In Vitro Conditions for a Successful Biolistics transformation System of Pineapples (ananas comosus merr.), in 'Contributing to a Sustainable Future'

In order to develop an efficient and reliable biolistics transformation system for pineapples parameters need to be optimised for growth, survival and development of explants pre- and post transformation. We have optimised in vitro conditions for culture media for the various stages of plant and callus initiation and development, and for effective selection of putative transgenic material. Shoot multiplication and proliferation is best on medium containing MS basic nutrients and vitamins with the addition of 0.1 mg/L myo-inositol, 20 g/L sucrose, 2.5 mg/L BAP and 3 g/L Phytagel, followed by transfer to basic MS medium for further development. Callus production on leaf base explants is best on MS nutrients and vitamins, to which 10 mg/L of BAP and NAA each was added. Optimum explant age for bombardment is 17-35 week old callus, while a pre-bombardment osmoticum treatment in the medium is not required. By comparing several antibiotics as selective agent, it has been established that a two-step selection of 2 fortnightly sub-cultures on 50 μg/mL of geneticin in the culture medium, followed by monthly sub-cultures on 100 μg/mL geneticin is optimal for survival of transgenic callus. Shoot regeneration from callus cultures is optimal on medium containing MS nutrients and vitamins, 5% coconut water and 400 mg/L casein hydrolysate. Plants can be readily regenerated and multiplied from transgenic callus through organogenesis. Rooting of shoots does not require any additional plant hormones to the medium. A transformation efficiency of 1 – 3.5% can be achieved, depending on the gene construct applied.

Polychaete-assisted sand filters – prawn farm wastewater remediation trial

Medium bedding sand which is commonly available in coastal sedimentary deposits, and a marine polychaete-worm species from Moreton Bay recently classified as Perinereis helleri (Nereididae), were deployed in a simple low-maintenance sand filter design that potentially has application at large scale. Previous work had shown that this physical and biological combination can provide a new option for saline wastewater treatment, since the worms help to prevent sand filter blocking with organic debris and offer a profitable by-product. To test the application of this new concept in a commercial environment, six 1.84 m2 Polychaete-assisted sand filters were experimentally tested for their ability to treat wastewater from a semi-intensive prawn culture pond. Polychaetes produced exclusively on the waste nutrients that collected in these gravity-driven sand filters were assessed for their production levels and nutritional contents. Water parameters studied included temperature, salinity, pH, dissolved oxygen (DO), oxidation/ reduction potential (redox), suspended solids, chlorophyll a, biological oxygen demand (BOD), and common forms of nitrogen and phosphorus. Pond water which had percolated through the sand bed had significantly lower pH, DO and redox levels compared with inflow water. Suspended solids and chlorophyll a levels were consistently more than halved by the process. Reductions in BOD appeared dependant on regular subsurface flows. Only marginal reductions in total nitrogen and phosphorus were documented, but their forms were altered in a potentially useful way: dissolved forms (ammonia and orthophosphate) were generated by the process, and this remineralisation also seemed to be accentuated by intermittent flow patterns. Flow rates of approximately 1,500 L m-2 d-1 were achieved suggesting that a 1 ha polychaete bed of this nature could similarly treat the discharge from a 10 ha semi-intensive prawn farm. Sixteen weeks after stocking sand beds with one-month-old P. helleri, over 3.6 kg of polychaete biomass (wet weight) was recovered from the trial. Production on a sand bed area basis was 328 g m-2. Similar (P>0.05) overall biomass production was found for the two stocking densities tested (2000 and 6000 m-2; n = 3), but survival was lower and more worms were graded as small (<0.6 g) when produced at the higher density (28.2 ± 1.5 % and approx. 88 %, respectively) compared with the lower density (46.8 ± 4.4 % and approx. 76 %, respectively). When considered on a weight for weight basis, about half of the worm biomass produced was generally suitable for use as bait. The nutritional contents of the worms harvested were analysed for different stocking densities and graded sizes. These factors did not significantly affect their percentages of dry matter (DM) (18.23 ± 0.57 %), ash (19.77 ± 0.80 % of DM) or gross energy 19.39 ± 0.29 MJ kg-1 DM) (n = 12). Although stocking density did not affect the worms’ nitrogen and phosphorus contents, small worms had a higher mean proportion of nitrogen and phosphorus (10.57 ± 0.17 % and 0.70 ± 0.01 % of DM, respectively) than large worms (9.99 ± 0.12 % and 0.65 ± 0.01 % of DM, respectively) (n = 6). More lipid was present in large worms grown at the medium density (11.20 ± 0.19 %) compared with the high density (9.50 ± 0.31 %) and less was generally found in small worms (7.1-7.6 % of DM). Mean cholesterol and total phospholipid levels were 5.24 ± 0.15 mg g-1 and 13.66 ± 2.15 mg g-1 DM, respectively (n = 12). Of the specific phospholipids tested, phosphatidyl-serine or sphingomyelin were below detection limits (<0.05 mg g-1), whilst mean levels of phosphatidyl-ethanolamine, phosphatidyl-inositol, phosphatidyl-choline and lysophosphatidyl-choline were 6.89 ± 1.09, 0.89 ± 0.26, 4.04 ± 1.17 and 1.84 ± 0.37 mg g-1, respectively (n = 12). Culture density generally had a more pronounced effect on phospholipid contents than did size of worms. By contrast, worm size had a more pronounced effect on total fatty acid contents, with large worms containing significantly higher (P<0.001) levels on a DM basis (46.88 ± 2.46 mg g-1) than smaller worms (27.76 ± 1.28 mg g-1). A very broad range of fatty acids were detected with palmitic acid being the most heavily represented class (up to 14.23 ± 0.49 mg g-1 DM or 27.28 ± 0.22 % of total fatty acids). Other heavily represented classes included stearic acid (7.4-8.8 %), vaccenic acid (6.8-7.8 %), arachidonic acid (3.5-4.4 %), eicosapentaenoic acid (9.9-13.8 %) and docosenoic acid (5.7-7.0 %). Stocking density did not affect (P>0.05) the levels of amino acids present in polychaete DM, but there was generally less of each amino acid tested on a weight per weight basis in large worms than in small worms. This difference was significant (P<0.05) for the most heavily represented classes being glutamic acid (73-77 mg g-1), aspartic acid (50-54 mg g-1), and glycine (46-53 mg g-1). These results demonstrate how this polychaete species can be planted and sorted at harvest according to various strategies aimed at providing biomass with specific physical and nutritional qualities for different uses.