Nutrient requirements of in vitro cultured Halophila ovalis and Posidonia coriacea: nitrogen source INTRODUCTION Halophila ovalis Contd. Posidonia ostenfeldii (den Hartog, 1970) OBJECTIVES • to establish the in vitro culture of P. coriacea and H. ovalis • examine the response of these two species to varying nutrient availability • to report the effect of increasing nitrogen concentration on growth of both species METHODOLOGY H. ovalis seeds collected from Swan River sediment, in metropolitan Perth Two apical rhizome sections of H. ovalis, consisting of 1–2 nodes each, were subcultured into fresh BMH from stock cultures surface sterilized in ultra pure water for 20 min five replicates were used for each treatment. Stock cultures were subcultured to fresh BMH supplemented with 5.0 lM 6furfurylaminopurine (kinetin) every 4 weeks for approximately 2 years prior to the start of the experiment 2% (v/v) benzalkonium chloride (in 10% (v/v) ethanol) for a further 20 min followed by three rinses in sterile distilled water. Seeds were germinated in 10 ml Basal Medium for H. ovalis (BMH) (control medium) Seedlings was subcultured into 250 ml polycarbonate tubs containing 100 ml BMH to produce stock cultures. Fruit of P. coriacea were collected from the Perth metropolitan area, on Parmelia Bank, Western Australia Each seedling culture was considered one replicate and ten replicates were used per treatment five replicates were used for each treatment. Spathes containing 20– 30 fruit removed from parent plants, transported to laboratory and maintained in aerated tanks containing seawater at 21C for a maximum of 48 h. Seedlings with no visual contamination were transferred to experimental media. BMP was used as the control medium Fruit were surface sterilised Seedling cultures were established by dissecting the seed from the fruit then transferring to 10 ml of basal medium After 3 weeks, the seedling cultures had two leaves, one root and were visually assessed for contamination each component for each case for both species with five concentrations (0, ¼, ½, 1 and 2 MS) The first experimental media tested three broad medium components of MS medium , MS macronutrients, MS micronutrients and MS vitamins • The second experiment involved varying the concentrations of NH4NO3 and the source of nitrogen, either as NH4+ or NO3 All cultures were maintained at 21 ± 1C and were subcultured every 4 weeks Total nitrogen (mM) ranged from 0 to 60 mM and was supplied as NO3 (KNO3 or Ca(NO3)2) alone or with NH4NO3.Halophila experiments ran for 4 weeks, Posidonia 8 weeks and were subcultured to fresh medium after 4 week Result & Discussion Halophila ovalis • Effect of MS macronutrients Parameter Effect Fresh weight • Sig. affect growth of H.ovalis • Fresh weight reduces with increasing conc. MS macro Total no. leaves No sig effect Total no. roots No sig. effect Chlorophyll ½ MS macro= highest chlorophyll level DO 1/2 MS macro= highest DO level • Same effect observed when MS micronutrients is used. • MS Vitamins no effect observed • Nitrogen treatment have sig. affect. Increases leaf no, no. roots, chlorophyll and DO level. But reduces length of root Contd. • All media have effect except MS vitamins due to: – Opportunistic in nutrient uptake – Have rapid turnover of rhizomes – Do not store carbohydrates to the extent Posidonia coriacea do. – Adaptable to changes or stresses Posidonia coriacea Fresh weight • Sig affect growth • Highest conc. MS macro= decrease growth Total no of leaves • Increases with increasing conc. of MS macronutrients. • At highest conc. MS macro= reduced no. leaves. • No sig. effect Total no of roots • No sig. effect • No effect of MS micronutrients and MS vitamins observed • No sig effect of Nitrogen on P.coriacea Contd. • MS macro have little effect on growth due to: – Slow growing nature Effect of nitrogen • pH in both cultures decreases at the end of experiment due to: – Plants actively absorbs NH4+,lowering the pH due to an exchange with H+ ion. – Plants favors NH4+ over NO-3 due to different oxidation state and ease of exchange Conclusion • For H.ovalis, chlorophyll content and DO level is optimum when ½ MS macronutrients and MS micronutrients are used. • ½ MS is the most suitable for both seagrass. • Highest nitrogen content=highest leaf and root number for H.ovalis.However root is short and no root hair when NH4NO3 is present. LABORATORY CULTURE OF THE SEAGRASS, Halophila ovalis (R.Br.) HOOKER f • 9 out of 12 genera of seagrasses; ThaZassia, HaZoduZe, HalophiZa, Posidonia, Zostera, Cymodocea, Syringodium, EnhaZus and ThaZassodendron have been successfully cultured in synthetic seawater and under controlled environmental conditions by McMillan (1976,1978, 1980a, 1980b). • facilitated studies involving the biological, ecological and phenological aspects of seagrasses (McMillan, 1980a; McMillan 1980b; McMillan et aI., 1981). • 14 species of seagrasses of which 5 species belong to Halophila and Halophila ovalis, being the most common occurred in lagoons, along the shallow inter-tidal coast and sub-tidal areas. Although common, its biology and phenology have not been examined (Japar Sidik et aI., 2006). OBJECTIVES: (i) the sustain growth and development of the H. ovalis population (ii) the reproductive biology (flowering and fruiting) (iii) the pattern of seedling development from seeds to mature plants METHODOLOGY • • • • 0.42 m x 0.42 m x 1.22 m glass aquarium provided with 3-4 em thick of substrate flooded with 45 liters of 30 psu ofInstant Ocean synthetic seawater. The substrate was flooded with distilled water for a month to reduce the nutrients in the H. ovalis • • • • natural Plugs of H. ovalis explants collected two types of substrate; H. ovalis native substrate of calcareous sandy mud from Merambong shoal and commercial artificial sand under separate light regimes of -90 ~lmollm2/sec six months, 25% by volume of the water was replaced to replenish the nutrients and to improve the water quality of the aquarium. • • • RESULTS Plugs of explants were successfully grown in natural substrate and under both light regimes of -90 lmol m2s' and -200 lmol m2sl The sequence of events on the growth and development, and reproductive biology of H. ova lis plug (Fig.2) to the formation of population Better growth performance of plugs was observed when grown in natural substrate and under the light regime of 200lmol m-2 S·I. INTRODUCTION • In the Mediterranean Sea, the most important seagrass • In this, Isolation of protoplasts = combination of cellulase Onozuka R-10, hemicellulase and pectinase Posidonia Oceanica Cymodocea nodosa Collect -At Ligurian Sea Exercised & Washed -Youngest leaves Cut tranversally -1-2mm² Rotatory shaker -Release protoplast Culture of Protoplast -petri dish -0.5x105 Sterelized Enzyme Mixture -10ml of 5g tissue Monitoring by fluorescence microscope -isolation -cell wall regeneration -7-10h Icubatiom -30 min METHODS Mesophyll protoplasts released from the cut end of a P. oceanica leaf Viable protoplasts of P. oceanica stained with fluorescein diacetate Callus-like structure in P. oceanica after 20 days in culture Mesophyll protoplasts from Cymodocea nodosa Protoplasts of P. oceanica proved. Cymodocea nodosa requires optimization. Affected by the developmental state and environmental conditions Electrophysiology and biophysics. Characterization of membrane properties and ion transport processes. Adaptations and stress resistance in these plants. RESTORATION OF CYMODOCEA NODOSA (UCHRIA) ASCHERSON SEAGRASS MEADOWS THROUGH SEED PROPAGATION. GERMINATION IN VITRO, SEEDLINGS CULTURE AND FIELD TRANSPLANTS Nor Suhada Zakaria 174578 INTRODUCTION • Concern has arisen over the decline of seagrass ecosystems worldwide as a result of adverse human activities in coastal areas (Walker and McComb 1992, Duarte et al. 2004, Orth et al. 2006, Duarte and Gattuso 2008, Hughes et al. 2009). • The use of seeds and seedlings germinated in vitro is widely considered to be a more cost-effective alternative (Thorhaug 1985, Orth et al. 1994, 2000, 2009, Balestri et al. 1998, Kirkman 1998, Harwell and Orth 1999, Granger et al. 2002, Pickerell et al. 2005, Ailstock and Shafer 2006). OBJECTIVE • To enhance biomass development, and outplanting in the wild under prevailing conditions ensuring enhanced survival rates • To improve the conservation of established meadows by introducing genetic variation. MATERIALS & METHODS Seed collection & culture methods Induction of germination Acclimation of seedlings Transplants of developed seedling to the field Results & discussion Figure 1 Cymodocea nodosa. (A–C) Three different stages of development: (A) Stage I, germinating seeds (arrow), bars0.5 cm; (B) Stage II, elongation of cotyledon, bars1 cm; (C) Stage III, development of first new leaves (arrow), bars1 cm; (D–G) Acclimation: (D–E) Seedlings acclimating in aquaria or tanks; (D) bars0.5 m; (E) bars0.4 m; (F–G) Seedlings with leaves attached to a young incipient rhizome with up to two leaves and root development after 30 days of acclimation; (H–J) Field transplant. Persistent patch formed from clustered seedlings transferred directly into the sediment: (H) Plants at day zero, bars5 cm; (I) Plants at day 30. Note plagiotropic Figure 2 Cymodocea nodosa: plant development during acclimation. (A–B) Leaf and root lengths over 45 days. (C–D) Shoot and root regeneration over 45 days seedling acclimation (values are means"SD, ns30). Figure 3 Cymodocea nodosa: field trials. (A) Survival (%). (B) Average leaf length (cm) of acclimated plantlets transferred to a natural meadow (27859920.440 N; 15822912.450 W,Gran Canaria, Spain) using three different transplant methodologies: a)seedlings scattered on nylon nets (rs3, ns60), b) seedlings clustered in biodegradable trays (rs3, ns108), and c) seedlings planted directly into the sediment (ns76). Seedling monitoring was performed over nine months (values are means"SE). THANK YOU!