125 A TWIN SEEDLING IN ZEA MAYS L. TWINNING IN THE GRAMINEAE i BY B. C. SHARMAN Department of Botany, University of Leeds (With Plate i and i figure in the text) Amongst some maize seedlings grown for class use, one was observed to have two embryos emerging from a single caryopsis (PI. i A, B). Because its appearance immediately caused speculation as to the exact relationship of the embryos to each other, it was pickled and sectioned (Text-fig, i A-J). The two embryos appear to be completely separate except for the scutellum, which offers an interesting condition. At the top and bottom (Text-fig, i A, B, C and J) it is completely separated into two parts, each being quite normal in appearance. In the region of the union of the epicotyl (Text-fig, i D, E, F and G) it is entire but indented, the epidermis being infolded to a considerable degree. At one point (Text-fig, i H) there is absolutely no sign of any bisection. From the section illustrated in J in Text-fig, i, it appears that both radicles were originally enclosed in a single coleorhiza. Although the right-hand embryo is placed a little lower in the caryopsis (PI. i A, and Text-fig, i A, E and G) the two appear to have been amazingly evenly balanced in their development and subsequent germination, and to have proved very well-matched rivals for the food supplies. Even as late in development as when photographed (PI. i A) they were still practically mirror images. This even development, the way in which the scutellum seems to have arisen as an essentially single unit and the obvious singleness of the endosperm, testa and pericarp, suggest that the twinning arose early in the life history, probably being caused by longitudinal division or constriction of the more or less spherical mass of cells constituting the 'pro-embryo' stage of Soueges (1924). There appears to be only a very limited literature bearing on the structure of twin seedlings in grasses. Kempton (1913) deals with a number of maize caryopses which have obviously arisen from two ovaries showing various degrees of 'fusion'. In extreme cases he shows examples of caryopses bearing embryos 'back to back' so that a germ appears on both sides of the grain. These have come from a single spikelet having two flowers, one of which is normally aborted but can, as on this occasion, be fertile. He thinks of his grains as resulting from the fusion of the two ovaries after fertilization, but his illustrations, even of the mature caryopses, would suggest that the union, if such it ever is, has arisen much earlier. He concluded, however, that ' the development of the two ovaries in one spikelet must be simultaneous, as a large number of cases have been found where the two seeds from one spikelet have grown together with a single pericarp. These connate seeds had been fertilized through a double silk which was attached to the pericarp near the union of the two seeds. Connate seeds are a distinct phenomenon from single seeds with
R.L..L. E.R. Text-fig. I. Transverse sections through the maize twin shown in PI. i A. A, above the union of the two embryos; J, just below the departure of the two radicles. C, coleorhiza; E, endosperm; /, indentation of scutellum; P, pericarp; E.L., E.R. epicotyls of left and right embryos; S.L., S.R. left and right portions of the scutellum; R.L., R.R. radictes of left and right embryos.
A twin seedling in Zea Mays L. Twinning in the Gramineae 127 a double embryo, two of which have been seen.' One of his photographs shows a caryopsis with two embryos growing out side by side in the same manner as the seedling described above. His example, however, is very deformed (probably through the way the caryopsis was lying when it germinated), and while left in the greenhouse to develop further, was eaten by a pest before it could be properly examined." Blaringhem (1920) described a strain of maize (called by him Zea Mays var. polysperma) in which many double and sometimes triple grains developed. Stratton (1923) examined this variety developmentally and showed that the double grains arose from two 'coalesced' flowers, the two flowers ofthe spikelet being fertile and developing, from the inception, in various degrees of 'fusion'. She describes connate and semi-connate grains, the former being kernels with the two embryos on opposite sides of the grain, arranged back to back and enclosed in a common pericarp formed from the coalesced ovaries of the two flowers. She states that in all the cases she examined the connate grains possessed two stigmas, which, however, were often close together and could only be seen as two entities with the aid of a lens. The semi-connate grains showed various degrees of fusion and were 'caryopses with the two seeds less coalesced than in the connate type, the pericarp extending more or less between them'. Jenkin (1931), when discussing grass breeding, remarks that twin seedlings occur frequently, and warns the breeder to watch for them, since the two plants arising have different characters; this suggests that he is dealing with a different type of twin from that described for Zea. Bledsoe (1929) describes double and triple kernels in florets of rye and wheat crosses, but in all cases his appear to be multiple caryopses arising from multiple ovaries, some -of which are shown from dissections of flowers. Nishimura (1922-3) states that in Poa pratensis and Agrostis alba numerous examples were found 'where two embryos appeared. These embryos were more commonly placed side by side but in some instances one of the embryos would occupy an oblique position to the median plane of the grain.' He also observed a number of triplets in Poa pratensis, one of which he illustrates: here each embryo apparently was quite normal and possessed its own scutellum. For Poa pratensis he describes the following seedlings: (i) two plumules with only one radicle; (2) two plumules in a single coleoptile with two radicles in a single coleorhiza (illustrated); (3) two plumules each with its own coleoptile, radicle and coleorhiza. In Poa he suggests that the polyembryony is connected with the massive suspensor and illustrates sections showing bud-like outgrowths on this as well as from the nucellus. He also states that in a seedling of corn (i.e. Zea) he observed a germinating caryopsis with ' two normal embryos, each with its coleoptile, scutellum, coleorhiza and root' (the italics are not in the original). By far the most signiflcant reference to twin grass embryos is that of Randolph (1936) who, in discussing the development of the maize embryo, states: 'Another type of anomalous kernel development is represented by paired or twin embryos arranged side by side in an otherwise normal kernel. Such grains are found not infrequently in different stocks of maize, and the normal frequency of occurrence
128 B. C. SHARMAN of this type of twinning is markedly increased by X-ray treatment. Since the pericarp and endosperm of these grains show no evidence of doubleness, the twin embryos presumably arise from a single embryo sac. Twin embryo sacs would be expected to produce twin endosperms as well as twin embryos. Furthermore, the twin plants produced by these kernels ordinarily are genetically identical, even in extremely heterozygous stocks, which indicates that they arise from a single zygote, presumably by a division of the entire embryo or of that portion of it from which the plant develops, at some relatively early stage in ontogeny." Seedlings with two plumules and a single primary seminal root undoubtedly owe their origin to an incomplete separation of the embryo into two parts, a separation which involves only the portion which forms the plumule meristem.' Four other fairly recent papers dealing with twinning in grass seedlings were traced, but all, however, are concerned with the occurrence of haploid, triploid and tetraploid plants amongst such pairs, and in none are there any observations on the anatomy of the caryopses from which the seedlings have arisen, so that the origin of the two embryos must remain obscure. Ramih, Parthasarthi & Ramanujam (1933) found a large number of twin seedlings in Orysa sativa. In one pure line the twin seedlings were as high as o-i %. One plant was found to be a haploid, all the rest being normal diploids. Namikawa & Kawakami (1934) found that twin seedlings occurred at the rate of about 0-05 % in common wheat. Of the twin plants, about a third had one of the twins with abnormal chromosome numbers. Miintzing (1937, 1938) reported twin seedlings from fourteen species of Gramineae as well as from Trifolium repens and Solanum tuberosum. Out of 2189 twin seedlings examined, ten had abnormal chromosome numbers in one of the twins. In two sets of Lolium perenne twins, both the embryos had the3w chromosome number. In Phleum pratense, he mentions two sets of triplets, all the embryos of which had 2w chromosome numbers. Unfortunately, these papers were not seen until after the maize twin had been fixed, which was done in a vacuome fixative. Attempts were made to observe the chromosomes in the root tips, but with no success. However, the shoot apices were sectioned, and here the nuclei, the cells and the whole apices were similar to each other in size, and were of the same dimensions as those observed in normal shoots. These observations, together with the obvious equality of the two embryos, would suggest that both were identical and were normal plants with the normal 2«number of chromosomes. SUMMARY The anatomy of a twin seedling of Zea Mays is described. Both embryos probably had the normal chromosome complement and appear to have arisen as the result of a constriction or partial longitudinal division of the early pro-embryo stage, giving two complete embryos attached to a single, partially divided scutellum and enclosed in a single caryopsis with a common mass of endosperm.
PLATE I A A. The twin maize seedlings. B. Part of A enlarged, showing tbe single caryopsis witb the two emerging embryos. E.L., R.L. epicotyl and radicle of left embryo; E.R., R.R. epicotyl and radicle of right embr>'o; C, split single coleorbiza. B SHARMAN A TWIN SEEDLING IN ZEA MA YS L. TWINNING IN THE GRAMINEAE
A twin seedling in Zea Mays L. Twinning in the Gramineae 129 REFERENCES BLARINGHEM, L. (1920). Production par traumatisme d'une forme nouvelle de mais a caryopses multiples, Zea Mays var. polysperma. C.R. Acad. Sci., Paris, 170, 677. BLEDSOE, R. P. (1929). Multiple kernels in wheat-rye hybrids. Double and triple kernels in florets of rye and wheat-rye crosses, jf. Hered. 2O, 137. JENKIN, J. H. (193I). Method and technique of selection, breeding and strain building in grasses. Bull. Bur. PI. Genet., Aberystw., no. 3. KEMPTON, J. H. (1913). Floral abnormalities in maize. Bull. U.S. Bur. PI. Ind. no. 278. MuNTZiNG, A. (1937). Polyploidy from twin seedlings. Cytologia, Tokyo, p. 211. MuNTZiNG, A. (1938). Notes on heteroploid twin plants from 11 genera. Hereditas, Lund, 24, 487. NAMIKAWA, SIGESUKE & KAWAKAMI, JIHO (1934). On occurrence of haploid, triploid and tetraploid plants in twin seedlings of common wheat. Proc. Imp. Acad. Japan, 10 (10), 668. NiSHiMURA, M. (1922-3). Comparative morphology and development of Poa pratensis, Phleum pratense and Setaria italica. Jap. J. Bot. I, 55. RAMIH, K., PARTHASARTHI, N. & RAMANUJAM, S. (1933). Haploid plant in rice (Oryza sativa). Curr. Sci. I, 277., RANDOLPH, L. F. (1936). Developmental morphology ofthe caryopsis in maize. J. Agric. Res. 53, 881. SoufeGES, R. (1924). Embryog^nie des Gramin^es. D^veloppement de l'embryon chez le Poa annua L. C.R. Acad. Sci., Paris, 178, 860. - STRATTON, MILDRED E. (I>923). The morphology of the double kernel in Zea Mays var. polysperma. Mem. Cornell Agric. Exp. Sta. no. 69. New Phytol. 41, 2