NOTES 200 1

podium (Freeberg and Wetmore 1957). The spores, ground or ilnground, rarely germinated on the surface of the unslanted medium. The best germination, although less than 0.1%, oc- curred by culturing the unground spores in the tubes with the slanted medium. The germinating spores or young gametophytes were usually found in the liquid at the base of the slant or between the agar and the glass sides of the culture tubes.

The early stages of germination (Figs. 1, 2) resembled those illustrated by Darnell-Smith (1917). Older gametophytes (Figs. 3, 4) were found, although they were small and immature. The endophytic fungus, which is present in Psiloturn gametophytes growing in soil (Holloway 1939; Bierhorst 1953), was not present in any of the gametophytes from axenic culture. Efforts are now underway to grow to maturity the gametophytes collected from these cultures.

Since the spores can be germinated in axenic culture, much call be learned about the factors controlling germination and prothallial growth. Light inhibition of spore germination in Psilotum is similar to the effect light has on spore germina- tion in Botrychiztm dissectum (Whittier 1973). The development of psi lo rut?^ gainetophytes in axenic culture will provide a better opportunity

for the observation of and experimentation on these ganletophytes than is possible with soil culture.

I thank Dr. James L. R i 0 ~ e l of the Uiliversitv of Virginia for providiilg the spores used in this investigation, and the Vanderbilt University Research Council for support of this research.

BIERHORST. D. W. 1953. S t ruc t~~re and develooment of the gametophyte of P.tilotrrt17 11rrclr1117. Am. 2. Bot. 40; 649-658.

1955. A note on spore germination in Psilotrun I I ~ I C ~ I I ~ I . Va. J . Sci. 6: 96.

DARNELL-SMITH, G. P. 1917. The gametop l~y teo fP~ i lo t~ r~~~ . Trans. R. Soc. Edinb. 52: 79-91.

DEMAGGIO, A. E. 1972. Induced vascular tissue dif- ferentiation in fern gametophytes. Bot. Gaz. 133: 311-317.

F~EEBERG, J. A., and R. H. WETMORE. 1957. Gameto- phytes of L)~copociir11~7 as grown irr vioo. Phytomorpho- logy (Delhi), 7: 204-21 6.

HOLLOWAY. J . E. 1939. The earneto~hvte. embrvo. and young rhizome of ~siiotlr11;tric~rre;r11~11 SW. A&.' Bot. (Lond.), 3: 3 13-336.

MARSDEN, M. P. F., and R. H. WETMORE. 1954. 111 vitro culture of the shoot tips of Psilotlon 11lrclrn11. Am. J . Bot. 41: 640-645.

WHITTIER, D. P. 1964. The effect of sucrose on apogamy in Cj~rfon1irt117 fnlccrfwj~ Presl. Am. Fern J. 54: 20-25.

1972. Gametophytes of Borq~cirirrt~r clissectro~7 as grown in sterile culture. Bot. Gaz. 133: 336-339.

1973. The effect of light and other factors on spore germination in Bor,:,~clri~l117 clissect~o~t. Can. J. Bot. This issue.

Identical and non-identical seedling twins in Pinus radiata

R. D. BURDON AND J. A. ZABKIEWICZ Forest Resenrcll I17stirrrre, New Zenlnnd Forest Service, Rotorlm, New Zenlcrnd

Received March 12, 1973

BURDON, R. D., and J. A. ZADKIEWICZ. 1973. Identical and non-identical seedling twins in Pblrrs radinra. Can. J. Bot. 51: 2001-2004.

Thirteen sets of twin seedlings emerging from single seeds of Pitzsls rarlinta were raised. Using morph- ology in 2 cases and oleoresin analysis in the remain~ng 11, six sets were accepted as genetically identical, one probably identical, two probably non-identical, and four definitely non-identical. Therefore, twins apparently result from both cleavage and archegonial polyembryony.

BURDON, R. D., et J. A. ZAUKIEWICZ. 1973. Identical and non-identical seedling twins in Pirrrrs rndiata. Can. J. Bot. 51: 2001-2004.

Les auteurs ont cultivi 13 paires de plantules jumelles, cl~acjue paire provenant d'une seule graine de Pinsrs rndinra. A partir d'6tudes morphologiques dans 2 cas et d'analyses d'oliorisines dans les 11 autres cas, six paires ont i t6 considerCes comme des paires de plantures ginitiqueme~lt identiques, ilne paire comme probablernent identique, deux cornrne probable~llent non identiques et quatre comme definitive- ment no11 identiques. Par consiquent, les plantules jumelles peuvent Etre formies par fragmentation de l'embryon aussi bien que par polyembryonie archigoniale. [Traduit par le journal]

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Introduction

It is often desirable to establish whether or not individuals are genetically identical. In this study the question arose with twin seedlings that emerged from occasional seeds of Pirzt~s rcrdiatn D.Don.

Seedling twins have been recorded in other conifers (Cayford and Waldron 1965; Ching and Simak 1971). It would be expected that in very many con if el.^, including Pinus, such twins could be either identical or non-identical, because both cleavage and archegonial polyembryony nor- mally occur. Twins resulting from cleavage polyembryony would be identical. In archegonial or polyzygotic polyembryony, however, the embryos derive from a single gametophyte geno- type but different pollen genotypes, so any resulting twins would be non-identical. With archegonial polyembryony the expectation is that if any one of several zygotes is genetically viable then the ovule itself should be viable. This would have important genetic implications (Sarvas 1968; Brarnlett and Popham 1971), par- ticularly concerning the breeding system. Against this, though, Orr-Ewing (1956, see Sorensen 1971), working with Pseudotsliga, concluded that the zygote formed in the most favorable position within the ovule would not, if it aborted, be replaced by another. On the other hand, any occurrence of non-identical twins would strongly suggest that Orr-Ewing's claim does not hold, at least for the species in question.

For determining whether or iiot twins are identical, a morphological examination may suffice in favorable circumstances. However, a powerful adjunct to this procedure is comparison by chemical analysis of plant constituents (Swain 1963; Mirov 1961), in this particular case, the monoterpenes present in the oleoresin of cortex near the shoot apex. The monoterpene composition differs widely among genotypes, but is very consistei~t within genotypes, provid- ing that the sampling site is standardized (Bernard-Dagan et nl. 1971; Zabkiewicz, un- published data for P. raclintn). This was the basis of the method used to determine whether or iiot the sets of twins were identical in all but two cases.

Material and Methods 111 a large genetic experiment with wind-pollinated

progenies of P. radirrtu, a very small proportion of the

seeds (about 0.1% overall, but apparently varying among populations) produced twin seedlings. Thirteen sets of twins were successfully raised, and were planted out in 1968. In each case, twins were planted next to each other.

Two sets of twins presented uncommon and grossly aberrant phenotypes. In one of these sets both seedlings were abnormal so it was classed as putatively identical, although there is a slender chance that a particular mutant gene could have come from two different pollen grains. The other set contained one normal and one abnormal seedling so it was definitely non-identical. None of the three abnormal seedlings could be sampled for oleoresin because they either died or grew too weakly. Among the normal seedlings it was impossible to decide, by morpho- logical examination during the early juvenile phase, whether sets of twins were identical. Juvenile trees of Pbilts mdiutcr are notorious for developmental instability of growth habit. Hence, the genotype is not closely reflected in the growth habit of an individual seedling.

Microliter volumes of oleoresin were taken from the cortex near the shoot apex on the leader or a prominent upper lateral of each seedling in the normal twins. Samples were taken from corresponding positions within each set of twins. Whenever practical, the standard posi- tion was about 15-20 cm from the tip of the leading shoot. If resin was difficult to obtain from this position, samples were taken from around the zone of bare cataphylls at the base of the current season's growth on a lateral. A 2-5mm transverse cut with a scalpel blade below the base of a cataphyll normally released resin from a prominent resin canal.

All these sets of twins were sampled in November, 1971, and resampled each subsequent month over a 6-month period. The monoterpenes from each sample were analyzed by means of gas-liquid chromatography (g.1.c.). Oleoresin from the shoot tips of P. rcrdintu can contain up to 11 monoterpene components (generally 8 or more), most of which are Icnown. Each component was expressed as a percentage of total monoterpenes on a peak height basis (cf. Smith and Green 1971), as elution times were similar. The proportions in which these monoterpenes occur are extremely variable between genotypes. A g.1.c. analysis gives a recorded trace which can be distinctive for a particular genotype, at least in relation to a specified sampling position (Bernard-Dagan et crl. 1971; Blight and McDonald 1964). Therefore, it is often possible to ascertain from a visual inspection of the peaks on such a trace whether twins are identical or not, even before statistical procedures are applied.

To test whether or not ally set of twins was identical. a balanced three-way analysis of variance was used, involving fixed effects, twins, peaks. and sampling dates. In some sets minor peaks appeared 011 isolated occasions, and such pealts were excluded from consideration. Because all peaks were expressed as a percentage of the total monoterpenes present, the differences between twins and between sampling dates, and the twins X sampling dates interaction, were 11~111 effects. Any difference be- tween twins was reflected in the statistical twins X peaks interaction while any difference in composition between assessments was reflected in the pealts X sampling dates interaction. Because only one sample was taken from each tree on each date, all effects had to be tested against

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the twins X peaks X sampling dates interaction mean square. In this mean square, any true interaction is con- founded with biological sampling error, and with the small amount of analytical error.

It proved appropriate to transform percentages to arcsin values, and when this was done the sums of peak values varied slightly. The result was that small sums of squares were obtained for the null effects, so these were pooled with the accepted error sum of squares without altering the degrees of freedom of the latter. Despite the likelihood of some bias in this statistical analysis, con- clusions could be drawn with confidence if tests were suficiently clear-cut.

Results and Discussion

Based on the methods described above, the evaluatio~i of the status of sets of twins is sum- marized in Table 1. The categorizing of twins is conservative. Reservations remain about accept- ing P = 0.27 as indicati~ig identical status, since the estimate of error could be inflated. With the "probably non-identical" twins the difference may have only represented environmental effects, even though the twins within a set grew next to each other. With two of the three "non-identical" sets, quite large differences were evident in par- ticular peaks, suggesting appreciable single-gene effects. Even in these cases, the differences were small in relation to the usual range of tree-to-tree differences; this would be expected in view of the coliilnoil female gametoplzyte genotypes. It is possible that non-identical twins may lack

detectable differences in monoterpene composi- tion, but such a coincidence is very unlikely to occur in 6 out of the 1 1 sets that were analyzed.

Differences between peaks were always very large, some components never being present in large amounts. In addition, the peaks X samp- ling dates interaction was generally very highly significant (P < 0.001), indicating differences in monoterpene composition between sampling dates. However, the significance generally re- flected numerous degrees of freedom rather than very large differences. With these juvenile seed- lings, more variation of uncertain cause was encountered than had been expected from pilot studies with adult clones. Further work is planned to minimize such variation.

The results indicate that genetically identical and lion-identical seedling twins can occur in P. racli~zln, and therefore that twins do result from both cleavage and archego~iial polyembry- ony. The results also illustrate moiloterpene analysis as a promising technique for establishing the genetic identity of individuals. Since mono- terpenes abound in all conifers and are easily analyzed, the applications of the technique could be widespread.

Acknowledgments

Thanks are due to C. B. Low, P. Allan, and Miss D. Blake for technical assistance: to Miss

TABLE 1

Status of sets of twins. The probability of twins being identical as determined from morphological characteristics, by inspection of the g.1.c. traces, and as calculated

from analysis of variance

Analysis of variance Mor1~11010gical G.1.c. (probability values

Twins traits inspection In parentheses) -

TI and T2 T3 and T4 T5 ant1 T6 T7 and T8 T9 and TI0 Ti l i n d T i 2 T13 and T14 T l5 and TI6 T17 and T18 T19 and T20 T21 and T22 T23 and T24 Identical T25 and T26 Non-identical

Total Identical 6 Non-identical 4

- - -

Inconclusive" Inconclusive'" Identical Inconclusive" Non-identical Inconclusive'' Non-identical Identical Inconclusive* Identical Identical

- - -

Identical (0.92) Prob. non-identical (0.02) Identical (0.86) Prob. non-identical (0.04) Non-identical ( <0.001) Prob. identical (0.27) Non-identical (<0.001) Identical (0.96) Non-identical (<O.OOl) Identical (0.69) Identical (0.65)

Probably identical 1 Probably non-identical 2

'Tlie "inconclusive" verdict was applied if inspection failed to give a clear-cut identical or non-iden- tical identification.

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S. Horne for the computations; and to G. B. MLROV, N. T. 1961. Composition of gun1 turpentines of pines. U.S. Dep. Agric. Forest Serv. Tech. Bull.

Sweet and F. Sorensen for criticizing the No. 1239. manuscript.

BEKN,\HD-D,\G~N, C., C. FILLON, G. P,\uLY, PH. BI\~.\~):\.r. i111d G. ILLY. 197 1. Teraenes of Piir~rs pillrrsrc~r:' biological and genetic characteristics. Ann. Sci. For. 28: 223-258.

BLIGHT, M. M., and I. R. C. MCDONALD. 1964. Sample reproducibility in Piirrrs essential oils. N.Z. J. Sci. 7: 2 12-220.

BR,\A,II.E.I.T. D. L., and T. W. POPHAM. 1971. Model relating unso~lnd seed and embryonic lethal alleles in self-pollinated pines. Silvae Genet. 20: 192-194.

C I \ ~ ~ ~ ~ ~ , J . H., and R. M. WALDKON. 1965. M~~lt ip le jack pine seedlings. Can. J. Bot. 43: 481482.

CHING. K.. ,and M. SIMAK. 1971. Competition among embryos In polyembryonic seeds of Pii1lr.s .sylvc.srris L. and Picerr abies (L.) Karst. Department of Re- fo~.estalion, Royal College of Forestry, Stockholnl. Kcsearch Note No. 30.

Onn-EWING, A. L. 1956. An investigation into the efiecls of self-pollinstion on Pserrdotslrga i~leilziesii (Mirb.) Franco. Ph.D. Thesis, University of British Columbia, Vanco~~ver , British Columbia. (Cited by Sorensen, p. 119.)

SARVAS, R. 1968. Investigations on the flowering and seed crop of Picecr nbies. Commun. Inst. For. Fenn. 67: 1-84.

SMITH, R. H., and L. E. GREEN. 1971. Use of peak heights from a fast column for rapid approsinlation of the normalised composition of pine resin. J. Chromatogr. Sci. 9: 755-756.

SOHENSEN, F. 1971. Estimate of self-fertility in Douglas fir from inbreeding studies. Silvae Genet. 20: 115- 120.

SWAIN, T. 1963. Chemical plant taxonomy. Academic Press. London and New York.

A bioassay for systemic fungicides using axenic cultures of the carnation rust fungus, Uromyces dianthi

D. R. JONES' Depcrrtineirt of Biology, Uirive,:sir)~ of Keele, Iceele, S~nfforrlslrire S T 5 5BG, Ei~gI(rircl

Received March 5, 1973

JONES, D. R. 1973. A bioassay for systemic fungicides using axenic cultures of the carnation rust fungus, Uroi~7yce.s dicrilrlri. Can. J . Bot. 51: 2004-2006.

The inhibitory action of three systemic f~~ngicides (Benlate, Hoe 6053, and Milstem) on the growth of axenic cultures of the carnation rust fungus, Uroin13ces rlinilrlri, was assessed. The rust- and smut-specific fungicide (Hoe 6053) was found to inhibit when present in the agar medium in very low con- centrations, while moderate concentrations of the wide range fi~ngicide (Benlate), and high concentra- tions of the powdery mildew-specific fungicide (Milstem) were needed for inhibition. The results indicated the possible use of U. tlinirrlri in future in vitro flngicide screening programs and mode-of-action studies.

JONES: D. R. 1973. A bioassay for systemic fungicides sing axenic cultures of the carnation rust fungus, Uroltiyces cliairrlri. Can. J. Bot. 51: 20042006,

L'auteur a mesurt I'action inhibitrice de trois fongicides systtmatiques (Benlate, Hoe 6053 et Milstem) sur la croissa~lce de cultures axiniques de la ro~~i l l e de l'oeillet, Uroinvces cIi(rirr11i. Le fongicide spicifique aux rouilles et aux charbons (Hoe 6053) s'est avtrt capable d'inlliber ia croissa~lce lorsqu'utilist en milieu gilost i~ des concentrations trEs faibles, alors que des concentrations modire~nent tlevies du fongicide no11 spicifique (Benlate) et de fortes concentrations du fongicide spicifique pour le mildiou poudreux (Milstem) Ctaient necessaires pour obtenir une inhibition. Les rtsultats ont ~n i s en evidence la possibilitk d'utiliser U. clioilrlri p o ~ ~ r de futurs programmes de dttection du potentiel et du mode d'action des fongicides. [Traduit par le journal]

Introduction temic properties of fungicides, in vitro tests are ~ l ~ h ~ ~ ~ l , illtense ill viva screening programs still useful, especially in mode of action studies.

are generally llecessary to investigate the sys- This in the past has proved difficult with obligate - parasites, as changes in their metabolism have

'Present address: Agriculture Canada, Research Insti- been almost impossible to distinguish from those tute, University Sub Post Office, London. Ontario. N6A 3K0, Canada. of their hosts. In vitro experiments also give a

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