3.2.1. In Vitro Culture Initiation

Four-year-old, healthy, certified mother plants of *Syngonium* imported from the Netherlands were used as donor plants. These plants were maintained in culture pots under controlled greenhouse conditions for more than 2 years to ensure that there was no phytosanitary contamination. It should be noted that using healthy mother plants is essential for starting plant meristem cultures according to Toshio Murashige and Folke Skoog [43]. Explants as stem fragments comprising the nodal segment (i.e., less than 5–6 cm in length) were well washed under running tap water for 2 h. Nodal explants were further immersed in an aqueous solution of Tween 20 (2–3 drops/L) under continuous shaking. After 10 min, all nodal explants were surface-sterilized in the laminar hood flow for 30 s using a sterile solution of 0.1% HgCl2, followed by three consecutive rinses in sterile water for 10 min, each under continuous shaking. Each nodal explant was placed on sterilized filter paper before taking the meristem under stereomicroscope [33,34].

Sterilized meristems were cultivated on a solidified, modified Murashige and Skoog (MS62) [43] culture medium and sub-cultivated after 10 days under the same conditions to prevent browning of meristems, implementing good laboratory practices to ensure a virusfree process similar to that used for carnations [44]. At this stage, the MS62 inoculation medium was supplemented with BAP (30 mg/L), IAA (1 mg/L), and sucrose (30 mg/L). Merck agar (7 g/L) was added before adjustment of the pH to 5.8, and after sterilization it was reduced to 5.4. The success rate for initiating meristem cultures was 100%, with all of the inoculated meristems being viable [33,34]. This was also due to the disinfecting method used, as well as to the skills of the staff members, who had almost 16 years of practice performing this activity. We should also note that some authors have successfully used antibiotics for in vitro micropropagation of *Syngonium* into liquid culture media at an industrial scale [45].

Meristematic domes with the first or second leaf primordia at a maximum size of 0.2 mm were taken under stereomicroscope in sterile conditions during springtime. *Syngonium* in vitro cultures have also been used by other professional groups [22,46–63] (see Table 1). In 1976, Lynn Miller and Toshio Murashige published the first protocol for micropropagation of *Syngonium*, starting with 0.2–0.4 mm axillary meristems taken under stereomicroscope [22].

**Table 1.** Balance of plant growth regulators and relevant observations related to in vitro micropropagation of *S. podophyllum* from our review of scientific articles whose full text was freely available on Google Scholar.



**Table 1.** *Cont.*

Abbreviations: IAA: indole-3-acetic acid; 2iP: 6-(γ,γ-dimethylallylamino)purine; Kin: kinetin; BAP: 6 benzylaminopurine; IBA: indole-3-butyric acid; NAA: 1-naphthaleneacetic acid; CPPU: (2-chloro-4-pyridyl)- N -phenylurea; TDZ: thidiazuron, or N-phenyl-N -1,2,3-thiadiazol-5-ylurea.

Further stages in the development of meristems are discussed in our previous papers [33,34]. After 12 weeks of cultivation in the same culture medium, it was generally possible to consider that all inocula were sufficiently developed (i.e., a diameter ranging between 0.5 and 1 cm) to the first multiplication stage by splitting clusters of buds into two to four pieces. The first multiplication stage was carried out in MS62 culture medium supplemented with BAP (1 mg/L), Kin (3 mg/L), and IAA (0.1 mg/L). After a total of 20 weeks from the initiation of the process (i.e., the day of meristem inoculation), it was possible to isolate clearly differentiated shoots (i.e., heights ranging between 10 and 15 mm) to begin the multiplication experiments, and only these shoots were recorded to assess the multiplication rate. All shoots and buds less than 10 mm in height were considered to be insufficiently developed to enter the economic workflow at the industrial scale. Consequently, these calli were transferred to new culture media in order to ensure further development of buds and elongation of shoots.

The balance of plant growth regulators used in our laboratory for meristem cultures was in favor of cytokinin (i.e., 30 mg of BAP and 1 mg of IAA) [33,34]. Other authors used different balances of plant growth regulators, as well as different regulators. For example, Lynn Miller and Toshio Murashige used a hormone balance in favor of auxin (i.e., 2 mg of 2iP and 3 mg of IAA) [22]. Other teams used a balance in favor of cytokinin [49,51,52,56–59]. Later, other authors used new synthetic regulatory substances, again more inclined towards cytokinin, such as 1 mg of NAA and 2 mg of (2-chloro-4-pyridyl)-N -phenylurea (CPPU) or 2 mg of N-phenyl-N -1,2,3-thiadiazol-5-ylurea (TDZ) [56,59] (see Table 1).

As shown Table 1, very different types and balances of plant growth regulators have been used in different laboratories to initiate meristem cultures, indicating that this species is very reactive towards different culture conditions and very easy to culture in vitro. We should also note the contributions to developing a micropropagation protocol by relevant researchers who are cited by others but whose articles were not available. For example, Scaramuzzi and coworkers [46] cited an unavailable paper published by Makino and Makino in 1978 (e.g., the article was entitled "Propagation of Syngonium podophyllum cultivars through tissue culture", and published in In Vitro, volume 14, page 357). Some other articles were also unavailable or written in languages that do not use the Latin alphabet.

The decision to incline the balance of the plant growth regulators in favor of cytokinin was based on our observations related to the well-expressed apical dominance of the main shoots of the pots' donor plants. Apical dominance has long been known to be supported by the high internal levels of auxin secreted by young organs [64]. However, the interruption of this auxin gradient flow can be achieved during the cutting-off of the meristem, and this process is confirmed by the further addition of auxin to the meristem's culture medium, as reported by Lynn Miller and Toshio Murashige [22]. This idea was not tested in our laboratory, and it should be taken into consideration for further studies.

#### 3.2.2. Micropropagation and Culture Media Testing

In the 28th week of in vitro cultivation, we began the testing of auxins and vitamins using an MS62 basal medium composition [33,34]. The highest recorded multiplication rate was ~9.17 shoots/explant in the case of an auxin–cytokinin ratio of 0.5 mg/L NAA to 1 mg/L BAP. It should be noted that only shoots over 7–9 mm in height were recorded.

In the 36th week of in vitro cultivation, we conducted the cytokinin tests, and based on our results the rate of multiplication was slightly higher than 9.85 shoots/explant [33,34]. This multiplication rate was slightly higher than that obtained on the same solidified MS62 culture medium by Lynn Miller and Toshio Murashige (i.e., 7.9 shoots/explant, with their minimum height not defined) [22]. However, Miller and Murashige obtained a higher multiplication rate when liquid culture medium was used (i.e., 26 –> 60 shoots/explant). Comparable multiplication rates were also reported by other authors [46,49,51,53,57,59,61,63].

In our laboratory, a multiplication rate of 9 shoots/explant was considered to be excellent for solid culture medium use when recording only shoots of over 7–8 mm in height, and we also found a low capacity for infection compared to liquid culture media. At the end of this stage, the height of the shoots used for industrial-scale micropropagation was set to be longer than 7–8 mm. Other authors obtained the same multiplication rate using different compositions of plant growth regulators and different cytokinin–auxin ratios [57]. The clusters comprising shoots shorter than 7 mm in length, as well as buds, were transferred for further multiplication and elongation of the shoots on the same culture medium, with a decreased cytokinin–auxin ratio [33]. However, increasing the shoots' multiplication rate to more than 60 shoots/explant should be more cost-effective for starting the technological flow and for producing a large number of shoots in a very short time. Some other teams were also successful in obtaining somatic embryos, completing all morphogenetic programs for the species, but without callus generation [54,55].

A multiplication rate of nine plantlets/explant up to the greenhouse stage generated total costs of 1 USD/100 pot plants, and in 1998 this was considered to be profitable. Under present conditions, such a value is no longer worthwhile; furthermore, the technology needs to be updated for the new energy requirements. Starting with the third stage of the technological flow, different experimental tests were initiated. Relevant stages related to Syngonium micropropagation are presented in Figure 1.

**Figure 1.** Different stages in the micropropagation of *Syngonium podophyllum* cv. 'White Butterfly'. From left to right, de novo shoots over 7 mm in height were separately transferred to solidified MS62 culture medium for elongation of shoots up to the moment of transfer for acclimation. The culture medium's composition is described in 2004 [33].
