Azolla - IRRI books - International Rice Research Institute

Table 4. Identification key for Azolla.1. Trichomes present on leaves as well asrhizomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21. Trichomes present only on leaves. . . . . . . . . . . . . . . . . . 32. lndividual plant deltoid ortriangular . . . . . . . . . . . . . . . . . . . . . A.pinnata var. imbricata2. Lateral branches regularly pinnate.secondary or tertiary branchesscattered along the plant periphery. . . A. pinnata var. pinnata2. Plants somewhat ascending, rootsin clusters on the rhizome nodes,sporocarps in clusters of four. . . . . . . . . . . A. nilotica3 Trichomes single-celled, glochidia withfew or no septae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43. Trichomes with 2 or 3 cells, glochidiawith several septae . . . . . . . . . . . . . . . . . . . . . . . . . . . A.mexicana,A. caroliniana,A. microphylla4. Trichomes clearly protruding . . . . . . . . . . . . . . . . A. filiculoides4. Trichomes less protruding . . . . . . . . . . . . . . . . . . . . . A. rubraTable 5. Codes for IRRl's Azolla accessions.SpeciesidentifierAccessionnumberSpecies or subspeciesPI >0 and 1000 and 2000 and 3000 and 4000 and 5000 and 6000 and 7000 and 8000 UnclassifiedConservation methodsAzolla are maintained in the vegetative stage becausea few strains form sporocarps. To maintaina healthy collection of strains in the vegetativestate, growth conditions must be adapted to allspecies and strains.Liquid medium for Azolla culture. Componentsof the liquid medium for Azolla culture arepresented in Table 6.Preparation of the liquid medium requirestrace elements solution and Fe-EDTA solution.1. To prepare the trace elements solution, dissolveeach trace element compound in asmall amount of water taken from 500 ml ofdeionized distilled water. Combine thedissolved compounds, and make up to 500ml. To prevent precipitation, add 10-15 ml6N H 2 SO 4 . Make up to 1 liter volume withdeionized distilled water.2. To prepare the Fe-EDTA solution, dissolveEDTA in 268 ml of 1N KOH. AddTable 6. Components of liquid medium for Azolla culture.Element/ Final Stock solutionstrace concentration Reagent (g/liter deionizedelement(ppm)water)P 20 NaH 2 PO 4 · H 2 O 89.0K 40 K 2 SO 4 89.1Ca 40 CaCI 2 ·2H 2 O 147.0MgFeMnMoBZnCuCo40 MgSO 4 ·7H 2 O 405.30.50 FeSO 4 ·7H 2 O24.9EDTA C 10 H 6 O 8 N 2 26.10.50 MnCI 2 ·4H 2 O1.800.15 Na 2 MoO 4 ·2H 2 O 0.380.20 H 3 BO 31.140.01 ZnSO 4 ·7H 2 O0.040.01 CuSO 4 ·5H 2 O0.040.01 COCI 2 ·6H 2 O0.04FeSO 1 · 7H 2 O and dilute to 1 liter. Aerateovernight or stand until a stable reddishbrown ferric complex is formed. The pHshould be 5.5. Store in an amber bottle.3. To prepare the liquid culture, add 1 ml oftrace elements solution (prepared from no.1), 1 ml each of the P, K, Ca, and Mg dissolvedin deionized water (see Table 6),and 0.1 ml Fe-EDTA per liter of deionizedwater. Stir after each addition. Adjust pH to5.5 using 1N NaOH or H 2 SO 4 solution. ForAnabaena -free Azolla, add 2 mM NH 4 NO 3 .Beaker culture. Put 150 ml of culture solutionin a 250-ml beaker coated with black paint fromthe 150 ml mark to the bottom. The black paintprevents algal growth. Wash actively growingAzolla with water, then inoculate it into thebeaker. At IRRI, cultures are grown under artificiallight with fluorescent lamps and incandescentlamps at 15 klx. The air temperature is maintainedat 26°C under light and at 18°C in the dark, thephotoperiod at a 12-h day, and relative humidityat 70-75%. Replace media 2-3 wk after washingthe Azolla.Shoot-tip culture. After washing healthyfronds. cut off 5-mm fragments that include meristemsand several leaves. Enclose cut fronds in asmall net bag and wash with running tap waterfor 30 min. Sterilize with 2% NaOCl and 1% TritonX 100 solution for 3 min. Wash with sterile waterten times. Inoculate 15-20 pieces of the cut tipportion into a 50-ml flask with 20 ml IRRI Azollamedium and 0.5% agar. For Anabaena-freeAzolla, add 2 mM NH 4 NO 3 .Incubate at 26/18°C under 5 klx. After 2-3 mogrowth, cut the top portion of the Azolla frond8