GENETIC AND PHYSIOLOGICAL STUDIES OF THE 'ALCOBACA' TOMATO RIPENING MUTANT BY MARIO LOBO A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1981 To the memory of my father, Manuel J. and to my mother, Ofelia; my wife, Yolanda and my children, Jorge Mario, Tatiana, and Juan Manuel. ACKNOWLEDGMENTS I wish to express appreciation to the members of my Supervisory Committee, Dr. L. C. Hannah, Chairman; Dr. M. J. Basset, Dr. C. B. Hall, Dr. D. D. Gull, and Dr. E. S. Horner, and former member, Dr. J. J. Augustine. Appreciation is also extended to the Rockefeller Founda- tion, for providing a graduate scholarship, to the Department of Vegetable Crops, University of Florida, to the Colombian government; and to the Instituto Colombiano Agropecuario (ICA) . Deepest gratitude goes to my lovely wife, Yolanda, for her understanding, encouragement, and patience, and to my children, Jorge Mario, Tatiana, and Juan Manuel, for the inspiration provided by their love and tenderness. in TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii ABSTRACT vi INTRODUCTION 1 LITERATURE REVIEW 3 Tomato Fruit Ripening 3 Ripening and Respiration 4 Ethylene and Ripening 7 Cell Wall Metabolism and Ripening 10 Softening and Hydrolytic Enzymes 12 Color and Ripening 15 Tomato Ripening Mutants 16 'Alcobaca' Tomato 23 MATERIALS AND METHODS 26 Pedigree of Studied Materials 26 Storage Life Studies 26 Color Determination 29 Firmness of the Fruits 30 Respiration Studies 31 Ethylene Evolution 32 Polygalacturonase Activity 32 Model To Relate Storage Life with Different Physiological Parameters 33 Genetic Studies 34 Inheritance Study 34 Functional and Recombinational Genetic Tests . 35 Inheritance of Storage Life 35 RESULTS 37 Storage Life Studies 37 Preliminary Observation 37 Storage Life of Commercial Varieties and 'Alcobaca' 39 Fruit Storage Life of Hybrids with Ripening Mutants 44 Fruit Storage Life of Hybrids Between 'Alcobaca' and the Ripening Mutants nor , rin , and rin-1 . 49 IV Page Respiration Study 55 Ethylene Evolution 55 PG Activity Study 63 Model To Relate Storage Life with Different Physiological Parameters » 70 Genetic Studies 70 Inheritance of the Abnormal Ripening ' Alcobaca ' ■ 70 Allelism Test Between 'Alcobaca' and rin .... 72 Allelism Test Between 'Alcobaca' and nor .... 72 Inheritance of Fruit Storage Life 79 DISCUSSION 83 Storage Life Indexes 83 Storage Life Studies 83 Respiration, Ethylene Evolution, and PG Activity . 87 Genetic Studies 92 SUMMARY AND CONCLUSIONS 97 LITERATURE CITED 99 BIOGRAPHICAL SKETCH 108 v Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GENETIC AND PHYSIOLOGICAL STUDIES OF THE 'ALCOBACA' TOMATO RIPENING MUTANT By Mario Lobo March 1981 Chairman: L. C. Hannah Major Department: Horticultural Science (Vegetable Crops) Genetic and physiological studies were conducted with the ripening tomato mutant 'Alcobaca'. A single recessive gene was found to be responsible for the abnormal ripening. This gene causes a ripening syndrome characterized by an attenuated climacteric pattern, delayed softening of the fruit, low polygalacturonase (PG) activity, and extended shelf life. When the mutant is heterozygous with the wild- type allele, these parameters of ripening are also affected although not to the extent observed in the homozygous con- dition. Thus the gene might be employed in the heterozygous condition to improve the shelf life of tomato. Allelism tests and examination of F^ progenies involv- ing the ripening mutants rin and nor showed that the mutant gene of 'Alcobaca' is allelic to nor . Here it is proposed A that the name nor be used to refer to the mutant allele of ' Alcobaca ' . vx The mutant allele nor^ is dominant to the standard recessive allele nor in that nor^ is climacteric. Also, A nor conditions higher levels of respiration, ethylene evolu- tion, and PG activity. These parameters are intermediate in A nor /nor heterozygotes. Thus there exist now three alleles at the nor locus. The wild- type allele conditions a normal climacteric and normal ripening. The norA allele has a climacteric but does not ripen normally. The recessive nor lacks a climacteric and normal ripening. These alleles should be useful in the study of regulatory mechanisms of ripening. Ethylene peak evolution and PG activity were re- duced to a greater extent in the double heterozygous genotype + A . + nor /nor , rin /rin than in single heterozygous genotypes nor / nor , indicating a gene dosage effect for these two mutants . Correlation studies of storage life and the different ripening parameters of normal and mutant ripening genotypes indicated that PG activity was the parameter that exhibited the most highly significant relationship to storage life. A quantitative genetic study of the fruit shelf life, employing as parents 'Alcobaca' and the normal ripening tomato 'Florida 1C', indicated that storage life fitted an additive-dominance model, and that the parents differed only at one locus. This, then, agrees with the more quali- tative study above in that 'Alcobaca' and 'Florida 1C' carry different alleles of nor. Vll INTRODUCTION The improvement of the tomato fruit storage life via different storage conditions has received considerable work. Low temperature (Tomkins 1963), controlled atmospheres (Eaves and Lockhart ( 1 9 6 6) Parsons et al. 1970, Salunkhe and Wu 1973), employment of ripening retardants or inhibitors (Babbit et al. 1973 , Kader ert al. 1973 , Dostal and Leopold 1967, Yang 1980), and use of plastic film wraps (Wu et al. 1972, Gilbert and Hening 1971) have been proposed to extend the shelf life of tomato fruit. Another possibility is genetic manipulation of the ripening process. The discovery of the tomato ripening mutants rin and and nor (Robinson and Tomes 1968, Tigchelaar et al. 1973) has been considered the cornerstone for possible genetic manipulation of the fruit storage life in tomato. 'Alcobaca', another ripening mutant was described pre- viously (Almeida 1961, Leal and Shimoya 1973). This material exhibits a very extended fruit shelf life (Almeida 1961, Leal and Mizubuti 1975, Leal and Shimoya 1973, Leal and Tabim 1974). However, there is a lack of information concerning the genetics and physiology of the abnormal ripening behavior of this material. These investigations were undertaken to study the storage life and some 1 2 physiological processes of fruit ripening in 'Alcobaca', in hybrids of this material with normal ripening tomatoes, and in hybrids between 'Alcobaca' and other ripening mutants. Also studies were done to elucidate the inheritance of the 'Alcobaca' abnormal fruit ripening and possible allelic relationships with another ripening mutants. LITERATURE REVIEW Tomato Fruit Ripening Tomato fruit ripening incolves a number of chemical and physical changes (Babbit et al. 1973, Hobson and Davies 1971) . The changes are highly synchronized as evidenced by the fact that respiration, ethylene evolution, carotene development, and flavor and textural changes occur in rapid succession during the relatively short period in which fruit ripens (Babbit et al. 1973, Khudairi 1972). There has been inconsistency in the literature in the use of the terms ma- turation and ripening. Kader and Morris (1976) stated that maturation occurs during the stage of development in which the fruit attains full growth. Most of the maturation pro- cesses occur on the plant, but they can continue after har- vest for partially mature fruits. Nagel, cited by Babbit et al . (1973) , defined a mature tomato as one that has reached nearly full external and internal development and has the capacity to ripen within a few days after harvest. Kader and Morris (1976) stated that ripening is a form of senes- cence that can occur on or off the plant, beginning when maturation is completed. Ripening, then, would include the many processes that make the fruit edible. 3 4 Hansen (1966) considered ripening to be a sequential phase in the life of the fruit. It requires induction in order to become active and, once ripening is induced, it proceeds according to a predeterminated pattern. Several investigators believe that the period of active ripening be- gins with the onset of the climateric rise (Biale 1960, Hansen 1966 ) . Hansen (1966), in an extensive review of postharvest physiology of fruits, stated that energy is required for ripening in contrast to an older view that the ripening pro- cesses were catabolic in nature. Tigchelaar et al. (1978a) reported that four clearly defined changes occur during normal ripening of tomato. These include a) chlorophyll degradation and carotenoid bio- synthesis, b) increased respiration and associated ethylene production, c) softening and associated increases in pecto- lytic enzyme activities, and d) seed maturation. In addi- tion, less well-defined changes in flavor, texture, and aroma are integral parts of the ripening process. Ripening and Respiration The modern study of the ripening processes stems from the investigation of the physiology of the apple fruit by Kidd and West in the early 1920s (Rhodes 1970). They found that after picking, the CC^ production of the fruit fell to a minimum value and then rose rapidly. Subsequently, the 5 respiration declined. They used the term climacteric to refer to this pattern of respiratory changes. This pattern of respiration is associated with the ripening of many other fruits. The respiratory rise was shown by Gustafson in 1920, cited by Rhodes (1970), for the tomato. Climacteric pat- tern of respiration on tomato has been described by a number of authors (Clendenning 1942, Biale 1950, Workman et a_l. 1957, Biale 1960) . Biale (1960) classified the fruits into two categories: climacteric and non-climacteric. In non-clamacteric fruits, changes in color and composition are not accompanied by a rise in ethylene or CC^ production. In climacteric fruits a large increase in respiration and ethylene production accom- panies ripening. Ethylene stimulates respiration and ripen- ing of mature unripe fruits, and once stimulated by exogenous or endogenous ethylene, climacteric fruits produce ethylene autocataly tically (Burg and Burg 1965) . Rhodes (1970) pointed out that it is difficult to deter- mine whether there is a distinct difference in the mechanism of ripening in the two types of fruits or whether events which occur slowly over a long period in the so-called non- climacteric types are merely telescoped in the climacteric fruits into a short dramatic period. Tigchelaar et al . (1978a) reported that the mechanisms by which ripening changes are initiated and synchronized remains largely un- known. The initiation of the respiratory rise and 6 associated ethylene are used to define the onset of ripen- ing for climacteric fruit such as tomato; and these dramatic changes are lacking and ripening is frequently protracted for non-climacteric fruit. Sacher (1973) postulated that considerable evidence in- dicates that some aspects of ripening are not dependent on the increase in respiration that occurs in climacteric fruits. Dostal and Leopold (1967) showed that ethylene stimulation of color development in tomatoes is prevented by gibberelic acid, but the stimulation of the respiratory climacteric was not affected. Quazi and Freebairn, cited by Sacher (1973), found that when tomato ripening was initiated with ethylene and the fruit given 2.5% , normal ripening took place in the absence of respiratory climacteric. Reid and Pratt, cited by Sacher (1973), suggested that the respiratory climacteric may be caused directly by the large amounts of ethylene produced during ripening rather than by the energy demands of other phenomena. This view was based on their demonstration that a respiratory rise comparable in magni- tude and duration to the climacteric occurred in the non- climacteric orange and potato tuber when each was treated with ethylene. They concluded that the critical difference between climacteric and non-climacteric fruits is in the amount of endogenous ethylene produced during ripening. 7 Ethylene and Ripening The role of ethylene in fruit ripening has been dis- cussed for a long time. There are two schools of thought concerning the role of ethylene in fruit maturation (Burg and Burg 1962) . The classic view of Kid and West (cited by Burg and Burg 1962) is that ethylene is a ripening hormone. Biale et al. , cited by Burg and Burg (1962), postulated that ethylene is a by-product of the ripening process. Burg and Burg (1965) reported data for several fruits, including tomato, which showed that ample ethylene accumu- lates before the onset of the climacteric. The quantity accumulated is larger than the threshold value for ripening. The rise in ethylene occurs at the onset of the climacteric rise, suggesting that ethylene might trigger ripening. When ripening is prevented by such conditions as low temperature, ethylene production is also inhibited (Galston and Davies 1970) , suggesting that ethylene is the natural fruit- ripening hormone. Abeles (1973) stated that there is no ex- ception to the fact that ethylene can promote ripening if the tissue is in a receptive state, although there is no reason to believe that ripening is controlled by ethylene alone . The onset of ripening is associated not only with a rise in the ability to biosynthesize ethylene, but also with a marked increase in ethylene responsiveness (Burg and Burg 1966). Work (1929) first demonstrated the relationship between maturity of fruit and rate of response to ethylene. Immature fruits show a slower response to ethylene than do mature fruit (Lyons and Pratt 1964 ) . Ethylene concentrations ranging from 100 to 2000 ppm are effective in stimulating fruit ripening (Harvey 1928, Saccher 1973). Lyons and Pratt (1964) stated that ethylene treatment induced a climacteric rise in respiration in tomato fruits of all ages and hastened or induced changes usually associated with ripening. Rosa (1925) first showed that gains of 5 to 8 days could be induced by ethylene in the ripening of mature green tomatoes. Similar data were pub- lished by Harvey and Prokosheva, and Babichev, cited by Pratt and Workman (1962). Pratt and Workmann (1962) found that ethylene treatment at 20°C caused mature-green tomato fruits to ripen early. The climacteric rise in respiration was induced immediately after initiation of ethylene treatments. The rise in respiration rate was more rapid and the maximum respiration rate was obtained sooner. In addition, the maximum rate of respiration was higher in ethylene-treated fruits then in control fruits. Similar results were re- ported by Hobson and Davies (1971), McGlasson (1970), Pratt and Workmann (1962), and Rhodes (1970). The role of ethylene in the biochemical processes occur- ring during fruit ripening has not been clearly established. Several hypotheses have been proposed to explain the action of ethylene. These include effects on enzymatic activities, 9 interaction with nucleic acids, effect on lipo protein membranes, and the formation of complexes with metallo- enzymes (McGlasson 1970) . Methionine was suggested as the precursor of ethylene since this amino acid is the biological precursor of ethylene in all higher plant tissues (Lieberman et al. 1966, Yang 1980) . Since ethylene production as well as the conversion of methionine to ethylene ceases in plant tissues placed in an anaerobic atmosphere, and the surge of ethylene produc- tion occurs upon return of the tissue to air, it was con- cluded that an intermediate accumulates during anaerobic in- cubation and is subsequently converted to ethylene upon exposure to oxygen (Burg 1962) . This intermediate was later identified as 1-amino-cyclopropane-l-carboxylic acid (ACC) (Yang 1980) . ACC content in preclimacteric fruits of avocado, banana, and tomato is quite low, but a massive increase occurred at the onset of rapid ethylene evolution, suggesting that the inablity of preclimacteric fruit tissue to produce ethylene is due to the lack of ability to form ACC (Hoffman and Yang, cited by Yang 1980) . Zobel (1973) indicated a divergence of mechanisms con- trolling ethylene synthesis in different tissues. The dia- geotropic mutant of tomato requires exogenous ethylene for normal vegetative growth and development, but fruit matura- tion and ripening and production of ethylene by flowers is normal. McGlasson et. al_. (1975b) found that aging leaf 10 segments of a non-climacteric mutant of tomato produced a normal pattern of ethylene evolution in contrast to the lack of increase in ethylene production during aging in whole fruits and only a very small rise in ethylene production in very old fruits of the mutant. Herner and Sink (1973) work- ing with the non-climacteric mutant tomato rin, found that ethylene production was induced by wounding the fruits. The response to wounding suggests that a) this stress, ethylene, was not produced through the same pathway of the climacteric or normal fruits, or b) cutting or wounding stimulated the synthesis of ethylene through the normal pathway, but exogenous ethylene is unable to do so in the mutant. Cell Wall Metabolism and Ripening Cell wall metabolism has recently been implicated in the regulation of the ripening process in tomato (Ng and Tigchelaar 1977, Tigchelaar and McGlasson 1977). An in- crease in soluble polyuronide has been well documented for a number of fruits and is generally attributed to hydrolysis of insoluble pec tic polysaccharides by polygalacturonase (Gross and Wallner 1979). Mohr and Stein, cited by Pressey (1974) , described the changes occurring in the tomato fruit parenchymatous outer pericarp. Enlargement of the cells during fruit develop- ment is accompanied by intercellular space enlargement and cell wall separation. The intercellular spaces are small 11 or nonexistent in very young fruits, but separation of cell walls begins within a week or two after fertilization. This separation starts at the intercellular spaces. The middle lamella surface next to the spaces is the last to separate. Increase in cell size and cell wall separation continues until the fruit approaches maturity. The tomato fruits at the edible stage are composed of senescing tissues. Further breakdown of the pectinaceous middle lamella occurs in mature pericarp tissue to the point where cells may be completely separated . Studying the degradation of cell wall polysaccharides. Gross and Wallner (1979) found that the only components to decline during tomato fruit ripening were galactose, arabi- nose and galacturonic acid. Isolated cell walls of ripen- ing fruit contained a water-soluble polyuronide, possibly a product of polygalacturonase action. The ripening-related decline in galactose and arabinose content appeared to be separated from polyuronide solubilization. This idea was supported by the fact that in the ripening mutant rin the loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. Further- more, Wallner and Bloom (1977) and Wallner and Walker (1975) observed that polygalacturonase was not capable of hydrolyz- ing the neutral sugars, which have been reported to lend rigidity and intracellular cohesion to cell walls (Knee 1974). Softening of the fruit during ripening was attri- buted to the solubilization of the pectic materials by 12 pectinases (Babbit et al. 1973) . Wallner and Walker (1975) reported that beta-1 , 3-glucanase , an enzyme capable of hydrolyzing the neutral sugars, was present in significant guantities in tomato fruits when polygalacturonase was largely absent. However, the major wall-softening modifica- tions occurred. Mattoo and Vickery (1977) postulated that changes in the degree to which enzymes are bound to menbranes may comprise one of the mechanisms by which the activities of the enzymes are controlled in tomato pericarp. Softening and Hydrolytic Enzymes Softening reflects chemical and physical changes in the cell walls (Kader and Morris 1976) . Early workers estab- lished that fruit ripening is accompanied by conversion of protopectin to soluble forms of pectin (Pressey 1974). Softening of the fruit during ripening is attributed to the solubilization of the pectin materials by pectinases (Babbit et al. 1973) . Polygalacturonase and pectinmethylesterase are the major hydrolases involved in fruit softening with cellulase as a secondary contributor (Kader and Morris 1976) . Gonzalez and Bretch (1978) suggested that two possible, related, mechan- isms of softening are present in tomatoes. The first one can be activated via exogenous ethylene and the second, which seems to operate without the effect of exogenous ethylene 13 occurs after normal tomatoes reach the turning stage of ripening . Softening is genetically controlled and affected by cultural practices, environmental conditions, picking stage, plant age and condition, cluster position in the plant, and incidence of diseases (Kader and Morris 1976) . Softening starts at the stylar end and proceeds toward the stem scar (Kader and Morris 1976) . El-Sayed et al . (1966) postulated that softening of fruit is controlled by a single major gene with complete dominance, but certain modifier genes might have an effect on whole fruit firmness. Hall (1964) found that firmness in tomato was affected by variety, harvest date, and the interaction variety-harvest date. Pectinesterase catalyzes the removal of the methyl ester groups of pectin (Pressey 1974). This enzyme is present in green tomatoes and increases about 4-fold during ripening (Kertesz 1938). A relationship between firmness and pectin- esterase activity was suggested by Hamson (1952a) . However, Hall and Denison (1960) and Hobson (1963) showed that there was no correlation between pectinesterase and softening. Pressey (1974) stated that attempts to identify the pectin- esterase role in fruit softening by measuring the enzymatic activity are of limited value because the enzyme is present at all stages of development. Evidence for the presence of isozymes of pectinesterase was presented (Pressey and 14 Avants, cited by Pressey (1974) stated that the presence of multiple forms of pectinesterase in tomatoes and variation in their levels during ripening might suggest specialized roles for the enzymes. Polygalacturonase (PG) acts to depolymerize or shorten the chain length of the esterified pectin (Ng 1976). Hobson (1964) found no PG activity in green tomato fruits. The activity rose exponentially to the time of the orange stage and increased further to the red stage. Enzyme activity continued to rise as fruit became overripe. Babbit et al. (1973) did not find PG activity in developing tomato fruits until after the fruit had initiated ripening. PG activity was detected at the onset of the climacteric and then in- creased rapidly. Increases of PG activity by a factor of 10 during tomato fruit ripening were found by McClendon et aJL. (1959). Hobson (1964) reported an increase in PG activity of up to 200 fold as the tomato fruits pass from the green to the red stage. McColloch et al. cited by Pressey (1974), were able to show a close relationship be- tween the level of red color and the level of PG activity in the tomato fruits. Very similar results were presented by Hobson (1964). Thus PG activity closely followed the normal sequence of coloration, suggesting that the enzyme is intimately connected with the ripening mechanism. PG activity in tomato is highest in the outer locule wall of the pericarp tissue, followed by the inner locule 15 walls and the placental tissue (Hobson 1964). No PG activity has been detected in the locular contents (Pressey 1974) . Reduced PG activity was found in areas of the tomato fruit affected with "blotchy ripening" (Hobson 1963) . A relation- ship between PG activity and firmness of fruits was demon- strated in tomatoes by Hobson (1965) . Color and Ripening The color of the mature tomato fruit can be largely attributed to its carotenoid pigments. These compounds increase markedly during ripening while chlorophyll is lost (Spurr 1976) . The common red tomato contains three pre- dominant carotenoids, i.e., lycopene, beta-carotene, and gamma-carotene (Tomes 1969, Harris and Spurr 1969). Lyco- pene accounts for about 90% of the carotenoid fraction (Harris and Spurr 1969) . McCollum (1955) showed that color depends upon the content of total carotenoids and the ratio lycopene to carotene. Rattan (1957) found that the stage of harvest modifies the final expression of color in the tomato fruits. Thus, fruit harvested at the mature-green stage did not attain as high color as fruits of other ripening stages. Khudairi (1972) affirmed that the process of color change in tomatoes during ripening is triggered by red light, followed by a chain of biochemical reactions within the color apparatus (chloroplast-chromoplast ) , which include 16 ethylene production, chlorophyll degradation, oxygen uptake, abcsicic acid synthesis, and the enzymatic transformation of carotenes to red color. Kirk and Tilvery-Basset , cited by Spurr (1976) , note that in the mature green fruit the plastids are chloroplasts , and as the fruit ripens, they are transformed into chromo- plas ts . Tomato Ripening Mutants The term ripening mutants has been used for single gene mutations with multiple effects on tomato ripening (Tigchelaar 1978). Before the discovery of the ripening mutants, a number of mutants that simply altered the ability to synthesize specific carotenoids were described (Tig- chelaar et al^. 1978a) , but the other ripening processes occurred normally (Mizrahi et al. 1976) . Three ripening mutants have been described: Never ripe (Nr), ripening inhibitor (rin) , and non-ripening (nor) . The Nr mutant was found by Harris, cited by Tigchelaar (1978) in a chimeral fruit. Part of the tissue was hetero- zygous for Nr. The Nr mutant is dominant and affects the intensity of fruit pigmentation (Rick 1956) . Polygalacturo- nase activity is reduced and, consequently, the softening rate (Hobson 1967). This gene does not totally inhibit ripening but rather retards the onset of ripening and at- tenuates the magnitude of specific ripening changes 17 (Tigchelaar et al. 1978a) . Hobson (1980) reported that Nr produced fruits with high acid and low sugar contents. The fruits exhibited a slow ripening and were very firm. Also there were very low activities of PG, phosphof ructokinase , and NADP-ma lie -enzyme in fully developed fruits. The low enzymatic activities offer an explanation for the weak climacteric respiration rise and slow ripening of Nr fruits (Hobson 1980) . The mutant rin, described by Robinson and Romes (1968) , was found as a spontaneous mutation in a line developed by H. M. Munger from a cross between 'Fireball' and his breeding line 54-149. Robinson and Tomes (1968) found that the gene affected color expression. The fruits were green when normal fruits turned red. The mutant fruit became yellow latter in development. Also, Robinson and Tomes (1968) found that the fruits were firmer when compared with normal fruits and that the storage life was extraordinarily in- creased . Herner and Sink (1973) and McGlasson et al. (1975a) reported that rin is a non-climacteric mutant. Herner and Sink (1973) classified the mutant as non-climacteric since a) it lacks a respiratory climacteric and a rise in ethylene production for up to 120 days from harvest, b) its response to exogenous ethylene resulted in enhanced respiratory activity only while ethylene was present, c) it showed repeated stimulations of C02 evolution by repeated ethylene 18 treatments, and d) it responded to the ethylene-analogue propylene where C02 production was stimulated but ethylene production was not. Herner and Sink (1973) concluded that the rin tomato mutant lacks the capacity for autocatalytic production of ethylene. Other characteristics exhibited by rin in relation to ripening include extended shelf life (Tigchelaar et al. 1978a) , normal pectinesterase activity (Buescher 1977) , reduced carotene content (Tigchelaar et al. 1978a) , reduced beta-carotene content (Tigchelaar et jil . 1978a), and trace amounts of lycopene (Tigchelaar et al. 1978a) . Poovaiah and Nukaya (1979) , who followed cell wall de- grading enzymes during development of normal and isogenic rin fruits, found no change in PG activity up to 100 days postanthesis in the rin mutant and that short-term ethylene treatment in rin increased cellulase activity but had no effect on PG activity. They suggested that the failure of rin fruits to ripen was related to their low PG activity dur- ing maturity. The fruit ripening mutant nor was described by Tigchelaar et al. (1973). It was found in an accession de- signated 'Italian Winter' obtained from W. A. Kerr, Vineland Station, Ontario, Canada. The mutant was classi- fied as a recessive gene non-allelic to rin. It exhibited the following characteristics: a) absence of softening during fruit maturation, b) high level of crack resis- tance, and c) abnormal carotene biosynthesis with orange 19 pericarp and reddish orange locular jelly and placental tis- sue . Ng and Tigchelaar (1977) stated that in many respects the nor mutant resembles rin. Both fail to undergo fruit softening or normal chlorophyll degradation, and both exhibit a prolonged storage life. Ng (1976) concluded that homozygous fruits of nor ex- hibit a ripening pattern typical of non-climacteric fruits. They lack a respiratory climacteric and concomitant rise in ethylene at a time when normal fruits undergo ripening. This condition persists up to 110 days after anthesis, twice the time required for normal fruits to mature and ripen. Color development occurs slowly over this period of time, and the fruit eventually attain a yellow appearance with a blush of red color at the blossom end. PG activity is present only in trace amounts in the fruit, and little softening occurs during what would constitute the ripening period of a normal fruit. Ng and Tigchelaar (1977) noted that phytoene, beta- carotene, and neurosporene were the major carotenes in fruits of this mutant, but in very mature fruits there was some lycopene development, although the content was less than 10% of normal fruits. In spite of the fact that the non-climacteric ripening mutants rin and nor have been reported to be recessive (Robinson and Tomes 1968, Tigchelaar et chL. 1973), it has been demonstrated that both mutants exhibit alteration of 20 several of the ripening processes in heterozygous condition (Buescher et al_. 1976, Tigchelaar et a_l. 1978a). Compared to the normal ripening parents, the hybrids have delayed softening, lower pectolytic enzyme activities, and decreased solubilization of pectic substances (Tigchelaar et al. 1978a) . Ng (1976) reported that ripening was delayed and shelf life was improved to a greater extent in nor heterozygotes than in similar rin heterozygotes. Similar results were found by Buescher et al. (1976) in that fruits produced from the nor heterozygotes displayed more than twice the storage life of the normal parent. Tigchelaar et. al. (1978b) showed that the mutants rin and nor each contributes additively as heterozygotes to sev- eral aspects of ripening: time from anthesis to respiratory peak, ethylene peak, carotene production, and polygalacturo- nase activity. The authors suggested that the mode of action of the nor and rin mutants is not directly through endogenous ethylene but probably via a slow change which pre- cedes the ethylene rise and other concomitant ripening changes. The researchers proposed that PG synthesis or activation may represent the primary genetic event which is inhibited in the mutants and attenuated in mutant hetero- zygotes . Ng and Tigchelaar (1977) reported that the ripening of nor and heterozygous nor resembles the ripening of normal tomatoes stored under low oxygen atmospheres, suggesting 21 that ripening is inhibited in the nor mutant by the limited availability of oxygen at sites of active oxidation within the tissue. The absence of PG activity in nor and rin, its attenua- tion in the mutant Nr, and its lowered activity in normal fruits with the physiological disorder "blotchy ripening," suggested to Ng and Tigchelaar (1977) that this enzyme plays a vital role in the initiation of the ripening process. Strand e_t a^. (1976) suggest that the absence of other ripen- ing changes in the mutant fruits may be secondary effects which may occur as a result of release of cell-wall-bound enzymes by PG. Continuous application of the ethylene analogue oleofin propylene to rin mutant tomatoes stimulated respiration in immature fruits. Although respiration reached rates similar to those during the climacteric of normal ripening fruits, there was no change in endogenous ethylene production, which remained at a low level. This suggests that the onset of ripening in normal tomato fruit is not controlled by endoge- nous ethylene, although increased ethylene is probably an integral part of the ripening process (McGlasson et al. 1975a) . By using reciprocal transplanting of tissues between normal fruits and the mutants rin and nor (Mizrahi et al. 1975d) and reciprocal grafting between normal genotypes and the ripening mutants rin and nor (Mizrahi et al. 1975c), it was concluded that the fruits of the mutants do not 22 contain translocatable ripening inhibitors or lack translo- catable ripening factors. However, the possibility of non- translocatable inhibitors in the mutants was not ruled out. More likely, however, is that the fruits lack the capacity to produce a key cellular component essential for ripening. Mizrahi et a^L. (1975a) found that applications of ethe- phon or ethylene gas to attached rin fruits induced ripen- ing as measured by lycopene development, fruit softening, increased total soluble solids, and tomato flavor. However, r fruits did not reach the fully ripe stage of normal tomato. Lycopene was 29% of fully ripe normals. Buescher (1977) found that the intensity of ethephon-induced color in rin fruits was dependent on the stage of maturity, elapsed time between harvest and treatment, and ethephon concentra- tion . Mizrahi et al . (1975b) concluded that endogenous levels of ABA and cytokinins did not account for the lack of ripen- ing in the rin fruit. Suwan and Poovaiah (1978) reported that the rin mutant contains higher levels of bound Ca dur- ing advanced stages of fruit development compared to normal fruits, which suggests more preserved membranes, a fact that may contribute to the delay in senescence observed in the rin mutant fruit. Poovaiah e t a 1 . (1975) recorded that rin did not show changes in water permeability through cellular membranes, but after the ripening process had been initiated in normal strains, there was an increase in the hydraulic 23 permeability of cell membranes which occurred after other parameters of ripening had already been altered. The above results were interpreted to mean that the increases in permeability are not causes of the onset of fruit ripening but rather may be involved in the progressive changes in- volved in ripening. Gonzalez et al. (1976) found similar free methionine levels in rin and normal isogenic tomato fruits, suggesting that the lack of ripening of rin fruits was not due to low methionine levels. 'Alcobaca' Tomato 'Alcobaca' tomato was first described by Almeida (1961) as a material with long fruit storage life, found in the Alcobaca region in Portugal. Leal and Shimoya (1973a) reported that 'Alcobaca' plants have excellent vegetative growth which show more intense leaf color than other cul- tivars. It has a potato-type leaf. The fruits are yellow and multilocular . The fruits remain on the plant in per- fect conditions longer than fruits of other cultivars. After a long period of storage they gradually begin to lose tur- gidity without becoming rotten and, in the final stage, they become mummified. Long tomato shelf life for this material has been reported by a number of researchers (Almeida 1961, Leal and Liberal 1971, Leal and Shimoya 1973b, Leal and Tabin 1974, Leal and Mizubuti 1975). Leal and Mizubuti (1975) described a variant from the yellow standard 'Alcobaca' which exhibit reddish fruits . 24 Leal and Mizubuti (1975) produced a series of reciprocal crosses between yellow 'Alcobaca' and normal materials, and conducted studies of fruit storage life. The researchers concluded that 'Alcobaca' is a genotype that exhibits long shelf life. Differences in storage life between direct and reciprocal hybrids were not found. The authors suggested that the inheritance of the long shelf life displayed by the fruits of 'Alcobaca' was quantitative in nature. Leal and Shimoya (1973b, 1973c) conducted anatomical studies on the pericarp tissue of 'Alcobaca' through storage of the fruits at room temperature conditions. The authors reported that many structural changes take place in the peri- carp region of the fruit as time of storage increased, which may account for the prolonged fruit shelf life of this material . Tigchelaar et al. (1976) noted that 'Alcobaca' is an aberrant ripening cultivar phenotypically distinct from both r in and nor , and suggested, based on unpublished data of allelism tests, that 'Alcobaca' may carry a third allele at the nor locus. Kopeliovitch et al. (1980) did not agree with the above allelism relationship but did not present data to support their argument. Kopeliovitch et al . (1980) studied several physiologi- cal parameters of 'Alcobaca' in comparison to a normal material. They found that the 'Alcobaca' fruits ripened on the vine were similar to those of the variety 'Rutgers' in flavor, pH, concentration of total soluble solids, titrable 25 acidity, and reducing sugars. The pattern of respiration and ethylene evolution was similar in the two genotypes, but the peak levels were reduced and delayed in time in the mutant 'Alcobaca'. The mutant fruits differed from normal in their prolonged shelf life, their relatively low PG and polymethylgalacturonase activities, and their low level of endogenous ethylene. Also, the authors reported that fruits of the mutant harvested before the onset of ripening failed to reach normal pigmentation and remained yellow; fruits harvested at the onset of the ripening reached an orange color, whereas fruits ripened while attached to the plant reached almost normal pigmentation. MATERIALS AND METHODS Pedigree of Studied Materials Seed of 'Alcobaca' and'Chonto' were obtained from the Colombian tomato breeding program of the Instituto Colom- biano Agropecuario (ICA). 'Alcobaca' was introduced to Colombia from the University of Vicosa (Brazil). 'Chonto' is a Colombian material registered in the tomato breeding pro- gram of ICA as MBT-1. The genotypes ' FTE #5', ' FTE #12', 'Tempo', 'Duke', and 'Castlex 1035' are commercial hybrids. Pedigrees and identification of the materials from the University of Florida tomato breeding program are included in Table 1. Storage Life Studies Four different experiments were conducted: a) prelimi- nary observation, b) fruit storage life of normal ripening materials in comparison to 'Alcobaca', c) fruit storage life of hybrids between normal ripening tomatoes and the mutants rin , nor , and 'Alcobaca', and d) fruit storage life of hy- brids between 'Alcobaca' and the ripening mutants rin and nor. For the preliminary observation, staked tomato plants were grown on raised beds of sandy soil and covered with plastic mulch at the Agricultural Research and Extension Center at Bradenton during the fall of 1978. Fruits were picked at the "turning" stage for the normal ripening 26 27 Table 1. Identification and materials included pedigree of experimental in the study. Name Pedigree 'Florida 1C' U.F. 645 ' Hayslip 1 U.F. 648 ' Burgis ' U.F. 71057 'Florida IB ' U.F. 631 ' Tropic ' ' Walter ' (479-6-1 X 18-D2-D4-8-1) F11 1544-3-4-1-Bk-Bk-Bk 'Walter PF ' U.F. 740985-3 'Florida MH-1' 24 31-1-1-SpBk-SpBk ' Flora-Dade ' U.F. 908 rin 'East Ithaca' rin-1 nor (rin X 'Florida MH-1')„ F8 'Italian Winter' rin F^ U.F. 648 X 'East Ithaca' rin-1 F U.F. 648 X rin-1 nor F^ U.F. 648 X 'Italian Winter' 'Hayslip' X rin U.F. 648 X 'East Ithaca' 'Burgis' X rin U.F. 71057 X 'East Ithaca' 'Hayslip' X nor U.F. 648 X 'Italian Winter' 'Florida 1C ' X nor (U.F. 645 X A816) A816 ('Walter' X 'Italian Winter') r ~ 28 materials as well as for the 'Alcobaca' tomato mutant, and at the inception of the yellow color for the ripening mutants rin and nor . The fruits were transported to Gainesville and, 24 hours after harvest, were rinsed with running water, air dried, reclassified for ripening stage, individually weighed, and stored on plastic trays in a ripening room at 20 °C. Loss of fruit weight was monitored twice weekly, and those fruits exhibiting decay or excessive softening were discarded. For the experiments b, c, and d, above, unstaked tomato plants were grown in plastic mulch beds at the University of Florida Horticultural Unit during the spring of 1980 in a ran- domized block design with four replicates. Each experimental unit was composed of 3 plants in a row. Fruits were har- vested at the breaker stage for those genotypes that exhibited a normal color-change pattern, and at the inception of the yellow color for the mutants rin , rin-1 , nor , and the hybrid nor X 'Alcobaca'. Within 24 hours, fruits were rinsed with running water, air dried, and selected for uniformity of rip- ening. Nine to 10 fruits of each genotype and each replica- tion, were selected with the exception of the hybrids between 'Alcobaca' and other ripening mutants (study d) , in which case each of the samples was composed by 5 to 6 fruits (in each replication) . The selected fruits were completely ran- domized on plastic trays and were stored in a ripening room at 20°C. The fruits having decay or excessive softening were discarded every 5 days. Fruit weight loss was evaluated every 10 days for study b, every 5 days for study c, and 29 every 10 days up to 30 days and then every 15 to 20 days for experiment d. Two measures of fruit shelf life were obtained: accumu- lative storage index (ASI) and mean storage life (MSL) , where j _ £ Percentage of remaining fruits X days in storage 100 and MSL equals the number of days required for 50% of the fruits to be discarded. ASI was calculated from the percen- tage of fruits remaining in storage every 10 days for studies b and c, and at 10, 20, 30, 45, 60, and 80 days for the experiment d. For analysis of variance, the percentage of discarded fruits and the percentage of fruit weight loss were trans- formed by using the angular or inverse sine transformation which is applicable to data expressed as percentages. This is particularly recommended when the percentages cover a wide range of values (Steel and Torrie 1960). Duncan's multiple range test with 95% of confidence was applied to compare the mean values. Color Determination The color of the fruits was evaluated with a Hunter Color and Color Difference Meter. The results reported are the radio a/b, where the higher the value, the more red the fruit (Hall 1964). A white plate with values L = 94.9, a = -1.2, and b = 2.2 was employed as a standard for calibration. Unstaked tomato plants were grown in plastic mulch beds at the University of Florida Horticultural Unit during the 30 spring of 1980. Fruits of all the genotypes were harvested the same date at the mature-green stage and stored at 20°C. After two days in storage, fruits at the inception of the yellow color for the ripening mutants rin and nor, and at the breaker stage for the remaining genotypes, were selected. Fruits were allowed to attain yellow color for the mutants rin and nor, reddish color for nor X 'Alcobaca' hybrid, and red color for the remaining genotypes. The above colors are considered to correspond to a fully ripened stage. Five samples of each genotype of 3 to 5 randomly selected fruits were analyzed. The fruits were sectioned and the stem scar was eliminated. They were then blended in a Waring blender and filtered through a double layer of cheesecloth. An anti-foam agent was added to the juice, and the samples were deaerated by partial vacuum. The deaerated juice was used to measure color. Firmness of the Fruits Firmness was determined with the Cornell Pressure Tester (Hamson 1952b) , using a 1.9 cm pressure plate and a load of 2000 g for 5 seconds. Pressure was applied to the equatorial section of the fruit, with only one determination for each fruit. Care was taken to align the fruit so that the plunger was applied over an inner wall as judged by external appearance (Hall 1964) . The range of the Cornell Pressure 31 Tester is 0 to 7 , where the higher the value, the softer the fruit (Hall 1964) . Unstaked tomato plants were grown in plastic mulch beds at the University of Florida Horticultural Unit during the spring of 1980. Fruits of all genotypes were harvested at the mature— green stage on the same date. They were stored at 20 °C and re-sorted when they reached the breaker stage. The selected fruits were allowed to attain red color for normal genotypes, reddish for 'Alcobaca' and the nor X 'Alcobaca' hybrids, and yellow color for rin and nor. At these stages the first determination of firmness was done on 5 randomly chosen fruits for each genotype. Subsequent determinations were done 1 and 2 weeks later. Respiration Studies Five fruits of each genotype were harvested at the mature green stage from plants growing in the greenhouse. The fruits were rinsed with running water, air dried, individu- ally weighed, and stored on plastic trays in a ripening room at 20°C. To determine the respiration, the fruits were enclosed daily in wide-mouth 500 ml glass jars supplied with a con- stant flow of atmospheric air at a rate of 50 ml per min. The evolved CC^ was monitored after 20 min. by passing the effluent air stream through an infrared analyzer Beckman Model 215. The results were recorded by a 24-point 32 Westronic recorder. A sample of the atmospheric air was monitored daily to correct the obtained readings for C02 content in the air circulating through the system. Respiration was expressed as milliliters of C02 pro- duced per kilogram of original fresh weight per hour. Ethylene Evolution Ethylene was determined by using a Hewlett Packard 4710A gas chromatograph with a flame ionization detector. A standard of 1 ppm ethylene in nitrogen was used daily to calibrate the results. Five fruits of each genotype were harvested at the mature-green stage from plants grown in a greenhouse, rinsed with running water, air dried, individually weighed, and stored in a ripening room at 20°C. Ethylene evolution was determined every 24 hours by placing the fruits inside 500-ml glass jars with a rubber stopper in the lid. After 20 min, 0.5 ml of volume was withdrawn from the jar and injected in the gas chromatograph. Ethylene was expressed as microliters of ethylene per kilogram of original fresh weight per hour. Polygalacturonase Activity Five fruits of each genotype from plants grown in the greenhouse were harvested at the mature-green stage, stored at 20°C, and sorted for ripening at the breaker stage. After 33 5 days, equatorial sections of the outer pericarp were ob- tained and stored at -30°C. The stored samples were weighed and then macerated with pestle and mortar in an aqueous solution of 5% NaCl and 1% polyvinylpyrrolidone. Three milliliters were used per gm of tissue. The solution was adjusted to pH 9.0, filtered through a double layer of cheesecloth, and after 30 min was centrifugated in a Sorvall Superspeed RC2-B centrifuge with an SS-34 rotor at 10000 rpm for 10 min. Polygalacturonase (PG) activity was determined by a modified method of the one described by Babbit et al. (1973), as described below. One milliliter of the enzyme extract was added to 5 ml of a 1.2% solution of polygalacturonic acid grade III (Sigma), buffered to pH 5.0 with 0.1 M sodium acetate in a Cannon #200 viscometer held at 31°C in a water bath. Initial read- ings of viscosity were obtained within 1 min of mixing. The change in viscosity was measured after 30 min. Activity was expressed as percentage loss in viscosity. For statistical analysis the percentage loss in viscosity was transformed by using the angular or inverse sine transformation which is applicable to data expressed as percentages (Steel and Torrie 1960) . Model To Relate Storage Life with Different Physiological Parameters A multiple regression model to determine the relation- ship of storage life, measured as ASI, and different 34 ripening parameters was carried out with the aid of the Northeast Regional Data Center in Gainesville. Two differ- ent procedures were used: R-square and Stepwise (SAS Institute 1979). Genetic Studies Inheritance Study In the study of inheritance of the abnormal ripening of 'Alcobaca', the normal ripening material 'Florida IC ' was used as a parent. The populations used were 'Florida 1C' (p]_) ' ’Alcobaca’ (P2), ’Florida 1C ' X 'Alcobaca' (F^ , 'Alcobaca' X 'Florida 1C (RF^, Fx X 'Florida 1C (BC^ , F1 X 'Alcobaca' (BC^, and 'Florida 1C' X 'Alcobaca' F2 1 fd Q r- LO O LO O co CD ' — I Q-i £ fd CO (D N i — I -H fd co •H -p •H £ H CD a. >i ■p o £ CD o LO LO LO CN LO 00 e'- er* o LO CN ' — I CN O LO 00 Oh LO CN Oh 00 co r- 00 r^ co ro oo CN 00 o 00 o LO o Oh o o LO 00 LO Oh CN CO o Oh LO CN CN LO CN LO fO CN O CO CD rT3 fd - - Q p 0 1 tT CD 4-1 rO 1 — 1 H -P c n 1 1 i — 1 • — 1 o O | | cd i 1 S-l u c c £ £ £ u 0 0 •H •H •H •H — — c g P P fd u fd o o I — I < Pedigrees included in Table 39 storage than nor , rin and 'Alcobaca'. At 166 days no dif- ferences were found in percentage of remaining fruits be- tween rin, rin-1 , nor, and 'Alcobaca' with 4, 4, 5, and 3% of the fruits of these genotypes remaining in storage. Fruit shelf life was improved to a greater extent in the nor heterozygotes than in the rin and rin-1 heterozy- gotes. After 30 days, 100% of the nor hybrid fruits remained in good condition in comparison to 81 and 82% of the rin and rin-1 hybrids. At 50 days 59% of the nor F fruits were in good condition in comparison to 38% rin F^ and 37% rin-1 F^ remaining fruits. After 90 days 24% of the fruits of the nor heterozygote remained in comparison to 0 and 9% from rin and rin-1 heterozygotes. Fruit weight loss during storage is included in Table 3. Significant correlations between storage life as percentage of remaining fruits and percentage of weight loss were not detected after 30 and 50 days in storage (r=0.17 and r=-0.25) respectively, but was detected after 70 days (r=-0.98). Storage Life of Commercial Varieties and 'Alcobaca' The storage life of the commercial varieties, experi- mental breeding lines, and 'Alcobaca' mutant used in the study is presented in Table 4. Significant differences in percentage of remaining fruits were found after 20 days in storage. At this time 'Florida IB ' , 'Burgis', ' FTE #12', and 'Duke' exhibited a significantly reduced number of Table 3. Fruit weight loss during storage of different tomato genotypes at 20°C. 40 o f" * m cn * * rH CM rH * Cf\ fN minmLnr^-^-^mLntn QJ l — I Qi £ cd in qj N i — I *H rd W •H +J •H c H r^moHr^LD'xinrHo H(NrH(NrHCNH(NrHm fruit . Table 4. Storage life of commercial varieties, breeding lines, and 'Alcobaca' held at 20 °C . (Fruits picked at the breaker stage.) 41 X in -p •H 3 p 44 tn C •H C ■iH 3 £ CD P 4-1 o o Cn 3 +J G cn C T\ cn Cn LO CD 00 00 CO 00 00 o CO IP s b i — 1 1 — 1 d o CO a, 3 1 — 1 a) - 05 a 3 3 - X, CM X u o P b i b b CM tn •H lO _ CD •H a) •H 3 •H •H CO •r-l 3t= _ o X} ■p a* 44 p P p p Cn U) a' Q, p o 0 0 ' — 1 o 0 0 0 CM P >1 w CP X E 6 c u 05 1 — 1 1 1 i — 1 r~ 1 3 a5 [H Eh 3 i X! (0 C e 3 • ■ i X o a) u b d d -H •H x (D -p pi ■H fC) • S P 0) O 4-1 d V -P O to ( — t •H ■p a; ai 3 > tn p -P 3 3 -p p ft3 O CL) i — I 4-) to 3 CO 3 0 3 0) O 0) S < S X >1 N 42 remaining fruits. At 30 days in storage and thereafter, 'Alcobaca' exhibited a higher percentage of remaining fruits than did the normal ripening materials. After 30 days in storage, the normal ripening materials with the longest storage life were ' Flora-Dade ' , 'Tempo', and 'Castlex 1035'. At 40 days no fruits remained of 'Tempo'. From 40 to 60 days, the normal genotypes 'Flora-Dade' and 'Castlex 1035' exhibited a significantly higher percentage of remaining fruits than did the other normal ripening mateirals. Significant differences in accumulative storage index (AS I ) and mean storage life (MSI) were detected between 'Alcobaca' and the remaining genotypes. The normal mate- rials 'Flora-Dade' and 'Castlex 1035' displayed the greatest AS I among the normal genotypes with values for ASI of 97.7 and 88.5. The normal genotypes with higher MSL were 'Flora- Dade', 'Castlex 1035', 'Tempo', and 'Florida MH-1 ' . The greatest weight loss after 10 days of storage was exhibited by the genotype 'Florida IB' (Table 5). After 25 days 'Florida 1C, 'Florida IB ' , Fl 2432', and 'Burgis' displayed the greatest weight loss. At 40 days the geno- types 'Fl 2432', 'Duke', and 'Castlex 1035' exhibited the greatest weight loss. No significant correlations were found between percentage of remaining fruits and fruit weight loss. 43 Table 5. Fruit weight loss of commercial varieties, breed- ing lines, and 'Alcobaca' held at 20°C. Percentage of weight loss2 Days after storage Genotype 10 25 40 1 Tropic ' 1. 98ab 3. 55bc ■ k * ** 'Walter PF 1 2 . 15abc 3 . 8 6cd ★ * 'Florida MH-1' 1.32a 2.25a * * ' Flora-Dade ' 1 . 67ab 2. 52ab 5.15a 'Florida 1C ' 2 . 09abc 4 . 88d * 'Florida IB' 3.22c 5. Old * FL 2432 (ug MH-1) 1. 92ab 5. 35d 8. 32d ' Burgis ' 2. 53bc 3. 96cd 6.64b ' Hayslip ' 2 . 20abc 3 . 14abc 6.60b ' FTE #5' 2. 46bc 4 . 09cd * * ' FTE #12' 2 . 0 6ab 3 . 4 Obc 6. 51bc ' Duke ' 2 . lOabc 2. 77ab 7. 37cd ' Tempo ' 2. 65bc 3 . 32abc ★ 'Castlex 1035' 2 . 22abc 4 . 35cd 7. lied 'Alcobaca ' 2 . 31abc 3 . 24abc 5. 83ab 2 Mean separation range test. within columns by Duncan ' s multiple *A11 fruits discarded. **Less than 10% of fruits remaining. 44 Fruit Storage Life of Hybrids with Ripening Mutants The experiment was carried out to evaluate the poten- tial use of hybrids involving ripening mutants and normal materials in order to improve the fruit shelf life. Also, the phenotypic appearance of the hybrid genotypes was monitored . Significant differences in storage life appeared 20 days after storage as shown in Table 6. At this time the materials with the highest percentage of remaining fruits were the 'Florida 1C' X nor and 'Hayslip' X rin hybrids. At 30 and 40 days the 'Florida 1C X nor hybrid had a signifi- cantly higher percentage of fruits remaining in storage than the other studied genotypes. After 50 days no differences in percentage of remaining fruits were detected between the 'Florida 1C' X nor and 'Hayslip' X nor hybrids. At this time the above hybrids exhibited the greatest percentage of remaining fruits. From 60 days on the 'Hayslip' X nor hy- brid had the greatest percentage of fruits remaining in storage. Differences in percentage of remaining fruits be- tween the studied hybrids and their normal parents were evident after 20 days in storage. The AS I was improved 3 to 4 fold and the MSL 2 to 3 fold in the hybrids with nor in comparison to the normal parents. The hybrids with 'Alcobaca' had an ASI and MSL with 2 to 3 and 2 fold, respectively, over the normal parents Table 6. Storage life of hybrids between normal ripening materials and ripening mutants held at 20°C. (Fruits picked at the breaker stage of maturity.) 45 N >1 P co S N X H C/) < O r- P P fd fd P P o O r- o o * o o r- CM ro CO CM LO vO i — i 00 ro 00 in ro ro CM p u p P fd fd P p TJ TJ U o o* o CM r- Oh CM LO vo vo CM r- oh vo VO cr> Oh p p 0 P TJ U P fd u u 0 0 o- CM LO o Oh CM o o ro VO r- o 1 — 1 P o o ro LO I — 1 i — 1 N w ■P •H 3 U 4n CT» C •H C •H fd e 0 MH 0 0 fd p C 0 O 0 0 fd O P 0 0 P P fd 0 !>1 fd Q o 0 T5 TJ u 0 p P fd O p 0 T5 o CO LO o LO LO o O Oh P r- o o- r- o LO P ro LO p CM T5 U •d' P TJ U r- ro o fd fd Oh o o CM o LO CM LO LO o p fd P VO LO o Oh o CO vo P P TJ TJ O o LO o LO ro CM LO CM ro P p Tf U U P Ti 0 o* O P o 00 LO CO LO ro CM CM O P P fd p VO o o VO vo o LO LO o- o r- p u p T5 P fd fd U p rH o O Oh o LO 00 Oh o O' p fd fd fd fd o o o o o o o o o o o o u P O TJ T5 O' CM P O ro r \ 00 O O' LO CM ro P O TJ TJ 0 O' CM LO o co VO CM o VO vO fd fd fd fd o o o o o o o o o o o o d) a >i P o c 0 o - _ fd _ X u X X - u o u I — 1 d5 p fd p - - - 0 p “ fd CT n3 fd 0 fd 0 •H TJ •H •rH XI u TJ •H i — 1 •rH SH P ■H 0 - r| p • H tX> 0 0 0 0 tn o u C Vi u g !>i c 0 c >i c I>1 rH 0 0 G -H fd •H 1 — 1 fd 03 < 1 — 1 1 — 1 CQ M X Vh X X re X SC - Cn X fr, “ ■■ - - - 0* •H i — I 0 >1 fd re p g c u fd fd U (D (D CSS X >i N iulative storage index, storage life. separation within columns by Duncan's multiple range test at 5% level. 46 and the hybrids with rin exhibited a 2- and 1.5-fold improve- ment of AS I and MSL respectively (Table 6) . As can be seen in Table 7, there were slight differ- ences in fruit weight loss between the hybrid genotypes during the 60 days of storage. Some differences were detec- ted only at 10 days in storage, but these differences disap- peared at 15 days. Differences between the hybrids and the normal parents were evident after 30 days of shelf life. No significant correlations were found between percentage of remaining fruits and percentage of fruit weight loss during the current experiment. Significant differences in fruit color as ratio a/b were found between genotypes (Table 8). The normal ripening material, ' Hayslip exhibited the highest a/b ratio followed by 'Florida 1C X 'Alcobaca', 'Florida 1C, and 'Hayslip' X 'Alcobaca' . These genotypes had a/b values above 2.0. The mutant ripening genotypes rin and nor exhibited the lowest a/b ratio value (0.2) followed by 'Alcobaca'. The hybrids between normal ripening parents and the ripening mutants rin and nor had a less intense red color. These genotypes dis- played a/b ratio values below 2.0. Significant variation in fruit firmness was found be- tween genotypes at the red stage and 7 and 15 days after the red stage (Table 8) . The fruits of the rin and nor mutants were significantly firmer than the remaining genotypes in all of the dates in which firmness was evaluated. 'Alcobaca' Table 7. Fruit weight loss during storage of hybrids between normal ripening materials and ripening mutants held at 20°C. (Fruits picked at the breaker stage of maturity.) 47 fd fd fd fd fd fd co kD o CN co r- • • • • • • r- r^* kO * -K in N to to 0 X X Cn •H CD 13 tD tn to x C (D O P tD ft a) Cn tti P O X to o P a) x X m to in CN >1 td Q in o tD d Pn X 0 c ai o fd fd fd fd td td X O (D ID (N CO CO Oh CN • • • • lo ^ LD LO •^r kO o 0\° 1 — 1 LO X td X X cn td x td td X td u X 1 o 0 >1 b td rp fd fd ' — 1 fd CQ X Pm PC PC d Pm PC tn g *H sc •P td e CD P to X ■P b p ip o -K 48 Table 8. Fruit color and firmness of normal ripening varieties, ripening mutants, and their parents. Genotype Firmness^2 Colorxz a/b Red stage 7 days after 15 days after 'Florida 1C' 2.1c - - — ' Hayslip ' 2.4a 2.8c 3.3b 2 . 2e ' Alcobaca ' l.lh 3 . 0c 3.2b 3 . 8bc rin 0. 2i 2 . 4ab 2 . 6a 2.6a nor 0. 2i 2 . 2a 2 . 3a 2.4a 'Burgis' X rin - 3 . 4cde 3. 9bcd 4. led 'Hayslip' X rin 1. 8e 3 . 3cde 3.4b 3. 6bc 'Hayslip' X nor 1. 8e 3 . 2cd 3.3b 3. 6bc 'Florida 1C' X nor 1. 7f 3. 2cd 3 . 6bc 4. led 'Hayslip' X 'Alcobaca' 2. Od 3 . 5cde 3 . 8bcd 4 . 4d 'Florida 1C X 'Alcobaca' 2.2b 3. 9e 4 . 4d 4 . 4d Higher value indicates a more intense red color, y Lower value indicates a firmer fruit, z Mean separation within columns by Duncan's multiple range test at 5% level. 49 fruits were firmer than those of the hybrid 'Florida 1C' X Alcobaca 1 at the red stage and 7 and 15 days after the red stage. 'Alcobaca' fruits were firmer than those of the 'Hayslip' X 'Alcobaca' hybrid at the red stage and 7 days after the red stage. 'Alcobaca' fruits were firmer than 'Hayslip' X 'Alcobaca' fruits 15 days after the red stage. 'Hayslip' did not differ from the 'Hayslip' X rin, 'Hayslip' X nor, and 'Hayslip' X 'Alcobaca' hybrids in fruit firmness at the red stage. Seven days after the red stage, 'Hayslip' fruits were as soft as those of the hybrid 'Hayslip' X nor. Fifteen days after the red stage 'Hayslip' exhibited the softest fruits of all of the studied genotypes. No signifi- cant correlation was found between firmness at the red state and 7 days after the red stage with storage life, but a significant correlation was obtained between firmness 15 days after the red stage and storage life. Fruit Storage Life of Hybrids Between 'Alcobaca' and the Ripening Mutants nor, rin, and rin-1 The experiment was carried out to evaluate the possi- bility of using hybrids between 'Alcobaca' and other ripen- ing mutants in order to improve the fruit storage life. Data of percentage of remaining fruits and ASI for the genotypes included in the study are presented in Table 9. Differences between genotypes for percentage of remain- ing fruits were detected at 30 days of storage. At this Table 9. Fruit shelf life of normal ripening varieties, ripening mutants, and hybrids of these mutants with 'Alcobaca' held at 20°C. (Fruits picked at the beaker stage. 50 N rd O 0 XI X u O in co Oh O 00 r- o 00 CM >1 • • • • • • • H o Oh 1 — 1 00 i — I i — i o C/3 in r- o o • — i kO Oh Oh c CM 1 — 1 • — i i — 1 i — i o o rd CO Ocd Oc 7b 7b 5d Ocd • • • • • • • t a o o in m o kO kO CM in Oh CM kO kO i — 1 CM N m x X g p 44 tn C *P C ■ H g £ o p 4-1 o Q) in (0 X G CD o p tro g Q o o m rd 00 3c 7c 8b 3b PO Ocd • • • • • • • • • o r- in 00 i — 1 O CO in in • — i Oh 00 CM CO rd X 0 X o X o X X o X X in in o o 00 ro in CO o CM (N o o in ro CO in o i — i r~- CO 00 00 kO g u u X X X g X X o in O CO O in kO CM O in nj o o in r- i — 1 Oh in kO r- o rH r- 00 o> r- rd rd rd rd rd rd rd o in o in o r- kO kO O in CM o o » — 1 rH i — l in Oh Oh o i — 1 00 Oh Oh Oh Oh 00 g _ _ o X g g g - o 0 X x x g g 0 1 X X u X p tn X G C tn 0 X • rH G a) X a> > g a, •H p ■p X g p g Q, i — 1 a) X g to g £ £ g G p o g 0 0 a) £ < £ X N within columns by Duncan's multiple range test at 5% level. 51 time the ripening mutant nor and the nor X 'Alcobaca' hybrid exhibited the highest percentage of remaining fruits. At 45 days in storage and thereafter nor displayed a signifi- cantly higher percentage of remaining fruits than any of the other studied genotypes. 'Alcobaca' had a significantly higher percentage or remaining fruits than did rin and rin-1 at 60 and 80 days in storage. Differences in percentage of remaining fruits between 'Alcobaca' and the nor X 'Alcobaca' hybrid were not found throughout the time of storage. Sig- nificant differences in percentage of remaining fruits were detected between 'Alcobaca' and the hybrid rin X 'Alcobaca' after 60 days in storage. Also, significant differences in this respect were found between 'Alcobaca' and the hybrid rin-1 X 'Alcobaca' after 45 days in storage. The normal ripening varieties, 'Florida MH-1' and ' Flora-Dade ', had a reduced percentage of remaining fruits after 30 and 45 days, respectively, and thereafter in comparison to the genotypes included in this study. 'Florida MH-1' and 'Flora-Dade' did not belong to the current experiment but they were stored at the same time in the ripening room. It should be noted that 'Flora-Dade' displayed the longest fruit shelf life of 14 normal ripening materials in a previous experiment (Table 4 ) . Significant differences in storage life as ASI were detected between genotypes. The mutant nor exhibited the highest ASI value. No differences in ASI were detected 52 between rin and rin X 'Alcobaca' and between rin-1 and rin-1 X 'Alcobaca'. 'Alcobaca' did not differ in ASI from the hybrid nor X ' Alcobaca ', but the ASI value of 'Alcobaca' was significantly higher than that of rin X 'Alcobaca' or rin-1 X 'Alcobaca'. The normal ripening varieties 'Florida MH-1 ' and 'Flora-Dade' displayed a very low ASI in compari- son to nor , rin, rin-1, 'Alcobaca' , and the hybrids nor X 'Alcobaca', rin X 'Alcobaca', and rin-1 X 'Alcobaca'. Variation in fruit weight loss was detected between genotypes (Table 10) . The mutant rin and the hybrid rin X 'Alcobaca' had the greatest weight loss. Differences in fruit weight loss were found between rin and rin-1 after 20 days in storage. The genotypes that exhibited the lowest fruit weight loss during storage until 80 days were nor , rin-1 X 'Alcobaca', and nor X 'Alcobaca'. The hybrids rin X 'Alcobaca' and rin-1 X 'Alcobaca' had fruits of normal color with an a/b ratio above 2.0 (Table 11). The hybrid nor X 'Alcobaca' had an a/b ratio 0.7, which was closer to the ratio of 'Alcobaca (1.1) than that of nor (0.2) . The hybrid fruits nor X 'Alcobaca' were firmer than the ones of 'Alcobaca' 7 and 15 days after the red stage and as firm as those of nor at the red stage and 7 and 15 days after the red stage (Table 11). The rin X 'Alcobaca' fruits were softer than the fruits of its parents (Table 11) . The fruits of the hybrid rin-1 X 'Alcobaca' were as firm as the ones of 'Alcobaca' and softer than rin-1 fruits. 53 Table 10. Fruit weight loss during storage of ripening mutants and hybrids of these mutants with ' Alcobaca ' . Percentage of weight loss2 Days after storage Genotype 10 20 45 60 80 nor X 'Alcobaca' 2 . 6c 3 . 4bc 5.7a 9.1b 13. lab rin X 'Alcobaca' 2 . 3bc 3 . 3bc 9. 5bc 13. 6d 17.5c rin-1 X 'Alcobaca' 1.3a 2.4a 6 . 3a 9.1b 11.2a nor 1 . 4a 2.2a 5.4a 6 . 6a 10.2a rin 2 . 4bc 4 . 3c 9 . lc 14. 5d 21. Id rin-1 1 . 8ab 2 . 8ab 6 . 7ab 9.2b 15 . 2bc ' Alcobaca ' 1.5a 3.8c 7 . 7bc 10.9c 16 . lbc zMean separation within columns by Duncan's multiple range test at 5% level. 54 Table 11. Fruit color and firmness of ripening mutants and hybrids of these mutants with 'Alcobaca'. FirmnessY2 Genotype T xz Color a/b Red stage 7 days after 15 days after nor X 'Alcobaca' 0. 7e 2. 5bc 2 . 6a 2 . 8a rin X 'Alcobaca' 2 . 0c 3. 8d 4.2c 4.5c rin-1 X 'Alcobaca' 2.1b 3 . 5cd 3. 7bc 4 . Obc nor 0. 2f 2 . 2ab 2 . 3a 2.4a rin 0. 2f 2 . 4ab 2 . 6a 2.6a rin-1 0. 2f 1.8a 2 . 0a 2.4a ' Alcobaca ' 1 . Id 3.0c 3.2b 3.8b ' Hayslip ' 2.4a 2.8c 3.4b 5. 2d XHigher values values indicate a more intense red color . ^Lower values indicate firmer fruit. ZMean separation within columns by Duncan's multiple range test. 55 Respiration Study The genotypes 'Florida 1C, 'Hayslip', and 'Chonto' had a typical climacteric pattern of respiration (Figures 1, 2, 3) . The 'Alcobaca' mutant also exhibited a climacteric pattern. This material did not display the sudden rise and fall in respiration of 'Florida 1C, 'Hayslip', and 'Chonto' but rather a smooth rise and gradual reduction (Figures 1, 2 3, 4, 5, 6). The hybrids between 'Alcobaca' and the normal ripening genotypes 'Florida 1C, 'Hayslip', and 'Chonto' showed a typical climacteric pattern but with a reduced CC>2 peak intermediate between 'Alcobaca' and the normal ripening parent (Figures 1, 2, 3; Table 12). The rin X 'Alcobaca' and rin-1 X 'Alcobaca' hybrid fruits had a respiration pattern similar to that of the nor- mal ripening genotypes (Figures 4, 5). The respiratory peak of the hybrids was greater than those of 'Alcobaca', rin , and rin-1 (Table 12). The nor X 'Alcobaca' hybrid had an attenuated climacteric respiratory pattern (Figure 6). The respiratory peak of this hybrid was similar to that of 'Alcobaca' (Table 12). The ripening mutants rin, rin-1 , and nor displayed a typical non-climacteric pattern or respira- tion (Figures 4, 5, 6). Ethylene Evolution 'Florida 1C, 'Hayslip', and 'Chonto' had a typical, climacteric pattern for ethylene evolution (Figures 7, 8, 9) 56 Alcobaca' Florida I C ' Alcobaca ' X 'Florida I C ' Figure 1. Respiration pattern of 'Alcobaca', 'Florida 1C, and 'Alcobaca' X 'Florida 1C fruits. EVOLUTION(mlx kg'1 xh'1 57 'Alcobaca ' 'Hayslip' 'Alcobaca’x 'Hayslip' DAYS AFTER BREAKER Figure 2. Respiration pattern of 'Alcobaca', 'Alcobaca' X 'Hayslip' fruits. 'Hayslip', and C02 EVOLUTION (ml X kg ' X h-') 58 'Alcobaca' Chonto' j* 'Alcobaca' x 'Chonto' DAYS AFTER BREAKER Figure 3. Respiration pattern of 'Alcobaca' and 'Alcobaca' X 'Chonto' fruits. ' Chonto C02 EVOLUTION (mlxkg-'xh-* 59 'Alcobaca' r]n 'Alcobaca' x rin DAYS AFTER BREAK Figure 4. Respiration pattern of 'Alcobaca', rin, and 'Alcobaca' X rin fruits. 60 'Alcobaca' rin— I 'Alcobaca' X rin - I DAYS AFTER BREAKER Figure 5. Respiration pattern of 'Alcobaca', rin-1 , and 'Alcobaca' X rin-1 fruits. EVOLUTION (ml x kg"1 x h"‘ ) 61 Alcobaca ' nor 'Alcobaca' x nor DAYS AFTER BREAKER Figure 6. Respiration pattern of 'Alcobaca1, nor, and 'Alcobaca' X nor fruits. Table 12. Tomato fruit maximum respiration, maximum ethylene evolution, and poly- galacturonase (PG) activity for different genotypes. 62 N 44 0) •H b 44 > rd •H XX b b •H CO LO ro CN 4-4 ft 0 • O o 0 a\ b 0 CO H i — 1 CM b •H a > fa o\° d) aj b o 44 44 co o co o CO o CM VO 00 r- i — 1 i — I rH 00 LO * * * • • -K -K -X O fa d> I Sb ft a) b -H O >i 4b 00 m O cn 00 o CM co b • 44 X rH • — i CM rH 1 — 1 1 — 1 1 — 1 ' — 1 o o d) + 1 +i +1 + 1 +1 +1 +1 +1 * +1 +i 1 1 CN (Ts CM i — 1 CO o LO CM £ i • b cn CM CO CM VO CO CM CD 00 i — i £ X •H CM i — 1 CO fa rH CM rH x x (0 S H 73. i — I a) > QJ C#i° in +J b .ration hour o 00 rH LD CO LO CO o LO CM o 44 b CD 44 d) tn •H • • c a, x rH • — 1 rH rH rH rH 1 — 1 1 — 1 rH i — 1 rH CN rH rd CO + 1 +1 +1 + 1 +1 +1 +1 +1 + 1 +1 + 1 + 1 + 1 ft (D i — 1 CO VO 1 — 1 ro 00 CO CO CD ' — 1 CM CN CD ft i • CD Cb CN 00 rH 00 i — 1 00 CM h LO 00 £ M b CM i — 1 CO rH i — 1 CM iH CN ' — 1 rH i — 1 a •H b £ x X b S £ b £ b b o b 4b b 0 u b o b o rH 4b b - i *H rH 44 •H i — 1 44 4b 44 ft b c ft b c X 1 — 1 X 0 o 0 I>i o 0 >i 0 1 O 1 sb rH b 4b 1 — 1 b 4b sb sb ft 1 1 sb £ d) fa fa u fa fa u •H •H 0 < •H •H 0 ft ft c ft ft sb b o b sb d) b • +J Q b U d) 144 44 .Q O d) d) b sb +J 0 O >1 •ft b 4-> 44 -r4 b b > ft a -h b 0 4J Sfafa O d) >i b b 4b 4J 0 C d) ft b dJ O o £ 2 S N * * * 63 'Alcobaca' also exhibited a climacteric pattern for ethylene production but, as in the case of respiration, 'Alcobaca' did not have the sudden rise and reduction of ethylene evolution but rather a gradual rise and reduction (Figures 7, 8, 9, 10, 11, 12). The "Florida 1C' X 'Alcobaca', 'Hayslip' X 'Alcobaca', and ' Chonto X 'Alcobaca' hybrids exhibited a climacteric pattern for ethylene evoluation (Figures 7, 8, 9) but with a reduced ethylene peak that was intermediate be- tween 'Alcobaca' and the normal ripening parent (Table 12). Ethylene evolution was attenuated in the hybrids rin X 'Alcobaca' and rin-1 X 'Alcobaca' (Figure 10, 11). In these hybrids the ethylene peak was less than the one of 'Alcobaca' (Table 12) . No ethylene was detected in the rin and rin-1 fruits. The nor X 'Alcobaca' hybrid fruits had a highly re- duced ethylene evolution (Figure 12) . The ethylene peak of this hybrid was lower than that of 'Alcobaca'. No ethylene was detected in the nor fruits. PG Activity Study No PG activity was detected in the mutants rin, rin-1, and nor. 'Alcobaca' had very low PG activity (Table 12). The 'Florida 1C' X 'Alcobaca', 'Hayslip' X 'Alcobaca', and 'Chonto' X 'Alcobaca' hybrids had reduced PG activity. These hybrids exhibited values intermediate between both parents for PG activity (Table 12) . The PG activity of the rin x 'Alcobaca' and rin- 1 X 'Alcobaca' hybrids was greater ETHYLENE EVOLUTION (/xl x kg-* x h" ) 64 'Alcobaca' 'Florida I C ' 'Alcobaca 'x'Florida 1C' DAYS AFTER BREAKER Figure 7. Ethylene production of 'Alcobaca', 'Florida 1C, and 'Alcobaca' X 'Florida 1C fruits. 65 'Alcobaca' Hayslip' Alcobaca' X Hayslip' DAYS AFTER BREAKER Figure 8. Ethylene production of 'Alcobaca', 'Hayslip', and 'Alcobaca' X 'Hayslip' fruits. ETHYLENE EVOLUTION ( /ml x kg'1 x h"' 'Alcobaca ' Chonto DAYS AFTER BREAKER Figure 9. Ethylene production of 'Alcobaca' and 'Alcobaca' X 'Chonto' fruits. ' Chonto ' ETHYLENE EVOLUTION (^l! xkg''xh‘‘) 67 'Alcobaca ' Alcobaca' x rjn rin DAYS AFTER BREAKER Figure 10. Ethylene production of 'Alcobaca' X rin, and rin fruits. ' Alcobaca ' 68 Alcobaca' 'Alcobaca' x rin- I rin - I X T a» -X X o i- o > LU UJ z UJ > X UJ DAYS AFTER BREAKER Figure 11. Ethylene production of 'Alcobaca', 'Alcobaca' rin-1, and rin-1 fruits. vr /v Alcobaca 'Alcobaca ' x nor ° — 1,0 — nor X 'a< JC X (- 3 _J O > LU UJ 2 UJ I > X h- LlI DAYS AFTER BREAKER Figure 12. Ethylene production of 'Alcobaca' , 'Alcobaca X nor, and nor fruits. 70 than that of 'Alcobaca' but much lower than that of hybrids between normal ripening materials and 'Alcobaca' (Table 12). The PG activity of the nor X 'Alcobaca' hybrid was inter- mediate between nor and 'Alcobaca'. Model To Relate Storage Life with Different Physiological Parameters A multiple regression model to relate fruit storage life measured as ASI against a/b ratio, firmness at the red stage, firmness 7 days after the red stage, firmness 15 days after the red stage, maximum respiration, maximum ethylene, and PG activity indicated that a high determination coefficient (R^) for one independent variable was obtained for PG activity. Once PG activity was included in the model, none of the other independent variables were significant enough to be included m the model. The obtained value for R in the PG-ASI model was 0.69. Genetic Studies Inheritance of the Abnormal Ripening 'Alcobaca' The abnormal fruit ripening 'Alcobaca' was found to be due to a single recessive gene. The segregating generations, BC2 and fit the model with a probability of 0.7-0. 5 (Table 13) . Although this mutant fit a single recessive gene model, it was found by studying the 'Florida 1C' X 'Alcobaca' hybrid Table 13. Inheritance of the abnormal tomato fruit ripening 'Alcobaca' 71 LD HO • • CM o o X i 1 1 1 i 1 1 r- o o p CD 0 -P ■H o l 1 O O O r— 1 1 — 1 O 4J 0 rd 1 — 1 o i — 1 i — 1 i — 1 i — 1 CO X W 4-1 in b c 4-> id o o o O o ro 0 b 4-) LO -H (TJ p 4J r— 1 S rti Q, o •H 44 0 •H in S4 i — 1 in 01 id rd t! E o o o o o CT\ • — 1 E V4 00 ro LD u P o i — i 2 2 44 0 in 4-1 V4 b o o o o o CM i — I aj id 00 00 i — I Q i — 1 CM S 04 3 2 CM 04 X . 1 — 1 1 — 1 C4 04 i — i — CM ' — ' 04 Cm - ' — ' - X u u b 1 — 1 - r— 1 CM 0 nj 04 •H id o id 4-> T5 rd t id •H pO •H 1 1 r— 1 U 0 04 P O o o a, 1 1 «— 1 1 — 1 o 1 — 1 CM 0 h < &4 a) u u CM C4 - - - oc CQ CQ p4 72 that in the heterozygous condition several of the ripening processes were affected. The respiratory and ethylene peaks were reduced 18.0 and 30 . 5%, respectively , PG activity was lowered 47.1%, and storage life (ASI) was increased more than 2 fold. The hybrid fruits had a normal appearance and good red color (Table 14) . Allelism Test Between 'Alcobaca' and rin Allelism tests between 'Alcobaca' and two different sources of rin (rin and rin-1) indicated that the mutant gene carried by 'Alcobaca' was not allelic to rin. The fruits of the plants were normal and the segregation was 9 normal : 7 mutant, indicating 2 non-allelic recessive genes for abnormal ripening (Tables 15, 17) . The F1 plants of the rin X 'Alcobaca' and rin-1 X 'Alcobaca' hybrids had normal red fruits (a/b/ ratio above 2.0), greater respiration peak, and greater PG activity than either parent. The ethylene peak was reduced and was similar to the one of 'Alcobaca' (Tables 16, 18). Allelism Test Between 'Alcobaca' and nor The nor X 'Alcobaca' F^ fruits had a mutant phenotype similar to that of 'Alcobaca' . The F^ segregated 3 ' Alcobaca '— type fruits : 1 nor (p=0.3-0.5), indicating that 'Alcobaca' carries another allele of nor. This allele is dominant to the standard nor recessive allele in phenotypic appearance (Table 19 ) . Table 14. Physiological parameters obtained from fruit of 'Florida 1C 'Alcobaca', and the hybrid between these genotypes. 73 N u X) rtf H CM cr> 03 • • • CM r- U0 o> 00 i — 1 >1 -P rtf jQ‘ O ■H O o 00 U > h CO h Cd -H • • i -P ON o r— l o i — i rH d N d £) rtf u 0 -Q in LD ID H \ • — i CM O 0 d • • • u CM CM i — 1 ai £ a d ai O 1 — 1 (T\ £ r-H CM 1 — 1 rH •H >1 i • • X -b ro ID i — 1 d P £ 03 CM rH i — 1 b 0 £'H rH i — 1 in d 4J ro CO CM £ d • • • *d d CM CO h X'H CM 1 — 1 rH d & £ w 03 d rtf O rtf rQ O 0 1 — 1 < X u u rH 1 — 1 - rtf a 3 rtf d O (£ mi rtf *H -H & 4-1 U d 0 o O O U a i — 1 i — i rH 03 (H Cm < u - - i — I 03 > o oV3 in -P d P cn 03 P 03 tn c d d cn 3 3 ■P 03 0 a -P •H 03 3 •p rH S 03 cu u •H M-l 4-1 O •H cn 3 rH cn CD 03 X3 S i — 1 § 3 U 3 0 2 2 o o o r- cn cn m o o o CN i — I 0 cn -P 3 3 CD 03 x m g Oj 3 2 o (N o CN o CN cx> rH CN Cn Cn 3 o •H p o3 T I 3 a. o (X 03 03 U V o3 03 X XI 0 0 o o o3 1 — 1 rH U < CO r- H • • • w r- o rH c o CTr CD H rH >1 4-1 •H rtf £ e> > * O in a •H * • • •P CO rH U rtf W >i P rtf 0 rtf u JQ •P m CO m • • • in rtf CM sj* CO r— 1 N U) U ) U) p Q) >1 0 rtf u X) £ rtf -P tD CM CM g MM • • • H rtf CM CO *H Pm CD rtf U £ Ttf tT> 00 o 0 rtf • 1 • ■P CM CO CO W N U 0 £) O rtf i — 1 \ CM O rH o rtf • • • o O CM H s a) £ e d) •H 1 — 1 X >1 o CM rtf X * • • s 4-1 CD H Q) H c g 0 H ■H s -P rH CM CM •H rtf . X P G> CO rtf •H ' — 1 H s ft w a) p rtf O rtf -Q 0 O - i — 1 0 0 < u CM 0 £ •P X 0 0 o £ £ a H 0 •H •H < O P M 0 > 0 o\° l D ■P 0 -P W 0 -p cu tr> £ rtf P 0 • — I a *r| -P i — I P s 0 £ rtf O Q l>i £ £ O ■H -P rtf P rtf CM 0 c n £ rtf CD £ N TJ 0 ■P O 0 0 T5 J>i 4J •H > ■H -P U rtf U CM O •K ■X Table 17. Allelism test between 'Alcobaca' and rin 76 CN X X) 1) o -P -H U -P 1 -p •H O > fit *H -P U ftf 0 0) 0 P •H Cm 0 P 0 rtf XI XI rtf •P O r- CM 4-t • rtf CM 00 co r- T) 0 rtf XI XI tJ> CO LD o 1 * » • X x: 00 i — 1 IT) 4J • — 1 a 0 £ rtf CM oo CM •H P • • X •H m CM IT) a 0 rH CM rH rtf O rtf XI O O i — I < i — I 0 > 0 H o\° LO -p rtf -P 0 0 •P (U tn P rtf P •r| -P ' — t e c it! O c d Q >1 n tn c o a c *r| si -p •H s c o •H -p rtf P rtf CU 0 0 P rtf 0 s XJ 0 -p o 0 -p 0 TJ >i -P •H > •H -P U rtf O 0^ o 2; * ■K Table 19. Allelism test between 'Alcobaca' and nor. 78 (N X T) 1 -P (0 o to i O b rH < 44 O W b b b O H • • • rH rH rH o r- 0 eg rH rH -p •H rtf rtf > He CO in o *rH ■K • • CM 4-> O i — i U rd in >i U rtf rtf X rtf ai 00 00 np 4-> • • • Mm CN CN ro N in rd 0) i — i W (D G p P X •H CN CN ro (fl N Ml U X rtf 0 n CN r^ i — 1 1 1 \ • . • 0 rd O o i — l u a) E a p 1 i — 1 X Xi rH rtf +J £ QJ a £ 0 3 •H CT> ■ — i CN 6 P • ■H rtf 00 X P rH rH rtf •H s cu in 0 p rtf U rtf X 0 O - rH rtf 0 < O rtf X P X 0 0 O G p p rH 0 o 0 < 0 G G i — I 0 > CD o\° LO P rtf P 0 0 P (D tX> G rtf P 0 rH ft •H P rH P s in G rtf O G P Q X O u G •H P •H S G O •H P rtf P rtf Qi 0 in G rtf N 70 CD P U (1) P 0 70 >i P •H > •H ■p a rd O CM o 2 * ■X Table 21. Joint Scaling Test (JST) for storage life inheritance. 81 1 >i • rd -P o X! -H 1 1 1 1 1 1 i 0 ft LO P ft • ft XI o r- CO CM w o o i — 1 O O O CM Sh rd O C -sT cr> CO cO O T5 P rd • • • • • • c P o 00 CO CT> ft E CM CO LO CO LO •xT X w TJ QJ > c CO ' — 1 o> CO CO p id • • • • • • Q) OJ CO IT) C50 LO « E CM P- LO CO LO X) o n W xi 2 (Tigchelaar et. ad. 1978a) , reduced lycopene content (Tigchelaar et al. 1978a) , and slow softening of the fruit (Hobson 1967) . 'Alcobaca' differs from rin and nor by the fact that these mutants are non-climacteric (Herner and Sink 1973, McGlasson et al. 1975a, Ng 1976, Ng and Tigchelaar 1977) . Similar results with rin and nor were obtained in this study. The hybrids between 'Alcobaca' and normal ripening materials have normal fruit color appearance, but several aspects of the ripening of these hybrids are modified. The respiratory peak and ethylene peak are reduced, and the amount of reduction depends on the normal parent. The 88 softening of the fruit takes place at a slower rate in com- parison to normal ripening genotypes, and the PG activity is reduced. The storage life’ of the hybrid fruit is improved. Quantitative modification of different ripening pro- cesses was reported for the rin and nor mutants (Buescher et al. 1976, Herner and Sink 1973, Sink et al. 1974, Ng 1976, Tigchelaar et al. 1976, Ng and Tigchelaar 1977) . Tigchelaar et al. (1978a) indicate that the ripening mutants in the heterozygous stage exert their effect largely on the rate at which specific ripening modifications take place. The ripening processes most affected in hybrids between 'Alcobaca' and the ripening mutant rin (rin and rin-1) were the ethylene pattern and PG activity. The ethylene peak was greatly reduced, in comparison to normal ripening material and lower than those of 'Alcobaca' and hybrids between 'Alcobaca' and normal ripening tomatoes. PG activity was higher than that of 'Alcobaca' but was reduced 66% in com- parison to normal ripening materials and was reduced 41% when compared with the PG activity obtained with hybrids between 'Alcobaca' and normal ripening genotypes. Respira- tion was less affected in hybrids of 'Alcobaca' and the ripening mutants rin and rin-1 with an average of 22% reduc- tion in comparison to normal ripening materials. The res- piratory peak was higher than the one of 'Alcobaca', rin and rin-1 . In the nor X 'Alcobaca' hybrid all of the ripening processes were affected. The respiratory peak was similar 89 to that obtained for ' Alcobaca ' , and the ethylene peak was reduced 63% in relation to that of 'Alcobaca'. The PG activity was lower than the PG activity obtained for ' Alcobaca ' . A comparison of the hybrids between 'Alcobaca' and normal ripening materials and the hybrids between 'Alcobaca' and the ripening mutants rin and rin-1 suggests gene dosage effects. In the hybrids between 'Alcobaca' and normal ripen- ing tomatoes, the ethylene peak was reduced 22% in comparison to normal ripening materials, and in the hybrids 'Alcobaca' and ripening mutants rin and rin-1, ethylene peak was reduced 66% in comparison to normal genotypes. PG activity was re- duced 33% in the hybrids normal X 'Alcobaca' and 60% in the hybrids rin and rin-1 X 'Alcobaca' in comparison to normal ripening tomatoes. Gene dosage effects for the genes nor and rin in double heterozygotes were reported by Tigchelaar et ad. (1978b) for respiration, peak ethylene, carotene production, and PG activity. In the present studies gene dosage effects were not found in the 'Alcobaca' X rin (rin-1) hybrids for respira- tion and color expression. These hybrids had normal red color (a/b ratio above 2.0). The dosage effects reported for 'Alcobaca' and the ripening mutants rin and rin-1 may have a certain degree of bias because isogenic lines were not used. 90 A multiple regression model to correlate storage life of the fruits with the different ripening parameters includ- ing color ratio a/b, firmness at the red stage, firmness 7 days after the red stage, firmness 15 days after the red state, maximum respiration, maximum ethylene, and PG activity indicated that the most closely related factor was PG activ- ity. Once this variable was included in the model, none of the remaining parameters was significant. The above results do not indicate that PG activity is the only factor that enhances fruit shelf life because the different ripening processes are not independent but appear to be highly syn- chronized (Tigchelaar et a^L. 1978a) . The close relationship between PG activity and storage life could be explained by the action of this enzyme on other ripening processes. Strand and Musell (1975) and Strand et al. (1976) indicated that PG induces release of cell wall-bound enzymes which are responsible for a number of processes such as ethylene production, carotene synthesis and respiratory control. Tigchelaar et al_. (1978a) postu- lated that both rin and nor mutants produce defective PG , and this enzyme (or enzyme complex) is required to condi- tion fruit tissue to undergo subsequent changes associated with the ripening process. Sacher (1962) and Salomos and Lati, cited by Tigchelaar et al. (1978a), indicated that this conditioning process may entail compartmentation changes required for the complex sequence of events 91 associated with the ripening process. Tigchelaar et al . (1978a) indicated that aberrancies in respiratory behavior, ethylene production, and carotene synthesis in the mutants represent secondary effects caused by failure of PG-induced modifications in cellular compartmentation . Based on previous studies in which applications of ethylene or the analogue oleoffin propylene did not induce normal ripening of the rin and nor fruits (McGlasson et al. 1975a), Frenkel and Garrison (1976) concluded that ethylene does not control the onset of ripening in tomato fruit. Tigchelaar et; Dwain D. Gull Associate Professor of Horticulture Science e -