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Monica David, Scientific Coordonator: Academician Nicolae N. Saulescu . Ways of genetic progress in improving winter wheat performance under drought. 2012.


This research was focused on the study of drought resistance of wheat plants. Research was aimed on supporting wheat breeding by studying the variability of physiological characters involved in plant response to stress conditions, improving methods and selection criteria for better efficiency in identifying genetic differences, in order to improve drought resistance. The
thesis contains 198 pages and is divided into nine chapters.
The general part (chapters I and II) is extended in two chapters and summarizes the main bibliographic database of scientific literature for the importance of wheat and abiotic stress
factors, concepts and mechanisms of drought resistance, improving drought resistance and physiological indicators used in the study of genetic variability of wheat. The second chapter
summarizes data of material and testing methods used in this study. As material I used 44 varieties of a large collection of wheat, from Romania and other countries, selected to represent
several ecotypes adapted to regions that are contrasting for water availability.

Research part (Chapter III-IX) includes six chapters, which include the results of systematic studies, performed on:
- genetic variability of cuticular transpiration indicators;
- genetic variability of osmotic regulation indicators;
- genetic variability of stem reserves remobilization indicators;
- stomatal frequency in flag leaves;
- genetic variability in seedling growth under induced water stress;
- possibilities of combining traits that could contribute to better performance under drought in wheat.
The final chapter presents the general conclusions. The research part is illustrated with 35 original figures and 30 tables. All researches are based on the study of. 147 bibliographic works.


Water supply is one of the environmental factors which influence most wheat production in Romania's conditions. Use of varieties with wide adaptability for contrasting environmental conditions and water supply can reduce the risks of decreased yields in unfavorable years.
If many physiological traits are involved in the determination of a complex character, as is the case of drought resistance, deliberate selection in order to combine the synergic traits could be more efficient and lead to obtaining results in a shorter period than if a more empirical
strategy is used. Varieties performance under drought depends on many traits and none of them can explain alone the observed differences among genotypes. But each of them can improve performance under drought and for this, it is necessary to know the available variability for each of these traits. The first condition for a successful introduction of physiological traits in a breeding program is to determine the degree of genetic variability for the trait (traits) of interest.
Previous research has suggested that several mechanisms can be used for improving drought resistance. These include: osmotic adjustment, assimilate remobilization, cuticular transpiration
and stomata frequency, which were approached in our research.


First step in improving physiological characters is to determine the degree of genetic variability for the character (characters) of interest. Therefore the objectives of this study were:
- to investigate the genetic variability of a set of widely diverse wheat cultivars for each of several physiological indicators involved in drought resistance, in order to identify potential parents for breeding programs in Romania, directed to improving wheat performance under drought.
- to identify varieties that could cumulate one or more of the studied traits and detect breeding opportunities for combining favorable traits.
To achieve these objectives, whenever necessary, we attempted to improve the
methodology for studying the respective traits.


Chapter III: The study of genetic variability of cuticular transpiration indicators in the studied wheat cultivars

Low rate of water loss (RWL) from excised leaves was proposed as a screening method of wheat cultivars (Triticum sp.) with better drought resistance (Clarke, 1992). In Romania, low rate of water loss (RWL) was investigated by Maria Balota in 1995 and Petcu Elena in 2005. Balota et al. (1995) found large variation among Romanian wheat cultivars for cuticular transpiration, the cultivars with good known performance under drought, having low or medium initial water content (IWC) and low water loss. I found that the dynamics of water loss was different both between years and cultivars. The data showed that differences between cultivars for initial water contents from excised leaves were strongly influenced by environmental conditions. Initial water content from excised leaves was not a determining character for performance under water stress.
There were significant differences between the cultivars tested for water loss in the first 5 hours, in the 3 years of study, but these differences do not appear to be associated with known differences in drought resistance. In the future, it is recommended to perform the readings for the parameters which characterize the dehydration rate of excised leaves within the first 5 hours.
This means a shorter period of experimentation and therefore represents an improvement to the
method of determination.

Chapter IV. The study of genetic variability of osmotic regulation indicators in the studied wheat cultivars

The research presented in this chapter was based on the concept developed by Patil and Ravikumar (2011) in their paper on osmotic adjustment in sorghum, to assess the effect of the two mechanisms of osmotic adjustment (soluble organic synthesis and accumulation of cations in the cytoplasm), using the test based on the expression of osmotic adjustment in pollen grain, proposed by Morgan in 1999. I used three indicators, based on estimating the intrinsic osmotic
adjustment (in the absence of cations intake), the induced osmotic adjustment (in the presence of potassium cation) and total osmotic adjustment (which includes both the intrinsic and induced
Immersing pollen grains in 55% or 65% PEG solutions, without adding KCl, induced modifications of both pollen grains shape and projected area, with large differences between varieties. The addition of KCl to the PEG solutions, in which pollen grains were immersed,induced modifications of pollen grains shape, either by returning to their original form, or
increasing or decreasing pollen grains projected area.
Separating the effects of osmotic adjustment mechanisms, expressed in pollen grains, revealed significant genotypic differences, both in the intrinsic and induced by adding potassium chloride, osmoregulation. As the intrinsic osmotic adjustment and the adjustment induced by absorption of inorganic osmolytes were not significantly correlated, they could be distinct and independent mechanisms. Therefore, combining high levels of both mechanisms by breeding might allow genetic progress in drought resistance. The total (overall) osmotic adjustment was determined almost equally by intrinsic (R = 0.68) and induced osmoregulation (R = 0.60).
Molecular analyses performed in the Molecular Genetics Laboratory of NARDI Fundulea, in 26 genotypes studied for osmotic adjustment capacity, revealed six types of polymorphism, one
corresponding to the variety Izvor, and other 5 more or less different. Although the limits of variation for osmotic adjustment capacity indicators largely overlapped, there was a tendency for cultivars from group 1 of polymorphism (similar to cultivar Izvor) to be superior to other groups
in intrinsic osmotic adjustment, while the differences between groups for induced osmotic adjustment were small. We concluded that the molecular marker Xwmc 603 can provide information mainly for intrinsic osmotic adjustment capacity, and less for the induced one.
Further research is needed to confirm this conclusion on a material more suitable for molecular genetic studies.

Chapter V. The study of genetic variability of stem reserves remobilization indicators in
the studied wheat cultivars

The experiments were conducted in 2009-2011, but the experience made in 2009 was affected by rainfall that fell during that period, and the results could not be taken into consideration. In our research, the amount of assimilates translocated from stem to grain was evaluated by the method of chemical desiccation. I identified cultivars in which the percentage of
assimilate translocated from the stem to the grain was high in both years, and cultivars in which the translocation was small, both in 2010 and 2011. The varieties, which translocated a larger
amount of reserves from stem to the grain, included both varieties known for their drought resistance and varieties which have not been characterized in this regard. Variety Izvor translocated a high amount of reserves, both in 2010 and 2011. In contrast, varieties Apache and
Murga, translocated relatively low amounts of assimilates in both years. Higher plant height was not associated with higher translocation of reserves from the stem to the grain. The highest
values of stem reserves contribution to grain filling was obtained in semi dwarf varieties with medium plant height. Estimation of translocation capacity of reserves from the stem to the grain,by applying leaf chemical desiccation, proved to be strongly influenced by the type of treatment
and environmental conditions. Therefore, it is necessary to repeat measurements many years, applying preferably the potassium iodide treatment. However, the obtained preliminary data allowed highlighting several genotypes that can be used in breeding programs for improving this

Chapter VI. Genetic variability of stomatal frequency in flag leaves in the studied wheat

Exploring the possibility of improving the characteristics of stomata, I determined the genetic variability of cultivars for stomata frequency in flag leaves, which can be considered one of
anatomical adaptation indicators that can help conserve moisture. Microscopic observation of the molds taken from the flag leaf showed significant differences among cultivars for stomata
frequency. Quantitative determination allowed a precise classification of cultivars in terms of stomata frequency on abaxial and adaxial epidermis. For the average of total stomata number on
flag leaf, the classification of the varieties was similar with the classification for the frequency of
stomata on adaxial epidermis. Based on research by Koy et al. (1972), it can be assumed that using as parents in breeding programs varieties characterized by a low frequency of stomata (such as TX86A8072, Milenka etc.), could help obtaining forms with lower rate of transpiration,
which can have improved performance under drought.

Chapter VII. The study of genetic variability in seedling growth under induced water stress

Based on research on six different F7 lines derived from the same hybrid combination in terms of osmotic adjustment capacity, Morgan (1988) suggested that coleoptile and roots length response to water stress can be used to identify differences for osmoregulation among wheat genotypes. Starting from this suggestion, we investigated differences root and shoot growth of seedlings exposed to water stress by gradual drying in a set of 33 genotypes of winter wheat, as
well as the correlations between these differences and osmotic adjustment as expressed in pollen grains. The relative growth of roots in seedlings exposed to water stress by gradual drying strongly differentiated the tested genotypes. Varieties with known lower performance under drought conditions had the lowest ratio between relative root and shoot growth.
Despite showing only a weak correlation with the total and intrinsic osmotic adjustment, the ratio between relative roots and shoots growth in seedlings exposed to water stress by gradual
drying, can provide preliminary information about the genotypic differences in osmotic adjustment and could be used for large scale screening in breeding wheat for drought resistance.

Chapter VIII. Possibilities of combining traits that could contribute to better performance under drought in wheat

The most promising way for improving the performance of wheat under water stress is the accumulation of as many characters that can positively affect plant performance under these
conditions (Richards et al., 2001). Thus, I examined to what extent the traits that I have studied
can be combined. In most cases, the studied traits were not significantly correlated. Intrinsic osmotic adjustment capacity was significantly correlated only with general osmotic adjustment capacity, in the composition of which it is included. Induced osmotic adjustment capacity was
also significantly correlated with total osmotic adjustment capacity.
In addition there was a significant correlation between osmotic adjustment capacity and cuticular transpiration. This correlation, which is probably not functional, can be advantageous in
view of combining the two traits.
Total (overall) osmotic adjustment capacity was significantly and negatively correlated with
frequency of stomata. As both smaller number of stomata and higher values of osmotic adjustment positively influence the performance under drought, this correlation is also advantageous for combining these two characters.
The capacity of assimilates translocation from stem to the grain did not correlate significantly
with any of the other characters studied. Also, correlations among the other characters were not statistically significant.


In this PhD thesis I obtained the following original results:
- Identification of a wide variability among the studied cultivars for four important traits that may influence the performance of wheat under drought, namely osmotic adjustment, remobilization of stem reserves, cuticular transpiration and stomata frequency. Best cultivars for
each of the examined physiological indicators can be used as parents in the breeding programs directed towards improving drought resistance, which could contribute to reducing the impact of climate change on wheat production;
- Identifying ways of genetic progress in improving winter wheat performance under drought by combining the favorable traits that can contribute to better performance of wheat under stress. Results demonstrated that there are possibilities for improving even the
performance of the cultivars that are recognized as drought-resistant, since none of them cumulated the highest levels for all physiological characteristics with favorable influence on resistance under drought.

Comparing the ranking of varieties for different traits involved in their
reaction to water stress suggested the ways to achieve further genetic progress in this direction.
This approach can be used to establish a strategy for improving drought resistance by combining in the same genotype as many traits that can contribute to a better performance under drought conditions;
- During the performed analyses we identified several ways of improving the methods,used for characterizing some traits involved in drought resistance. Thus:
- We demonstrated the possibility of shortening the period of experimentation to determine the rate of water loss from the cuticle of the leaf flag. This allows increasing the productivity of this method.
- We introduced, for the first time in wheat, the quantification, of changes in the cytoplasm areas of pollen grains for 3 types of osmotic adjustment (intrinsic, induced and total), after the model suggested for sorghum in 2011. The analyses were performed using a high performance IT program, which proved to be quick, efficient and precise in determining the areas of cytoplasm of pollen grain under the microscope. This improved methodology will allow a significant increase of precision in estimating the osmotic adjustment expressed in the pollen grains, opening new perspectives for breeding programs.
- The chemical desiccation method was also improved, for better characterization of cultivars regarding the indicator of assimilate translocation.
- Starting from Morgan's suggestion in 1988, we adapted the method for measuring the relative growth of seedlings exposed to water stress by gradual drying, in order to obtain information about genetic differences in osmoregulation, earlier in the breeding process.

Keywords: drought, breeding, wheat, pollen grain


Posted by Monica David


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