Genetic Stability of In vitro Multiplied Phalaenopsis gigantea Protocorm-like Bodies as Affected by Chitosan

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant.


Introduction
The Orchidaceae family is considered as one of the most diverse flowering plant families, comprising of 25,000 species and more than 800 identified genera.Orchids contribute around 8% of global floriculture trade (Chugh et al., 2009).Phalaenopsis gigantea is one species found in the lowland forests of the state of Sabah, Malaysia.Deforestation and over-collections have resulted in near extinction of this species (Rodrigues and Kumar, 2009).This tropical orchid is commonly known as Elephant's Ear orchid with its enormous leaves.Phalaenopsis gigantea has the potential of producing beautiful hybrids.This species is usually propagated through the formation of new buds induced at the bases of mature plants.But the number of new buds initiated by a mature plant is very low (Shu-guo, 2008).The induction of protocorm-like bodies (PLBs) or callus from the protocorm using liquid media and shake cultures has become a reliable method for mass propagation due to the great number of PLBs that can be achieved with short labor time.The aeration system of liquid medium and the close contact of explants with medium may facilitate the uptake of oxygen, nutrients, phytohormones and consequently lead to increase the rate of plant regeneration (Sandal et al., 2001).
Chitosan is a cationic polymer and N-deacetylated product derivative of chitin which is present in shells of crustaceans and cell wall of fungi (Devlieghere et al., 2004).This component is an environmentally friendly carbohydrate polymer and has been reported to stimulate growth of some plant species, including orchids (Nge et al., 2006).It also has been reported that the supplementation of chitosan for in vitro regeneration of Dendrobium 'Eiskul' did not induce somaclonal variations (Pornpienpakdee et al., 2010).Phalaenopsis gigantea has produced outstanding novelty hybrids and has become a much sought after species that result in over-collection from its natural habitat leading to eventual extinction.This species is inherently difficult to propagate in the nature.This species is usually propagated through the formation of new buds at the bases of mature plants.However, this method

Experimental design and statistical analysis
The study on in vitro multiplication of PLB was laid out in a factorial combination of treatments based on randomized complete block design (RCBD).The recorded data were analyzed using the analysis of variance (ANO-VA) and means separated using Duncan's New Multiple Range Test (DNMRT).All statistical data were tested at the 5% level of significance for comparison between treatment means.In this study 5 experimental units were considered for each replicated and the experiment consisted of 3 replicates.The results are presented as means ± the standard error.

DNA extraction
Leaves of in vitro mother plant were used to isolate genomic DNA for PCR amplification and termed as MP.
To study the genetic stability among MP and regenerated PLBs after 2, 4, 6, and 8 weeks of culture, the secondary PLBs obtained at the end of every two weeks of sub-culturing (S1, S2, S3 and S4) were randomly used to isolate genomic DNA.Samples from MP, S1, S2, S3 and S4 were first washed with tap water and sterilized in 10% (v/v) Clorox® solution for 5 minutes.The PLBs were rinsed three times with distilled water, wrapped with aluminum foil and stored at -80ºC for DNA extraction.Genomic DNA was extracted using cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990) with minor modification.Quality and quantity of DNA was monitored by spectrophotometry and gel inspection.

PCR amplification and gel scoring
Eight primers were selected from a total of 10 ISSR primers for assessment of genetic fidelity (Tab.1).PCR amplification was carried out in a total volume of 25 µL including DNA templates (20, 30, 40 and 50 ng/µL) and ISSR primers (1µM), 12.5 µL DreamTaq™ Green PCR Master Mix (Fermentas, Inc, Hanover, USA) and 10.4 µL nuclease free water.Amplification was performed in a programmable Thermal Controller (MJ Research Inc., USA) which was consisted of an initial denaturation step at 94ºC for 5 minutes, followed by 35 cycles of 30 s denaturation at 94°C, annealing temperature at 54.8-60.5ºCfor 90 s (Tab. 1) and continued at 72ºC for 90 s with a final extension at 72ºC for 5 minutes.Amplified products were separated on 1.5 % (w/v) agarose gels.The molecular size of amplified PCR products were estimated using 1 kb DNA ladder (Fermentas, Inc, Hanover, USA).The digital image files were analyzed using UVIDoc software v.99.01 and fragment sizes were estimated based on DNA ladder.All the reactions were repeated three times.Only well-separated bands with high intensity were scored as present or absent for ISSR markers.The scoring of bands was done as 1 for presence and 0 for the absence of DNA bands in the gel.Electrophoretic DNA bands of low visual intensity that could not be readily differentiated as present or absent were considered ambiguous markers and were not scored. of propagation is very inefficient as the number of new buds produced by a plant is very low.In the current study efforts to enhance PLB multiplication using different liquid media supplemented with chitosan and assessment of genetic stability across regenerated PLBs and mother plant were undertaken.

Plant materials and culture conditions
Young leaves from in vitro donor plant (mother plant) were used for induction of initial PLBs.The leaf tip segments (1.5 cm in length) were excised and cultured on semi solid New Dogashima medium (NDM) (Tokuhara and Mii 1993;1998) supplemented with 0.1 mg/L thidiazuron (TDZ) and 1.0 mg/L naphthalene acetic acid (NAA).The protocol was selected based on an earlier report on Phalaenopsis gigantea (Niknejad et al., 2011).The cultures were placed under a 16-h photoperiod at an irradiance of 30 μmol m ² s ¹ and temperature of 25±2°C.The earliest morphological sign of PLB formation appeared as swellings on the adaxial side of leaf segments within 4-6 weeks of culture; small round bodies (small initial PLBs) were transferred to hormone free NDM and used as plant material (Fig. 1a).

Media and chitosan preparation
The interaction of different media types and chitosan concentrations on PLB multiplication of P.gigantea was investigated.For this purpose, liquid VW (Vacin and Went, 1949) and New Dogashima medium (NDM) (Tokuhara and Mii 1993;1998) were used and each liquid medium was supplemented with 20% coconut water and different concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L).Chitosan powder labeled as low molecular weight and 75% degree of deacetylation (Sigma Aldrich) was selected for the preparation of stock solution.One gram of chitosan powder was dissolved and stirred in 1% acetic acid and heated to 35ºC, constantly agitating for 24 hours.The pH of chitosan stock was adjusted to 5.5 using 2 M NaOH and the solution was stirred for 4 hours.Ultimately, the pH of media was adjusted to 5.4 followed by autoclaving for 20 min at 121ºC and 15 psi.

Proliferation conditions
For evaluating the multiplication of secondary PLB, five initial PLBs (0.25-0.3 g) induced from leaf segments of donor plant were cultured in 200 ml Erlenmeyer flasks containing 100 ml of growth media (VW and NDM).The PLBs produced in each flask were transferred to freshly prepared medium every 14 days for two months.The cultures were shaken at 60 rpm on a rotary shaker under 16-h photoperiods using fluorescence lighting of 30 μmol m ² s ¹ per day and the cultures were maintained at 25±2°C.The number of PLBs and their fresh weights were determined at the end of the experiment.

Effects of different chitosan concentrations and media types on PLBs proliferation
Initial response of primary PLBs to different chitosan concentrations and media types was observed 2 weeks after culturing and it was consisted of protocorms swellings (Fig. 1b).The nodular structure from the swelling regions was observed after 3-4 weeks of cultivation (Fig. 1c), and these nodular tissues ultimately developed further and formed more secondary PLBs.The results exhibited significant differences (p ≤ 0.05) in PLB multiplication efficiency among different treatments (Fig. 2a).All treatments including the control were able to induce secondary PLBs with mean number of PLBs varying from 31 to 177 after 8 weeks of culturing.Regardless of media used, the best response was recorded at concentrations ranging from 5 to 15 mg/L of chitosan supplementation in both media and most PLBs were between 4-5 mm in diameter.Further increasing the amount of chitosan did not significantly improve the formation of secondary PLBs at 20 mg/L.Chitosan at a concentration of 10 mg/L produced the highest mean number of PLBs in VW (177) and NDM (147), showing no remarkable difference in terms of PLB mean number (Fig. 1d).Conversely, NDM at 0, 20 and 25 mg/L chitosan induced the lowest mean number of secondary PLBs compared to the other treatments, since only 40-32 PLBs per flask were observed after 8 weeks of cultivation.VW at 25 mg/L chitosan did not show further prolifera-tion compared to the control.It was also observed that the effect of NDM at 15 mg/L chitosan on PLB proliferation was not statistically different (p ≤ 0.05) with VW at 20 mg/L chitosan (Fig. 2a).

Effects of chitosan on leaf organogenesis
The earliest morphological changes and differentiation of PLBs occurred after 5 weeks of culturing.Some tiny secondary PLBs were converted to mature PLBs during the first five weeks of culturing and ultimately the small emerging leaves appeared on the apical region of differentiated PLBs after 7-8 weeks of cultivation (Fig. 1e).The highest differentiation potential of PLB and leaf formation were observed in liquid NDM at a chitosan concentration of 20 mg/L with a significantly larger mean number of developing leaves (66) compared to the other treatments during the same period of culture.Interestingly, there was no significant difference (p ≤ 0.05) in mean number of leaves produced by NDM at 15 mg/L chitosan (42) and VW at 10 mg/L of supplemental chitosan (32).The highest number of new leaves in liquid VW treatments was observed at 10 mg/L chitosan supplementation (Tab.2).Subsequently, the result showed that in the presence of chitosan in liquid culture, the success of leaf formation largely depended on the type of growth medium.The results showed that the interaction of NDM and chitosan was more effective on differentiation and conversion of mature PLBs to leaves when compared with the interaction of VW and chitosan.

Assessment of genetic stability using ISSR analysis
Optimization of ISSR protocol and selection of the primers exhibited that the ISSR bands were reproducible.Of the 10 random ISSR primers used for initial screening, only 8 primers gave more than four clear and scorable bands (Tab.1).The genetic stability was assayed for PLBs obtained from each subculture stage in both media supplemented with 10 mg/L chitosan (optimal concentration).ISSR molecular technique generated 55 band classes and the number of bands for each primer varied from 5 to 11, with an average of 6.9 bands per ISSR primer.A total of 275 bands were generated by ISSR techniques, giving rise to monomorphic patterns across mother plant and secondary PLBs produced during the process of sub-culturing.Fig. 3 represents the monomorphic band classes generated by ISSR across mother plant and the PLBs achieved by the subculture stages of optimum chitosan (10 mg/L) in VW and NDM.Ultimately, the monomorphic banding model indicated that addition of 10 mg/L of chitosan in both media did not induce any detectable somaclonal variation.

Effects of different chitosan concentrations on PLB growth
Varying the concentrations of chitosan in VW and NDM led to differences in total fresh weight.The increase in total fresh weight of PLBs and initiated leaves was registered after eight weeks of culture.The results showed that treatment with 10 mg/L chitosan induced the highest mean total fresh weight in VW (8.4 g) and NDM (7.3 g) compared to other treatments.After eight weeks of culture, liquid VW with 10 mg/L chitosan exhibited a stimulated 30-fold increase in total fresh weight in comparison with PLB weight (0.25-0.3 g) at the start of the experiment.Moreover, VW with 20 and 25 mg/L chitosan did not significantly increase mean fresh weight when compared with the chitosan free control.NDM with 25 mg/L chitosan and control cultures showed a non-stimulated increase in weight of 4-5-fold.In general, liquid VW alone or when supplemented with chitosan was more effective in enhancing the growth of PLBs when compared with liquid NDM (Fig. 2b). of 15 mg/L chitosan in liquid VW medium was optimal for PLB multiplication (Nge et al., 2006).
According to the above observations and the findings of the present study, the effectiveness of chitosan depended on molecular weight, frequency of application, the concentration, the ratio of sugar carbons to glucosamine and N-acetyl-glucosamine (Uthairatanakij et al., 2007).However, the PLB proliferation response to different chitosan concentrations seems to be variable from species to species.Regardless of the similar effects of 10 mg/L chitosan in both media on the mean number of secondary PLBs, the comparative analysis showed that the interaction of liquid VW and chitosan was more efficient for PLB proliferation in comparison with NDM supplemented with chitosan.Various media have been used for mass proliferation of orchid PLBs.Some reports showed that VW medium was more effective for Phalaenopsis and Dendrobium PLB proliferation (Baker et al., 1987;Kalpona et al., 2000).Ishii et al. (1998) also reported that VW with the addition of 20% CW was suitable for PLB multiplication in Phalaenopsis.

Effects of chitosan on leaf organogenesis
It has been previously reported that the presence of 20% CW in tissue culture media were effective for PLBs growth and plantlet regeneration (Ng and Saleh, 2010).This natural compound is rich in cytokinins that are mostly used for PLB multiplication in the plant tissue culture

Effects of different chitosan concentrations and media types on PLBs proliferation
There have been only a few reports on in vitro effects of different chitosan types, molecular weights, deacetylation degree, polymerisation and concentration on orchid propagation (Nge et al., 2006;Pornpienpakdee et al., 2010).Various comparative analyses of plant responses to different chitosan concentrations have shown that the presence of chitosan in tissue culture medium induced higher frequencies of PLB formation and multiplication in different orchid species such as Dendrobium phalaenopsis (Nge et al., 2006), Dendrobium 'Eiskul (Pornpienpakdee et al., 2010) and Grammatophyllum speciosum (Sopalun et al., 2010).The study presented here is in conformity with other reports indicating that optimal PLB proliferation response was observed at 5-15 mg/L of chitosan range (Nge et al., 2006;Sopalun et al., 2010).Pornpienpakdee et al. (2010) observed that in VW medium supplemented with 10 mg/L polymeric chitosan of 70% (p-70) degree of decetylation, the average number of PLBs ( 541) was almost 2-fold higher than the number of PLBs (278) produced by chitosan free medium after 3 months of cultivation.PLB proliferation of Grammatophyllum speciosum, in ½MS liquid medium supplemented with 15 mg/L chitosan led to a 7-fold increase in PLB growth (Sopalun et al., 2010).Similarly, in the case of Dendrobium phalaenopsis the addition sessment of somaclonal variation and genetic fidelity of regenerants.Different studies have shown the genetic stability of in vitro raised plants such as gerbera regenerated from tissue culture of capitulum, leaf and shoot tips (Bhatia et al., 2009), the plantlets generated from in vitro cultured banana regenerated from rhizomes (Lakshmanan et al., 2007), almond plantlets regenerated from axillary branches (Martins et al., 2004), a monopodial orchid hybrid with several shoots initiated from seedlings (Kishor and Devi, 2009) and Cymbopogon flexuosus initiated from somatic embryogenesis (rhizomatous explants) (Bhattacharya et al., 2008).The proliferation system reported here is efficient and capable of producing large numbers of genetically uniform Phalaenopsis gigantea PLBs within a relatively short period of time.The PLBs obtained using this procedure could be proliferated further in a largescale bioreactor system.

Conclusions
In summary, the present report expresses the establishment of a promising in vitro culture system to stimulate PLBs proliferation without causing a somaclonal variation rate.VW medium supplemented with 10 mg/L chitosan was most ideal for multiplication of phalaenopsis gigantea PLB.The results of study showed that leaf formation from secondary PLBs in liquid culture supplemented with chitosan largely depends on medium composition.The present protocol of PLB multiplication, as outlined in this paper will be efficient means for commercial proliferation of phalaenopsis gigantea within a relatively short period of time (8 weeks).
industry (Huan et al., 2004).However, the synthesis of cytokinins cannot completely substitute the effect of CW because other phytohormones such as auxins, gibberellins and undefined chemical components in CW may exert synergistic effects with cytokinin (Yong et al., 2009).After the formation of secondary PLBs and at the time of organogenesis, the endogenous PGR synthesis system may be activated and the increased levels of endogenous PGRs in PLBs may affect differentiation of the cells in the absence of exogenous PGRs (Smith and Krikorian, 1990).Nge et al. (2006) reported the formation of juvenile leaves during multiplication of Dendrobium phalaenopsis PLBs, in liquid tissue culture medium supplemented with chitosan and the initial PLB's from meristematic buds were treated with different chitosan concentrations.The optimum fresh weight of PLB's was reported at 15 ppm oligomer chitosan during the first three weeks of culturing.The excised meristem tissue initially increased in size and was converted into small round bodies, and the juvenile leaves appeared after five weeks of culturing.It has been previously indicated that in D. formosum, the effect of chitosan on growth and leaf induction depended on the growth medium composition (Limpanavech et al., 2003).The results of the present study showed that the liquid NDM supplemented with chitosan was more effective for leaf regeneration.Chitosan seems to be an appropriate growth stimulator for orchid micropropagation which may play a role in increasing the growth and development of explants by some signaling pathway similar to auxin biosynthesis via a tryptophan-independent pathway (Uthairatanakij et al., 2007).

Assessment of genetic stability using ISSR analysis
Most orchid researchers prefer to use PGR-free media to obtain genetically stable PLBs (Huan et al., 2004).As the propagation process does not involve constant exposure to exogenous PGRs, secondary PLBs with the lowest chance of somaclonal variation can be obtained.During in vitro culture of plants, variations can happen due to different reasons such as modifications in DNA methylation, gene amplification, chromosomal abnormality and point mutation (Saker et al., 2000).The use of synthetic plant growth regulators in growth media even at suboptimal concentrations was also found to induce somaclonal variations in some tissue cultured plants (Martins et al., 2004).The presence of CW in tissue culture media results in considerable plant cell multiplication without enhancing the number of undesirable mutations (Arditti, 2008).Pornpienpakdee et al. (2010) also reported that the addition of 10 and 20 mg/L chitosan in liquid media did not induce any somaclonal variation.Molecular analysis is being commonly used for monitoring genetic fidelity of in vitro raised plants.DNA based markers provide an effective procedure to determine tissue culture induced variations since these markers are not influenced by environmental factors (Peredo et al., 2009).PCR-based techniques such as SSR, ISSR, RAPD and AFLP have been used for as-

Fig. 3 .
Fig. 3. ISSR banding pattern in multiplied PLBs and mother plants (lane MP is mother plant, lanes 1-4 are multiplied PLBs obtained after subcultures 1-4 with optimal chitosan).(A, B and C), Genetic stability of PLBs obtained in VW at 10 mg/L chitosan.(D, E and F), Genetic stability of PLBs in NDM at 10 mg/L chitosan Tab. 1. List of 10 ISSR primers used for PCR amplification and assessment of genetic stability across mother and secondary PLBs obtained during Sub-culturing process According to analysis of variance (ANOVA), Values are exhibited as means ± standard errors (SE); different letters within a column represent significant differences at (p<0.05)