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).
Table 1
The effect of TDZ and lighting conditions on regeneration from leaf explants of Rhododendron mucronulatum. Means followed by the same letter are not significantly different according to the Duncan test (p = 0.05). Means ± SE
Lighting conditions
|
TDZ concentration, µM
|
% Regeneration
|
Shoot number per explant
|
Shoot, length, mm
|
light
|
0
0.1
0.25
0.5
1.0
2.5
5.0
10.0
|
0
14
15
11
0
4
4
4
|
-
7.0 ± 2.4 de
40.6 ± 5.7 b
21.8 ± 4.9 c
-
0
0
0
|
-
10.0 ± 0.7 c
23.3 ± 5.8 a
19.8 ± 4.6 ab
-
-
-
-
|
darkness
|
0
0.1
0.25
0.5
1.0
2.5
5.0
10.0
|
0
24
37
75
40
23
0
0
|
-
5.3 ± 1.1 e
8.2 ± 1.1 d
51.7 ± 12.1 a
42.2 ± 6.2 ab
0
-
-
|
-
11.0 ± 1.0 c
10.5 ± 4.5 c
20.2 ± 4.7 ab
17.8 ± 5.8 ab
-
-
-
|
After 12 wk of cultivation on TDZ-containing AM, the regenerants represented the clusters of fused short shoot primordia or embryo-like 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 previously shown that 1.0 µM TDZ is an effective trigger of direct morphogenesis in the leaf explant culture among evergreen rhododendrons such as R. tomentosum, R. catawbiense ‘Grandiflorum’ and semi-deciduous wild species such as R. sichotense (Jesionek et al. 2016, Zaytseva et al. 2016). However, Tian et al. (2020) used 0.36 µM TDZ to achieve direct organogenesis and higher regeneration rate in evergreen R. delavayi leaf explants. In the present study on the regeneration potential of 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, that allows considering this species as recalcitrant to de novo regeneration. 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. As a result, the use of 0.25 µM TDZ and dark treatment increased the percentage of regeneration more than two-fold compared to cultivation only in the light (Table 1). However, the maximum regeneration rate (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. The use of dark treatment coupled with TDZ triggered direct morphogenesis on the petiole of the leaf explants which was identical to that under cool-fluorescent light. Darkness-derived shoots clusters were white color (Fig. 1a). Transferring clusters to AM0 for elongation under cool-fluorescent light forwarded recovery of normal coloring de novo shoot (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 (Afshari et al. 2011; Dietz 2015; Kissoudis et al. 2017). In this study, the reason for this increased adventitious bud proliferation may be due to increasing natural auxin levels at lower light intensity or in darkness (Chory et al. 1994). 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 (Taranto et al., 2017), 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 divisions were found to occur in the epidermal cells of leaf next to petiole the adaxial side of the region near the main vein of leaf explant. The small protrusions on the surface of the explant were resulted from intensively divisions of cells (Fig. 2а). On the 12th day of the experiment, the laying of meristematic centers in the protrusions was noted (Fig. 2b). At the same time, meristematic centers formed directly from epidermal cells of explants (Fig. 2c). On day 20, the embryo-like structures was developed from the meristematic centers (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 developing of shortened bud clusters noted on 57th day of the cultivation on TDZ-containing AM, the formation of the same embryo-like structures were 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 made such structures morphologically similar to somatic embryo (Fig. 2d, 3а). However, based on the morpho-histological analysis, studied morphogenesis pathway was referred to direct organogenesis.
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 localization of cells competent to regeneration is the most important challenge of histological analysis of morphogenesis from leaf explants. The cells competent to regeneration were found to locate directly in the tissues of epidermis on the adaxial side of petiole of R. mucronulatum leaf explant next to cut side. The same location of cells competent to regeneration has been revealed in R. sichotense and R. catawbiense ‘Grandiflorum’ leaf explants (Zaytseva et al. 2016). In R. delavayi, another scenario of TDZ-induced morphogenesis from partly incised leaf lamina was shown. The regeneration of adventitious shoots started from the swelling cells at the site of wounding leaf blade what can be identified as dedifferentiation stage, and then redifferentiation and acquisition of regeneration competence by the swelled cells resulted in the formation of protrusions and further development of meristematic cell clusters (Peng et al. 2022). Moreover, the epidermal cells in petiole and the base of the leaf blade of R. delavayi were found not to able to regenerate de novo shoots. The wounding-triggered molecular cascade leading to cell reprogramming has been extensively studied in Arabidopsis (Ikeuchi et al. 2017, Iwase et al. 2017). Thus, the morphogenesis scenario in the leaf explant culture and the localization of its initial stages can be modulated not only by the TDZ, but also mechanical impact during explant isolation.
A high morphogenic potential of epidermal cells of deferent plant organs have been shown in wide range of plant taxa (Chen and Chang 2006, de Araújo Silva-Cardoso et al. 2020, Zhao et al. 2022). Moreover, the TDZ-induced formation of protrusions from epidermal tissues of leaf explants is frequent finding not only in rhododendrons and has been also recorded in Agave angustifolia (Monja-Mio et al. 2015), Cibotium barometz (Yu et al. 2017), Pteris aspericaulis (Yu et al. 2021). The TDZ-derived protrusions were formed by small cells, these cells did not contain big nuclear inherent in meristematic cells, and they are dissimilar to callus cells. So, the tissue affiliation of these formations has not been fully clarified still and yet to be studied. It has been found that the сells of these protuberances were arranged neatly and stained deeply, indicating they harbored pluripotency (Peng et al. 2022). Besides, there has been reported that high TDZ concentrations initiate somatic embryogenesis, and low – de novo organogenesis in African violet (Lo et al. 1997). From the result obtained it may be deduced that the TDZ depending on concentration is capable to make explant cells either totipotent or pluripotent. 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 (Fritsche et al. 2022). Thus, G. brasiliensis protrusion cells exhibited both pluri- and totipotency under the action of TDZ. In R. mucronulatum, tested TDZ concentration triggered pluripotency in epidermal cells of lef explants as well as in cells of de novo protrusions. The studying capacity for epidermal reprogramming, that is the transformation of fully differentiated epidermal cells into meristem cells and de novo organization, is a new trend in developmental biology of plant (Morinaka et al. 2021). The ability of TDZ to change the genetic program of plant cell development offers wide opportunities for plant tissue culture and requires additional in-depth study of its action mechanism on plant cells genome.
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 the tip of the shoot 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 correct identification of the morphogenesis pathway (Bassuner et al. 2007, Woo and Wetzstein 2008, Dobrowolska et al. 2017).
The development of embryo-like structures under the action of TDZ has been observed in Crocus sativus (Sharifi et al. 2010), Lilium spp. (Bi et al. 2015), Portulaca pilosa (Chen et al. 2020), Hedychium forrestii (Ma et al. 2022), and Heliotropium foertherianum (Yu et al. 2022). In the presented study, histological analysis showed that direct regeneration of adventitious shoots from leaf explant of R. mucronulatum regeneration also occurred through formation of embryo-like structures. Moreover, TDZ-induced embryo-like shoot structures were noted during regeneration from leaf explants of R. sichotense и R. catawbiense ‘Grandiflorum’ (Zaytseva et al. 2016), and from floral explants of R. dauricum (Zaytseva and Novikova 2022). Moreover, regardless of the type of explant, parenchymal and epidermal tissues of TDZ-derived embryo-like shoot primordia in rhododendrons were completely separated from explant tissues, but the connection with explant tissues was maintained through a thin vascular bundle (Fig. 3b, c). Thus, the histological studies is especially important when TDZ was used for triggering morphogenesis since this PGR leads to the development of abnormalities in regenerants structure even in the early stages (Dewir et al. 2018) which, apparently, resulted to formation of embryo-like phenotype. Considering that TDZ is a defoliant due to its property of initiating the formation of the leaf abscission zone, that its characteristic can be one of the reasons for the formation of an embryo-like phenotype since TDZ may also initiate the "abscission" of regenerant tissues from explant at the late stages of morphogenesis. Thus, the formation of embryo-like structures can be included in the list of TDZ-induced abnormalities in plant tissue culture presented by Dewir et al. (2018).