In Vitro Secondary Embryogenesis Derived from Meta-Topoline Treatment on Mass Propagation of Phalaenopsis ‘ AMP 17 ’

Phalaenopsis ‘AMP 17’ is an important orchid commodity in Indonesia with high market demand; however, scaling up the orchid commercially is constrained by the availability and sustainability of qualified seedlings. To overcome the problem, a reliable in vitro propagation protocol, especially via secondary embryogenesis, was undertaken. In the present study, in vitro secondary embryogenesis derived from meta-topoline (mT) treatment on mass propagation of Phalaenopsis ‘AMP 17’ was successfully established. Embryos, as explant sources, were prepared by culturing meristem tips of flower stalk shoots on Murashige and Skoog (MS) medium containing 1.5 mg/L thidiazuron (TDZ) and 0.25 mg/L N-benzylaminopurine (BAP) for ± 3 months. High secondary somatic embryo (SSE) formation up to 64.90% with 12.30 SSEs regenerated per embryo was determined on half-strength MS augmented with 0.5 mg/L BAP and 2.5 mg/L mT. The combination also stimulated the result of high multiplication rate of SSE formation, up to 10.1 fold on the third subculture, maintained low conversion rate of germinated-embryos down to 55% and improved qualified-growth of the germinated embryos. The mT treatment produced 86% survival plantlets with high qualified-performance. The system could be applied as an alternative method to step forward towards an improved propagation protocol, commercially efficient due to high productivity. Detail findings in each step were discussed.


Introduction
Phalaenopsis is one of important orchid commodities in the world, with economically impact in Indonesia.The orchids have various flower types, sizes, colours and in some cases fragrance (Tang and Chen, 2007).Phalaenopsis are generally sold as cut flowers and pot plants with different prices depending on cultivar and decorative quality, respectively.The orchid varieties cultivated and commercially produced in Indonesia are mostly imported, 89% are consumed by local market, while 11% are exported to Japan, USA, Netherlands, Singapore, Australia and Canada (Wisdya, 2009).
Development of the Phalaenopsis in a commercial scale is significantly constrained by the availability and sustainability of high qualified-seedlings.Thus, a reliable mass propagation protocol for the moth orchid shall be addressed.
However, in vitro mass propagation protocols for Phalaenopsis were successfully established using variedexplants, basal media, concentrations and combinations of plant growth regulators (PGRs), with good results for different varieties (Murdad et al., 2006;Sinha and Jahan, 2011;David and Bala, 2012;Samarfard et al., 2013;Feng and Chen, 2014;Rittirat et al., 2014).Each plant genotype and explant has specific behaviour within in vitro culture in response to medium composition and other factors.Therefore, each genotype needs specific in vitro propagation route system; one of the route systems is somatic embryogenesis, as established by Kuo et al. (2005), Chen and Chang (2006), Gow et al. (2010) and Feng and Chen (2014), while specific studies concentrating on secondary somatic embryogenesis are scarce.The formation of secondary somatic embryo (SSE) on Phalaenopsis was regenerated on basal part of somatic embryos (SEs) (Kuo et al., 2005), nodular masses of primary embryos (Chen and Chang, 2006) and embryo section (Park et al., 2010), especially under thidiazuron (TDZ) treatment with a pivotal role in the formation of SSEs.However, long term application of TDZ induced hyperhidricity, morphological alteration and somaclonal variation as reported for in vitro culture of banana (Bidabadi et al., 2010) and Phalaenopsis (Khoddamzadeh et al., 2010).However, meta-topolin (mT) and its advantages in stimulating SSE are still to be studied.
The objective of the present study was to establish a reliable in vitro mass propagation protocol of Phalaenopsis 'AMP 17' via secondary somatic embryogenesis based on mT treatment on selected media.The study was undertaken starting with the initiation of SSE formation, followed by multiplication and conversion till acclimatization.Interesting findings in several studies using the mT treatment are discussed in this paper.

Materials and Methods
Planting material and sterilization Phalaenopsis 'AMP 17' was used as donor plant in the study.The harvested floral stalks were pre-treated by cleaning and weeping the floral stalk with 96% alcohol wetted cotton, placing them under tap water for 60 min, immersing in 1% detergent for 30 min and rinsing with distilled water 4-5 times (3 min each).The stalks were then cut individually (at each node) and prepared as explant sources.The explants were then separately disinfected by soaking them in 0.05% and 0.01% mercury chloride (HgCl2) for 5 min and finally rinsed with sterile distilled water for 5 times (aprox.5 min each).The sterile explants were subsequently cultured on the explanting medium.

Explant preparation
Preparation of embryos as explant sources was carried out by culturing the prepared nodes on MS medium supplemented with 0.5 mg/L TDZ, 0.25 mg/L BAP, 20 g/l sucrose and 7 g/l Swallow agar and incubated under light incubation for 16 h photoperiod under cool fluorescent lamp with 13 µmol/m 2 /s light intensity at 24 ± 2 °C for ± 8 weeks.The meristem tips of the shoots derived from the node explants were then carefully isolated by removing scale-like leaves that covered them, one by one, using sharp tissue culture blade.After removing all scale-like leaves, the small shoot tip (0.3 mm in length, width and height) was sliced and cultured on MS medium fortified with 1.5 mg/L TDZ, 0.25 mg/L BAP, 20 g/l sucrose and 2.0 g/L gelrite for ± 1.5 months, in dark conditions; here after, the explants were transfer to light incubation till vigor embryos were produced (± 1.5 months).The embryos derived within this stage were then used as explant sources for the current study.

The effect of selected media and mT on SSE formation
The two selected media tested in the hereby experiment were SM1 (half-strength MS full vitamins) and SM2 (half-63 strength MS supplemented with 0.5 mg/L BAP), while the various concentrations of mT were 0.0, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 mg/L.All media used in the study contained 2% sucrose and 2.0 g/L gelrite.The experiment was arranged in a randomized complete block design (RCBD) with 4 replications.Each treatment consisted of 2 petri dishes (5 cm in diameter).Each petri dish contained 5 clustered embryos and each cluster had ± 5 embryos.Cultures were incubated in dark condition for ± 8 weeks.
Periodical observations were carried out to note explants alteration during the incubation interval.Parameters observed in the experiment were percentage of SSE formation and the number of SSE per embryo, and were counted ± 8 weeks after culture establishment.

Determining the effect of selected media and mT concentrations on multiplication and conversion of SSEs
Two selected media and different concentrations of mT as described above were applied in the experiment.The embryos were sub-cultured for 4 times and observations were registered monthly until embryo germination (S1, S2, S3, S4).Parameters recorded were (1) embryos fresh weight (g), ( 2) number of SSE and (3) frequency of SSEs conversion (%).

Determining the effect of selected media and mT concentrations on plantlet growth
Small plantlets (± 0.4 cm plant height; ± 2.0 leaves; ± 0.41 cm 2 leaf area; ± 0.6 roots; ± 0.5 root length) were used in the study.The two selected media were (1) SG1: half strength MS medium with full vitamins and without activated charcoal (AC) and (2) SG2: half strength MS medium augmented with 1.5 g/L AC.Similar concentrations of mT (as for SSE formation) were applied within the experiment.The factorial experiment was arranged in RCBD with 4 replications.Each treatment consisted of 2 jam bottles (7 cm in diameter, 12 cm in height and 30 ml of medium).Each bottle contained 5 plantlets.Parameters observed were (1) number of shoots, (2) plant height (cm; measured from the basal part of plantlet to shoot apical meristem), (3) number of leaves per plantlet, and (4) number of roots per plantlet.All data were collected after ± 8 weeks of culture.

Determining the effect of mT concentrations on plantlets acclimatization
Rooted plantlets (± 0.4 cm height; ± 2 leaves in average, ± 0.85 cm 3 leaf area, ± 1.5 root, ± 1.0 cm root length and ± 40 SPAD unit of leaves chlorophyll content) derived from the different mT treatments established within the experiment were used in the acclimatization stage.The acclimatization of plantlets was started by taking plantlets from the jam bottles, cleaning the root plantlet from remains of agar, soaking the roots in 1% pesticide solution (50% benomil and 20% kanamycin sulphate) for 3 min, then planting them separately based on mT treatments in plastic trays (2 cm in diameter).The trays were then placed in glass house under low light (87-118 µmol/m 2 /s) and humid area (65-80%) without watering for 7 days.Each treatment contained 5 replications, and each tray contained 5 plantlets, thus obtaining a total of 125 acclimatized plantlets.After 8 weeks of acclimatization, (1) percentage of survivability (%), (2) vegetative growth indicator, represented by plant height (cm), (3) number of leaves per plantlet, (4) leaves area (cm 2 ), (5) number of roots per plantlet, (6) root length (cm), and (7) leaves chlorophyll content (SPAD unit), measured by SPAD meter (SPAD -502, Konica Minolta Co.Ltd, Osaka, Japan) were noted.

Data analysis
Data collected from the experiments was interpreted using analysis of variance (ANOVA) determined with SAS Release Window 9:12.If there were significant effects between treatments, mean values of the treatments were further tested using Duncan multiple range test (DMRT) at p=0.05 (Mattjik and Sumertajaya, 2006).

Results and Discussion
The effect of selected media and mT on SSE formation Under periodical observation it was clearly revealed that initiation of SSE was obviously at ± 6 weeks after establishing the culture.The SSE formation was initiated by the emergence of several protrusions in the most surface body of primary embryos, especially in the basal to middle part of them.The protrusions continued to grow and then produced mature SSEs ± 2.0 months after culture establishment.The number of SSEs varied from 4 up to 25 SSEs per primary embryo cultured, thus the result was affected by selected medium and mT concentration.
In the first phase of the experiment, the two selected media and different mT concentrations gave significant different effects on SSE formation; however, there was no significant interaction effect from the treatments, in all parameters observed.Among the two selected media tested, medium SM2 was more suitable to produce a higher number of SSEs.
The medium induced high frequency of SSEs, up to 83.8%, with 12.0 per embryo (Table 1).Even more, 2.5 mg/L was the most appropriated mT concentration in order to stimulate high SSE production, with 64.9% SSE formation and 12.3 SSEs per embryo (Table 2).Saharan et al. (2011) reported that the use of 0.1 mg/L mT in combination with 1.0 mg/L 2,4-D in MS medium 64   successfully induced a high percentage of SE, up to 68.9%, with maximum number of SEs (98.2 ± 2.3 per g callus culture) in in vitro culture of Balanites aegyptiaca Del. (L.) derived from root explants.Application of 1.2 mg/L mT in MS medium stimulated high callus formation of Aloe polyphylla (Bairu et al., 2007), while 0.5 mg/L mT in N6 medium stimulated high embryogenic callus formation for Citrus clementina 'Monreal Rosso' derived from microspore with 1.4 embryos per petri dish (Chiancone et al., 2015).The results revealed that the higher the concentration of mT, the higher SSE regenerated, but a concentration of mT higher than 2.5 mg/L gradually reduced SSE formation; even so, Meyer et al. (2009) reported that the application of 1.3-3.6 mg/L mT in MS medium gave high impact on callus initiation and shoot regeneration of Hypericum frondosum.

Effect of selected media and mT concentrations on multiplication and conversion of SSEs
Under periodical subculture of SSEs, it was clearly recorded that fresh weight and number of SSEs produced in each subculture gradually increased (Fig. 1 A, B).However, multiplication rate (MR) of SSEs formation increased in the first and second subculture and decreased afterwards (Fig. 2).The highest MR of SSEs formation, with 10.1 fold in the third subculture and 55% CF, was recorded at 2.5 mg/L mT treatment on SM2 medium.Wojtania (2010) reported that a high multiplication rate of shoot formation (2.7-4.7 fold, depending on genotype) on Pelargonium × hortorum and Pelargonium × hederaefolium was noted on MS medium containing 0.5-1.0mg/L mT.In pineapple, 15.75 fold of multiplication rate of shoot formation was established on MS liquid medium supplemented with 0.6 mg/L mT      (Teklehaymanot et al., 2010); 12.92 multiplication rate of embryo regeneration of Paulownia fortunei on MS medium augmented with 1.0 mg/L mT (Clapa et al., 2014).

Effect of mT concentrations on acclimatization of plantlets
Application of different mT concentrations did not gave significant effect on acclimatization of plantlets.All treatments resulted within plantlet survivability percentage of 80-90%, with the highest percentage of survivability recorded on mT free medium, while application of mT tended to reduce the survivability plantlets during the acclimatization stage.
In A. polyphylla, more than 90% of plants treated with mT acclimatized successfully, compared to 65% survival rate recorded with BA treated plants (Bairu et al., 2007).Ninety four percent (94%) of L. meyenii plantlets were easily acclimatized to ex vitro environment in a mixture of peat, soil and sand (Polzerova et al., 2011), as well as 96% survival plantlets of B. aegyptiaca in a mixture of vermiculite, soil and sand (2:1:1, v/v/v) (Saharan et al., 2011).
Though the mT treatments had negative effect on survivability of plantlets, the mT treatments accelerated with significant effect the growth of plants and gave the highest chlorophyll content of 86.75 SPAD unit, as seen in the case of 2.5 mg/L mT treatment (Table 6).

Conclusions
In vitro protocol was successfully established for mass propagation of Phalaenopsis 'AMP 17' via secondary embryogenesis.Half-strength MS supplemented with 0.5 mg/L BAP and 2.5 mg/L mT induced high secondary somatic embryo (SSE) formation up to 64.9%, with 12.3 SSEs regenerated per embryo.High multiplication rate of SSE formation up to 10.1 fold, low conversion rate of germinated-embryos as 55% and qualified-growth of the germinated embryos were also recorded in the combination treatment.Survival plantlets, up to 86%, with high qualified-performance were determined in the mT treatment.Establishment of the current protocol may increase the number of high qualified-seedlings obtained and influence their sustainability, in order to propagate Phalaenopsis in a commercially efficient manner.
Fig. 1.The effect of selected media and different mT concentrations on (A) fresh weight of SSEs (g) and (B) number of SSEs within the 4 subcultures SM1: half-strength MS full vitamin; SM2: half-strength MS supplemented with 0.5 mg/L BAP; S1: first subculture; S2: second subculture; S3: third subculture; and S4: fourth subculture

Fig. 4 .
Fig. 4. The effect of meta-topolin (mT) application on propagation of Phalaenopsis 'AMP17', via induction of secondary somatic embryos (SSEs).(a) Embryos derived from meristem tip of Phalaenopsis 'AMP17' used as explant sources; (b) direct SE from shoot tip culture ± 6 weeks after culture; (c) initiation of SSEs from germinated primary somatic embryos ± 6 weeks after culture; (d) cluster of SSEs on medium containing mT ± 12 weeks after culture; (e) a single converted/germinated embryo; (f) maturation of SSEs on SG2 medium containing 2.5 mg/L mT in initial germination ± 6 weeks after culture; (g) germinated-embryos ± 8 weeks after culture in the similar medium and mT treatment; (h) growth of plantlets on the similar treatment ± 8 weeks after culture; (i) plantlets prepared for acclimatization stage; (j) acclimatized-plantlets on tray with C. rumphii bulk; (k) growth of single plant derived from mT treatment 6 months after individually repotting; (l) flower performance of Phalaenopsis 'AMP 17'

Table 1 .
The effect of selected medium on SSE formation Half-strength MS full vitamin; SM2: Half-strength MS supplemented with 0.5 mg/L BAP.Means followed by the same letter in the same column are not significantly different (Duncan multiple range test, P=0.05)

Table 2 .
The effect of different concentrations mT on SSE formation

Table 3 .
The effect of mT application on mass propagation and conversion of Phalaenopsis somatic embryos within the 4 subcultures Half-strength MS full vitamin; SM2: Half-strength MS supplemented with 0.5 mg/L BAP.Means followed by the same letter in the same column are not significantly different (Duncan's multiple range test, P=0.05)

Table 4 .
The effect of media on plantlet growth after 6 weeks on culture vitamin half strength MS medium; SG2: half strength MS medium augmented with 1.5 g/L activated charcoal (AC).Means followed by the same letter in the same column are not significantly different (Duncan's multiple range test, P=0.05).

Table 5 .
The effect of different mT concentration on plantlet growth after 6 weeks on culture Means followed by the same letter in the same column are not significantly different (Duncan's multiple range test, P=0.05).

Table 6 .
The effect of different mT concentration on the growth of plants after 8 weeks on acclimatization Means followed by the same letter in the same column are not significantly different (Duncan's multiple range test, P=0.05).