By Ken Hough, OCPA Director of Research and Market Development

egular readers of the Ontario Corn Producer will know that OCPA has been sponsoring initial research over the past two years aimed at enhancing the chilling tolerance of corn during the grain filling period (silking to maturity).

University of Guelph Research:
• The procedure developed to induce chilling and evaluate plant response provided very useful results. The procedure involved growing corn plants hydroponically, and moving these plants into a cold room (either 4o or 10oC) for chilling treatment for either one night or three consecutive nights. Appropriate unchilled ‘check’ treatments were undertaken. Plant growth and development parameters were monitored, and showed that chilling treatments did reduce subsequent photosynthetic activity (capacity) and dry matter accumulation. Thus the procedures developed will form the basis of a method to assess the chilling tolerance response of a broader range of corn lines in future studies.
• The chilling treatments on representative corn hybrids (one ‘old-type’ representative of hybrids grown about 30 years ago, two ‘modern-type’ hybrids) demonstrated that corn hybrids differ in their response to chilling temperatures during the grain filling period (Table 1). This sets the stage for evaluating a broader selection of corn hybrids for response to chilling treatments, and assessing the plant physiological mechanisms responsible for the response and enhanced tolerance levels.
• Further, these hybrids did not show differences in their response to low temperature during the seedling stage (Figure 1). This means that screening inbreds or hybrids for late season chilling tolerance must be an independent procedure from screening for early-season tolerance to cold.

ECORC Research:
• Laboratory-based chilling studies were undertaken to identify a repeatable procedure for determining differences among corn lines (genotypes) for chilling tolerance and photo-inhibition of photosynthesis (disruption of photosynthesis due to high light intensities, which often occur under clear atmospheric conditions subsequent to cold late summer/autumn nights). A set of treatment conditions were established which were adequate to screen germplasm for chilling-stress and photo-inhibition differences.
• Studies verified that certain ECORC corn breeding lines are more susceptible to chilling than others, confirming genetic diversity and identifying lines with some tolerance or sensitivity to chilling. Future studies will expand the number of genotypes being assessed, in order to develop an understanding of the tolerance mechanisms.
• These studies indicated that photo-inhibition of corn and vigour of seedlings exposed to low-temperature stress are independent traits, which need to be screened for independently. Further research to confirm this finding will be undertaken.
• Initial steps in genetic manipulation provided some transgenic lines incorporating candidate ‘cold-tolerance genes’ (OXO, GPAT). Compared to normal corn lines, OXO transgenics recovered faster from chill-induced damage to photosynthesis, and GPAT transgenics exhibited significantly reduced chill-induced photo-inhibition. GPAT-enhanced chilling tolerance was similar at both pre-silking and grain filling growth stages.

Future Objectives
We are now trying to mount a comprehensive research program for the next four years to develop enhanced cold tolerance during corn grain filling. Funding is now being pursued for research proposals from the UoG and ECORC that focus on separate but complementary aspects of research towards achieving the overall goal. The primary objectives for the proposed research are:
University of Guelph:
• Combine basic plant physiology and gene identification technology to identify a battery of genes in corn that permit photosynthesis to proceed at or near optimum levels in the presence of cool temperatures
• Sequence the coding of genes, and create ‘libraries’ of these genes, enhanced for grain filling cold tolerance
• Develop a mapping population, and map QTLs (quantitative trait loci, i.e., segments of DNA that exhibit a measurable effect) for genes involved in grain filling chilling tolerance
• Enhance the understanding of mechanisms underlying grain filling chilling tolerance.

ECORC:
• Develop a large-scale, rapid, photo-inhibition-based screening methodology, by building on existing ‘small-scale’ protocols
• Evaluate chilling tolerance of a broader spectrum of germplasm and across a wider range of chilling stresses, including in-field ‘chilling’ conditions
• Create and evaluate additional ‘chilling tolerant’ transgenics (using the ECORC corn transformation system), building on key photosynthetic enzymes already identified, along with the GPAT and OXO transgenics already developed
• Develop novel stress-tolerant germplasm, and identify unique pathways of gene control.

The total funding required for the proposed research is about $260,000 per year, for each of the next four years. Requests for funding have been sent to several seed companies that have participated in or shown interest in the earlier cold tolerance research, with the hope that we can secure about $130,000 annually from industry and OCPA combined. Our plan is to match the industry/OCPA funds with either NSERC (for the UoG research) or AAFC-MII (for ECORC) grant funding. (Earlier this year, we were unsuccessful in obtaining matching grant funds from the CanAdapt program).

In the past, OCPA and companies have found excellent value in similar partnership research projects, such as the ‘Fusarium Ear Mould Resistance Through Genetic Engineering’ project, which also involves UoG and ECORC researchers. We hope the same level of success will be achieved with this endeavour.

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