Molecular Breeding Foods

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Research Signpost37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Advances in Agricultural and Food Biotechnology, 2006: 263-288 ISBN: 81-7736-269-0Editors: Ramn Gerardo Guevara-Gonzlez and Irineo Torres-Pacheco

12Molecular breeding for

nutritionally and healthy food

components

S. Horacio Guzmn-Maldonado and Alejandra Mora-Avils

Instituto Nacional de Investigaciones Forestales, Agrcolas y Pecuarias (INIFAP).

Unidad de Biotecnologa del Campo Experimental Bajo

AbstractProtein quality is the capacity of a protein to meet

nutritional and functional requirements for essential

and non-essential amino acids and its relation to

human amino acid requirements. The problem ofprotein and amino acids requirements, is one of the

most difficult in the science of nutrition. One of the

most interesting specialist uses of transgenic crops for

human nutrition is that of expressing various human

milk proteins in edible plants. This chapter will detail

several aspects related to molecular breeding to

improve nutritional and healthy components in plant

foods.

Correspondence/Reprint request: Dr. S. Horacio Guzmn-Maldonado, Instituto Nacional de InvestigacionesForestales, Agrcolas y Pecuarias (INIFAP). Unidad de Biotecnologa del Campo Experimental Bajo

E-mail: [email protected]


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S. Horacio Guzmn-Maldonado & Alejandra Mora-Avils264

Plant foods are a good and relatively cheap source of proteins, vitamins,

minerals, carbohydrates and lipids. Recently, some plant components raised as

phytochemicals, might provide additional health benefits. Concerns such as

the actual nutritional effect on human health of staple foods in developingcountries, and the constant necessity of increase the nutritional content of

plant foods, are some discussed issues. With this in mind, we revised and

analyzed the state of art in plant nutrients and some antinutritional

components, as well as the most novel research reports with biotechnology

approaches to increase nutritional quality and content in main foods.

IntroductionAnimal foods are consumed in low proportions in developing countries,

where cereals and legumes are the most important sources of food supply.

These plant foods are a very good and relatively cheap source of proteins and

contribute a significant amount to the total protein in the diet. Maize, rice,

common bean, soybean, and faba beans are some of the most important food

crops in the world, and the major cereals and legumes used in developing

countries as a direct source of food. Protein quality is the capacity of a protein

to meet nutritional and functional requirements for essential and non-essential

amino acids and its relation to human amino acid requirements (4, 5). The

problem of protein and amino acids requirements, is one of the most difficultin the science of nutrition (6). Several approaches have been carried out using

transgenic plants, in order to improve the nutritional quality of plant foods.

Among these improvements are for example, the use of the gene encoding the

beta-casein protein can be introduced into potato under the control of an auxin

inducible promoter and that the human protein can be detected in the leaves

and tubers of the transgenic plants at a level of 00.1% soluble protein (120).

Similarly, the human alpha-lactalbumin (121) and lactoferrin (122) proteins

have been expressed in transgenic tobacco.

1. Nutritionally and healthy important componentsin plants1.1 Protein quality and amino acid requirements

Animal foods have been recently projected in negative terms because these

foods contribute dietary saturated fats, cholesterol and high caloric intake,

three well-known factors in the etiology of heart-related, cancer and diabetes

disorders (1, 2, 3). Animal foods are consumed in low proportions in

developing countries, where cereals and legumes are the most important

sources of food supply. These plant foods are a very good and relatively cheapsource of proteins and contribute a significant amount to the total protein in the

human diet. Maize, rice, common bean, soybean, and faba beans are some of


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Food biotechnology 265

the most important food crops in the world, and the major cereals and legumes

used in developing countries as a direct source of nutrients (Table 1).

The relatively poor quality of cereal and legume proteins, is of hardly any

importance in Western diets; in this countries, animal proteins comprise asubstantial portion of the diet. In the developing countries, however, animal

proteins are either too expensive (Latin America and Africa) or not readily

accepted (India) (1).

Table 1. Protein content of some food-crop plants.

Crop Protein content

(% on dry matter)

Cereals

Barley 7.0 - 14.6

Maize 8.8 - 11.9

Oats 8.8 - 14.6

Rice 8.3 - 10.1

Rye 7.0 - 14.0

Triticale 11.7 - 16.3

Wheat 8.0 - 17.5

LegumesChickpea 17.1 - 21.0

Common bean 17.0 - 30.0

Peas 20.0 - 26.0

Soybean 38.0 - 40.0

Faba bean 26.0 - 34.0

Lentil 22.0 - 25.0

Peanuts 27.0

Lupin 28.0 - 45.8

Emerging protein-seeds sources

Oilseeds Safflower

Seed 10.8 - 19.1

deffated meal 18.8- 53.7

Sesame

Seed 20.0 - 25.0

deffated meal 57.7 - 63.4

Sunflower

Seed 3.7 - 23.4

deffated meal 37.2 - 56.4

Pseudo-cereals

Amaranth 13.7 - 17.8

Quinoa 11.0 - 13.7___________________________________________________________________________

Adapted from (1)


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Protein quality is the capacity of a protein to meet nutritional and

functional requirements for essential and non-essential amino acids and its

relation to human amino acid requirements (4, 5). The problem of protein and

amino acids requirements, is one of the most difficult in the science ofnutrition (6). When food is available the appetite normally takes care of the

energy requirements, and will automatically supply adequate protein and

amino acids (7). In developing countries, protein-energy malnutrition is a

major cause of ill-health both directly and indirectly; it occurs at all ages but its

incidence is greater, and the results more serious, in the weaning and

immediate post-weaning periods (1).

When a deficiency of a nutrient results in a specific behavior, such as the

appearance of beri-beri in thiamin deficiency, it is possible to measure with some

precision how much is needed to prevent or cure the deficiency signs (1). This isnot true for proteins nor to amino acids. Long-term studies become necessary

and current methods are based on replacing body losses of protein or maintaining

nitrogen balance (7). International expert committees estimate that a daily adult

need of protein varies from not less than 0.57 g/kg of body weight to 1.0 g/kg (7, 8).

Only the essential amino acids (histidine, isoleucine, leucine, lysine, methionine,

threonine, phenilalanine, tyrosine, triptophane, valine) are required from

exogenous sources because human body can not synthesize them (5). However,

two additional aminoacids, tyrosine and cisteine, have been incorportated to the

list, given their role in support of maintenance, growth and tissue repair (9).Inadequate levels of essential amino acids (EAA) result in depression of food

intake and retardation of growth; these consequences may be seen among the

world's poor where protein deficient diets are common. The essencial aminoacid

requirements for humans has been reevaluated by the Massachusetts Institute of

Technology (MIT) (Table 2) (Young, 1994). Tentative new amino acid

requirements values given in Table 2 represent a better apoproximation of the

minimal physiological needs for well nutrished adult subjects in North America.

Then it is legitimate to ask whether the amino acid requirements of

individuals or populations in developing regions of the world are the same,particularly where protein and/or dietary lysine intake are likely to be less

generous (9). Some studies are carried out designed to confirm if the estimates of

the lysine requirements in healthy adults is applicable to other populations.

Figure 1 shows amounts of plant seeds that are required to satisfy such

amino acids for a human adult (the higher the quantity of plant food is needed,

the lesser essential amino acid content in such food). It is very difficult get some

one to prolong for a long period of time a daily intake of some of the plant foods

presented in Figure 1.

In practice, most poorest areas of developing counties do not have a diet

which is exclusively from cereals but is mixed to varying extents with legumes

and in exceptional cases vegetables, milk and meat (1).


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Table 2. Comparison of amino acid requirements (mg/kg/day) and their patterns (mg/g

protein) as proposed for adults in 1985 by FAO/WHO/UNU and by the group of the

Massachusetts Institute of Technology (MIT).

FAO/WHO/UNU (6) MIT

Amino acid (mg/kg day) (mg/g protein) (mg/kg day) (mg/g protein)

Lysine 12 16 30 50

Aromatic 14 19 39 65

Sulphur 13 17 15 25

Valine 10 13 20 35

Leucine 14 19 39 65

Isoleucine 10 13 23 35

Threonine 7 9 15 25

Tryptophan

3.5 5 6 10

Total 84 111 187 310

Source (9)

0

10

20

30

40

50

60

70

80

Grams

perday

Figure 1. Amount of seed-crop that be consumed inorder to meet the minimum daily

requirements of some essential amino acid. Met + CysLle Lys Trip

Adapted from (1).


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1.2 VitaminsIndividual members of vitamins are designated also by alphabet letters A,

B1, B2, C, etc. Vitamins are also classified according to their solubility as water

soluble (C, B1, B2, B6, B12, niacin, pantotenic acid, biotin) or fat soluble (A, D,E, K). In early times, long sea voyages deprived sailors of fresh fruits and

vegetables, often resulting in death from scurvy. In addition to riboflavin,

vitamin B2preparations in early times were reported also to contain another

factor named pyridoxine or B6 (1).

A question often asked is which and how much vitamins in the diet can

assure us no health problem? Unfortunately, science has not advanced enough

to give a precise answer to this question (10). The National Research Council

(NRC), has recently published the Tenth Edition of the Recommended Dietary

Allowances (RDA). This organization established RDA for eleven vitamins:A, B1 B2 B6, B12 C, D, E, K, folic acid and niacin. Determination of the RDA,

involved considerations like the amount of a particular vitamin required for

elimination of its deficiency symptoms (11).

It is generally believed that people in Western countries have adequate

(as defined by RDA) vitamins intake. There are, however, susceptible groups

within the general population who may have inadequate vitamin intake. Suchgroups include dieters, people on medication including oral contraceptives,

pregnancy, alcoholics, teenagers and people with diabetes and other chronic

ailments (12). In developing countries very little information existsconcerning the nutritional status; nevertheless, Barclay et al. (13) found that

in Ecuador, an average country of Latin America, adults satisfy 72% thiamin

(B1), 62% niacin and 42% retinol (A) those allowances recommended by

RDA; meanwhile, intakes of riboflavin (B2) and ascorbic acid (C) reaches

98% and 175%, respectively, those daily allowances recommended. High

vitamins B2 and C intake could be because high amounts of daily intakes of

cereals (195g), vegetables (88 g) and fruits (102 g), and a reasonable daily

intake of legumes (23 g). On the other hand, Calloway et al. (14) compared

the food consumption in Mxico during one year with standard requirements,and found inadequacies for retinol (A) (68%), ascorbic acid (C) (63%) and

riboflavin (B2) (52%).

1.3 MineralsDiverse minerals contained in foods are essential nutrients for humans.

These minerals can be categorized in two types, macrominerals and trace

elements (15). Macromineral consists of minerals that have clearly been

demonstrated to be deficient in the diets of large segments of populations in

the developed and developing nations. The trace elements include mineralnutrients that may be potentially deficient in populations, but whose

deficiencies are difficult to establish (1).


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In developing countries cereal grains and some legumes are the primary

and least expensive source of minerals (16, 17). Wheat contain the highest

amounts of iron (6-15 mg/100 g) and zinc (3 mg/100g) and differ significantly

from other cereals (1). Raw common bean is also a good source of minerals,including calcium 70-210 mg/100g), iron (3.3-8.0 mg/100 g), zinc 1.9-6.5

mg/100g), and potassium (1320-1780 mg/100g) (16); nutrient retention values

during cooking vary from 78.9% (Cu) to 100% (Ca) (16, 18). Iron is present in

amaranth (17 mg/100g) approximately two to five fold than in cereals and

legumes (19).

The mineral content of food crops should be considered in conjunction

with its bioavailability (20). Bioavailability of minerals refers to the proportion

in food that is absorbed and utilized by the body (21). Until a nutrient is

absorbed from the gastrointestinal tract and enters the systemic circulation, it isnot available for utilization. Usual absorption of minerals ranges from less than

1% to over 90%. The bioavailability of dietary minerals must be considered

when determining whether the diet contains enough, too little, or too much.

Mineral content should be considered also in conjunction with phytic acid,

oxalic acid, proteins, polyphenols, and complex polysaccharides such as

starch, crude fibre and lignin which can complex with dietary essential minerals

(22, 23).

Calcium and iron are the only minerals that are mandatory for labeling

because potential nutritional deficiencies are most likely for such minerals(15). Dietary calcium deficiency has been linked epidemiologically to

several chronic diseases, including osteoporosis, hypertension and colon

cancer (15). Several natural history or retrospective studies have

demonstrated positive linkages between adequate lifetime calcium intakes

and greater bone mass measurements or reduced fracture rates (24, 25, 26).

Other lines of evidence support an adverse effect of amino acid on calcium

reabsorption suggesting that excessive protein intakes from animal products

can be calciuretic and responsible for the high fracture rates in Western

nations (27, 28).Iron deficiency anemia, rather than simple iron deficiency, is characterized

by decrements in red blood cell function, as well as in depressed function of

numerous other cellular activities because of inadequate oxygen delivery (15).

Several epidemiologic lines of evidence suggest that high intakes of iron may

contribute to cholesterol diseases because of the speculated increase in iron-

estimulated oxidation of low density lipoproteins. Similar evidence on excess

iron consumption has been gathered for several types of cancer, butspeculations about possible mechanisms remain unsupported by experimental

data (15).

The percentage of anemic subjects (26%) in Ecuador was higher than

those observed in European (10.9%) and US individuals (8.0%) (13). Data


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revealed that anemia in infants and young women was predominantly caused

by iron deficiency. In the cited studies, although iron intakes were generally

satisfactory, as was calcium, the bioabailavility of both dietary minerals was

probably low because the reaction with phytic acid (13, 29).

1.4 CarbohydratesCarbohydrates constitute three-quarters of the biological world and about

80% of the caloric intake of humankind (30). The most abundant carbohydrate

is cellulose, the principal structural component of trees and other plants. The

major food ingredient consumed by humans is starch, providing 75-80% of the

total caloric intake. However, other carbohydrates such resistant starch and

fiber and oligosaccharides are of importance because the proved contribution

to health.While most starch consumed by humans is degraded in the upper segment

of the gastric apparatus, some 10% of starch consumed may be resistant to

human digestive enzymes and may enter the colon in a similar manner to the

no-starch polysaccharides of dietary fibre (31, 32). Resistant starch (RS) can

be classified in: RS1, physically inaccessible starch; this starch may be present

in those grains coarsely ground because the larger the particle size of starch theless starch is digested by pancreatic amylase in vivo (1). RS2, starch that is

resistant because of the crystalline nature of the starch granules. RS3,

retrograded starch. Granules swell and are disrupted under a heating processknown as gelatinization; when the starch is cooled, retrogradation occurs,

converting the starch to a crystalline form that is resistant to digestion (33).

Oligosaccharides are short-chain polysaccharides which have a sucrose

moiety plus one (raffinose), two (stachyose) or three (versbascose) galactose

moieties. Major emphasis is placed on investigations relating this family of

sugars because they are the most common oligosaccharides present in legumes

at low levels. These compounds need to meet two specific requirements: 1)

they are not digestible by human digestive juices and, 2) they are preferentially

consumed by beneficial intestinal bacteria, bifidobacteria, in the colon (1).There are two main effects that varying reduced starch digestion, fiber and

oligosaccharides in the small intestine can have: 1) an effect on the glycemic

response and 2) an effect on the amount of starch entering the colon and the

implications this has for health. Beneficial action of resistant starch, fibre and

oligosaccharides for colon is given by Stephen (31) and Anderson et al. (34).

1.5 LipidsGlycerol esters of fatty acids, which make up to 99% of the lipids of

plant origin, are traditionally called fats (solid) and oils (liquid) (35): fatsare solid at room temperature and oils are fluid. Dietary lipids play an

important role in nutrition. They supply calories and essential fatty acids act


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as vitamins carriers (36), and increase the palatability of food. The most

abundant class of food lipids are the acylglycerols, which dominate the

composition of vegetable fat and oils. Of these, triglycerides (a glycerol

residue molecule plus three fatty acid residues), are the principal componentsof fats and the most concentrated form of energy among the macromolecules

of the diet, providing 9 cal/g (37).

Fatty acids are classified by 1) their length: short chain (less than 6

carbons), 2) medium chain (6 to 11 carbons), and long chain (12 or more

carbons); and in terms of their degree of saturation: saturated fatty acids lack

double bonds, monounsaturated fatty acids have a single double bond and

polyunsaturated fatty acids have two or more double bond. Palmitic and stearic

acids are the major saturated fatty acids in foods (meat and dairy products)

(37). Meanwhile, oleic acid (18:19) is the major monounsaturated fatty acidin the diet. Plants, not humans, can insert additional double bonds into oleic

acid and form linoleic and linolenic acid with three double bonds. Linoleic

acid is known to lower cholesterol content in human blood and thus helps to

prevent atherosclerosis and heart attacks. The proportion of oleic acid andlinoleic acids determines the quality of the oil and its use (1).

Consumers have been made aware of the importance to health of

polyunsaturated fatty acids in the diet. Maize oil is an excellente source of the

essential polyunsaturated fatty acid, linoleic acid (C18:26) (35). Among the

commercial vegetable oils, only safflower (80% per g of oil) and sunflower (upto 73 % per g of oil) oils have higher percentages of polyunsaturated fatty acid

than maize oil (59% per g of oil) (38). Sesame oil is dominated also by linoleic

acid (up to 46 % per g of oil) (39). The dominant vegetable oil in the world,

soybean oil, has a lower level of oleic and linoleic oils, but a major problem is

its higher content of linolenic acid (40).

Beneficial compoundsApproximately 90% of all cancer cases correlate with environmental

factors, including one's dietary habits. Manipulating dietary intakes appears tobe one of relatively few realistic approaches to bring about significant cancer

risk reduction. While major limitations exist in defining the precise role of

food constituents in the cancer process, published data suggest that about 60%of cancers in women and more than 40% in men are related to food habits.

Cardiovascular diseases (CVD) have been also correlated to dietary habits.

Similar relationships (diet and disease) have been reported in obesity, immune

function, diabetes and aging, among multiple diseases. The influence of dietary

components of plant foods in human health has been extensively examined.

Epidemiological studies suggest that the intake of some specific foodcomponents influence in preventing specific diseases. Discussion of some of

these food-crop compounds and their relation to health is given in reference of


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Guzmn-Maldonado and Paredes-Lpez (41) and Guzman-Maldonado y

Paredes-Lopez (1).

From an antinutritional point of view, low protein digestibility is an

important nutritional problem of plant foods, which is often attributed topresence of trypsin inhibitors (TI), tannins, lectins and phytic acid (16, 42). In

addition, TI also diminish the protein efficiency ratio (43), reduce the gain/feed

rate (44) and are an occupational inhalant allergen (45). Tannins are also

responsible for decreases in feed intake, growth rate and mineral

bioavailability (42, 46). Pure lectins apparently induce pancreatic growth (47)

and produce ulceration and necrosis of the intestinal epithelium in rats (48).

Despite the negative aspects of antinutritional factors, these and other

compounds currently referred to as phytochemicals (41), might provide

additional health benefits. Scientific evidence continues to accumulate insupport of the role of phytochemicals such as TI, tannins and lectins in the

prevention and treatment of human diseases, particularly in soybean (49); the

potential health benefits of consuming common bean have largely been

overlooked. Trypsin inhibitors confer protection against rotavirus (50), inhibit

carcinogenesis (51) and can be used as chemopreventive agents (52).

Moreover, trypsin inhibitors have not produced detectable clinical or

laboratory toxicity when provided at high doses (52). Bean lectins diminishes

growth of non-Hodgkin lymphoma (53) and can be used as diagnostic markers

in tumors (54). Resolution of genetic and environmental factors affecting theaccumulation of phytochemicals as well as their consequences in foods and

health, may enable breeders to select for cultivars that have a balance of health

promoting activities with antinutritional effects. Such cultivars could be used

for direct consumption or for clinical or laboratory studies.

Phenolics have been also traditionally considered antinutrient factors,

because of their ability to reduce the digestibility of proteins and subsequent

increase in fecal nitrogen excretion (55). Some phenols may effectively

precipitate proteins, while antinutrient effects may be a consequence of the

ability of other phenols to inhibit the activity of digestive enzymes and otherimportant enzymes (e.g. hydrolases, isomerases, oxygenases, etc.) and to form

complexes with metal cations such as iron and copper, among others (56).

However, scientific evidence continues to accumulate in support of the role of

phenolics in the prevention and treatment of human diseases such as cancer

and diabetes, and coronary heart disease. Condensed and hydrolysable tannins

have been shown to be effective antioxidants (42, 57), anticarcinogens (58)

and antimutagens (59, 60).Given that phenolic acids account for approximately one-third of the

dietary phenols, interest in their antioxidant behavior and potential health

benefits associated with these simple phenolics has been increased in resent

years (61). Beyond the protective antioxidant behavior, other biological


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activities of phenolic acids have been reported. Caffeic acid and some of its

esters might possess antitumor activity against colon carcinogenesis (62). A

series of phenolic acids (p-cumaric, ferulic, sinapic, caffeic, protocatecuic and

gallic acids) have linked with the inhibition of AP-1 transcriptional activity(14). AP-1 is an active protein implicated in the processes that control

inflammation, cell differentiation, and proliferation. Gallic acid has been

indicated as a potent inhibitor of mutagens (63). On the other hand, since the

flavonoid epigalocatechin galate (EGCG) reduces blood glucose by an

unknown mechanism, the evaluation to examine the effect of EGCG on insulin

pathways, gene expression, and glucose production was carried out (Walter-

Law et al., 2002). Apparently changes in the redox state may have beneficial

effects for the treatment of diabetes and suggest a potential role for EGCG, or

derivatives, as an antidiabetic agent.It is suggested various reviews as excellent sources for information about

the classification, structure and the beneficial effects on human health of

phenolic acids and flavonoids (61, 56, 64, 65, 66, 67).

2. Genetic manipulation of food components2.1 Essential amino acids

Although total yield is still the first priority for both traditional plant

breeding and contemporary plant genetic engineering in developing countries,the goal of improving the nutritional value of plants is receiving increasing

attention. Modifications of the levels of several essential aminoacids, lipids,

fatty acids, minerals, nutraceuticals, antinutritional compounds, and aromas

have been reported by research groups applying genetic modifications,

mutations, RNAi among others in order to increase, decrease or modify levels

of nutritional compounds as well as antinutritional elements.

Zeins are the predominant proteins in maize grain which belong to a

family of alcohol-soluble prolamin storage proteins and account for more

than 50% of total seed proteins, however, are deficient in several aminoacids

such as lysine, tryptophan and methionine. Corn mutants with reduced levels

of zein such as opaque-2 have improved nutritional quality. The O2 gene

encodes maize basic leucine zipper transcriptional factor that controls the

expression of a subset of storage proteins, including the 22-kDa -zein

storage protein and lysine ketoglutarate reductase (LKR) (68, 69,18).

Although it is rich in lysine, the opaque 2 mutant is not very useful in

agriculture because of its adverse effects on seed quality and yield. An o2

mutant typically shows a marked increase in non-zein proteins, which in

association with the decrease in lysine-poor -zein proteins, leads to a

greater percentage of lysine and tryptophan. Furthermore, the loss of LKR

activity results in increased levels of free lysine (70).


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According to Gibbon and Larkins (71) an alternative approach to

understand the relationship between zein synthesis and the origin of the opaque

endosperm phenotype is to perturb zein accumulation. With the introduction

of sense and antisense zein genes approach it seems possible to improvenutritional values (72). These authors knocked down the 19-kDa -zeins and

previously (73) did the same to the 22-kDa -zeins by RNA interference.

Both groups observed that the lysine content was increased in the

transgenic lines by 1520%. Notably, this increase is well below the >100%

increase that is often observed in o2 genotypes, however, it does not affect

additional endosperm gene function. These experiments showed that transgenic

approaches could be useful to increase kernel lysine content. It is noteworthy

that the 22-kDa -zein RNAi lines showed a more pronounced opaque

phenotype than the 19-kDa -zein RNAi lines. By contrast, down regulation ofthe maize lysine-poor 22-kDa zein gene via RNAi does not alter the general

functions of O2, but generates quality and normal maize seeds with high levels

of lysine-rich proteins (74).Some promising transgenic approaches for improving nutritional quality

of seed proteins involved a chimeric gene encoding a Brazil nut methionine-

rich seed protein. This gene was introduced into tobacco which signified a

30% increase in methionine levels in the seed protein of transgenic plants.

This represents a feasible method for improving nutritional quality of seed

proteins particularly in legumes whose seeds are deficient in the essentialsulphur-containing amino acids (75). Similarly, transformation of

V. narborensis (76, 77), tobacco (78), alfalfa and trefoil (79, 80) with theBrazil nut 2S protein gene improved methionine content. Sulphur rich maize

seed storage protein delta zein was introduced in white clover for forage

quality by Agrobacterium-mediated transformation and the protein is

processed similarly as in maize (81, 82). Similarly, grain legumes were also

transformed to improve methionine and lysine contents (83, 84) as well as

rice to improve lysine (Zhenweizg et al., 1995), methionine (85), iron (86)

and aminoacid content (80), and rapeseed to improve methionine, lysine, andcysteine contents (87, 88).

Lysine is known to promote the uptake of minerals. In rice this essential

aminoacid is present in limited amounts. Datta and Bouis (89) reported that the

introduction of two bacterial genes DHDPS (dihydrodipicolinic acid synthase)

and AK (aspartokinase) enzymes encoded by the Corynebacterium dapA gene

and a mutant Escherichia coli lysC gene enhanced lysine about fivefold in

canola, corn, and soybean seeds (89). According to Yu et al. (90) seed specific

expression of lysine rich protein was obtained in maize by introducing the

sb401 gene from potato (Solanum berthaultii) encoding a pollen specificprotein with high lysine content under control of a maize seed-specific

expression storage protein promoter. Quantification of expression showed an


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increase in 16.1 to 54.8% for lysine and 11.6 to 39% for protein content;

keeping high levels for over six continuous generations indicating heritability

of the new trait (90).

Improving crops for feeding has acquired importance. According toGuenoune et al. (91) storage proteins of soybean (S-VSPs), improved

nutritional quality of vegetative tissues. S-VSPs contain lysine and accumulate

high levels inside the vacuoles (7%) as well as in chloroplasts (10%) of

vegetative tissues of tobacco (92).

2.2 Vitamins and mineralsMicronutrient malnutrition vitamin A, iron and zinc deficiencies among

others afflict over 40% of worlds population, increasing in many developing

countries. Staples are not considered an important source of minerals in thediet. However, because of high staple consumption, any increase in mineral

concentration might well have a significant effect on human nutrition and

health. Fourteen mineral elements are considered as essential, these are N, S,

P, K, Ca, Mg, Cl, Fe, Zn, Mn, Cu, B, Mo, and Ni. Of these 14 elements,human essentiality has been confirmed for all but B and Ni (93).

-Carotene, a precursor of vitamin A (retinol), does not occur naturally

in the endosperm of rice. Ye et al. (94) have reported transgenic rice that

produces grain with yellow-coloured endosperm. According to Ye et al.

(94), the genes plant phytoene synthase psy (cloned from Narcissuspseudonarcissus), bacterial phytoene desaturase cryt1 (cloned from Erwinia

uredovora); and lycopene cyclase lyc have been introduced into the rice,

driven by the endosperm specific rice glutelin promoter (Gt1). The crt1 was

fused to the transit peptide (tp) sequence by the pea Rubisco small subunit to

lead the accumulation of lycopene in the endosperm plastids. The reported

level of-carotene in 1 g of the transformed rice is 1.6 g in the heterozygous

form.

An improved understanding of the biosynthetic pathway for vitamin C has

led to claims for its over expression in plants. For example, it was suggested(95) that the level of this vitamin can be increased by expressing the gene

encoding the L-galactonoa- lactone dehydrogenase enzyme (96).

Micronutrients enrichment traits are available within the genomes of

these major staple crops such as bean, wheat, maize, and rice; that could

allow for substantial increases in iron, zinc, and provitamin A carotenoids

without compromising crop yield (97), in fact in all crops studied, it is

possible to combine the high micronutrient/density trait with high yield,

unlike protein content and yield that are negatively correlated (98). A key

issue is the level of bioavailability of total Fe or Zn intakes and whether thepercentage bioavailability will remain constant as additional Fe and Zn is

added to the diet during biofortification (99). According to Welch et al. (100)


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and Glahn et al. (101), in vitro and rat studies indicated that bioavailable Fe

and Zn remains relatively constant across cereal genotypes with high and low

micronutrients density. It was reported by CIAT researchers a very highly

significant positive correlation of 0.52 between the concentrations of Fe andZn across different common bean genotypes. Thus, genetic factors for

increasing Fe are cosegregating with genetic factors for increasing Zn.

Therefore, selecting for a higher Fe level in bean seeds will also tend to

select for increased Zn levels in the seeds (98). Genetic studies showed

evidence of significant genotype X environment interactions that can

ultimately affect Fe and Zn concentrations in extreme environments (102,

98). Variations in Fe and Zn in the seed were attributed to both genetic

differences and environmental conditions.

It is clear that the transgenic approach to enhance mineral content inspecific plant tissues must include not only trans membrane movement trough

the whole plant, but also the long distance pathways that serve to move

absorbed minerals throughout the plant (93). According to Grusak (93) the

more successful (and quantitatively significant) transgenic strategies should be

those directed towards enhancing the supply processes, however, there have

been efforts to manipulate storage tissues directly in order to enhance their

mineral content.

Vasconcelos et al. (103) reported improvement of the iron content of rice

by transferring soybean ferritin gene into indica rice. The introduced ferritingene was expressed under the control of a rice seed-storage protein glutelin

promoter to mediate the accumulation of iron specifically in the grain. Authors

reported as much as 71 g/g iron and 55.5 g/g of zinc in unpolished transgenic

seeds and as much as 37 g/g iron in polished seeds. However, authors

suggested bioavailability studies.

Similarly, Lucca et al. (2001) reported a two-fold increase in iron content

from transgenic Japonica rice variety Taipei 309 seeds using a ferritin gene

derived from Phaseolus vulgaris with the highest iron level (22.07 0.70 g/g

seeds). Lucca et al. (2001) also reported transformation with cystein richmetallothionein-like protein as cystein peptides are the major enhancers of iron

absorption that metallothionine was also expressed in the rice grain, increasing

the cysteine content sevenfold. Still, it is not known if the cysteine-containing

peptides formed on digestion of metallothionine in the human gut have a

similar enhancing effect on iron absorption as those formed on digestion of

muscle tissue (89).

2.3 Carbohydrates

Beside modifications in protein quality, tailoring of carbohydrates is andimportant area in molecular breeding programs in three different aspects,increase primary production, analysis and modification of allocation of


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photosynthates and engineering of storage carbohydrates. In this sense, a new

metabolic pathway has been transferred in to potato that leads to the

production of inulin, a fructose-based carbohydrate polymer that increase

nutritional quality of transgenic plants (104).The major sources of starch are cereals (maize, rice, wheat, etc.) although

roots and tubers are also good providers (potato, sweet potato, yam, etc.). It is

believed that one of the limitations to endosperm filling in wheat at

temperatures above 20 C is the temperature sensitivity of starch synthase.

Burrell (105), reports that ATC Ltd. and Biogemma produced transgenic wheat

containing the E. coli glgA gene encoding glycogen synthase. Measurements

of total starch synthase activity showed that between 8 and 15 d post-anthesis

(dpa) the transgenic lines (79.42a and 72.11b) had an increased activity

associated with an increased flux of carbon into starch. Although at 20 C thisincreased activity did not lead to an increase in starch stored, the decrease in

seed weight at the higher temperature was not as large in the transgenic lines as

in the controls (105).

Raemakers et al. (2003) reported cloning the granule-bound starch

synthase gene (gbss) from cassava in the antisense orientation under control of

the CaMV 35S promoter and the gbss promoter from potato. A total of 50

transgenic cassava plants with the gbss antisense gene were recovered, two of

which were subsequently shown to produce and store amylase-free starch in

their thickened, secondary roots (Raemakers et al., 2003). On the other handIhemere et al. (106) and Lloyd et al. (107) reported up-regulating overall

starch biosynthesis by expressing a modified version of theE. coli glgC gene

under control of the tuber-specific patatin gene from potato. glgC encodes

AGPase which is the rate-limiting step in cassava starch biosynthesis.

Transgenic cassava plants expressing the transgene were recovered and shown

to have a greater than 65% increase in AGPase activity compared to controls.

These plants were shown to generate significantly increased shoot and root

biomass and to accumulate almost twice the dry weight starch content as the

nontransgenic mother plants.

2.4 LipidsFatty acids

Conventional breeding of quantitative traits requires specific molecular

markers. By means of biochemical experiments, it was established that the

induced variation in linolenic acid content in rapeseed is associated with the

fad3 gene encoding the microsomal 15 desaturase. Using a pair of primers

specific to this gene and a doubled haploid progeny derived from a low

linolenic x high linolenic acid F1 hybrid, authors identified a polymorphismof the fad3 alleles between the low- and the high-linolenic acid genotypes(108).


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RNAi technology has also been successful in genetic modification of the

fatty acid composition of oil. RNAi mediated by a hairpin RNA has been

used in cotton to down regulate two key fatty acid desaturase genes encoding

stearoyl-acyl-carrier protein D9-desaturase and oleoylphosphatidylcholineu6-desaturase (109). Knockdown of these two genes in cotton leads to an

increase in nutritionally improved high-oleic and high-stearic cottonseed

oils, which are essential fatty acids for health of the human heart.

2.5 Beneficial compoundsTrypsin inhibitor

There are numerous naturally occurring compounds which interfere with

nutrient absorption. Enzyme inhibitors have received much attention as legume

albumin proteins with negative attributes. Hedemann et al. (1999) reportedfrom studies of near-isogenic pea lines, a difference in the quantity of trypsin

inhibitor (TI) proteins. Mutants and/or transgenic knockouts could also

provide answers to the question of whether or not TI proteins are essential for

seed/plant viability (110). In soybean, analysis of TI proteins is confounded by

the existence of two unrelated gene families (Kunitz and Bowman-Birk).

Mutants have been described for both of these. Such mutants may proveadvantageous to the processing of soybean, in that the high temperatures

required for inactivation of inhibitors for animal feed purposes may be

avoided. On the other hand, it is believed that trypsin inhibitors are involvedin protection against and regulation of proteolysis as well as to act as defense

mechanism against insects and diseases (111).

Phytic acid

Phytate (phytate, myo-inositol 1, 2, 3, 4, 5, 6-hexakisphosphate) is a regular

compound in seeds. During germination, inorganic phosphorus is released via

the action of the hydrolytic enzyme phytase (myo-inositol hexakisphosphate

phosphohydrolase). Phytic acid forms complexes or chelates proteins and

minerals to form phytates not readily digested in humans and non-ruminantsand thereby lowers the bioavailability of these minerals (111). According to

Cheryan (112) phytic acid readily forms complexes with Zn2+, Ni2+, Co2+,

Mn2+, Ca2+ and Fe2+ in decreasing order of stability.

One way to overcome the effect of phytic acid is through recombinant

phytase, however, in soybean, phytase is not able to withstand the high

temperatures necessary to inactivate proteinacious antinutritional factors (111).

On the other hand phytin reduction may compromise P levels as phytin is the

primary storage. Low levels of P would affect germination and seedling

growth (113). Selecting for seed and grain crops with low phytin contentscould have a deleterious effect especially in areas where soils are poor in P

(114).


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Concerning reduction of phytate and raffinose-family-oligosaccharides

(RFOs), in common bean RFOs (-1,6-galactosyln-sucrose) cannot be

metabolized because of the absence of- galactosidase in the human digestive

system and are therefore considered as major determinants of flatulence, whichmay accompany bean consumption (115). RFO levels could be reduced

through antisense or cosuppression constructs targeted at the genes encoding

the -galactosyl transferases that catalyze RFO synthesis. Alternatively,

specific -galactosidases could be expressed in a seed-specific manner.Similarly, reduction of phytate levels could be achieved by seed specific

expression of a phytase gene (116). It has been shown that intestinal digestion

of the -galactosides can be increased if animal diets are supplemented with

exogenous -galactosidase. The presence of -galactosides in the colon,

however, may have a beneficial effect by increasing the bifidobacteriapopulation. These bacteria produce short-chain fatty acids that reduce the

incidence of colon cancer in humans (117). RFOs also constitute a defense of

the plant towards biotic or abiotic stresses such as in cold and desiccation

tolerance (115).

The phytase that does exist in rice seeds will hydrolyse phytic acid if seeds

are left to soak in water. However, boiling destroys the phytases that occur

naturally in rice. Lucca et al. (118) reported introducing a transgene for a heat-

stable phytase from Aspergillus fumigatus, which increased the level of

phytase by 130-fold. Unfortunately, after being expressed in the grain, it wasno longer stable to heat and lost its activity on boiling.

Phytoesterols

It has been known for quite some time that phytosterols have the potential

to reduce cholesterol in humans by 1015% by interfering with cholesterol

absorption in the gastrointestinal tract. Indeed products containing these

phytosterols such as Benecol TM and Take ControlTM are beginning to appear in

the market to assist individuals in managing their cholesterol levels more

aggressively. Phytosterols are not currently available in adequate quantities inthe foods that we ordinarily consume. It has been known for some time that

expression of genes in the phytosterol pathway in plants increases the sterol

content of plant tissues (119).

2.6 High expression and new characteristicsOne of the most interesting specialist uses of transgenic crops for human

nutrition is that of expressing various human milk proteins in edible plants. For

example, it has been shown that the gene encoding the beta-casein protein can

be introduced into potato under the control of an auxin inducible promoter and

that the human protein can be detected in the leaves and tubers of the


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transgenic plants at a level of 001% soluble protein (120). Similarly, the

human alpha-lactalbumin (121) and lactoferrin (122) proteins have been

expressed in transgenic tobacco. These findings provide the basis for the

reconstitution of human milk in plants as a replacement for bovine milk inbaby foods which are used either for the general improvement of infant

nutrition or for the prevention of gastric and intestinal diseases in children

(96). A human lysozyme was introduced in rice (Taipei 309) by particle

bombardment. The gene was under control of the promoter and signal peptide

sequence for rice storage protein Glutelin 1. Expression levels of lysozyme

riched 0.6% of the brown rice weight or 45% of soluble proteins. Biochemical,

functional and biophysical comparisons of native and recombinant human

lysozyme revealed identical N-terminal sequence, molecular weight, specific

activity, pH and thermal stability (123). Further expression and clinical dietswith chicks revealed the potential of rice expressing lysozyme and lactoferrin

to serve as an alternative to antibiotics in broiler diets. The antibacterial

properties of lysozyme and lactoferrin evolved in the presence of commensal

and pathogenic microflora, and there is no indication that bacteria have

become resistant to these proteins in nature. However, the development of

bacterial resistance after prolonged feeding of these proteins has yet to be

examined (124).

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