COMPARATIVE LEAF STRUCTURAL ANALYSIS OF Nepeta nuda L. PLANTLETS, REGENERATED FROM CRYOPRESERVED SHOOT MERISTEM AND ex vitro -ADAPTED PLANTS

The leaf anatomy and chloroplast ultrastructure of Nepeta nuda L. plantlets regenerated in vitro from cryopreserved shoot apical meristem and in vitro -micropropagated plantlets were studied comparatively to assess whether cryoprocedure affected leaf morphogenesis. Both postcryo and in vitro plantlets failed to develop a distinguishable palisade layer, making the mesophyll appear homogeneous. Significant damage to the chloroplast envelope and substantial thylakoid ruptures were also observed. We assumed that the specific in vitro conditions more likely affected the structures than the cryotreatment itself. Light and transmission electron microscopy observations were also carried out on newly formed leaves of ex vitro -adapted plants. The examined leaf features were similar to those in the in situ plants – bifacial leaf lamina, double-layered palisade parenchyma, loosely arranged spongy parenchyma cells, and chloroplasts with intact envelope and evenly distributed throughout the stroma internal membrane system. The obtained histological and ultrastructural results revealed the retained morphogenetic potential of N. nuda plants and proved cryopreservation as a suitable method for long-term storage.


INTRODUCTION
. Comparative leaf structural analysis of Nepeta nuda l. Plantlets, regenerated from cryopreserved shoot meristem and ex vitro-adapted plants, Acta Sci. Pol. Hortorum Cultus, 22(4), 135-142. https://doi.org/10.24326/ asphc.2023.4791 2020, O'Brien et al. 2021]. Germplasm cryostorage has been reported for over 100 plant species in recent decades [Kaviani and Kulus 2022]. Currently, the scientific interest in this field has become even more comprehensive. Cryopreservation -the storage of plant germplasm in liquid nitrogen -is especially applicable for species in which micropropagation can be initiated from shoot tips [Zhang et al. 2023]. The shoot tips comprise organized structures and are genetically programmed to develop into "true-to-type" plants. Cryopreservation for long-term conservation of in vitro germplasm results in the exposure of tissues to physical, chemical, and physiological stresses that cause cryoinjury, so it is of great importance to assess the genetic stability of plants surviving cryostorage to determine if they are "true-to-type" after the procedure [Roque-Borda et al. 2021]. Moreover, cryopreservation induces processes associated with reactive oxygen derivatives, which may manifest later in regenerated plant tissues, causing cytological and histological alterations [Ren et al. 2021, Whelehan et al. 2022] and leading to problems for shoot development and whole plant recovery after cryostorage treatments [Kulus et al. 2018]. Some histological and genetic studies on surviving cryotreated shoot tips confirmed their potential for regeneration was preserved, but they did not further research the extent to which this potential is achieved [Halmagyi et al. 2017]. Ganeva et al. [2009] supposed that the cell and tissue structure of differentiated leaves from plants cultivated in vitro from successfully cryoprotected plant material was insufficiently investigated. Later studies of in vitro regenerated plants after cryopreservation found that in the most critical photosynthetic organs, the leaves, there are deviations in the mesophyll structure and chloroplast ultrastructure, and these alterations are species-specific, which confirms the need to continue the research. These works revealed that examinations at the histological, cellular, and subcellular levels were necessary for evaluating plant regeneration capacity in vitro and during ex vitro acclimatization [Stoyanova- Koleva et al. 2013Koleva et al. , 2015. Nevertheless, many open questions that refer to the structural and functional status of the plants regenerated from recovered after cryo-treatment meristem remain and deserve further analysis.
In this study, we aimed to evaluate the morphogenetic capacity of N. nuda plantlets regenerated in vitro from cryopreserved shoot tips and then adapted ex vitro to assess whether this method was suitable for long-term storage of N. nuda germplasm. To achieve this goal, we examined and compared the leaf histological features and the chloroplast ultrastructure of plantlets regenerated in vitro from cryopreserved meristems, ex vitro-adapted plants, and in situ plants.

Plant material
Nepeta nuda L. plants in the flowering phase were collected from a natural locality in Lozen Mountain [42.584722, 23.5167524], Bulgaria. The voucher specimen SO-105807 was deposited in the Herbarium of Sofia University "St. Kliment Ohridski." Plant multiplication and maintenance in vitro, cryopreservation by the vitrification method, postcryo in vitro regeneration, and ex vitro adaptation were carried out in the Laboratory of Plant Biotechnology, Department of Plant Physiology, Faculty of Biology, University of Sofia "St. Kl. Ohridski" [Dragolova et al. 2014. The examined variants were as follows: 1) in situ N. nuda plants (in situ plants); 2) in vitro cultivated 5-week-old plantlets (in vitro plantlets); 3) 5-week-old plantlets recovered in vitro from cryopreserved meristems (postcryo plantlets); 4) plants adapted ex vitro in a greenhouse from in vitro cultivated plantlets approximately three months after transferring them to soil (ex vitro plants); and 5) plants adapted ex vitro in a greenhouse from regenerated in vitro after cryopreservation plantlets approximately three months after transferring them to soil (postcryo ex vitro plants).

Statistical analysis
Ten microphotographs of each variant were used to measure the thickness of the leaf lamina (LL), palisade parenchyma (PP), spongy parenchyma (SP), adaxial (AdE), and abaxial (AbE) epidermises. Thirty measurements for each parameter and each variant were made with ImageJ software. The statistical analysis was performed using a one-way analysis of variance (ANOVA), followed by the comparison of group means (Tukey test, p < 0.05) with the program SigmaPlot.

Transmission electron microscopy (TEM)
For ultrastructural analysis of the mesophyll chloroplasts, leaf segments (1-2 mm 2 ) were fixed in 3% (m/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) and postfixed in 1% (m/v) KMnO 4 in the same buffer for 2 h at room temperature. After dehydration by increasing concentrations of ethyl alcohol (from 25 to 100% v/v), the samples were embedded in Durcupan (Fluka, Switzerland) and sectioned with a Reichert-Jung (Wien, Austria) ultramicrotome. The ultrathin (50-60 nm thickness) samples were mounted on copper grids and double-stained with uranyl acetate and lead citrate. A JEM-2100 (JEOL Ltd. Tokyo, Japan) electron microscope performed observations and documentation of the results.

RESULTS
The transverse section of in situ N. nuda leaves revealed bifacial leaf lamina with true-to-type organized mesophyll (Fig. 1A1). The palisade parenchyma was double-layered, composed of cells with a typical cylindrical shape and many peripherally located chloroplasts. The cells were longer in the upper layer than in the lower layer. The spongy parenchyma consisted of 3-4 rows of loosely arranged cells with irregular shapes. Despite the large intercellular spaces, the spongy tissue was 23% less thick than the palisade parenchyma. Both adaxial and abaxial epidermises were composed of ordinary cells, stomata, and two types of trichomes -non-glandular and glandular (Fig. 1A2). The outer walls of the ordinary epidermal cells of the adaxial epidermis were convex. Additionally, the thickness of the adaxial epidermis was greater than that of the abaxial epidermis. According to the measurements, the in situ plants developed the thickest leaf blade, palisade, and spongy parenchyma (Tab. 1).
Predictably, there were significant differences in the histological organization and the measured features between the in situ plants and both types of in vitro plantlets but not between them (Fig. 1B, 1C, Tab. 1). The leaf lamina was approximately threefold thinner in the in vitro plantlets and three and a half times thinner in the postcryo plantlets. In both in vitro variants, the palisade parenchyma consisted of only one row of concise cells with round or irregular shapes. The spongy tissue comprised 2-3 rows of compactly arranged oval-shaped cells. However, the thickness of the spongy parenchyma was 66% greater than that of the palisade parenchyma. The observed mesophyll organization was difficult to accept as bifacial. The epidermises were thinner than in the in situ leaves, and usually, the outer walls of the adaxial ordinary cells were flat and even slightly concave.
The leaf histological organization of the ex vitroadapted plants' two variants was very similar and slightly different from that of the in situ plants (Fig. 1D,  1E). The palisade parenchyma was also double-layered, but the cells in the lower row were relatively short and often hourglass-shaped, especially in the postcryo ex vitro plants. However, the thickness of the palisade parenchyma was almost the same as that in the in situ plants. In both ex vitro variants, the spongy parenchyma was almost twofold thinner than the palisade tissue. Compared with the in situ leaves, the spongy cells were more compact, reducing the volume of the intercellular spaces. The latter decreased the lamina thickness of the ex vitro leaves compared with the in situ leaves (Tab. 1). The thickest abaxial epidermises were measured in both ex vitro variants, although marked differences in the structure of the epidermis in comparison with the in situ plants were not observed.
TEM analysis of the mesophyll cells from the in situ plants identified chloroplasts with a typical oval shape. The internal membrane system occupied the entire volume of the stroma and was composed of medium-high grana (7-15 lamellae) connected by parallel-oriented long stromal thylakoids. Starch grains were observed in the stroma. Plastoglobules were absent ( Fig. 2A).
The chloroplasts in the in vitro plantlets were elongated to oval-shaped. The chloroplast envelope was impaired. The thylakoid membranes in the central zone of the stroma were destroyed, so it was not easy to distinguish apparent granal stacking. The granal thy-Stefanova, M., Ganeva, T., Koleva, D., (2023). Comparative leaf structural analysis of Nepeta nuda l. Plantlets, regenerated from cryopreserved shoot meristem and ex vitro-adapted plants, Acta Sci. Pol. Hortorum Cultus, 22 (4)  Stefanova, M., Ganeva, T., Koleva, D., (2023  lakoids in the periphery were fused, while the stromal thylakoids were partially preserved. (Fig. 2B). The chloroplasts in the postcryo plantlets' mesophyll cells were also elongated to oval. The chloroplast envelope membranes appeared intact, while the thylakoid membranes were almost completely fused or destroyed. Only a few stromal thylakoid membranes in the chloroplast periphery seemed partially preserved (Fig. 2C).
The ex vitro-adapted plants had typical oval-shaped chloroplasts. The chloroplast envelope was entire, and the internal membrane system was evenly distributed throughout the chloroplast stroma. The grana were of different heights and consisted of 3-4 to 17-20 thylakoids. Few stromal thylakoids were observed. Single starch grains were present in the stroma (Fig. 2D). In the postcryo ex vitro plants, the chlo-roplast structure was also somewhat restored during ex vitro adaptation. The internal membrane system occupied the entire volume of the stroma except for the space in which there were starch grains. The grana were low, some thylakoids were fused, and the stromal thylakoids were few and loosely situated in the stroma (Fig. 2E).

DISCUSSION
In fully secured shoot tips, the dome meristem and the leaf primordia can survive storage in liquid nitrogen [Kulus et al. 2019]. The possible injuries that might occur at the cellular and subcellular levels during cryopreservation may become evident sometime later and Stefanova, M., Ganeva, T., Koleva, D., (2023). Comparative leaf structural analysis of Nepeta nuda l. Plantlets, regenerated from cryopreserved shoot meristem and ex vitro-adapted plants, Acta Sci. Pol. Hortorum Cultus, 22(4), 135-142. https://doi.org/10.24326/ asphc.2023.4791 could directly affect morphogenesis during subsequent regeneration and ex vitro acclimatization [Halmagyi et al. 2017[Halmagyi et al. , 2022. For example, histological studies of regenerated Hypericum rumeliacum and Orthosiphon stamineus plantlets after cryopreservation reported cell plasmolysis, the collapse of the photosynthetic tissue, and destruction of some intracellular membranes [Ganeva et al. 2009]. However, in our study, such severe damage to leaf tissues was not observed in the postcryo N. nuda plantlets. The anatomical characteristics of the postcryo leaves were strongly reduced thickness of the lamina and its tissues, dense uniformly structured mesophyll -poorly differentiated palisade parenchyma, and devoid of intercellular spaces spongy parenchyma. Compared to the in situ N. nuda leaves, these characteristics were significantly different but not specific -the same histological organization had the leaves of N. nuda in vitro plantlets. In our opinion, that structure was probably a consequence of the specific in vitro conditions. Moreover, similar morpho-and histological variations, such as a thin palisade layer and dense spongy parenchyma, have been observed in some other species propagated in vitro, which was an adverse effect of the high sugar content and addition of plant growth regulators in the medium [Sáez et al. 2012, Suárez et al. 2019.
Transmission electron microscopy of the chloroplasts expanded the analysis of mesophyll organization and provided information about the regenerative capacity of proplastids in the leaf primordia and the extent to which they could differentiate into normal chloroplasts. In N. nuda postcryo plantlets and in vitro plantlets, the chloroplasts were slightly flattened and smaller compared with the in situ ones. Although the shape remained typical, significant damage to the chloroplast envelopes and substantial thylakoid ruptures were observed. The specific in vitro cultivation conditions had an apparent adverse effect on the structural organization of the membranes. According to Sáez et al. [2012], impaired chloroplast ultrastructure was probably a consequence of cultivation under insufficient irradiance in vitro or exogenous sucrose in the medium.
In N. nuda cryo-plantlets, the damage to the chloroplasts was more prominent. That was not a unique phenomenon. Similar structural disorders, such as altered chloroplast shape, destroyed envelope, a relatively small volume of the thylakoid system, and even destroyed granal and stromal thylakoids or specific spatial orientation without any destruction of the thyla-koid membranes, were observed in the leaf mesophyll of Hypericum plantlets regenerated in vitro after cryopreservation [Skyba et al. 2012, Stoyanova-Koleva et al. 2013, Stoyanova-Koleva et al. 2015. We presumed that cryo treatment of the apical meristem negatively affected the proplastids and that they could not differentiate into chloroplasts with proper structure. Halmagyi et al. [2017Halmagyi et al. [ , 2022 considered that the histological and ultrastructural analysis of shoot tip cells of recovered plants after ex vitro acclimatization will bring added value to the existing studies. Proceeded by the authors, TEM observations on tomato shoot apex cells from acclimatized plants showed a regular ultrastructure, which suggested regular histo-and organogenesis. Our study of N. nuda went further by examination of ex vitro differentiated leaves. Despite the atypical structure of the mesophyll in vitro and the fact that the chloroplasts in both variants were entirely damaged, N. nuda plants showed high plasticity and regenerative ability and adapted ex vitro. The histological analysis of the newly formed leaves of the ex vitro plants revealed that their anatomical structure corresponded to that found in plants in situ. The only significant difference between the in situ and the ex vitro plants was the smaller lamina thickness, palisade, and spongy parenchyma in the latter variants.
According to Khoshravesh et al. [2022], the average structural characteristics of the mesophyll suggested the probability of photosynthetic efficiency and better water regulation capacity of the plants. The chloroplast ultrastructure of N. nuda ex vitro and postcryo ex vitro plants was generally similar to that of the in situ plants. As a confirmation of preserved photosynthetic capacity, starch grains in their stroma were observed.

CONCLUSIONS
We could assume that the impaired mesophyll structure and chloroplast integrity in vitro were not a result of cryo-damage of the meristematic cells but most likely were caused by the cultivation conditions themselves. The structural changes of the leaves in vitro were overcome in the ex vitro adaptation process. The observed leaf organization and chloroplast ultrastructure of all ex vitro-adapted plants could be considered a structural marker for regular histogenesis Stefanova, M., Ganeva, T., Koleva, D., (2023). Comparative leaf structural analysis of Nepeta nuda l. Plantlets, regenerated from cryopreserved shoot meristem and ex vitro-adapted plants, Acta Sci. Pol. Hortorum Cultus, 22(4), 135-142. https://doi.org/10.24326/ asphc.2023.4791 of the newly formed leaves and a sign of remarkable phenotypic plasticity of the species. Cryopreservation of N. nuda meristems with high recovery capacity could benefit large-scale micropropagation projects for this plant of great medicinal interest.

SOURCE OF FUNDING
This research received no external funding.