INHERITANCE OF BETA-CAROTENE IN TOMATOES’ RALPH E. LINCOLW AND JOHN W. PORTERa Purdue Agricdtural Experiment Station, Lafayefte, Indiana Received September 15, 1949 ETA-carotene is the principal vitamin A active carotenoid found in to- B mato fruits. This pigment, however, constitutes only about 5 percent of the total carotenes present in commercial red-fruited varieties. Almost all of the remaining 95 percent of carotene is lycopene. In spite of the relatively small percentage of beta-carotene occurring in red-fruited varieties, tomatoes are classified, nevertheless, as a “good” source of vitamin A for the human diet (HEINZ1942). In 1942 a study aimed at increasing the vitamin content of tomato selections by breeding was initiated at this station. Progress toward the production of varieties genetically constituted to produce high concentrations of provitamin A and ascorbic acid has been reported (LINCOLNet al. 1943, 1949; KOHLERet al. 1947). It also has been reported that selections were made during the course of this work in which beta-carotene constituted 95 percent of the total carotenes (KORLERet al. 1947). This paper reports the results of studies of the inheri- tance of beta-carotene in crosses between high lycopene (low beta-carotene) and high beta-carotene tomato selections. PREVIOUS LITERATURE The results of several studies on the inheritance of tomato flesh and skin color have been summarized by BOSWELL(1937). Most commercial varieties are red fleshed with yellow skin and therefore carry the dominant alleles RTY of yellow flesh (r),tangerine, orange flesh (t) and colorless skin (y). LE ROSEN et al. (1941) have shown that flesh and skin color are genetically and chemically unrelated. The R gene for red flesh color affects only the red and yellow plastid pigments, chiefly lycopene, but to a smaller extent other carotenes and xan- thophylls, while the Y gene for yellow skin has no effect on plastid pigments but causes a ten-fold increase in an alkali soluble epidermal pigment. Recently LESLEYand LESLEY(1947) presented evidence that three or more genes deter- mine the red-yellow color series in subgeneric crosses of Lycopersicon. In the initial paper, LINCOLNet al. (1943) reported obtaining a selection con- taining 67.5 y/ gm of beta-carotene or nine times as much as the content of the highest commercial variety. The same group, KOHLERet al., (1947), later re- ported individual plant fruits containing as much as 115 y/gm of beta-carotene (95 percent of total carotenes), while others of commercial fruit size contained Journal Paper No. 416 of the Purdue Agricultural Experiment Station, Lafayette, Indiana. This investigation was supported in part by a grant from the Nutrition Foundation, Ink. Formerly Associate Geneticist. Now Microbiologist, Camp Detrick, Frederick, Maryland. Formerly Associate Chemist. Now Group Head of Botany, General Electric Co. Richland, Washington. GENETICS35: 206 March 1950. BETA-CAROTENE IN TOMATOES 207 83 y/gm. LESLEYand LESLEY(1947) reported individual plants containing 75 and 90 percent of total fruit pigments as beta-carotene but did not give absolute quantities. METHOD Crosses to produce Fl seed were made in the greenhouse. FZseed was obtained byselfing Fl plants in the greenhouse. F3 seed was obtained from open-pollinated flowers of Fz plants grown in a field block. Genetic populations were grown, sampled and the fruits were analyzed chemically as described in detail in a previous publication (ZSCHEILEand POR- TER 1947). In brief, representative fruits were homogenized in a Waring Blendor, and a weighed aliquot was extracted with acetone and hexane (75:60). After filtration acetone was removed from the hexane by washing with HzO. Carotenols and esters were then removed from the hexane with 90 percent methanol and 20 percent KOH in methanol. The hexane solution was washed three times with water and then brought to a volume of 100 ml. Spectroscopic readings (for beta-carotene and lycopene determinations) were made directly on the resultant hexane solution with a Beckman spectrophotometer. Check values for beta-carotene content were obtained by chromatographically sepa- rating this pigment from other carotenes on a MgO-Super Cel column. The beta-carotene solution was then analyzed spectroscopically. RESULTS The high beta-carotene parent (4079-5012-9-13) used in these studies was an Fe single plant selection of the cross Indiana BaltimoreXFl (Rutgers XL. hirsutum P.I. 126445). The percentage of beta-carotene of 25 sib plants from which this plant was selected ranged from 93 to 98 percent of the total caro- tenes of the fruits. However, fruits of these plants varied from 54 to 11.5 r/gm in beta-carotene content. Cuttings were made of the desired plant which had a fruit size of 35 grams and contained 115 y/gm of beta-carotene which was 96 percent of the total carotenes present. The low beta-carotene parent was a single plant from an Indiana Baltimore seed stock. It had a fruit size of 180 grams and a carotene content of 112 y/gm, of which 6 percent was beta-carotene. Cuttings of the two parental plants were rooted and grown to maturity in the greenhouse. F1 seed was produced with the Indiana Baltimore selection as the seed parent. FZseed was obtained in the greenhouse from 8 F1 plants. Two fruits from each plant were used as the seed stock from which the FZpopulation was grown. Analyses were made of the parental, F1 and FZpopulations grown in the field in 1946. The results of these analyses expressed as percentage of total carotenes present as beta-carotene are given in figure 1. Chromatographic analyses showed that lycopene and beta-carotene were the only carotenes pres- ent in quantities greater than a trace in these populations. The average per- centage of beta-carotene in the high beta-carotene parent was 93, in the low beta-carotene parent, 10, and in the FI, 61. This would indicate that the factor (or factors) responsible for the synthesis of beta-carotene is an incompletely dominant one. The mean percentage of beta-carotene in the entire F2 popula- 208 RALPH E. LINCOLN AND JOHN W. PORTER tion was 55 percent, almost exactly the mean value of the two parents. There are three distinct peaks in the distribution curve of the 209 F2 plants analyzed. These peaks occur at 12, 55, and 88 percent of carotenes as beta-carotene and coincide almost exactly with the mid-point for the distribution in Indiana Baltimore, the F1 and the 4079-5012-9-13 population. The distribution of this curve fits the 1: 2: 1 ratio that would be expected if the genetic constitution of the two parents as regards factors for carotene synthesis differed by a single incompletely dominant gene. The distribution of observed and expected plants on the basis of 0-24.9 percent, 25-74.9 percent, and 75-100 percent beta-caro- PERCENTAGE OF BETA-CAROTENE .- . - . -. -. Indiana Baltimore ___________ FBpopulation .. .- . -. 4079-5012-9-13 .............FZ population FIGURE1. Distribution of results of analyses for percentage of befa-carotene for the parental, I', and Fz populations, of a cross between high and low beh-carotene parents-Fa Sel. 4079= 5012- 9 13 by Indiana Baltimore. tene is given in table 1. The observed values closely approach that expected on a 1: 2 :1 hypothesis. The x2 value for these data is 0.79 .The simple correla- tion between fruit weight and total carotenes was 0.137, between fruit weight and lycopene, 0.047, and between total carotenes and lycopene, 0.105. The multiple R for these characters was 0.151. It is apparent that in this cross no significant correlation exists between these characters. In 1947, 169 Fa progenies of 9 plants each were grown from seed of open- pollinated fruits. The progenies were classified visually into 39 orange-fruited progenies, similar to the high beta-carotene parent, 92 intermediate or se- gregating progenies, and 38 red-fruited progenies. This classification included BETA-CAROTENE IN TOMATOES 209 in the parent-like groupings those progenies that had one plant classified as intermediate with all other plants of the parental class. This was considered reasonable inasmuch as open-pollinated F3 seed was used. Unreported work has shown that although the average percentage of outcrossing may be only about 1 percent, individual fruits may have up to 100 percent of crossed seeds. The observations on the Fa population, as classified, again fit the hypothesis of a monofactorial, incompletely dominant gene for beta-carotene production. These data had a x2 value of 1.34. The above visual observations were checked with chemical analyses to de- termine the accuracy of visual classification. An ll percent discrepancy was TABLE1 Number of observed and expected plants in an F2 population with the designated percentage of beta-carotene and x2 value for the fit of these data to a 1 :2: 1 hypothesis. PERCENT OBSERVED EXPECTED X2 beta-CAROTENE NUWER NUMBER ~~ ~ 0- 24.9 57 52.25 0.24 2s- 74.9 105 104.50 0.02 75-100 47 52.25 0.53 Total 209 - 0.79 observed between the two classifications on 60 samples classified by both systems. The gene present in the 4079-5012-9-13 stock, which is characterized by its production of beta-carotene, has been designated B. It is incompletely epistatic to R. It is believed B functions in the formation of beta-carotene from lycopene and therefore the B gene requires the R gene to express its effect. An alter- native view would suggest that B is a member of an allelic series of genes for carotene formation. There is little evidence available at present to support this suggestion. There is no evidence from the breeding program with either Rutgers or Indiana Baltimore as recurrent varieties that the increased beta-carotene con- tent in the high beta-carotene selections is due to an increase in the total caro- tenes.