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conditions of the cuttings on 2020/21 and 2021/22 were related to their nutritional status and plant hydration (unpublished data).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 species [6, 7, 13].
Our study indicated that the chlorophyll fluorescence data were not consistent with the chlorophyll content data. Positive correlation coefficients between the OJIP test parameters and the SPAD values were observed only when the measurements were taken directly after cold storage or after planting and not after one week of growth. This indicates that the rooting process changes the relationship between the chlorophyll content and chlorophyll fluorescence. 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 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 stem [14]. 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 present [15].
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 stress [16]. However, in our experiments, the rooting cuttings were extensively watered and fogged to avoid water stress.
According to the results of the present paper, the different nutritional statuses of plants can alter the relationship between their chlorophyll content and chlorophyll fluorescence. In the 2021/22 experiment, a clear negative correlation was detected between all the parameters, indicating that photochemical energy transfer and chlorophyll content were efficiently related; this was not observed in 2020/21. 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 plants [17, 18]. Additionally, in the paper of [19], 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.
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 apparatus [20], 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 reported [7, 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 transients [22, 23]. PCA of OJIP parameters was successfully demonstrated to be useful for analysing and indicating differences between plant accessions affected by mineral deficiency [7], de-acclimation response during winter [24] or waterlogging [25].