In vitro Embryogenesis Derived from Shoot Tips in Mass Propagation of Two Selected-Clones of Phalaenopsis

Phalaenopsis is of high economic value and market demand in Indonesia; however, orchid products are mostly imported from other countries. ‘Kristina Dwi’ (KD) 69.274 and ‘Dedeh’ (D) 802.28 are two selected clones with high potential utilized and developed commercially. To support their commercialization, a reliable in vitro propagation protocol is essential.  In the current study, an in vitro mass propagation protocol for KD 69.274 and D 802.28 clones was successfully established using shoot tips as explant sources. A high number of embryos, up to 8.2 embryos per explant, with 58.5% explant regeneration, and 3.5 regenerated-explants in average were regenerated from shoot tips of KD 69.274 clone cultured on  half-strength Murashige and Skoog (MS) medium, with full strength micro, Fe-chellate and vitamin containing 0.5 mg/L thidiazuruon (TDZ) and 0.25 mg/L N6-benzyladenine (BA). The initial embryos were proliferated by culturing embryos individually on half-strength MS medium with 0.13 mg/L TDZ and 0.25 mg/L BA and resulted in high embryo regeneration up to 91.4%, with 10.2 embryos per explant and no embryo browning. The embryos were multiplied under periodical subcultures of 3 months each, resulting in gradual increasing number of embryos from the first subculture till the fifth subculture, with 23.6 embryos produced, then declined afterward. The embryos were easily germinated on half-strength MS medium with full strength of vitamin and hormone free, with 73.9% embryo germination and 14.9 germinated embryos. Healthy plantlets were stimulated on the same medium with 2 g/L activated charcoal (AC) and successfully acclimatized on Cycas rumphii bulk, with 88.3% survival plantlets. Finally, it can be summarized that a new in vitro mass propagation protocol, as new alternative choice for Phalaenopsis propagation, was successfully established.


Introduction
Phalaenopsis (Orchidaceae), commonly known as moth orchids Gow et al., 2009;2010), is one of the most popular and high economical orchid in the world for floriculture market. The Phalaenopsis is generally sold commercially as pot plant and cut flowers with the Netherland (70%) and Taiwan (23%) as the main producers of orchids (Griesbach, 2002;Liu et al., 2006;Plasmeijer and Yanai, 2012). The orchid, involving in a monopodial orchid, is difficult to propagate vegetatively (Ishii et al., 1998), while generative propagation derived from seeds do not produce uniform regenerants (Park et al., 2002), thus propagation through tissue culture is desirable. However, utilization of shoot tips harvested from mother plants as explant source for in vitro culture of Phalaenopsis may lead to the loss of the mother plants (Ishii et al., 1998), therefore alternative explant sources shall be addressed for the purpose.
Recently works on the in vitro embryogenesis of Phalaenopsis were successfully established previously. Kuo et al. (2005) used entire leaf surface cultured on half-strength MS medium containing 1.0 mg/L TDZ to produce high embryos (55 embryos per explant). Cut end from leaf explant cultured on the half-strength modified MS supplemented with 3.0 mg/L N 6 -benzyladenine (BA) and 1.0 mg/L N 6 -(2isopentenyl) adenine (2-iP) was the most effective for embryogenesis of P. amabilis and P. 'Nebula' respectively (Gow et al., 2008) and 3.0 mg/L TDZ with sucrose free for P. amabilis and P. 'Nebula' (Gow et al., 2010). Sub-culturing embryos in the half-strength MS medium fortified with 3.0 mg/L TDZ were successfully applied for secondary embryogenesis in P. amabilis var. formosa (Chen and Chang, 2006). Two months old seedlings of P. aphrodite easily produced embryos in the distal part of shoots on modified-halfstrength MS augmented with 1.0 mg/L TDZ (Feng and Chen, 2014). All this being considered, it was noted that there is no in vitro mass propagation method established for Indonesian Phalaenopsis, especially selected clones of 'Kristina Dwi' (KD) 69.274 and 'Dedeh' (D) 802.28, both having high potential to produce commercially and substituting import products. Establishment of the in vitro mass propagation protocol for the clones has important effect on commercialization of the Indonesian Phalaenopsis.
A reliable in vitro mass propagation via embryogenesis of KD 69.274 and D 802.28 clones involving induction and proliferation of embryos derived from shoot tips, followed by embryo germination, plantlet preparation and acclimatization, were the main objectives of the study. Interesting findings in each stage are discussed in this paper.

Plant material and explant preparation
Inflorescence flowers were harvested from the Phalaenopsis mother plants of two selected-clones (KD 69.274 and D.802.28; of ±1.5 years old) and used as explant sources. The inflorescence surfaces were then pre-treated by gently wiping all surfaces with wetted-cotton using 96% alcohol. After the pre-treatment, all nodus without flower buds were cut at ± 3 cm length (2.5 cm bottom and 0.5 upper part of nodus). The explants were put under tap water for 1 h, soaked in 1% Tween 20, then in 1% pesticide solution of benomil 50% and 20% streptomycin 318 sulphate, followed by manual shaking for 30 min, then finally rinsed with distilled water 4-5 times (@ 3 min each). The explants were then sterilized by immersing 0.05% mercury chloride (HgCl2) for 10 min. followed by rinsing 5-6 times using sterile distilled water (@ 3 min. each) and then put on sterile Petri dish with sterile towel tissue.
After sterilization, damage tissue in each nodus was sliced using tissue culture blade. The nodus was then cultured on halfstrength MS medium containing 1.5 mg/L TDZ, 0.25 mg/L BA, 20 g/L sucrose and 2 g/L gel rite, for 1.5 months for shoot initiation, under light incubation with 16 h photoperiod under cool fluorescence lamp with light intensity of ~13 µmol/m 2 /s and 24 ± 1 °C (Fig. 4A). After initiated-shoots in ± 0.5 cm in length, the shoots were used as explant source of shoot tips (Fig. 4B and 4C).
Isolation of shoot tips was carried out with less than 40 × magnification of Wild Herbrugg stereo microscope using blund pinset and sharp Aesculap tissue culture blade. Using the tools, the 4-5 scale like leaves covered the shoot tip were carefully removed one by one. After all the scale like leaves were removed, the shoot tip was sliced vertically in four positions as close as possible to the tip position using the blade, with final size of ± 0.3 × 0.3 × 0.3 mm in length-width and height (Fig. 4D). The shoot tips were then cultured on initiation media (Fig. 4E).

Induction of embryos derived from shoot tips
Induction of embryos was carried out by culturing shoot tips (Fig. 4F) of the two selected-clones KD. 69.274 and D. 802.26 (as main plot) on different initiation media (IM, as sub-plot) of (1) half-strength MS containing 0.75 mg/L TDZ and 0.25 mg/L BA (IM-1), (2) half-strength macro MS and full strength  complete block design with 3 replications. Each treatment consisted of three bottles. Each bottle contained 5 explants cultured. The cultures were incubated in dark for 1.0-1.5 months and then transferred to light condition in 16 h photoperiod under cool fluorescent lamp with ~13 mmol/m 2 /s and 24 ± 2 °C till 3 months of incubation.

Multiplication of embryos derived from shoot tips
Multiplication of embryos derived from shoot tips was carried out by individually culturing the embryos on two multiplication media (MM): half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BA (MM-1) and half-strength MS medium with full strength of vitamin containing 150 ml/L CW (MM-2).
Each treatment consisted of 10 bottles; each bottle contained 10 embryos and was replicated 5 times. The cultures were incubated in dark for 1.0-1.5 months and light condition till 3 months of incubation. The periodical subculture of embryos was done till optimal multiplication rate of them was noted, and declined thereafter.

Germination of embryos and plantlet preparation
Germination of embryos and plantlet preparation were carried out by culturing clusters of embryos on half-strength MS medium with full vitamin hormone free. In the experiment, the treatment consisted of ten bottles and was replicated 4 times. Each bottle contained 4 clusters of embryos. Each cluster had ± 20 embryos.
Variables observed in this experiment were percentage of germination and number of germinated-embryos per cluster. Plantlet preparation was done by culturing germinated-embryos individually in half-strength MS medium with full vitamin containing 2 g/L AC. In the experiment, the treatment consisted of ten bottles and was replicated 4 times. Each bottle contained 5 germinated-embryos.
Acclimatization of plantlets Well-rooted plantlets (90 days old) with 4-6 leaves and 3-4 roots were selected and prepared for acclimatization. Plantlets were carefully removed from culture vessels and remaining agar 319 for micro, Fe-chellate and vitamin supplemented with 0.5 mg/L TDZ and 0.25 mg/L BA (IM-2), (3) half-strength MS for macro, micro and Fe-chellate and full strength for vitamin augmented with 0.5 mg/L TDZ and 0.25 mg/L BA (IM-3), (4) half-strength MS for macro and full strength for micro, Fechellate and vitamin containing 150 ml/L coconut water (CW) (IM-4) and (5) half-strength MS for macro, micro and Fechellate and full strength for vitamin supplemented with 150 ml/L CW (IM-5).
The split-plot experiment was arranged in a randomized complete block design with four replications. Each treatment consisted of 2 bottles and each bottle contained 5 explants. Cultures were incubated in dark condition for 1.0-1.5 months. Embryos successfully regenerated were then transferred to light till 3 months of incubation.

Proliferation of embryos derived from shoot tips
Proliferation of embryos derived from shoot tips was carried out by culturing three types of explants as main plot and using different proliferation media as sub-plot. Three different types of explants applied in the step were (1)  The split-plot experiment was arranged in a randomized was washed off gently with tap water. Plantlets were then immersed in a 1% pesticide solution of 50% benomyl and 20% streptomycin sulphate for 3 min to reduce root rot caused by Erwinia sp. Small plants 4-6 cm in canopy height were cultivated in plastic pots (30 cm in diameter) containing Cycas rumphii bulk. Approximately 35 plantlets were planted in each plastic pot. A total of 210 plantlets derived from the root formation experiment were acclimatized in six plastic pots, which were covered with transparent plastic foil for 7 days and placed in a glass house under reduced light intensity (100-120 µmol/m2/s) using 50% shading net. Relative humidity during plantlet acclimatization was 85-95% when plantlets in plastic pots were covered by transparent plastic and 70-90% after removing the plastic.

Variables or parameters
Variables observed in these experiments were (1) percentage of explant browning (%), calculated by counting number of browned-explants divided by total explant cultured time by 100%, (2) percentage of explant regeneration (%), calculated by counting number of regenerated-explants divided by total explant cultured time by 100%, (3) number of regeneratedexplants, (4) number of embryos per explant, (5) multiplication rate of embryos, total number of embryos-regenerated per explant in the end of culture divided by number of embryos culture in the initial culture, (6) percentage of germination (%), calculated by counting number of germinated-embryos divided by total embryos cultured times by 100%, (7) number of germinated embryos per cluster, (8) percentage of survivability (%), calculated by counting number of survival plantlets divided by total plantlets cultured time by 100%, and (9) number of survival plantlets. Periodical observation was carried out to note all alteration occurred during culture incubation. Data were recorded ± 3 months after culture for initiation til multiplication experiment and ± 2 months after culture for others.

Data analysis
All data collected from the experiments were analyzed by analysis of variance (Anova) with SAS program Release Windows 9.12. Significant differences between means were assessed by Tukey's Studentized Range (HSD) at p = 0.05 (Mattjikand Sumertajaya, 2006).

Results and Discussion
Induction of embryos derived from shoot tips Under periodical observation it was known that initial embryo formation was taken place 15-20 days after culture (Fig. 4G). The initial embryos continually grew and were easily observed 30-40 days after culture (Fig. 4H). After transferring embryos derived from shoot tips under light incubation, the white-yellow embryos turned green 5-10 days after transferring culture under light. After 2.5-3.0 months of culture, number of embryos produced per explant varied between 1-9 embryos (Fig. 4I).
From statistical data analysis it was clearly known that the type of clones and initiation media gave significant effect on embryo regeneration in all variables observed (p=0.05). The two treatments also induced high significant interaction effect, whereas the type of media had higher effect compared to clones. KD 69.274 was the responsive clone in embryo formation and gave significant effect, especially in number of embryos regenerated, compare to D 802.28 clone. Though the clone had lower percentage of explant regeneration and number of regenerated-explant, it produced a number of embryos per explant up to 4.2 embryos (Fig. 1A). IM-2 (half-strength MS medium with full strength micro, Fe-chelatte and vitamin containing 0.5 mg/L TDZ and 0.25 mg/L BA) was the most appropriate medium for embryo formation, though there was no significant different compared to IM-1 (Fig. 1B). The medium induced higher number embryos up to 5.7  embryos per explant and the value indicated significant effect compared to IM-3 to IM-5, while the lowest medium effect on embryo regeneration was exhibited by IM-5.
Treatments of clones and media also gave significant interaction effect on embryo formation in all variables observed. Clone of KD 69.274 cultured on IM-2 medium was the most suitable combination, though it was no statistically different compared to others in percentage of explant regeneration and number of regenerated-explant. The combination successfully induced higher embryo regeneration up to 8.2 embryos per explant (Table 1, Supplementary Tables 1 and 2). The second best combination was performed on D 802.28 and KD 69.274 clones cultured on IM-1, while the lowest combination effect was indicated by D 802.28 cultured on IM-5 or IM-4, along with KD 69.274 cultured on IM-5.

Proliferation of embryos derived from shoot tips
Based on periodical observation it was known that initiation of embryos was recorded within 13-21 days after culture. In single embryo culture, especially, embryo formation was initiated by immersing protrusions in middle to basal part of single embryo body (Fig. 4K). Number on initial embryos varied among 4-25 embryos per explant (Fig.  4L) that were regenerated faster (13-15 days) than initial embryos derived from tTCL and lTCL (15-21 days). Number of embryos induced from tTCL and lTCL was only 1-6 embryos per explant.
In the experiment, types of explant and proliferation media gave significant effect on embryo formation. The two treatments also exhibited significant interaction effect, whereas types of explant stimulated higher effect compared to proliferation media. tTCL and lTCL stimulated higher number of embryos initiated per explant; the two treatments caused lowering number of regenerated-embryos and stimulating high explant browning. Percentage of explant regeneration derived from the treatments was approximately 20-50%, with higher percentage of explant browning up to 75% and number of embryos produced per explant of 1.0-1.8 embryos ( Fig. 2A). Culturing single embryo individually led to reducing explant browning down to 6%, increasing percentage of explant regeneration up to 83% and inducing high number of embryos regenerated up to 5.2 embryos per explant. Whereas PM-5 medium (half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BA) was a suitable medium for embryo proliferation. Though there was no significant different of percentage of explant browning and regeneration, the medium stimulated higher number of embryos per explant up to 4.3 embryos (Fig. 2B).
Interaction effect of explant types and proliferation media was also significantly revealed in the experiment. Single embryo cultured in PM-5 (half-strength MS medium added with 0.13 mg/L TDZ and 0.25 mg/L BA) was the most suitable combination treatment compared to other combinations. The combination reduced explants' browning down to 0%, increased percentage of explant regeneration up to 91.4% with 10.2 embryos regenerated per explant (Table 2,  Supplementary Tables 3 and 4). The second best combination was single embryo cultured on PM-7 (halfstrength MS with full strength of vitamin augmented with 150 ml/L CW). Combination of tTCL and lTCL expected could produce high results, which were due to leading high 321 Fig. 4. Development of in vitro propagation protocol via shoot tip explant embryogenesis of Phalaenopsis selected-clones. A. Inflorescent node in the initial culture, selected to regenerate shoot as explant source; B. Regeneratedshoot 1.5 months after culture; C. Cross section longitudinally of shoot isolated as explant source; D. Initial isolated-shoot tip prepared and used as explant source; red circle indicate area where the shoot tip explant isolated; E. Size of shoot tip (± 3 × 3 × 3 mm; length, width and height) used for explant source; F. Initial condition of shoot tip cultured on initiation medium; G. Initial embryogenic callus derived from shoot tip on half-strength Murashige and Skoog (MS) medium with full strength micro, Fe-chellate and vitamin containing 0.5 mg/L thidiazuruon (TDZ) and 0.25 mg/L M6benzyladenine (BA) ± 15 days after culture; H. Development of initial embryos 1.5 months after culture; I. Mature embryos derived from shoot tip ± 3 months after culture; J. Single embryo used as explant source in proliferation and multiplication step; K. Single embryo with initial secondary embryos on half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BA 1.5 months after culture; L. Mature embryos derived from single embryo culture on the selected proliferation medium ± 3 months after culture; Black arrow indicates the originated embryo; M. Germinated embryos on half-strength MS medium with full strength of vitamin and hormone free ± 2 months after culture; N. Plantlets prepared for acclimatization on half strength MS medium with full strength of vitamin, 2 g/L AC and hormone free ± 2 months after culture; O. Plantlet performance prepared for acclimatization; P. Well growth performance of acclimatized plantlets on C. rumphii bulk 3 months after acclimatization; Q. Performance of individual acclimatized-plant ± 4 months after repotting on the same medium. explant browning with lower regeneration of embryos. Potential embryo formation up to 3.2 embryos per explant was performed by lTCL cultured on PM-4.

Multiplication of embryos derived from shoot tips
Interested-information was clearly revealed in the stage under periodical subculture of single embryo in two multiplication media [half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BAP (MM-1), and half-strength MS medium with full strength of vitamin containing 150 ml/L CW (MM-2)] with different results. Number of new embryos derived from single embryo cultured increased gradually in each subculture period from first subculture till the fifth subculture and declined thereafter (Fig. 3). In MM-1 medium, single embryo in the first subculture period (SP-1) successfully produced secondary embryos up to 8.4 embryos, increased to be 10.8 embryos the SP-2, 12.1 embryos in the SP-3, 17.8 embryos in the SP-4, 26.6 embryos in the SP-5, then lowered to 23.6 embryos in the SP-6. While in the MM-2, secondary embryos produced in each subculture period were less than in MM-1. From the study, it was also clearly observed that application of synthetic hormone, TDZ and BAP in the experiment, was pre-requisite culture medium condition needed to produce higher secondary embryos for Phalaenopsis clones, while application of natural hormone derived from CW induced lower secondary embryos compared to the synthetic one.

Germination of embryos, plantlet preparation and its acclimatization
Germination of embryos was successfully carried out by culturing embryos in half-strength MS medium with full strength of vitamin and hormone free. The treatment induced high percentage of embryos germination from 65-89% (73.9% in average) with 13-18 numbers of germinated embryos (14.9 germinated-embryos in average) (Fig. 4M). Well plantlets were successfully prepared by culturing germinated-embryos individually in half-strength MS medium with full strength of vitamin, 2 g/L AC and hormone free. Under the treatment, plantlets had 4-6 leaves, 0.5 -4.1 cm leaf length (1.94 cm in average), 0.4 -1.6 cm in width (0.93 cm in average), 3 -4 number of roots and 1.3 -4.2 cm root length (2.47 cm in average) ( Fig. 4N and 4O). Whereas in acclimatization stage of plantlets, percentage of plantlet survivability was 74.3 -91.4% with 88.3% in average and number of survival plantlets were 26-32 plantlets, with 29.2 plantlets in average (Fig. 3P). After repotting individually, healthy and well growth of plantlets was established (Fig. 4Q).
From the study, it was clearly revealed that germination of embryos, plantlet preparation and acclimatization were easily and successfully carried out with promising results. Each step of in vitro plant culture needs specific and unique prerequisite condition (Chen et al., 2007;Winarto et al., 2013). High responsive genotype or explant supported by optimal medium in initiation till well plantlet preparation will produce a reliable in vitro propagation protocol (Kuo et al., 2005;Sinha and Jahan, 2011;. From the hereby research, it was successfully proved an in vitro propagation protocol for two selected Phalaenopsis clones. The protocol can enrich alternative choices of protocol in producing high quality and productivity of qualified seeds in Phalaenopsis. Initiation of embryo is the most critical step for in vitro plant culture (Sinha and Jahan, 2011;Winarto et al., 2013). This phase can be affected by several factors and some of them are genotype or explant, culture medium and incubation culture (Gow et al., 2009;2010;Winarto et al., 2013). Culturing small shoot tips on the half-strength MS medium with full strength of micro elements, Fe-Chellate and vitamin containing 0.5 mg/L TDZ and 0.25 mg/L BA and incubating in dark condition for one month, then transferring to light incubation, produced high embryos up to 8.2 per explant, with 58.5% explant regeneration for ± 2.5 months. While Tokuhara and Mii (2001) found 44.4% plb formation with 24.4% callus formation by culturing shoot tips of P. [(Baby Hat × Ann Jessica) × Equestris] on New Dogashima medium (NDM) containing 0.1 mg/L αnaphthaleneacetic acid (NAA), 1.0 mg/L 6benzylaminopurine (BAP) and 10 g/L sucrose under light incubation with 14 h photoperiod provided by fluorescent lamp at 33 µmol/m 2 /s for 4 months. The plb formation was enhanced by substituting 10 g/L sucrose with 10.5 g/L glucose (Tokuhara and Mii, 2003). In other studies, utilization of apical meristem of ginger cultured on MS medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg/L BA resulted in high embryogenic callus formation up to 93.3% with 32.8 embryos per g of callus (Rostiana and Syahid, 2008); the similar explant of Vitis vinifera cultured on MS medium fortified with 1.0 mg/l indole-3-butyric acid (IBA) and 1.5 mg/l BA successfully regenerated 5.4 shoots per explant (Aazami, 2010); the meristem of Aloe barbadensis cultured on MS medium supplemented with 0.25 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 1.0 mg/L Kinetin (Kin) induced embryogenic callus formation up to 21% (Garro-Monge et al., 2008) and lateral meristem of Manihot esculenta 'Kibaha' cultured on MS medium containing 10 mg/L BAP stimulated 77.6% embryo formation with 5.8 embryos per explant (Opabode et al., 2013).
Those above studies revelead that though meristematic cells having high tot potency and competency to be initiated and stimulated to form embryogenic callus and/or embryos (Henry et al., 1994;Gaj, 2004), embryogenesis derived from the cells, in fact, was varied in results. High compatibility and balance effect between the explant, culture medium, culture condition and other factors kept playing important roles in supporting in vitro plant culture success. The optimal condition successfully induced high number of embryos up to 12.8 embryos per explant in peagon pea (Krishna et al., 2011), inversely it unsuccessfully regenerated embryo formation in the most of cassava varieties (Opabode et al., 2013). Thus it is worth mentioning that in the current study, high-moderate result of embryos was noted.
Successful in regeneration of embryos in initial stage of plant tissue culture will not give high effect on production and preparation of qualified-seeds, when the initial regenerated-embryos fails to be proliferated. Proliferation of the embryos or repetitive embryogenesis was significantly affected by media and/or explant type (Chen and Chang, 2007;Gow et al., 2010;Feng and Chen, 2014). Chen and Chang (2007) reported that high proliferation of embryos up to 13.8 embryos per explant was established on half-strength 322 MS supplemented with 3.0 mg/L TDZ on P. amabilis. Abaxial side down of P. amabilis embryos on half-strength MS containing the similar TDZ concentration induced high repetitive embryos up to 15.6 embryos per explant (Gow et al., 2010), then improved by incubation of embryos in dark condition for 60 days (Gow et al., 2010). The half-strength MS medium augmented with 0.5 mg/L TDZ stimulated embryos up to 19.2 embryos (Feng and Chen, 2014). Whereas in the hereby study, it was revealed that high repetitive embryogenesis up to 10.2 embryos per embryo cultured with 91.4% explant regeneration were established by culturing ad axial side down of single embryos in half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BAP in dark condition for 30 days then transferred to light incubation for ± 1.5 months. Furthermore, TCL, either tTCL or lTCL, successfylly applied and was reported on Aranda 'Deborah' (Lakshmanan et al., 1995), Rhynchostylis gigantean (Bui et al., 1999), Cymbidium Twilight Moon 'Day Light' (Teixeira da Silva et al., 2005); Teixeira da Silva and Tanaka, 2006) induced low embryo proliferation and high explant browning for Phalaenopsis clones studied in the research.
Browning explants is due to several factors and in the study it was presumably induced by stress of explant after slicing and lateness culturing them on culture media. The condition let occurring phenolic compounds easily released and in the existence of oxygen the phenolic compounds were oxidized by phenoloxidase (Ndakidemi et al., 2014), then produced quinone compounds that have negative effect on cell growth and can result in death/necrosis of cells (Ozygit et al., 2008;Rittirat et al., 2012). In other studies, browning explants were occurred due to surface damage caused by liquid culture rotation, as reported on Dendrobium 'Zahra FR 62' (Winarto et al., 2013), by unsuitable culture medium as reported on Phalaenopsis cornu-cervi on New Dogashima medium (Tokuhara and Mii, 1993) supplemented with 4% sucrose and without sucrose that induced plb browning up to 30% (Rittirat et al., 2012), or by condition of culture incubation as reported in culturing leaf explants in the dark for 15 days followed by 45 days under light which induced browning of the explants up to 90% of embryos after 60 days (Gow et al., 2009).
Multiplication of embryos under periodical subcultures on selected medium generally indicates different growth patterns (from acceleration till deceleration) and capacities in each subculture. In the hereby study, it was noted that production of embryos increased gradually starting from the first, second, third, and fourth subculture, then declined afterwards with 16.6 multiplication rate (MR), especially on half-strength MS medium containing 0.13 mg/L TDZ and 0.25 mg/L BA. Other studies showed similar trend, as recorded in D. 'Zahra FR 62' plb proliferation on half-strength MS medium containing 0.05 mg/l BAP with 11.99 MR (Winarto et al., 2013), in D. 'Gradita 31' 2.9 MR established on half-strength MS medium supplemented with 150 ml/L CW . Different growth pattern and capacity were also reported by  on multiplication of Eustoma grandiflorum adventitious shoots, Rumohra adiantiformis (Winarto and Teixeira da Silva, 2012) and Ruscus hypophyllum (Winarto and Setyawati, 2014). From those studies, it was revealed that each explant and plant has unique and specific growth pattern and capacity in the multiplication explant derived from initiation step.
High germination of embryos (73.9%) and well, healthy and vigor plantlet performance led to high survival rate of plantlets (88.3%) in transferring them in ex vitro condition as established in the current studym, using half-strength MS medium with full strength of vitamin, 2 g/L AC and hormone free, and C. rumphii bulk. The healthy and vigor plantlets were presumably become important key factor leading to high acclimatization results. The condition was also evident and reported in: P. cornu-cervi with high germination rate, well plantlet performance and 100% survival plantlets using MS medium supplemented with 150 ml/L CW and 2.0 g/L AC for development plbs into complete plantlets of Sphagnum moss (Rittirat et al., 2014); P. amabilis 'Golden Horizon', 100% plb conversion and 85% survival plantlets, half strength MS medium supplemented with 2 g/L peptone, 2 g/L banana pulp powder, 20 g/L sucrose, 100 ml/L CW, 1 g/L activated charcoal and coconut husk (Sinha and Jahan, 2011); Aerides odorata, well plantlets performance, 95% survivability of plantlets, half-strength MS medium containing 0.5 mg/L NAA and mixture of brick chip and charcoal pieces (1:1, v/v) (Devi et al., 2013); Cymbidium lowianum, 100% plantlet formation, 92% of survivability, half-strength MS medium 1.5 mg/L NAA, 0.1 mg/L BA, 3.0 g/L AC, and moss (Wang et al., 2013); Orchis catasetum, high plb germination, plantlet preparation, 100% of plants survived, MS medium supplemented with 0.5 mg/L BA and 0.5 mg/L NAA, perlite + wood pieces + ionolite + mineral cartridge shell (1:1:1:1, v/v/v/v) (Fakouri-Ghazani et al., 2014); Coelogyne flaccida, high plb conversion, well plantlet performance, 80% of survival plantlets, MS medium with 2 mg/L NAA and 2 mg/L Kin, and coconut husk (De and Sil, 2015). These results indicated that plb germination, plantlet preparation and high acclimatization were easily established for orchid in vitro propagation protocols.

Conclusions
Finally, it can be summarized that a new in vitro mass propagation protocol, as new alternative choice for Phalaenopsis propagation, was successfully established. High embryo formation was determined by culturing shoot tips on halfstrength MS medium with full strength micro, Fe-chellate and vitamin containing 0.5 mg/L TDZ and 0.25 mg/L BA. High proliferation of embryos was proved by culturing embryos individually on half-strength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BA. Under periodical subcultures, the embryos were successfully multiplied on halfstrength MS medium supplemented with 0.13 mg/L TDZ and 0.25 mg/L BA; number of embryos increased gradually from the first subculture till the fifth subculture, then decreased thereafter. The embryos were easily germinated on half-strength MS medium with full strength of vitamin and hormone free. Well and healthy plantlets were stimulated on the same medium with 2 g/L AC and successfully acclimatized on C. rumphii bulk with high survival plantlets. Table 1 3.5 a 2.9 a 2.5 b 2.3 a 2.6 a ==================================================================== Means followed by the same letter in the same column are not significant different based on HSD (p=0.05).