergence tolerant rice cultivars of North-East India by early co-cultivation of Agrobacterium with pre-cultured callus. Physiol Mol Biol Plants 23, 115-123

Kumar, S., Pandey, P., Kumar, K., Rajamani, V., Padmalatha,K. V., Dhandapani, G., Kanakachari, M., Leelavathi, S., Kumar, P.A., Reddy, V.S. 2016. Delineating the glycoproteome of elongating cotton fiber cells. Data Brief 5, 717–725 PubMed link

Kumar, S., Kanakachari, M., Gurusamy, D., Kumar, K., Narayanasamy, P., Padmalatha, K. V., Solanke, A., Gamanagatti, S., Hiremath, V., Katageri, I. S., Leelavathi, S., Kumar, P.A., Reddy, V.S. 2015. Genome-wide transcriptomic and proteomic analyses of bollworm infested developing cotton bolls revealed the genes and pathways involved in the insect pest defense mechanism. Plant Biotechnol J 14, 1438-55 PubMed link

Patents

Method of expressing foreign protein in plastid. US Patent number US 9,175,300

“Produzione di cellulasi ricombinanti di Chaetamium globosum in piante ransplastomic di tobacco.” Italian  Patent application no. MI2010A002240. Patent granted.

“Produzione di cellulasi termostabili in piante transplastomiche di tabacco e loro impiedo per la digestione di substrati cellulosici.” Patent No 0001423979/Italy.