In Vitro Stress Selection And Phenotypic Characterization Of Drought Resistant Somatic Hybrids And Derivate Between Cultivated Potato And Solanum Chacoense

In vitro stress-selection and phenotypic characterization of drought resistant somatic hybrids and derivate between cultivated potato and Solanum chacoense

Imola Molnár1, Raluca-Alina, Mustata, Tunde Denes1, István Vas2, András Cseri2, Imre Vass2, Elena Rakosy-Tican1

1 Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, “Babes-Bolyai” University, Romania

2 Biological Research centre, HAS-RSDS Szeged, Hungary

Corresponding author: Elena Rákosy-Tican, Cluj-Napoca, Romania, Clinicilor str. 5-7 nr, RO-400006, e-mail: [anonimizat], Telephone: 0040-264-431878, Fax: 0040-264-591906

Abstract

Water is essential for plant survival and development. Drought stress have the most adverse factor of plant growth and productivity. Cultivated potato is considered as drought sensitive species, and therefore moderate water shortages can lead to significant yield losses. Due to the increased frequency of drought periods, development of water-stress resistant potato are indispensable to reduce the devastating agronomical and social effect of drought. Modern biotechnological tools, like somatic hybridization made possible to transfer multigenic properties from wild Solanum species into cultivated potato. The aims of our study were to assess drought tolerance ability of somatic hybrids and backcross progenies between potato and Solanum chacoense, respectively S. chacoense with MMR deficiency using in vitro stress-selection with PEG. Also, plant response to drought stress was evaluated by phenotypic characterization and by photosynthesis efficiency determination of drought stressed plants. The complex metabolomic and phenotypic evaluation of plants made possible a more confident selection of drought resistant plants. Based on our results we concluded that somatic hybridization procedure contributed to produce from sensible parent’s protoplasts drought tolerant hybrid plants. Also we observed that deficiency in DNA repearing system in one of the parents species, increase the possibility to develop drought resistance ability in their derivates.

Introduction

Fresh water shortage became an increasing worldwide problem, which is the result of climate changes, increased pollution and increased human pretension and overuse of water. The fresh water scarcity doesn’t affect only the accessibility of drinking water but lead to food shortage also. Water deficit reduce the growth and threaten crop productivity.

As a consequence of drought stress the water potential and turgor of plants will be reduced, which leads to carrying out with difficulties the normal physiological functions. Plants suffer from water deficit in the case when the rate of transpirations higher than water uptake (Bray 1997). When plants roots recognize the soil dryness, the level of abscisic acid (ABA) in plant increases which led to stomatal closure. Therefore the first physiological response to drought stress is the limitation of gas exchange, which leads to transpirational reduction (Hu and Schmidhalter, 2005).

As a result of the declined stomatal aperture the CO2 assimilation of plants are reduced. By the inhibition of photosynthetic activity the generation of reactive oxygen species (ROS: O2-, 1O2, H2O2) increases, which attack the valuable macromolecules within the cell. In plants several mechanism exist to detoxify the produced active oxygen species (Reddy et al 2004). During drought stress the accumulation of solutes in the extracellular matrix and cytosol increases, which contain ions such as K+, Na+ and Cl- or organic compounds like different amino acids (proline), polyamines and glycine betaine to prevent water loss of cells and to maintain the turgor of leaves (Tamura et al 2003).

In the case when water deficit persists for a long time, cells lose their turgor, the cell volume will be reduced, cell-division enlargement and differentiation will be limited which reduces the plant growth (Donnelly et al, 2003).

As a negative effect of drought stress, total leaf area decreases, which leads to reduced crop yield through reduction in CO2 assimilation (Rucker et al 1995).

Cultivated potato uses water relatively efficiently, but it is considered to be sensible to moderate levels of water deficit, which causes yield losses. Its sensibility is attributed to superficial location of roots (Dalla Costa et al, 1997). Jefferies et al (1993) demonstrated that during drought stress potato leaf length has linear relation with leaf water potential. Leaves which expand during water-deficit period grow smaller and have lower specific leaf area than control ones. Moreover linear correlation between CO2 assimilation and potato plant height and tubers production were observed (Shalhevet et al 1986, Feddes 1987). A water shortage after stolon and tuber initiation greatly reduces the produced tuber size and yield (Vos and Haverkort, 2007).

Selection procedures of drought tolerant plants

Increasing in drought periods which affect the most agriculturally important areas, stimulate breeders to select drought tolerant cultivars to avoid yield losses. As selection agent in the case of in vitro selection experiments of drought-tolerant plants Polyethylene glycol (PEG) is frequently used (Hassanpanah 2010, Pino et al 2013. PEG proved to be effective to select drought tolerant potato cultivars, like Agria, Kennebec, Sante (Pino et al 2013) and also to select resistant transgenic potato varieties (Mustata et al, under publication).

Phenotypic characterization of drought stressed plants helps to determine the morphology and physiology effects of the induced stress. Phenotyping platforms made possible to monitor the development of plants during water deficit, by determination of in vivo biomass accumulation of plants, without physiological damaging (Feher-Juhasz et al 2013).

Drought stress effect on plants photosynthesis

As an effect of drought stress, the photosynthesis rate and with this the CO2 accumulation decrease in plants (Kaiser 1987, Chaves et al 2009). This plants response can be attributed to stomatal closure (Cornic 2000).

Inhibition of photosystem II (PSII) activity during water deficit, disrupts the balance between generation and utilization of electrons, which leads to reactive oxygen species production. ROS damage the most sensitive biological macromolecules, which can cause the cell death. But before PSII inhibition occur, which trigger the cell damaging cascade, several neutralizing mechanisms try to protect the reaction center of photosynthesis by elimination of the excess energy through cyclic electron transport (Chaves et al 2009).

These mechanisms decrease the efficiency of photosynthesis (Jefferies 1994), therefore evaluation of photosynthesis of plants could provide information about the early response of plants to drought stress.

The goals of our research were to assess drought tolerance ability of somatic hybrids and backcross progenies between potato and Solanum chacoense, respectively S. chacoense with MMR deficiency using in vitro stress-selection with PEG. Also, plant response to drought stress was evaluated by phenotypic characterization and by photosynthesis efficiency determination of drought stressed plants.

Materials and methods

Plant material

Somatic hybrids were produced by using protoplast electrofusion technique. Thieme et al (unpublished data) used mesophyll cells of S. tuberosum cv. Delikat and S. chacoense 1G from Gross Lüsewitz Genebank (ex. SH 1552/1). BC1 plants were obtained after sexual backcrossing of SH 1552/1 with S. tuberosum cv. Sonate (ex. 1552/1/2). The second generation of backcrosses were produced between BC1 1552/1/7 and S. tuberosum cv Romanze (ex. 1552/1/7/1). Rakosy et al (2004, 2015) used as parents in somatic hybridization Delikat (Dk) and Desiree (De) cultivars and the highest leptine producer S. chacoense accession (PI 458310) from NPGS Sturgeon Bay, USA. The obtained SH were noted as De.C#. MMR deficient S. chacoense production was performed using Agrobacterium-mediated transformation. The AS construct contained the 1 kb fragment of the Atmsh2 cDNA in antisense orientation. The DN construct contained the Atmsh2 coding sequence with a mutation converting a strongly conserved Gly codon at position 697 to an Asp codon (Ispas, 2004). One transgenic line for AS and two for DN (DN5 and DN11), and S. tuberosum cv. Delikat and Desiree was used in protoplast electrofusion to produce MMR deficient somatic hybrids. The hybridity nature of regenerated plants with or without MMR deficiency was validated using SSR and RAPD molecular markers (Unpublished data).

In vitro stress-selection experiments

In vitro stress-selection of somatic hybrids with or without MMR deficiency and backcross progenies was performed using RMB5 media supplemented with Polyetylenglicol (PEG) (Table 1). The plants were exposed to 2 type of drought conditions: a moderate stress simulated with 5 % PEG and a severe drought condition through 15 % PEG in culture media. Potato plants were maintained for three weeks in a growth chamber at 21 °C with a photoperiod of 16 hours and a light intensity of 90 µmol*m2*s-1. After three weeks the tested plants were evaluated for viability, regeneration ability, shoot and root growth rate. Plants proline concentration was also determined, which is closely related with plant’ tolerance ability to drought stress (Mustata et al, 2014). For proline determination Bates et al (1973) protocol was used.

Ex vitro stress-selection experiments

Drought tolerant plant selection in ex vitro conditions was performed using Phenotyping Platform HAS-RSDS in Biological Research Centre Szeged, Hungary.

Eight somatic hybrids and two BC1 was sorted from previously analyzed genotypes. One week old in vitro grown plants were planted in 50 % sandy (Maros) soil and 50 % Tera peat soil. Pots have 30 cm height and 15 cm diameter and contained unique radio-frequency identifier (RI). In drought conditions where water shortage was created, the stressed plants were watered to 20 %, while control plants were watered to 60 % compared to 100 % soil water capacity. The adjusted water limitation corresponded to moderate drought conditions. Irrigation was performed automatically, pots were measured and when the computer detected water loss, automatically administered the lacking water amount. Every second day the growth of plants was photographed using Olympus C-7070WZ (Olympus Ltd., UK) digital cameras. Pots were placed onto rotating tray and each plants were photographed in eleven position while the pots span around.

Plant biomass accumulation ability was used to select drought tolerant genotypes. Plants biomass were calculated using green pixels amount of photos with Matlab software, Image Processing Toolbox (The MathWorks Inc., Natick, MA, USA)

After six weeks the crops were harvested. In the case of each genotype and separately for both treatment groups (stressed, control) the collected tubers quantity and weight were recorded.

Determination of photosynthesis efficiency

To select of drought tolerant genotypes firstly morphological (biomass accumulation and yield) changes of stressed plants were evaluated, secondly physiological effects of drought were followed up. Therefore in our experiments chlorophyll fluorescence emission was determined because it is a good, sensitive indicator of the photosynthetic reaction changes in photosystem II.

Chlorophyll fluorescence emission was measured with pocket Plant Efficiency Analyzer (PEA) chlorophyll fluorimeter (Hansatech Instruments) and pulse amplitude modulation fluorometer (PAM-2000 Heinz Walz Gmbh). The upper surface of the third completely developed leaf was used in both measurements.

Statistical analysis

Statistical analysis was performed using Microsoft Excel Statistics and R statistical software. The morphological aspects, proline concentration, biomass accumulation, tubers data and photosynthesis parameters of stressed and control plants were compared using Student T test and ANOVA.

Results and Discussion

Nowadays the frequency of drought periods are increased, which is one of the undesired result of climate changes. Unfortunately, in the near future it does not look to improve, on the contrary, drought periods will increase and more area will be affected by water shortages. As an effect of drought stress the sustainability of crop production have been compomised. Lobell et al (2008) predicted that until 2030 in southern Africa maize production will decrease up to 30 % and South Asia could lose more than 10 % of economically important crops (rice, millet, maize) production. Potato is considered as a drought sensitive crop, with high quantity of yield reducton during water shortages (Fleisher et al, 2015). Therefore development of drought tolerant cultivated plants with more efficient water-usage are indispensable to reduce the devastating agronomical and social effect of drought (Feher-Juhasz et al 2014).

In vitro stress-selection of drought tolerant plants

Stress-selection of drought tolerant SHs with or without MMR deficiency and BC progenies was performed both in vitro and ex vitro conditions. In the case of in vitro stress-selection the drought stress was induced with different concentration of PEG (5 % and 15 %), which simulated mild and severe drought conditions.

During moderate drought stress (5 % PEG) the majority of the analyzed genotypes developed weaker than control plants, these stressed plants shoot length were significantly smaller (t. test, p<0.05), the number of developed leaves were significantly fewer (t. test, p<0.05) than the control ones. Shao et al (2008) revealed similar results. They associated the plant height and leaf area reduction during drought stress with decrease of cell enlargement and with increased leaf senescence.

The analyzed genotypes responded differently to the induced drought stress, therefore we classified them in three groups (Table 1):

Table 1 Classification of parental lines, SHs and BCs by morphological appearance after moderate drought stress

Group 1: contained plants which developmental level was similar to the control plants. No significant differences between root (t. test, p= 0.683) and shoot (t. test, p= 0.491) length between the treated and control group were observed.

Group2: included genotypes with well-developed root system (t. test, p= 0.129, no significance differences between stressed and control plants root length), but the shoot length of plants was significantly smaller (t. test, p< 0.05) than the control plant’s shoot length.

Group 3: containded plants which developed very weakly. Both shoot and root length were significantly smaller than in the case of control plants.

In the case of cultivated potato and S. chacoense HL only the root system were developed normally, they shoot did not grew as efficiently like control plants shoots, therefore they were included in second group. S. chacoense 138 developed poorly, therefore it was classified into the 3th group. Besides that, can be observed that SHs with or without MMR deficiency and BCs have been proportionally divided among the created groups.

Based on our morphological differences of the stressed plants we can conclude that the genotypes from Group 1 managed efficiently the water deficit during moderate drought stress. In their case only the number of leaves was significantly fewer than in the control group, which means that these plants highly tolerate the moderate drought condition.

The members of the second group tolerate in lesser degree the water shortage. They invest all of their reserves in root system development in order to increase the possibility to reach the water stores located in deeper layers of the soil before the adverse effects of drought stress damages the plant.

The genotypes in the third group survived the drought stress, but they were not capable to overcome on the negative effects created by water deficit. This plants were sensible for moderate drought conditions. In this case, if the water deficit have been persisted for a longer time, the plants most probably were died. The plants shoot and root system did not developed, which would be essential for surviving, and also in many instances, the bottom leaves and the edges of the upper leaves were withered. Only in the case of this group some stressed plants were withered and died: one plantlets died from the analyzed five from BC2 1552/1/7/2, SH 1913/10, Dk.DN5.25, De.DN5.5 and Dk.DN5.17.

In the second experiment when the plants were exposed to severe drought conditions which was induced with 15 % PEG supplementation in culture media, all of the genotypes developed weaker, their shoot length were significantly lower than the control plants.

The stressed plants root system was not so rich in ramification than the control ones, and a large part of the analysed genotypes did not developed more than 0.5 mm long root.

During severe drought contition the newly grown leaves were visibly smaller than those leaves which the plants possessed at inoculation and smaller than control plants’ leaves. Shao et al (2008) observed that leaf area was negatively affected during water stress, which lead to decreased crop yield due to photosynthesis reduction. Also greater degree of leaf senescence were observed at drastical stressed plants than in the case of moderate stress condition.

The analyzed genotypes can not be classified similarly as in the case of moderate stress. None of the SHs and BCs can be included in the first category (Group 1). The majority of the sressed plants belonged to the third group and only one-third of the analysed SHs and BCs were included in Group 2 (Table 2).

Table 2 Classification of parental lines, SHs and BCs by morphological appearance after severe drought stress. The number of † mark after different genotypes represents the died plants.

During severe water stress more plants were withered than in the case of moderate drought condition. Parental lines were sensible to severe water shortage.

In the case of moderate stress approximately two-third of SHs and BCs tolerated the drought stress (Group 1 and 2), while during severe water shortage only one-third of the analysed genotypes tolerated the induced stress (Group 2). During severe water deficit, genotypes which belonged to Group 2 in moderate drought stress were transferred in Group 3, while most of the Group 1’s plants were moved to Group 2. Based on this observation we can conclude that genotypes from Group 2 in moderate water stress had weaker drought-tolerance strategy than Group 1’s plants, which preserved their resistance ability during severe water shortages.

Based on different morphological responses of stressed parental lines and resistant SHs and BCs, we concluded that during somatic hybridization drastic genetical changes may take place within the fused cells, which may result in the appearance of new, useful properties of the regenerated plant. The parental lines were susceptible to drought stress, but a large number of their hybrids and backrosses were highly resistant to moderate and severe drought conditions.

Taking into account the fact that both type of SHs with or without MMR deficiency can be found proportionally in susceptible and tolerant group, we concluded that somatic hybridization had greater influence on resistance trait development than the deficiency in mismatch repair system. The effectiveness of drought tolerance properties is higher in the case of MMR deficient SHs than in the case of Solanum genotypes without MMR deficiency. MMR deficient SHs were developed significantly richer (t. test, p< 0.05) and longer (t. test, p< 0.05) root system during severe drought stress, than MMR proficient SHs and BCs. Higher proportion of MMR deficient SHs (50 %) were effectively tolerated severe drought stress also from moderate drought resistant MMR deficient group, than wild type SHs and BCs, from which only 40 % tolerated both type of drought stress.

Proline accumulation

The degree of proline accumulation during drought stress influences the plant’s stress tolerance ability. Proline protects the integrity of essential proteins and enzymes and has an important role in harmful ROS detoxification (Chaves, 2009). Szabados et al (2010) and Mustata et al (under publication) demonstrated, that drought tolerant plants accumulated high amount of proline during drought stess, while sensible plants accumulated in lesser extend this amino acid. Survival rate of plants with high concentration of proline was significantly higher than in the case of drought-susceptible plants (Mustata et al under publication).

After phenotipic responses evaluation of stressed plants, the accumulated proline concentration in control and both type of water stressed group were determined. Based on control group analyses we observed that proline concentration was genotype dependent, therefore in order to compare without distortion the analysed genotypes drought responses we used a prolin concentraion rate of stressed and control group, thereby we were able to follow up the proline concentration changes during water stress.

All of the analysed genotypes without SH De.DN5.5, accumulated significantly more (t. test, p< 0.05) proline as a response of moderate water stress, but in the accumulation of proline levels visuable differences can be obseved (Figure 1). Some hybrids accumulated 7-8 fold more proline during water deficit (1552/1, Dk.DN5.11, etc), while the others accumulated only 2-3 fold more proline than control plants (Dk.DN5.17, De.DN11.29).

Figure 1 Proline concentration changes during moderate water stress of different SHs with or without MMR deficiency, BC progenies and parent lines (S. tuberosum, S. chacoense)

In order to establish the proline drought-tolerance role in the analysed Solanum genotypes the relationship between morphological responses and proline concentration were determined. Group 1 contains plants which highly tolerated water stress, members of Group II moderately tolerated, while Group 3 contains genotypes which were susceptible to water shortage. Among the created three groups, proline accumulation differences can be observed: the members of first group accumulated the most of proline, an average of 7.55 fold more than control ones, the second group 5.5 fold more than control plants, the third group accumulated the lowest concentration of proline, only 4.52 fold more than control plants. Genotypes from Group 1 accumulated significantly more proline during moderate water stress than susceptible plants from Group 3 (t. test, p< 0.05).

During severe water deficit a large part (2/3) of the stressed plants were not able to efficiently manage their resousces which would helped to overcome on the negative effects created by drought, therefore these genotypes became susceptible to severe water stress.

The drought tolerant group (Group II based on morphological traits) accumulated significantly more proline than susceptible genotypes (t. test, p< 0.05) (Group III). (Figure 2).

Figure 2 Proline concentration changes during severe water stress of different SHs with or without MMR deficiency, BC progenies and parent lines (S. tuberosum, S. chacoense)

Among the resistant plants: SH 1913/6, 1552/1, BC1 1552/1/2, Dk.DN5.11, Dk. DN11.24 and Dk.AS10.40 accumulated the most proline during severe water stress, approximately twice as much than the other genotypes. Some genotypes from the susceptible group (De.DN5.5, Dk.DN5.17) synthetised less proline than control ones, wich can be explained with the fact that these genotypes tolerated the least the induced stress, four out of five stressed plants were withered and the remained plants were suffered also (yellowed leaves), therefore these plants were unable to carry out the vital metabolic processes.

Among both type of somatic hybrids with or without MMR deficiency can be found in drought- resistant group, which supports our hypothesis, that somatic hybridization have a greater influence in drought-tolerance ability development, than the deficiency in DNA repair system.

Proline accumulation allowed the plants to survive and fairly develop in the water deficit period. In the case of SH 1913/6, 1552/1, BC1 1552/1/2, Dk.DN5.11, Dk. DN11.24 and Dk.AS10.40 resistant plants, the proline amino acid proved to be the main contributor which maintained the macromolecule integrity and osmotic pressure, while in the case of SH De. C7, Dk.DN5.3, Dk.DN11.34, Dk.AS10.13 and Dk.AS10.43 near proline accumulation probably another mechanism contributed to drought-tolerance ability.

Plants biomass accumulation under drought condition

Based on in vitro stress-selection results, from drought tolerant group: 6 SH and 1 BC1, from susceptible group: parental lines (S. tuberosum, S. chacoense HL, 1 G), 2 SH and 1 BC1 were selected and were further analysed their drought-resistance ability in ex vitro condition. Among the analyzed SHs both type: MMR deficient and MMR provicient were selected. Several researches proved that the phenotyping platform is effective to monitorize morphological and physiological changes of plants during water stress (Wegener et al 2015).

In our experiment, the impact of drought on morphological traits of stressed plants were determined using biomass accumulation differences between control and drought stressed plants. Biomass accumulation of plants were calculated from green pixels quantity of RGB images, which corresponds to surface area of plants shoot. Therefore plants with high amount of green pixels represents extended surface area of shoot (Figure 3), which is directly proportional to the drought tolerance ability of plants.

Figure 3 Biomass accumulation of the analysed Solanum genotypes during drought stress

Based on the control plants biomass we observed that cultivated potato had the most extended surface area of green shoot, while SHs and BC1 progenies biomass accumulation were between the parent lines biomass level, which proves their hybridity nature, namely that these genotypes were regenerated form a combined cell of S. chacoense and cultivated potato.

As an effect of drought stress all of the analysed plants in except of Dk.AS10.13 accumulated significantly less biomass (ANOVA, p< 0.05) than control ones. Obidiegwu et al (2015) obtained similar results, drought negatively affected plants development, which resulted in reduced foliage extension and decreased tubers yield quantity and quality. Water deficit had negative effect on Dk.AS10.13 also, but this genotype was able to catch up the level of control plant’s biomass until the sixth week of the investigation (ANOVA, p=0.122). Therefore this genotype proved to be the most resistant among the analyzed plants (Figure 4).

Figure 4 Biomass accumulation in SH DK.AS10.13 during moderate drought conditions

The slightest difference between stressed and control plant’s biomass were observed in the case of SH Dk.DN5.11, Dk.DN11.24, Dk.AS10.13 and Dk.AS10.40 (Figure 3). These genotypes responded better to drought stress than parent lines. Their shoot surface biomass greatly approached the control plant’s biomass, which proves their drought-tolerance ability. All of the drought-tolerant plants have deficiency in DNA repair system, none of them were from MMR provicient plants group which proves that MMR deficiency increases the transfer of drought-tolerance trait.

Drought stress effects on tuber production

Drought stress negatively affects foliar extension of plants, which led to tuber yield reduction. Not only the number of tubers can be affected but also the tubers weight can be reduced as a consequnce of drought condition during tuber formation (Wishart et al 2013, Basu et al 1999). Therefore tuber development and yield can be considered as the most economically important drought-tolerance indicator of water stressed plants.

After two months of water shortage the control and stressed plants tubers were harvested and mean weight of tubers were calculated (Figure 5).

Figure 5 Mean weight of tubers after six week of water shortage

S. chacoense HL and 138 do not developed tubers even not under control conditions, which is due to this wild species is weaker tuber-forming species and it is more sensible to artificial conditions (like to grow in pots) than cultivated potato. S. tuberosum, Dk.AS10.40 and Dk.AS10.51 developed the largest tubers in control condition, but under drought stress only Dk.AS10.40 were able to develop large tubers (mean weight of 10.906 g/tuber). Only in the case of this hybrid no significant differences between control and stressed plants tuber’s mean weight were observed (t.test, p=0.348) (Figure 5).

Besides that genotype in the case of SH 1913/10, Dk.DN5.11 and Dk.DN11.24 slight decline of tuber weight were recorded during water-stress. Unfortunately in control conditions SH 1913/10 developed also small sized tubers, therefore this genotype were considered to be not suitable to use in breeding program.

Comparing tuber yield and biomass accumulation changes during water stress we observed that plants which suffered less from drought and accumulated approximaltey as much green biomass than control plants, were able to develop good quality of tubers, in contrary plants with reduced foliar extension developed small-sized tubers. Therefore we can conclude that foliar extension detrmination can be used as indirect method to predict the success rate of yield without distroying the plant. The only exception were observed in the case of Dk.AS10.13. This genotype accumulated same level of green biomass than control plants, but the tuber yield was much less than control plant’s yield. This observation can be explained with the fact that in order to the plant develop extended green biomass the other parts of plants grow weaker, in our case the quality of tubers declined, because of plants need to compensate somewhere the invested energy in shoot development.

After ex vitro stresselection we can conclude, that all of the analysed genotypes tolerated the induced moderate drought stress, none of them were withered during the experiment, but significant differences in biomass accumulation and tuber yield and quality were observed between drought-sensible (S. tuberosum, S. chacoense, 1552/1, 1552/1/2, 1552/1/7, 1913/6, 1913/10, Dk.AS10.51) and drought-tolerant or resistant (Dk.DN5.11, Dk.DN11.24, Dk.AS10.13, Dk.AS10.40) genotypes (ANOVA, p<0.05).

In ex vitro stresselection we obtained similar results as in the case of in vitro experiments. Plants which were sensible to water stress in the in vitro conditions were sensible in ex vitro stress-selection also, but among drought-resistant genotypes from in vitro experiment only MMR deficient genotypes were resistant to water shortage in ex vitro stress conditions.

Drought stress effect on photosynthesis

Photosynthesis is the most important biological process of plants, which is essential in biomass accumulation (Arabzadeh 2013). Drought stress proved to reduce the growth and yield of potato by affecting the kinetics of chlorophyll fluorescence (Jefferies 1992). First response of plants after drought perception is stomatal closure, which have the role to reduce the transpiration and to increase the water use efficiency of plants (Lei et al 2006). As a result of this defensive mechanism, the intercellular CO2 concentration will be reduced, which will led to declined photosynthetic carbon assimilation.

The chlorophyll fluorescence measurements are rapid, non-destructive methods, which provide information about the integrity and functionality of the electron transport chain (Strasser et al 2004) and proved to be a sensitive indicator of different abiotic stresses, for example caused by low temperature (Greaves and Wilson, 1986), heat (Havaux et al 1988), nutrient deficiency (Conroy et al 1986) and was suitable to detect the influence of drought stress onto the photosynthesis (van Kooten and Snel 1990).

In our study, plants were exposed to long-term moderate drought stress and its impact on chlorophyll fluorescence were investigated in the beginning of the exposure to stress (2nd week) and at the end of the experiment (5th week).

On both occasions, after 15 minutes of dark adaptation the initial fluorescence (F0) and maximum fluorescence (Fm) were measured from which the maximal quantum efficiency (Fv/Fm) was determined. From light-adapted state of leaves the effective quantum efficiency (Fv’/Fm’) was also calculated. In addition to these ratios, which efficiently indicates the early stress-symptoms of plants, several other parameters (e.g. Fm/F0, F0/Fv, Fv/2, Area, PI, NPQ) were also determined, which provide a more detailed view about the functionality of electron transport chain.

After both measurement we observed that the maximal quantum efficiency of stressed plants were not decreased under drought conditions. In the all analyzed genotypes the calculated ratio fell within the optimal range (0.75-0.84) like in the case of control, unstressed plants. From this results we can conclude that the induced moderate water stress had not negative effect on the PSII photochemistry in dark adapted leaves. Flagella et al. (1998) observed that the quantum efficiency of PSII have been reduced only in the case of drastic water shortage. Lu and Zhang (1999) experiments supports our results also, they observed that drought doesn’t destabilized the PSII functionality in drought stressed wheat plants. Jefferies et al (1992) obtained similar results in the case of potato, F0 and Fv parameters were reduced in drought condition but the yield of fluorescence was not affected.

Photosynthesis occurs on thylakoid membranes, therefore these membranes integrity are essential for normal, healthy functioning of electron transport. Thylakoid membranes of plants, grown up in drought condition were uneffected, Fm/F0 ratio didn’t decreased to 1 value, it was between 4 and 5, which characterize the expected value of healthy, unstressed organism. The water splitting complex integrity (F0/Fv) were also not damaged in water scarcity, no significance differences (t.test, p=0.325) were observed between control and stressed plants in both measurement occasion. Shapendonk et al (1989) demonstrated that only severe drought stress affects the functionality of these systems.

Furthermore, we determined the plastoquinone pool fluorescence (Fv/2) which positively correlates with the pigment number around the reaction centers (Li et al 2015). Based on our result we can conclude, that as an effect of drought the number of pigments increased in the second measurements. which suggests that stressed plants reorganised their pigments (Fodorpataki 2004) in such a way that the pigments have been accumulated around the active reaction centres (Jaleel et al 2009).

From the measured parameters, the plastoquinone pool size was also determined from the area above the fluorescence curve, and we observed that plastoquinone pool size was unaffected in the first, and was significantly increased (t.test, p< 0.05) in the second fluorescence measurements. This result confirms the previous observations, that the induced moderate water deficit, have not affected the electron transport of stressed plants.

In the next step the effectiveness of the photosynthesis were evaluated by calculating the performance index (PI), which proved to react more sensible to drought stress than the maximal quantum efficiency (Cavender-Bares and Bazzaz 2004). As the other calculated parameters, in this case also, the PI value of drought stressed plants was similar to control group in the first investigation, and was significantly higher (t.test, p<0.05) in the second measurements (Figure 6).

Figure 6. Performance index of SHs, BC1s and parental lines (S. tuberosum cv. Delikat, S. chacoense)

This index provides a complex image about the entire process of photosynthesis (Zivcak et al 2008) by quantifying the most important steps of photosynthesis in PSII reaction center complex: light energy absorption, the excitation energy trapping and the conversion and transmission of this energy to the electron transport chain (Strasser et al 2004). The increase of PI index in the second measurements, suggests that the analyzed plants were able to adapt with time to the applied moderate drought stress. Therefore if this parameter was not reduced during water stress, it is possible that the induced water scarcity was not strong enough to have a negative impact to this trait.

In the next step the light adapted values of initial and maximal fluorescence were used to determine the effective quantum yield (Fv’/Fm’), which provides information about the effectiveness of light energy transformation into chemical energy (Murchie and Lawson 2013). After the first measurements significantly decreased of Fv’/Fm’ rates in drought stressed plants were observed (Figure 7A), which can be explained by increased light-induced non-photochemical quenching or by photochemical quenching like photorespiration (Jefferies 1994). Both type of mechanism have the role to dissipate the excess of non-radiative energy in the PSII system, thereby provide protection against photo damage of electron transfer chain (Fracheboud and Leipner 2003). In the second measurements the effective quantum yield of stressed plants approached the control plants Fv’/Fm’ rates, no significant differences (t.test, p>0.05) between the two experimental groups was observed (Figure 7B). The obtained results can be explained by the fact that the plants successfully adapted to the induced drought condition, and were able to utilize more effective the light energy to CO2 assimilation, than in the earlier stage.

Figure 7 The effective quantum yield of SHs, BC1s and parental lines in control and drought conditions: A: first fluorescence measurements, B: second measurements

Non-photochemical quenching (NPQ) of the chlorophyll fluorescence indicates the level of light-energy dissipation through fluorescence re-emission or heat releasing in the PSII reaction center (Fracheboud and Leipner 2003). Stern-Volmer NPQ was calculated using the formula Fm/Fm’-1. Large increase in NPQ lead to xanthophyll cycle activation, which can down-regulate the photosynthesis (Flexas et al 1999). NPQ level of drought stressed plants were significantly higher in the early stage of water scarcity (2nd week), then during second measurements. This observation explains the lack of similar amount of biomass accumulation in stressed plants compared to control ones. Large amount of the harvested light energy was lost by non-photochemical quenching, therefore the effectiveness of carbon fixation was reduced. Non-photochemical quenching was a necessary evil solution of plants to protect themselves from photo damaging, thereby the stressed plants were able to survive and to maintain their vital functions without noticeable losses.

Based on the second measurements results we can conclude, that stressed plants adapted to the induced stress conditions. Non-photochemical quenching level were reduced, which yielded visible resulted in biomass accumulation. Carbon fixation of stressed plants began to work more efficiently, in some cases stressed plants biomass level caught up (Dk.AS10.13) or approached (Dk.DN5.11, Dk.DN11.24, Dk.AS10.40) the control plants biomass level.

Conclusion

Based on the obtained results we can conclude that proline accumulation during water stress greatly contributed to plant tolerance ability. During moderate stress, the resistant plants were developed normally, while in severe drought condition the tolerant plants were able to grow roots, which are essential to achieve water storages in natural conditions.

Parent lines were sensible in both type of drought stress, but between somatic hybrids highly resistant plants to water shortages can be observed. The results of photosynthesis analysis suggests that plants are able to overcome the negative impact caused by moderate drought stress.

Based on this observation we can conclude that somatic hybridization process increases the possibility to develop new important traits in the generated plants, including multigenic regulated properties like drought-resistance (Bartels and Philips 2009). Besides that MMR deficient drought-resistant properties were more stable than MMR provicient plants resistance ability. The MMR deficient SHs were highly resistant to the both type of drought conditions (in vitro and ex vitro), while normal SHs and BCs were resistant only in controlled conditions to water stress.

Acknowledgements

The funding from Ministry of Education and Research, the national project CNCS PNII-ID-PCE-2011-3-0586 is acknowledged.

Szeged….

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