Pinus radiata D. Don is one of the most important forestry species in the world in terms of annual wood production, with almost 25% of the world’s production [1] . Today, this production depends on the ability to generate selected clones in the short term. However, as in many woody species, in Pinus radiata, there is a decline in morphogenetic ability across maturation. Therefore, upgrading forestry programs face the problem of selecting interesting traits during the mature age, while vegetative propagation is only possible during juvenile phases of development [2] . Thus, the decrease of morphogenic capacity with the ontogeny of the source material [3] , affect among others the rooting ability of plant tissue [4] being often a barrier for plant multiplication or regeneration. Several studies conducted in Pinus sp. indicate an ontogenetic gradient of shoot tips morphogenic competence, determined by the vertical localization in the tree crown, where basal meristematic shoot tips present higher morphogenic capacities than apical ones [5] - [7] . It has been suggested that the decline in morphogenic capacity could be due to the loss in competence at the cellular level and it is highly likely that this phenomenon causes changes in gene transcription [8] . In vitro propagation methods may induce reinvigoration of advanced physiological tissue and renewal of ontogenetically adult P. radiata [3] [6] , and in this way avoid the problems associated to the lost of morphogenic capacity. This reinvigoration includes anatomical, molecular and epigenetic changes which reflect characteristic juvenile individual’s protein patterns, DNA methylation and polyamine content [6] [9] . Altogether, the reversion of several adult phenotypic traits towards more juvenile stages and the recovery of tissue morphogenic capabilities allow the cloning of selected adult trees [6] [9] [10] . One tissue reinvigoration techniques is the in vitro micrograft, which has been tested to reinvigorate ontogenetically adult vegetative buds on juvenile rootstocks [11] and can be a solution for cloning adult trees of several species, in which sprouts present deficient rooting and lack of vigor [3] [4] .

It is known that during plant ontogenetic development, apical meristems exhibit significant morphophysiological changes, which reflect modifications in cellular competence to perceive or respond to external and internal signals, such as growth regulators [12] - [14] and environmental conditions [15] - [17] . These variations in cell sensitivity induce modifications in the capability to form adventitious buds and roots, but also involve changes in leaf anatomy, growth rates and photosynthetic functioning [4] [16] . Photosynthetic capacity has been commonly considered as plant overall performance indicator [18] - [20] . Light energy absorbed by chlorophyll can drive charge separations at photosystem II (PSII) reaction centers, triggering photosynthetic electron transport, which mostly drive carbon uptake. These reactions can be probed by chlorophyll a fluorescence measurements through a process called photochemical quenching. In this context, fluorescence measurement has become a physiological tool commonly used to evaluate plant stress responses [15] [21] . Specifically, maximal photochemical efficiency of PSII (Fv/Fm) is a parameter that quantifies the fraction of absorbed light that is used in photochemistry after a dark period long enough to obtain all PSII reactions centers to reach an open state (reaction center reduced and acceptors completely oxidized), allowing the determination of the maximal photochemical light use capacity [22] . Hence, Fv/Fm has been commonly assumed as a proxy of photosynthetic capacity, reflecting the photosynthetic apparatus condition and at the same time is considered an ideal monitor of plant health and viability [18] [20] [21] [23] - [25] . Recently chlorophyll fluorescence has become a common tool to assess the photosynthetic performance of in vitro cultured plants [26] - [29] . Thus, [30] reported higher photosynthesis rates in juvenile and rejuvenated than adult in vitro grown Sequoia sempervirens shoots. Photosynthetic performance has also been positively correlated with growth rates and survival of in vitro plants and with their viability to establish in ex vitro conditions [31] and has been used as an indicator of better in vitro acclimatization treatments [26] . We hypothesize that variations in fluorescence parameters such as Fv/Fm, ETR and NPQ are good indicators of micrograft’s viability and phenological stage. Accordingly, we inquire into the effects of the position from which the scion is obtained (basal vs. apical) in the ortet and the utility of fluorescence as an early estimate of P. radiata micrografts viability. To our knowledge, this is the first study that specifically addresses the early prediction of micrograft viability through the detection of scion’s photochemical capacity along in vitro reinvigoration process.

The micrograft’s viability varied between vertical positions of shoot tips in the ortet. Micrografts made from apical shoot tips shows higher viability than basal ones (P = 0.029; Figure 2, statistical details in Table 1 supplementary material). Thus, the 35% lower establishment of basal shoot tips may be due to several reasons; among them the effectiveness of asepsis of plant tissues introduced in in vitro conditions usually have a deep impact in their viability [36] . In this context, is important to note the different morphology exhibited between apical and basal scions (Figure 1(A) and Figure 1(B)). Apical scions present longer and less compact needles (Figure 1(A)). This may favor disinfection efficiency inducing greater micrograft viability and therefore establishment. On the contrary, basal scions showed evidently higher abundance, aggregation and shorter needles (Figure 1(B)). Otherwise, the concentrations of growth regulators in the shoot tips vary depending on their position in the ortet. [37] Reported higher concentration of auxins and cytokinins in apical shoot tips than basal ones, which may improve tissue responses to the in vitro introduction, specifically during P. radiata micrograft establishment [38] .

Regarding to maximal photochemical efficiency, significant differences in shoot tips Fv/Fm values were detected between the establishment and consolidation phases (Figure 3, statistical details in Table 2 supplementary material). Only during the first day of in vitro introduction, a decrease in Fv/Fm was observed in basal scions (P = 0.01; Figure 3). Such small Fv/Fm reduction could be attributable to the physiological stress imposed by the excision from ortet, and then the in vitro introduction [39] [40] . Despite this, we find a fast scions Fv/Fm recovery, due to the in vitro culture conditions, which also exert the surpass of Fv/Fm values measured in the ortet

from the second day. During the first two weeks of in vitro culture, in average basal scions had a more variable Fv/Fm and sometimes slightly lower than apical ones (P = 0.045; Figure 3), which agreed with their lowest micrograft establishment (Figure 2). Thereafter, during the beginning of consolidation phase (around 13 day), the trend was reversed. Apical scions significantly decreased their Fv/Fm (P < 0.001; Figure 3), even to lower values than those observed in the ortet. On the contrary, along in vitro culture basal scions displayed higher Fv/Fm during consolidation than apical ones (P < 0.001; Figure 3).

Additionally, during establishment PSII light energy partitioning of apical and basal scions were similar (lowest P = 0.435; Figure 4, statistical details in Table 3 supplementary material). Conversely during consolidation these ontogenetic younger (basal) and more plastic scions were able to increase ETR in 43% and NPQ in 100% (P = 0.046 and P = 0.019, respectively). The latter both are save valves to dissipate higher PSII excitation pressures, which are indicators of higher capacity to handle and take advantage of higher PPFDs. Increments by more than 100% in thermal dissipating capacity of excess light energy has been reported in ventilated vessels respect to traditional in vitro condition [29] . Regardless of vertical location in the ortet, shoot tips show similar values of ETR, indicating that in the field they display similar capacities to conduct absorbed light forwards photochemical processes. Interestingly, apical shoot tips in the ortet exhibited higher NPQ, therefore higher capacity to safely dissipate the excess absorbed energy as heat. A commonly response to high light is the increment in xanthophyll pool size (VAZ), allowing them manage the excess of absorbed light by means of heat dissipation [41] [42] . Together, these parameters could determine a greater ability to withstand the stress of transfer to ex vitro conditions [31] and may therefore be an indicator of greater potential physiological performance once a micrograft coming from basal section in the ortet is consolidated. Unfortunately, the low efficiency of micrografting of these basal scions is a big constraint for a reinvigoration program. Further studies on the behavior of these micrografted plants during transference to ex-vitro conditions are needed to fully address this problem.

The different responses among in vitro culture phases suggest that it is more feasible to establish micrografts in vitro from apical shoot tips than that from basal shoot tips. Hence they are physiologically younger, and theoretically they are synthesizing auxins and therefore are able to faster cellular differentiation and division, favoring rapid adaptation to in vitro conditions [13] [14] [37] [43] . This was reflected in a higher viability and photochemical efficiency during establishment. Basal shoot tips, even though they are ontogenetically younger, they are physiologically and chronologically older, have lower endogenous auxins concentration, and slower cellular de-differentiation rates [13] [14] [37] . However, once basal scions are established, they achieve greater consolidation, concomitant with greater Fv/Fm at the end of this phase, concomitantly with greater photochemical ca-

pacity, manifested through higher ETR and NPQ values. This higher photochemical activity is a proxy of higher CO₂ assimilation capacity, and therefore of greater photosynthetic performance and potentially faster growth [18] [20] [21] . Additionally higher Fv/Fm reflects an ontogenetic age gradient of shoot tips morphogenic competence, where basal meristematic shoot tips present higher morphogenic capacities than apical ones [5] [6] . Finally, based on our results, we conclude that Fv/Fm can be an indicator of the micrograft’s development according to the shoot tips position in the ortet and can be a useful indicator of the physiological stage of scions during micrograft transition from establishment to consolidation. This method is suggested to be suitable at low scale or laboratory characterization. It needs further scaling up if intended to use at larger scale such as productive forest nurseries.

The investigation was supported by MECESUP AUS 0103, MECESUP UCO 0214 grants. Materán MH thanks Universidad Nacional Experimental de Guayana-Venezuela and Escuela de Graduados Universidad de Con- cepción-Chile for scholarship. The Chilean National Commission of Scientific and Technological Research project PDA-24 through which RC was inserted into the academy.