Member SpotlightsCollaborative Studies in Citrus Plant Genetics Madhugiri Nageswara Rao, Ph.D. and Jaya R. Soneji, Ph.D. SAB Members Since April 2007  Madhugiri Rao and Jaya Soneji at the International Society for Horticultural Science (ISHS) conference. This week The Science Advisory Board had the opportunity to interview two SAB members at the same time, Madhugiri Rao, Ph.D., and Jaya Soneji, Ph.D., of the University of Florida's Institute for Food & Agricultural Sciences (IFAS). They have both worked at IFAS as Post-Doctoral research associates since 2004 in the Citrus Research & Education Center. Rao started his career in industry, with a chemist position at Karnataka Breweries and Distilleries (KBD) Pvt. Ltd., before switching to academic research with the University of Agricultural Sciences, Bangalore, India. He received his M.S. in Biochemistry from Bangalore University, and his Ph.D. in Genetics & Tree Propagation from the Forest Research Institute of the Indian Council for Forest Research & Education. Soneji taught classes at Ramnivas Ruia College in Mumbai, India, before becoming a research associate at the Indian Institute of Horticultural Research. She received her M.S. in Botany, specializing in cytogenetics, plant breeding, and molecular biology from the University of Mumbai. She continued research at the University of Mumbai and received her Ph.D. in Botany, with a specialization in nuclear agriculture and biotechnology, from the Bhaba Atomic Research Centre. IFAS research efforts, including those of Rao and Soneji, have been featured in newspaper articles and television reports. You may have seen the recent FOX News broadcast on seedless citrus fruit development at IFAS on October 16, 2007. This research was also mentioned in Genome India International on October 20, 2007, in which Rao and Soneji were specifically mentioned. Both Rao and Soneji belong to the International Society of Horticultural Sciences and the International Citrus Genome Consortium. Soneji is also a member of the Horticultural Society of India, and Rao belongs to the Society for Conservation Biology and the Forest Conservation Genetic unit at Ashoka Trust for Research in Ecology and the Environment. Additional interests include education outreach programs & biological conversation. This research duo belongs to the Samatva Trust, which endeavors to promote education in India'a rural areas. They also belong to the Biodiversity Conservation and Research Trust. Rao enjoys photography, hiking, cricket and badminton. Soneji likes to pencil sketch, listen to music, swim, and cook. The following questions have been jointly answered by Rao & Soneji, except where noted. Rao is mainly involved with functional genomics of citrus, whereas Soneji works with genetic engineering of citrus and other plants. Your team is currently involved in many different aspects of citrus research. Please describe your individual and collaborated projects. We are currently working on functional genomics, disease resistance and genetic improvement of citrus using advanced molecular and bioinformatics tools. Citrus is among the top 3 fruit crops in the world and the US is the second largest producer of citrus. Florida alone accounts for more than 65% of the total citrus production in the US. Rootstock genetic improvement project: Rootstock is a major contributor to tree performance and longevity and influences tree size, yield, fruit quality and the tolerance of scion to various abiotic and biotic stresses. To establish a comprehensive citrus rootstock genetic improvement program, it is very important to distinguish zygotic (sexual) from nucellar seedlings. Traditional plant breeding methods of screening are based on whole plant performance and are very time consuming (5-7 years after planting). To address such problems efficiently and quickly, we have successfully used high throughput molecular marker approaches to characterize closely related and morphologically similar individuals. We have identified two elite rootstock candidates that produce a high number of nucellar seedlings, thus improving the accuracy of early selection in a citrus rootstock-breeding program. These tools would greatly enhance the citrus breeders’ abilities to make precise multi-trait selections in a reasonable timeframe, ultimately hastening release of new and genetically improved rootstocks. Functional genomics studies for the identification of gene transcripts responsible for apomixis in citrus: Apomixis in citrus results in uniform clonal progeny having the maternal genetic constitution essential for reliable tree performance. There is no information concerning the genomic processes that regulate apomixis and their relationship to sexual reproductive pathways in citrus. To gain this insight, we have successfully standardized a protocol for the extraction of total RNA from different developmental stages of citrus ovaries and for differential gene expression of mRNA gene transcripts. We have used RT-PCR to amplify the cDNA fragments. At the moment we are assessing the comparative gene transcript expression studies that are differentially expressed. Once the genes are identified they can be introduced into those citrus varieties, which do not produce apomictic seeds. This has great potential in crop improvement. In fact, this will be beneficial for other plants where apomixis is desirable. Developing genomic resources for the promotion of fundamental and applied research on Coreopsis: Coreopsis is Florida's state wildflower and 11 out of 14 species are native only to Florida. With the increase in demand and production, a number of practical questions frequently arise related to quality of commercially, mass-produced seeds. Where seeds of a local ecotype should be reproduced? Are there any population genetic structures occurring among ecotypes? What ecotype seeds should be effectively used in beautification, restoration, or mitigation projects? To address these critical questions, we are working on genetic diversity analysis among the natural production and introduced populations of Coreopsis species. We are examining the SSR primer pairs developed for various Asteraceae species for cross genome amplification in Coreopsis. We have identified 10 markers which would be helpful in determining the gene flow events and genetic differentiation of populations in Coreopsis. In addition, this genus provides an intriguing opportunity for fundamental research on patterns of endemism. This area is of interest to NSF and NRI and we are developing potential proposal(s) to secure competitive grant(s). Genetic engineering of citrus for resistance to Citrus tristeza virus (CTV): Genetic transformation is currently the only available targeted approach for genetic improvement of most citrus scion cultivar types for disease resistance. We have designed a genetic transformation strategy and introduced two candidate gene fragments (identified from Poncirus trifoliata, a close relative of citrus, having broad resistance against all strains of the CTV studied), individually, into the epicotyl segments of various elite citrus cultivars using Agrobacterium-mediated transformation with both candidate gene sequence(s) and gfp (green fluorescent protein) or GUS (â-Glucuronidase) as a marker gene. We have hardened the putative transgenic plants by PCR analysis and grafted them onto CTV infected rootstock plants in the greenhouse. At this moment, we are screening these grafted transgenic plants by ELISA, to test their resistance. We have also attempted to introduce both the candidate gene fragments together into the epicotyl segments of citrus by co-culturing them with a mixture of two different binary vectors harboring these gene fragments. We have identified putative transgenics containing both the gene fragments by PCR analysis. We will be challenging them with CTV and testing their resistance using ELISA. Controlled crossing & triploid hybrid embryo rescue to produce seedless citrus varieties: Production of triploid hybrids is the most promising approach to obtain cultivars that do not produce seeds and have great commercial potential. We carried out in vitro hybrid embryo rescue technique to rescue the immature hybrid embryos to reduce the risk of their abortion and competition with nucellar embryos. As the embryo developmental stage influences their germination capacity, we harvested the fruits at 80, 120, 160 and 200 days post pollination (dpp) to determine the influence of time of harvest on hybrid embryo rescue. The hybrid plantlets will be evaluated in the field. Citrus genome mapping and sequencing (Rao only): I am involved in other projects which are part of large national and international efforts to develop genetic maps that support citrus genomics and breeding. The goal of this project is to develop integrated physical and genetic maps of gene regions related to disease resistance and stress tolerance in the citrus genome. To address the various types of problems confronting the citrus industry, International Citrus Genomics Consortium involving the US Department of Energy Joint Genome Institute has been set up. The goal of this consortium is to sequence the whole genome of citrus. As a team member, I was involved in probing the BAC library of sweet orange to identify specific clones containing heterozygous SSR markers. The specific gene information derived through citrus genome sequencing will help us to develop new tools that make variety improvement, production and harvest management more efficient and predictable. Contributions to forest genetics at University of Agricultural Sciences, Bangalore, India (Rao only): My work at UAS, Bangalore, India has contributed significantly to the areas of population, evolutionary, conservation and forest genomics. My work focused on a) the impacts of human disturbance on the genetic diversity of medicinal plant forest species, b) identification of ‘hot-spots’ of genetic variability of economically important plant species, c) effect of fragmentation on the population genetics of forest plants, d) the role of protected areas in conserving the genetic diversity of forest plants, e) identification and characterization of bioactive compounds in forest plants. Based on outcome of these results we have proposed a new model called ‘Forest Gene Banks’ which is being considered by many researchers for effective conservation of rare, endangered and threatened plant species. My studies have important implications for the formulation of effective conservation strategies for the rapidly declining genetic resources of forest plant species and are perused by people from different backgrounds including genetic resource managers, forest officials, scientists and policy makers. Regeneration from transverse thin cell layers of mature stem segments of citrus (Soneji only): Regeneration from mature plant material of citrus species has to be achieved to overcome the long juvenile period (5-7 years) and also for the successful introduction of desired candidate genes, via transformation, into important cultivars. We have standardized a system for in vitro plant regeneration from transverse thin cell layers (1-2 mm) using mature stem segments of citrus. We either micrograft the regenerated shoots onto Carrizo citrange rootstock seedlings or subculture them onto elongation medium and subsequently graft them ex vitro onto Carrizo citrange rootstock seedlings. We are now working on standardizing the protocol for Agrobacterium-mediated gene transformation of mature stem segments. Contributions to plant research while in India (Soneji only): My research in India involved (a) In vitro strategies for the disease-free clonal propagation of citrus and caladium, (b) Biotechnological investigations for the propagation and improvement of mango, (c) In ovulo embryo culture and induction of multiple shoots in axillary bud in grape, (d) Transverse thin cell layer (t-TCL) culture in banana, (e) Tissue culture studies in pineapple & molecular characterization of its variants, (f) Tissue culture studies in neem and potato, & (g) Induction of mutations by exposing the seeds as well as etiolated seedlings to gamma rays & somatic embryogenesis in seedling explants of eggplant. As a lecturer in India, I taught Plant Physiology & Genetics courses. What motivated you both to pursue a career in citrus genetics? The strong urge to understand the nature of things we see around us has led us to obtain a diverse education. Our academic qualifications, coupled with strong training in multidisciplinary areas, have helped us to get a broader perspective in the philosophy of science. Our early years in the backdrop of a natural environment in rural India and education in urbanized surroundings drew us to inquire into the complexity and diversity of life and human influences on it. Through these experiences, we started to quickly realize how germplasm diversity and their genomes play a major role in food quality and safety, which has a great influence on human health, international trade and the global environment. These are important not only for the developing countries but also for the developed countries. Various traditional efforts have been taken to improve food quality. However, these efforts are time-consuming and the limited genetic resources of most food crops have left us little room for continued improvement by these means. Recent advances in the fields of genomics and bioinformatics offer great potential to revolutionize the fields of breeding and genetics through targeted manipulations of important traits. The desire to improve the lives of farmers and cultivators as well as mankind motivated us to pursue this field of research, which will lead to the improvement of the food crops by making them tolerant to various abiotic and biotic stresses, and also by improving their quality. We were always inclined to do this kind of research as it will go a long way in answering the many interesting questions we had in our minds and will also, hopefully, be helpful in improving the lives of the people around us. What would you like to achieve with your research in the future? Our goal is to establish a career in academia aimed at research and teaching. We hope to utilize our graduate, post-graduate, and doctoral as well as post-doctoral research and training to realize our goals and make personal contributions in the fields of food and agricultural biotechnology for the betterment of human health and the global environment. We wish to live science rather than just doing it. The following are publications by Rao and Suneji that are relevant to this Member Spotlight. Nageswara Rao M, Jaya Soneji, Chunxian Chen, Shu Huang and Fred G. Gmitter Jr., 2008, Characterization of zygotic and nucellar seedlings from citrus rootstock candidates using RAPD and EST-SSR markers, Tree Genetics and Genomes, 4:113-124. Chunxian Chen, Kim D. Bowman, Young A. Choi, Dang Phat, Nageswara Rao M, Shu Huang, Jaya Soneji, Greg McCollum and Fred G. Gmitter Jr., 2008, EST-SSR genetic maps for Citrus sinensis and Poncirus trifoliata, Tree Genetics and Genomes, 4:1-10. Fred G. Gmitter Jr., Chunxian Chen, Nageswara Rao M and Jaya Soneji, 2007, Citrus fruits, pp 265-279, Fruits and Nuts, Genome Mapping & Molecular Breeding in Plants, Volume (4), Chitta R Kole, (ed), Springer, Heidelberg. Jaya R Soneji, PS Rao and M Mhatre. 2002. Germination from synthetic seeds of pineapple (Ananas comosus L., Merr.). Plant Cell Reports, 20: 891-894 Jaya R Soneji, PS Rao and M Mhatre. 2002. Suitability of RAPD analysis of “spined” and “spineless” variant regenerants of pineapple (Ananas comosus L., Merr.). Plant Molecular Biology Reporter, 20(3):307a-307i. Jaya R Soneji, PS Rao and M Mhatre. 2002. Plantlet regeneration from leaf ex-plants of pineapple (Ananas comosus L., Merr.). Journal of Plant Biochemistry & Biotechnology, 11(2):117-119. ### << Previous Next >> [ View All Member Spotlights ] |
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