Shoot tips derived-somatic embryogenesis in mass propagation of Dendrobium Indonesia Raya ‘ Ina ’

*Corresponding author: Agus Purwito, Bogor of Agriculture University,Kampus IPB Darmaga, Bogor, West Java-Indonesia. E-mail: apurwito@gmail.com Received: 05 May 2015; Revised: 12 October 2015; Accepted: 18 October 2015; Published Online: 20 October 2015 Emirates Journal of Food and Agriculture. 2015. 27(x): 1-10 doi: 10.9755/ejfa.2015.05.212


INTRODUCTION
Dendrobium is one of important orchid commodities, widely cultivated and high market demand in Indonesia.The orchid is sold as cut flower and potted plants.The plant is sold as cut fl ower and potted plants with USD $ 0.20-0.50per stalk and USD $ 3.00 -4.00 per pot, respectively.In 2012, the orchid product reached ± 20.714.137stalks per year with ± 1.209.938m 2 total cultivated areas (ICBS, 2013).Export of the product can reach 34% from total production of Indonesian orchids, especially to Japan and one of the high market demand of the Dendrobium is Dendrobium Indonesia Raya 'Ina' (DGPMAP, 2014).D. Indonesia Raya 'Ina' was resulted from conventional hybridization between D. Kim Bora x D. Wee Lian and released as a new superior hybrid in 2009 under the Indonesian Center for Plant Variety Protection and Agricultural Permit.The cultivar fl owers less than 2.5 year after acclimatization, has more than 80 cm plant height, produces four fl ower stalks at once with 18 -29 fl ower buds per stalk, longer vase-life (more than 2.5 months), no discoloration, upright position with spiral fl ower, high disease resistance, and adaptability in low to medium land (The Indonesian Center for Plant Variety Protection, 2009).Although the Dendrobium has high market demand, commercialization of the variety in larger scale is constrained by availability of qualifi ed-seedlings.
Conventionally, the Dendrobium is propagated sexually by seeds and asexually by division of off shoots and keikies.The sexual propagation result in heterozygous seedling progenies with no warrant true-to-type plants, while the asexual one produce plants in a very limited number of 2 -4 plants per year with a low quality and multiplication rate (Nasiruddin et al., 2003, Martin andMadassery, 2006).Therefore high promising and potential system, i.e. in vitro propagation, shall be addressed.There were several mass propagation system established previously such as via protocorm like body (plb) formation of Dendrobium hybrids (Khatun et al., 2010), dwarf Dendrobium (Sujjariturakarn and Kanchanapoom, 2011), D. aphyllum (Dutta et al., 2011), D. 'Zahra FR 62' (Winarto et al., 2013); via axillary shoot proliferation of D. nobile 'Emma White' (Asgar et al., 2011), D. longicornu (Dohling et al., 2012), D. Sonia 'Earsakul' (Kumari et al., 2013); while somatic embryogenesis studies in Dendrobium are still limited.
Several in vitro mass propagation protocols of the Dendrobium via somatic embryogenesis in producing high qualifi ed-seedlings were successfully published previously for D. 'Chiangmai Pink' (Chung et al., 2005 and2007), and D. 'Gradita 10' (Rachmawati et al., 2014).Chung et al. (2005) successfully found the best condition in inducing high direct embryos up to 33.6 embryos per explant of D. 'Chiangmai Pink' by culturing leaf explants on half-strength MS (Murashige and Skoog, 1962) medium containing 4 mg/L TDZ incubated under light for 60 days.The embryos were multiplied on the half-strength MS medium PGRs free till plantlet formation and then transferred the plantlets in ex vitro condition on sphagnum moss in 50% shaded green house.The previous study was signifi cantly improved by applying 1 mg/L α-naphthalene acetic acid (NAA) and 3 mg/L thidiazuron (TDZ) for high multiplication rate up to 5.17 (Chung et al., 2007).The leaf explants of D. 'Gradita 10' cultured on the halfstrength MS containing 1 mg/L TDZ and 0.5 mg/L N 6benzylaminopurine (BAP) were the most suitable explant sources and medium to induce high EC up to 80% with EC initiation period as short as 26.3 days after culture.The EC was signifi cantly proliferated on half-strength MS augmented with 0.3 mg/L TDZ and 0.1 mg/L NAA; converted into somatic embryos on the half-strength MS containing 0.05 mg/L BAP, easily germinated on the halfstrength MS fortifi ed by 0.05 mg/L BAP (Rachmawati et al., 2014).However in vitro mass propagation of D. Indonesia Raya 'Ina' using shoot tips as explant source via somatic embryogenesis is not reported yet.
The studies aimed to establish in vitro mass propagation protocol for D. Indonesia Raya 'Ina', especially to prepare qualifi ed-seedlings continuously for commercial purposes of the variety.The researches were initiated by selecting of explant types and medium for initiation of EC, improving EC proliferation, conversing EC to SEs, germinating SEs and acclimatizing well growth-plantlets.Interestedfi ndings in each stage of the study are detail discussed in the paper.

Plant material and explant preparation
Plantlets of D. Indonesia Raya 'Ina' (D.Kim Bora x D. Wee Lian) (90 days old) with 3-4 leaves and 3-5 cm height used as donor explants were obtained from Edward and Frans Orchids, Prigen, Pasuruan, East Java.Explants employed in this study were shoot tips (± 0.3 mm in length), lateral buds (± 2 mm in length), and youngest leaves (± 4 mm in length).The explants were prepared by removing leaves gently, one by one, using a tissue culture blade under a stereo microscope.After removing all leaves, the three explants were isolated and then cultured in penicillin bottles (Ø 2.5 cm 5.5 cm in height) that containing 2.5 ml of treatment media for EC induction.

Effect of explant types and media on initiation of EC
In the initiation stage of EC, two treatments were tested i.e. explant types and media.Three types of explant investigated in the experiment were shoot tips (± 0.3 mm in length), lateral buds (± 2 mm in length), and youngest leaves (± 4 mm in length), while eight compositions of initiation media (IM) were presented in Table 1.A factorial experiment was arranged in a randomized completely block design (RCBD) with three replications.Each treatment consisted of ten bottles, each bottle contained one explants cultured on semi-solid media.The cultures were exposed to 12 h photoperiod under cool fl uorescent lamps with ~13 μmol/m 2 /s light intensity at 23.5 ± 1.1 °C.The light incubation was also applied to other experiments.
All media were supplemented with 20 g/l sucrose, adjusted in pH 5.8, solidifi ed with 2 g/L gelrite and sterilized for 20 min at 121 °C and 15 psi.The EC derived from shoot tips established in the study were then proliferated by slicing the EC and culturing the pieces of EC periodically (3 times, one month each) on the half-strength MS supplemented with 1.5 mg/L TDZ, 0.5 mg/L BAP and 150 ml/L CW in petridish (5 cm in diameter) to prepare suffi cient EC for the proliferation experiment.

Effect of TDZ-BA combination and EC densities on proliferation of EC
The half-strength MS medium containing 0.3 mg/L TDZ and 0.1 mg/L NAA was used as basic medium in the experiment.Varied combinations of the TDZ and BAP concentrations of 0, 0.1, 0.3, and 0.5 mg/L; 0, 0.05, and 0.1 mg/L, respectively were tested in the study.All liquid media were supplemented with 150 ml/L (v/v) CW and 20 g/L sucrose, 25 ml of media were poured in 100 ml erlenmeyer.EC densities investigated in the proliferation stage were 1, 2, 3, and 4 g/25 ml of liquid medium.All cultures were monthly subcultured periodically four times.A factorial experiment was arranged in a RCBD with three replications.Each treatment consisted of three erlenmeyers.The liquid cultures were placed on an orbital shaker on incubation racks at 80 -100 rpm under light incubation.
Parameters observed in this experiment were: (1) EC fresh weight (g), (2) EC fresh weight added (g per month), (3) multiplication rate, calculated by weighting total fresh weight of EC at the end of the culture divided by total fresh weight of EC at the initial culture, and (4) proliferation responses (percentage of EC, SEs formation and germinated-SEs; %).The growth and development of EC or SEs during culture incubation was observed periodically.Data was recorded every month for 4 months of incubation period, but only data in the end of culture were analyzed and exhibited in text.

Effect of times and temperatures of desiccation on conversion of EC into SE and germination of SEs.
EC harvested from the previous experiment were used as explants sources, while half strength MS containing 0.05 mg/L BAP was applied as basic medium.Desiccation times applied in the experiment were 3, 7, 10, and 14 days, while three desiccation temperatures were 5 ± 2 °C, 18 ± 2 °C, and 24 ± 2 °C.A factorial experiment was arranged in a completely randomized design (CRD) with three replications.Each treatment consisted of three petridishes (9 cm in diameter) and each petridish contained ± 15 EC clumps (± 0.5 cm diameter).
The parameters observed in this experiment were: (1) the percentage of EC contamination (%), ( 2) the percentage of EC browning (%), (3) time of initial SEs formation (DAI), ( 4) percentage of SEs formation (%), ( 5) number of SEs per EC clump, (6) time of initial germination of SEs (DAI), ( 7) percentage of SEs germination (%), and ( 8) number of germinated-SEs per EC clump.Periodical observation were carried out to follow the growth and development of EC clump till germination of SEs during culture incubation.All parameters were observed and measured 2 months after incubation.
Periodical observations were carried out to follow the alteration of mini-plantlets during culture incubation.All parameters were assessed 3 months after incubation.

Acclimatization of plantlets
Well-rooted plantlets (± 90 days old) with 3 -5 leaves were selected and prepared for acclimatization as described by Winarto et al. (2013).Total plantlets acclimatized were 500 plantlets, planted in fi ve pots (15 cm in diameter) and then the pots were placed in glass-house.After one week, acclimatized plantlets were watered once a day in the morning (07.00-08.00 am) and fertilized twice a week with 1 g/L of NPK (20: 20: 20).Two months after acclimatization the survival-plantlets were repotted individually in pots (15 cm in diameter) containing wood charcoal and C. rumphii bulk (1: 1, v/v).The percentage of plantlet survivability (%) and number of survival plantlets were recorded after 2 months of acclimatization.

Data analysis
All data collected from the experiments were analyzed by analysis of variance (ANOVA) with SAS program Release Windows 6.12.All percentage data were arcsin transformed before analysis.Signifi cant differences between means were assessed by Tukey's Studentized Range (HSD) at P = 0.05 (Mattjik and Sumertajaya, 2006).

Effect of explant types and media on initiation of EC
Based on periodical observation it was clearly revealed that initial EC formation was observed ± 9 days after culture.Initial formation of EC was generally occurred after browning of explants observed (Fig. 1).The initial EC grew and increased in size 1.0 to 4.5 mm 3 in diameter with varied-fresh weight values of 0.1 to 4.0 g EC (Fig 4A

Effect of TDZ-BA combination and EC densities on proliferation of EC
In the experiment, TDZ-BA combinations and EC densities affected on the growth and proliferation of the EC.The most optimal growth and proliferation of EC were determined on PM-12 liquid medium and 3 g per 25 ml EC density.The PM-12 medium and EC density of 3 g per 25 ml of liquid medium were the best combination for optimal growth and proliferation of EC with 11.91 g EC  Note: Average data is derived from observations of 30 explants.DAI-day after incubation.IM-1 to IM-4 used half-strength MS medium with 1 mg/L TDZ and 0.5 mg/L BA (IM-1), 1 mg/L TDZ and 1 mg/L BA (IM-2), 1.5 mg/L TDZ and 0.5 mg/L BA (IM-3), 1.5 mg/L TDZ and 1 mg/L BA (IM-4); IM-5 to IM-8 used VW supplemented with 1 mg/L thiamine, 2 mg/L TDZ and 1 mg/L KIN (IM-5), 2 mg/L TDZ and 0.5 mg/L KIN (IM-6), 1 mg/L TDZ and 1 mg/L KIN (IM-7), and 1 mg/L TDZ and 0.5 mg/L KIN (IM-8).Means followed by the same letter in the same column are not signifi cantly different based on Tukey test at p=0.05 fresh weight, 3.97 multiplication rate and 0.63 g EC dry weight (Table 3; Fig. 4F).Other treatments reduced the growth and proliferation of EC.
There were different proliferation responses of EC in different media.The highest percentage of EC up to 91.7% was maintained in PM-10 medium, but high conversion of EC to SEs of 32% was occurred in PM-2 and high germination of SEs as high as 13.4% was recorded in PM-1 medium.In the study it was clearly revealed that different combination and concentration of TDZ and BAP leading to different morphogenesis responses (Table 4).

Effect of times and temperatures of desiccation on conversion of EC into SEs and germination of SEs
Times and temperatures of desiccation statistically gave signifi cantly effect on the conversion of EC into SEs and germination of SEs.Three days and 18 ± 2 °C were the most suitable desiccation time and temperature for high conversion of EC into SEs and germination of SEs, however desiccation of the EC clump higher than seven days and 18 ± 2 °C lead to increasing explant contamination and browning (Fig. 2A   Note: Average data is derived from observations of 9 erlenmeyers.All media used half strength MS medium supplemented with 150 ml/L CW.Varied combinations of the TDZ and BAP concentrations of 0, 0.1, 0.3, and 0.5 mg/L; 0, 0.05, and 0.1 mg/L, respectively.Means followed by the same letter in the same column are not signifi cantly different based on Tukey test at p=0.05

Effect of media on acceleration of mini-plantlets growth
The growth of mini-plantlets as high as 7.07 cm with 4.3 leaves per shoot, 5.15 cm length of leaf, 1.36 cm width of leaves, 8.67 number of roots, 5.30 cm length of roots, 0.71 g fresh weight of plantlets, and 0.076 g dry weight of plantlets were established on AM-5 medium (Table 6; Fig. 4K dan 4L).Other media derived from compound fertilizers Hyponex ®, Rosasol ® or Growmore ® stimulated better growth of mini-plantlets compared to MS and VW media having analytical grade components.

Acclimatization of plantlets
In the acclimatization stage, 500 plantlets were successfully transferred to ex vitro condition in C. rumphii bulk with high survival rate of 91.6% (Fig. 4M).Two months after acclimatization, the survival and well growth of plantlets were repotted individually in plastic pots (15 cm in diameter) containing wood charcoal and C. rumphii bulk (1:1, v/v) for further growth.After 4 months, vigorous and healthy plants were established (Fig. 4N).

DISCUSSION
The new fi nding in in vitro mass propagation of D. Indonesia Raya 'Ina' using somatic embryogenesis pathway was successfully developed (Fig. 4).The protocol that was initiated from selection of explants and media, followed by proliferation till acclimatization has almost similar protocol established previously for D. 'Gradita 10' with slightly alteration, especially for selected medium for EC proliferation (Rachmawati et al., 2014).
The shoot tips cultured on the half-strength MS semisolid medium supplemented with 1.5 mg/L TDZ and 0.5 mg/L BAP was the best explant and medium to induce EC initiation time as short as 9.3 days with 100% explant regeneration; while in D. 'Gradita 10' 80% of EC formation in 23.6 days after culture was derived from leaf explants cultured on the same basal medium fortifi ed by 1.0 mg/L TDZ and 0.5 mg/L BAP (Rachmawati et al., 2014).Roy and Banerjee (2003) successfully stimulated high EC formation up to 66.7% after 2 weeks of culture derived from shoottip explants of D. fi mbriatum Lindl.var aculatum Hk. f. cultured on modifi ed Knudson C medium (Knudson, 1946) containing 0.5 mg/L NAA, 1 mg/L BAP, 10% (v/v) CW, 0.5 mg/L niacin, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamin HCl.In D. chrysotoxum, the 69% callus formation regenerated from the similar explants was signifi cantly proved by applying 0.44 mg/L TDZ or BAP in the Knudson C basal medium (Roy et al., 2007), 68% the EC formation derived from PLBs slicing for D. 'Sonia 28' for six weeks after culture was on the halfstrength MS medium augmented with 1 mg/L NAA and 0.1 mg/L 2,4-D and 2 mg/L tryptone (Mei et al., 2012), 100% EC formation for less than 20 days after culture for D. sabin was on MS medium supplemented with 5% of sucrose (Rafi que et al., 2012).All reported studies were clearly revealed that high EC formation was signifi cantly affected by the suitability of explant selected and initiation medium established.
Varied-EC proliferation media with their modifi cations have unique and specifi c effect on proliferation of the EC in different Dendrobium in vitro cultures.Density of 3 g EC per 25 ml and half strength MS liquid medium fortifi ed by 0.50 mg/L TDZ, 0.10 mg/L BAP, and 150 ml/L CW was the best treatment for EC proliferation.In the D.
Application of TDZ and BAP individually and/or in combination in the study giving high signifi cant effect on morphogenesis responses of EC was successfully revealed.Application of TDZ in higher concentration (more than 0.3 mg/L) and combination of TDZ-BAP in higher concentration signifi cantly maintained EC in the similar condition more than 80%, while removing TDZ and applying TDZ-BAP in lower concentration induced SEs formation and SEs germination up to 32% and 13%, respectively.Utilization of TDZ and BAP combination to maintain EC condition and SE formation was also recorded in the previous study for D. 'Jayakarta', D. 'Gradita 31' and D. 'Zahra FR 62' (Winarto, 2012), D. 'Gradita 10' (Rachmawati et al., 2014), D. Gradita 31' (Winarto and Rachmawati, 2013), D. 'Zahra FR 62' (Winarto et al., 2013).
The desiccation of EC and/or SEs in varied-ways would reduce water content; reserve accumulated carbohydrates, lipids and proteins; increase stability of SEs; harden SEs and accelerate maturation of SEs leading to high SEs germination rate with high quality performances (Etienne et al., 2006;Srinivas et al., 2006).Under the treatment alteration of the pattern of gene expression from a maturation program to germination, breaking dormancy and post-germination growth was occurred (Gray, 1989).Furthermore combination of 7 days and 18 ± 2 °C EC desiccation successfully induced the highest conversion of  (Singh, 2014), exposing embryos to 2.6 mg/L abscisic acid (ABA) followed by 2 h desiccation at 10 °C increased SEs germination up to 56% in Musa spp cv.Rasthali (AAB) (Srinivas et al., 2006).
Contamination and browning of explants in desiccation step that are important problem in in vitro culture of orchids were also recorded in the study with 0 -100% and 0 -86%, respectively.The high contamination occurred due to desiccation process during exposing EC in limited-air free in sterile petridishes at 24 ± 2 °C more than 7 days.Longer exposing EC, higher contamination and browning explants occurred with 100% and 86% of them recorded at 14 days of desiccation mostly due to bacteria.The higher contamination up to 24% was also reported in in vitro Phalaenopsis (Lesar et al., 2012) and 26% on Pleurothallis glumacea (Alvarez-Pardo, 2006).Furthermore browning explants are occurred due to accumulation of phenolic compounds causing the loss of growth capacity and tissue death during culture (Rittirat et al., 2012).In the study the browning explants were presumably induced by light surface damage of EC during active shaking in liquid medium and exposing to air-free during desiccation.
In the study, SR up to 91.6% was successfully established.

CONCLUSIONS
From the studies, it can be summarized that in vitro mass propagation protocol via somatic embryogenesis derived from shoot tips of plantlets for D. Indonesia Raya 'Ina' was successfully determined.High EC formation was established by culturing shoot tips on half-strength MS semi-solid medium supplemented with 1.5 mg/L TDZ and 0.5 mg/L BAP.The EC was easily proliferated by culturing 3 g EC per 25 ml in half-strength MS liquid medium fortifi ed by 0.50 mg/L TDZ, 0.10 mg/L BAP, and 150 ml/L CW.Culturing of EC after 7 days of desiccation at 18 ± 2°C on half-strength MS containing 0.05 mg/L BAP signifi cantly induced the highest conversion of EC into SEs and SEs germination.The best performance of plantlet growth was noted on 2 g/L Hyponex ® medium containing 150 ml/L CW.The plantlets were successfully acclimatized on C. rumphii bulk with 91.6% SR.

Fig 1 .
Fig 1. Interaction effect of explant types and media on percentage of explant browning (%).

Fig 2 .
Fig 2. Effect of times and temperatures of EC desiccation on percentage of contamination (a) and explant browning (b) b a

Fig 4 .
Fig 4. Mass propagation protocol of D. Indonesia Raya 'Ina' via somatic embryogenesis pathway.(a) Shoot tip isolated from plantlets usedas explants source.(b) Initial-EC was successfully regenerated ± 2 weeks after culture on half strength MS containing 1.5 mg/L TDZ and 0.5 mg/L BAP.(c) The growth of regenerated-EC ± 1 months after culture.(d) Early proliferation of EC ± 1.5 months after culture.(e) Early proliferation of EC ± 2 months after culture.(f) Proliferation of EC (5 months old) in liquid medium.(g) Conversion of EC into SE on semi solid medium.(h) Growth SEs derived from 7 days of EC desiccation at 18 ± 2 °C ± 20 days after culture.(i) Initial germination of SEs derived from the similar desiccation ± 1.5 months after culture.(j) Germinated SE's performance ± 2 months after culture.(k) The growth of plantlets on AM-5 medium ± 3 months after culture.(l) Well rooted-plantlets harvested ± 3 months after culture.(m) Acclimatized-plantlets 1 month after transferring in ex vitro condition.(n) Individual plants after repotting ± 4 months after acclimatization.(o) Well-bloomed donor plant of D. Indonesia Raya 'Ina' ± 2.5 years olds.

Table 4 : Proliferation responses of D. Indonesia Raya 'Ina' in different media Proliferation media (PM) Proliferation responses TDZ (mg/L) BAP (mg/L) Percentage of EC Percentage of SEs formation
Average data is derived from observations of 9 erlenmeyers.All media used half strength MS medium supplemented with 150 ml/L CW.Means followed by the same letter in the same column are not signifi cantly different based on Tukey test at p=0.05 Note:

Table 5 : Interaction effect of temperatures and times of desiccation on conversion of EC into SEs and germination of SEs of D. Indonesia Raya 'Ina' Times of storage Conversion time of EC into SEs (DAI*)
Note: Average data is derived from observations of 30 EC clumps.Means followed by the same letter in the same column are not signifi cantly different based on Tukey test at p=0.05

Table 6 : Effect of different media on acceleration of mini-plantlets growth D. Indonesia Raya 'Ina' after three month was subcultured on solid media Acceleration media (AM) Height of plantlet (cm) Number of leaves per plantlet
(Lee et al., 2001)derived from observations of 25 plantlets.Means followed by the same letter in the same column are not signifi cantly different based on Tukey test at p=0.05EC into SEs and SEs germination (Tabel 5).In different study, 5 days EC starvation at room temperature and culturing on half-strength MS resulted in 20-fold increase in SEs production, enhanced maturation and germination of Daucus carota SEs(Lee et al., 2001), 48 h desiccation in combination with chilling leading to 58.3% SEs formation of Citrus jambhiri Note