Formation of Megachromosomes from Heterochromatic

A heterochromatic block (HB) from N. otophora occasionally undergoes great enlargement to form a “megachromosome” in hybrids and hybrid derivatives with N . tabacum. This paper shows that the two large HB’s of closely related N . tomentosiformis, and perhaps a smaller one, also have the same capability. The evidence that both large HB’s form megachromosomes is twofold. In a segregating backcross population from a parent possessing the two large HB’s, all segregants with one block produced megachromosomes at metaphase or large heterochromatic clumps at interphase. Second, those segregants which possessed both heterochromatic blocks produced megachromosomes of two visibly different types. Proliferation to make megachromosomes thus may not be the property of merely one particular segment but a more common property of heterochromatin in a hybrid or otherwise disturbed background. N previous papers (BURNS and GERSTEL 1969,1972; GERSTEL and BURNS 1966, I 1967, 1972) we have shown that the introduction of a particular heterochromatic block (HB) from Nicotiana otophora into N . tabacum leads to instabilities of two kinds, First, the alien HB undergoes breakage which causes chromatin loss; this can be observed cytologically and also phenotypically by the variegation it causes. Second, in some cells chromosomes with the HB from N . otophora proliferate enormously to many times their normal length, forming what we have called megachromosomes. In this paper we seek to determine whether heterochromatin from another species, N . tomentosiformis, can also enlarge. Furthermore, the question remains to be solved whether only one particular HB from a given species is involved in formation of megachromosomes, or whether this is a more general property of alien heterochromatin. HOEGERMAN (1969, 1972) demonstrated that at least three out of five HB’s from N . otophora generate megachromosomes; however, his evidence was indirect and based on rates of transmission in interspecific hybrids. MATERIALS AND METHODS Three species were used in the investigation: N . tabacum L. (2n = e), N . tomentosiformis Goodsp. (2n = 24) and N. sylvestris Speg. et Com. (2n = 24). The coral line of Red Russian tobacco served as the N . tabacum parent. There are no large blocks of heterochromatin in N. ‘Paper No. 4113 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. Genetics 75: 497-502 November, 1973. 498 J. A. BURNS A N D D. U. GERSTEL FIGURE 1 .-Late prophase cell from root tip of N. iomeniosiformis showing heterochromatin. Below, the three pairs with the largest blocks. (The black spot in the middle of the rightmost chromosome is due to an overlap). FIGURE 2.-“Extra-heterochromatin” (left top cell) compared at interphase with normal HB’s (below). FIGURE 3.--Somatic metaphase containing the two different HB-carrying chromosomes (arrows point a t centromeres). FIGURE 4 . 4 o m a t i c metaphase with two megachromosomes having short arms of differing lengths. (FIGURES 1-2 ~ 1 2 0 0 ; FIGURES 3-4 x1800). MEGACHROMOSOMES IN NICOTIANA 499 tubacum, but only scattered small knobs which can be seen clearly only at pachytene. The U.S.D.A. line of N . tomentosiformis which was used possesses a haploid complement with two large HB’s at the distal ends of their chromosomes and one medium-sized intercalary block. In addition, there are several very small HB’s or knobs. Figure 1 shows a late somatic prophase. At interphase the large HB’s are large enough to be visible as darkly stained bodies while the medium and smaller ones cannot be identified. The Catamarca race of N . syluestris which was used has no HB’s but only small knobs like N. tabacum. Thus, all the large HB’s encountered in the study were derived from N . tomentosiformis. The aim was to analyze the behavior of HB’s from N . tomentosiformis on a background with N . tubacum. Since N. tubacum is an allopolyploid and N. tomentosiformis a diploid, we circumvented the complications of aneuploidy by first synthesizing an amphidiploid from the ancestors of N . tabacun, viz. N. tomentosiformis and N. syluestris. The amphidiploid was crossed to N . tabucum to give a triple hybrid, N . tabacum-sylvestris-tomentosiformis. This triple hybrid is quite fertile, since the chromosomes of N . syluestris pair with those of one genome of N . tubacum and the N. tomentosiformis chromosomes pair with those of the other N . tubacum genome (GOODSPEED 1954). The triple hybrid was backcrossed to N . tabucum to derive the plants used in the investigation; in this generation the HB’s from N . tomentosiformis were separated by assortment. The details of the cytological methods used were described earlier (BURNS 1964.; BURNS and GFZSTEL 1969). All fixations were made of young petal tissue or root tips in a Carnoy mixture. Prophase and metaphase preparations were pretreated in 8-hydroxyquinoline, fixed, hydrolyzed in 10% concentrated HCl and stained with acetocarmine. This treatment made the heterochromatin visible at late prophase. Preparations for the study of interphase were neither pretreated nor hydrolyzed; nearly enhre, young and unfixed corollas were placed on slides in acetocarmine solution, heated, covered and then scanned under the mlcroscope. This permitted observation of very large numbers of cells. It was essential to find out which plants were capable of forming megachromosomes. At interphase these are visible as very large blocks of heterochromatin (“extra heterochromatin” = XHC; Figure 2). A piece of heterochromatin was scored as XHC if it was at least four times the size of a normal HB. As pointed out previously (GERSTEL and BURNS 1966) megachromosomes are not transmitted intact through cell division and are found only in widely scattered cells or groups of cells. In some plants it was sufficient to score a single corolla or part of a corolla to find five or more separate cells or small groups of cells with XHC, in which case the plant was diagnosed as XHC positive. In other plants more than one corolla had to be scanned before five separate cases of XHC were found. Negative diagnoses were based on at least three coroilasi.e. if three corollas showed no XHC the plant was judged unable to make megachromosomes. In two doubtful plants (see below) interphases of five corollas were scanned.