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In VitroCell.Dev.Biol.--Plant35:200-205,May-June1999 © 1999Societyfor In VitroBiology 1054-5476/99 $05.00+ 0.00

OPTIMIZATION OF IN VITRO CONDITIONS FOR STIGMA-LIKE-STRUCTURE PRODUCTION FROM HALF-OVARY EXPLANTS OF CROCUS SATIVUS L. A. V. LOSKUTOV,1 C. W. BENINGER,T. M. BALL, G. L. HOSFIELD, M. NAIR, ANDK. C. SINK Department of Horticulture, Plant and Soil Sciences Building, Michigan State University, East Lansing, Michigan 48824 (A. V. L., T. M. B., M. N., K. C. S.), USDA Agricultural Research Service, Sugarbeet and Bean Research, Crop and Soil Sciences, Plant and Soil Sciences Building, Michigan State University, East Lansing, Michigan 48824 (C. W. B., G. L. H.), and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824 (M. N.)

(Received 25 August 1998; accepted 12 January 1999; editor E. C. Yeung)

SUMMARY Studies were conducted to optimize the in vitro production of stigma-like-structures (SLS) that yielded the important biochemical constituents responsible for the color, taste, and aroma naturally found in the stigmas of autumn crocus. Immature half-ovary explants were evaluated for the frequency of proliferation of SLS by culture on five basal media supplemented with different combinations of plant growth regulators and vitamins. The optimum proliferation of SLS was observed on B5 basal medium containing (x-naphthalene acetic acid (NAA) (5.4 ~M), N6-benzyladenine (BA) (44.4 ~tM), MS organics, casein hydrolysate (0.05%), and L-alanine (11.2 mM) 5 0 ~ 0 d after inoculation. Some explants formed other structures (roots, corms, petals, leaves), the growth and development of which substantially reduced the development of SLS. Removal of brown tissues and other tissues during subculture (3-wk intervals) allowed continuous culture of halfovary explants for 9-10 mo. SLS that had deep red pigment and reached more than 0.5 cm length were removed from the explants and with concomitant reculturing of the ovaries, enabled SLS to be harvested three times. Activated charcoal (1%) added to B5 basal containing NAA (5.4 ~M), BA (44.4 ~M), and sucrose (3%) was found to be a helpful addendum to prevent browning of explants and accelerate the initiation, growth, and development of SLS. The amounts of crocin, crocetin glucosyl esters, picrocrocin, and safranal in SLS, as determined by high performance liquid chromatography analysis, were found similar to those in natural saffron. Key words: activated charcoal; browning; crocetin; crocin; picrocrocin; saffron; safranal; secondary products.

saffron in callus tissues (Hori et al., 1988; Visvanath et al., 1990). All these studies demonstrated the feasibility for producing saffron in vitro. However, problems encountered in the in vitro production of saffron are low frequency of regeneration of SLS on explants (Fakhrai and Evans, 1990; Sarma et al., 1990), low concentration of secondary metabolites produced in comparison with the natural spice (Himeno and Sano, 1987; Sano and Himeno, 1987; Hori et al., 1988; Sarma et al., 1990), slow growth of callus tissues (Hori et al., 1988), short life of explants, browning of explants, formation of non-SLS structures, and single harvest. The aim of this stud); therefore, was to determine a culture medium and method to increase the harvest of in vitro-produced saffron with a high concentration of the main secondary metabolites.

INTRODUCTION Saffron is prepared from dried, bright red stigmas of Crocus sativus L. and its value is determined by the color compounds, carotenoids, crocin and other crocetin glucosyl esters, slightly bitter flavor, picrocrocin, and pleasant aroma, safranal. Saffron has been used as a spice in cooking and baking for hundreds of yr. Furthermore, crude extracts of stigmas of C. sativus L. and/or purified compounds also have been found to have beneficial medicinal properties (Madan et al., 1966; Gainer and Jones, 1975; Basker and Negbi, 1983; Duke, 1985; Nair et al., 1991; Wang et al., 1991; Sugiura eI al., 1994; Escribano et al., 1996). Despite wide use of this unique spice, availability is limited due to its price, about $2000 per kilogram (Plessner et al., 1989). The high cost is due to the fact that it takes 150 000-200 000 flowers and over 400 h of hand-labor to produce 1 kg of saffron. The search for alternative economical ways to produce saffron include tissue culture methods. One approach is in vitro production via the stigma-like-structures (SLS) produced directly on cultured floral explants and/or indirectly through callus tissues (Himeno and Sano, 1987; Sano and Himeno, 1987; Koyama et al., 1988; Fakhrai and Evans, 1990; Sarma et al., 1990; Kohda et al., 1993). Another approach is the induction of the essential secondary metabolites of

MATERIALSAND METHODS Plant material. Corms of autumn crocus, Crocus sativus L., were obtained from The Daffodil Mart (Gloucester, VA) and planted 9-10 cm deep in the field (sandy loam) at the MSU Horticulture Research Center at the beginning of September. During October through December, corms with floral buds 1 d prior to anthesis were collected. They were brought to the laboratory and thoroughlywashed with runningtap water. The floral buds were removedfrom the corms and leaves, washed in soapy water for 10 min, and rinsed with distilled water. They were then surface sterilized by being dipped consecutively in 70% ethanol for 1 rain, 15% aqueous Clorox (5.25% sodium hypochlorite) plus 3-4 drops of Tween 80 per 500 ml for 15 min, and rinsed

~Towhom correspondence should be addressed. 200

201

IN VITRO STIGMA PRODUCTION IN CROCUS four times in sterile, double-distilled water. Half-ovaries, previously reported as the optimum C. sativus L. explant for in vitro organogenesis ofSLS (Himeno et al., 1988; Fakhrai and Evans, 1990; Kohda et al., 1993), were used. The immature ovaries were dissected out and sliced into three or four crosssections (1.5-2 mm) that were placed on the various culture media. Culture media. Murashige and Skoog (1962) medium (except glycine) plus casein hydrolysate (CH) (0.05%) and L-alanine (11.2 mM) (Otsuka et al., 1992) or Linsmaier and Skoog (1965) medium both supplemented with different combinations of NAA (1.1, 2.7, 5.4, 10.8, 16.2, 21.6, 27.0 laM) and BA (0.09, 0.44, 0.88, 2.2, 4.4, 22.2, 44.4 p.M) were used for SLS induction during the first experiments. Subsequently, basal media used were those of Murashige and Skoog (1962) (MS), Heller (1953), Gamborg et al. (1968) (B5), Nitsch and Nitsch (1969) or White (1963), all with NAA (5.4 p-M), plus BA (44.4 pM), and MS organics [sucrose (3%), thiamine (0.33 p-M), pyridoxine (0.27 p-M), nicotinic acid (0.41 pM), i-inositol (0.01%), CH (0.05%), and Lalanine (11.2 raM)]. In addition, two media reported in the literature were evaluated (Fakhrai and Evans, 1990; Kohda et al., 1993). The first medimn was White plus sucrose (3%), NAA (21.6 laM), zeatin (18.2 p-M), coconut water (2%), and glutamine (1.4 mM). The second medium was B5 plus sucrose (2%), NAA (10.3 ~1/), and BA (29.9 p-M). To optimize the in vitro conditions to prevent browning of the explants, various addenda including glycine (13.3-99.8 p-M),citric acid (4.8-23.8 p-M), ascorbic acid (56.8-283.8 p.M), coconut water (5%), cysteine (82.5-495.0 p-M), L-alanine (0-5.6 p-/t,/),CH (0-0.05%), i-inositol (0-0.01%), sucrose (14%), activated charcoal (AC) (1%), vitamin-free and their combinations were tested by addition to B5 basal containing NAA (5.4 p.M) and BA (44.4 p-M). B5 basal containing sucrose (3%) supplemented with NAA (5.4 p_M)and BA (44.4 ~M) was used to evaluate the influence of AC (1%). All media were solidified with Phytagel (Sigma Chemical Co., St. Louis, MO) (0.35%), and the pH was adjusted to 5.8 before autoclaving. L-alanine, glutamine, and coconut water were added to autoclaved and cooled (80 ° C) media through a Millex-GS filter (0.22 lam). The media were autoclaved at 1.45 kg/cm2 for 20 min. Each plastic petri dish (100 × 20 ram) contained 30 ml of medium. Five or six half-ovary explants were placed in each dish. There were 50 half-ovary-replicates for each test medium. The explants were subcultured every 3 wk and all cultures were maintained in the dark at 25 ° C. Harvesting and drying of SLS. SLS that had primarily red pigmentation, along with some intense yellow or orange and were 5 mm long or longer, were cut from the ovary explants. The trimmed ovary explants were then suhcultured on fresh medium to obtain the next cycle of SLS. The harvested SLS were sieved (mesh size 1.0 mm2) and dried at 80 ° C for 30 min (Himeno and Sano, 1987). Dry weight was measured immediately thereafter. The dried SLS and natural saffron (dry stigmas) obtained from Sigma Chemical Co. (St. Louis, MO) or McCormick & Co. (Hunt Valley, MD) were stored in the dark at 4 ° C. Extraction procedure. The method described by Iborra et al. (1992) with some modifications was used for extraction of crocin, crocefins, and picrocrocin from SLS or natural saffron. Ten milligrams (dry wt) of each sample was ground, resuspended in 1 ml of 50% ethanol and stirred for 1 h at 26 ° C in the dark, and microcentrifuged at 14 000 rpm for 10 rain. The supernatant was collected and the residue was extracted with 50% ethyl alcohol/ H20 (2 × ; 1 ml each time). The combined extracts were filtered (Millex-GS, 0.22 p-m), freeze-dried, and stored at - 2 0 ° C until use. For safranal, 10 mg of dry SLS or natural stigmas (Sigma) were ground, resuspended in 1 ml of 100% acetonitrile, and shaken vigorously for 1 h at 26 ° C (Loskutov et al., unpublished). Each sample was centrifuged at 14 000 rpm for 10 min and the supernatant was collected in a syringe and passed through a filter (0.22 p-m). After filtration, the extracts were analyzed immediately by high performance liquid chromatography (HPLC). Standards. Crocetin pyridine salt (ca. 95%), was obtained from Sigma. Safranal (ca. 88%) was obtained from the Aldrich Co. (Milwaukee, WI). Crocin and picrocrocin were obtained by thin layer chromatography (TLC) from the Spanish saffron (McCormick & Co.) extracted in 50% (vol/vol) ethanol in water. A crocin spot at Rf value 0.32 was resolved on TLC plates with 0.5mm-thick silica gel (GF plates, Anahech Inc., DE) by the solvent system nbutanol-acetic acid-water (4:1:1 vol/vol/vol) (Anonymous, 1978). A picrocrocin spot of Rf 0.73 was resolved on TLC plates (Sigma-Aldrich, Steinheim, Germany) with the method of Iborra et al. (1992). Crocin was visibly detected in white light as an orange spot, whereas picrocrocin was identified under UV (254 nm) as a dark brown spot. For elution, crocin and picrocrocin bands were removed, redissolved in water, microcentrifuged at 14 000 rpm for 10

rain, filtered (0.22 p.m) [crocin was additionally purified through Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden)) and then freeze-dried. High performance liquid chromatography (HPLC). Crocin, crocetin, picrocrocin, and safranal were identified and quantified by HPLC with a Waters (Milford, MA) Autosampler Model 717, a Model 510 pump, and a Model 996 Photodiode Array Detector (PDA) interfaced with NEC powermate Pentium PC and Millennium 2010 software. A Shiseido C~s (5 p-m particle size; 120A pore size) column (250 mm long × 4.6 mm I.D.) was used. Three methods were used for analytical and quantitative determination of saffron compounds: (i) croein and croeetins--linear gradient acetonitrile: water (total run time was 40 min), from Sujata et al. (1992); (ii) pierocrocin--isocratic condition according to Iborra et al. (1992); (iii) safranal--isocratic condition using 100% (vol/vol) acetonitrile at a flow rate of 0.5 ml/min, and detection at 308 nm and total run time of 8 min (Loskutov et al., unpublished). The HPLC profiles of the purified crocin and picrocrocin standards were compared with those published for these compounds (Iborra et al., 1992; Sujata et al., 1992; Tarantilis et al., 1994, 1995). The purity of the croein standard was estimated as 93-95% all-tram digentiobiosyl ester of croeetin (erocin) and 5-7% its cis-isomer. The purity of the pieroerocin standard was estimated as 96%. The croein standard and the dry extracts of test samples were dissolved in 50% (vol/vol) ethanol in water. Standards of croeetin and picrocrocin were dissolved in ethanol and water, respectively. The safranal standard was dissolved in acetonitrile and HPLC grade solvents were used. Unless otherwise indicated, all samples were filtered through a 0.22-p-m filter before injection. The amounts of emcin, pierocrocin, and safranal in SLS or in natural stigmas (Sigma or McCormick & Co.), were measured from their peak areas at 440, 250, and 308 nm, respectively, from calibration curves made with the standards. Statistical analysis. All metabolite concentrations are reported as the mean of three replicates + SE. Results were statistically analyzed with Student's t-test. P values less than 0.05 were considered significant. RESULTS AND DISCUSSION In the first series of experiments, half-ovary explants cultured on 24 different media were evaluated for frequency of induction and proliferation of SLS, and for degree of pigmentation. The first response observed in vitro was a three- to five-fold expansion of the explants within 30 d from the start of culture. Initiation of SLS was apparent 10-20 d later on all media except MS plus NAA (5.4 btM), BA (44.4 btM), CH (0.05%), and L-alanine (11.2 mM) on which no SLS were seen. This observation further confirmed that a wide range of basal media and concentrations and combinations of various addenda will initiate and produce colored SLS on half-ovary explants (Himeno and Sano, 1987; Sano and Himeno, 1987; Fakhrai and Evans, 1990; Kohda et al., 1993). However, in our experiments, the optimum response of SLS initiation, as evaluated by number of explants that produced at least 1 SLS with intense color at about 60 d of culture, was observed on only two media: 1) MS plus NAA (5.4 ~M), BA (44.4 I.tM), CH (0.05%), and L-alanine (11.2 mM), (herein CR.NAA5.4) on which 45.0% of half-ovary explants had 1-10 SLS (5 mm or longer) per explant (Fig. 1 A); and 2) MS plus NAA (10.8 p.M), BA (44.4 ~M), CH (0.05%), and L-alanine (11.2 mM), (herein CR.NAA10.8) on which 44.8% half-ovary explants had 1-8 SLS (5 mm or longer) per explant. When the combination of White plus NAA (21.6 btM), zeatin (18.2 ~M), coconut water (2%), glutamine (1.4 raM), and sucrose (3%) from Fakhrai and Evans (1990) was included as a control, 40.0% explants had 1-5 SLS per explant; similar to what Fakhrai and Evans obtained. However, extensive brown to dark brown coloration rapidly developed throughout the explants on this medium; whereas on CR.NAA5.4 and CR.NAA10.8 media, browning occurred at a slower rate.

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B

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FIG. 1. In vitro responses of half-ovary explants of cultured Crocus sativus. A, Initiation of SLS on CR.NAA5.4 after 60 d of culture. B, SLS after 4 mo. of culture on CR.NAA5.4. C, Typical brown tissues. D, Callus and leaf-, petal-, and root-like structures. E, Morphological structures: A) Spanish saffron (McCormick & Co.); B) saffron (Sigma Chemical Co.), C) in vitro-produced and dried SLS. F, SLS after 50 d of culture on B5-CR.NAA5.4 (no vitamins). G, SLS after 50 d in culture on B5-CR.NAA5.4 + 1% AC (no vitamins).

After 4 mo. culture on CR.NAA5.4 and CR.NAA10.8, the number of SLS on the majority of explants ranged from 20--40 per explant (Fig. 1 B). A similar frequency (16 or more) of SLS per explant of half-ovaries after culture for 4 too. was observed by Kohda et al. (1993) on B5 or LS supplemented with a relatively wide range of levels of NAA or indoleacetic acid or indolebutyric acid and BA. In our experiment on the B5 plus NAA (10.3 ~M), BA (29.9 ~M), and

sucrose (2%) of Kohda et al. (1993) we observed a frequency of 8 12 SLS per explant and the least intensity of browning of explants. Whereas SLS occurred on media containing a wide range of auxin/ cytokinin levels, the optimum response was observed when relatively low NAA (5.4; 10.8 ~tM) and high BA (44.4 ~tM) levels were used. Thus, our observations on CR.NAA5.4 and CR.NAA10.8 differ from those of Sano and Himeno (1987) who reported that a combination

IN VITRO STIGMAPRODUCTIONIN CROCUS

203

TABLE 1 QUANTITYOF SECONDARYMETABOLITESIN NATURALSAFFRON AND SLS FROM THREE HARVESTS FROM B5-CR.NAA5.4a Source and date

Main cis-trara$ crocetin glucosyl esters (total)

Crocin

Picroerocin

Safranal

[Mg compound/lO mg dried plant tissue (mean + SE)]~'

Saffronc (1 control) First harvest of SLS March 11 (2 control) Second harvest of SLS April 9 Third harvest of SLS May 7

0.434 0.585 0.478 0.461

+ 0.018 + 0.031~ _+ 0.025" _+ 0.039"

0.863 1.116 0.851 0.748

+ 0.037 + 0.076 + 0.044~ _+ 0.097~

0.757 2.188 t.994 1.338

_+ 0.046 -+ 0.129 a - 0.147 a + 0.319

0.0013 "4- 0.0001 0.0013 + 0.0001 0.0021 + 0.0001 a'o 0.0021 +- 0.0007

"B5-CR.NAA5.4 is B5 basal medium containing NAA (5.4 p.M), BA (44.4 pM), CH (0.05%), L-alanine (11.2 raM), and MS organics [sucrose (3%), thiamine (0.33 ~tM), pyridoxine (0.27 ~M), nicotinic acid (0.41 ~tM), I-inositol (0.01%)]. bn = 3 replications. cFrom the Sigma Chemical Co. ap < 0.05 (1 control). "P -< 0.05 (2 controls).

of high IBA or NAA and relatively low BA or kinetin levels, respectively, induced the maximum number of SLS. At the same time, they observed proliferation of SLS from half-ovary explants when much lower levels of NAA (0.54 or 5.4 ~M) and kinetin (23.2 ~M) were used. These variable findings may be due to different preculture plant environmental practices or variable environmental conditions for the culture of explants. Browning of explants and formation of non-SLS structures occurred and inhibited development of SLS. Browning occurred on all media and it started where the explant was in contact with the medium and then extended into the explant (Fig. 1 C). Also, some explants formed other structures (root-, corm-, petal-, leaf-like tissues) (Fig. 1 D). On CR.NAA5.4 and CRNAA10.8, where optimum response of SLS was noted, other tissues formed more extensively on the latter medium. Initially, the deleterious effects of browning and non-SLS structures were both controlled by removal at subculturing. Subsequently, the influence of basal medium on the degree of browning of explants was examined. The basal media MS (control), Heller, B5, Nitsch, and White, all with CR.NAA5.4 organics, were tested. By subjective evaluation, the explants on B5 had less browning after 3 mo. Next, SLS production was continued with the explants on B5-CR.NAA5.4, and brown tissues and all other tissues were removed during each subculture. This technique allowed maintenance of explants in culture for a 9-10 mo. period, yielding three harvests of SLS. These SLS had morphology similar to that of natural saffron (Fig. 1 E). HPLC quantification results indicated that the concentrations of saffron components produced in vitro were equivalent to those in natural stigmas (Sigma) (Table 1). The contents of crocin (0.585 rag) in the first harvest of SLS, picrocrocin in the first (2.188 mg) and second (1.994 mg) harvests, and safranal (0.0021 mg) in the second harvest were higher than in natural stigmas (0.434, 0.757, and 0.0013 mg, respectively). Crocetin glucosyl esters in the SLS tissue from the first harvest (1.116 rag) were higher than in the second (0.851 mg) and third (0.748 rag) harvests. Safranal in SLS from the second harvest (0.0021 rag) was higher than in the first harvest (0.0013 mg) of SLS. Crocetin concentration in all samples was very low. While the half-ovary exptants on B5-CR.NAA5.4 medium produced SLS with a relatively high response and good quality, the need to periodically remove brown tissues remained labor intensive.

Thus, in 1997, a further study testing the vitamin components of B5-CR.NAA5.4 and various addenda including glycine, citric acid, ascorbic acid, coconut water, cysteine, and activated charcoal (AC) on browning of explants was conducted. These studies showed that explants did not exhibit browning for the first 3 wk on B5CR.NAA5.4 (without vitamins) or B5-CR.NAA5.4 plus AC (1%) media (without vitamins). After 4 wk on these two media, initiation of red SLS on the latter medium with AC (1%) occurred. Seven to 10 d later, initiation of yellow SLS was noted on the medium without AC. The other factors studied, namely glycine, citric acid, ascorbic acid, coconut water, cysteine, and the vitamins at the concentrations stated in the Materials and Methods, singly and in combination, had no effect on browning. Subsequently, medium B5-CR.NAA5.4 was compared to B5CR.NAA5.4 plus AC (1%), both without vitamins, to examine the influence of AC (1%) on control of browning and development and growth of SLS over 3 mo. with subculture every 3 wk. Paired halfovary explants were used to minimize explant effects. Intensive redcolored SLS developed and grew in 50 d on B5-CR.NAA5.4 plus AC (1%), earlier than on B5-CR.NAA5.4 (Fig.1 F and G). No browning of tissues was evident for 3 mo. on the medium with AC (1%), and although some browning did occur on medium without AC, it was not sufficient to require removal. SLS structures occurred at about the same frequency as was noted in earlier studies on both media. Attempts to continue the cultures in this study after the single harvest of SLS at 3 mo. were not possible due to the onset and spread of browning into explants and the formation of non-SLS structures. On the medium with AC (1%), the non-SLS were only root-like. Thus, the use of AC (1%), while it did not eliminate the browning, was found to inhibit its onset through the first harvest. HPLC analysis of SLS indicated that the addition of AC (1%) increased the levels of secondary metabolites (Table 2). The concentrations of main cis-trans crocetin glucosyl esters (total), crocin (in particular), and picrocrocin increased, respectively, 32.7%, 22.2%, and 69.3% on B5-CR.NAA5.4 plus AC (1%) versus B5-CR.NAA5.4, both without vitamins. The concentrations of saffron compounds in these SLS were comparable to those of natural Spanish saffron (McCormick & Co.) (Table 2). Interestingly, similar concentrations of the color compounds (crocin and main cis-trans crocetin glucosyl esters) were found in the

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LOSKUTOV ET AL. TABLE 2

EFFECT OF 1% ACTIVATED CHARCOAL ADDED TO B5-CR.NAA5.4 (WITHOUT VITAMINS) ON SECONDARY METABOLITE CONTENT IN SLS Crocin

Main cis-tran.~ crocetin glucosyl esters (total)

Sample

Picrocrocin

[Mg compound/t0 rag dried plant tissue (mean _+ SE)]~

SLS from medium without AC SLS from medium with 1% AC Saffrond

0.582 __+_0.009 0.711 + 0.009 b 1.172 -4- 0.033

1.244 _+ 0.088 1.651 + 0.007" 3.084 + 0.047

0.693 + 0.080 1.173 + 0.052" 2.057 -4- 0.220

an = 3 replications ~'P