Influence of nutritional media and photoperiods on in vitro asymbiotic seed germination and seedling development of Dendrobium aqueum

Dendrobium aqueum Lindley is an epiphytic orchid decreasing in nature owing to environmental disturbance and inherent lower germination rates of mature seeds. To conserve this species, in vitro asymbiotic media and photoperiod screening were experimented. Twenty asymbiotic orchid seed germination media were examined to get the optimal medium for germination and seedling development under 16/8 hrs photoperiod. Highest seed germination occurred in 1 /2 MS medium (93.41%) followed by mMS (91.04%) and m 1⁄2 MS (90.23%) in 6 to 7 weeks. The biggest protocorms were observed in MS medium (788.8 μm) followed by 1⁄2 MS (615.60 μm). The yellowish embryos during germination exhibit varied colours from yellow to pale green, dark green, yellow, brown, black and white. MS, 1⁄2 MS, mMS, 1⁄2 mMS, and 1⁄4 MS media only promote fully developed seedlings within 126 to 143 days. The influence of five different photoperiods with four media was also examined separately. 24/0 hrs (L/D) photoperiod provide highest germination (97.75%) on MS followed by 1⁄2 MS (96.01%). Dark Photoperiod 0/24 h (L/D) supports up to protocorm stage but with poor seedling growth. The well rooted seedlings were hardened and successfully reintroduced in its natural habitat with 56% survival rate.


INTRODUCTION
Orchidaceae is one of the largest families of flowering plants.The genus Dendrobium is the largest genus of Orchidaceae having mostly epiphytic or occasionally lithophytic habits and comprising of about 1500 species worldwide (Carlsward et al., 1997) with 116 species in India (Misra, 2007).Wide range of flower colors, size and shapes, year-round availability, and long flowering life by several weeks to month are the prime attraction of this genus (Adelheid, 2007).*Corresponding author.E-mail mvrao_456@yahoo.co.in.Tel: +91-9442147460.
Dendrobium aqueum Lindley is an epiphytic, sympodial orchid endemic to Eastern Ghats, found at an altitude of 1300 to 1600 m on Shola trees in deep shade (Matthews, 1983).This species is being reported under Near Threatened category by the conservation assessment and management plan workshop (CAMP, 2001).Continuous pressure of forest degradation, deforestation, shifting cultivation, forest fire, tree cutting, lopping, biological invasion, and indiscriminate exploitation of natural resources in Eastern Ghats affect and destroy the native flora completely of which orchids are the primary victims (Reddy et al., 2005).D. aqueum population has been declining due to loss of habitat and inherent lower germination rate in nature.In vitro studies will immensely aid conservation measures of this orchid species.
Mass propagation of the orchid seedlings in vitro could be achieved with a suitable basal medium devoid of growth regulators as orchid seeds have sufficient growth hormones to germinate and develop into seedlings in nature (Arditti, 1979).Also, seedlings raised through this growth regulator free experiment would help as a source of explants to produce multiples of desired healthy plantlets.It is suggested that epiphytic species can germinate both in the light and dark and seem to require light only for the induction or improvement of shoot and or root formation (Arditti, 1979).Both media and photoperiods could affect the germination (Dutra et al., 2009).Therefore, separate experiment was carried out focusing on nutrient media screening (devoid of growth regulators) and the effect of different photoperiod to assess germination, protocorm and seedling development of D. aqueum.

Seed source, sterilization procedure
Green, mature capsules collected from Kolli hills, Eastern Ghats of Tamil Nadu were used during experiments.A single green capsule measures ca.4.4 × 1.6 cm length and width (Figure 1a).The seeds measured ca.273 to 340 x 61 to 106 µm in size (Figure 1b) and numbered around 900 to 1000 seeds per one mg of weight.The embryos were elliptical and yellowish in colour.
Our regular study on seed viability of green mature capsules often to have about 80 to 90%.For both the experiments single green capsule was washed in running tap water with 1 to 2 drops of soap solution (Teepol) for 15 min.Then the capsule was transferred to sterilized laminar hood.The capsule was air dried for a while and dipped in 70% alcohol for few seconds and burnt off in seconds.This was repeated two to three times.Finally the capsule was cut opened longitudinally with a sterile surgical knife and all the seeds were carefully extricated and mixed well before inoculation and viability test.

Seed viability test
Seed viability was tested by using TTC (2, 3, 5-triphenyl tetrazolium chloride) test (Waes and Debergh, 1986a).10 ml of 1% TTC solution with the pH 7.0 was taken in a screw cap tube with the seeds.Then the solution was kept in dark at 20 ± 2°C for 24 h.After 24 h, the solution was drained and the seeds were washed in sterile distilled water for 3 times.Red stained viable seeds and yellowish unstained seeds were counted under a light microscope.

Asymbiotic media selection and culture condition
Twenty basal media successfully reported in orchids seed germination studies were used for the media screening experiment (Table 1).MS medium (Murashige and Skoog, 1962) diluted and modified into, ½ macro strength (½ MS), quarter macro strength (¼ MS), MS vitamin replaced with B5 vitamin (mMS, m ½ MS, m ¼ MS) were also used.The entire medium was set with their appropriate pH and sugars and solidified with 8% agar before autoclaved at 121°C and 1.5 kg cm -2 for 20 min (Table 1).The sterilized media were poured into sterile petriplates.After the media cooled, the seeds were sown and the plates were sealed with 2 to 3 layers of parafilm.Cultures were maintained under white fluorescent lamp with the standard protocol of 16 hrs photoperiod with a temperature of 23 ± 2°C.

Photoperiods on germination and development of seedlings
Light is one of the factors which influence orchid seed germination (Arditti, 1967).To observe the effect of different photoperiods on germination, protocorms and seedling development, five different Light/Dark (L/D) photoperiods, 0/24, 8/16, 12/12, 16/8 and 24/0 h were tried.The best four media (MS, ½ MS, mMS and KC) out of the twenty screened were used in combination with the aforementioned photoperiods.Illumination was given with cool white fluorescent lamps (30 µmol m -2 s -1 , Philips, India) and the cultures were maintained at 23 ± 2°C.

Acclimatization and reintroduction
Seedlings derived from in vitro asymbiotic seed germination, were subcultured on the same medium (MS) with reduced macro nutrient at ¼ strength.Sucrose level was reduced to 1% for 2 weeks and thereafter stopped.After a month, seedlings were transferred to a mixture of compost, coir pith, bricks and charcoal for two weeks.Well acclimatized seedlings were reintroduced in its habitat.Live mosses were used to hold the roots intact over the bark and tied gently with a hard thread.Macro nutrients (MS) at ¼ strength without sucrose were sprayed twice a month.

Experimental design and data collection
For both the experiments, a minimum of eight replicates with about 300 to 400 seeds were treated.Cultures were observed periodically every week under a dissection microscope.The day of start of each stage (about 40 to 50% of seeds) was observed and tabulated.Germination percentage was calculated by dividing the number of germinated seeds with total number of seeds inoculated followed by multiplying with 100 on 46th day after inoculation where maximum germination attained in most media.Data related to orchid seed germination stages was adopted from Stenberg and Kane (1998 (Duncan, 1955).

Effect of media on germination and PLB development
Seed viability was around 92.67% as confirmed by triphenyl tetrazolium chloride (TTC) test.Stage 1 that is, enlarged seeds with ruptured testa (Figure 1c), later turned green and got partially or fully ruptured/without testa (Figure 1d).Embryos at this stage were considered as germination and calculated.Among the media tested, half strength MS medium supplemented with 3% sucrose observed highest percentage of germination of about 93.41% ( The color of the germinating seeds and the protocorms differed between media.Four major colorations were observed during the germination viz., green, white, brown and black.The yellow colored seeds turned to green and developed seedlings on MS, mMS, ½ MS, ¼ MS, m ½ MS and m ¼ MS media.The reason could be that the nutrients at macro and micro level along with vitamins were sufficient for development.Whereas, the seeds on RE, KP, KC-O, Curtis and Pf media turned to pale green (whitish green).On KC, Th-Gd, V and W and WS media seeds and protocorms remained yellow and turned brown.Fast and Mitra media seemed to be not suitable for D. aqueum where the seeds turned white and brown color respectively.Inconsistencies in germination of the seeds, amongst various media experimented may represent insufficiency of the nutrients.

Effect of nutrient media on seedling development
Some media contributed to protocorms development but failed in further seedling development.This may be because of the inappropriate nutrient level or compositions.The embryos swelled and resulted in the rupture of testa either partially or completely in about 36 to 46 days in all media.Embryos in NP, Liddell, Curtis, Fast, Mitra and W and S media failed to develop further.Some of the media (N 3 F, RE, V and W, KC and KP / 57, 83, 90, 107 and 143 days respectively) showed not only delayed development of protocorms but also struggled to promote further development (Figure 2a).N 3 F and Th-Gd promoted seedlings in a very low number (5 and 6 respectively) (Table 2).The protocorms grew and developed leaf primordium (Stage 4) (Figure 1f) with numerous rhizoidal hairs within 12 to 15 weeks in ½ MS, m ½ MS, m ¼ MS media.Further, the seedling with an average of 2 leaves with developing roots (Figure 1g) were observed in ½ MS, MS, ¼ MS, mMS, ½ mMS, Pf and Th-Gd medium in about 16 to 17 weeks.Seedlings of about 1 cm in length with more than 3 leaves and well developed roots (Figure 1h) of about 1 to 1.5 cm in length were observed in ½ MS, MS, mMS medium by 18 to 20 weeks.¼ MS, Pf and Th-Gd developed seedlings of smaller in size ca.<1 cm or with stunted growth.

Effect of photoperiod on seed germination and development
Photoperiods significantly influences the germination irrespective of nutrient medium used.Both light and dark promotes germination irrespective of nutrient media used.Increase of seed germination was observed as the photoperiod increases and vice versa.The highest germination 97.75% was observed in 24/0 h L/D photoperiod (Table 3 and Figure 2e) combination with full strength MS medium followed by half strength MS medium (96.01%) within 46 days (Table 3).Least germination (31.36%) was observed in ½ MS medium (Figure 2a) followed by mMS (48.10%) in dark (0 h L/D) photoperiod.Photoperiods 8, 12, 16 and 24 h L/D supports the green protocorms grew into seedlings with well developed roots on all medium except KC.In complete darkness 0/24 h L/D, the germinated white embryos grew up to the stage of protocorms along with rhizoids where further development was arrested in all medium.The germination stages were similar with effect to media whereas the germination responses (percentage) differed with effect to photoperiods.The roots were developed well in 12 and 16 h of photoperiods compared to 0, 8 and 24 h of photoperiods (Figure 2).
Seedlings were acclimatized well in both in vitro and in glass house conditions.Thereafter they were reintroduced successfully with 56% survival up to 3 months (Figure 3).

DISCUSSION
Orchid seeds are known to be dependent on symbiosis for germination in nature.It was believed that fungal partner (Mycorrhiza) supply not only nitrogen (Magrou, 1944), but it also supplies carbohydrates and phosphorus (Dearnaley, 2007) to support germination.Also the increased osmotic concentration and pressure initiate germination (Arditti, 1979) in orchid seeds.In nature, epiphytes are provided with lesser nutrient by tree barks and the carbon source from litter compost deposited on  the bark.If the seeds are spread on a moist substratum, the undifferentiated embryos absorb water, swell slightly and may burst the testa and sometimes produce epidermal hairs.Even though, the embryo does not develop further unless it receives an exogenous supply of carbohydrate or is infected by a compatible mycorrhizal fungus (Smith et al., 1997).After the success of asymbiotic seed germination by Knudson (1921), new media were devised with various proportions of mineral nutrition.Generally, terrestrial orchids prefers low concentration of nutrients or more dilute media (Voth, 1976) for germination whilst epiphytes prefer high nutrition or concentrated media (Arditti, 1979) and carbon source for germination and development.McKendrick (2000) had also suggested that, when attempting to germinate a new species it is good to try with a different media at both full and half strengths in order to determine which is best for that species, so as to test the best medium for in vitro asymbiotic seed germination of D. aqueum, medium varied in concentration and types of minerals were tested.
The results showed varied germination and developmental responses among the media.Here the germination and seedling growth was not uniform in all successive stages of development because of variation in quality and quantity of the nutrient used.Half strength MS medium offered sufficient nutrients required for germination and seedling development of D. aqueum.The highest germination was achieved only in ½ MS medium followed by full strength MS medium which is higher than the germination previously reported by Robinson et al. (2009) in KC medium with an organic supplement.At a shorter period of 126 to 161 days, seedlings with more than 3 leaves with well developed roots were achieved on the same medium.Similar result on ½ MS and MS medium were reported in D. parishii (Buyun et al., 2004), D. transparens (Alam et al., 2002) Coelogyne cristata (Naing et al., 2011), D. tetrachromum and D. hamaticalcar (Ali et al., 2011).Arditti (1979) infers that, orchid seed germination to be higher in media low in calcium.½ MS contains low amount of calcium (220 mg l - 1 ) compared to other media used and this could be the optimum level for the seeds to germinate, whereas an increase or decrease from this level showed lesser germination.It is suggested that, orchid seedlings might react favorably to an increase in nitrogen as ammonium ion than the nitrate form in seedling growth (Wynd, 1933) since the uptake of ammonium is faster than that of nitrate.In this experiment, nitrogen source in both the forms are noticeably effective in germination and development.Of the different forms of nitrogen used, media with KNO 3 and NH 4 NO 3 at higher concentrations 950 and 825 mg l -1 respectively increased germinability of the seeds and the seedling development.Previous works have reported that inorganic nitrogen may limit germination, possibly due to low nitrate reductase activity during seed germination and early protocorm development Parthibhan et al. 391 (Raghavan and Torrey, 1964;Waes and Debergh, 1986b;Malmgren, 1992;Dutra et al., 2008).Whereas in this species, nitrogen in both forms even at higher concentrations can be used by the plants.This may be because of higher nitrate reductase activity in the seeds during germination and development.Hew and Yong (1996) and Johnson et al. (2011) stated that both mineral uptake and growth are enhanced when the culture media are supplemented with sucrose.Addition of sucrose at 2 to 3% might help the uptake of nutrient in germinating seeds and seedlings.The pH about 4.5 to 8 have also been reported to increase the availability of macro nutrients (Arditti, 2008) such as nitrate, phosphate, calcium and micro nutrients such as Fe, boron, copper, zinc, etc.Many orchids including some Dendrobium species have been reported to germinate and grow well at pH 5 to 6 (Piria et al., 2008).In this study, pH 5.7 could be the optimum level for nutrient availability to D. aqueum.Microelements along with iron reported stimulatory effect on plant growth (Ichihashi and Yamashita, 1979).Arditti (1979) has reported that niacin is the only vitamin that could increase germination.The higher germination and better seedling achievement in ½ MS could also be the effect of micro nutrient and vitamins.
In general, orchid seeds vary in their light requirement for germination and development (Darnell, 1952).Several experiments on the effect light on orchid seed germination recorded that, light is a limiting factor with/without the presence of suitable nutrients (Dutra et al., 2008(Dutra et al., , 2009;;Kauth et al., 2006;Vasudevan and Van Staden, 2010;Wang et al., 2011).In this experiment, light is not a limiting factor for early stages of seed germination but is necessary for the seedling development.Irrespective of the media used, differential light exposure times resulted great variation in both germination and development (Figure 2).
As the photoperiod increases, the germination rate also increases.The day of response to initial stages of seed germination (up to protocorms) was similar in all photoperiods (Figure 2b to f).The seeds/protocorms cultured under dark (0/24 h) photoperiod were white in colour and eventually produces lesser leafy shoots (Stage IV) than in light.They were turned into green in 2 to 4 days when transferred to light condition and further development commenced.This shows that, light is not a limiting factor for germination of D. aqueum seeds but is essential for seedling development.

Conclusion
The present study reports the best nutrient composition/media and photoperiod for in vitro asymbiotic seed germination of D. aqueum.The results concurred that D. aqueum is sensitive to specific nutrients and photoperiod in germination and seedling development.

Figure 1 .
Figure 1.In vitro asymbiotic seed germination of Dendrobium aqueum.a) D. aqueum capsules; b) Seeds; c) Imbibed seeds with ruptured testa; d) Protocorm with pointed shoot; e) Protocorm with leafy shoot; f) seedling with developing leaves and root initial; g) seedling with well developed roots; h) Germination of seeds under different photoperiods.

Figure 2 .
Figure 2. Days of start of six stages of germination on twenty media and five different photoperiods.a) Six stages of seed germination achieved by 20 media; b -f) Stages observed on five different photoperiods with four different media.

Figure 3 .
Figure 3. Acclimatization and reintroduction.a) D. aqueum seedlings subcultures in ¼ MS (5 to 6 month old); b) Seedlings in potting mixtures; c) Reintroduced seedlings on the host tree fixed with mosses; d) Rejuvenation of new shoots after a month of reintroduction.

Table 1 .
Nutrient composition of media used for in vitro asymbiotic seed germination of Dendrobium aqueum.

Table 2
).This was almost similar to the viability tested and thus the TTC viability test proved best for this species.Significant germination was also observed on MS media µm) which was ca. 4 times larger than the normal embryo size, followed by ½ MS medium (615.60 µm) which was ca. 3 times larger.The smallest protocorms (307.04 µm) measured were in Thomal-Gd medium.The protocorms on ¼ MS callused and further development was arrested.

Table 2 .
Effect of nutritional media on in vitro asymbiotic seed germination and seedling development of D. aqueum 1 1 Data were scored every week;2 Mean ± std.error; the mean values followed by the same letter are not significantly different at P = 0.05 (Duncan's new multiple range test); # indicates no enlargement of embryos; * indicates no germination.

Table 3 .
In vitro asymbiotic seed germination of Dendrobium aqueum on different photoperiods.

Media Germination response on 46 day (%) Color of protocorms Protocorms response
*indicates no further development.