Introduction

Pelargoniums are commonly used ornamental species for commercial purposes as balcony and bedding plants. They belong to the Geraniaceae family and are xerophytes native to South Africa and Australia. Popular modern varieties of pelargoniums are most commonly propagated through vegetative cuttings for mass-market distribution. Nowadays, stock plant nurseries of bedding plants, including pelargonium, are located in countries with suitable climate conditions, such as Kenya, Ethiopia, Israel, Colombia and Mexico. Unrooted softwood stem cuttings severed from stock plants are imported to Europe and the United States1. After collection, cuttings are packaged in plastic bags, gradually cooled and transported to commercial producers within 2–3 days, where they are rooted and cultivated. In the Northern Hemisphere, bedding plants are mainly propagated and rooted in winter (from mid-January to mid-March). The success of vegetative propagation depends on various factors, eg, the quality of the stock plants, plant nourishment, their phytosanitary state, production temperature and light intensity etc.

European pelargonium farms face challenges because wintertime cuttings delivered from Southern countries (Africa, Israel, Turkey) are often of low quality, other due to stock plant cultivation problems. The concern is also the extended transportation time, which affects cuttings' viability and rooting ability after they reach Europe. Additionally, differences in peak stock plant nursery productivity and peak demand for unrooted cuttings were observed over time. To match the peak of nursery productivity (December) and the peak of market demand for cuttings (late February/March), we measure if it is possible to store freshly planted cuttings in a cold environment without considerable loss of rooting efficiency. This would make it possible to obtain cuttings of the size preferred by customers at the time of peak demand. However, we assumed that length of effective storage depends on both the species and cultivar of pelargonium and the initial physiological state of the cuttings (nutrition and water supply).

Chlorophyll a fluorescence is considered a reliable technique for assessing photosynthetic electron transport and related photosynthetic processes2. To analyse a high number of photosynthetic samples, a fast chlorophyll a fluorescence transient analysis was developed3,4. This method involves high-frequency recording of chlorophyll fluorescence signals in dark-adapted leaf samples during exposure to high-intensity actinic light. The resulting fluorescence signal, which is polyphasic and ranges from the minimal F0 to the maximal Fm value, provides information about the structural and functional state of photosystems, mainly photosystem II (PSII)3,5. The mathematical model, known as the OJIP test, describes this fluorescence transient and allows the calculation of parameters characterizing the functioning of photosystems6. Changes in chlorophyll fluorescence transients have been observed in plants under different types of environmental stress or nutrient status7,8,9,10. Additionally, many studies have shown that the OJIP test is useful for studying differences in photosynthetic performance among plant accessions11,12. However, the OJIP test has never been used in studies related to rooting of plants in production systems.

Pelargonium, a staple in ornamental horticulture, is primarily propagated through vegetative cuttings. This process is significantly influenced by the physiological status of the cuttings, which is affected by factors such as nutrient availability, water supply, and storage conditions. The primary objective of this study is to explore whether chlorophyll fluorescence, particularly through the OJIP test, can serve as a reliable indicator of cutting fitness and rooting potential. By analyzing six varieties of Pelargonium under different initial conditions (including nutritional status) and length of low-temperature storage, this research aims to establish a link between photochemical activity and rooting success, thereby offering a valuable tool for commercial propagation.

Materials and methods

Plant material

Six different Pelargoniums’ (geranium) varieties, namely, 'Champion Fire Red', 'Flower Fairy White Splash', 'Ville de Paris Rot' ('VDP Rot'), 'Flower Fairy Berry', 'Ruby' and 'Victor', were used in the experiment during the two production seasons of 2020/21 and 2021/22. The plant material was obtained from Savanna Flowers Company, Kenya, as shown in Table 1. These varieties are known mainly for their versatile decorative qualities and are used commercially for gardening and landscaping. The experiment was conducted at the Production Farm “Plantpol” in Zaborze (Poland) to study the effect of rooting efficiency and the physiological responses of geranium cuttings by assessing and analysing them under different experimental conditions.

Table 1 List of Pelargonium cultivars studied in the experiment in the two cultivation seasons 2020/21 and 2021/22.
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The cuttings were prepared from stock plants in Kenya at weekly intervals (weeks 48, 49, 50, and 51) in 2020 and 2021. All terminal stem cuttings, each of which were adorned with three to four leaves (including one fully developed), underwent a meticulous process. These unrooted cuttings, totalling 100 per batch, were rapidly chilled to less than 8 °C, encased in perforated polyethylene bags, parcelled into carton boxes, and started their journey by truck to Nairobi airport. During the night, they embarked on a direct cargo flight bound for Amsterdam airport in the Netherlands. Subsequently, the cuttings continued their route by truck to their final destination: the Production Farm Plantpol in Zaborze, Poland. This entire logistical voyage, from the Kenyan nursery to the Polish production site, spanned four days (60–72 h), transpiring in a dim environment with temperatures rigorously maintained between 2.5 and 15 °C (averaging 8 °C). The unrooted cuttings, which were delivered on specific dates (6th, 13th, 20th, and 30th of December 2020 and during the corresponding weeks of 2021 and 2022, respectively), were placed in paperpot plugs filled with a Hawita SoMi537 commercial mixture (pH 5.3) and arranged in 104 plastic plug trays at a density of 52 plugs per tray for stability. The trays, which were placed in an innovative greenhouse with climate control managed by SERCOM SC800 and SercoVision software, were subjected to consistent environmental conditions. Storage involved temperature control at 8 °C (day) and 6 °C (night), with an average air temperature of 7 °C and 80–90% relative humidity sustained by a high-pressure fogging system. Watering by sprinklers every 2 h was followed by ebb and flow irrigation. The shading intensity was set at 30 klx. Fertilization included Granusol WSF 17-10-17 NPK fertilizer, which was applied through both foliar and fertigation methods. Preventive fungal protection was administered using the Signum 33 WG fungicide. The same storage conditions were maintained for each batch in both cultivation seasons. After storage, the trays were moved to a greenhouse under optimal rooting conditions (20 °C in January, 80–90% relative humidity, natural light). The rooting process included a dwarfing procedure using CCC (Stabilan750SL) at a concentration of 0.1% weekly. The rooting response was assessed after 4 weeks, which indicated the percentage of well-developed liners with visible root systems. The steps of the experiment with cold storage of pelargonium cuttings are presented on Supplementary Figure.

Chlorophyll content measurements

Chlorophyll meter readings (SPAD-502 plus, Konica Minolta, Osaka, Japan) were taken at the centre of the adaxial side of the leaves with major veins avoided. The measurements were taken in situ between 11:00 and 13:00, with 120 replications per variety/treatment.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence was measured in the plants of the six geranium varieties during two subsequent production seasons. After the plants were transferred from the nursery, the cuttings were planted in peat plugs and subjected to chilling treatment (6 °C in the glasshouse) to extend the production cycle for 1, 2 and 3 weeks. The cuttings were subsequently transferred to a heated glasshouse (23 °C) to initiate growth and rooting. The measurements were taken directly after the plants were transferred to a warm environment (day 1) and after 7 days (day 7). The induction of a chlorophyll fluorescence signal was measured after 30 min of leaf dark adaptation with a Handy-PEA (Plant Efficiency Analyzer, serial number 1009, Hansatech, Kings Lynn, UK) chlorophyll fluorimeter. The measurements were replicated 10 times (10 leaves from different plants). The OJIP test parameters were calculated according to (Strasser et al., 2004). The calculated parameters are defined as follows:

(1) Quantum yields and efficiencies: Fv/Fm—maximum quantum yield for primary photochemistry; ψ0—efficiency/probability for electron transport (ET), i.e., efficiency/probability that an electron moves further than QA; φEo—quantum yield for electron transport; δRo—efficiency/probability with which an electron from the intersystem electron carriers in photosystem II (PSII) moves to reduce end electron acceptors at the PSI acceptor side (RE); φRo—quantum yield for reduction of the end electron acceptors at the PSI acceptor side (RE). (2) Performance indices (potentials) for energy conservation from excitons to the reduction of intersystem electron acceptors in PSII (PIABS), QA (PICS0), plastoquinone (PICSm), and PSI end acceptors (PItotal). (3) Phenomenological energy fluxes (per excited leaf cross-section—CS) for absorption (ABS/CS), trapping (TR0/CS), electron transport after QA- (ET0/CS) and dissipation (DI0/CS). (4) Minimal (RC/CS0) and maximal (RC/CSm) numbers of QA-reducing PSII reaction centres (RCs) were observed during fluorescence induction. (5) Specific energy fluxes (per QA-reducing PSII reaction centre (RC)) for ABS/RC absorption, TR0/RC trapping, electron transport after QA- (ET0/RC), dissipation (DI0/RC) and electron flux reducing end electron acceptors at the PSI acceptor side (RE0/RC).

Statistical analysis

Statistical analysis was performed with Statistica 13.1 software (Dell, Round Rock, TX, USA) using ANOVA (for the calculation of standard errors and confidence intervals of means), multiple regression (for calculation of linear correlation coefficients) and multidimensional (for principal component analysis, PCA) modules. PCA was performed on the basis of the correlations.

Statement regarding IUCN policy

This study was conducted using plant species that are not listed on the IUCN Red List of Threatened Species. Additionally, all plant species used are not listed under the Convention on the Trade in Endangered Species of Wild Fauna and Flora (CITES). All experiments adhered to ethical guidelines, ensuring no endangered or threatened plants were involved.

Results

The experiment was performed in two production seasons, 2020/21 and 2021/22. The growth conditions of the mother plants affected the photochemical performance of the cuttings after planting (Fig. 1). The negative effect was similar for all the varieties studied but was clearly stronger for 'Champion Fire Red' and 'Victor'. A decrease in photochemical performance was visible for the whole electron transport chain (PItotal) and for end acceptor-related processes (φRo, δRo, RE0/RC). Additionally, the performance index for energy conservation from exciton to the reduction of intersystem electron acceptors in PSII (PIABS) was reduced, which indicated decreased performance during the initial steps of energy flow in PSII. As a result, the energy dissipation per leaf cross-section (DI0/CS) clearly increased in 2021/22.

Fig. 1
figure 1

Relative changes in OJIP test parameters during transient chlorophyll fluorescence induction measured directly after planting in six pelargonium varieties planted in 2021/22 relative to those in 2020/21.

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After planting, the cuttings were placed into cold storage media to slow growth and extend the shelf life of the cuttings. The effect of cold storage on the chlorophyll fluorescence parameters differed between the two production seasons (Figs. 2, 3). In 2020/21, clear increases in PICS0, PICSm and PIABS as well as in ET0/RC were observed during cold storage, and the same was still visible after seven days of recovery (Fig. 2). In 2021/22, cold storage negatively affected PItotal, but this effect differed among the different varieties (Fig. 3). After 7 days of recovery, an increase in PItotal and other parameters dependent on the efficiency of the energy transfer upstream PSII was observed in 'VDP Rot' and 'Victor', and an increase in ET0/RC and PIABS was observed in cold-stored cuttings after one week of recovery (Fig. 3).

Fig. 2
figure 2

Changes in OJIP parameters during chlorophyll fluorescence induction between cold-stored pelargonium cuttings (6–8 °C) for 1, 2 and 3 weeks and control pelargonium cuttings measured 1 day after the transfer to rooting-promoting temperature (recovery at 22 °C) and after 7 days of rooting, and after one week of recovery in the 2020/21 experiment.

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Fig. 3
figure 3

Changes in OJIP parameters during chlorophyll fluorescence induction between pelargonium plants cold-stored (6–8 °C) for 1, 2 and 3 weeks and control plants measured 1 day after the transfer to rooting-promoting temperature (recovery at 22 °C) and after 7 days of rooting, and after one week of recovery, in the 2021/22 experiment.

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It may be assumed that the latter effect may be connected to the initiation of rooting, which started in the cold in 2020/21 due to better rooting potential associated with the different initial conditions of the cuttings. The PIABS values were significantly lower in 2021/22 than in 2020/21, regardless of the variety or length of cold storage and recovery (Fig. 4).

Fig. 4
figure 4

Changes in the values of the performance indices (potentials) for energy conservation from exciton to intersystem electron acceptor (PIABS) reduction in plants of six pelargonium varieties triggered by cold storage (6–8 °C) of newly planted cuttings. The plants were stored for 1, 2 or 3 weeks. The measurements were performed 1 day after the plants were transferred to a rooting-promoting temperature (22 °C) and after 7 days of rooting. The means ± confidence intervals were calculated for P = 0.05.

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The increase in the PIABS after recovery was statistically significant in 2021/22, except for 'Ruby' and 'FF White Splash', which were cold-stored for one week, and in 'VDP Rot', which was cold-stored for two and three weeks in 2020/21. Some differences were also observed between cultivars, especially directly after cold storage in 2021/22. PItotal was not affected by recovery and only in some cases depended upon the variety in 2020/21, when a clear decrease in PItotal was observed with increasing length of cold storage (Fig. 5).

Fig. 5
figure 5

Changes in the performance indices (potentials) for energy conservation from excitons to the reduction of PSI end acceptors (PItotal) in plants of six pelargonium varieties triggered by cold storage (6–8 °C) of newly planted cuttings. The plants were stored for 1, 2 or 3 weeks. The measurements were performed 1 day after the plants were transferred to a rooting-promoting temperature (22 °C) and after 7 days of rooting. The means ± confidence intervals were calculated for P = 0.05.

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In 2021/22, the PItotal values were lower than those in 2020/21; however, the PItotal values increased after recovery but did not change with increasing length of cold storage. Principal component analysis (PCA) of all the chlorophyll fluorescence parameters calculated during the experiment clearly distinguished the plants analysed in 2020/21 from those analysed in 2021/22 (Fig. 6).

Fig. 6
figure 6

PCA biplot showing the results of chlorophyll fluorescence transient measurements and the OJIP test performed for six pelargonium varieties directly after planting the cuttings on 2020/21 and 2021/22.

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It is also remarkable that the differences between years were the highest for the cultivar 'Victor'. When a similar analysis was performed for cold-stored plants for different durations, differences between the years were still visible, especially for the measurements taken before recovery (one day after the start of heating; Fig. 7).

Fig. 7
figure 7

PCA biplot showing the results of chlorophyll fluorescence transient measurements and the OJIP test performed for six pelargonium varieties cold stored (6–8 °C) for 1, 2 and 3 weeks after planting the cuttings on 2020/21 and 2021/22. The measurements were performed 1 day after the plants were transferred to a rooting-promoting temperature (22 °C) and after 7 days of rooting.

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The effect of the length of cold storage on the chlorophyll fluorescence parameters was more obvious in 2021/22 than in the other months and after seven days of recovery. However, these effects were not detected in 'VDP Rot', the variety in which no detrimental effect of cold storage on rooting efficiency was observed in 2021/22—92% efficiency after 3 weeks of storage (Supplemental Table). The opposite was observed for the 'Victor' variety; in 2021/22, after 3 weeks of cold storage, the rooting efficiency decreased to 22% (from 99% in the control), and the differences shown in the biplot were the greatest. The calculated values of the OJIP test were correlated with rooting efficiency and chlorophyll content in leaves, as measured using SPAD, as presented in Table 2 and Supplemental Table (A and B).

Table 2 The effect of initial cuttings conditions (seasons) and their physiological state (after cold storage for 1, 2 or 3 weeks and after one week of subsequent recovery under greenhouse conditions optimal for rooting) on the linear correlation coefficients between OJIP parameters, relative chlorophyll content (SPAD) and rooting efficiency in six cultivars of pelargonium.
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The only OJIP parameter that was significantly correlated with rooting efficiency in both seasons was ET0/RC, and this correlation was negative. In 2020/21, similar negative correlations were also observed for TR0/RC. A negative correlation between the OJIP parameters, which describe energy flow efficiencies in thylakoids, and the chlorophyll content (SPAD) was observed in cuttings from worse conditions (2021/22). Only Fv/Fm was negatively correlated with SPAD in 2020/21. On the other hand, irrespective of the season, phenomenological energy fluxes for leaf cross-sections and the number of active PSII reaction centers in the not-excited state (RC/CS0) are greater when plants contain more chlorophyll. Notably, in 2021/22, ET0/RC was negatively correlated with rooting efficiency and chlorophyll content.

Discussion

This study examined the correlation between chlorophyll fluorescence and the rooting efficiency of Pelargonium cuttings under different conditions. The results emphasize the potential of chlorophyll fluorescence, particularly through OJIP test parameters, in assessing and predicting the physiological state and rooting success of these cuttings. The results of our study clearly indicated that the measurement of chlorophyll fluorescence transients followed by an OJIP test can indicate the initial conditions of geranium cuttings prior to planting. In our study, the different conditions of the cuttings in 2020/21 and 2021/22 were related to their nutritional status and plant hydration (unpublished data). In 2020/21, the cuttings contained much (30–50%) less K, Mg and Ca; however, they contained approximately 10% more total N. As a result, in 2020/21, the rooting efficiency was higher than that in 2021/22 in the case of cold storage of newly planted cuttings but not in the case of the control plants, which rooted directly after insertion in the growing media (always almost 100%, data not shown). In our study, the poor condition of the cuttings affected almost all of the calculated chlorophyll fluorescence parameters, especially the performance indices (PIs), for the different steps of photochemical energy transfer. The effectiveness of OJIP tests and other methods of chlorophyll fluorescence transient analysis for detecting nutritional status has been reported previously for many species6,7,13.

A key finding is the inconsistent relationship between chlorophyll content (measured by SPAD values) and chlorophyll fluorescence parameters. While a positive correlation was observed immediately after cold storage, this relationship diminished after one week of growth. This indicates that the rooting process changes the relationship between the chlorophyll content and the photochemical efficiency of photosynthesis indicated by chlorophyll fluorescence measurements. This may result from the increasing demand for photoassimilates observed during rooting. Thus, the efficiency of the photochemical side of photosynthesis is regulated during rooting by assimilate demand but not by chlorophyll content. This underscores the importance of evaluating photochemical parameters rather than relying solely on chlorophyll content to predict rooting potential.

This observation is in line with many studies showing changes in photosynthetic activity during rooting. It was reported that in poinsettia (Euphorbia pulcherrima), photosynthesis was low in cuttings before roots were visible and increased rapidly as roots visibly emerged from the base of the stem14.

According to these authors, cuttings appear to initiate root growth independently of the photosynthetic rate, with photosynthesis increasing upon visible root elongation. The same effect, as indicated by the OJIP test parameter, was observed in our experiment during the recovery phase after cold storage. In the newest study on the propagation of poinsettias, photosynthesis rates remained consistently low throughout the first week after propagation, after which plants began to recover by day 21 during propagation when roots were present15.

In contrast, Acer rubrum stem cuttings rooted under greenhouse conditions exhibited low photosynthetic rates, and the stomatal conductance of the cuttings during rooting was associated with water stress16. However, in our experiments, the rooting cuttings were extensively watered and fogged to avoid water stress.

The nutritional status of the cuttings significantly influenced their chlorophyll fluorescence parameters. For instance, in 2020/21, cuttings had lower levels of K, Mg, and Ca but higher N levels, correlating with better rooting efficiency post cold storage compared to 2021/22 (data not shown). This suggests that nutrient imbalances can affect the initial photochemical performance and subsequent rooting success. Furthermore, cold storage impacted the chlorophyll fluorescence parameters differently between seasons, with the 2021/22 season showing a greater negative impact on PItotal, highlighting the variability in response due to initial cutting conditions. This can be explained by the fact that the 2021/22 cuttings were generally less rich in some macro- and micronutrients but contained more nitrogen, and the chlorophyll content is considered an indicator of the nitrogen content in plants17,18. Additionally, in the paper of19, the authors reported that chlorophyll meter data, including SPAD-502 data used in our study, indicate that mineral deficiency at various accuracies depends upon the species. In maize, high accuracy for potassium and nitrogen deficiency and low accuracy for phosphorous and magnesium deficiency were observed; however, in tomatoes, the accuracy for calcium, potassium, and iron deficiencies was high, while that for phosphorus was low.

The study showed a significant negative correlation between ET0/RC values and rooting efficiency, indicating that higher electron transport flux per reaction center is associated with lower rooting efficiency. This relationship was consistent across different varieties and cold storage durations. Interestingly, other parameters, such as TR0/RC and the performance indices (PIABS and PItotal), also reflected changes in the physiological state of the cuttings, particularly in relation to their nutritional status.

In the present study, the correlation between the single OJIP parameters and the differences in rooting efficiencies associated with variety and cold storage were nonsignificant in most cases. The only exceptions are two parameters describing energy fluxes in single, active PSII reaction centers measured after one week of recovery (one week of growth under optimum rooting conditions): ET0/RC—flux for electron transport and TR0/RC—flux for energy trapping in the reaction center, but only in 2020/21. The correlations are negative. This is connected with the clear increase in the values of ET0/RC (and TR0/RC) after increasing the length of cold storage. However, rooting efficiency decreases with increasing duration of cold storage. This increase in ET0/RC and TR0/RC may be connected to the compensatory after-effects of cold-induced damage to the photosynthetic apparatus20, which was indicated by the decreasing number of active RCs after the increase in cold storage time, especially during 2021/22.

The very low correlation between single OJIP parameters and rooting efficiency excludes the usefulness of single OJIP parameters for predicting the difference in rooting efficiency between varieties connected with cold storage, which is crucial for application in production systems. On the other hand, such differences were clearly visible after the principal component analysis of the measured OJIP parameters. Taken together, these findings clearly indicated that when rooting efficiency decreased after cold storage, the spatial coordinates of the plant varieties markedly changed. To be reliable, the measurements should be taken after one week of recovery. The necessity of secondary fluorescence data processing, using multivariate analyses such as PCA or machine learning methods in the analysis of chlorophyll fluorescence signals in more complex cases, which cannot be efficiently analysed by traditional methods, was previously reported7,21. In the case of such complex analysis, it was even suggested that the chlorophyll fluorescence transient can be treated as a “fingerprint”, which can even be applicable for identifying individual species from their fluorescence transients22,23. PCA of OJIP parameters was successfully demonstrated to be useful for analysing and indicating differences between plant accessions affected by mineral deficiency7, de-acclimation response during winter24 or waterlogging25.

The study established that while single OJIP parameters like ET0/RC and TR0/RC can indicate rooting potential, comprehensive analysis using multivariate approaches, such as PCA, is more reliable for predicting varietal differences in response to cold storage. This approach captures the complex interactions and compensatory mechanisms within the photosynthetic apparatus under stress conditions. While chlorophyll content measurements alone were insufficient, fluorescence parameters provided early indications of physiological status and potential rooting success. Future research should explore the integration of chlorophyll fluorescence analysis with other physiological and molecular markers to enhance the prediction accuracy for rooting efficiency. Additionally, investigating the underlying mechanisms linking nutrient status, chlorophyll fluorescence, and rooting dynamics could provide deeper insights into optimizing cutting propagation practices for commercial cultivation.

Conclusion

Chlorophyll fluorescence analysis through the OJIP test proves to be a valuable tool in predicting the rooting success of Pelargonium cuttings. The imposed rooting ability affects the demand for photoassimilates in pelargonium cuttings, which can be detected very early by chlorophyll fluorescence analysis but not by chlorophyll content measurements. Chlorophyll fluorescence measurements and single OJIP test parameters, such as the performance indices PIABS and PItotal, appear to be useful for predicting differences in rooting efficiency associated with different nutritional statuses; however, for predicting the effects of cold storage, especially varietal differences in the cold-storage response to rooting efficiency, multivariate analyses of OJIP parameters, such as principal component analysis (PCA), are needed. The study highlights the necessity of considering both single fluorescence parameters and multivariate analyses to account for varietal and storage condition differences. This approach can significantly enhance the efficiency of vegetative propagation in commercial Pelargonium cultivation.