In Vitro Cell. Dev. Biol. 26:425-430,April 1990 1990Tissue Culture Association 0883-8364/90 $01.50+0.00

THE ESTABLISHMENT OF CELL SUSPENSION CULTURES OF GLADIOLUS THAT REGENERATE PLANTS

KATHRYN KAMO, JANET CHEN, ANDROGER LAWSON

United States Department of Agriculture, Florist and Nursery Crops Laboratory, Beltsville Agricultural Research Center, Beltsville, .Maryland 20705

(Received 27 September 1989; accepted 27 January. 1990~

SUMMARY Inflorescence stalks from greenhouse-grown Gladiolus plants of the cuhivars 'Blue Isle' and 'Hunting Song' cultured on a nurashige and Skoog basal salts medium supplemented with 53.6 /~M l-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars {'Peter Pears,' 'Rosa Supreme,' 'Jenny Lee') propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar 'Peter Pears' after the suspension cells had been cultured on solid medium. Key words: flower bulb crops; monocot cell suspensions.

INTRODUCTION Regeneration of Gladiolus has been reported from The ability to regenerate plants from cell suspensions floral explants ~33). The explants formed either a thin and protoplasts is important for future experiments in layer of callus or no callus prior to plant regeneration. In genetic engineering. Monocots have been relatively addition, Wilfret ~31) reported that shoot tips grown in difficult to manipulate in culture, although there has liquid medium developed callus more readily than on been much progress recently, particularly with crops of solid medium, but plant regeneration from the callus was agronomic significance. Emphasis has been placed on the not reported. Simonsen and Hildebrandt {24) regenerated establishment of cell suspensions which regenerate plantlets from callus cultures derived from cormel stem plants because it has been found with almost all monocots tips. This is the first report for Gladiolus of plant that protoplasts isolated from regenerable suspension regeneration from a well-established callus used to cells have the capacity to grow back to plants. Protoplasts establish a cell suspension culture that maintained the isolated from suspension cells have been regenerated capacity to regenerate plantlets. back to plants from Pennisetum spp. (27,30), Oryza sativa MATERIALS AND METHODS {23,26~, Dactylis gtomerata (9), Panicum spp. (8,16~, Saccharum officinarum 125), Zea mays ~20,21,22), Festuca Tissue culture of intact plant organs. Tissue explants arundinacea (5), Triticum aestivum (7), and Lolium were obtained from the following Gladiolus cultivars: perenne (5). Suspension cells capable of plant regenera- 'White Friendship', 'Sierra Snow', 'Blue Isle', 'Hunt- tion but for which protoplasts have not yet been ing Song', 'Pandion', 'Oscar'. Plants were grown in a regenerated to plants include Musa spp. 118) and greenhouse and flowered May through August. Plant Dioscorea ~1). organs were sterilized in various concentrations of a Many genera of ornamental bulbs are monocots, but Clorox solution tClorox Company, Oakland, CA) contain- with the exception of Hemerocallis (12), ornamental bulbs ing 20 drops Tween 20 per liter, followed by 3 rinses of have not yet been manipulated in culture to undergo sterile, distilled water, 5 min each. plant regeneration from cell suspensions or protoplasts. Young bases and tips. Corms were germinated on Bulb crops have mainly been manipulated in vitro by moist paper towels in the dark at 26 ~ C until the shoot was micropropagation of shoots for commercial cultivation about 3 cm long. Most of the corm was removed, leaving (10,14,25,32). In Gladiolus micropropagation occurred by only about 1 cm2 of corm and the shoot which was surface the development of axillary buds into plantlets when sterilized 15 min in 0.525% sodium hypochlorite (10% cultured on a medium containing a (11 ). (v/v) Clorox). The excised leaf bases were 0.5-1.0 cm long.

425 426 KAMO ET AL.

Excised leaf tips were 0.5-1.0 cm long and were placed tip TABLE 2 up so that the cut surface was directly in contact with EFFECT OF HORMONES AND LENGTH OF FLORAL Murashige and Skoog's tMS) basal salts medium (17) STALK ON THE FORMATION OF REGENERABLE supplemented with 3.0% (w/v) sucrose, 0.2% (w/v) CALLUS (R) AND THE NUMBER OF PLANTLETS Gelrite (Merck & Co.), and the following in mg/h glycine, REGENERATED FROM THE GLADIOLUS 2.0; thiamine, 1.0; pyridoxine, 0.5; nicotinic acid, 0.5; CULTIVARS 'BLUE ISLE' AND myo-inositol, 100.0; pH 5.8 and various hormones as 'HUNTING SONG' indicated in the tables. All media were autoclaved at 121 Length o! % Explants No. C/20 psi/20 rain. The hormones 2,4-dichlorophenoxyacetic Floral Stalk No. Hormone with R Plantlets/ qcm; Explants ~MF Callus Explant acid (2,4-D), kinetin, and l-napthaleneacetic acid (NAA) were autoclaved, and indole-3-acetic acid (IAA) was added by 1-60 45 2.2 2,4-D 0 0 filter-sterilization to sterile media. Callus and plantlets were 1-60 58 4.5 2,4-D 0 0 subcultured every 3-4 wk to fresh medium. 1-60 61 9.0 2,4-D 0 0 10-30 28 18.0 2,4-D 0 0 from mature corms. Corms were placed in a 10-30 23 36.0 2,4-D 0 0 saturated solution (5.0 g/50 ml water) of Zineb wettable 1 2 53.6 NAA 0 0 garden fungicide powder (Dragon Chemical Corp.) for 30 2-3 46 53.6 NAA 26 1 min, then placed on moist paper towels in the clark at 26 ~ 4 18 53.6 NAA 73 1 C until the shoots were about 3 cm long. The shoot and 5 5 53.6 NAA 100 9 10 9 53.6 NAA 0 0 approximately 1 cm 2 of attached corm was sterilized in 15 6 53.6 NAA 17 0 10% Clorox for 15 min, 5% Clorox for 10 min, 0.5% 60 48 53.6 NAA 40 3 Clorox for 5 min, Outer were removed from the Tissue slices from floral stalks were cultured 4 mo. on solidified MS shoot and the was exposed by alternately basal salts media containing the various hormones and concentrations cutting away outer leaves (2-3 at a time) and washing with above. Plantlets were transferred to MS basal salts medium lacking 0.5% Clorox for 5 min until the meristem region was hormones. exposed. An apical meristem region of about 5 mm was excised and placed into 50 ml flasks containing 15 ml of liquid MS basal salts medium with supplements as epidermis of the inflorescence stalk was removed, and 2 described previously. Flasks were cultured at 26 ~ C on a mm nodal sections were transversely cut. The surface gyratory shaker at 100 rpm under a 16 h light/8 h dark distal to the inflorescence base was placed in contact with the photoperiod at 75 wE m -2 sec-t. medium. Inflorescence stalk height was measured from the Mature corms. Mature corms were dipped in 70% base of the inflorescence stalk to the tip of flowers. ethanol and placed in a 50% Clorox solution for 15 min. Intact plantlets and cormel slices. Three cultivars, Whole corms were cut into 4 mm transverse slices and the 'Peter Pears', 'Jenny-Lee', 'Rosa Supreme' of virus- cut surface placed in contact with MS medium supple- indexed plantlets, clonally-propagated through tissue mented with various hormones. culture, on MS basal salts medium with 9.3 t~M kinetin Inflorescence stalks. The basal portions of inflores- (14), were supplied by Oglesby Plant Laboratories. cence stalks were removed by cutting into the corm. The Cormels cut into 2 mm transverse slices were cultured inflorescence stalk was sterilized with three washings as on either MS basal salts medium as described previously described previously for meristems of mature corms. The or on N6 basal salts medium (4) with 2.0% (w/v) sucrose, 20 mM L-proline, 0.2% Gelrite, and the following in mg/h TABLE 1 glycine, 1.0; thiamine, 1.0; pyridoxine, 0.5; nicotinic acid, 0.5; casein hydrolysate, 200.0; various hormones as THE EFFECT OF EXPLANT FROM INTACT GREENHOUSE- indicated in the tables, pH 5.8. GROWN GLADIOLI ON CALLUS THAT REGENERATED PLANTS Plantlet regeneration. Plantlets about 4 cm long which had regenerated from callus were removed and placed on No. of No. ot Callus with Plant solidified MS basal salts medium lacking hormones. Explant Explants Cuhivars Regeneration About one month later plantlets were transferred to a vermiculite/charcoal mixture according to Logan and Mature corm slices 31 1 -- Unfertilized ovules 66 2 -- Zettler (14) to further stimulate root formation. About one Fertilized ovules 51 3 -- month later, plantlets were transferred to soil in a Filaments 49 2 -- greenhouse. Young leaf bases 15 1 -- Suspension cells. Approximately 2 g fresh weight of Young leaf tips 18 1 -- Inflorescence stalks 359 5 + friable callus was placed in 10 ml MS basal salts liquid medium supplemented with 4.5 or 9.0 t~M 2,4-D, sugars, Meristems from mature corms 16 3 -- and vitamins as described above. Cultures were placed on Meristems from mature corms were cultured in MS basal salts liquid a gyratory shaker at 150 rpm in the dark at 26~ C. After media containing either 1~ 4.5 ~M 2,4-D. 2~ q.0 ~M 2.4-D. 3) 26.8 one week 10 ml liquid medium was added. Two weeks CM NAA, 4~ 53.6 ~M NAA. All other explants were cultured on MS after culture initiation the cells were diluted 1:3, and basal salts solid media containing either 1) 2.2 ~M 2.4-D. 21 4.5 ~.%/ there was 30 ml total volume in a 125 ml Erlenmeyer flask. 2,4-D, 3) 9.0 ~M 2.4-D. 41 36.0 ~M 2A-D. or 5) 53.6 ~M NAA as the hormtme. The total number of each explant varied depending .n Cultures were routinely transferred once a week at a 1:3 availability and success in sterilizing it. dilution. CELL SUSPENSIONS OF GLADIOLUS 427

FIG. 1. Plant regeneration from Gladiolus. A, Friable callus that has begun to develop numerous globular structures resembling somatic embryos. B, Globular embryo-like structures that have developed a coleoptile opening (arrow). X19. C, Coleoptile emerging from the opening of the globular embryo-like structure. X38. D, Regenerated shoot that was attached to callus mass. E, Plantlet that was removed from callus and beginning to form roots. F, Cell clusters present in a 'Peter Pears' suspension culture. X77. G, Scanning electron micrograph of regenerable callus showing a structure resembling a globular embryo (left side arrow} and the beginning growth of two shoots (right side arrows). X25. H, Scanning electron micrograph of regenerated shoots present on callus initiated from cormel explants. X 19. 428 KAMO ET AL.

For plantlet regeneration, 1 ml of suspension cells was TABLE 4 spread out on a Whatman No. 1 filter paper (7 cm THE EFFECT OF VARIOUS MEDIA ON INITIATING diameter) and placed on medium solidified with Gelrite. EITHER COMPACT (C) OR FRIABLE (Ft CALLUS Excess liquid was removed with a Pasteur pipet. The CAPABLE OF PLANTLET REGENERATION~ medium was MS basal salts medium supplemented with % Cormel Slices Forming Callus That either 13.4 or 53.6 ~M NAA or 2.2, 4.5, or 9.0 ~M 2,4-D, or Regenerated Plantlets no hormones. Cultures were kept in the dark until shoots Media Peter Jenny Rosa began to develop at which time they were placed in 16 h ~uM Hormottel Pears Lee Supreme light/8 h dark. MS 2.2 2.4-D 24 IF) 29 (C) 0 Scanning electron microscopy. Samples showing sever- MS 4.5 2,4-D 26 (F) 38 (C) 0 al stages of differentiation were cut from tissue cultures MS 9.0 2,4-D 10 (C) 53 (C) 21 (F) 5 (F) 0 and fixed in 2% glutaraldehyde and 1.5% acrolein in 0.05 M Na2HPO,-KH2PO4 pH 7.0, for 2 h in a vacuum. MS 53.6 NAA 0 0 64 (C) Following dehydration in ethanol, the samples were critically point dried, mounted on double-sided tape and N6 2.2 2,4-D 0 5 (C) 2 (C) attached with silver paint to SEM stubs. A coating of N64.52.4-D 0 0 0 N6 9.0 2,4-D 0 0 0 gold-palladium was applied with a sputter coater and the N6 18.0 2,4-D 0 0 0 tissues were viewed in a scanning electron microscope. ~ explants were cormel slices of three Gladiolus cu|tlvars. In one experiment at least 10 cormel slices were cultured for each RESULTS cultivar on MS basal salts medium with each hormone concentration, Most explants from intact greenhouse-grown plants of and the experiment was repeated once. Five cormel slices were the six cultivars tested formed little or no callus when cultured for each cuhivar on N6 basal salts medium with each hor- mone concentration, and one experiment was done. All cormel slices cultured on solid media (Table D. Typically, roots were cultured four months. formed rather than shoots. Meristems from mature corms cultured in liquid media formed a large ball of compact cultured on MS basal salts medium with NAA (53.6 taM) callus about 4 cm in diameter, but the callus did not but not 2,4-D (Table 2L The maximum frequency of regenerate plantlets after it had been cultured on inflorescence explants that formed callus capable of plant solidified MS medium lacking hormones. Only inflores- regeneration occurred when the inflorescence stalk was cence stalk explants of the cultivars 'Blue Isle' and only about 4-5 cm long. About three months after the 'Hunting Song' formed a compact callus that subse- inflorescence stalk explants were placed on solid quently differentiated to regenerate plantlets l Table 11. medium, creamy white callus formed from the vascular Inflorescence explants from seven out of sixteen bundle regions. The callus was compact and its surface different plants of the cultivars 'Blue Isle' and 'Hunting composed of smooth bumps (Fig. 11. Globular structures Song' formed callus capable of plant regeneration when resembling somatic embryos quickly formed over all of the bumpy callus and within about two weeks they TABLE 3 regenerated shoots. Once plant regeneration occurred, THE EFFECT OF VARIOUS CONCENTRATIONS OF callus proliferation ceased, even though NAA was present HORMONES ON INITIATION OF EITHER COM- in the medium. The callus from inflorescence stalk PACT (CI OR FRIABLE (F) CALLUS CAPABLE explants capable of plant regeneration was always very. OF PLANT REGENERATION FROM compact and its cells would not separate to form a cell INTACT PLANTLETS OF THREE suspension. The regenerated plantlets formed roots when GLADIOLUS CULTIVARS transferred to a MS basal salts medium lacking hormones. Cultivar Callus capable of plant regeneration was initiated from Hormone Pe~r Jenny' Rosa (~M) Pears Lee Supreme the basal region of intact plantlets approximately 5 cm tall of the three cultivars tested that were propagated in vitro 13.4 NAA O O C (Table 3L Presumably this callus was initiated from the 26.8 NAA O O O 53.6 NAA O O C cormel and basal shoot meristems. Callus growth was observed only after about three months. There were 5.7 IAA O O O genotypic differences in the ability of the intact plantlets 11.4 IAA O O O to form a friable callus capable of plant regeneration. 22.8 IAA O O C 'Rosa Supreme' formed abundant amounts of compact 2.2 2,4-D O O O or friable callus capable of plant regeneration when 4.5 2,4-D O F C cultured on NAA, IAA or kinetin whereas the two 9.0 2,4-D F F F cultivars 'Peter Pears' and 'Jenny Lee' did not respond to any of these hormones. Explants from all three 18.6K O O O 37.2 K O O O genotypes had to be grown on MS medium containing at 55.8 K O O C least 4.5-9.0/aM 2,4-D {Table 3) to initiate a friable callus that would form a cell suspension. For each hormone concentration a cluster of 5-6 intact plantlets was cultured, and there were two replicates done. Plantlets were grown 4 Cormei slices and basal meristems cut transversely of months. 'Peter Pears' and 'Jenny Lee' readily formed the CELL SUSPENSIONS OF GLADIOLUS 429 friable type of callus within 1-3 months when cultured on D ISCUSSION basal salts medium with 2,4-D ITable 4}. N6 basal salts medium containing 2,4-D was inhibitory to friable callus formation (Table 4}. Callus formation on N6 medium was typically minimal, and roots usually formed rather than Embryogenic callus from most monocots cultured thus shoots for all three cultivars. Friable callus from 'Peter far, including Panicum sp., Pennisetum sp., Zea mays, Pears' was watery and soft whereas friable callus of Oryza sativa, Triticum, has been initiated primarily from 'Jenny Lee' and 'Rosa Supreme' was hard, dry and immature embryos cultured on 2,4-D. We were unable to crumbly. Friable callus from all three genotypes has been initiate any callus from immature seeds of Gladiolus maintained in culture on solid medium for over one year cultured on MS medium containing 2,4-D. Inflorescence and approximately doubled in volume once a month. stalks of cuhivars 'Blue Isle' and 'Hunting Song' As soon as the friable callus appeared there were cultured on MS medium containing NAA, not 2,4-D, were globular structures resembling somatic embryos present the only source of callus from intact plants of Gladiolus on it (Fig. 1 A}. A coleoptile opening developed at the top capable of plant regeneration. Others have obtained of the globular structure within a few days (Fig. 1 B}, embryogenic callus and regenerated plants from inflores- followed by a small coleoptile that emerged from the cence stalks of various monocots such as Panicum sp. and opening 2-3 days later lFig. 1 C}. This coleoptile Pennisetum sp. 129L Old World bluestem and bermuda developed into a green shoot (Fig. 1 D}. Roots developed grasses ~2~, Triticum sp. ~6,19}, rice ~13}, and sorghum 131. from the shoots after they were transferred to a basal salts These monocots required the atLxins 2,4,5-trichioro- medium lacking hormones. Plantlets 5-6 cm tall phenoxyacetic acid, dicamba, or 2,4-D for embryogenic callus regenerated from floral stalks of 'Blue Isle', intact tissue initiation. culture plantlets and cormel slices of 'Peter Pears', Gladiolus callus obtained from inflorescence stalks of 'Jenny Lee', and 'Rosa Supreme' have been transplant- 'Blue Isle' and 'Hunting Song' was unsuitable for ed to soil in a greenhouse. The stages of plant establishing cell suspensions, The callus was very regeneration from friable callus were similar to develop- compact and would not dissociate in liquid medium, the mental stages from compact callus tFig. 1 G, HL Because amount of callus formed was limited, and plantlets the regenerated plantlets originated from a well- formed quickly after callus initiation. Lu and Vasil 115} established, friable callus clearly separated from the and Vasil and Vasil (28~ have been successful at explant, the regenerated planflets did not directly establishing cell suspensions of Panicum maximum and develop from axillary buds already present in the Pennisetum americanum from the compact callus corrects. derived from inflorescences. Instead we were able to Cell suspension cultures have been easily established identify a friable type of callus which had the ability to from the friable callus, and the cell suspensions immediately fall apart in liquid medium as well as approximately tripled in volume once a week over a regenerate plants. This friable type of callus was initiated two-month period. The cell suspensions consisted of from cormels or tissue culture plantlets of Gladiolus of small cell clusters consisting of only several cells lFig. 1 the cultivars 'Peter Pears', 'Rosa Supreme', and F}. When suspension cells of 'Peter Pears' were cultured 'Jenny Lee' cultured on 2,4-D tTables 3,4~. It was on a solid medium, they formed callus structures especially easy to obtain friable callus from the cuhivar resembling globular, somatic embryos followed by plant 'Peter Pears.' In the establishment of cell suspensions of regeneration. Suspension cells cultured on 2,4-D typical- maize, it was found that the selection of a fast-growing ly continued to form callus or regenerated relatively few embryogenic type of friable callus was necessary. In plantlets compared to cells cultured on media lacking maize this friable type of callus was initiated from hormones or with N AA l Table 5}. immature embryos at a low frequency (0.2%) t22) compared to 26% for 'Peter Pears.' It was fortuitous that friable callus of Gladiolus was derived from cormels and TABLE 5 tissue culture plantlets which are readily available THE EFFECT OF VARIOUS HORMONES ON PLANTLET throughout the year and do not require several months REGENERATION FROM SUSPENSION for plant growth as do the inflorescence stalks. CELLS OF 'PETER PEARS' Establishing a cell suspension of the ornamental bulb,

Hormone No. Plantlets/ Daylily, occurred by culturing unopened flower buds on (/~M~ ml Cells MS basal salts medium with 2,4-D to obtain a compact 0 8 type of callus 111}. Eventually the compact callus formed 2.2 2,4-D 0 a cell suspension capable of plant regeneration. Unlike 4.5 2,4-D 1 Daylily, unopened flower buds of Gladiolus cultured on 9.0 2,4-D 0 MS basal salts medium with 2,4-D never formed callus 13.4NAA 0.6 ~Table 1}. There appears to be no hormone and tissue 53.6 NAA 5 explant that will universally result in a monocot cell suspension. This is the first report of Gladiolus tissue Suspension cells of the cultivar 'Peter Pears' cultured on solidified MS basal salts medium for 4 too. formed callus followed by plantlets. culture methods for producing cell suspensions capable of One ml of cells was cultured per plate, and there were 3-5 plates for plant regeneration and a step towards manipulating each hormone concentration. Gladiolus for biotechnology purposes. 430 KAMO ET AL.

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The authors would like to thank Beth Logan for her helpful conversations.