Pollen of Sarawakodendron (Celastraceae) and some related genera, with notes on techniques Ding Hou Summary A for of small of material is described 1. simple technique acetolysis quantities polliniferous (herbarium) and notes on pollen photomicrography are presented. 2. Pollen grains of Sarawakodendron and six related genera, consisting of twenty-nine mostly Malesian species, have been examined and recorded. 3. The result of pollen study on Kokoona and Lophopetalum agrees with the generic delimitation based on gross morphology. least four have been found in the examination of all the 4. At pollen types genus Lophopetalumon species involved. The of Sarawakodendron shows resemblance that of the related 5. pollen a great to genera Xylonymus and Kokoona. 6. The pollenofHedraianthera and Brassiantha resembles that of Sarawakodendron,Kokoona , and Xylonymus in aperture configuration, but differs in sculpture and shows in this respect similarity to the pollen of the African Salacighia. In Kokoona of reticulate correlated with anther characters. This 7. coarseness sculpture appears genus also be from its can easily distinguished Lophopetalum by single pollen grains. 8. Parallels are found between the pollen types in Lophopetalum and those in Hippocratea (sens. str.). Introduction has been shown of the Sarawakodendron closely allied to Salacia Hippocrateaceae on one hand andto KokoonaandLophopetalum ofthe Celastraceae on the other by its gross morphol- link between these related families which ogy. It represents an important two closely have Celastraceae already been treated as one family, the (Hou 1963, 1964, 1967; Robson examined 1965). The pollen grains of Sarawakodendron were already by myself, and it be if those would interesting of its related genera, e.g. Salacia, Kokoona, Xylonymus, could also be studied for Hedraianthera, Brassiantha, and Lophopetalum, etc., comparison. A. C. Smith and I. W. Bailey (1941), in connection with their studies on Brassiantha of A. C. Sm., stated that the pollen of the Celastraceae (sensustr.) exhibits a similar range that of variability in size, form, and structure as the Hippocrateaceae. Erdtman (1952) also mentionedthat similar in more or less pollen grains occur these two families. Later, other in addition to the publications concerning pollen of these families by workers, Van Campo and Halle (1959) made a comprehensive study of African representatives of the with the of in of Hippocrateaceae very interesting finding grains polyads consisting their has with four tetrads in Hippocratea (sensu Hallé 1962); work been illustrated forty plates of beautiful drawings and photographs facilitating comparison. Recently, Waanders, Skvarla, and Pyle (1968) studied the fine structure of pollen walls of four American Hippocrateaceae; they recorded polyads consisting of 4 tetrads in Hippocratea and volubilis L. (erroneously ascribed to 'Stehle Quentin'). 97 98 BLUMEA VOL. XVII, No. I, 1969. the So far, only pollen grains of a few representatives of the Celastraceae (sensu str.) connection with taxonomic work for the Flora I have been studied. In my Malesiana, could collect polliniferous samples of all the species accepted by me. of familiarize In order to acquire some reading knowledge pollen morphology and myself with its terminology and techniques for preparations and photomicrography, I those mentioned started to work on the pollen of genera above. I have not studied pollen African ofSalacia and have relied for comparisons on the descriptions and figures ofWest species published by Van Campo & Halle (1959). of of I am fully aware of the limitations using simple equipment and my restricted for confine ability, being a taxonomist, studying pollen morphology. So I just myself to the description of those features of pollen grains which have taxonomic significance of information available. and to the presentation the of In the course of this study, literature on techniques pollen preparations and photomicrography has been reviewed. After having tested rather simple equipment, I and made have prepared a working procedure for do-it-yourself acetolysis some notes those on photomicrography. I hope that this information will be of use to interested in such techniques. DO-IT-YOURSELF ACETOLYSIS For pollen preparations the fundamentalprinciples of the well known acetolysis method have devised by Erdtman (1943, 1952, i960) have been followed (cf. Traverse 1965). I the small tried to reach goal by using simple equipment, quantities of chemicals and, above all, a minimumof pollen bearing (herbarium) material, as in the 'micro-method' and applied and described by Punt (1962) some others before him (cf. Faegri & Iversen have 1964, p. 79). By trial and error, satisfactory microscopic slides been obtained. My working procedure is as follows: or anther evena of soften the material (1) Boil a flower, a flower-bud, an (or part it) to for dissection. the material in in few (2) Put boiled a watch-glass (about 7 cm diam.), containing a under the binocular. If the anthers drops of (distilled) water, are big enough, remove from small the pollen grains them by using a pair of fine forceps and a needle. For very and break the material into For anthers, take one or more of them pieces. type or scant sometimes obtain and still be able save the material, one may even enough grains to empty anther(s) or flower(s). the thenadd about fresh solution (3) Let the pollen in watch-glass dry, 1\ cc acetolysis of of and of (a mixture 9 parts acetic anhydride 1 part conc. sulphuric acid, having mea- a with scales marked down to 0.1 cc). sured each acid with respective pipette, 5 cc, kinds be used. the which (4) Two of heating apparatus can Try one is more convenient to you. with (a) Alcohol lamp or Bunsenburner. Hold the watch-glass containing pollen grains rather low flame of alcohol Bunsen- a pair of coarse forceps rotating over a an lamp or dark brown. burner until the solutionturns brown or Keep the solution below boiling point. Then proceed to 5). the (b) Electric tea-warmer. In order to standardize acetolysis process, an electric tea- has been used. has switch for and hot and warmer It a two-way warm (60 W) (120 W), is with marked scale from connected to themain by an Electric EnergyRegulator a 0—10. For rather with a metal thermometer fixed obtaining a even temperature, a copper plate, to it, is placed on the warmer. (Fig. 1). Pollen Sarawakodendron and related Ding Hou: of (Celastraceae) some genera 99 Fig. 1. Heating apparatus and slide mounting card. Place the watch-glass containing pollen material on thewarmer. Turn on the Regulator the scale of with the switch of the warmer on 'hot'. Set the Regulator to let the tempera- about ture gradually rise from room-temperature to 100° C in 15 minutes. The glass the should be rotated once and again. After the temperature has reached 100° C, adjust the Regulator to keep temperature at that degree for about three minutes and then turn it off. (5) Remove the watch-glass from the flame or warmer for cooling and then place it under the binocular to examine the grains which would appear brown or dark brown colour ofthembecomes after If take ofthe solution (the lighter washing). necessary, a drop from the glass, prepare a slide, and thenexamine the grains under the microscope. With the some experience, one may judge the conditionof acetolysis by taking a look at colour of the solution left in the glass. (6) Add some alcohol (about 95 %) to the solution in the watch-glass with an eye- it is wash acid dropper and rotate for washing. It unnecessary to with glacial acetic After few (Erdtman, i960)! a minutes, the grains gather together in the center of the and at the a ofsolution will surface. a glass same time rim appear on its upper inner Use of soft for off the rim and the solution in the piece paper wiping drawing away glass as much as possible. Wash the pollen at least once more. of alcohol and mixture (7) Add a few more drops one or two drops of a of 1 part of and ofalcohol the and glycerine 1 part (about 95 %) to solution rotate it. (8) After the alcohol has evaporated, the grains will either stick to the watch-glass coated of the with a layer glycerine or gather together in the glycerine in center. They are ready to be transferred to a slide for mounting. If the and theircolour dark which grains are over-acetolyzed appears too may obscure 100 BLUMEA VOL. XVII, No. i, 1969. features for observation, add a few more drops of the mixture of glycerine-alcohol to and with dish. After of the the watch-glass cover it a Petri one day or more, the colour willbe should grains faded justlike bleached ones. One realize, however, that theoxidation does clear the exines, but at the same timewill also cause the grains to swell (cf. Faegri & Iversen, 1964, p. 79). (9) Attach a small piece ofglycerine jelly to a needleand touch the grains in the watch- glass. Glycerine jelly has been used as mounting medium. For convenience, it is melted in a thin about Petri dish (about 6 cm in diam.) to form a layer ij mm thick and then cut with a razor blade into strips each about i| mm wide. Whenever needed, use a needle to cut off a piece of the size desired. besides If there are enough acetolyzed grains, using glycerine jelly, sometimes one may use as medium. This kind of has been try to methyl-green glycerine jelly mounting jelly prepared according to the procedure of Wodehouse (1935) (cf. Staining Procedures, i960) by adding saturated methyl-green solution (in alcohol 50 %) gradually, drop by drop, to the melted glycerine jelly. For staining the grains, I have triedto use safranin (1 %; prepared with alcohol or water).