THE ELONGATION of the pollen tube in flowering plants is exceedingly rapid and its requirements, in general, seem quite unimpressive, i.e., water, oxygen and a suitable osmotic milieu. Despite extensive attempts to hasten this growth process with the conventional host of growth factors, few have met with convincing success. Boron, as borate, is the only factor previously shown to enhance growth markedly in a wide range of species. Its role is completely enigmatic. It is the purpose of this report to show that calcium ion plays a similarly prominent role in pollen germination and pollen-tube growth. The role of calcium, like that of boron, remains an intriguing enigma. A preliminary report of these findings has been made (Kwack and Brewbaker, 1961).MATERIALS AND METHODs-Several hundred flowering plant species were used in these tests. Most intensive studies were made with 2 selfincompatible species, Petunia infiata and Ornithogalum virens, which were brought to flower in controlled growth chambers. Ornithogalum virens (n = 3) was used in all studies; like many of its liliaceous relatives, it has large pollen grains, rapid pollen germination and growth, and clearcut responses to growth constituents.The basal medium for most studies included 10% sucrose and 100 ppm boric acid in deionized ABSTRACT BREWBAKER, JAMES L., and BEYOUNG H. KWACK. (U. Hawaii, Honolulu.) The essential role of calcium ion in pollen germination and pollen tube growth. Amer. Jour. Bot. 50 (9): 859-865. lIlus. 1963.-A pollen population effect occurs whenever pollen grains are grown in vitro. Small pollen populations germinate and grow poorly if at all, under conditions which support excellent growth of large pollen populations. The pollen population effect is overcome completely by a growth factor obtained in water extracts of many plant tissues. This factor is shown to be the calcium ion, and its action confirmed in 86 species representing 39 plant families. Other ions (K+, Mg"", N a") serve in supporting roles to the uptake or binding of calcium. The high requirement of calcium (300-5000 ppm, as Ca (N03),·4H20, for optimum growth) and low calcium content of most pollen may conspire to give calcium II governing role in the growth of pollen tubes both in vitro lind in situ. It is suspected that ramifications of this role extend to the self-incompatibilities of plants lind to the curious types of arrested tube growth distinguishing, for example, the orchids. A culture medium which proved its merit in II wide variety of pollen growth studies included, in distilled water, 10% sucrose, 100 ppm H 3B03, 300 ppm ell (NO,)2·4H20, 200 ppm MgS04·7H20 lind 100 ppm KNO,. distilled water; this is referred to as the 10: 100 solution. Pollen grains were sown in standing drops of 1/50 ml on cover slips. The cover slips were then placed on moist filter paper in Petri dishes, and normally left in room temperature during growth (up to 24 hr). Extensive studies were made of refinements of this technique (sterilization, humidity control, etc.), and of other techniqu...

Brewbaker, James L., and Beyoung H. Kwack. (U. Hawaii, Honolulu.) The essential role of calcium ion in pollen germination and pollen tube growth. Amer. Jour. Bot. 50(9): 859–865. Illus. 1963.—A pollen population effect occurs whenever pollen grains are grown in vitro. Small pollen populations germinate and grow poorly if at all, under conditions which support excellent growth of large pollen populations. The pollen population effect is overcome completely by a growth factor obtained in water extracts of many plant tissues. This factor is shown to be the calcium ion, and its action confirmed in 86 species representing 39 plant families. Other ions (K+, Mg++, Na+) serve in supporting roles to the uptake or binding of calcium. The high requirement of calcium (300–5000 ppm, as Ca (NO3)2·4H2O, for optimum growth) and low calcium content of most pollen may conspire to give calcium a governing role in the growth of pollen tubes both in vitro and in situ. It is suspected that ramifications of this role extend to the self‐incompatibilities of plants and to the curious types of arrested tube growth distinguishing, for example, the orchids. A culture medium which proved its merit in a wide variety of pollen growth studies included, in distilled water, 10% sucrose, 100 ppm H3BO3, 300 ppm Ca (NO3)2·4H2O, 200 ppm MgSO4·7H2O and 100 ppm KNO3.

Studies were conducted to determine cellular site of Ca action in promoting pollen growth of Crinum asiaticum and a few other species. The following experimental results have strongly indicated that Ca binding takes place in pectins of the pollen tube walls. This appeared to increase the wall rigidity and to regulate permeability of the pollen cells thereby enhancing pollen growth. Radioactive Ca incorporation was observed exclusively in the pollen tube wall regions. The promoting action of Ca on pollen growth disappeared when pectinase was supplemented to the media. This was not the case with cellulase and other enzymes used. Methyl donors promoted pollen growth, and the promotion was more than doubled if Ca ions were present. Ethionine, on the other hand, inhibited tube elongation and exhibited no Ca effect. Growth of pollen tubes in oscillaled liquid media during elongation was poorer than growth in standing media. The Ca effect was also reduced when pollen was oscillated. The observations made of the reduced rate of 45Ca incorporation when pollen was washed in water, and the hydroxylamineferric chloride test, have indicated that a considerable portion of these pectins are cold‐water soluble.

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