MORPHOLOGICAL DIFFERENTIATION IN CHROMOSOMES OF DATURA STRAMONIUM

THE ACETO-CARMINE method adopted since 1920 in this laboratory for research on Datura has been successfully applied in a large number of problems (1). Using this quick and easy technique by which P.M.C., pollen and root-tips become both fixed and stained, it was found that the twelve chromosomes forming the karyotype in Datura differ considerably in length. Belling arranged the twelve meiotic chromosomes as seen in P.M.C. into six classes according to their length (2). Later work led to a slight modification of the number of chromosomes in two of the classes. Our present formula is one extra large (L), two large (1), five large medium (M), two small medium (m), one small (S) and one extra small (s) chromosomes. A numerical system was adopted in 1930 which involved giving a number to each of the two ends of the twelve chromosomes, beginning with the largest (L) which was designated 1-2. The second largest is 3-4 and so on until the smallest (s) is 23 24. A secondary chromosome, which consists of a doubled half chromosome, has the same number for each end. Thus 1 1 and 2-2 are secondaries of the 1-2 chromosome, 3 3 of the 3 4 chromosome and so on to include the 19 19 secondary of the 19 20 chromosome. Only four chromosomes (1 2, 5-6, 910 and 1314) have so far given both complementary secondaries and for the smallest two (21-22 and 23 24) no secondaries are known (5). In P.M.C., terminal humps were found at one end of eight of the twelve Line 1 chromosomes. At that time the suggestion was m.ade that these humps might be the equivalents of satellites or knobs which Lewitzky had described from mitotic divisions in root-tips (4). Chromosomes, when fixed and stained with acetocarmine, do not show any detailed morphological differences, such as Navashin (10), Sakamura (11), Taylor (12), Heitz (6) and many others have demonstrated for mitotic chromosomes by the use of other techniques. If properly fixed and stained, the chromosomes of many plants can be recognized individually by certain constant characteristics such as the location of the insertion region, or primary constriction, which divides the chromosome into two arms of equal or unequal size; the secondary constriction which separates satellites from the rest of the chromosome, other achromatic regions, and the relative size of the individual chromosomes, etc. These permit one to recognize and to label chromosomes in many plants. That such differentiation in chromosomes is also present in Datura was shown by Lewitzky, who comments on the "uncommon variety and fineness of their morphological differentiation" (9). He found that in root-tip cells certain chromosomes of the karyotype are almost equal armed, others are divided by the insertion region into two obviously un1 Received for publication January 27, 1941. equal arms. Some chromosomes have satellites attached to the end of one of their arms, and an achromatic region separating an appendage from the rest of the body was found in one of the large chromosomes. Lewitzky also measured the length of the chromosomes and his results correspond to those obtained by Belling, although the latter based the measurements on meiotic chromosomes (3, 9). TECHNIQUE.-Lewitzky thus made an important contribution to our knowledge of Datura chromosomes. But it is only when all the necessary chromosomal testers are at hand, such as haploid plants, primary and secondary 2n+1 forms, prime types (PT's) and other chromosomal types, that each chromosome can be identified in the karyotype from its size and differentiation. Their lengths and differentiation were studied at the metaphase stage of mitosis in haploid root-tip cells and in young pollen grains, where the number of chromosomes is also haploid. Root-tips were fixed in Lewitzky's solution (10 per cent formalin, 4 parts; 1 per cent chromic acid, 5 parts). This proportion was recommended by Lewitzky for Datura (9). Sections 5 , in thickness were stained with iron haematoxylin. Division occurs in young pollen grains when the flower buds are 2-3 cm. long. Anthers were smeared on slides, fixed in Allen's B-15 solution and stained with crystal violet and orange G. according to Newton's method (8). The Feulgen reaction was also used in some cases. METHODS OF STUDY.-The drawings of chromosomes at the metaphase stage shown in figure 1 were made from a cell in a haploid root-tip. For clarity, they were drawn somewhat more separated than they occurred in the cell, but their relative positions were not disturbed. Each chromosome is divided at the insertion region into two unequal arms. In some chromosomes the difference in the length of the arms is not as marked as in others (chromosomes 12 or 5 6 as compared with 7-8 or 910). Seven of the twelve chromosomes have a secondary constriction and six of these bear satellites at the end of one of the arms. In this figure some satellites are split (910, 19 20), others are undivided (7 8, 11 12, 21 22, 23 24). The seventh chromosome with a secondary constriction (3 4) has a large achromatic region which separates an appendage from the rest of the arm. This chromosome, when fixed and stained in the pollen grain by other methods, looks different (fig. 2, 3) in that there is no achromatic region and the appendage has a distinct connection with the arm. It differs from the satellites by having the same diameter as the chromosome and by the absence of a connecting thread. In another chromosome (13 14) the arms are very unequal in size and joined by a thin chromatic region so that near the insertion point a piece of chromosome seems to be missing. Further compari-