Direct regeneration and morpho-histological study of de novo shoot development from leaf explants of Rhododendron mucronulatum Turcz.

Rhododendron mucronulatum Turcz., a rare medicinal and ornamental deciduous multi-branched shrub native to northern parts of East Asia, is a valuable genetic resource for breeding and biotechnological producing bioactive metabolites. To create propagation system of R. mucronulatum, an e�cient method of direct shoot regeneration from leaf explants based on thidiazuron (TDZ) and dark treatment was developed, and detailed histological analysis for revealing localization of cell competent to morphogenesis was carried out for the �rst time. The highest regeneration rate (75%) and number of shoots per explant (an average of 51.7) were achieved under 0.5 µM TDZ and dark treatment. The dark treatment was found to increase the regeneration rate more than two-fold compared to culturing under light conditions. The histological analysis showed direct organogenesis pathway. The competent to morphogenesis cells were located in epidermal tissue of leaf explants on adaxial side of petiole next to main vein. Two sites of beginning of TDZ-induced adventitious shoot formation were revealed: the shoot formation directly from epidermal tissues and shoot formation from de novoepidermis-derived protrusions. The formation of embryo-like structures was observed on the late morphogenesis stages. It was discovered that parenchymal and epidermal tissues of embryo-like structures were separated from explant tissue but its vascular bundle was connected with vascular bundle of explant. These structures gave rise to development of adventitious shoots. The result obtained can promote further establishment of e�cient and stable systems for genetic transformation and biomass production of R. mucronulatum.


Introduction
Rhododendron mucronulatum Turcz. is a rare medicinal and ornamental deciduous multi-branched shrub native to northern parts of East Asia.In traditional and folk medicine, R. mucronulatum flowers and leaves have been known to have high pharmacological potency due to rich content of bioactive compounds such as phenolic compounds and essential oils [1][2][3].It blooms before foliage expansion; it's single-flowers gathered in 3-6 at the ends of the shoots.R. mucronulatum corolla color is varied from pale reddish purple, lilac-pink to white among different individuals.Moreover, R. mucronulatum is frost-resistant species what makes it promise genetic resource for breeding.
Application of genomic techniques provides great tools for the improvement of Rhododendron traits [4,5].The leaf explant culture is the most promising platform for genetic improvement through Agrobacterium-mediated transformation, isolation and transfection of protoplasts or microprojectile bombardment [6].For obtaining transgenic genotypes, it is necessary to transfer new genes to the leaf cells being able to de novo morphogenesis [7].In this regard, the study of the regenerative potential of leaf explants, the identification of the localization of competent cells and the possibilities of managing the program of further morphogenesis is a necessary condition for developing the standardized protocols of genetic transformation and large-scale propagation of new genotypes with improved properties.At the same time, the success of in vitro regeneration is depended on plant genotype as well as exogenous plant growth regulations (PGRs), which reprogram the fate of explant cells to the ability to differentiate with subsequent implementation of either the de novo organogenesis or somatic embryogenesis.
Among the PGRs used, thidiazuron (TDZ) is considered the most powerful synthetic PGR for in vitro reprogramming cell developmental fate initiating a wide variety of morphogenic responses [8,9].It was noted that TDZ-induced in vitro morphogenesis from Rhododendron leaf explants could occur through direct [10,11], indirect organogenesis [12,13] as well as somatic embryogenesis [14].Moreover, it was reported that TDZ can simultaneously induce the development of somatic embryos and adventitious shoots on leaf explants of Gaylussacia brasiliensis from Ericaceae family [15].It is well known that a variety of morpho-physiological anomalies in microshoots arise during TDZ-induced regeneration [16], therefore, it is often not possible to exactly identify the morphogenesis pathway when using morphological observations only [17].The histological studies of initial morphogenesis stages of R. catawbiense cv.Grandiflorum showed the formation of embryo-like strictures and revealed the connection of its vascular system with vascular system of leaf explant and the lack of a root apex [18].The histological analysis of TDZ-induced regeneration from leaf explants of evergreen R. delavay showed that the direct morphogenesis was also accompanied by the formation of globular structures connected with explant by jointed vascular system [19].Thus, although these de novo structures looked like somatic embryos under the stereo microscope, histological examination revealed only direct regeneration of adventitious shoots.
Rhododendrons were known to include evergreen, semi-deciduous and deciduous plants, but successful standardized protocols based on triggering TDZ-induced morphogenesis from leaf explants were developed mainly for evergreen varieties [12, 19 -21].According to our data, histological studies of regeneration from leaf explants of wild deciduous rhododendrons such as R. mucronulatum have not been carried out.TDZ-based regeneration systems for conservation and in vitro propagation of R. mucronulatum from nodal explants [22,23] as well as seedlings [24] have already been developed, but deep research of the regenerative potential of R. mucronulatum leaf explant under TDZ has not been conducted.
During in vitro regeneration, PGRs not only affect the shoot regeneration and biomass production, but also, they have a great impact on the synthesis of a variety of bioactive compounds [25].In this regard, there has recently been a tendency to use PGRs for enhancing secondary metabolism and increasing biomass production in tissue culture of medicinal plants.Among PGRs tested, TDZ was one of the most powerful elicitors for accumulation of bioactive secondary metabolites in a broad range of medicinal plants including Tecoma Stans [26], Linum usitatissimum [25], and Plectranthus amboinicus [27] as well as R. mucronulatum [28].Therefore, TDZ-induced regeneration protocols from leaf explants may be a start platform for research into secondary metabolism physiology in R. mucronulatum.
The present study is aimed to develop an efficient method of direct regeneration and micropropagation of R. mucronulatum using leaf explants, to identify the localization of competent cells and to conduct a detailed morpho-histological analysis of the morphogenesis pathway.The results obtained can promote further establishment of efficient and stable genetic transformation system to support the application of genetic engineering in Rhododendron genus as well as development of protocols for R. mucronulatum biomass production with high concentration of target bioactive compounds.

Materials and Methods
Plant materials and culture conditions.In vitro shoots of R. mucronulatum cultivated on Anderson's medium (AM) [29] supplemented with 1.0 μM zeatin.Before starting the experiment, the microshoots were maintained on PGRs-free Anderson's medium (AM0) for 4 wk.
Initiation of shoot regeneration from leaf explants.The two upper leaves with petioles were cut out from the R. mucronulatum microshoots under stereoscopic microscope (Lomo, MSP-1 var.1,St. Petersburg, Russia) and used as the leaf explants for current experiments.The leaf explants were placed horizontally by adaxial side up on AM supplemented with different TDZ (plant cell culture tested, BioReagent, Sigma-Aldrich®) concentration (0.1; 0.25; 0.5; 1.0; 2.5; 5.0; 10.0 μM) or AM0 for the control.The explants incubated either in the darkness (dark treatment) or under cool-white fluorescent light (40 µmol m -2 s -1 ) at 16-h photoperiod.After 12wk cultivation, the regeneration percent was counted.Then all explants were transferred to AM0 under cool-white fluorescent light for elongation.After 8 wk of elongation, the number of shoots (over 5 mm height) per explant and shoot length were estimated.
Morpho-histological analysis.For morpho-histological study, the leaf explants cultured on AM supplemented with 0.25 µM TDZ were collected at 0, 4, 7, 12, 20, 46, and 56 days from start of experiment and at 18th day of elongation stage.Sampling and preparing microslides for histological observations were realized according to previous studies [23].The samples were fixed in mixture of glacial acetic acid, formalin, and ethyl alcohol (7:7:10), dehydrated in ethanol series and embedded in Paraplast (Sigma-Aldrich, USA).Embedded samples were sectioned by microtome (HM-325 Microm, Walldorf, Germany) at 5 μm and stained with 0.05% toluidine blue (Sigma-Aldrich, USA) for 1 min.Axioskop-40 equipped by AxioCam MRc5 with AxioVision 4.8 software (all from Carl Zeiss, Germany) was used for analyzing stained samples.Statistical analysis.Three replications consisted of 15 explants were analyzed in all experiments.Collected data were examined by oneway analysis of variance (ANOVA) with use of STATISTICA 8 software (StatSoft Inc., Tulsa, OK).All data were presented as means with standard errors (M ± SE) and processed by Duncan's means separation test (P = 0.05) for revealing significance between the means.

Results and Discussion
The effect of TDZ on direct shoots regeneration.Under cool-white lighting conditions, the maximum morphogenic response (15%) was obtained in the presence of 0.25 µM TDZ (Table 1).The regeneration rate of explants reduced with increasing TDZ concentration in AM.Tested TDZ concentrations induced direct morphogenesis pathway.The clusters of adventitious buds and structures morphologically similar to somatic embryos were formed on petiole (Fig. 2e).
After 12 wk of cultivation on TDZ-containing AM, the regenerants represented the clusters of fused short shoot primordia or embryolike structures (less than 5 mm in height).However, subsequent 8-week elongation of de novo clusters on AM0 resulted to the development and growth of shoot with normal structure.The maximum number of shoots per explant (an average of 40.6) with shoots reaching an average of 3.3 mm in height was obtained under 0.25 µM TDZ (Table 1).
It was shown that 1.0 µM TDZ was the best trigger of direct morphogenesis in the leaf explant culture among evergreen R. tomentosum, R. catawbiense 'Grandiflorum' and R. s semi-deciduous wild species [21,18].It was reported that 0.36 µM TDZ promoted direct organogenesis and higher regeneration rate in evergreen R. delavayi leaf explants [30].In R. mucronulatum leaf explants, which belongs to deciduous representatives of the Rhododendron genus, the effectiveness of a low concentration of TDZ (0.25 µM) was demonstrated, while the presence of 1.0 µM TDZ in the medium did not cause regeneration and led to explant tissue necrosis.Moreover, the regeneration potential of R. mucronulatum leaf explants were found to be significantly lower than of rhododendron genotypes previously studied.The obtained data confirm the occurrence of genotypic differences of in vitro morphogenic responses among evergreen, semi-evergreen, and deciduous rhododendron groups.
The effects of dark treatment on TDZ-induced shoot regeneration.The cultivation in darkness for 12 wk (dark treatment) was used to enhance the morphogenetic response along with TDZ treatments, since TDZ-induced regeneration from R. mucronulatum leaf explants had a low rate of regeneration.Direct shoot regeneration without callus formation was observed under darkness and tested TDZ concentration.As a result, the use of 0.25 µM TDZ and dark treatment increased the percentage of shoot regeneration more than twofold (up to 37%) compared to cultivation only in the light condition (Table 1).However, the maximum regeneration percentage (75%) and the number of shoots per explant (on average 51.7) were obtained under 0.5 µM TDZ (Fig. 2b).The same TDZ concentration led to the development of the maximum height of shoots after elongation stage under lighting conditions.Darkness-derived shoots clusters were white color (Fig. 1a).After transferring to AM0 under cool-fluorescent light, de novo shoot clusters elongated and turned green (Fig. 2b).Thus, the dark treatment significantly enhanced the effect of TDZ on direct regeneration rate from R. mucronulatum leaf explants as well as number of shoots per explant compared to the cultivation only in lighting conditions.It is generally known, that changes in plant physiology and morphogenesis depending on light intensity or quality are regulated by plant hormones [31][32][33].In this study, enhance in adventitious bud proliferation may be due to increasing natural auxin levels at lower light intensity or in darkness [34].In addition, the process of oxidation of phenolic compounds, which takes place during isolation of explants and at the first stages of in vitro cultivation, is significantly reduced in the dark [35], which leads to a decrease in necrotic changes of explant tissues and, consequently, an increase in their survival and regenerative potential.
Morpho-histological analysis of morphogenesis pathway.The histological analysis revealed that TDZ at the tested concentrations induced direct morphogenesis of adventitious buds and structures similar to somatic embryos from R. mucronulatum leaf explant.The initial cell divisions occurred in the epidermal tissue in adaxial side of petiole.Further intensively divisions of these cells led to formation of small protrusions on the leaf explant (Fig. 2 ).On the 12th day of the experiment, the meristematic centers originated from upper layers of protrusions (Fig. 2b).Simultaneously, the meristematic centers were formed directly from epidermal cells of explants (Fig. 2c).On day 20, the divisions of cells of meristematic centers gave rise to formation of embryo-like structures (Fig. 2d).When analyzing a series of histological sections, it was revealed that these structures had joining vascular system with the explant, and root apex was absent (Fig. 2e).Thus, the bipolar structure typical for somatic embryo was not formed.On 46th day, the formation of shoots primordia and activation of de novo axillary meristems were noted (Fig 2g to e).Moreover, while further cultivating bud clusters on TDZ-containing AM, the formation of embryo-like structures was detected (Fig 3a to c).A characteristic property of TDZ-induced embryo-like structures on leaf explants of R. mucronulatum was found to be the anatomical isolation of epidermal and parenchymal tissues of de novo shoot primordium from explant tissues (Fig. 2f, 3b), which was made such structures morphologically similar to somatic embryo (Fig 2d, 3 ), however, further development of these structures resulted in direct shoot formation only.After cultivation on a TDZ-containing medium, de novo bud clusters were transferred to hormone-free media for elongation.On TDZ-free medium, further formation of adventitious buds as well as activation of axillary meristems of newly formed adventitious shoots occurred bypassing both protrusions and embryo-like structures development (Fig 3d to f).As a result, the secondary formation of adventitious shoots and the activation of axillary meristems during elongation stage significantly increased the multiplication efficiency.
The identification of cells competent to morphogenesis is the most important challenge of histological analysis.The cells competent to regeneration were found to locate directly in the epidermal tissues of adaxial side of petiole of R. mucronulatum leaf explant next to cut side.The same location of cells competent to morphogenesis has been revealed in R. sichotense and R. catawbiense 'Grandiflorum' leaf explants [18].However, in R. delavayi, the development of meristematic cell clusters started from the swelling cells at the site of wounding leaf blade, but epidermal cells in petiole and the base of the leaf blade of R. delavayi were found not to able to regenerate de novo shoots.[19].The stimulatory effect of wounding was found to be due to initiating wounding-triggered molecular cascade of cell reprogramming well-studied in Arabidopsis [36,37].In presented study, the localization of initial cell division on petiole of R. mucronulatum leaf explants could also be associated with wounding during explant isolation.Thus, the localization of its initial stages of morphogenesis can be modulated not only by the TDZ treatment, but also wounding during explant isolation.
A high morphogenic potential of epidermal cells of various plant organs has been shown in wide range of plant taxa [38][39][40].In R. mucronulatum leaf explants, two sites of beginning of TDZ-induced adventitious shoot formation were revealed: (1) the shoot formation directly from epidermal tissues of explant and (2) shoot formation from de novo epidermis-derived protrusions.In Rhododendron genus, shoot morphogenesis mediated by TDZ-induced protrusion formation was observed in R. sichotense [18], R. dauricum [23] and R. delavayi [19].Moreover, the TDZ-induced formation of protrusions from epidermal tissues of leaf explants has been also recorded in Agave angustifolia [41], Cibotium barometz [42], Pteris aspericaulis [43].It is not yet completely clear which tissue cells form these protrusions.Contrary to callus or meristematic cells, the protrusions were formed by small cells contained a small nucleus.However, it was found that the ells of these protrusions were arranged neatly and stained deeply which indicated its pluripotency [19].It has been reported that high TDZ concentrations initiate somatic embryogenesis, and low -de novo organogenesis [44].Thus, varying TDZ concentration can obtain totipotent or pluripotent cells.Moreover, in Gaylussacia brasiliensis leaf explants, the same concentrations of TDZ induced a dual morphogenesis pathway, when small protrusions on the adaxial leaf surface developed either into adventitious shoots or somatic embryos [15].Thus, G. brasiliensis protrusion cells exhibited both pluri-and totipotency under the action of TDZ.Thus, epidermal reprogramming occurred since fully differentiated epidermal cells transformed into meristem cells or de novo structures [45].In R. mucronulatum, tested TDZ concentration triggered pluripotency in epidermal cells of leaf explants as well as in cells of de novo protrusions.
In the presented study, histological analysis revealed direct regeneration of adventitious shoots from R. mucronulatum leaf explant; however, the shoot regeneration also occurred through formation of embryo-like structures.It is well known that the main distinguishing features of somatic embryos are the formation of a bipolar structure including the apex of shoot and root, as well as the presence of its own vascular system separated from the explant tissues.However, the embryo-like structures are only morphologically similar to somatic embryos, and at the histological level differ from them by the presence of only shoot apex and the vascular bundle connected to the explant.Thus, the further growth of embryo-like structures results in organogenesis and development of adventitious shoots.Therefore, the execution of histological analysis of regeneration processes is a necessary condition for identification of the morphogenesis pathway [17,46,47].The development of embryo-like structures under the action of TDZ has been observed in Crocus sativus [48], Lilium spp.[49], Portulaca pilosa [50], Hedychium forrestii [51], and Heliotropium foertherianum [52].Moreover, TDZ-induced embryo-like structures were noted during regeneration from leaf explants of R. sichotense R. catawbiense 'Grandiflorum' [18], and from floral explants of R. dauricum [23].Regardless of the type of explant, parenchymal and epidermal tissues of TDZ-derived embryo-like shoot primordia in rhododendrons were separated from explant tissues, but the connection with explant tissues was maintained through a thin vascular bundle.The formation of an embryo-like structure can be associated with triggering TDZ-induced "abscission" of parenchymal and epidermal tissues, since TDZ has been found to have a property of initiating formation of leaf abscission zone as familiar defoliant [9].Thus, the formation of embryo-like structures during TDZ-induced morphogenesis from R. mucronulatum leaf explants is an abnormality of adventitious shoot development.

Conclusions
To summarize, e cient method of direct regeneration and in vitro propagation of deciduous R. mucronulatum from leaf explants using TDZ and dark treatments was developed, as well as detailed histological analysis of morphogenesis pathways was carried out for the rst time.The maximum regeneration rate and the number of shoots per explant were obtained under the action of 0.5 µM TDZ and dark treatment.The cultivation in the darkness doubled the effect of TDZ and increased the e ciency of regeneration.Histological analysis revealed direct regeneration of adventitious shoots from R. mucronulatum leaf explant.The cells competent for morphogenesis were located in the epidermal layer of the adaxial side of the leaf explant in the petiole.Two sites of beginning of TDZ-induced adventitious shoot formation were revealed: (1) the shoot formation directly from epidermal tissues of explant and (2) shoot formation from de novo epidermis-derived protrusions.The formation of embryo-like structures with joined vascular bundle with explant was noted.However, the further growth of embryo-like structures resulted in organogenesis and development of adventitious shoots.The result obtained can promote further establishment of e cient and stable genetic transformation system to support the application of genetic engineering in Rhododendron genus as well as development of protocols for R. mucronulatum biomass production.

Declarations
Tables Table 1.The of TDZ and lighting conditions on regeneration from leaf explants of Rhododendron Figures