Influence of Nutritional Media and Photoperiods on in Vitro Asymbiotic Seed Germination and Seedling Development of Dendrobium Aqueum Lindley

Full Length Research Paper

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

S. Parthibhan1, J. H. Franklin Benjamin2, M. Muthukumar1, N. Ahamed Sherif1, T. Senthil Kumar3 and M. V. Rao1*

1Department of Plant Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India. 2Botanical Survey of India, Southern Regional Center, TNAU campus, Coimbatore-641 003, Tamil Nadu, India. 3Department of Industry University Collaboration, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.

Accepted 25 September, 2012

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 1 development under 16/8 hrs photoperiod. Highest seed germination occurred in /2 MS medium (93.41%) followed by mMS (91.04%) and m ½ MS (90.23%) in 6 to 7 weeks. The biggest protocorms were observed in MS medium (788.8 µm) followed by ½ 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, ½ MS, mMS, ½ mMS, and ¼ 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 ½ 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.

Key words: Dendrobium aqueum, asymbiotic seed germination, nutritional media, photoperiods, seedling development, reintroduction.

INTRODUCTION

Orchidaceae is one of the largest families of flowering Dendrobium is economically valuable in countries like plants. The genus Dendrobium is the largest genus of US, Taiwan, Thailand, Germany, Netherland, and Japan Orchidaceae having mostly epiphytic or occasionally as potted plants (Puchooa, 2004). Several species of lithophytic habits and comprising of about 1500 species Dendrobium have also been used in Chinese medicine worldwide (Carlsward et al., 1997) with 116 species in (Hu, 1970) and in Indian system of medicine (Misra, India (Misra, 2007). Wide range of flower colors, size and 2007) to cure cough, cold etc. Species of this genus were shapes, year-round availability, and long flowering life by being reported to produce a variety of secondary several weeks to month are the prime attraction of this metabolites such as alkaloids (Hu, 1970), sterols (Chang genus (Adelheid, 2007). et al., 2001), gum (Li et al., 2007), Bibenzyl derivatives (Zhang et al., 2006), flavanoids, cyanogenic glycosides and tannin (Maridass et al., 2008). Dendrobium aqueum Lindley is an epiphytic, sympodial *Corresponding author. E-mail [email protected]. Tel: orchid endemic to Eastern Ghats, found at an altitude of +91-9442147460. 1300 to 1600 m on Shola trees in deep shade 384 Afr. J. Plant Sci.

(Matthews, 1983). This species is being reported under viability test. Near Threatened category by the conservation assessment and management plan workshop (CAMP, 2001). Continuous pressure of forest degradation, Seed viability test deforestation, shifting cultivation, forest fire, tree cutting, Seed viability was tested by using TTC (2, 3, 5-triphenyl tetrazolium lopping, biological invasion, and indiscriminate chloride) test (Waes and Debergh, 1986a). 10 ml of 1% TTC exploitation of natural resources in Eastern Ghats affect solution with the pH 7.0 was taken in a screw cap tube with the and destroy the native flora completely of which orchids seeds. Then the solution was kept in dark at 20 ± 2°C for 24 h. After are the primary victims (Reddy et al., 2005). D. aqueum 24 h, the solution was drained and the seeds were washed in sterile population has been declining due to loss of habitat and distilled water for 3 times. Red stained viable seeds and yellowish inherent lower germination rate in nature. In vitro studies unstained seeds were counted under a light microscope. will immensely aid conservation measures of this orchid species. Asymbiotic media selection and culture condition In vitro asymbiotic seed germination is considered much faster and effective method for conservation and Twenty basal media successfully reported in orchids seed mass multiplication of threatened and endangered germination studies were used for the media screening experiment (Table 1). MS medium (Murashige and Skoog, 1962) diluted and orchids. Several workers have successfully reported modified into, ½ macro strength (½ MS), quarter macro strength (¼ asymbiotic seed germination in Dendrobium [D. MS), MS vitamin replaced with B5 vitamin (mMS, m ½ MS, m ¼ strongylanthum (Kong et al., 2007), D. lituiflorum (Vyas et MS) were also used. The entire medium was set with their al., 2009), D. parishii (Lyudmila et al., 2004), D. appropriate pH and sugars and solidified with 8% agar before -2 transparenes (Alam et al., 2002), D. tosaense, D. autoclaved at 121°C and 1.5 kg cm for 20 min (Table 1). The moniliforme, D. tosaense (Lo et al., 2004) and D. sterilized media were poured into sterile petriplates. After the media cooled, the seeds were sown and the plates were sealed with 2 to 3 fimbriatum (Sharma et al., 2005)]. layers of parafilm. Cultures were maintained under white Mass propagation of the orchid seedlings in vitro could fluorescent lamp with the standard protocol of 16 hrs photoperiod be achieved with a suitable basal medium devoid of with a temperature of 23 ± 2°C. growth regulators as orchid seeds have sufficient growth hormones to germinate and develop into seedlings in Photoperiods on germination and development of seedlings nature (Arditti, 1979). Also, seedlings raised through this growth regulator free experiment would help as a source Light is one of the factors which influence orchid seed germination of explants to produce multiples of desired healthy (Arditti, 1967). To observe the effect of different photoperiods on plantlets. It is suggested that epiphytic species can germination, protocorms and seedling development, five different germinate both in the light and dark and seem to require Light/Dark (L/D) photoperiods, 0/24, 8/16, 12/12, 16/8 and 24/0 h light only for the induction or improvement of shoot and or were tried. The best four media (MS, ½ MS, mMS and KC) out of the twenty screened were used in combination with the root formation (Arditti, 1979). Both media and aforementioned photoperiods. Illumination was given with cool photoperiods could affect the germination (Dutra et al., white fluorescent lamps (30 µmol m-2 s-1, Philips, India) and the 2009). Therefore, separate experiment was carried out cultures were maintained at 23 ± 2°C. focusing on nutrient media screening (devoid of growth regulators) and the effect of different photoperiod to assess germination, protocorm and seedling Acclimatization and reintroduction development of D. aqueum. 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 MATERIALS AND METHODS thereafter stopped. After a month, seedlings were transferred to a mixture of compost, coir pith, bricks and charcoal for two weeks. Seed source, sterilization procedure Well acclimatized seedlings were reintroduced in its habitat. Live mosses were used to hold the roots intact over the bark and tied Green, mature capsules collected from Kolli hills, Eastern Ghats of gently with a hard thread. Macro nutrients (MS) at ¼ strength Tamil Nadu were used during experiments. A single green capsule without sucrose were sprayed twice a month. 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 Experimental design and data collection embryos were elliptical and yellowish in colour. Our regular study on seed viability of green mature capsules For both the experiments, a minimum of eight replicates with about often to have about 80 to 90%. For both the experiments single 300 to 400 seeds were treated. Cultures were observed periodically green capsule was washed in running tap water with 1 to 2 drops of every week under a dissection microscope. The day of start of each soap solution (Teepol) for 15 min. Then the capsule was transferred stage (about 40 to 50% of seeds) was observed and tabulated. to sterilized laminar hood. The capsule was air dried for a while and Germination percentage was calculated by dividing the number of dipped in 70% alcohol for few seconds and burnt off in seconds. germinated seeds with total number of seeds inoculated followed by This was repeated two to three times. Finally the capsule was cut multiplying with 100 on 46th day after inoculation where maximum opened longitudinally with a sterile surgical knife and all the seeds germination attained in most media. Data related to orchid seed were carefully extricated and mixed well before inoculation and germination stages was adopted from Stenberg and Kane (1998) Parthibhan et al. 385

0 h0 8 h 0 12 h 0

16 h 24 h

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.

and Mariat (1952), Stage 1: Imbibed seed, swollen and greening leaves and one or developing roots; Stage 6: Seedling with evident still covered or partially covered by testa; Stage 2: Enlarged seeds roots and two or more leaves. 15 protocorms randomly selected without testa (considered as germination); Stage 3: Protocorms with thrice from each treatment to measure the size (width) of the pointed shoot apex and rhizoids; Stage 4: Protocorms with protocorms. Comparisons of protocorm size between treatments developing leaves and rhizoids; Stage 5: Seedling with one or more were made with Duncan’s new Multiple Range Test (DMRT) 386 Afr. J. Plant Sci.

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

Nutrient compositions MS mMS ½ MS ¼ MS m½ MS m¼ MS KnC Kc-O Np Th-GD Pf N3F V&W RE Fast WS Mitra KP Macro elements (mgl-1) Ca(NO3)2.4H2O - 1000 347.5 637.6 800 1000 150 80 613 200 1000 CaCl2.2H2O 440 440 220 110 220 110

Ca3 (PO4)2 200 KH2PO4 170 170 85 42.5 85 42.5 250 250 462.7 300 200 250 300 80 250

K2 HPO4 250 KNO3 1900 1900 950 475 950 475 424 400 200 525 400 70 180 250 KCl 250 100 250 160 142

Mg(NO3)2.6H2O 256.4 110 100 MgSO4.7H2O 370 370 185 92.5 185 92.5 250 122.1 200 250 250 80 1550 200 250 NaH2PO4.H2O 400 250

Na2 SO4. 10H2O 1388 (NH4)2SO4 500 500 303.9 60 250 500 150 100 NH4NO3 1650 1650 825 412.5 825 412.5 500 82 370 400 160

NaNO3 4800 Total mg/l 4530 4530 2265 1132.5 2265 1132.5 2000 1969.33 2166.6 1240 1500 2000 1725 1500 560 8963 930 1750

Micro elements (mgl-1) AlCl3 0.03

Co (NO3)2. 6H2O 0.05 CoCl2. 6H20 0.025 0.025 0.025 0.025 0.025 0.025 0.012 CuSO4.5H2O 0.025 0.025 0.025 0.025 0.025 0.025 0.04 0.5 0.03

CuSO4.7H2O 0.05 Cupric sulphate.5H2O 0.012 FeSO4.7H2O 27.8 27.8 27.8 27.8 27.8 27.8 25 25 27.8 20 25 20 25 20 16.71

Fe3 (C2H4O6)3 28 28 H3BO3 6.3 6.3 6.3 6.3 6.3 6.3 0.056 3.1 0.5 1.0 3.2 0.06 KI 0.83 0.83 0.83 0.83 0.83 0.83 0.415 0.01 1.6 0.03 MnSO4.H2O 5.682 11.2 MnSO4.4H2O 22.3 22.3 22.3 22.3 22.3 22.3 7.5 0.5 7.5 0.1 0.5

MnSO4.7H2O 0.4 Na2EDTA 37.3 37.3 37.3 37.3 37.3 37.3 37.3 5.5 Na2EDTA. 2H2O 22.35

Na2MoO4.2H2O 0.025 0.025 0.025 0.025 0.025 0.025 0.05 NiCl2. 6H2O 0.03 ZnSO4.7H2O 8.6 8.6 8.6 8.6 8.6 8.6 0.331 4.3 0.5 1.0 3.2 0.05 Parthibhan et al. 387

Table 1. Continued.

Vitamins (mgl-1) Glycine 2.0 2.0 2 Myo-Inositol 100 100 100 100 100 100 100 Nicotinic acid 0.5 1.0 0.5 1.0 1.0 0.5 1.25 Pyridoxine HCl 0.5 1.0 0.5 1.0 1.0 0.5 0.3 Thiamine HCl 0.1 10.0 0.1 10.0 10.0 0.1 0.1 0.3 Riboflavin 0.05 Biotin 0.5 Folic acid 0.3

Sugars (mgl-1) Fructose 10000 20000 5000 Glucose 10000 10000 10000 Sucrose 30000 30000 20000 20000 20000 20000 20000 20000 20000 20000 20000 12000 20000 30000

pH 5.7 5.7 5.7 5.7 5.7 5.7 5 4.5 4.5 5.1 5.5 5.3 5.2 5.2 5.5 5.5 5.6 5.8

(Duncan, 1955). fortified with B5 vitamins, mMS (91.04%), m½MS Thomal-Gd medium. The protocorms on ¼ MS (90.23%) and MS (84.40%). Protocorms, the callused and further development was arrested. globular green cell masses (Figure 1e) considered The color of the germinating seeds and the RESULTS as the early stages of seedling development in protocorms differed between media. Four major orchids enlarge in size. Stage 3 or protocorms colorations were observed during the germination Effect of media on germination and PLB were observed in all media (Table 2) except in viz., green, white, brown and black. The yellow development KP, Liddell, Curtis, Fast, Mitra and W&S medium. colored seeds turned to green and developed The embryos formed in these media observed seedlings on MS, mMS, ½ MS, ¼ MS, m ½ MS Seed viability was around 92.67% as confirmed were different in color (Table 2) due to insufficient and m ¼ MS media. The reason could be that the by triphenyl tetrazolium chloride (TTC) test. Stage nutrients to produce chlorophylls and so no further nutrients at macro and micro level along with 1 that is, enlarged seeds with ruptured testa development could be observed. This may be vitamins were sufficient for development. (Figure 1c), later turned green and got partially or caused by the deficiency of nutrients in those Whereas, the seeds on RE, KP, KC-O, Curtis and fully ruptured/without testa (Figure 1d). Embryos media. Protocorms with dark green pointed shoots Pf media turned to pale green (whitish green). On at this stage were considered as germination and (Stage 3) were considered for measurement. The KC, Th-Gd, V and W and WS media seeds and calculated. Among the media tested, half strength sizes of the protocorms varied with respect to protocorms remained yellow and turned brown. MS medium supplemented with 3% sucrose nutrient media used. The largest protocorms were Fast and Mitra media seemed to be not suitable observed highest percentage of germination of observed only in full strength MS media (788.8 for D. aqueum where the seeds turned white and about 93.41% (Table 2). This was almost similar µm) which was ca. 4 times larger than the normal brown color respectively. Inconsistencies in to the viability tested and thus the TTC viability embryo size, followed by ½ MS medium (615.60 germination of the seeds, amongst various media test proved best for this species. Significant µm) which was ca. 3 times larger. The smallest experimented may represent insufficiency of the germination was also observed on MS media protocorms (307.04 µm) measured were in nutrients. 388 Afr. J. Plant Sci.

Table 2. Effect of nutritional media on in vitro asymbiotic seed germination and seedling development of D. aqueum1

Germination 2 Nutrient Color of the Embryo/ Size of the Protocorms response Protocorms response medium protocorm (width in µm) (at 46th day) (%) MS 84.4 Green 788.75 ± 2.13a Developed seedlings ½ MS 93.41 Green 615.75 ± 1.10b Developed seedlings ¼ MS 75.56 Light green to yellowish 608.5 ± 1.32c Developed seedlings mMS 91.04 Green 504.75 ± 2.95f Developed seedlings ½ mMS 90.23 Green 598.75 ± 1.18d Seedlings with basal Callus ¼ mMS 63.20 Green 614.25 ± 2.42b Leafy shoots with basal Callus Pf 65.58 Green 530.5 ± 1.32e Developed seedlings Th-GD 67.69 Pale green 307.25 ± 2.78l Developed seedlings in very few no. (2-5) k N3F 74.58 Yellow / Pale green 350.5 ± 2.21 Developed seedlings in very few no. (2-5) KC 71.73 Pale green to yellowish 527.5 ± 1.70e No seedling KC-O 27.78 Green/white 480.5 ± 1.32g No seedling NP 32.39 Brown to black 376.75 ± 1.37j No seedling KP 42.00 Yellowish 402.75 ± 1.75i No seedling V&W 50.40 pale yellow 348.25 ± 1.31k No seedling RE 48.41 White 422.25 ± 2.86h Leafy shoots with no response Liddel. 26.95 Yellow/White # No seedling Curtis * White # No seedling Fast * White # Nil Mitra * Brown/Black # Nil W&S * Pale yellowish # Nil

1Data were scored every week; 2Mean ± 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.

Effect of nutrient media on seedling development Effect of photoperiod on seed germination and development Some media contributed to protocorms development but failed in further seedling development. This may be Photoperiods significantly influences the germination because of the inappropriate nutrient level or irrespective of nutrient medium used. Both light and dark compositions. The embryos swelled and resulted in the promotes germination irrespective of nutrient media used. rupture of testa either partially or completely in about 36 Increase of seed germination was observed as the to 46 days in all media. Embryos in NP, Liddell, Curtis, photoperiod increases and vice versa. The highest Fast, Mitra and W and S media failed to develop further. germination 97.75% was observed in 24/0 h L/D Some of the media (N3F, RE, V and W, KC and KP / 57, photoperiod (Table 3 and Figure 2e) combination with full 83, 90, 107 and 143 days respectively) showed not only strength MS medium followed by half strength MS delayed development of protocorms but also struggled to medium (96.01%) within 46 days (Table 3). Least promote further development (Figure 2a). N3F and Th-Gd germination (31.36%) was observed in ½ MS medium promoted seedlings in a very low number (5 and 6 (Figure 2a) followed by mMS (48.10%) in dark (0 h L/D) respectively) (Table 2). The protocorms grew and photoperiod. Photoperiods 8, 12, 16 and 24 h L/D developed leaf primordium (Stage 4) (Figure 1f) with supports the green protocorms grew into seedlings with numerous rhizoidal hairs within 12 to 15 weeks in ½ MS, well developed roots on all medium except KC. In m ½ MS, m ¼ MS media. Further, the seedling with an complete darkness 0/24 h L/D, the germinated white average of 2 leaves with developing roots (Figure 1g) embryos grew up to the stage of protocorms along with were observed in ½ MS, MS, ¼ MS, mMS, ½ mMS, Pf rhizoids where further development was arrested in all and Th-Gd medium in about 16 to 17 weeks. Seedlings of medium. The germination stages were similar with effect about 1 cm in length with more than 3 leaves and well to media whereas the germination responses developed roots (Figure 1h) of about 1 to 1.5 cm in length (percentage) differed with effect to photoperiods. The were observed in ½ MS, MS, mMS medium by 18 to 20 roots were developed well in 12 and 16 h of photoperiods weeks. ¼ MS, Pf and Th-Gd developed seedlings of compared to 0, 8 and 24 h of photoperiods (Figure 2). smaller in size ca. <1 cm or with stunted growth. Seedlings were acclimatized well in both in vitro and in Parthibhan et al. 389

a 200 StageI I 150

StageII II

100 StageIII III)

No. of days of No. Stage 50 StageIV IV( StageV V 0

O VI

Pf Stage VI

N3F

Fast

W&S

mMS

V&W

¼ MS ¼

½ MS ½

Mitra

Curtis

Liddel. ¼ mMS ¼ mMS ½ Nutrient media

b 160 140 c 160 120 140 100 MS 120 80 100 MS 60 ½ MS 80 No. of daysof No. ½ MS

40 MMS 60 No. of daysof No. 20 40 MMS KC 20 0 0 KC StageI I StageII II StageIII III StageIV IV StageV V StageVI VI StageI I StageII II StageIII III StageIV IV StageV V StageVI VI 0(Stage) hrs (Stage) 8 hrs 0 hrs 8 hrs

d e 160 160 140 140 120 120 100 100 MS MS 80 80 ½ MS ½ MS

60 60 No. of daysof No. No. of daysof No. 40 MMS 40 MMS 20 KC 20 KC 0 0 IV VI StageI I StageII II StageIII III Stage IV StageV V Stage VI StageI I StageII II StageIII III StageIV IV StageV V StageVI VI 12(Stage) hrs (16Stage) hrs 12 hrs 16 hrs

f 160 140 120 MS 100 80 ½ MS 60

No. of daysof No. MMS 40 20 KC 0 StageI I StageII II StageIII III StageIV IV StageV V StageVI VI 24(Stage) hrs 24 hrs

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.

glass house conditions. Thereafter they were for germination in nature. It was believed that fungal reintroduced successfully with 56% survival up to 3 partner (Mycorrhiza) supply not only nitrogen (Magrou, months (Figure 3). 1944), but it also supplies carbohydrates and phosphorus (Dearnaley, 2007) to support germination. Also the increased osmotic concentration and pressure initiate DISCUSSION germination (Arditti, 1979) in orchid seeds. In nature, epiphytes are provided with lesser nutrient by tree barks Orchid seeds are known to be dependent on symbiosis and the carbon source from litter compost deposited on 390 Afr. J. Plant Sci.

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

Photoperiods (L/D) (h) Media Germination response on 46 day (%) Color of protocorms Protocorms response MS 70.74 White No response ½ MS 31.36 White Leafy shoots* 0 MMS 48.18 White No response KC 53.07 White No response

MS 83.78 Green Seedling developed ½ MS 48.50 Green Seedling developed 8 MMS 65.96 Green Seedling developed KC 73.16 Green Developing seedlings*

MS 85.45 Green Seedling developed ½ MS 67.43 Green Seedling developed 12 MMS 75.68 Green Seedling developed KC 85.47 Green Seedling developed

MS 91.05 Pale green Seedling developed ½ MS 72.66 Pale green Seedling developed 16 MMS 91.04 Pale green Seedling developed KC 88.27 Pale green Leafy shoots*

MS 97.75 Pale green Seedling developed ½ MS 96.01 Pale green Seedling developed 24 MMS 91.27 Pale green Seedling developed KC 90.69 Pale green No response

*indicates no further development.

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. Parthibhan et al. 391

the bark. If the seeds are spread on a moist substratum, (Raghavan and Torrey, 1964; Waes and Debergh, the undifferentiated embryos absorb water, swell slightly 1986b; Malmgren, 1992; Dutra et al., 2008). Whereas in and may burst the testa and sometimes produce this species, nitrogen in both forms even at higher epidermal hairs. Even though, the embryo does not concentrations can be used by the plants. This may be develop further unless it receives an exogenous supply of because of higher nitrate reductase activity in the seeds carbohydrate or is infected by a compatible mycorrhizal during germination and development. fungus (Smith et al., 1997). After the success of Hew and Yong (1996) and Johnson et al. (2011) stated asymbiotic seed germination by Knudson (1921), new that both mineral uptake and growth are enhanced when media were devised with various proportions of mineral the culture media are supplemented with sucrose. nutrition. Generally, terrestrial orchids prefers low Addition of sucrose at 2 to 3% might help the uptake of concentration of nutrients or more dilute media (Voth, nutrient in germinating seeds and seedlings. The pH 1976) for germination whilst epiphytes prefer high about 4.5 to 8 have also been reported to increase the nutrition or concentrated media (Arditti, 1979) and carbon availability of macro nutrients (Arditti, 2008) such as source for germination and development. nitrate, phosphate, calcium and micro nutrients such as McKendrick (2000) had also suggested that, when Fe, boron, copper, zinc, etc. Many orchids including attempting to germinate a new species it is good to try some Dendrobium species have been reported to with a different media at both full and half strengths in germinate and grow well at pH 5 to 6 (Piria et al., 2008). order to determine which is best for that species, so as to In this study, pH 5.7 could be the optimum level for test the best medium for in vitro asymbiotic seed nutrient availability to D. aqueum. Microelements along germination of D. aqueum, medium varied in with iron reported stimulatory effect on plant growth concentration and types of minerals were tested. (Ichihashi and Yamashita, 1979). Arditti (1979) has The results showed varied germination and reported that niacin is the only vitamin that could increase developmental responses among the media. Here the germination. The higher germination and better seedling germination and seedling growth was not uniform in all achievement in ½ MS could also be the effect of micro successive stages of development because of variation in nutrient and vitamins. quality and quantity of the nutrient used. Half strength MS In general, orchid seeds vary in their light requirement medium offered sufficient nutrients required for for germination and development (Darnell, 1952). Several germination and seedling development of D. aqueum. experiments on the effect light on orchid seed The highest germination was achieved only in ½ MS germination recorded that, light is a limiting factor medium followed by full strength MS medium which is with/without the presence of suitable nutrients (Dutra et higher than the germination previously reported by al., 2008, 2009; Kauth et al., 2006; Vasudevan and Van Robinson et al. (2009) in KC medium with an organic Staden, 2010; Wang et al., 2011). In this experiment, light supplement. At a shorter period of 126 to 161 days, is not a limiting factor for early stages of seed seedlings with more than 3 leaves with well developed germination but is necessary for the seedling roots were achieved on the same medium. Similar result development. Irrespective of the media used, differential on ½ MS and MS medium were reported in D. parishii light exposure times resulted great variation in both (Buyun et al., 2004), D. transparens (Alam et al., 2002) germination and development (Figure 2). Coelogyne cristata (Naing et al., 2011), D. tetrachromum As the photoperiod increases, the germination rate also and D. hamaticalcar (Ali et al., 2011). Arditti (1979) infers increases. The day of response to initial stages of seed that, orchid seed germination to be higher in media low in germination (up to protocorms) was similar in all calcium. ½ MS contains low amount of calcium (220 mg l- photoperiods (Figure 2b to f). The seeds/protocorms 1) compared to other media used and this could be the cultured under dark (0/24 h) photoperiod were white in optimum level for the seeds to germinate, whereas an colour and eventually produces lesser leafy shoots increase or decrease from this level showed lesser (Stage IV) than in light. They were turned into green in 2 germination. It is suggested that, orchid seedlings might to 4 days when transferred to light condition and further react favorably to an increase in nitrogen as ammonium development commenced. This shows that, light is not a ion than the nitrate form in seedling growth (Wynd, 1933) limiting factor for germination of D. aqueum seeds but is since the uptake of ammonium is faster than that of essential for seedling development. nitrate. In this experiment, nitrogen source in both the forms are noticeably effective in germination and development. Of the different forms of nitrogen used, Conclusion media with KNO3 and NH4NO3 at higher concentrations 950 and 825 mg l-1 respectively increased germinability of The present study reports the best nutrient the seeds and the seedling development. Previous works composition/media and photoperiod for in vitro asymbiotic have reported that inorganic nitrogen may limit seed germination of D. aqueum. The results concurred germination, possibly due to low nitrate reductase activity that D. aqueum is sensitive to specific nutrients and during seed germination and early protocorm development photoperiod in germination and seedling development. 392 Afr. J. Plant Sci.

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