Although there are several species of ginseng, the commercially important species, P. ginseng grows in an area of 30-48° north latitude such as Korea (220). The cultivation of ginseng in the field requires four to seven years, and it is impossible to plant consecutively for 20 to 50 years but the demand for the plant has increased dramatically in the world, and its price has soared. These are reasons why many researchers have tried to produce ginseng cells through plant tissue cell cultures.

Furuya et al. at Kitasato University (221, 222) have studied P. ginseng callus tissues since the early 1970's. Meiji Seika Kaisha in Japan investigated the large-scale production of the cells established by Furuya using various types of fermentors. According to their patent published in 1973 (223), crown gall calli, callus tissues and redifferentiated roots of P. ginseng were able to accumulate saponins and sapogenins known from the intact plant. The callus tissues and roots were cultivated on both MS solid and liquid media containing vitamins, sucrose, 2,4-D and suitable natural nutrients such as soybean powder or beef extract for several weeks at 25-28° C. The concentrations of crude saponins in the callus (21.1%), in the crown gall (19.3%) and in the redifferentiated root (27.4%) were much higher than those in the natural root (4.1%). The saponins were found to contain ginsenoside-Rb and -Rg. To obtain high saponin-producing cells, mutagenesis was conducted using nitrosoguanidine and ?-ray as mutagens and a variant cell line of the drown gall induced by ?-ray irradiation was shown to accumulate 25.5% of saponins.

Staba of the University of Minnesota also obtained cultured cells of P. ginseng containing ginsenosides (224). Choi of Korea Ginseng & Tobacco Research Institute (220) has investigated in vitro culture of P. ginseng extensively. He indicated that plant growth regulators such as 2,4-D and kinetin in the medium affected the levels of saponins in callus and suspension cultured cells. For example, 3.62% of total saponins was detected in the callus cultivated in MS medium containing 5 mg/L 2,4-D and 1 mg/L kinetin, while 8.78% was produced in 10 mg/L 2,4-D and 1 mg/L kinetin medium.

After the Meiji Seika abandoned development of the ginseng project, another Japanese company, Nitto Denko Corporation, constructed a 20 KL fermentor to scale-up cultivation of ginseng cells in collaboration with Furuya in the middle of the 1980's. Ushiyama et al. (225) of the company have optimized various environmental conditions using 30 L jar fermentors for the cell line having partly differentiated tissues originally induced by Furuya et al. Glucose in the medium promoted cell growth in the initial stages of the fermentation and sucrose fed during the growth cycle stimulated the productivity of saponins. Although a higher NO₂/NH₃ ratio was favorable to the growth, it decreased the concentration of saponins in the cells. The growth was suppressed by moderate agitation, but the yield of saponins increased. The highest cell mass, 19 g/L on a dry weight basis was obtained using a 2 KL fermentor and the production rate of the cell mass was approximately 700 mg/L/day.

Table 13 shows a comparison of the chemical variation between mother plants of P. ginseng and its tissue cultured cells. Ushiyama indicated that their cultured cells contained basically the same constituents as those in the intact plant. Acute virulence tests, Ames tests and dietary tests with livestock feed containing 12.5% of dried cultured cells for 6 months did not show any abnormalities in animals.

In 1988, Nitto Denko was approved by the Ministry of Welfare and Health in Japan to market the cultured ginseng cell mass as a food additive. The product has been used as an additive for wines, tonic drinks, soups, herbal liqueurs and others. The company is expecting that the cultured ginseng cells will be approved as a drug by the Ministry within a couple of years.

Rosmarinic acid, or a-0-caffeoyl-3,4-dihydroxyphenyllactic acid has been found in the families Laminaceae and Boraginaceae. Rosmarinic acid and some related compounds were reported to have physiological or pharmaceutical activities. Oxidized rosmarinic acid was reported to show antithyrotropic activity and rosmarinic acid itself has been shown to effectively suppress the complement-dependent components of endotoxin shock in rabbits (226), however these compounds have not yet been used as commercial drugs.

Cultured cells of several plant species such as Coleus blumei (227), Anchusa officinalis (227) and Lithospermum erythrorhizon (228) were found to accumulate rosmarinic acid. It is of interest that production of the acid is stable, and high levels are produced. Ellis described that the production of rosmarinic acid appeared to be constitutively expressed in C. blumei cells, some of which had been in continuous culture for 10 years with no reduction in metabolite yield.

Dedifferentiated cell cultures of C. blumei and A. officinalis accumulated almost exclusively rosmarinic acid with the levels higher than in the intact plants when both types of cells were cultivated in a Gamborg and Eveleigh's B5 medium. The yield of the acid, before optimization, was about 1.4 g/L for C. blumei and 0.7 g/L for A. officinalis (227). Both species grew very well and reached up to 16 g-d.w./L within 30 to 40 hours of the culture period.

Zenk et al. (229) reported in 1977 that increasing the sucrose concentration in B5 medium up to 7.5% greatly stimulated both cell growth and rosmarinic acid formation in C. blumei cultures. The yield of the acid was approximately 3.6 g/L and the cell mass was 27 g-d.w./L. The yield shown seems to be one of the highest productivities of secondary metabolites in plant cell cultures.

Culture conditions and correlation between cell growth and production of rosmarinic acid were investigated extensively by De-Eknamkul and Ellis (227) using both cell lines, for example NAA was the most favorable auxin to produce the product for A. officinalis cells, while 2,4-D was the most effective in C. blumei cultures.

Alfermann and his colleagues in Germany (230) have been investigating the biosynthetic pathway of rosmarinic acid and found two new enzymes, i.e., hydroxyphenylpyruvic acid reductase which catalyses the reduction of 4-hydroxyphenyl pyruvic acid to the corresponding lactic acids, and rosmarinic acid synthetase (caffeoyl-CoenzymeA:3,4-dihydroxyphenyllactic acid caffeoyl transferase), the enzyme transferring the caffeoyl moiety from caffeoyl-CoA to 3,4-dihydroxyphenyllactic acid. This is the crucial enzyme in rosmarinic acid biosynthesis forming the ester linkage between the caffeic acid moiety and the 3,4-dihydroxyphenyllactic acid moiety.

To confirm localization of rosmarinic acid accumulated and its biosynthetic enzymes in the cells, the same group prepared protoplasts from C. blumei cultured cells. Rosmarinic acid as well as the enzymes were mainly found in vacuoles. They also purified some of the enzymes.

Recently Mizukami and Ellis (231) isolated three isoforms of tyrosine aminotransferase (TAT) from Anchusa officinalis cell suspension cultures and indicated two (TAT-1, TAT-4) out of three isoforms were involved in the synthesis of rosmarinic acid. They suggested that TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.

Determination of the biosynthetic pathway of secondary products will undoubtedly contribute to the further improvement of producing cells using recombinant DNA technology. This technology should be applied more extensively in the future.

Ulbrich et al. (232) of A. Nattermann & Cie. GmbH in Germany investigated large-scale production of rosmarinic acid by a C. blumei cell culture process. In order to increase oxygen supply in the medium and to operate at high cell density, they designed a special module spiral stirrer. It was built of six modules, and a special end module each consisting of one plain metal ring (spiral blade) fixed to the stirrer shaft with two spokes and two rings. The rotation speed of the module spiral stirrer ranged between 50-100 rpm without significant cell damage. Standard rotation speed was set at 100 rpm. The inoculum of C. blumei cells was cultivated in a fed-batch process and then about 30-50% culture broth was transferred from the seed fermentor to the production fermentor with sucrose solution (50 g/L) as the production medium.

Using this procedure the yield of rosmarinic acid increased to 5.5 g/L or 910 mg/L/day, representing 21% of the dry weight. According to Ulbrich's experiments, the process needs only 14 days to produce in total 200 g of 97% purity of rosmarinic acid in two parallel production batches from one inoculum fermentor (32 L working volume). It is not necessary to separate the cell mass from the growth medium, and they would recycle it as a part (30% v/v) of the simple and cheap production medium (sucrose 50 g/L). It is hoped that some commercial utility for rosmarinic acid will be found in the future.

Arbutin is widely distributed in various plants of the Ericaceae such as Arctostaphylos uva-ursi and Vaccinium vitisidaea. The level of arbutin in the bark of Pyrus communis reaches up to 28%. Arctostaphylos uva-ursi has been used as a urethal disinfectant and its major active principle, arbutin, was shown to suppress the synthesis of melanin in human skin (233). A Japanese cosmetic company, Shiseido, has developed arbutin as an additive for the company's product lines because of its preventive activity toward pigmentation of skin.

Although arbutin is commercially available by a chemical process, researchers of Shiseido have investigated an alternative process using plant cell cultures including biotransformation (234). Tabata et al. (235) showed that cultured cells of Datura innoxia had a remarkably high capability for glucosylation of hydroquinone to form arbutin, and hydroquinone was totally converted to arbutin within 10 hours after administration. The glucosylation is catalyzed by an enzyme, uridine diphosphate glucose (UDPG)-hydroquinone glucosyltransferase. Yokoyama et al. (234) selected C. roseus cells as a producer of the enzyme since arbutin was formed efficiently when hydroquinone was added into the suspension culture.

They have optimized various components in Linsmaier-Skoog's basal medium for production of arbutin and found that higher levels of sugars such as sucrose in the medium, up to 6%, gave higher yields of arbutin. Concerning this sugar effect, Yokoyama suggested that sucrose was a scavenger, and therefore it protected cultured cells from the damaging activity of hydroxy radicals of the substrate. This was also supported by their finding that some antioxidants such as ascorbic acid, gallic acid, cysteine, tannin and phytic acid increased the level of arbutin.

One of the high producing cell lines, C. roseus strain B was cultivated in a 5 L jar fermentor equipped with modified paddle-type impellers and spargers of porous sintered metal which prevents to some extent the adhesion of cells to the inner surface of the fermentor. Glucose was used as a carbon source instead of sucrose for economic reasons for mass production of arbutin. In order to increase the cell density in the medium, 10 times concentration of the medium components was fed to the medium during the fermentation. It was prerequisite to keep the level of hydroquinone in the medium as low as possible to avoid damage of the cells by the substrate. Therefore, hydroquinone was fed at 6 mM in the beginning of the fermentation and after its concentration had decreased to around 0 mM in the medium, they started to feed hydroquinone continuously at 1.4 mmol per hour. Under these conditions, 9.2 g/L of arbutin which corresponded to 45% of dry cell weight was obtained in 3 to 4 days after administration of hydroquinone. Similar yields was also obtained using larger scale fermentors.

Since the production continued until cell death, arbutin was accumulated extracellularly and its concentration reached approximately 1% in the culture filtrate at the end of the production phase. Therefore, arbutin was easily extracted from the filtrate. The total cultivation period was approximately 18 days including 2 weeks for high density cell culture and 3 or 4 days for the biotransformation process.

According to Yokoyama (234), the chemical synthesis of the compounds requires at least 3 step reactions and the production cost of arbutin by a plant cell culture process is comparable to the chemical process.

As the author described previously, biotransformation is one of the most feasible processes in terms of industrial application of plant tissue and cell cultures. It is advantageous that the yield of arbutin is high and the cost of hydroquinone is inexpensive enough as a substrate, accordingly the author believes that the process will be employed for commercial manufacturing arbutin in the near future.

7.2 Agricultural Drugs

A large number of chemically synthesized compounds and natural molecules have been examined for their inhibitory effects on plant viruses and some of them have potent activity as protectors against virus infection (236).

In order to screen high producing cultured cells of plant viral inhibitors, a variety of callus extracts were examined the inhibitory activity towards tobacco mosaic virus (TMV) infection using a tobacco disc method by Misawa et al. of Kyowa Hakko in Japan (237). Among these extracts Phytolacca americana callus was selected as the most potent producer of the inhibitor. The level of the inhibitor accumulated in the suspension cultured cells and reached maximum level on the 9th day of culture using MS medium containing 1 mg/l 2,4-D. The cell suspension of P. americana was homogenized and the supernatant was diluted up to 100 times with water. The diluted solution was found to inhibit TMV infection on tobacco and tomato plants significantly.

Further studies in the same laboratory used Agrostemma githago, a more potent producer of the plant virus inhibitor (238). The growth of suspension cultured cells of A. githago was somewhat faster than P. americana.

Active principles of P. americana and A. githago were isolated with Column-lite chromatography and electrofocusing (239). At least four basic proteins were obtained from P. americana cells whose molecular weights were 1.10X10⁴ to 3.15X10⁴ Among them the highest molecular weight component contained sugars in the molecule. On the other hand, only one basic protein was isolated as the principle from A. githago culture and its molecular weight was 2.5X10⁴. The proteins obtained from P. americana have been widely investigated because of their activities to HIV.

Ikeda et al. of Japan Tobacco Inc. (240) also screened various plants and selected Mirabilis jalapa as a producer of an anti-plant virus protein. The callus was induced from leaves of M. jalapa and its suspension cultured cells established were found to accumulate the protein intracellularly. Optimization of the production and the cell growth as well as selection of high producing cell lines were conducted extensively. One of the lines produced 95 mg/L of the protein in the optimized medium based on MS medium on the 7th day of the cultivation. Its molecular weight was 24 KD and the amino acid sequence was determined which had 24% homology with a ribosome-inactivating protein, ricin D-A chain.

7.3 Food Additives

Shikonin and its derivatives such as acetyl shikonin and isobutyl shikonin accumulated in roots of Lithospermum erythrorhizon Sieb. & Zucc. are reddish purple pigments and have been used in traditional dyeing. The plant has also been used as a herbal medicine. Because of a shortage of this plant, Fujita et al. at Mitsui Petrochemical in Japan investigated mass cultivation of L. erythrorhizon cells to produce shikonin compounds (19, 241, 242). Using the cell line established by Tabata's laboratory of Kyoto University (243), they optimized its culture conditions extensively to increase the level of the products using flasks and various types of fermentors including a rotating cylindrical fermentor designed by Tanaka et al. of Tsukuba University (37).

Fujita et al. found that L. erythrorhizon produced shikonins in White's medium but the cell growth was poor in the same medium. On the other hand, Linsmair-Skoog's (LS) medium was recognized to support the growth but not shikonin production. Therefore, they used a two-stage culture for mass production of shikonin compounds. Namely, to proliferate the cells, LS medium was used at the first stage of the fermentation, and then the cells were transferred into White's medium for production of shikonin compounds. In order to improve the yields further, optimization of components in both media was carried out extensively, and MG-5 and MG-9 media were established,

Since most cultured cells in liquid and solid media occur as aggregates, selection of high-producing cell lines from the aggregated cells is not effective and labour-intensive. The Mitsui group prepared protoplasts from the cultured cells with appropriate enzymes and selected high shikonin compounds-producing protoplasts using a cell sorter. The selected protoplasts were generated to cell lines and cultivated in suspension. From 48 cell lines, they obtained a cell line having 1.8 fold the productivity of the parent line. The cell line showed stable production of shikonin compounds.

To produce the compounds more efficiently, the same group attempted to employ a high-cell density culture process in the second stage of the two-stage cultures. By feeding the nutrients into M-9 medium, the level of cell mass increased and that of the compounds produced increase twice as much as without feeding. Shikonin and its derivatives are being manufactured commercially by the company. A major application of the pigment is for lipsticks.

Shimomura et al. (244) established a hairy root culture of L. erythrorhizon with Agrobacterium rhizogenes. The hairy root culture did not produce shikonin on solid MS medium but produced the pigment in the root culture medium and also secreted it into the medium. Addition of absorbents; XAD-2, XAD-4, charcoal and so on increased the concentration of shikonin produced. The roots were cultivated in a 2 L air-lift type fermentor connected to a XAD-2 column (25 g) through a peristatic pump and 5 mg/day of shikonin was continuously produced during a period of more than 220 days.

Anthocyanins are the large group of water-soluble pigments responsible for many of the bright colours seen in flowers and fruit. They are composed of an aglycone (anthocyanidin) and more than one sugar moiety and normally change colour over the pH range due to the existence of four pH-dependent forms. Thus, at low pH they are red and at higher pH value (over 6) they turn blue. They are commonly used in acidic solutions in order to impart a red colour to soft drinks, sugar confectionary, jams and bakery toppings. The major source of anthocyanins for commercial purposes are grape pomaces and waste from juice and wine industries, but other potential sources have been investigated.

Crude preparations of anthocyanins are used extensively in the food industry and it has been claimed that pure anthocyanins are priced $1,250-2,000/kg, but crude materials are rather inexpensive.

Commercial exploration of cell cultures for anthocyanins therefore, has not been tackled seriously. Although there have been many papers describing the production of anthocyanins using cultured cells of various plant species (145-248), most of them seem to use an anthocyanin-producing cell line as a model system for secondary product production because of their colour which allows production to be easily visualized.

Among them, Yamamoto et al. of Nippon Paint Co. in Japan have studied production of anthocyanins intensively (248). They induced callus from Euphorbia millii leaves on MS medium containing 2,4-D, NAA, natural sources such as malt extract and yeast extract. As a major component in the callus, they identified cyanidin-3-arabinoside. The callus consisted of cell aggregates was cut to small pieces and cultivated on solid agar media. High producing cell aggregates were selected visually and they were transferred to fresh agar media. This procedure was repeated 28 times and one of the cells was determined to produce 1.32% d.w. anthocyanins in the cells. The levels of the pigments in flowers and leaves were 0.28% and less than 0.01%, respectively. They also established suspension cultures of E. millii.

Accumulation of anthocyanins was enhanced by a high osmotic potential in Vitis vinifera L. (grape) cell suspension cultures (249). They added sucrose or mannitol in the medium to increase the osmotic pressure and found the level of anthocyanins accumulated was increased to 1.5 times, 550 µg/10 cells.

This yellow pigment obtained from the floret of the safflower plant (Carthamus tinctorius L.), is also known as Mexican saffron or American saffron, although it has no relation to genuine saffron (250). The major pigment is carthamin, which exists at levels of up to 30% in the flowers, and there is also a red pigment in concentrations of about 0.5%. Carthamin is the quinoid form of isocarthamin, the glucoside of 2',3',4',6'-tetrahydro-chalcone. Safflower yellow is not approved for use in the U.S. or in the E.E.C., but regulations do permit its use in Japan. It is stable to heat and light and is used in baked goods and beverages.

The production of carthamin from safflower callus cultures has been described by Kibun Co. in Japan (251, 252). The callus was obtained from flower bud explants and could also be put into suspension. Medium optimization has been performed. The production of alpha-tocopherol (the tocopherol with the highest vitamin E activity) has been described for safflower cultures (253, 254). Selection with various media components and precursor feeding experiments have enhanced the production. Kusaka et al. (252) found that addition of cellulose, chitin or chitosan increased production of the red pigment. These polysaccharides appeared to show an eliciting activity. Addition of 1 mM D-phenylalanine and removal of Mg alone or both Mg and Ca from the culture medium also increased the production.

Plant cell cultures cannot presently be used for the production of these metabolites, given the high cost of the technology and the low value products, ie. $50-$80 and $48 for tocopherol and carthamin respectively.

Saffron is the name of the spice which is made from the stamens of Crocus sativus and are prized for their use as a flavoring and colourant. The stigma of the plant contains crocin (yellow pigment), safranal (a fragrance) and picocrocin (bitter substance). The plant is grown mainly in Spain and India and it requires about 30,000-35,000 hand-picked blooms to produce 1 lb of dry saffron.

Crocin, being a glycoside, is water-soluble and is not soluble in oils and fats. Saffron is sensitive to pH changes and is unstable towards light and oxidative conditions, but it is moderately resistant to heat. It is used in baked goods, soups, meat and curry products, cheese, confectionary and as a condiment for the rice of Spanish and Indian foods. Saffron is also reputed to have medicinal value for stomach ailments.

The very high value of the product is due mainly to the fact that the life of the flowers is very short, making harvesting difficult. Thus, this is an ideal target for plant tissue cultures. Ajinomoto of Japan have approached this problem through the propagation of saffron stigma-like structures in vitro (255). Further studies showed that crocin and picrocrocin were present and, after heat treatment (as done with field-grown stigmas), safranal was produced. The composition of these phytochemicals corresponded with that of similarly-treated young, intact stigmas (256).

Rubia tinctorum (Rubiaceae) is a perennial plant, madder, originated from the coastal regions of the Mediterranean and its roots have been used as red dyes in western Europe. The major components in the pigment are alizarin, purpurine and its glycoside, ruberythric acid. Pure alizarin is an orange crystal and is soluble at 1 part to 300 in boiling water and in other solvents. The R. tinctorum pigment, so-called madder colorant, shows a yellow color in acidic to neutral pH and tends to be reddish with increasing pH (257). It is highly resistant to heat and light which is favorable to food industry.

The callus of R. tinctorum was induced from the root of a germ-free plant by Odake et al. (258) of San-Ei Chemical Industries in Japan and was grown on LS agar medium containing 2,4-D (10^-5 M) and 0.2% gellan gum. Through the selection of high-producing cell lines and successive transfers, yellow pigment-producing cells were obtained which were then transferred into a liquid LS medium containing 10^-6 M 2,4-D, 10^-6 M kinetin and 3% sucrose. After 21 days cultivation in a 100 L jar fermentor, approximately 1.5 g of the pigment was extracted.

In order to remove auxins such as 2,4-D and IAA which are not desirable for food industry, a hairy root culture of R. tinctorum was established by the same group using Agrobacterium rhizogenes. They used a 5 mm diameter disc obtained from a leaf of R. tinctorum. It was incubated with cells of A. rhizogenes in suspension. After 78 hours at 25° C in the dark, the leaf disc was transplanted to a hormone-free solid LS medium containing claforan (0.05%), sucrose (3%) and gellan gum (0.2%). Fourteen days later, hairy roots were found to produce the pigment. Using a 100 L bubble-column type jar fermentor, the hairy roots were cultivated for 21 days and approximately 800 mg of the pigment was obtained.

Chicle is the most important raw material in chewing-gums and is made from the latex of Achras sapota Linn. Chicle contains approximately 60% of resin and 15% of rubber. The resin consists of lupeol, a-amyrin and ß-amyrin, and the rubber fraction contains cis- and trans-1,4-polyisoprene, however the biosynthetic pathway of chicle and its regulation mechanisms have not been elucidated although components of chicle are suggested to be synthesized through the mevalonic acid pathway.

Itoh et al. of Lotte Central Laboratory Co. Ltd. (259), has tried to manufacture chicle by A. sapota tissue cultures. The callus induced from young shoots on a Linmaier-Skoog's medium was shown to produce lupeol acetate, palmitate and stearate neither a,ß-amyrin nor rubber. On the other hand, the cells cultivated in suspension produced triterpenes as well as phytosterols such as campesterol, cholesterol, ß-sitosterol and stigmasterol.

In spite of many efforts for optimization of the culture conditions in suspension, the yield of triterpenes was not high enough for commercial application of the cell culture, therefore the researchers induced callus tissue of Dyera costulata and Couma macrocarpa Bars Rodr., both of which were known to produce latexes for chicle as well. The callus tissues of both plants produced triterpenes at higher levels than those in the intact plants, respectively, but not the rubber.

Polysaccharides produced by Astragalas gummifer have been used as additives of ice cream and edible dressings.

Isa et al. (260) of Q.P. Corp. in Japan used a hairy-root culture technology in order to establish an alternative method of production of mucilages of A. gummifer because of high cost and unstable supply of natural gums.

A. rhizogenes was inoculated in the stem of the in vitro plantlet of A. gummifer, which was incubated for approximately 14 days. Hairy root tips induced at the inoculated site were excised and cultivated on a hormone-free MS medium solidified with 0.2% Gelrite containing 500 mg/L Clafovan, a cefotaxim antibiotic. After successive transfers on the solid medium, the hairy roots were transferred into 30 ml of hormone-free liquid medium in a 100 ml Erlenmyer flask and cultivated at 25° C in the dark with constant agitation at 60 r.p.m.

The cells transformed by A. rhizogenes were found to produce opines as well as several types of water soluble mucilages. The composition of monosaccharides such as glucose, arabinose, galactose and xylose in the mucilages obtained from different hairy root lines varied widely, but the chemical structure of mucilages in the mother plant is much more complicated.

Hernandulcin is a sesquiterpene compound having strong sweet taste isolated from Lippia dulcia (Verbenaceae), but it has not yet been approved by FDA in the U.S.A.

Sauerwein and Shimomura (261) induced hairy roots of this plant using A. rhizogenes. The roots cultivated in MS liquid medium supplemented with 2% sucrose under 16 hr/day light accumulated 0.25 mg/g-d.w. of hernandulcin. Addition of 0.2-10 mg/L chitosan into the medium increased its level up to 5 times. The axenic shoot culture of L. dulcia on MS solid medium containing 2% sucrose was shown to produce a high concentration of hernandulcin, 2.9%-d.w. (262).