Original article

Cytogenetic study of diploidand spontaneous triploid oaks, Quercus robur L

AK Butorina

Department of Genetics and Bioecology Central Research Instituteof Forest Genetics and Breeding, Voronezh, Russia

Summary — Data are presented on the cytogenetics of 2 unusually large oak trees found in theVoronezh region of Russia. In both trees, cells with 2n = 3x = 36 chromosomes were predominant,with occasional observations of diploid hypoaneuploid and hyperaneuploid cells. Functionally, thetrees can be considered triploids, although in a strict sense, they are mixoploids. Meiosis in micro-sporogenesis of these trees is very disturbed and, as a consequence, pollen with unbalanced chro-mosome numbers are produced. Correspondingly, the progeny from each tree were very different inmorphological characteristics and cytogenetic constitution. These progeny can be used in genemapping studies and for other basic research purposes. Studies on some diploid oaks reveal thepresence of 2n pollen, formed by parallel spindles in the 2nd meiotic division. Methods for producingadditional oak triploids that have a potential for heterosis are discussed.

oak / triploid / mixoploid / meiosis / meiotic mutant / progeny

Résumé — Cytogénétique de chênes diploïdes et triploïdes spontanés (Quercus robur L).Des données cytogénétiques relatives à 2 chênes de très grande taille situés dans la région de Vo-ronezh (Russie) sont présentées dans cet article. Des cellules comprenant 2n = 3x = 36 chromo-somes sont prédominantes dans chaque arbre. Des cellules hypoaneuploïdes et hyperaneuploïdesont également été rencontrées. Au plan fonctionnel, les 2 arbres peuvent être considérés comme tri-ploïdes, plus précisément mixoploïdes. La méiose durant la microsporogenèse est très perturbée etproduit des grains de pollen au nombre de chromosomes déséquilibré. Les descendants de cesarbres manifestent des caractéristiques morphologiques et une constitution cytogénétique très va-riables. Ces familles peuvent être utilisées pour des études de cartographie génétique et d’autres re-cherches fondamentales. Des études similaires faites sur des chênes diploïdes mettent en évidencedes grains de pollen à 2n chromosomes formés par des fuseaux parallèles lors de la seconde divi-sion méiotique. Des méthodes de production de chênes triploïdes en vue de générer de l’hétérosissont discutées.

chêne / triploïde / mixoploïde / méiose / mutant méiotique / descendance

INTRODUCTION

The Voronezh region in south central Rus-sia is famous for oak stands producing ex-cellent quality lumber. Rich Voronezhchernozem provide optimum edaphicconditions for oak species. Two triploidtrees of Quercus robur L were discoveredin the Voronezh region by the scientific re-searchers of the Central Research Insti-tute of Forest Genetics and Breeding, VVlevlev and TI Pletmintseva. The trees weremore than 100 years old and differed fromoaks of a similar age. They were of gigan-tic height, weak fertility and exhibitedunusual morphological and anatomicalfeatures. These characteristics have oftenindicated a polyploid nature in many plantspecies.

In order to obtain objective informationabout the cytogenetic nature of these 2trees, we analyzed various cytologicalcharacteristics, including chromosome

number, meiosis in microsporogenesisand the development of the male gameto-phyte.

MATERIALS AND METHODS

For the cytological investigations, branches weretaken from each putative triploid and a number ofputative diploid trees at the appropriate samplingperiod for meiotic observations. The brancheswere placed into water vessels and kept in a coldroom. Somatic chromosome counts were made

using vegetative buds that were removed fromthe branches and placed in a damp penicillin bot-tle under freezing conditions for 1-2 h to inhibitspindle fiber formation. The young leaves werethen fixed in aceto alcohol.

For the study of meiosis in microsporogene-sis and the process of development of the malegametotype, the flower buds were fixed from thestage of green cone up to the flowering.

All materials were stained in acetohaemotac-silin. The squash technique was used to prepareslides for microscopical examination.

RESULTS

Cells containing 2n = 3x = 36 chromo-somes were prevalent in leaf meristematictissues in both trees, confirming the suspi-cions of a polyploid nature (fig 1a,b). In ad-

dition to the triploid cells, each tree hadmeristematic cells with other chromosomenumbers. Diploid, hypoaneuploid and hy-peraneuploids cells were found. Conse-

quently, these trees could be classified asmixoploids in a strict sense.

Meiosis in both trees was abnormal, astypically found in plants with unbalancedchromosome numbers. At metaphase I, abroad spectrum of chromosomal configura-tions, from 36 univalents to 12 trivalents,were observed. Lagging chromosomeswere frequent in anaphase I (fig 2a,b).Some chromosomes were delayed at theequatorial plate, while others were locatedoutside the achromatic spindle (fig 2b). Oc-casionally, metaphase plates were formedand only one stage was observed: meta-anaphase (fig 2c). In such instances, thedistribution of chromosomes in metaphaseII was unequal (fig 2d,e). In some micro-

sporocytes, aggregation of chromosomesinto separate groups was observed (fig2e).

The chromosomal disturbances in thesecond meiotic division were similar to ab-normalities in the first division (fig 2f). Cor-respondingly, many unbalanced micro-

spores were formed (fig 2g), that

subsequently resulted in pollen grains withdifferent chromosome numbers (fig 3a-c).

The number of cells with meiotic distur-bances in both oak trees varied over differ-ent years. The maximum percentage of ab-normal divisions was 98% of the totalnumber of dividing microsporocytes.

Although the 2 triploid oak trees hadmany common meiotic characteristics,they also had specific peculiarities. Cyto-mixis was observed in one tree (fig 3a).Preliminary divisions of the nuclei in telo-

phase II were found in the other tree, re-

sulting in unbalanced pollen (fig 3b). Themeiotic irregularities caused diversity in

pollen chromosome number and distur-bances in male gametophyte development

(fig 3d-g). The same anomalies may be ex-pected in the female gametophyte, sincethe progeny of these trees have variable

morphological characters (levlev and Plet-mintzeva, personal communication).

Meiosis in pollen mother cells of diploidoak trees was also investigated. This pro-cess was found to be essentially normal inthese trees, with approximately 5% of themicrosporocytes exhibiting abnormal divi-

sion. However, among these trees, severalindividuals were found to form 2n pollengrains, comprising 5-10% of the micro-

spores (fig 4a). The 2n pollen was found tobe formed by parallel spindles (sensu, Mokand Peloquin, 1975) in the second meioticdivision (fig 4b).

DISCUSSION

Chromosomal variation in progeny fromthe triploid trees can be the basis for fur-ther cytogenetic research. In particular,analyses of aneuploid offspring from thetriploid trees would be an excellent methodto genetically map oak chromosomes. Un-fortunately, only these 2 triploid oaks areknown to exist. Other mature triploids of

oak have not been discovered. A possiblecause may relate to dysgenic selectionthat was conducted in forests of this regionfor many years. It would be desirable to

study additional trees in order to gain abetter understanding of the mechanism(s)of origin for triploid oaks.

Other triploid Q robur have been ob-served in studies of twin seedlings byJohnsson (1946) and Burda and Schepo-tiev (1973). These scientists respectivelyspeculated that polyembryony could be re-sponsible for the triploid condition. Unfortu-nately, there have been no additional re-

ports on the triploid seedlings identified in

their studies. Our data suggests that trip-loid oaks may originate by participation of

2n gametes as found in other plant spe-cies, eg, Populus tremula.

The observed mechanisms of 2n pollenformation by parallel spindles ensure highheterozygosity in the resulting progeny.This type of pollen formation could be theresult of a meiotic mutant gene(s) as

shown in Solarum (Mok and Peloquin,1975) and Medicago (Vorsa and Bingham,1979). In these 2 crop species, the trip-loids formed in this manner expressed het-erosis due to the high heterozygous 2ngametes. Correspondingly, the meiotic

mutant oaks detected in this study or theuse of 2n pollen induced by thermoshock(cf Mashkina et al, 1989) could be used toproduce additional triploids that have a po-tential for heterosis resulting in increased

yield.Triploid oaks appear to occur rarely,

however, more thorough surveys need tobe conducted. More observations are

needed to determine the relative growthrates and development of triploid versus

diploid trees. Triploid seedlings may be ascompetitive as diploid seedlings in early

development, which is critical to establish-ment of oaks in forest setting. It is possiblethat triploid oak seedlings have some ab-normalities in the development of their rootsystem as in polyploid seedlings of pine(Isakov et al, 1977). When triploid seed-lings are detected, morphological observa-tions should be compared with those of

diploid seedlings for possible identificationof distinguishing features.

ACKNOWLEDGMENTS

The author expresses her gratitude to VV levlevand Tl Pletmintseva who selected triploid treesby their morphological characteristics and pro-vided materials for cytological analysis and toLS Muraya for her contribution to cytologicalstudy of the first triploid oak tree.

REFERENCES

Burda RP, Schepotiev FL (1973) Spontaneouspolyploids in germinative oak seeds (Quer-

cus robur L) Cytol Genet 7, 140-143 (in Rus-sian)

Isakov Yu N, Butorina AK, Muraya LS (1977)Genome mutants of Conifers. Third Meetingof the All-Union Society of Geneticists andBreeders named after Ni Vavilov, Collectionof reports 1, 196 (in Russian)

Johnsson H (1946) Chromosome numbers of

twin plants of Quercus robur and Fagus sil-vatica. Heriditas 32, 469-472

Mashkina OS, Burdaeva LM, Belozerova MM,V’yunova LN (1989) A method of inducingdiploid pollen in woody species. Lesovedenie1, 19-25 (in Russian)

Mok DWS, Peloquin SJ (1975) Three mecha-nisms of 2n pollen formation in diploid pota-toes. Can J Genet Cytol 17, 217-225

Vorsa N, Bingham ET (1979) Cytology of 2n pol-len formation in diploid alfalfa, Medicago sati-va. Can J Genet Cytol 21, 525-530