Asymbiotic germination of immature embryos of a medicinally important epiphytic orchid Acampe papillosa (Lindl.) Lindl

The immature embryos (28 weeks after pollination) were inoculated on M (Mitra et al., 1976), and PDA (Potato Dextrose Agar) media, with and without different growth additives. The seeds showed positive germination response in both the nutrient media but the frequency and onset of germination response and associated morphogenetic changes leading to seedling development varied with the nature of growth stimulus. In basal M medium, about 40.75±0.75% seeds germinated, while in basal PDA medium, 21.25±1.25% seeds reacted positively to germination (P<5%). M medium supplemented with coconut water (CW) (15%), supported early and highest germination (70.75±0.75%) and induced protocorm multiplication; complete seedlings were obtained in 131.50±1.73 days. Additional presence of activated charcoal (AC) (0.2%) in the PDA medium inhibited the seed germination, while use of CW (15%) or yeast extract (YE) (2 g/L) in the medium, favoured enhanced and early germination response and differentiation of protocorms. YE, favored development of profusely hairy protocorms formation and healthy seedlings were obtained within 176.25±1.25 days.


INTRODUCTION
Orchid seeds are unique in being exceedingly small, dust like in appearance, and more or less fusiform in shape; these lack endosperm and have undifferentiated embryos enclosed within transparent seed coats.Their germination in nature is dependent upon a suitable association with a mycorrhizal fungus.Their fungal requirement can, however, be compensated by supply of sugars and other mineral nutrients in vitro, and several orchid species from diverse habits and habitats have successfully responded to asymbiotic germination (Arditti et al., 1982a;Hossain et al., 2009;Pathak et al., 1992Pathak et al., , 2001Pathak et al., , 2011)), much, however, still remains to be learnt *Corresponding author.E-mail: hsalar1970@gmail.com.about the nutrient requirements of commercially important and / or endangered orchid species, keeping in view of the large size of the orchid family.
Acampe papillosa, a species of epiphytic orchids, has ornamental potential for its evergreen, clustered foliage and multiflowered, pendulous racemes, with yellow green flowers.Its therapeutic utility is also well documented; its roots are used to cure rheumatism (Bi et al., 2005;Lawler, 1984).Its natural populations are on decline due to commercial collection and habitat destruction.The protocols for its mass propagation are, however, yet to be developed.
In the present study an attempt was made to assess the in vitro asymbiotic germination potential of immature embryos of A. papillosa, a medicinally important epiphytic orchid.

MATERIALS AND METHODS
The green and undehisced capsules, harvested 28 weeks after pollination (WAP) from live plants served as source for young seeds with immature embryos.These were scrubbed with 'teepol' (1%), washed thoroughly under running tap water for 15 to 20 min, and surface sterilized for 7 min with HgCl₂ solution (0.1%), with 1 to 2 drops of 'teepol' as a wetting agent prior to washing with sterilized distilled water.These were also treated with streptomycin (0.03%) for 5 min and repeatedly washed with sterilized double distilled water so as to remove all the traces of sterilizing agents, and subsequently, these were dipped in 70% ethyl alcohol for 30 s, flame sterilized, and were split open longitudinally with a sterilized blade to scoop out the immature embryos, under aseptic conditions.The effect of two different media [M (Mitra et al., 1976), and PDA (Potato Dextrose Agar)] was tested on in vitro seed germination and subsequent seedling development in A. papillosa.Effect of different growth additives [AC (activated charcoal; 0.2%), CW (coconut water; 15%), YE (yeast extract; 2 g/L)] was also assessed during the experimentation.The seeds were inoculated on different media in cultures vessels, and incubated at 25±2°C under 12 h photoperiod provided by cool white fluorescent tubes (40 µmol m -² s -¹).Eight replicates were used for each treatment.The cultures were examined regularly and the observations such as germination frequency, and on the number of days taken for onset of germination, spherule and protocorm formation, chlorophyll synthesis thereof, emergence of leaf as well as root primordia and seedling development were recorded.Subculturing was done at four week intervals.The well-developed seedlings with 2 to 3 leaves and 1 to 2 roots were gradually subcultured on hormone free and subsequently on one half and one fourth strength nutrient medium, respectively for 3 months as a part of hardening procedure.These were then removed from culture vessels and thoroughly washed with lukewarm water to remove adhering medium completely without causing damage to the roots.Subsequently, the seedlings were treated with a mild fungicide (Bavistine; 0.01%) solution and streptomycin (0.03%) for 5 min.These were then transferred to green house and potted in clay pots with potting mixture of charcoal pieces, Sphagnum moss, pine bark and brick pieces (1:1:1:1); these showed 70% survival rate.
One -way analysis of variance (ANOVA) which is used to compare two or more or means using the F-distribution was performed with respect to each response (average ± standard error against each additives is mentioned in Tables 1 and 2).The results of ANOVA showed no significant differences of additives at 5% level of significance.Various groups of additives showing identical/similar responses were statistically formed.To this end, Tukey test was performed at 5% level with respect to showing identical/similar responses are denoted in Tables 1 and 2 by putting the same alphabet as superscript on the respective means.In order to observe if the difference between means of the two groups is significant or not (two culture media), a t-test for independent samples was used.

RESULTS
The immature embryos in young seeds (28WAP) procured from the green undehisced capsules (Figure 1A) showed positive germination response in both the nutrient media that is M and PDA but the frequency and onset of germination response and associated morphogenetic changes leading to seedling development, varied with the nature of growth stimulus; a Piri et al. 163 differential response was, however, observed on these media (Tables 1 and 2).

M medium
In basal M medium (control), about 40.75±0.75%seeds were germinated.During germination, the embryos swelled (Figure 1B), assumed spherical outline, and emerged out of the seed coats through apical/lateral slits as chlorophyllous spherules (Figure 1C 1G).In YE supplemented medium, favoured     development of profusely hairy protocoms and healthy seedlings were obtained within 176.25±1.25 days.With CW in the medium, 31.25±1.25%seeds germinated and the protocorms were obtained in 61.50±0.87days; this additive favoured better and early seedling growth and complete rooted seedlings were obtained in 161.50±0.87days (Figure 1H), but with a significantly inferior germination frequency as compared to CW when added to M medium.Complete seedlings thus developed were transferred to green house and these subsequently showed 70% survival rate (Figure 1I).

DISCUSSION
Since the orchid seeds contain negligible quantity of reserve material for the development of embryos (Leroux et al., 1997) and further lack metabolic machinery (glyoxysomes) for conversion of their lipidaceous reserve food material into more utilizable forms (Harrison, 1977;Vij and Sharma, 1983), these require a symbiotic association with a mycorrhizal fungus, in nature (Bernard, 1904).Ever since Knudson (1921Knudson ( , 1922Knudson ( , 1925) ) germinated the seeds of Cattleya, Laelia, and Epidendrum on a sugar rich medium in vitro, thus bypassing their fungal requirement, the technique of asymbiotic seed germination has been positively tested in a large number of orchid species and hybrids (Arditti et al., 1982a, b;Buyun et al., 2004;Chang et al., 2005;Kauth et al., 2008;Pathak et al., 1992Pathak et al., , 2005Pathak et al., , 2011;;Vij and Pathak, 1988).In the present investigation, the young seeds with immature embryos (28WAP) were used because it has been reported that upon maturation the seeds of many orchid species show decrease in viability or become dormant (Arditti et al., 1981).The fertilized ovules from few weeks old capsules are capable of germinating in vitro (Arditti et al., 1982a;Vij and Pathak, 1988).The technique reduces the time lapse between pollination and sowing of seeds, saves them from exposure to sterilizing agents and favours production of a large number of seedlings.Though literature studies reveal that the immature seeds germinate better than the mature ones, the earliest stage at which the embryos can germinate varies with the species, genus, hybrid and culture conditions in orchids (Arditti et al., 1982a).The easy germinability of immature seeds can be attributed to their distended testa cells, metabolically awakened embryos, and absence of dormancy factors (Linden, 1980;Yam and Weatherhead, 1988).In the presently investigated species, seeds successfully germinated on chemically defined (Mitra et al., 1976) and undefined [Potato Dextrose Agar (PDA)] media under asymbiotic conditions indicating thereby that their fungal requirement was compensated by providing an appropriate nutrient regime in vitro.Though the seeds showed positive germination response in both the nutrient media, but the frequency and onset of germination response and associated morphogenetic changes leading to seedling development varied with the nature of growth stimulus.M medium has been successfully used earlier in a number of orchid species (Pathak et al., 2001(Pathak et al., , 2011)).M medium supplemented with AC, enhanced frequency of, and slightly advanced onset of germination, promoted growth and multiplication of protocorms, and advanced differentiation thereof and subsequent seedling development, whereas, its presence proved inhibitory for seed germination in PDA medium.Protocorms whenever obtained, develop absorbing hair.The absorptive potential of protocorms, however, varied with the chemical stimulus in the nutrient pool; protcorms obtained in YE enriched PDA medium were profusely hairy.The profuse growth of absorbing hair observed presently in YE enriched PDA supplemented cultures, and their profuse growth in dark incubated cultures reported earlier by Ichihashi (1990) in Bletilla striata seedlings indicates their relation with stress (nutritional as well as physical).
in the columns nos. 2 and 3 are mean ± S.E.; same alphabetical letter in the superscript denotes that the corresponding means are in the same group using Tukey's multiple range test at 5%. a-b, Written horizontally as superscripts on right side of the data represent significantly difference between two media.

Table 1 .
Effect of different nutrient media on the frequency and onset of seed germination, and spherule and protocorm formation during asymbiotic germination in A. papillosa.
Entries in the columns nos. 2 and 3 are mean ± S.E.; same alphabetical letter in the superscript denotes that the corresponding means are in the same group using Tukey's multiple range test at 5%. a-b, Written horizontally as superscripts on right side of the data represent significantly difference between two media.AC, Activated charcoal; CW, coconut water; YE, yeast extract.

Table 2 .
Effect of different nutrient media on chlorophyll synthesis, emergence of leaf and root primordia and seedling development during asymbiotic germination in A. papillosa.