Rick, C. M.
Publication of the latest summary of our work with the trisomics (Genetics 39:640-666, 1954) did not include certain aspects of our routine, which might be of interest and aid to others who might wish to apply the trisomic method for locating genes.
In respect to methods of handling generations, we have found it most expedient to grew the trisomic material only during the main growing season. With greenhouse cultures during the winter season the strong morphological differences between the trisomics and diploids -- one of the most useful features of the tomato trisomics--- largely disappear. To be sure, trisomic segregates can be identified by chromosome counts, and in our "eager" early experience we grew F1's in the greenhouse and made counts of all material, but the shortening of the time required to grow the necessary generations scarcely seemed to justify all the additional time and effort invested in counting chromosomes. Also, the slower growth rate of some of the trisomics makes it somewhat more difficult to squeeze two generations into one year. By analyzing all of the material in the summer it has been possible to identify the trisomic segregates by their phenotype in nearly all lines, whether in pure stocks, F1 hybrids, or even in widely segregating F2's.
As to hybridization technique, it has been our experience that crosses with most of the trisomics can be made reliably without emasculation. Triplo-4, and especially 8, which tend to self naturally, should be emasculated, but with the rest, heavy pollinations of nearly opened or freshly opened flower buds result in a very high proportion of hybrid progeny. Our attitude in this matter is probably colored to some extent by the fact that, since our trisomic stocks are of San Marzano type and the genetic tester stocks with which they are hybridized usually in a background of one of the larger round-fruited tomatoes, hybrids can be readily distinguished from selfs.
For various reasons we are moving more and more to the summer greenhouse for making controlled pollinations. The benefits from this change were first realized when the very unsatisfactory behavior of triplo-1 forced us to carry out crosses in the greenhouse. Another advantage of the greenhouse is the freedom from insect pollination. We often plant the families in the field at the regular time, and after the trisomic types can be readily identified, move them back into the greenhouse where they are grown for the remainder of the season in 4 gallon containers. Under summer conditions at Davis it is necessary to provide heavy shade and to use humidifying coolers. Even under optimum conditions, some of the trisomics, notably triplo-2 ard 12, are reluctant to set fruit and these should consequently be pollinated on a grand scale. The use of Twist-Em strips or other materials (Science 106:645, 1947) has saved us lots of time in marking the pollinated flowers. Growth regulating substances as aids for setting fruit are used sparingly since we have found that these tend to induce much more parthenocarpy in trisomics than in diploids.
A shortcut that we have adopted is concerned with elimination of undesired diploids. In stock cultures and F1 hybrids, the only plants that are wanted are the trisomics. Since the rates of transmission are low for many of the trisomics, elimination of most or all of the diploids before the time of transplanting means more effective utilization of field or greenhouse space. Later emergence and slower growth are characteristic of the trisomics, but additional characteristics, enumerated below, can assist in identifying and eliminating most of the diploid segregates.
Compar- Color
Triplo- ative of true Shape of true leaves and cotyledons
size leaves
1 1/4 Very narrow, distorted and convex.
Most distinct of all trisomic
seedlings.
2 1/2 Pale Fewer and more elongate segments.
3 1/3 Pale Very elongate cot.; true leaves more
incised and more elongate.
4 1/2 Pale Less dissected than normal.
5 1/2 Normal First leaves diminutive and appear
whorled; very distinct stunted
appearance.
6 1/2 Pale Elongate segments.
7 2/3 Very Slender; granular surface on 4th and
pale younger leaves.
8 1/2 Pale Broad.
9 1/2 Dark Same general shape but fewer
segments.
10 1/3 Dark Deeper incisions, larger sinuses,
rougher surface.
11 1/2 Pale Very broad segments; very distinct.
12 1/3 Dark First two leaves remain very small;
very slow seedling.
One additional practical consideration deals with fertility of the genetic
tester stocks. Since the rate of transmission of the extra chromosome is very
low through pollen, it is much more efficient to use the trisomics as female
parents in making crosses. As a result the method is limited to the location
of genes that do not interfere severely with pollen production, unless the
mutant heterozygote were used as the male parent, in which case about seven
times as many progenies would be needed as in the use of a homozygous parent.