Biotechnology for Improved Foods and Flavors - American Chemical

Chapter 10

In Vitro Tailoring of Tomatoes To Meet Process and Fresh-Market Standards 1

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M. L. Weaver , H. Timm , and J. K. Lassegues 1

Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany,CA94710 Department of Vegetable Crops, University of California, Davis,CA95610

Downloaded by COLUMBIA UNIV on January 21, 2018 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch010

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Expansion of genetic variability, by providing accessability to hidden genetic traits, has been accomplished by development of somaclonal/feedback-inhibition technology. This technology allows processors and breeders to access new genetic traits to improve the fresh-market quality of tomatoes, to minimize the degree of processing and to develop new high-quality products. Through selection of specific chemical and stress environments that challenge the feed-back-inhibition system of plants, over 300 new genetic variants were created with total solids contents of 7.0% to 15.0%. The increase in total-solids content is primarily due to an increase in soluble-solids content. The soluble solids/total solids increased from about 81% to 90% as total solids increased from about 8% to 13%. Insoluble solids decreased from about 22% to 10%. Reducing sugar/soluble-solids, however, remains constant at about 63.5%, over the same range of total solids. Fructose/glucose ranged from 1.25 to 1.13. Other traits of commercial importance: high juice consistency; firmness; different flavors; extended shelf life of 2 to 5 weeks; and different fruit shapes and sizes have been created and isolated in in-vitro created variants. To improve the potential of high-solids variants for commercial exploitation other technologies were developed: pollen screening for disease-resistance and environmental-stress tolerance, phytotoxin screening for disease resistance; and nonenzymatic release of pollen protoplasts for direct transfer of genetic traits. Raw material quality is the key to the development of high-quality fresh-market tomatoes, and to increasing the efficiency and minimizing the degree of processing. It is virtually impossible to minimize processing unless raw-material composition can be standardized and controlled. Creating these desirable changes in raw0097-6156/96/0637-0109$15.00/0 © 1996 American Chemical Society

Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS

material composition requires an expansion of genetic variability. Standard breeding technology, however, often does not allow accessibility to important quality, cultural, and biotic- and abiotic-stress traits even though considerable genetic variability may already exist in the explant tissue. Access to these valuable hidden genetic traits can now be made available through new biotechnology procedures. Induced and spontaneous alterations have been uncovered through somaclonal variations (7,2) resulting in several novel and agriculturally useful genetic variants (3-6). Somaclonal variation is the result of preexisting genetic traits and cell-culture induced variations. Genes can be uncovered, or modified, by specific chemicals in a culture medium, or they can be expressed in response to biotic or environmental stresses applied to the whole plant, or explants, prior to, or after, cultures have been made. This technology will allow processors to actively participate in the creation and development of raw materials more specifically amendable to meet their particular processing standards. Creation of High-Solids Content, Tissue-Culture Induced Tomato Variants A modified somaclonal/feedback-inhibition challenge technology was used to control and direct the in-vitro creation of tomato variants. Small leaves taken from 4- to 6-week old plants of well accepted tomato cultivars were sterilized in a 7% Clorox solution for 10 to 15 minutes, and then rinsed 3 times in sterile, glassdistilled water. Leaf rachis were prepared by trimming leaflets to about 0.6 cm on each side of the midrib, cutting off the leaf tip and leaving a small piece of the petiole. The midrib was then cut in half and each cut half was placed with the cut side down on to the culture medium (7) in magenta tubes. The medium consisted of MS salts (Gibco); nicotinic acid (1.0 mg/1); thiamine (1.0 mg/1); pyridoxine-HCl (1.0 mg/1); BA (2.5 mg/1); IAA (1.2 mg/1); sucrose (3.0%); myoinositol (100 mg/1); Bacto agar (0.8%). The pH of the mixture was adjusted to 5.8 prior to adding agar. Fifteen ml of medium was dispensed into magenta tubes and autoclaved at 121 °C for 25 minutes. To challenge the explant, selected metabolites and antimetabolites of the sugar pathway, various enzyme inhibitors and inorganic chemicals in ascending concentrations were tested. Individual plant were then exposed to heat stress. A specific protocol is now being prepared for patent consideration and cannot be frilly described at this time. As a starting point, a concentration that just started to inhibit the formation of shoots was determined. Then a new challenge medium was made starting at this concentration with further increases in the concentration by small increments until all but 1 or 2 of the explants failed to produce shoots. The few explants with shoots were kept in culture until rooted, then transplanted in coil into 10 cm pots until they reached the 3-true-leaf stage, then retransplanted into 10 to 20 liter pots and grown in a greenhouse at 25°C until fruits matured. Fruits were photographed whole and cut in half. A drop of juice was then squeezed onto a differential refractometer (Atago PR-1, NSG Precision Cells, Inc., 195G Central Ave., Farmingdale, N.Y. 11735) to determine the % soluble solids. Fruits with a reading of less than 7.0% were discarded. Seed of fruits with more than 7.0% was saved and stored. Plants generated from these seeds were grown to maturity and then fruit evaluated for


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soluble-solids content as before. This process was repeated for several generations to test fruit stability. When found to be stable, new explants were made and rechallenged for the creation of new variants. Juice of variants with soluble-solids contents 7.0% were evaluated for total-solids content using a CEM microwave oven (CEM AVC-80, CEM Corporation, 3100 Smith Farm Road, Mathews, N.C. 28105). The difference between total solids and soluble solids was expressed as insoluble-solids content.


Evaluation of High-Solids Tomato Variants Over 300 high-solids, tissue culture variants were created with total-solids contents from 7.0 to 15.0%. As the total solids increased, soluble solids also increased and insoluble solids decreased (Table I). Table I. Change in Soluble-Solids/Total Solids and Insoluble Solids/Total Solids as Tomato Variants Increase in Total-Solids Content Soluble

Insoluble

Sol. Solids (%) Insol. Solids (%)

Total Solids Solids (%) Solids (%) Tot. Solids (%) Total Solids (%) 7.8 8.8 9.7 10.8 11.5 12.7

6.3 7.3 8.2 9.2 10.2 11.4

1.5 1.5 1.5 1.6 1.3 1.3

80.8 83.0 84.5 85.2 88.7 89.8

19.2 17.0 15.5 14.8 11.3 10.2

As the soluble-solids content increased from 6.3% to 11.4%, soluble-solids/totalsolids increased from about 81% to 90%, and the insoluble-solids/total-solids decreased from about 19% to 10%. Although a high soluble-solids content can result in an increase in the pounds of tomato paste, or sauce, per ton of tomatoes, this does not always mean a big advantage for processors that produce highconsistency products. Often the recovery is greater from thick-juice cultivars that express the highest insoluble-solids content. High-Solids Content and Reducing-Sugar Content in Tomato Variants Solids content continues to be a major selection factor in tomato breeding evaluations. As solids content increases, so does the sugar content. Using a Waters HPLC system equipped with Millenium 3.1 software, the reducing-sugar contents of hundreds of high-solids, tissue-culture variants were determined. Twenty ml of tomato juice was extracted in boiling 80% ethanol for 1 h in a crudefiber extracter. The extract was centrifuged at 600 rpm for 15 min, then filtered twice through a 0.22jan filter to assure all sediment was removed. Five ml of the


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clarified extract was then run through a Sep Pak-C18 column. Triplicate 10/d samples were then injected into the HPLC. The parameters used were: flow rate of 3.8 cm /min at a column temperature of 35°C; attenuation 4X; run time 15 min using a Waters 510 pump; detector was a Waters 410 differential refractometer. A carbohydrate 3.9 X 300 mm column was used for separation. The mobile phase was acetonitrile/water (85:15, v/v). Only curves for fructose and glucose were detected and peak height was used for quantitation. The sum of peak heights of fructose and glucose, determined from standard curves, was used to calculate the percentage of reducing sugars in the total soluble solids. While soluble solids increased as a percentage of total solids (Table I), the reducing-sugar content, as a percentage of soluble solids, tended to remain fairly constant in variants from 7.0% to over 11% soluble solids (Table II).


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Table II. Relationship of Reducing Sugar Content to Soluble-Solid Content in High-Solids Tomato Variants Reducing Sugar Total Soluble Solids (%) 7.0 8.4 9.6 10.0 11.6

Total Soluble Solids Fructose (%) (%) 62.5 61.9 62.5 64.0 64.7

2.5 2.8 3.2 3.4 4.0

Fructose Glucose (%) 2.0 2.4 2.8 3.0 3.5

Glucose 1.25 1.17 1.14 1.13 1.14

The average reducing sugar/soluble solids content was 63.5% The average ratio of fructose/glucose was 1.17. Both values compare with those of standard commercial tomato cultivars (8). Other Important Genetic Variants Created. A wide range of fruit sizes and shapes varying from 15-g cherry tomatoes to large 500-g fresh-market variants and 65 to 125 g processing types in elongated, round, and oval shapes were created. Variants with almost solid interiors and with very small locule areas were evaluated for dehydration as dices and slices. Dried products maintained a better shape, and rehydrated to a more natural piece form, than did those from commercial lowsolids cultivars. Most variants were extremely firm when harvested fully ripe and could be held at room temperature (21°-25°C) for 2 to 5 weeks without softening and shriveling. With few exceptions, variants had pH values at commercially acceptable levels of 4.0 to 4.4. High-solids variants were very sweet with several distinctly different flavors, especially in cherry types. Juice consistency of some variants was so thick it had to be scraped off the pulper and could not be poured


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from the beaker. High-solids cherry-tomato variants created from high-solids variants had a firm processing-type interior, not the typical soft interior of most commercial cherry tomatoes. Field Trial of High-Solids Variants. In cooperation with tomato breeders from industry, several high-solids variants were grown in a commercial field and their fruit was compared with a high-solids commercial cultivar, UC204C (Table III). Fruit of most hybrid crosses among high-solids variants were as large or larger than the commercial standard. The pH of all variants was at, or below, 4.4 and all were lower than that of the commercial standard. All hybrids were higher in soluble solids than the commercial standard. It is usually accepted that as fruit size increases solids content decreases. In this study however, the largest fruit had the highest solids content, 45% more than the standard tomato cultivar. Also, there is usually a negative relationship between high-solids content and juice consistency. Bostwick readings indicated that with fruit of many high-solids variants this relationship may not hold true. While all hybrids had more soluble solids than the standard, all but three had better fruit consistency (lower Bostwick readings) than the control. Table III. Field Evaluation of Hybrids Made From U.S.D.A. High-Solids Hybrids

Hybrid 1 2 3 4 5 6 7 8 9 10 UC204C (Avg. of 3 trials)

Fruits /lb

Bostwick (cm/30 sec)

pH

Brix (%)

6.6 8.0 6.6 7.3 5.3 6.2 10.0 7.3 9.0 7.3

4.40 4.33 4.26 4.41 4.38 4.30 4.34 4.36 4.30 4.38

7.7 7.9 7.8 7.4 9.3 7.2 7.1 8.3 7.2 7.0

13.5 20.5 15.0 12.0 24.0 17.0 24.0 14.5 15.0 15.0

7.2

4.60

6.4

20.60

Other Technologies to Improve the Commercial Potential of High-Solids Tomatoes To assist in enhancement of the commercial potential of high-solids variants, work on other important genetic traits is needed. Other somaclonal variations, (both


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desirable and undesirable) can occur along with high-solids variations. Desirable cultural traits such as: optimal foliar cover; plant size; and determinate and indeterminate growth are now being incorporated into high-solids variants. These high-solids variants will also have to be screened for environmental stresses and disease resistance. With modern advances in biotechnology now used to uncover hidden genetic traits, and to transfer these traits to plants of the same and other diverse species, rapid and accurate screening strategies must be developed to identify stress response in new variants. Pollen Screening for Heat Tolerance. Since reproductive organs are very sensitive to diseases and environmental stresses (9,10), pollen activity was chosen as an appropriate screening tool. Pollen germination and tube growth are representative of the growth of the male gametophyte and have been correlated with the growth and function of the sporophyte (11,12). Pollen screening has the added advantage of being able to treat hundreds of pollen grains in a single petri dish and reading results in 1 to 3 hours. Pollen can be exposed to many stress factors using little space and low-cost equipment, when compared to expensive greenhouse facilities, or utilizing field studies. Under controlled laboratory conditions pollen can easily be germinated and tube growth initiated in simple and defined media of any time of the year. Pollen function has been used to screen for salt tolerance (75), metal tolerance (14), and high-temperature tolerance (15,16). Using a heat-tolerance, pollen-screening test developed at WRRC (16), tomato hybrids created by crossing the heat-tolerant (HT) Grivorski cultivar with U.S.D.A. high-solids variants were exposed to 50 C for 2 h. Data showed (Table IV) that high-temperature tolerance was present in all hybrids in varying degrees. Table IV. Pollen Screening for Heat Tolerance of High-Solids Tomatoes Created from Grivorski and Exposed Two Hours at 50° C.

Tomato Selection Grivorski Hybrid 1 Hybrid 2 Hybrid 3 Hybrid 4 79N45

Germination (%) 78 80 53 82 22 0

Pollen Tube Length (fim) 2950 3050 1500 3100 240 0

A tomato selection, 79N45, obtained from the Department of Vegetable Crops, University of California, Davis, showed no pollen viability after two hours at 50° C, which is the normal response of pollen of most commercial tomatoes.



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Pollen Screening for Disease Resistance Using Phytotoxins. A liquid shake culture was made using a 4:1 (V-8 juice: glass distilled water) medium. The pH was adjusted to 5.8, and the medium sterilized at 121°C for 15 min. Two hundred and fifty ml of medium in 500 ml Erlenmeyer flasks was inoculated with Fusarium and Verticillium races supplied by Dr. Ken Kimball, Moran Seed Co., Davis, CA, and shaken for 7 days at 24°C. The medium was then centrifuged at 1000 rpm to separate out the mass of spores and filaments. The filtrate was run through a 0.45^tm filter and 0.25 ml was applied by pipette to several locations on a solidagar pollen-germination media (76) and held overnight. Pollen from different tomato variants and hybrids was shaken onto the areas where toxin had been applied. After 3-6 h, at 100% relative humidity, the areas of agar medium where pollen was applied were removed by cork borer and placed on a microscope slide. A drop of phyloxin-methylgreen dye (76) was applied to the agar. A cover glass was placed on the agar and the slide was placed on a heated hot plate. As soon as the agar began to melt the slide was quickly removed. The slide was then microscopically evaluated to determine the percentage of pollen germination and to measure the length of pollen tube growth. Table V. Pollen Screening of High-Solids Variants and Hybrids Made Among High-Solids Variants for Disease Resistance

Tomato Selection

Fusarium oxysporum Pollen Germination (%)

Variant 1 Variant 2 Variant 3 Variant 4 Hybrid 1 Hybrid 2 Hybrid 3 Ace Ace 55

65 5 72 82 15 5 67 8 76

Verticillum albo-atrium Pollen Tube Length (fim) 2 35 80 85 78 10 25 15 82

In cooperation with industry tomato breeders, pollen-screening tests for disease resistance were initiated using commercial cultivars and breeding selections with known disease resistance. These tests indicated that a relationship existed between pollen-screening tests and standard root-inoculation tests. High-solids variants, or hybrids, were not evaluated at that time because their disease resistance was not known. Based on these studies, preliminary pollen-screening tests were conducted on high-solids tomato variants and hybrids among them (Table V). The data showed that there were differences in pollen function of both variants and hybrids


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in response to toxins from both Fusarium and Verticillium cultures. Pollen of two cultivars, 'Ace' known to be susceptible and 'Ace55' resistant to both disease organisms, showed a positive relationship between pollen function and known disease resistance. As soon as storage tests on the filament/spore mass are completed the mass will be ground and used for root inoculations of seedlings of the same variants and hybrids to determne their disease response and compare it with that of pollen function response to the phytotoxin. Other Methodologies Developed to Create and Commercialize New HighQuality Tomato Variants. When this study to tailor raw material to improve the quality of tomatoes was initiated, it was believed that pollen protoplasts could be used to accept new genetic traits to create and transfer genes from one plant source to another. Pollen is basically a protoplast covered with a thick cell wall. Once the cell wall is removed, DNA can enter the protoplast by direct uptake (79), or by electroporation into the pollen tube (20). The obstacle to using pollen protoplasts is the cell wall which is composed of sporopollenin, one of the most impervious natural materials known. This material cannot be disintegrated by any of the common cell-wall degrading enzymes without causing protoplasm disfunction. While searching for alternative methods of protoplast release, it was found (18) that viable pollen protoplasts of beans could be removed in less than five minutes using dilute salt solutions. By using a solidified agar medium with different mineral contents, protoplasts can be released from the pollen of tomato, pepper, zucchini, peas, alfalfa, cucumber (27). Since pollen tubes are basically extended protoplasts they can also be used to take up DNA. By varying the concentration and type of mineral, primarily calcium, in the protoplast-release medium, it was possible to stop the growth of the pollen tube just as it emerged from the cell wall creating a partial protoplast. By holding the partial protoplast in a DNA solution followed by pollination of the stigma with the partial protoplast, or by insertion of the DNA-impregnated partial protoplast directly into the ovary some fertilization occurred, but as yet the transfer of genetic traits has not been definitely confirmed. The procedures are continually being modified. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Evans, D . A . ; Sharp; W.R.; Medina-Filko, H.P. Amer. J. Bot. 1984, 71, 759774. Larkin, P.S.; Scowcraft, W.R. Theor. Appl. Genet. 1981, 60, 197-214. Brettelli, R.I.; Ingram, D.S. Biol. Rev. 1979, 54, 329-345. Heinz, D.J. In Mutation breeding of vegetatively propagated perennial crops; G A O / A E P : Vienna, 1972, pp. 53-59. Chaleff, R.S. Science 1983, 219, 676-682. Nickel, L . G . Crop Sci. 1977, 17, 717-719. McCormick, S.; Niedermeyer, J . ; Fry, J . ; Barnason, A . ; Horsch, R.; Fraley, R. Plant Cell Reports 1986, 5, 81-84. Kadar, A.A.; Morris, L.L.; Stevens, M . A . ; Albright-Holen, M . J. Amer. Soc. Hon. Sci. 1978, 103, 6-13.



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9. El Ahmadi, A . B . ; Stevens, M . A . J. Amer. Soc. Hort. Sci. 1979, 104, 691696. 10. Sugizama, T . ; Iwahor, S.; Takahashi, K . Acta. Hort. 1966, 4, 63-69. 11. Tanksley, S.D.; Zamir, D . ; Rick, C . M . Science 1981, 213, 453-455. 12. Mulcahy, D . L . Science 1971, 171, 1155-1156. 13. Sacher, R . F . ; Mulcahy, D . L . Plant Physiol. 1981, 67, 67-96. 14. Searcy, K . B . ; Mulcahy, D . L . Amer. J. Bot. 1985, 72, 1695-1699. 15. Weaver, M . L.; Timm, H . ; Silibernagel, M . J.; Burke, D. W. HortScience 1985, 110, 797-799. 16. Weaver, M . L.; Timm, H . HortScience 1989, 24, 493-495. 17. Owczarak, A . Stain Technology 1952, 27, 249-251. 18. Weaver, M . L.; Timm H . ; Breda, V . ; Gaffield, W. J. Amer. Soc. Hort. Sci. 1990, 115, 640-643. 19. Kanji, O. O.; Gamborg, I.; Miller, R. A . Can. J. Bot. 1972, pp. 2077-2080. 20. Href, A . ; Abdul-Baki, J. A . ; Saunders, B. F.; Pillarelli, E . A . Plant Sci. 1980, 7, 181-190. 21. Weaver, M . L . 1992. Methods to obtain intact viable protoplasts from pollen grains. U.S. Patent #5, 169, 777.


Biotechnology for Improved Foods and Flavors - American Chemical

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