**3. Results and discussion**

As previously indicated (**Tables 1** and **2**), climatic conditions in our region suggest the need for research, which has the potential to enhance selection of genotypes more tolerant to drought.

#### **3.1. Experiment under semi-controlled conditions in greenhouse**

### *3.1.1. Sugar beet genotype classification based on physiological tests in semi-controlled conditions*

Sugar beet genotypes in semi-controlled conditions showed different reactions to 5-day water deficiency. As expected, decline in turgor was observed in all genotypes. Number of leaves was significantly different between treatments and respective controls. Concentrations of photosynthetic pigments and leaf area varied between genotypes and standard normal distribution was not observed here. Therefore, the data were subjected to Johnson's data transformation which proved to be very effective [55]. This procedure allowed assessing differences in concentrations of photosynthetic pigments between different genotypes (**Figure 1**).

Secession in water supply caused water loss from plant tissues within both sensitive and tolerant genotypes. Due to this fact, sugar beet genotypes may be divided on the basis of tested parameters and following treatments (**Figure 2**).

The results obtained in semi-controlled conditions (experiment 1) were compared to previous field observations (**Figure 3**). Proline concentration increased in all genotypes after exposure to water deficit as well as % of DM (except for genotypes 9 and 11). Changes within treatments with respect to control, referring to dry weight were less pronounced than changes referring to % of DM and RWC of root, stem, and leaf. Plants subjected to stress conditions had in average three leaves less, 4% higher % of DM, and seven times higher proline content.

The relationships between two effects on measured traits were assessed by mixed model (**Figure 4**). Crossed pink lines in diagrams represent average impact on genotypes in control

**Figure 1.** Genotype separation on the basis of pigment concentration and leaf area for variables normalized according to Johnson's data transformation (jlarea—leaf area; jcar—carotenoids; jca—chlorophyll a; jcb—chlorophyll b; jcab chlorophyll a + b).

Effects of drought were observed in case of F<sup>v</sup>

**3.2. Experiment in tissue culture (***in vitro***)**

obtained in the field.

(**Figure 6**).

tolerance towards water deficit in field conditions (**Figure 5**).

and Fm, but not for Fv

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largest differences between genotypes were obtained. In addition, overlap of intervals of interaction between stress and genotype indicates stress, which caused differences, similar for all genotypes. The influence of water deficiency on fluorescence may be related to plant

**Figure 2.** The separation of the sugar beet genotypes based on experiments in greenhouse with a highlighting treatment (control, drought) for a variable normalized by Johnson's transformation (jrwc—relative water content; jpcdw—dry weight; jdwproli—free proline; jpcdwleaf—leaf dry matter; jpcdwstem—stem dry matter; jpcdwroot—root dry matter).

Plant development may be inhibited in different ways in field conditions. It may be affected by interactions among drought and other ecological stresses, precipitation, and temperature availability as well as interactions with different micro-organisms [36]. On the contrary, semicontrolled conditions may only eliminate interference of other factors with plant development. Therefore, it is necessary to compare results obtained in the greenhouse with those

Increased PEG concentration decreased growth of axillary buds with respect to control

/Fm ratio, where the

(x axis) and stress effect (y axis). There is a nearly perfect negative correlation between the unstressed value and the response to stress for root DM and a similar, but weaker one, for leaf number. Genotypes showing positive scores for the slope effect (y axis) are less affected than the average by (more tolerant to) water stress for the involved trait, and vice-versa. Genotypes showing positive scores for both effects are both higher scoring in absence of stress and less affected than the average by (more tolerant to) stress for the involved trait, and vice-versa.

Conventionally, results of chlorophyll fluorescence indicate a high sensitivity to influence of ecological factors. Therefore, it is often used as an indicator of functioning of photosystem II.

According to Fv /Fm, sugar beet genotypes were compared on the basis of photosynthetic characteristics. Water deficit did not cause significant variations in fluorescence indicators (**Figure 5**).

**Figure 2.** The separation of the sugar beet genotypes based on experiments in greenhouse with a highlighting treatment (control, drought) for a variable normalized by Johnson's transformation (jrwc—relative water content; jpcdw—dry weight; jdwproli—free proline; jpcdwleaf—leaf dry matter; jpcdwstem—stem dry matter; jpcdwroot—root dry matter).

Effects of drought were observed in case of F<sup>v</sup> and Fm, but not for Fv /Fm ratio, where the largest differences between genotypes were obtained. In addition, overlap of intervals of interaction between stress and genotype indicates stress, which caused differences, similar for all genotypes. The influence of water deficiency on fluorescence may be related to plant tolerance towards water deficit in field conditions (**Figure 5**).

Plant development may be inhibited in different ways in field conditions. It may be affected by interactions among drought and other ecological stresses, precipitation, and temperature availability as well as interactions with different micro-organisms [36]. On the contrary, semicontrolled conditions may only eliminate interference of other factors with plant development. Therefore, it is necessary to compare results obtained in the greenhouse with those obtained in the field.

#### **3.2. Experiment in tissue culture (***in vitro***)**

(x axis) and stress effect (y axis). There is a nearly perfect negative correlation between the unstressed value and the response to stress for root DM and a similar, but weaker one, for leaf number. Genotypes showing positive scores for the slope effect (y axis) are less affected than the average by (more tolerant to) water stress for the involved trait, and vice-versa. Genotypes showing positive scores for both effects are both higher scoring in absence of stress and less affected than the average by (more tolerant to) stress for the involved trait,

**Figure 1.** Genotype separation on the basis of pigment concentration and leaf area for variables normalized according to Johnson's data transformation (jlarea—leaf area; jcar—carotenoids; jca—chlorophyll a; jcb—chlorophyll b; jcab—

Conventionally, results of chlorophyll fluorescence indicate a high sensitivity to influence of ecological factors. Therefore, it is often used as an indicator of functioning of photosys-

characteristics. Water deficit did not cause significant variations in fluorescence indicators

/Fm, sugar beet genotypes were compared on the basis of photosynthetic

and vice-versa.

chlorophyll a + b).

78 Plant, Abiotic Stress and Responses to Climate Change

According to Fv

tem II.

(**Figure 5**).

Increased PEG concentration decreased growth of axillary buds with respect to control (**Figure 6**).

**Figure 3.** Effects of drought stress on growth traits and proline production of greenhouse grown plants of sugar beet genotypes (1–11) from three classes of visually field-assessed drought tolerance (DT). Observed values of three replicates (circles, 10 plants each), average genotype positions (numbered gray lines), and class means with 95% confidence intervals (crossbars). The drought stress was induced by suspension of watering to test plots after 3 months of culture and observations were made after 5 more days [28].

variability in dry weight was recorded in the group of drought sensitive group (according to field observations), but the same trend as in the other two groups of genotypes remained. Tissue water content linearly decreased following the increase in PEG concentration, the average drop in presence of 5% PEG was 6%, and was followed by the low average difference among groups of different tolerance and higher difference among genotypes of one group

**Figure 4.** Genotype effects from mixed model analyses for traits of greenhouse experiment (deviations for values in unstressed condition on the x axis, deviations for stress effects on the y axis. Genotypes are identified by the

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Proline accumulation under stress conditions increased under treatments in both experiments. In tissue culture, it was 6 times increased and in greenhouse 16 times with respect to

If taking into account the genotypes tolerance in the field, in relation to the parameters obtained from the analysis of plants in tissue culture and in experiment in the greenhouse, dry matter, in relation to the water content and the concentration of proline is not significantly

Recorded differences between genotypes show that there are two approaches for the separation of sugar beet genotypes in relation to response to water stress, which cannot substitute each other. On one hand, proline content in plants grown in tissue culture enabled to match their grouping with respect to observations in the field. On the other hand, experiment in greenhouse was less efficient in that sense (**Figure 3**). The main cause of this may be the fact

(**Figure 6**).

numbers).

corresponding controls.

different among groups of the tolerance (**Figure 7**).

that stress in the field was not continuous as it was in the greenhouse.

Number of axillary shoots may be indicator of the influence of different PEG concentrations, which cause water deficit, on micropropagation potential of genotypes. Average number of axillary shoots of 11 subjected genotypes showed 2.2 times decreased number of shoots in the presence of 3% PEG and 2.7 times in the presence of 5% of PEG.

The degree of tolerance to drought observed in the field corresponded to tolerance recorded in the experiments performed in the greenhouse and in tissue culture (**Figure 6**). The most prominent criterion for estimation of genotype tolerance to drought was found to be concentration of free proline [28]. Proline concentration was significantly increased in leaves exposed to drought and axillary buds and it was positively correlated with PEG concentration, which is in accordance with the results of other researches [56].

PEG treatment decreased total dry weight and number of axillary shoots by more than twice, while presence of 3% PEG in the substrate increased total fresh weight. Furthermore, PEG caused decrease in water content in tissues and decreased number of buds, but increased bud weight and % of DM. The highest values were recorded in control (0% PEG) for total fresh weight, in the presence of 3% PEG for proline concentration and fresh weight of axillary buds and in presence of 5% PEG for % of DM. Fresh weight of plants grown in presence of 3 and 5% PEG decreased (**Figure 6**). Average dry weight of the plants was the highest in the presence of 3% PEG. However, in the presence of 5% PEG, it was almost in line with the control. Higher

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**Figure 4.** Genotype effects from mixed model analyses for traits of greenhouse experiment (deviations for values in unstressed condition on the x axis, deviations for stress effects on the y axis. Genotypes are identified by the numbers).

variability in dry weight was recorded in the group of drought sensitive group (according to field observations), but the same trend as in the other two groups of genotypes remained. Tissue water content linearly decreased following the increase in PEG concentration, the average drop in presence of 5% PEG was 6%, and was followed by the low average difference among groups of different tolerance and higher difference among genotypes of one group (**Figure 6**).

Number of axillary shoots may be indicator of the influence of different PEG concentrations, which cause water deficit, on micropropagation potential of genotypes. Average number of axillary shoots of 11 subjected genotypes showed 2.2 times decreased number of shoots in the

**Figure 3.** Effects of drought stress on growth traits and proline production of greenhouse grown plants of sugar beet genotypes (1–11) from three classes of visually field-assessed drought tolerance (DT). Observed values of three replicates (circles, 10 plants each), average genotype positions (numbered gray lines), and class means with 95% confidence intervals (crossbars). The drought stress was induced by suspension of watering to test plots after 3 months of culture

The degree of tolerance to drought observed in the field corresponded to tolerance recorded in the experiments performed in the greenhouse and in tissue culture (**Figure 6**). The most prominent criterion for estimation of genotype tolerance to drought was found to be concentration of free proline [28]. Proline concentration was significantly increased in leaves exposed to drought and axillary buds and it was positively correlated with PEG concentration, which

PEG treatment decreased total dry weight and number of axillary shoots by more than twice, while presence of 3% PEG in the substrate increased total fresh weight. Furthermore, PEG caused decrease in water content in tissues and decreased number of buds, but increased bud weight and % of DM. The highest values were recorded in control (0% PEG) for total fresh weight, in the presence of 3% PEG for proline concentration and fresh weight of axillary buds and in presence of 5% PEG for % of DM. Fresh weight of plants grown in presence of 3 and 5% PEG decreased (**Figure 6**). Average dry weight of the plants was the highest in the presence of 3% PEG. However, in the presence of 5% PEG, it was almost in line with the control. Higher

presence of 3% PEG and 2.7 times in the presence of 5% of PEG.

and observations were made after 5 more days [28].

80 Plant, Abiotic Stress and Responses to Climate Change

is in accordance with the results of other researches [56].

Proline accumulation under stress conditions increased under treatments in both experiments. In tissue culture, it was 6 times increased and in greenhouse 16 times with respect to corresponding controls.

If taking into account the genotypes tolerance in the field, in relation to the parameters obtained from the analysis of plants in tissue culture and in experiment in the greenhouse, dry matter, in relation to the water content and the concentration of proline is not significantly different among groups of the tolerance (**Figure 7**).

Recorded differences between genotypes show that there are two approaches for the separation of sugar beet genotypes in relation to response to water stress, which cannot substitute each other. On one hand, proline content in plants grown in tissue culture enabled to match their grouping with respect to observations in the field. On the other hand, experiment in greenhouse was less efficient in that sense (**Figure 3**). The main cause of this may be the fact that stress in the field was not continuous as it was in the greenhouse.

**Figure 5.** Maximal (Fm) and variable (Fv ) chlorophyll fluorescence and their ratio (F<sup>v</sup> /Fm) in sugar beet genotypes grouped according to their field-assessed drought tolerance (ctrl—control; drought; DT—drought tolerance).

**3.3. Analyses of changes in expression of genes involved in reactions to water stress** 

Changes in the expression of 13 candidate genes in 11 different sugar beet genotypes were followed in leaves of plants grown in the greenhouse. Expression pattern corresponding to BI543243 differed in plants exposed to drought in comparison with corresponding controls in genotypes 1, 10, and 11 (**Figure 8**). Therefore, it may serve to develop molecular marker useful

**Figure 8.** Expression pattern of gene corresponding to BI543243 in sugar beet leaves (c—template cDNA deriving from control plants; d—template cDNA deriving from plants exposed to drought). Amplification on genomic DNA served as

**Figure 7.** Water deficit effect on dry weight, water content, and free proline concentration in greenhouse and in tissue

│c d g │ c d g │ c d g │ c d g │c d g│M│ **7 8 9 10 11** 

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˗˗˗ 1000 bp ˗˗˗ 500 bp

Tolerance to drought is very complex. Experiments in three different environments (tissue culture, greenhouse, and field) with 11 genotypes, where many different parameters were followed, revealed that it is not easy to find single criteria for classification with respect to drought tolerance. However, the results suggest that free proline accumulation may be used as a reliable parameter. The classification based on changes in concentration of free proline in plants exposed to drought in greenhouse and tissue culture corresponded to classification made on the bases on field observations. Therefore, similar fast tests, conducted with young plants and possibly aided by the use of molecular markers, can be useful for estimation of breeding material with respect to tolerance to water deficiency, which will significantly enhance sugar beet breeding for expected future changes in climate.

**(plants from greenhouse experiment)**

additional control (g). M—100 bp DNA ladder size marker.

│c d g │c d g│ c d g │c d g │c d g│c d g│M **1 2 3 4 5 6** 

**4. Conclusion**

culture experiment [28].

to differentiate genotypes with respect to drought.

**Figure 6.** PEG effect on growth traits and free proline concentration of plants cultivated in tissue culture [28].

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**Figure 7.** Water deficit effect on dry weight, water content, and free proline concentration in greenhouse and in tissue culture experiment [28].

**Figure 8.** Expression pattern of gene corresponding to BI543243 in sugar beet leaves (c—template cDNA deriving from control plants; d—template cDNA deriving from plants exposed to drought). Amplification on genomic DNA served as additional control (g). M—100 bp DNA ladder size marker.

#### **3.3. Analyses of changes in expression of genes involved in reactions to water stress (plants from greenhouse experiment)**

Changes in the expression of 13 candidate genes in 11 different sugar beet genotypes were followed in leaves of plants grown in the greenhouse. Expression pattern corresponding to BI543243 differed in plants exposed to drought in comparison with corresponding controls in genotypes 1, 10, and 11 (**Figure 8**). Therefore, it may serve to develop molecular marker useful to differentiate genotypes with respect to drought.
