Author Institution: Biology Department, Wittenberg College, Springfield, Ohi

Norstog, Knut J.

The Ohio Journal of Science

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RESPONSES OF THE OAT COLEORHIZA TO VARIOUSTREATMENTS IN CULTURE1KNUT J. NORSTOGBiology Department, Wittenberg College, Springfield, OhioThe coleorhiza or root sheath of the grasses has received little attention fromplant physiologists and experimental morphologists. Nevertheless it is of im-portance in the germination of the oat seed and is interesting in its responses tocertain factors in its immediate environment.In a number of cultures of excised oat embryos, extreme coleorhizal elongationswere observed. Such elongations occurred regularly in liquid media. However,the coleorhizae of oat embryos cultured on the surface of nutrient agar elongatedonly slightly. Instead they produced numerous epidermal hairs which appearedto be identical to root hairs. These phenomena have been reported by otherinvestigators. Nishimura (1922) and Howarth (1927) consider that such coleorhizalhairs serve to supply water to the embryo during germination. Dietrich (1924)discovered that greatly lengthened coleorhizae were developed in subsurfacecultures of excised oat embryos. LaRue (1936) observed that immature, excisedembryos of certain grasses formed coleorhizal hairs in vitro.Normally the coleorhiza is the first part of the oat embryo to break throughthe seed coat during germination. At this time it further elongates and forcesitself through the husk or lemma surrounding the seed. When this is accomplishedcoleorhizal elongation ceases. Hairs then are often formed on the parts of thecoleorhiza exposed to air. The following account deals with oat coleorhizaesubjected to various treatments.MATERIAL AND METHODSSeeds of oats, Avena sativa L. variety Andrew, were obtained from the NorthDakota Agricultural Experiment Station. Some of these were planted to provideembryos of various stages of development. Other seeds were cultured in sterilewater or on moist filter paper. A simple apparatus was used in the latter cul-tures to obtain various dilutions of air with nitrogen gas. Excised embryos, ofdifferent ages, were cultured in White's solution (White 1943). All seeds weresterilized in a 10 percent solution of Clorox for ten minutes prior to culture.RESULTSColeorhizal growth in liquid culture. Coleorhizal elongations were noted inexcised embryos cultured for a week in White's solution but not in those culturedin sterile water alone. Coleorhizal elongations also occurred when intact seedswere placed either in White's solution or in sterile distilled water. Because ofthe ease with which they could be cultured in sterile water, whole seeds ratherthan excised embryos were used in the experiment reported in table 1. Thisexperiment was performed in order to discover whether or not coleorhizal elonga-tions were due solely to the presence of water. The effect of indole-3-acetic acidon coleorhizal elongation was also tested.The effect of various factors on the growth of oat coleorhizae. As a result of ex-perimentation, the data of which appear in table 1, some factors affecting coleorhizalbehavior have been determined. Elongation of the coleorhiza was found to beinversely proportional to the amount of air present in the immediate environment,the greatest length being attained in liquid media. Hair formation was pro-a dissertation submitted in partial fulfillment of the requirements for the degree ofDoctor of Philosophy in the Rackham School of Graduate Studies of the University of Michi-gan, Ann Arbor. Much of this work was done during the tenure of a fellowship granted by theNational Science Foundation, Washington, D. C.THE OHIO JOURNAL OF SCIENCE 55(6): 340, November, 1955.No. 6 RESPONSES OF OAT COLEORHIZA 341portional to the amount of air present and was stimulated by the applicationof IAA. Lower concentrations of IAA (0.1 to 1.0 mg/lit.) in liquid culturesstimulated coleorhizal elongation, but higher concentrations (5.0 to 10.0 mg/lit.)of this substance proved inhibiting. The greatest length, 10 mm., was obtainedin cultures with water and 0.1 mg/lit. IAA. Mitotic figures were not observedin aceto-carmine smears of the coleorhizae of cultured embryos. Coleorhizalgrowth in these instances, whether characterized by elongation or hair formation,occurred only by the enlargement of existing cells.TABLE 1The effect of various factors on the growth of oat coleorhizae.Treatment20%20%IAA10%4%2%H2OH2OH2OH2OH2OO2 + 80% N2O2 + 80% N2 +O2 + 90% N,O2 + 96% N2O2 + 98% N»+ 0.1 IAA2+ 1.0 IAA+ 5.0 IAA+ 10.0 IAANo. ofembryos20212020204722273233Mean coleorhizalInitial30.8 mm.0.80.80.80.80.80.80.80.80.8Final2.6 mm.2.53.23.75.06.17.17.15.75.4length<rV n±0.07±0.10±0.11±0.07±0.13±0.16±0.29±0.28±0.26±0.22Lengthincrease225%212%300%362%525%662%787%787%612%575%Hairs4moderateconsiderableslightnonenonenonenonenonenonenone^resoaked 24 hours in water and 10 mg/1. IAA.2The figures given for IAA are milligrams per liter.3Initial lengths based on measurements of section embryos.4Based on the amount of coleorhizal surface covered by hairs.TABLE 2The effect of adenine sulfate on the growth of coleorhizae of 0.8 mm. oat embryos.Medium No. of embryos Mean coleorhizal lengthInitial FinalIncreasein lengthWhite's agar.White's agar + adeninesulfateWhite's agar + adeninesulfate + coconut milk. .4628210.24 mm.0.240.240.74 mm.*1.581.96208%558%771%*Coleorhizal hairs were produced by some of these embryos.In order to discover whether elongation would occur in the absence of oxygen,oat seeds also were placed on moist filter paper in several bottles. The oxygenthen was removed from the air in the bottles by means of pyrogallol. Coleorhizalelongations were not observed in these cultures and germination was suppressed.Cultures of detached coleorhizae. Coleorhizae were also dissected from matureembryos and placed in culture on White's agar. In these cultures hairs wereformed as in the preceding cultures, but coleorhizal elongation did not occur.Other detached coleorhizae did not grow at all when cultured in White's liquid.It was found that not all coleorhizal cells were capable of forming hairs. Thecoleorhiza is a hollow organ made up of several layers of parenehymatous cells.342 KNUT J. NORSTOG Vol. LVA number of coleorhizae were removed from embryos, slit open and flattenedout on nutrient agar. Those which were placed so that the epidermal cells wereexposed to the air formed hairs. Others, placed so that the epidermis lay incontact with the agar and the cells of the inner surface were exposed to the air,formed no hairs. Thus it appears that only the epidermal cells are able to producecoleorhizal hairs.The growth of coleorhizae of smaller embryos. Many small oat embryos wereexcised from unripened seeds and placed in culture. The largest of these wereless than half the size of full term embryos. Embryos in the size range of 1.0 to1.5 mm. exhibited elongated coleorhizae in liquid media and produced coleorhizalhairs when grown on nutrient agar. Smaller embryos did not develop lengthenedcoleorhizae in liquid media. Only one instance of coleorhizal elongation wasobserved in the 125 0.8 mm. embryos grown in White's solution. Yet hairswere usually produced even by 0.8 embryos when they were cultured on nutrientagar. Combinations of certain growth substances were added to White's medium.Yeast extract, IAA and coconut milk had little effect on coleorhizal development.However, in two separate sets of cultures on media containing adenine sulfate,definite coleorhizal elongations and hair suppression were observed. The first ofthese two sets of cultures was made on White's agar to which 40 mg/lit. of adeninehad been added. The second set contained coconut milk in addition to 40 mg/lit. adenine sulfate. Coconut milk was found to promote the overall growth ofexcised oat embryos but did not of itself stimulate coleorhizal elongation, sincemarked elongations were not observed in cultures made on coconut milk mediawithout adenine sulfate.The effect of adenine sulfate on the growth of coleorhizae of 0.8 mm. oat embryos.Not all cultures of 0.8 mm. embryos exhibited coleorhizal elongations when treatedwith adenine. Larger embryos did not respond to this substance at all. Thereason for these failures is not clear at present.DISCUSSIONThe behavior of the oat coleorhiza during germination seems to depend ondifferences in oxygen tension inside and outside of the seed and its coverings.Low concentrations of oxygen (down to at least 2%) stimulate coleorhizal elonga-tions, while higher concentrations promote the formation of epidermal hairs.Neither elongation nor the formation of the "root hairs" occurred, however, atextremely low concentrations of oxygen. The hairs of the coleorhiza are a regularfeature of the surface cells of this structure. That they are not simply adven-titious . outgrowths is shown by the failure of sub-surface cells to produce them.Indole-3-acetic acid appears not to initiate but rather to determine the extentof such responses as hair formation and coleorhizal elongation.Skoog and Tsui (1951) report that adenine sulfate may be used to inducedifferentiation of buds in cultures of tobacco callus on nutrient agar. They alsoobtained such differentiation without adenine in callus masses submersed in liquidmedia. Thus adenine sulfate used with surface cultures seems to duplicate theeffects obtained with subsurface cultures. Adenine sulfate also seems to substi-tute for liquid media in embryo cultures at least to the extent that it may inducecoleorhizal elongation.REFERENCESDietrich, K. 1924. Uber Kulture von Embryonen ausserhalb der Samen. Flora 117: 379-417.Howarth, W. O. 1927. The seedling development of Festuca rubra L. var. tennuifolia Mihi.and its bearing on the morphology of the grass embryo. New Phyt. 26:46-57.LaRue, C. D. 1936. The growth of plant embryos in culture. Torrey Bot. Club Bull. 69: 332-341.Skoog, F., and C. Tsui. 1951. Growth substances and the formation of buds in plant tissues.Plant Growth Substances. Univ. Wis. Press, Madison.White, P. R. 1943. A Handbook of Plant Tissue Culture. Ronald Press, New York.

Responses of the Oat Coleorhiza to Various Treatments in Culture

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Responses of the Oat Coleorhiza to Various Treatments in Culture

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