TDZ and 2,4-D on in vitro propagation of panda plant from leaf explants

The panda plant ( Kalanchoe tomentosa Baker) is a popular ornamental succulent. The optimal method for in vitro plantlet propagation was investigated. Effects of plant growth regulator on growth and development of leaf explants were determined using various concentrations of Thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.01, 0.1, 0.2 and 0.5 mg L -1 . A concentration of 0.01 mg L -1 2,4-D gave the highest plantlet quality derived from direct organogenesis. Higher concentrations of 2,4-D promoted callus proliferation. The lowest concentration of 0.01 mg L -1 TDZ induced shorter shoots, while higher concentrations resulted in greater callogenesis and inhibit root production. After in vitro culture, plantlets from the optimal treatment were acclima-tized by exposure to growth in sand, sand with coconut husk (2:1), sand with potting soil (2:1) and sand with perlite (2:1). Highest survival percentage (100%) was found in plantlets grown in sandy soil, the most well-drained material of those selected materials. Results demonstrated an alternative production method for panda plantlets using usando técnicas de cultura de tecidos de plantas. Palavras-chave: Kalanchoe tomentosa , propagação in vitro, regulador de crescimento vegetal. aclimatação de plântulas, planta suculenta.


Introduction
The panda plant, Kalanchoe tomentosa Baker, belongs to the family Crassulaceae that comprises 35 genera and some 1400 species. This perennial succulent has distinct hairy velvet leaves and is found naturally in rocky areas of central and southern central Madagascar. The attractive characteristic of the leaves has promoted K. tomentosa as an indoor ornamental plant. This succulent is usually used as potting plant and container gardening (Smith et al., 2019). Alternatively, the panda plant has an important substance that can be applied for medicinal uses such as anticancer and antidiabetes (Aisyah et al., 2015;. propagation of K. tomentosa is usually handled by leaf and stem cutting, however, the donor plants can be deteriorated. With an increasing demand on ornamental succulents (Stead, 2016), the plantlet production may be inadequate. An alternative method that can produce numerous plantlets in short time is archived by plant tissue culture. In vitro propagation has been reported in several Kalanchoe species (Kulus, 2015;Kale et al., 2018;Cui et al., 2019) but there are few reports for panda plant (Frello et al., 2002;Khan et al., 2006). For success in plant micropropagation in vitro, suitable techniques must be followed as many factors affect plant tissue culture including the effects of plant growth regulators (PGRs).
Thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) as synthetic PGRs are widely used for tissue culture in many plant species. Optimal concentration of PGRs enhances high crop yield. In Kalanchoe species, TDZ applied to induce the production of numerous shoots depending on concentration and explant cultivar (Sanikhani et al., 2006). TDZ also induced explants of a Kalanchoe species to regenerate callus (Kertrung and Junkasiraporn, 2018); however, the effect of TDZ concentration on in vitro culture of K. tomentosa Baker has not yet been examined. Another synthetic PGR, 2,4-D is also used for callus induction in many plant species (Machakova et al., 2008) and its effects have not been reported on in vitro culture of K. tomentosa. Understanding about the effects of those PGRs on explant responses may improve the plantlet production in vitro of the panda plant. One of the limitations on plant tissue culture is plantlet exposure ex vitro because high mortality may be occurred in this process. For the successfully propagation, the plantlet acclimatization ex vitro should be investigated (Vahdati et al., 2017).
Thus, here, the effects of different concentrations of TDZ and 2,4-D were examined on in vitro propagation of K. tomentosa leaf explants to optimize plantlet production. Also, the optimal planting materials for plantlet acclimatization were investigated. The improvement of K. tomentosa plantlets by plant tissue culture techniques was reported here.

Plant materials and media preparation
Plant materials were purchased from a local market and grown in a greenhouse for one week. Four leaves were collected from each plant as the fourth to the seventh leaves from the shoot apex. The leaves were washed in tap water with 1% (v v -1 ) commercial surfactant (Sunlight ® ) and rinsed in running tap water for 30 min. Then, the cleaned leaves were surface-disinfected by a series of 70% (v v -1 ) ethanol for 1 min, 0.01% (w v -1 ) mercuric chloride solution with 1% (v v -1 ) Tween 20 for 5 min, and 10% commercial hypochlorite solution (Clorox ® ) for 10 min. disinfected leaves were washed aseptically three times in distilled water. Ventral sides of the leaves were placed on the surface of semi-solid MS (Murashige and Skoog, 1962) basal medium supplemented with 0.5 ml L -1 plant preservative mixture (PPM TM ; Plant Cell Technology). The explants were then incubated at 25 ± 2 °C under 40 μmol m -2 s -1 light intensity (provided by cool white LED tubes) and 12 h light photoperiod. Regenerated shoots were subcultured on MS basal medium and aseptic plants were used as explant material in further experiments.

Effects of plant growth regulator on growth and development of leaf explants
Leaf explants at about 5×5 mm 2 from aseptic culture were randomly applied in the experiment, and cultured on MS media supplemented with different concentrations (0, 0.01, 0.1, 0.2 and 0.5 mg L -1 ) of TDZ and 2,4-D. Each treatment was applied to one explant per bottle. All treatments were incubated at 25 ± 2 °C at 40 μmol m -2 s -1 light intensity and 12-h light photoperiod. Growth and development parameters of the explants were observed after culture for 4, 8 and 12 weeks.

Plantlet acclimatization ex vitro
Healthy plantlets (new shoots with active root), induced by MS with 0.01 mg L -1 2,4-D, about 2 cm high from the previous experiment were applied in this experiment ( Figure 4a). Each plantlet individuals were separated carefully from each other by scalpel. The plantlets were rinsed in running tap water and then submerged in 0.5% (w v -1 ) fungicide solution for 15 min. Disinfected plantlets were transplanted into four different planting materials including sand, sand with coconut husk (2:1), sand with potting soil (2:1) and sand with perlite (2:1). A total of 25 plantlets were randomly assigned for each treatment. After transplantation, the plantlets were grown in a greenhouse (27 to 30 °C ambient temperature, 50% to 60% relative humidity and 50% (sunlight) transparency). The plantlets were watered by a spray pump for 1-h twice a day. Survival rate and plantlet quality were recorded weekly for 10 weeks.

Statistical analysis
All growth and development parameters of the in vitro experiment were analyzed by one-way ANOVA and posthoc using Duncan's new multiple range test (DMRT) with SPSS version 22. To normalize the data distribution, the percentage data were arcsine transformed before analysis.

Effects of plant growth regulator on growth and development of leaf explants
After culture for 4 weeks, direct organogenesis was observed in most explants except those cultured on MS medium supplemented with 0.2 and 0.5 mg L -1 TDZ. Direct organogenesis was displayed as the occurrence of root(s) from the cut end of the explants (Figure 1). After culture for 8 weeks, shoot regeneration ranging from 53.3 to 83.3% was found in 0.01 mg L -1 TDZ, 0.01, 0.1, 0.2 and 0.5 mg L -1 2,4-D and the control treatments ( Figures. 1 and 2).
Highest shoot number (1.9 shoots) was observed in the 0.01 mg L -1 2,4-D treatment; however, there were no significant differences between 0.01 mg L -1 TDZ, 0.1 and 0.2 mg L -1 2,4-D and the control treatments (data not shown). Bars and error bars show mean ± SE of three replications (ten explants per replication at week 8 and nine explants per replication at week 12). Different letters above the bar indicate significant differences by DMRT at p ≤ 0.05.
Callus formation was observed in all treatments, except for 0.01 and 0.1 mg L -1 2,4-D and the control. The percentage of callus formation varied from 4.2 to 100% (Figure 2). The root regeneration without shooting was found on explants treated by MS medium with 0.1 mg L -1 TDZ. The three treatments that induced the highest plant height, leaf number and rooting percentage were 0.01 and 0.1 mg L -1 2,4-D and the control but with no significant differences. MS medium supplemented with 0.01 mg L -1 2,4-D (Figure 3a and Table  1) induced the healthiest plantlets of K. tomentosa with a significant difference in root numbers per explant.

Plantlet acclimatization ex vitro
Ten weeks after the acclimatization of healthy plantlets of K. tomentosa, survival percentage varied from 50% to 100%. Highest survival percentage (100%) was found for plantlets grown in sand, while survival percentages of plantlets grown in sand with coconut husk, sand with potting soil and sand with perlite were 88, 76 and 56%, respectively. Plantlet quality in different planting materials is depicted in Figure 4b  Throughout the acclimatization, survival percentages in each treatment were decreased except plantlets grown in sand. On 2:1 ratio of sand per perlite, the survival percentage dramatically decreased in week 5 after acclimatization while other treatments the percentage slightly decreased to stable (Table 2). Table 2. Weekly survival pecentage of Kalanchoe tomentosa on different planting materials in the acclimatization for 10 weeks (n = 25).

Planting Materials
Week (

Discussion
Low concentration (0.01 mg L -1 ) of TDZ induced new shoot regeneration, however, using TDZ resulted in shorter plantlets than MS without PGR treatment (Table 1). Higher concentrations of TDZ gave increased callus formation with faster callogenesis (Fig. 2). The same result was found in other ornamental succulents, where TDZ promoted callus formation (Liu et al., 2016;. In K. rhombopilosa Mannoni & Boiteau, higher TDZ concentration increased the callogenesis rate and decreased the differentiation rate (Kertrung and Junkasiraporn, 2018). Callus induction might be due to that TDZ stimulated cell division in plant cell more than other PGRs. TDZ also increase the level of endogenous cytokinin by reducing catabolism, increasing synthesis, and conversing inactive cytokinin to active forms (Dinani et al. 2018). By contrast, TDZ induced shoots regeneration in Kalanchoe blossfeldiana Poelln. and Sedum sarmentosum Bunge, while callus formation was not recorded (Sanikhani et al., 2006;Kim and Sivanessan, 2016). In K. tomentosa Baker, MS medium supplemented with 0.25 mg L -1 TDZ and 0.4 mg L -1 indole-3-acetic acid (IAA) induced new shoot regeneration at only 30% (Frello et al., 2002). Responses of Kalanchoe species varied with TDZ and the optimal quantity should be examined in each application. In our study, the present of root without shoot regeneration found on MS medium supplemented with 0.1 mg L -1 TDZ might be due to the consequence of explants development. An evidence was supported by the organogenesis at week 4 that root was the first occurrence organ in most explants of this study. From our result also revealed that higher TDZ concentration reduced root development.
Low concentration (0.01 mg L -1 ) of 2,4-D promoted the highest quality plantlets, while higher concentrations increased the callogenesis rate ( Figure 2). 2,4-D is usually used with cytokinins for callus induction. The induction of callogenesis was also confirmed in K. blossfeldiana Poelln. and Digitalis lanata Ehrh. (Kale et al., 2018;Bhusare et al., 2020). However, our results suggest that 2,4-D at low concentration may induce plantlet formation. 2,4-D is an auxin and plays a primary role in cell elongation and root initiation (Machakova et al., 2008). The response of explants in this study might be due to the auxin-cytokinin balance derived from exogenous auxin (2,4-D) and endogenous auxin cytokinin in plant cells.
Plantlet acclimatization ex vitro results indicated that planting material affected survival rate. Proper planting material should be a major concern for each application. Many reports of Kalanchoe species on plantlet acclimatization ex vitro from in vitro culture have indicated over 90% survival rate (Khan et al., 2006;Sanikhani et al., 2006). Our results indicated that the best planting material for K. tomentosa was sandy soil. High plantlet quality observed in sand insists that well-drained planting material is appropriate for growing panda plant (Smith et al., 2019) while higher water absorbed materials reduce survival rate.

Conclusions
Our result demonstrated an alternative production method for panda plantlets using plant tissue culture techniques. Experimental results identified 2,4-D as a candidate plant growth regulator (PGR) for leaf explant culture in vitro of panda plant. 2,4-D gave a highly quality plantlet with increased plant height, leaves and root numbers per explant than TDZ. The suggested medium was MS medium supplemented with 0.01 mg L -1 2,4-D. Our protocol produced 2-4 shoots per explant but might be compensated by lower disturbance on plant individuals when leaf explants were deployed. To produce calluses of panda plants, MS medium supplemented with 0.2 mg L -1 TDZ is recommended. For ex vitro acclimatization, sand is suitable for promoting more survival rate.

Author Contribution
WP: conceptualization, experiment development, data analysis and interpretation, manuscript writing and corrections, ON: data collection, preliminary analysis and interpretation, AK: review and editing.