Photosynthetic responses of subtidal seagrasses to a daily light cycle in Torres Strait: A comparative study

In this study, we examined the photosynthetic responses of five common seagrass species from a typical mixed meadow in Torres Strait at a depth of 5–7 m using pulse amplitude modulated (PAM) fluorometry. The photosynthetic response of each species was measured every 2 h throughout a single daily light cycle from dawn (6 am) to dusk (6 pm). PAM fluorometry was used to generate rapid light curves from which measures of electron transport rate (ETRmax), photosynthetic efficiency (α), saturating irradiance (Ek) and light-adapted quantum yield (ΔF/F′m) were derived for each species. The amount of light absorbed by leaves (absorption factor) was also determined for each species. Similar diurnal patterns were recorded among species with 3–4 fold increases in maximal electron rate from dawn to midday and a maintenance of ETRmax in the afternoon that would allow an optimal use of low light by all species. Differences in photosynthetic responses to changes in the daily light regime were also evident with Syringodium isoetifolium showing the highest photosynthetic rates and saturating irradiances suggesting a competitive advantage over other species under conditions of high light. In contrast Halophila ovalis, Halophila decipiens and Halophila spinulosa were characterised by comparatively low photosynthetic rates and minimum light requirements (i.e. low Ek) typical of shade adaptation. The structural makeup of each species may explain the observed differences with large, structurally complex species such as Syringodium isoetifolium and Cymodocea serrulata showing high photosynthetic effciciencies (α) and therefore high-light-adapted traits (e.g. high ETRmax and Ek) compared with the smaller Halophila species positioned lower in the canopy. For the smaller Halophila species these shade-adapted traits are features that optimise their survival during low-light conditions. Knowledge of these characteristics and responses improves our understanding of the underlying causes of changes in seagrass biomass, growth and survival that occur when modifications in light quantity and quality arise from anthropogenic and climatic disturbances that commonly occur in Torres Strait.

Developments in controlled green-water larval culture technologies for estuarine fishes in Queensland, Australia and elsewhere

Since 1989, researchers with the Department of Primary Industries and Fisheries (DPI&F) in Queensland, Australia, have successfully used controlled low-water exchange green-water cultures to rear the larvae of estuarine fishes and crustaceans through to metamorphosis. High survivals and excellent fry condition have been achieved for several commercially important endemic species produced for various projects. They include barramundi or sea bass, Lates calcarifer, Australian bass, Macquaria novemaculeata, dusky flathead, Platycephalus fuscus, sand whiting, Sillago ciliata, red sea bream or snapper, Pagrus auratus, banana prawn, Fenneropenaeus merguiensis, and others. The consistent success of our standardised and relatively simple approach at different localities has led to it being incorporated into general fingerling production practices at several establishments in Australia. Although post-metamorphosis rearing methods have differed for each species investigated, due to various biological and behavioural traits and project requirements, these larval rearing methods have been successful with few species-specific modifications. Initially modelled on the Taiwanese approach to rearing Penaeids in aerated low-water exchange cultures, the approach similarly appears to rely on a beneficial assemblage of micro-organisms. Conceptually, these micro-organisms may include a mixture of the air-borne primary invaders of pure phytoplankton cultures when exposed to outdoor conditions. Whilst this would vary with different sites, our experiences with these methods have consistently been favourable. Mass microalgal cultures with eco-physiological youth are used to regularly augment larval fish cultures so that rearing conditions simulate an exponential growth-phase microalgal bloom. Moderate to heavy aeration prevents settlement of particulate matter and encourages aerobic bacterial decomposition of wastes. The green-water larval rearing approach described herein has demonstrated high practical utility in research and commercial applications, and has greatly simplified marine finfish hatchery operations whilst generally lifting production capacities for metamorphosed fry in Australia. Its potential uses in areas of aquaculture other than larviculture are also discussed.

Optical peanut yield monitor: Development and testing

An optical peanut yield monitor was developed, fabricated, and field-tested. The overall system includes an optical mass-flow sensor, a GPS receiver, and a data acquisition system. The concept for the mass-flow sensor is based on that of the cotton yield-monitor sensor developed previously by Thomasson and Sui (2000). A modified version of the sensor was designed to be specific to peanut mass-flow measurement. Field testing of the peanut yield monitor was conducted in Australia during the May 2003 harvest. After subsequent minor modifications, the system was more extensively tested in Mississippi in October of 2003 and November of 2004. Test results showed that the output of the peanut mass-flow sensor was very strongly correlated with the harvested load weight, and the system's performance was stable and reliable during the tests.

Effect of defoliation interval and height on the growth and quality of Arachis pintoi cv. Amarillo

The effect of defoliation on Amarillo (Arachis pintoi cv. Amarillo) was studied in a glasshouse and in mixed swards with 2 tropical grasses. In the glasshouse, Amarillo plants grown in pots were subjected to a 30/20°C or 25/15°C temperature regime and to defoliation at 10-, 20- or 30-day intervals for 60 days. Two field plot studies were conducted on Amarillo with either irrigated kikuyu (Pennisetum clandestinum) in autumn and spring or dryland Pioneer rhodes grass (Chloris gayana) over summer and autumn. Treatments imposed were 3 defoliation intervals (7, 14 and 28 days) and 2 residual heights (5 and 10 cm for kikuyu; 3 and 10 cm for rhodes grass) with extra treatments (56 days to 3 cm for both grasses and 21 days to 5 cm for kikuyu). Defoliation interval had no significant effect on accumulated Amarillo leaf dry matter (DM) at either temperature regime. At the higher temperature, frequent defoliation reduced root dry weight (DW) and increased crude protein (CP) but had no effect on stolon DW or in vitro organic matter digestibility (OMD). On the other hand, at the lower temperature, frequent defoliation reduced stolon DW and increased OMD but had no effect on root DW or CP. Irrespective of temperaure and defoliation, water-soluble carbohydrate levels were higher in stolons than in roots (4.70 vs 3.65%), whereas for starch the reverse occured (5.37 vs 9.44%). Defoliating the Amarillo-kikuyu sward once at 56 days to 3 cm produced the highest DM yield in autumn and sprong (582 and 7121 kg/ha DM, respectively), although the Amarillo component and OMD were substantially reduced. Highest DM yields (1726 kg/ha) were also achieved in the Amarillo-rhodes grass sward when defoliated every 56 days to 3 cm, although the Amarillo component was unaffected. In a mixed sward with either kikuyu or rhodes grass, the Amarillo component in the sward was maintained up to a 28-day defoliation interval and was higher when more severely defoliated. The results show that Amarillo can tolerate frequent defoliation and that it can co-exist with tropical grasses of differing growth habits, provided the Amarillo-tropical grass sward is subject to frequent and severe defoliation.

Post capture necrosis of the feet caused by soft-catch traps – incidents, cause and prevention.

When recapturing satellite collared wild dogs that had been trapped one month previous in padded foothold traps, we noticed varying degrees of pitting on the pads of their trapped paw. Veterinary advice, based on images taken of the injuries, suggests that the necrosis was caused by vascular compromise. Five of six dingoes we recaptured had varying degrees of necrosis restricted only to the trapped foot and ranging from single 5 mm holes to 25% sections of the toe pads missing or deformed, including loss of nails. The traps used were rubber-padded, two–coiled, Victor Soft Catch #3 traps. The springs are not standard Victor springs but were Beefer springs; these modifications slightly increase trap speed and the jaw pressure on the trapped foot. Despite this modification the spring pressure is still relatively mild in comparison to conventional long spring or four-coiled wild dog traps. The five wild dogs developing necrosis were trapped in November 2006 at 5-6 months of age. Traps were checked each morning so the dogs were unlikely to have been restrained in the trap for more than 12 hours. All dogs exhibited a small degree of paw damage at capture which presented itself as a swollen paw and compression at the capture point. In contrast, eight wild dogs, 7-8 month-old, were captured two months later in February. Upon their release, on advice from a veterinarian, we massaged the trapped foot to get blood flow back in to the foot and applied a bruise treatment (Heparinoid 8.33 mg/ml) to assist restoring blood flow. These animals were subsequently recaptured several months later and showed no signs of necrosis. While post-capture foot injuries are unlikely to be an issue in conventional control programs where the animal is immediately destroyed, caution needs to be used when releasing accidentally captured domestic dogs or research animals captured in rubber-padded traps. We have demonstrated that 7-8 month old dogs can be trapped and released without any evidence of subsequent necrosis following minimal veterinary treatment. We suspect that the rubber padding on traps may increase the tourniquet effect by wrapping around the paw and recommend the evaluation of offset laminated steel jaw traps as an alternative. Offset laminated steel jaw traps have been shown to be relatively humane producing as few foot injuries as rubber-jawed traps.

Row spacing and planting density effects on the growth and yield of sugarcane. 2. Strategies for the adoption of controlled traffic.

Controlled traffic (matching wheel and row spacing) is being promoted as a means to manage soil compaction in the Australian sugar industry. However, machinery limitations dictate that wider row spacings than the standard 1.5-m single row will need to be adopted to incorporate controlled traffic and many growers are reluctant to widen row spacing for fear of yield penalties. To address these concerns, contrasting row configuration and planting density combinations were investigated for their effect on cane and sugar yield in large-scale experiments in the Gordonvale, Tully, Ingham, Mackay, and Bingera (near Bundaberg) sugarcane-growing regions of Queensland, Australia. The results showed that sugarcane possesses a capacity to compensate for different row configurations and planting densities through variation in stalk number and individual stalk weight. Row configurations ranging from 1.5-m single rows (the current industry standard) to 1.8-m dual rows (50 cm between duals), 2.1-m dual (80 cm between duals) and triple ( 65 cm between triples) rows, and 2.3-m triple rows (65 cm between triples) produced similar yields. Four rows (50 cm apart) on a 2.1-m configuration (quad rows) produced lower yields largely due to crop lodging, while a 1.8-m single row configuration produced lower yields in the plant crop, probably due to inadequate resource availability (water stress/limited radiation interception). The results suggest that controlled traffic can be adopted in the Australian sugar industry by changing from a 1.5-m single row to 1.8-m dual row configuration without yield penalty. Further, the similar yields obtained with wider row configurations (2 m or greater with multiple rows) in these experiments emphasise the physiological and environmental plasticity that exists in sugarcane. Controlled traffic can be implemented with these wider row configurations (>2 m), although it will be necessary to carry out expensive modifications to the current harvester and haul-out equipment. There were indications from this research that not all cultivars were suited to configurations involving multiple rows. The results suggest that consideration be given to assessing clones with different growth habits under a range of row configurations to find the most suitable plant types for controlled traffic cropping systems.

The performance of irrigated mixtures of tall fescue, ryegrass and white clover in subtropical Australia. 1. The effects of sowing mixture combinations, nitrogen and oversowing on establishment, productivity, botanical composition and persistence.

In the subtropics of Australia, the ryegrass component of irrigated perennial ryegrass (Lolium perenne) - white clover (Trifolium repens) pastures declines by approximately 40% in the summer following establishment, being replaced by summer-active C4 grasses. Tall fescue (Festuca arundinacea) is more persistent than perennial ryegrass and might resist this invasion, although tall fescue does not compete vigorously as a seedling. This series of experiments investigated the influence of ryegrass and tall fescue genotype, sowing time and sowing mixture as a means of improving tall fescue establishment and the productivity and persistence of tall fescue, ryegrass and white clover-based mixtures in a subtropical environment. Tall fescue frequency at the end of the establishment year decreased as the number of companion species sown in the mixture increased. Neither sowing mixture combinations nor sowing rates influenced overall pasture yield (of around 14 t/ha) in the establishment year but had a significant effect on botanical composition and component yields. Perennial ryegrass was less competitive than short-rotation ryegrass, increasing first-year yields of tall fescue by 40% in one experiment and by 10% in another but total yield was unaffected. The higher establishment-year yield (3.5 t/ha) allowed Dovey tall fescue to compete more successfully with the remaining pasture components than Vulcan (1.4 t/ha). Sowing 2 ryegrass cultivars in the mixture reduced tall fescue yields by 30% compared with a single ryegrass (1.6 t/ha), although tall fescue alone achieved higher yields (7.1 t/ha). Component sowing rate had little influence on composition or yield. Oversowing the ryegrass component into a 6-week-old sward of tall fescue and white clover improved tall fescue, white clover and overall yields in the establishment year by 83, 17 and 11%, respectively, but reduced ryegrass yields by 40%. The inclusion of red (T. pratense) and Persian (T. resupinatum) clovers and chicory (Cichorium intybus) increased first-year yields by 25% but suppressed perennial grass and clover components. Yields were generally maintained at around 12 t/ha/yr in the second and third years, with tall fescue becoming dominant in all 3 experiments. The lower tall fescue seeding rate used in the first experiment resulted in tall fescue dominance in the second year following establishment, whereas in Experiments 2 and 3 dominance occurred by the end of the first year. Invasion by the C4 grasses was relatively minor (<10%) even in the third year. As ryegrass plants died, tall fescue and, to a lesser extent, white clover increased as a proportion of the total sward. Treatment effects continued into the second, but rarely the third, year and mostly affected the yield of one of the components rather than total cumulative yield. Once tall fescue became dominant, it was difficult to re-introduce other pasture components, even following removal of foliage and moderate renovation. Severe renovation (reducing the tall fescue population by at least 30%) seems a possible option for redressing this situation.

Cynodon dactylon, Green Couch Grass 'TL1'

Chance seedling: observed in about 1989 as a distinctly coarser textured, densely matting, darker green mutant bermuda grass plant growing among the hybrid ‘Tifgreen’ on the eighth green at the Townsville Golf Course. Although ‘TL1’ was selected from a sward of the hybrid Bermuda grass ‘Tifgreen’, its inflorescence structure (4, not 3, racemes per inflorescence), agronomic attributes (e.g. its tolerance to certain herbicides), and its DNA profile are consistent with a chance seedling of Cynodon dactylon rather than a mutant plant of hybrid (C. dactylon x transvaalensis) origin. Selection criteria: exceptionally short stolon internodes resulting in an extremely tight knit stolon mat under close (c. 5-6 mm) but not very close (c. 3-4 mm) mowing; very deep, strong rhizome system; very dark green colour; tolerates shade better than other Australian bermuda grass varieties of common knowledge (except for ‘Plateau’A); and remains low growing under heavy tropical cloud cover even after 6-8 months. Designated ‘TL1’ by Tropical Lawns Pty Ltd and trialed successfully during the late 1990s and early 2000s in high wear situations (e.g. golf tees) in north Queensland. Propagation: vegetative. Breeder: Barry McDonagh, Townsville, QLD. PBR Certificate Number 2638, Application Number 2002/267, granted 24 February 2005.

Zoysia matrella, Manila Grass 'A-1'

‘A-1’ was selected from a breeding population of 40 seedling Zoysia matrella plants from various parts of Southeast Asia (Japan, Philippines, China, Korea, Vietnam and Thailand). The original plants were vegetatively propagated and evaluated first in pots. A shortlist of selected genotypes was expanded to field plantings at Sheldon, QLD and evaluated against existing Z. matrella and Z. matrella x Z. japonica hybrid cultivars under mowing heights from 10 to 25mm and under shade levels ranging from 0 to 80%. ‘A-1’ showed higher tiller density and a more prostrate growth habit than the parent ecotype, and was selected from the subsequent breeding population on the basis of its superior turf colour and quality under mowing for 6 years and its shade tolerance as shown by its ability to maintain density of the mown sward under greatly reduced light levels (70-80% shade). Additional observations regarding climatic adaptation were made in Cairns, QLD, and Melbourne, VIC, respectively. Breeder: Donald S Loch, Alexandra Hills, QLD. PBR Certificate Number 3649, Application Number 2008/091, granted 16 December 2008.

Morphological and developmental comparisons of seven greens quality hybrid bermudagrass (Cynodon dactylon (L.)Pers.x C transvaalensis Burtt-Davy

Fine-textured hybrid bermudagrass [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] cultivars have been widely used for golf putting greens and lawn bowls greens in warm-climate areas for more than 40 years. During the past decade, the choice of cultivar for professional turfgrass managers has been expanded by a range of secondgeneration hybrid bermudagrasses, which differ from the first-generation cultivars ‘Tifgreen’ and ‘Tifdwarf ’ in their management requirements. In this paper, we present comparative morphological and developmental data for seven cultivars (Champion Dwarf, FloraDwarf, MS-Supreme, Novotek, Tifdwarf, TifEagle, Tifgreen) grown in spaced plant and sward experiments at Cleveland, Australia (27º32’S lat, 153º15’E long, 25 masl). The four ‘ultradwarf ’ cultivars (Champion Dwarf, MS-Supreme, FloraDwarf, TifEagle) showed slower vertical extension and produced fewer inflorescences than Tifdwarf, Tifgreen, and Novotek. However, in terms of the length of stolon internodes and their overall rate of lateral spread, Champion Dwarf, FloraDwarf, and TifEagle were comparable to Tifdwarf; MS-Supreme (with longer internodes) spread faster laterally, though slower than Tifgreen (which had the longest stolon internodes). In unmown swards, the four ultradwarfs produced shorter leaves than Tifgreen, Tifdwarf, and Novotek, but only Champion Dwarf produced significantly narrower leaves than Tifgreen, Tifdwarf, and Novotek, with TifEagle leaves also significantly narrower than those of Tifgreen and Novotek. Minimum threshold temperatures for growth were approximately 9° to 10°C (air temperature) and 15° to 16°C at 10 cm soil depth.

Best management practices for sustainable and safe playing surface of Australian Football League sports fields

In 2002, AFL Queensland and the Brisbane Lions Football Club approached the Department of Primary Industries and Fisheries (Queensland) for advice on improving their Premier League sports fields. They were concerned about player safety and dissatisfaction with playing surfaces, particularly uneven turf cover and variable under-foot conditions. They wanted to get the best from new investments in ground maintenance equipment and irrigation infrastructure. Their sports fields were representative of community-standard, multi-use venues throughout Australia; generally ‘natural’ soil fields, with low maintenance budgets, managed by volunteers. Improvements such as reconstruction, drainage, or regular re-turfing are generally not affordable. Our project aimed to: (a) Review current world practice and performance benchmarks; (b) Demonstrate best-practice management for community-standard fields; (c) Adapt relevant methods for surface performance testing; (d) Assess current soils, and investigate useful amendments; (e) Improve irrigation system performance; and (e) Build industry capacity and encourage patterns for ongoing learning. Most global sports field research focuses on elite, sand-based fields. We adjusted elite standards for surface performance (hardness, traction, soil moisture, evenness, sward cover/height) and maintenance programs, to suit community-standard fields with lesser input resources. In regularly auditing ground conditions across 12 AFLQ fields in SE QLD, we discovered surface hardness (measured by Clegg Hammer) was the No. 1 factor affecting player safety and surface performance. Other important indices were turf coverage and surface compaction (measured by penetrometer). AFLQ now runs regularly audits affiliated fields, and closes grounds with hardness readings greater than 190 Gmax. Aerating every two months was the primary mechanical practice improving surface condition and reducing hardness levels to < 110 Gmax on the renovated project fields. With irrigation installation, these fields now record surface conditions comparable to elite fields. These improvements encouraged many other sporting organisations to seek advice / assistance from the project team. AFLQ have since substantially invested in an expanded ground improvement program, to cater for this substantially increased demand. In auditing irrigation systems across project fields, we identified low maintenance (with < 65% of sprinklers operating optimally) as a major problem. Retrofitting better nozzles and adjusting sprinklers improved irrigation distribution uniformity to 75-80%. Research showed that reducing irrigation frequency to weekly, and preparedness to withhold irrigation longer after rain, reduced irrigation requirement by 30-50%, compared to industry benchmarks of 5-6 ML/ha/annum. Project team consultation with regulatory authorities enhanced irrigation efficiency under imposed regional water restrictions. Laboratory studies showed incorporated biosolids / composts, or topdressed crumb rubber, improved compaction resistance of soils. Field evaluations confirmed compost incorporation significantly reduced surface hardness of high wear areas in dry conditions, whilst crumb rubber assisted turf persistence into early winter. Neither amendment was a panacea for poor agronomic practices. Under the auspices of the project Trade Mark Sureplay®, we published > 80 articles, and held > 100 extension activities involving > 2,000 participants. Sureplay® has developed a multi-level curator training structure and resource materials, subject to commercial implementation. The partnerships with industry bodies (particularly AFLQ), frequent extension activities, and engagement with government/regulatory sectors have been very successful, and are encouraged for any future work. Specific aspects of sports field management for further research include: (a) Understanding of factors affecting turf wear resistance and recovery, to improve turf persistence under wear; (b) Simple tests for pinpointing areas of fields with high hardness risk; and (c) Evaluation of new irrigation infrastructure, ‘water-saving’ devices, and irrigation protocols, in improving water use and turf cover outcomes.

The role of tillage, fertiliser and forage species in sustaining dairying based on crops in southern Queensland 1. Winter-dominant forage systems.

Field studies were conducted over 5 years on two dairy farms in southern Queensland to evaluate the impacts of zero-tillage, nitrogen (N) fertiliser and legumes on a winter-dominant forage system based on raingrown oats. Oats was able to be successfully established using zero-tillage methods, with no yield penalties and potential benefits in stubble retention over the summer fallow. N fertiliser, applied at above industry-standard rates (140 vs. 55 kg/ha.crop) in the first 3 years, increased forage N concentration significantly and had residual effects on soil nitrate-N at both sites. At one site, crop yield was increased by 10 kg DM/ha. kg fertiliser N applied above industry-standard rates. The difference between sites in fertiliser response reflected contrasting soil and fertiliser history. There was no evidence that modifications to oats cropping practices (zero-tillage and increased N fertiliser) increased surface soil organic carbon (0-10 cm) in the time frame of the present study. When oats was substituted with annual legumes, there were benefits in improved forage N content of the oat crop immediately following, but legume yield was significantly inferior to oats. In contrast, the perennial legume Medicago sativa was competitive in biomass production and forage quality with oats at both sites and increased soil nitrate-N levels following termination. However, its contribution to winter forage was low at 10% of total production, compared with 40% for oats, and soil water reserves were significantly reduced at one site, which had an impact on the following oat production. The study demonstrated that productive grazed oat crops can be grown using zero tillage and that increased N fertiliser is more consistent in its effect on N concentration than on forage yield. A lucerne ley provides a strategy for raising soil nitrate-N concentration and increasing overall forage productivity, although winter forage production is reduced.

Damage and physiological stress to juvenile eastern rock lobster (Sagmariasus verreauxi) discarded after trapping and hand collection

Large numbers of Sagmariasus verreauxi are trapped and hand collected in Australia, but discarded due to size and quota restrictions, and under the unevaluated assumption of few impacts. To test the validity of enforced discarding, trapped and hand-collected S. verreauxi (49-143. mm carapace length - CL) were examined for external damage, placed into cages, transferred to aquaria and monitored (with controls) over three months. Haemolymph was non-repetitively sampled immediately and at one, three, and seven days to quantify stress. Most trapped (64%) and hand-collected (79%) specimens were undersized (<104. mm CL), with the latter method yielding broader ranges of sizes and moult stages. Within-trap Octopus tetricus predation caused the only mortalities (3.3%). Hand collection resulted in much greater antennae and pereopod loss than trapping (53 vs. 4%) but, compared to controls, both methods evoked benign physiological responses that resolved within a week. While most wounded S. verreauxi regenerated all or some missing appendages post-moult, their mean CLs were less than those from intact conspecifics. Simple strategies, including larger mesh sizes, and/or installing modifications to reduce bycatch in traps, careful hand collection, and appropriate release techniques might minimise impacts (including predation) to unwanted S. verreauxi, and help to control stock exploitation. © 2012 Elsevier B.V.

Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium

The development of a more efficient in vitro regeneration system for somatic embryos (SEs) of avocado (Persea americana) would facilitate the development of new superior cultivars for this valuable horticultural crop. In this study, we report a new and efficient method for maintenance and regeneration of avocado SEs. Avocado SEs of four cultivars remained healthy and viable in vitro for 11 months on a medium used for mango somatic embryogenesis, compared with 3-4 months on Murashige and Skoog medium. Various supplements and media modifications were investigated to improve the low conversion rate of regenerated plants from avocado SEs reported previously. The one-step system for regeneration of white-opaque somatic embryos (WOSEs) used solid medium only over a period of 12-14 weeks (sub-culturing every 6 weeks). Addition of praline and glutamine improved the total regeneration from 0 to 17.5% and 10.5%, and plant/shoot recovery from 0 to 12.5% and 5%, respectively. A two-step culture system involving the transfer of WOSEs of cultivar 'Reed' after 6 weeks on solid to liquid medium for 12-15 days as an intermediate step, followed by subculturing again onto solid medium for 6 weeks improved total regeneration to 29% and plant/shoot recovery to 18.3 from 0% when regenerated by subculturing on solid medium only. Supplementation with proline in the solid as well as liquid medium in the two-step culture system at 0.4 g/L increased total regeneration to 35% and plant/shoot recovery to 20%. We were able to achieve highest regeneration using glutamine at 1 g/L in the two-step culture system in terms of both total regeneration (58.3%, including 43.3% bipolar regeneration) and plant/shoot recovery (36.7%) rates, which were significantly higher than in any other treatment investigated. (C) 2013 Elsevier B.V. All rights reserved.