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The Establishment of Cell Suspension Cultures of <Emphasis Type= 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 leaf 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 cytokinin (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 Meristems 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 leaves were removed from the Tissue slices from floral stalks were cultured 4 mo. on solidified MS shoot and the meristem 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.
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