Quality Factors of Fruits and Vegetables - ACS Publications

Chapter 30

to

the

Applications

of

Improvement

of

Biotechnology Quality of

Fruits

and

Vegetables W. R. Romig and T. J. Orton

Downloaded by UNIV LAVAL on April 8, 2016 | http://pubs.acs.org Publication Date: September 7, 1989 | doi: 10.1021/bk-1989-0405.ch030

DNA Plant Technology Corporation, Cinnaminson, NJ 08077

The integration of emerging biotechnologies with conventional breeding will greatly facilitate the modification of quality or "value-added" attributes of fruits and vegetables, (i.e. appearance, organoleptic, nutrition, physiological benefit and safety). Modern plant genetics will make possible the specific control of enzymes contributing to the development and deterioration of produce quality or the accumulation of secondary metabolites which provide added-value to a specific crop. However, to fully exploit this potential, an understanding of the biochemical and genetic control of these value-added attributes is necessary. Following is a brief discussion of the application of biotechnology to the improvement of the quality of fruits and vegetables. Plant breeding has played a dominant role in providing the diversity and quality of fruits and vegetables enjoyed by the consumer and processor today, and will continue to make improvements related to production, enhancing such agronomic traits as pest resistance, yield, and stress tolerance. These production contributions by "supply-side" genetics affect the availability and cost of raw materials, whereas, "utilization" or "value-added" genetics determine the processibility, nutrition, overall quality, and functionality of the raw materials if subjected to processing. Traditionally, the food industry has used available raw materials as commodities and built in "added-value" through processing and packaging. The food industry is now requesting improved functionality of raw materials in the form of nutritional and physiological benefit, chemical and physical properties, and 0097-6156/89/0405-0381$06.00/0 ο 1989 American Chemical Society

Jen; Quality Factors of Fruits and Vegetables ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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improved preservation; the consumer i s demanding convenience, premium quality, year-round a v a i l a b i l i t y , and the absence of p e s t i c i d e s . Concomitantly, s c i e n t i s t s and consumers are developing a better understanding and awareness of the n u t r i t i o n a l , p h y s i o l o g i c a l , and even medicinal benefits of the d i f f e r e n t components of f r u i t s and vegetables. Produce q u a l i t y i s defined or referenced i n several d i f f e r e n t ways depending upon what portion of the food production and d i s t r i b u t i o n chain i s being referred to, i . e . harvest, marketing, shipping, storage, or eating. These d e f i n i t i o n s can be summarized into f i v e major q u a l i t y factors: (1) appearance, (2) f l a v o r , (3) n u t r i t i o n and well being, (4) texture, and (5) safety, which are influenced by such preharvest and postharvest factors as: (1) genotype, (2) c u l t u r a l and c l i m a t i c conditions, (3) harvesting practices, including the physiological state of the crop, (4) postharvest treatment, and (5) the various interactions among these factors. Although substantial progress has been made i n the q u a l i t y improvement of f r u i t s and vegetables which reach the consumer, emerging biotechnologies w i l l greatly broaden the scope of products targeted by plant breeding. Modern c e l l u l a r and molecular genetic approaches w i l l provide new strategies to improve those value-added c h a r a c t e r i s t i c s mentioned. However, the r e a l i z a t i o n of t h i s p o t e n t i a l i s greatly dependent on progress i n the understanding of functional t r a i t s at the biochemical and genetic l e v e l s . Thus, the following perspective i s based on a l i m i t e d number of examples of r e a l i z e d applications, work i n progress, and speculation. EMERGING BIOTECHNOLOGIES AND

CROP IMPROVEMENT

This section w i l l provide a very b r i e f review of the biotechnological state of the a r t as of t h i s writing. Any present treatment i s rendered obsolete by rapid advances. The interested reader i s urged to consult one or more excellent recent volumes on t h i s subject (1-2). Biotechnology consists of a body of i n t e r r e l a t e d methods wherein sub-organismal units, e.g. organs, tissues, c e l l s , organelles, chromosomes, genes, etc, are manipulated. The f i e l d of plant c e l l and tissue culture has i t s o r i g i n i n the early 20th century (3). The finding by Skoog and M i l l e r (4) that developmental changes i n cultured tissues could be influenced by phytohormones i n i t i a t e d an era of rapid advances: i n v i t r o meristern propagation f o r v i r u s elimination, micropropagation, protoplast i s o l a t i o n , culture, and regeneration, microspore culture, and transformation with T-DNA vectors v i a i n f e c t i o n with the crown g a l l pathogen (Agrobacterium tumefaciens^. Most of the methods of plant c e l l and tissue culture were derived empirically. In f a c t , we s t i l l understand very l i t t l e of the mechanisms which underlie developmental transformation. Experiments i n the early to mid 1970's quickly established that plant species and even genotypes within species varied tremendously i n the r e l a t i v e ease with which they could be cultured and regenerated. For example, the genotypes A188 and Black Mexican Sweet of maize would regenerate while other genotypes were r e c a l c i t r a n t (5-6). U n t i l recently, regeneration from c e l l and tissue culture of c e r t a i n important crops such as cotton (7) and soybean (8) was only demonstrated i n c l o s e l y r e l a t e d



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wild species. Persistence i n exploring new empirical approaches has been l a r g e l y successful i n overcoming r e c a l c i t r a n c e . However, while i t i s clear that predisposition for c e l l p r o l i f e r a t i o n i n v i t r o and regeneration has a genetic component, no absolute b a r r i e r s e x i s t i n the development of successful protocols. Numerous successful applications of c e l l culture technologies to crop improvement have now been reported. Most notable among these are protoplast-mediated alloplasmic conversion, doubled haploids, somaclonal v a r i a t i o n , and i n v i t r o mutant s e l e c t i o n . The doubled haploid approach (9) i s u n i v e r s a l l y lauded by plant breeders, because the p o s s i b i l i t y of proceeding d i r e c t l y from a hybrid to an inbred harboring f i x e d recombinant gene assortments eliminates the need for inbreeding, unfortunately, gaps i n our knowledge and c a p a b i l i t i e s i n gametophyte development are numerous. Culture and regeneration of microspores or megaspores i s successful i n only a handful of plant species (e.g. Nicotiana. Brassica. Datura. Orvza. Capsicum) and the l i s t i s not growing very quickly (9) . In c e r t a i n rare cases, natural or g e n e t i c a l l y mediated processes during gametogenesis give r i s e to progeny derived from the egg only, thus imparting the same end r e s u l t as anther culture (10) . Also, i n barley, i t i s possible to recover haploid plants by hybridizing with the wild r e l a t i v e Hordeum bulbosum (11). Alloplasmic conversion i s a strategy which emerged from c a p a b i l i t i e s developed with plant protoplasts. O r i g i n a l l y touted as a method f o r the recovery of exotic i n t e r s p e c i f i c hybrids (12), protoplast fusion has proven to be an excellent way to approach the wholesale manipulation of mitochondrial genes (13). The predominant t r a i t of note i s cytoplasmic male s t e r i l i t y (cms), which i s of interest to the seed industry as a t o o l to f a c i l i t a t e hybrid seed production. L i t t l e or no impact on possible value-added t r a i t s impacting on food q u a l i t y , however, i s anticipated from alloplasmic conversion. Somaclonal v a r i a t i o n i n regenerated plants has been observed and reported since c e l l culture technology was very young (14). Early reports of such v a r i a t i o n were dismissed as exceptional or a r t i f a c t u r a l because the notion c o n f l i c t e d with the widely accepted view of genetic f i d e l i t y i n both eukaryotic and prokaryotic c e l l cultures. Larkin and Scowcroft (15) were noteworthy i n f i r s t suggesting that genetic i n s t a b i l i t y i s an i n t r i n s i c feature of plant c e l l cultures. Mounting evidence has much supported t h i s view. The o r i g i n a l Larkin and Scowcroft paper also espoused the exploitation of 'somaclonal v a r i a t i o n ' i n crop improvement. I t has been argued that using tissue culture to induce mutations i s operationally and perhaps even mechanistically equivalent t o mutagenesis v i a chemicals or i o n i z i n g r a d i a t i o n . A small number of papers have recently appeared, however, which demonstrate that the frequency and spectrum of mutants generated v i a mutagens vs. c e l l culture are indeed d i f f e r e n t (16). The a p p l i c a t i o n of somaclonal v a r i a t i o n to crop improvement i s a t t r a c t i v e because i t i s cheap, simple and requires few assumptions as compared to more directed approaches (17). The g l a r i n g disadvantage of the phenomenon i s that i t i s not q u a l i t a t i v e l y controllable. I f one possesses a l i n e which i s g e n e t i c a l l y superior i n a l l respect with one or few d e f i c i e n c i e s , somaclonal v a r i a t i o n can t h e o r e t i c a l l y be induced to mitigate the d e f i c i e n c i e s while



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leaving the desirable f r a c t i o n of the genotype undisturbed (17). There are, unfortunately, few concrete examples where t h i s has been accomplished convincingly. C e l l s e l e c t i o n i s a process wherein medium composition or physical environment are engineered to d i f f e r e n t i a t e the growth rate of mutant vs. non-mutant cultured c e l l s . This i s a t t r a c t i v e because the p r o b a b i l i t y of success i s much enhanced as compared with screening large populations of random somaclones. Moreover, searching for mutants i s much more e f f i c i e n t among populations of cultured c e l l s than f o r whole plants. Numerous examples of the successful application of t h i s approach e x i s t i n the l i t e r a t u r e and within commerce (18). The strategy i s most successfully applied when information exists regarding the biochemical pathways which culminate i n compounds of i n t e r e s t . For example, tryptophan auxotrophs can be selected by exposing c e l l s to 5-methyltryptophan (19). Exposing normal c e l l s to one amino acid involved i n a branch synthetic pathway i s i n h i b i t o r y because the control enzyme i s i n h i b i t e d r e s u l t i n g i n d e f i c i e n c i e s . C e l l s containing mutants which render the enzyme insensitive to feedback i n h i b i t i o n can grow under these conditions. Such c e l l s should also accumulate free amino acids by v i r t u e of acquired feedback i n s e n s i t i v i t y . Direct pertinence of molecular approaches to crop improvement was f i r s t demonstrated by Fraley et a l . (20), who successfully used T-DNA of Agrobacterium tumefaciens to introduce foreign DNA into a plant i n a permanent and heritable fashion. Subsequently, methods have been devised for the d i r e c t introduction of foreign DNA sequences (21-24) into the genomes of higher plants. At t h i s juncture, i t would appear reasonable to conclude that no t h e o r e t i c a l b a r r i e r s exist to the stable integration and expression of foreign DNA sequences or transformation into higher plants. U n t i l recently, major issues have imparted l i m i t a t i o n s which much constrain the widespread u t i l i t y of higher plant transformation. F i r s t , how can genes which condition a desirable genetic outcome be captured? Secondly, conventional thought holds that most economically important characters are conditioned by many genes which interact i n a spacial and developmental matrix. Moreover, many t r a i t s whose inheritance i s simple are either codominant or recessive. New techniques have now been forged which mitigate these l i m i t a t i o n s . Progress i n the i s o l a t i o n and capture of desirable genes o f economic importance has been much f a c i l i t a t e d by transposon mutagenesis (25). U n t i l recently i s o l a t e d genes of known function were r e l a t i v e l y rare because properties of polypeptide, mRNA, or nuclear sequence abundance were necessary. High abundance of genes or gene products makes i t possible to i s o l a t e desired DNA sequences d i r e c t l y from l i b r a r i e s or to work backward enzymatically from mRNA to i s o l a t e clones from a random genomic l i b r a r y . I f the polypeptide which conditions a desired t r a i t has been i s o l a t e d and sequenced, i t i s also possible to deduce the nucleotide sequence of an oligomer which could encode a part of the protein. The oligomer can then be used to locate the genomic sequence based on binding a f f i n i t y . Most aspects of phenotype are, however, encoded by a gene or genes whose products are not r e l a t i v e l y abundant, and i t i s not generally possible to ascertain the contribution of i s o l a t e d polypeptides and mRNAs based on sequence alone. An exception to



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t h i s i s f o r genes which are highly conserved over long periods of evolutionary history. Thus, i f one obtains a gene from a prokaryote which encodes an enzyme i n a fundamental biochemical pathway, a p o s s i b i l i t y exists that s u f f i c i e n t homology remains within the corresponding gene of higher eukaryotes to permit i s o l a t i o n by binding a f f i n i t y (26). New methods of gene i s o l a t i o n , which take advantage of natural polymorphisms and genetic transformation c a p a b i l i t i e s , have now been developed which transcend limitations imposed by the aforementioned techniques. I f a plant c e l l heterozygous Aa (respectively active and inactive a l l e l e s ) at a given locus i s transformed, the sequence or multiple copies thereof integrate into the host nuclear (and organelle) genome presumably at random. I f the sequence integrates i n or near the active a l l e l e , i t w i l l be turned o f f , thus changing the phenotype of c e l l s or plants from dominant A to recessive a. The transforming sequence can then be used to locate t h i s gene from a mixture of random DNA fragments. Since plant genomes are very large, and the l i k e l i h o o d of desired events low, transformation cassettes containing known transposable elements, such as AG (maize) and Tam-1 (AnthirrhinunO have been employed to improve such p r o b a b i l i t i e s (27). Examples of t r a i t s which have thusfar been recovered by t h i s approach include nuclear male s t e r i l i t y , a l t e r e d seed f a t t y acid composition, and c o r o l l a pigmentation. Recently, i t has been demonstrated that the T-DNA of A. tumefaciens can a f f e c t the i n s e r t i o n a l i n a c t i v a t i o n of genes by random integration. Implicit i n any molecular transformation approach to crop improvement i s the need to modulate the expression of the foreign gene. I t has been well documented that DNA sequences upstream from the 5* end of coding genes, or promoter, are necessary f o r normal t r a n s c r i p t i o n . While some promoters confer constitutive t r a n s c r i p t i o n of the corresponding coding sequence, most appear to respond to a stimulus such that they are only expressed at the proper place and time. Examples of such promoters include developmental, t i s s u e - s p e c i f i c , and signal mediated (28). I t has been demonstrated that polypeptides are produced following environmental or development stimuli which bind s p e c i f i c a l l y to 5' sequences and permit t r a n s c r i p t i o n to i n i t i a t e and proceed. At least some promoters have been shown to function s i m i l a r l y i n divergent plant species (29). The fact that most economic t r a i t s are inherited i n a recessive or complex fashion i s a much more daunting problem than finding simply inherited genes. Recessive t r a i t s appear to be at least p a r t i a l l y approachable by transformation of targeted gene l o c i with sequences which encode an mRNA complementary to that of the native coding sequence, or "antisense RNA" (30). Antisense RNAs t h e o r e t i c a l l y inactivate sense mRNAs by forming duplex structures which interfere with normal t r a n s l a t i o n . Many processes can be postulated, however, which might antagonize the desired r e s u l t . More evidence on the e f f i c a c y of the antisense approach to recessively inherited t r a i t s w i l l be necessary before any judgement regarding effectiveness i s possible. Recent findings about t r a i t s exhibiting complex inheritance are very encouraging from the standpoint of a c c e s s i b i l i t y to molecular approaches. A general assumption i n the f i e l d of quantitive genetics i s that t r a i t s are conditioned by a large number of genes with equal, additive e f f e c t s . Cosegregation experiments involving



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r e s t r i c t i o n fragment length polymorphisms (RFLPs) suggest that complex t r a i t s such as y i e l d and s o l i d s content are controlled by genes with d i f f e r e n t quantitative effects (31). In f a c t , most of the phenotypic v a r i a t i o n may be accounted f o r by fewer than f i v e d i s t i n c t genes. I f t h i s i s the case, and i f one was able to i s o l a t e desirable a l l e l e s of genes exerting major e f f e c t s on quantitative t r a i t s , then transformation would appear quite possible f o r the manipulation of such t r a i t s (32). As mentioned above, RFLPs have been very useful f o r dissecting the genetic components of very complex characters. RFLPs are differences i n base sequence r e s u l t i n g i n the addition or deletion of r e s t r i c t i o n enzyme recognition and cutting s i t e s , or other changes which a f f e c t sequence length between two r e s t r i c t i o n s i t e s . Mutations of t h i s type apparently occur r e l a t i v e l y frequently i n that they are abundant even among some c l o s e l y related genotypes (31). Since they occur randomly within the genome and are p h y s i o l o g i c a l l y s i l e n t , many can be detected simultaneously within a single organism, thus f a c i l i t a t i n g studies of linkage and attempts to derive genetic " f i n g e r p r i n t s " f o r proprietary protection. Commercializing these biotechnological strategies w i l l require a c a r e f u l integration with plant breeding, postharvest physiology, food science, and packaging. Transformation, alloplasmic conversion, somaclonal v a r i a t i o n , and mutant s e l e c t i o n are a l l powerful techniques to broaden and/or manipulate germplasm. RFLPs and dihaploids are methods to a s s i s t the breeder i n reducing the time and e f f o r t necessary to achieve desired recombinants. A thorough understanding of the product targets i s e s s e n t i a l to r e a l i z a t i o n of the f u l l economic potential of biotechnological strategies. VALUE-ADDED GENETICS Although the improvement of functional properties can be greatly enhanced by complementation of conventional breeding with the new technologies, progress i s s t i l l limited by the lack of knowledge regarding the biochemical control of s p e c i f i c attributes and the fact that many such t r a i t s are multigenic, which greatly complicates improvement strategies. Even with these complications, however, there w i l l be increased emphasis on the development of raw materials g e n e t i c a l l y designed f o r the processor and consumer. A few examples of f r u i t s and vegetables can be c i t e d , where focus has been on the improvement of composition or functional properties. A f a i r l y thorough understanding of the biochemical and genetic control of carbohydrate metabolism i n sweet corn has allowed considerable progress i n the development of genotypes with an array of sweetness, texture, color, and rates of sugar to starch conversion (33). In spite of poor seedling vigor, d i f f e r e n t i a t e d sweet com v a r i e t i e s , based on the sh2 gene (endosperm gene « shrunken-2), are being grown commercially. Over 90 percent of the sweet corn produced i n F l o r i d a i s sh2 hybrids. Increasing the solids content of tomato i s a demonstration of the modification of multigenic t r a i t s by modern biotechnology. Although various i n s t i t u t i o n s have u t i l i z e d several approaches, only somaclonal v a r i a t i o n has proven successful, with DNA Plant



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Technology Corporation (DNAP) receiving a PVPA c e r t i f i c a t e ( C e r t i f i c a t e of Plant Variety Protection No. 8400146) f o r DNAP-9. DNAP-9, a derivative of UC82B, a standard open p o l l i n a t e d processing v a r i e t y , was obtained through the regeneration of plants from tissue culture. Data indicated that the solids increase of approximately 20% was the only p r i n c i p a l difference from the parental material. Although DNAP-9 did demonstrate the u t i l i t y of a somaclonal v a r i a t i o n approach, i t has not proven to be competitive with recently developed hybrids with higher y i e l d s and comparable s o l i d s content. The most e f f e c t i v e means of leveraging biotechnology f o r the q u a l i t y improvement of f r u i t s and vegetables i s to complement the t r a d i t i o n a l sciences. To provide unique high q u a l i t y f r u i t and vegetable products w i l l require synergies provided by optimizing the raw material, and the c u l t u r a l and environmental conditions f o r production, processing, packaging, and d i s t r i b u t i o n conditions. The V i d a l i a onion i s an example of the combination of genotype and s o i l properties that provides a unique product. V a r i e t i e s of some crops such as apples respond d i f f e r e n t l y to controlled and modified atmosphere. The maturity of f r u i t s and vegetables i s also a f a c t o r in determining t h e i r response or s u s c e p t i b i l i t y to preservation environments. This suggests that f o r increased shelf l i f e , new v a r i e t i e s may be selected for biochemical modifications i n t h e i r response or resistance to s p e c i f i c environments. An example i s VegiSnax vegetable s t i c k s , which DNAP and DuPont, A g r i c u l t u r a l Products D i v i s i o n w i l l market. This product i s based on optimizing the q u a l i t y of the raw celery and carrots through v a r i e t y improvement, then packaging i n a modified atmosphere to provide a high q u a l i t y product with a shelf l i f e of 30 days under r e f r i g e r a t e d distribution. R

NEW GENOTYPES WITH UNIQUE PROPERTIES WHICH ENHANCE THE QUALITY OF FRESH PRODUCE Because of increasing public concern regarding p e s t i c i d e a p p l i c a t i o n and p e s t i c i d e residues, insect and disease resistance, t r a d i t i o n a l l y considered as part of the "supply side" genetics, may now be accounted f o r on the "value-added" genetics column. Because of i t s economic importance, tomato i s the major vegetable crop which biotechnology and a g r i c u l t u r a l products firms have targeted to further improve resistance to insects and pathogens. This has resulted i n recent f i e l d studies with g e n e t i c a l l y engineered tomato plants exhibiting resistance to TMV and lepidopteran insects. Postharvest losses due to the presence of plant pathogens are s i g n i f i c a n t . Recent reports (34) regarding the i s o l a t i o n of a chitinase gene, suggests the p o s s i b i l i t y of transforming plants with genes encoding f o r fungal c e l l wall degrading enzymes. Health and Well Being. F r u i t s and vegetables have always had a strong r e l a t i o n s h i p to human health and n u t r i t i o n . Such benefits as dietary f i b e r and several e s s e n t i a l nutrients are contributed with minimal c a l o r i c contribution and no c h o l e s t e r o l . Recently, because of the established p h y s i o l o g i c a l benefit of dietary and soluble f i b e r ( i . e . the reduction of c h o l e s t e r o l ) , f r u i t s and vegetables have taken on additional s i g n i f i c a n c e as one



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of the major sources of dietary f i b e r . There has also been some interest i n the p o t e n t i a l of h o r t i c u l t u r a l crops as possible sources of Omega-3 f a t t y acids associated with reduced incidence of heart disease, (35). Vegetables, although low i n f a t , are a source of alpha-linolenic acid, the precursor of eicosapentaenoic a c i d (EPA) and docosahexaenoic acid (DHA). Linolenic acid predominates i n the chloroplasts of green plants while seeds are generally r i c h e r i n l i n o l e i c acid, precursor to archidonic acid. Currently there are no studies directed toward the evaluation of breeding green vegetables to enhance Omega-3 f a t t y acid content. There i s also interest i n crops f o r anticarcinogenic compounds. Consumption of cruciferous vegetables has been associated with a reduction i n the incidence of some s p e c i f i c s i t e cancers, i . e . colon and stomach (36); and t h e i r increased consumption has been endorsed by The American Cancer Society (37). Epidemiological studies have suggested that higher intake of carotene may reduce the r i s k of cancer (38). For example, carrots (Daucus carota) are thought to be the most important source of pro-vitamin A carotenes, and at the same time are a source of high q u a l i t y f i b e r . Strawberries contain e l l a g i c acid, a substance speculated to protect normal c e l l s from becoming cancerous. T r a d i t i o n a l l y , a n t i v i r a l , antithrombotic, and diuretic, benefits have been associated with s p e c i f i c metabolites i n a range of spices. Allium species, for example, have a long h i s t o r y and f o l k l o r e pertaining to t h e i r a b i l i t y to i n h i b i t s p e c i f i c diseases. Other herbs have been associated with a d i v e r s i t y of remedies, generally attributed to secondary metabolites. Quantitative changes in secondary products produced by plant c e l l s are quite responsive to modification by plant c e l l culture. This often r e s u l t s i n the regeneration of plants exhibiting abnormally high concentrations (39). This suggests that components exhibiting such benefits could be induced to accumulate at higher concentrations i n new v a r i e t i e s . Breeding programs are slow, expensive, and very time consuming, even with the complementation of modern biotechnology. However, rapid a n a l y t i c a l screening procedures, nondestructive analysis, i . e . NMR and h a l f seed analysis, integrated with a breeding program make genetic modification increasingly e f f e c t i v e . As the p h y s i o l o g i c a l and anticarcinogenic benefits of these plant components become c l a r i f i e d , and considering the general absence of undesirable compounds such as cholesterol and saturated f a t t y acids, the selection and development of v a r i e t i e s with such enhanced properties may become appropriate. Key metabolites which exhibit p h y s i o l o g i c a l and medicinal attributes could be enhanced i n new v a r i e t i e s . The American Heart Association has indicated that i t w i l l endorse c e r t a i n types of foods, a p o s i t i o n they have avoided i n the past, perhaps providing incentive to develop v a r i e t i e s with such enhanced properties, or unique combination of t r a i t s previously unattainable. Texture. Flavor, and Color of F r u i t s and Vegetables. Considerable v a r i a b i l i t y i s found i n the texture, f l a v o r , and color of the edible portion of h o r t i c u l t u r a l crops. Obviously much of t h i s v a r i a b i l i t y i s due to preharvest c u l t u r a l and environmental conditions, postharvest treatment, and the stage of maturity at consumption. However, there i s also considerable genetic v a r i a b i l i t y which accounts f o r the range of v i s u a l and organoleptic properties.



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Strawberries range from almost white to deep red centers; from a pale red exterior to almost black due to the high concentration of anthocyanins; the texture of r i p e strawberries can range from mushy to c r i s p . The texture and f l a v o r of apple v a r i e t i e s d i f f e r due to genotype and the varied responses to postharvest storage environments. Cauliflower and b r o c c o l i d i f f e r i n concentrations of glucosinolates, which are responsible f o r o f f and pungent f l a v o r s . In most cases conventional breeding has provided adequate d i v e r s i t y i n these q u a l i t y attributes f o r the consumer. However, further genetic modification of key components could f a c i l i t a t e the d i s t r i b u t i o n of q u a l i t y produce as one envisions greater d i s t r i b u t i o n ranges, and longer term storage or remote production areas to provide year-round a v a i l a b i l i t y . The improvement of f l a v o r , through the enhancement of key f l a v o r components, l e v e l s of reducing sugars and key amino acids or the removal of undesirable components can be f a c i l i t a t e d by biotechnology. Secondary metabolites such as f l a v o r compounds are quite responsive to c e l l culture manipulation, with somaclonal variants generated with decreased or increased levels of targeted components (39) . By p r e c i s e l y regulating starch biosynthetic enzymes, antisense technology may o f f e r the opportunity to elevate sucrose l e v e l s of sweet corn without the concurrent reduction of phytoglycogen associated with shrunken-2 mutants (40). Phytoglycogen i s responsible f o r providing much of the mouth f e e l of normal sugary sweet corn v a r i e t i e s . Key enzymes involved i n the depolymerzation of c e l l wall components or the development of f l a v o r could be modulated up or down. The use of developmentally regulated promoters would allow the precise regulation of such biochemistry. For example, transformation with an active cloned gene f o r up-modulation of lipoxygenase a c t i v i t y could a l t e r the f l a v o r c h a r a c t e r i s t i c s o f f r u i t tissue. A reduction i n postharvest a c t i v i t y of lipoxygenase would be u s e f u l i n minimally processed vegetables such as peas (41). Color and f l a v o r deficiencies greatly reduce the q u a l i t y of tomatoes. Through the enhancement of the levels of β-carotene, lycopene, key f l a v o r components and a more s t r i c t control of the processes of ripening and senescence, the q u a l i t y of non-seasonal tomatoes can be greatly improved. The i s o l a t i o n of genes responsible f o r pigment accumulation i n plant materials and subsequent transformation with the active genes f o r up-modulation could enhance s p e c i f i c colors. The potential to modify such q u a l i t y attributes i n plant tissue w i l l be greatly accentuated by biotechnology; the cost/benefit r a t i o w i l l be the determining factor as to whether such u t i l i t y w i l l be r e a l i z e d . MAINTENANCE OF HARVEST QUALITY Quantitative and q u a l i t a t i v e losses i n produce are due to microbial degradation; deterioration due to the a c t i v i t y of endogenous enzymes associated with maturation, ripening, and senescence; enzymatic a c t i v i t y r e s u l t i n g from physical injury and the subsequent decompartmentalization of substrate and enzymes; and the i n t e r a c t i o n of these various a c t i v i t i e s . The extent of such b i o l o g i c a l a c t i v i t y determines the appearance, texture, and f l a v o r of the plant tissue when i t reaches the consumer. To what degree these deteriorative



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processes can be controlled through the genetic modification of the raw material or the use of microbial pesticides developed through modern biotechnology remains to be seen. There are s u f f i c i e n t examples of genetic v a r i a b i l i t y among genotypes i n t h e i r rates of b i o l o g i c a l maturation and d e t e r i o r a t i o n to suggest that v a r i e t i e s which progress perhaps more slowly through maturation, ripening, and senescence or which exhibit a reduction i n the rate of key biochemical events could be developed. The genetic d i v e r s i t y i n the conversion rate of sucrose to starch among sweet corn genotypes i s well documented (33). Genotypes incorporating the bt ( b r i t t l e ) or sh2 gene not only provide elevated l e v e l s of sucrose but also exhibit a reduction i n the conversion rate of sucrose to starch. Several spontaneous mutants of tomatoes have been i d e n t i f i e d s p e c i f i c a l l y as "ripening" mutants. The ripening i n h i b i t o r ( r i n ) and non-ripening (nor) mutations r e s u l t i n non-climacteric f r u i t s (42) . These f r u i t s f a i l to produce system 2 ethylene and thus do not ripen normally. Other mutant a l l e l e s such as "never r i p e " (NR), "green r i p e " (GR), and "alcobaca" cause a l l ripening to proceed much more slowly and to occur over an extended period of time. These slow-ripening mutants with a depressed and extended c l i m a c t e r i c accumulate pigment slowly or not at a l l , and often ununiformally. They also tend to exhibit reduced polygalacturonase a c t i v i t y , which very slowly causes a depolymerization of the c e l l walls and subsequent softening of the tissue. Cultivars of apples Vamos-Vigyazo et a l . , 1976) and apricots (43) , and avocados (44) demonstrated d i f f e r e n t rates of enzymatic browning. Such browning tendencies were dependent upon substrate levels as well as the concentration of polyphenol oxidase i n the f r u i t tissue (45). Quality a t t r i b u t e s such as appearance, texture, and f l a v o r are susceptible to change a f t e r harvesting, but very l i t t l e has been accomplished i n establishing the e f f e c t of genotype. During storage, plant tissue pigments can undergo considerable change depending upon the storage environment and the endogenous biochemistry of the plant tissue. The more common c a t a l y t i c agents of carotenoid degradation appear to be peroxidases and lipoxygenases. Anthocyanin synthesis i s stimulated by l i g h t and i s often effected by temperature. One of the more obvious changes which occurs i n senescing tissue i s the loss of c h a r a c t e r i s t i c green color. Peel and often pulp degreening i s associated with the ripening of most f r u i t and "yellowing" i s a common c h a r a c t e r i s t i c of many detached l e a f , stem, or flower tissue consumed as vegetables. The highly r e s p i r i n g f l o r e t s of b r o c c o l i are very susceptible to postharvest loss of chlorophyll and the generation of o f f and pungent f l a v o r s . Although one would expect such enzyme mediated processes to be effected by genotype, the differences among e x i s t i n g genotypes i s apparently so subtle that they are many times accountable to preharvest growing conditions. V a r i a b i l i t y among b r o c c o l i genotypes with regards to the rate of color change has not been observed. Uniformity of ripening, and rate of ripening w i l l provide targets f o r the a p p l i c a t i o n of emerging technology. Polygalacturonase (PG) i s the major enzyme responsible f o r the depolymerization of c e l l walls and the subsequent softening of the



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tissue during the ripening of fruits such as tomato (46). With the anticipation that reduced PG activity would result in improved shelf l i f e and processing quality, scientists at the university of Nottingham and ICI have reported the inhibition of the expression of endogenous developmentally regulated gene for PG in transgenic tomato expressing antisense mRNA (47). However, there was no observed reduction in softening as measured by compressibility. In the case of avocado, a fruit which does not ripen while attached to the tree, cellulase probably plays a major role in the solubilization of the c e l l wall polymers (48). Further understanding of the interaction of ethylene and cellulase accumulation may offer other opportunities for the control of c e l l wall deterioration in specific tissue. The control of ripening through the use of tissue specific promoters for the control of ethylene mediated ripening and senescence processes could yield substantial rewards in the uniformity of ripening and postharvest stability. Ethylene synthesis is generally limited by the supply of the immediate precursor, 1-aminocyclopropane-l-carboxylic acid (ACC). The control of ethylene induced ripening would necessitate the control of ACC synthase or the ethylene forming enzyme (EFE). The other possibility would be to genetically modify plant tissue to be devoid of ethylene receptors. Blecker et al. (49) recently reported a dominant mutation in Arabidopsis which appears to affect the ethylene receptor. DIAGNOSTICS As a related area, biotechnology will greatly impact the protection of the food supply. Immuno-diagnostic techniques will provide simple, rapid, and inexpensive means for monitoring a wide range of variables including: plant pathogens, food pathogens, aflotoxins, pesticides, etc. As the consumer continues to demand freshness and convenience, and the processor attempts to meet such demands, quality control will take on increasing significance. SUMMARY The maintenance of harvest quality through control of the physiology of the harvested tissue, pathogens, and interaction of the commoditywith the environment provides one of the greatest opportunities for modern plant genetics to have an impact. The enhancement of quality components of fruits and vegetables (appearance, texture, flavor, nutrition and physiology, and safety) is made more feasible by biotechnology. Diagnosis or monitoring of food pathogens and pesticides will be one of the f i r s t applications of biotechnology to fruits and vegetables. Advances will depend on the integration of biotechnology with traditional sciences, and an understanding of the biochemical basis of the targeted attributes. LITERATURE CITED 1. Biale, J.B. and Young, R.E. In: The Biochemistry of Fruits and Their Products; Part I. A.C. Hulme, Ed. Academic Press: NY, 1971; pp. 1-63. 2. Green C.E., Sommers, D.A., Hackett, W.P., Biesboer, D.D., Plant Tissue and Cell Culture; Alan R. Liss: NY, 1987; pp.520. 3. Haberlandt, G. 1902. Kulturversuche mit isolierten


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