diethylstilbestrol and in vitro morphogenesis of ...

Proc. Fla. State Hart. Soc. 109:8-11. 1996.

DIETHYLSTILBESTROL AND IN VITRO MORPHOGENESIS OF IMMATURE COCONUT (COCOS NUCIFERA L.) LEAF TISSUES JOHN L. GRIFFIS, JR. ', RICHARD E. LITZ 2 Al'\ID THOMAS SHEEHAN 3

J.

1

Citrus and Environmental Horticulture Department Florida Southern College Lakeland, Florida 33801 2

Tropical Research and Education Center University of Florida Homestead, Florida 33031

3

Department ofEnvironmental Horticulture University of Florida Gainesville, Florida 32611

Additional index words. Palms, micropropagation, tissue cul-

ture, growth regulators. Abstract. In vitro morphogenesis of selected 'Puerto Rico' coconut palm seedling leaf tissues in response to a novel plant growth regulator, diethylstilbestrol (DES), was studied. Callus initiation was stimulated by 2,4-dichlorophenoxyacetic acid (2,4-D) and DES in modified Eeuwens Y3 plant growth medium. Some leaf explants formed callus on their cut edges, while others formed roots or somatic proembryos directly. Haustoriallike tissues were associated with the somatic embryos, and the structures were biploar. Development of the somatic embryos to maturity and subsequent germination were not observed.

Several studies have been reported for developing an in vitro clonal propagation method for coconut palm (Griffis, 1992). Plant tissues and organs that have been utilized as explants have included newly emerged roots, root initials, stem pieces, immature inflorescences, anthers, filaments, unfertilized ovaries, immature fruits, endosperm, and zygotic embryos. Unfertilized coconut ovary explants have been the subject of a previous report (Griffis et a!., 1995); however, clonal palms were not produced in the earlier studies. In all earlier experiments, only tissues that could be removed from mature coconut palms without destroying the plants were used as explants. In the current studies, immature leaves of seedling palms were used, and the plants were sacrificed . Several reports have indicated that embryogenic cultures could be induced from monocotyledonous leaf tissues, including immature leaves (Coconut Research Institute, 1985; Conger eta!., 1987; De Touchet and Pannetier, 1990; Drira, 1983; Gupta eta!., 1984;Jones, 1983, 1984; Nwankwo and Krikorian, 1983; Omar eta!., 1990; Pannetier and Buffard-Morel, 1982a, 1982b; Raju eta!., 1984). A combination of techniques and plant growth media developed in earlier trials were utilized in the current study in order to provide additional insight into coconut regeneration. Materials and Methods

The seedling leaf trials involved the use of 'Puerto Rico ' coconut palms sprouted and growing in 10-inch bulb pans Florida Agricultural Experiment Station journal Series No. N-01373.

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(donated by Margo Farms of Homestead, FL). The coconut seedlings were carefully removed from the nuts at the union of shoot and nut with a sharp budding knife . Care was taken not to break them off too far from the nut, thereby exposing either the immature leaf tissues or the growing point. The expanded leaves were cut off except for the clasping portion of the petiole, leaving a large cylindrical piece of tissue containing the stem base and the growing point as well as numerous immature leaves sheathed by a few older leaf bases. These cylinders of palm tissue were washed repeatedly with detergent and deionized water to remove any soil particles and debris. Subsequently, they were dipped in acetone and then surfacedisinfested by submersion and continuous agitation in a 15 % Clorox™ (v/v) solution for 15 min. In the laminar flow hood, the sterilant was decanted and the tissues were rinsed two times with sterile deionized water. The plant growth medium consisted of modified Y3 medium (Eeuwens, 1976, 1978) composed of the following salts mixture: KN0 3 (2020 mg/ liter); KCl (1492 mg/liter); NH 4 Cl (535 mg/liter); MgS0 4 ·7Hp (247 mg/liter); CaC1 2 ·2Hp (294 mg/ liter); NaH 2P0 4 2Hp (312 mg/liter); MnS0 4 4Hp (11.2 mg/liter); H 3 B0 3 (3.1 mg/liter); ZnS0;7Hp (7.2 mg/ liter) ; KI (8.3 mg/liter); CuS0 4 5Hp (0.16 mg/liter); CoC1 2 ·6Hp (0.24 mg/liter); NiCI 2 ·6Hp (0.24 mg/liter); NaMo0 4 2Hp (0.24 mg/ liter); and Fe·EDTA (32.5 mg/liter). In addition, Y3 medium was supplemented with myoinositol (100 mg/liter), thiamine HCl (0.5 mg/liter), adenine hemisulfate (40 mg/ liter), pyridoxine HCl (0.5 mg/liter), glycine (2.0 mg/liter), nicotinic acid (0.5 mg/liter) , 0.3% activated charcoal (w/v), and sucrose (3% w/v). Several combinations of plant growth regulators were investigated (Table 1). Birth control pills containing 5 mg of DES in an inert carrier were added to certain media formulations (Table 1) . The various media combinations were solidified with either 0.8% (w/v) Difco Bacto agar or 0.2% (w/v) gellan gum. The pH of each medium was adjusted to 5.7 before the addition of the activated charcoal and solidifying agent. Each 25 mm x 150 mm test tube contained 10 ml plant growth medium. The various media were autoclaved at 121C and 1.1 kgcm 2 for 20 min. Individual cylinders of coconut palm tissue were dissected under axenic conditions. The outer leaf bases were removed with a sterile scalpel, revealing the off-white, unexpanded, immature leaves. These immature, folded leaves were cut at the Table 1. Growth regulators added to modified Y3 medium for immature coconut leaflet cultures. Treatments Growth regulators, (mg/ liter) 2,4-D" BAP'

51' S1des1' S1des3'

52 S2des1 S2des3

53 S3des1 S3des3

54 S4des1 S4des3

55 S5des1 S5des3

0.0 0.0

10.0 0.0

25.0 0.0

50.0 0.0

25.0 3.0

•Media S1-S5 were not supplemented with DES. rMedia coded with "des1" were supplemented with 5 mg/ liter of DES. •Media coded with "des3" were supplemented with 15 mg/ liter of DES. •2,4-Dichlorophenoxyacetic acid '5-Benzylaminopurine

Proc. Fla. State Hart. Soc. 109: 1996.

point of attachment and sliced across the veins at 1-cm intervals. Leaf tissue was then sliced longitudinally into several explants, each containing three or four folds of immature leaf tissue. Some immature petioles were also used as explants. One leaf explant was placed on medium in each 25 x 150 mm test tube. Only the ridges of the folds were in contact with the medium. Seven seedlings yielded sufficient explants for five replicates of each treatment; there were 150 explants. Cultures were maintained in darkness in an incubator at 26 ± 1C. After 7 days, cultures were exposed to indirect light for a 16hr photoperiod. Cultures were evaluated after 10 weeks in culture and then transferred to fresh plant growth medium (15 ml medium in 15 x 100 mm plastic Petri dishes sealed with Parafilm™). Leaf explants were maintained in a darkness in an incubator at 26 ± 1C for the remainder of the trial. Leaf explants were evaluated again after 8, 16, 24, and 32 additional weeks in culture and were transferred to fresh medium at each evaluation. The experiment was repeated using eight seedlings, which yielded sufficient leaf explants for 10 replicates of each treatment. There were 300 palm explants in the second experiment. Cultures were maintained in darkness at ca. 26 ± 2C. After 3 weeks, the temperature was increased to 34 ± 2C for the remainder of the trial. Then they were returned to 26 ± 2C for the remainder of the trial. Leaf explants were evaluated after 12, 16, 20, and 24 weeks, and were transferred to fresh growth medium in test tubes at weeks 12 and 20. Individual leaf sections were examined for the development of callus on either the cut leaf edges or the surface of the leaves. Differentiation of roots, shoots, nodules, somatic embryos or other structures arising from the callus and from the explants were noted.

The spherical nodules in cross section were composed almost entirely of small, isodiametric, highly cytoplasmic cells with prominent nuclei. The larger nodules were more highly organized (Fig. 1c), and were composed of several distinct layers of cells and cell types (Fig. 1d, 1e). These structures became biploar, and possessed meristematic regions that consisted of highly cytoplasmic cells with prominent nuclei (Fig. 1d). Many cells in the meristematic zones were actively dividing. Spherical nodules and other adventitious structures originated primarily near vascular bundles, in accordance with observations of other researchers who have reported somatic embryogenesis from coconut leaf explants (Pannetier and Buffard-Morel, 1982a, 1982b; Schwendiman et al., 1988, 1990). Somatic proembryos were associated with leaf vascular tissues or were in tissues adjacent to vascular bundles or traces. The entire embryogenic culture was composed of somatic proembryos, ranging in size from a few cells to well-defined globular structures. Both 2,4-D and DES were present in the induction medium of all leaf cultures that were embryogenic. In most reports of somatic embryogenesis of palm species, 2,4-D has been utilized in the plant growth medium (Branton and Blake, 1983; Gupta et al., 1984; Srinivasan et al., 1985), although anti-auxins have also been reported to be effective (Hanower and Hanower, 1984). Except for our previous reports (Griffis, 1992; Griffis et al., 1995), DES has not been utilized to induce embryogenic culture. DES is known to inhibit plasmalemma ATPase activity and thereby increase sucrose efflux in isolated plant tissues (Secor, 1987). In Dionaea muscipula cultures, 2,4-D increased the rate ofH• efflux while the addition of DES caused the rate to decrease (Rea, 1983). It is not clear in the current study how DES might interact with 2,4-D during induction of embryogenic competence. Activated charcoal was also present in the plant growth medium and could have affected the growth Results and Discussion response in some way (Ebert and Taylor, 1990). The second experiment confirmed many of the above obJuvenile phase tissues of perennial plants cultured in vitro are often more easily manipulated than adult phase tissues. servations. Approximately 25% of the explants remained offThere have been several reports of juvenile coconut palm white or pale green, and were alive after nearly 6 months after leaves as explants for initiating embryogenic cultures (Panne- explanting; 8.3% produced some form of adventitious tier and Buffard-Morel, 1982a, 1982b; Raju et al., 1984; Ver- growth. Ten of the explants, including three grown on medideil et al., 1989). Excised shoot tips from seedling coconuts um containing 50 mg/liter 2,4-D but without DES, produced have also been reported to produce multiple shoots a small amount of callus either on the cut edges of the leaf explant or on the leaf surface. Morphogenesis also occurred in (Pavithran and Shyla, 1984). Very few coconut seedling leaf explants produced callus the presence of2,4-D and DES on the other 15 explants. Simduring the first experiment, although many explants did ilar observations were reported by Paranjothy et al. (1990) show considerable expansion in vitro. Small amounts of callus with oil palm leaf tissues. Leaf explants grown on modified Y3 formed along the cut edges of some explants (Fig. 1a), and on medium supplemented with 25-50 mg/liter 2,4-D, 15 mg /lithe surface of a few vascular ridges. Only four of the 150 co- ter DES, 0-5 mg/1 BA demonstrated the optimum morphoconut leaf explants demonstrated any other growth response, genic response. In addition to the induction of embryogenic and all were on formulations containing both 2,4-D and DES. cultures, adventitious shoot (Fig. 1f) and root formation was One leaf explant developed several nodules on the surface of occasionally observed from callus. Roots also formed from a vein on the underside of the explant. After ca. 6 months, a haustoria-like tissues that developed from callus. Direct root single nodule continued to enlarge, and developed a root formation occurred from the leaf explants. The choice of gel(Fig. 1b). Another single explant developed numerous pale ling agent, i.e., either agar or gellan gum, did not significantly yellow globular structures on both sides of the leaf explant. affect results. The induction of embryogenic and organogenic cultures Some of the globular structures increased in size for 42 weeks, whereas others became hyperhydric and eventually necrotic. from coconut leaf explants on modified Y3 medium containSeveral nodules developed on both sides of the midrib and ing 2,4-D and DES has been described. Although normal sopetiole of one explant and enlarged over time (Fig. 1c). matic embryo maturation and germination were not These nodules appeared to be similar to the early stage coco- observed, this embryogenic pathway represents a new apnut somatic embryos reported elsewhere (Pannetier and Buf- proach that can be investigated for clonally propagating coconut in vitro. Studies are currently underway that address some fard-Morel, 1982a, 1982b; Verdeil et al., 1989). Proc. Fla. State Hart. Soc. 109: 1996.

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Figure la. Callus formed in vitro on cut edge of immature coconut leaflet. Figure lb-c. Various adventitious structures, including "nodules" and roots, arising in vitm from immature coconut lealets. Figure ld-e. Microscopic examination of some of these various adventitious structures reveals tissue differentiation and organization. Figure lf. Adventitious shoot arising in vitm from immature coconut leaflet.

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of the problems associated with maturation of coconut somatic embryos. Literature Cited Branton, R. L. and]. Blake. 1983. Development of organized structures in callus derived from explants of Cocos nucijlrra L. Ann. Bot. 52:673-678. Coconut Research Institute. 1985. Unilever's success in coconut tissue culture . Coconis Newsletter 19:1. Conger, B. V., F.J. Novak, R. Afza and K Erdelsky. 1987. Somatic embryogenesis from cultured leaf segments of Zea mays. Plant Cell Rep. 6:345-347. De Touchet, B. , Y. Duval and C. Pannetier. 1990. Oil palm (Elaeis guineensis Jacq.) regeneration from embryogenic suspension culture, p . 249. In: Abstracts Vlith international congress on plant tissue and cell culture. IAPTC, Amsterdam. (abstr.) Drira, N. 1983. Multiplication vegetative du palmier (Phoenix dactylifera L.) par Ia culture in vitro de bourgeons axillaires et de feuilles qui en derivent. C. R. Hebd. Seances Acad. Sci., Ser. III 296:1077-1082. Ebert, A. and H . F. Taylor. 1990. Assessment of the changes of 2,4-dichlorophenoxyacetic acid concentration in plant tissue culture media in the presence of activated charcoal. Plant Cell Tis. Org. Cult. 20:165-172. Eeuwens, D.J. 1976. Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms (Cocos nucifera) and cultured in vitro. Physiol. Plant. 36:23-28. Eeuwens, D.]. 1978. Effects of organic nutrients and hormones on growth and development of tissue explants from coconut (Cocos nucifera) and date (Phoenix dactylifera) palms cultured in vitro. Physiol. Plant. 42:173-178. Griffis,]. L.,Jr. 1992. Morphogenesis of the coconut palm (Cocos nucifera L.) in vitro. PhD Diss., Univ. of Fla., Gainesville. Griffis, Jr.,]. L., R. E. Litz and T.]. Sheehan. 1995. Diethylstilbestrol and in vitro morphogenesis of unfertilized coconut (Cocos mucifera L.) ovaries. Proc. Fla. State Hort. Soc. 108:10-15. Gupta, P. K , S. V. Kendukar, V. M. Kulkarni, M. V. Shirgurkar and A. F. Mascarenhas. 1984. Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro. Plant Cell Rep. 3:222-225. Han ower,]. and P. Han ower. 1984. Inhibition et stimulation, en culture in vitro, de l'embryogenese des souches issues d 'explants foliares de palmier a huile. C. R. Hebd. Seances Acad. Sci., Ser. D. 298:45-48. Jones, L. H. 1983. Development of high quality oil palm clones by tissue culture propagation, pp. 1-9. In: A. C. Cassells and]. A. Kavanagh (eds.) .

Plant tissue culture in relation to biotechnology. Royal Irish Academy, Dublin. Jones, L. H. 1984. Novel palm oils from cloned palms.JAOCS 61:1717-1719. Nwankwo, B. A. and A. D. Krikorian . 1983. Morphogenetic potential of embryo-and-seedling-derived callus of Elaeis guineensis Jacq. var pisifera Becc. Ann. Bot. 51:65-76. Omar, M. S., M. K Hameed and M.S. Al-Rawi. 1990. In vitro propagation of the date palm, p. 121. In: Abstracts Vlith international congress on plant tissue and cell culture. IAPTC, Amsterdam. (abstr.) Pannetier, C. and]. Buffard-Morel. 1982a. Premiers resultats concernant Ia production d 'embryons somatiques a partir de tissus foliaires de cocotier, Cocos nucifera L. Oleagineux 37:349-354. Pannetier, C. and J. Buffard-Morel. 1982b. Production of somatic embryos from leaf tissues of coconut, Cocos nucifera L. , pp. 755-756. In: A. Fujiwara (ed.). Plant tissue culture 1982. Proc. 5th Int. Plant Tiss. Cell Cult. Marzuen, Tokyo. Paranjothy, K , R. Othman and C. S. Tan. 1990. Developmental aberrations in oil palm clones, p . 263. In: Abstracts Vlith international congress on plant tissue and cell culture. IAPTC, Amsterdam. (abstr.) Pavithran, K and R. Shyla. 1984. Observations on 'midget' and 'suckering' coconut palms.]. Plantation Crops 12:85-89. Raju, C. R., P. P. Kumar, M. Chandramohan and R. D. Iyer. 1984. Coconut plantlets from leaf tissue cultures.]. Plantation Crops 12:75-78. Raju, C. R., P. P. Kumar, M. Chandramohan and R. D. Iyer. 1984. Coconut plantlets from leaf tissue cultures.]. Plantation Crops 12:75-78. Rea, P. A. 1983. The dynamics of H+ efflux from the trap lobes of Dionaea muscipula Ellis (Venus's [sic] flytrap) . Plant Cell Env. 6:125-134. Schwendiman, J., C. Pannetier and N. Michaux-Ferriere. 1988. Histology of somatic embryogenesis from leaf explants of the oil palm Elaeis guineensis. Ann. Bot. 62:43-52. Schwendiman, J., C. Pannetier and N. Michaux-Ferriere. 1990. Histology of embryogenic formations during in vitro culture of oil palm Elaeis guineensis]acq. Oleagineux 45:409-418. Secor, ]. 1987. Regulation of sucrose efflux from soybean leaf discs. Plant Physiol. 83:143-148. Srinivasan, C., R. E. Litz,J. Barker and K Norstog. 1985. Somatic embryogenesis and plantlet formation from christmas palm callus. HortScience 20:278-280. Verdeil,J. L.,J. Buffard-Morel and C. Pannetier. 1989. Embryogenese somarique du coco tier (Cocos nucifera L.) a partir de tissus foliaires et inflorescenciels. Bilan des recherches et perspectives. Oleagineux 44:403-411.

Proc. Fla. State Hart. Soc. 109:11-14. 1996.

EFFECT OF LATE GROWTH REGULATOR APPLICATIONS AT FLORAL BUD EMERGENCE AND STRETCH ON EASTER LILY DEVELOPMENT GARY J. WILFRET AND R. A. REISER Gulf Coast Research and Education Center University of Florida, IFAS 5007 60th St. East Bradenton, FL 34203

Additional index words. Lilium longi.florum, paclobutrazol, uniconazole, ornamental plants.

ancyrnidol,

Abstract. Easter lily cv. Nellie White (Lilium /ongiflorum Thunb.) bulbs (7/8 size), which had been stored at 40 F for 7 weeks, were planted in 6-inch diameter pots on 28 Dec. 1995. Plants were grown in a glasshouse with 40% light exclusion and amFlorida Agricultural Experiment Station journal Series No. N-01353.


bient temp. to a minimum of 45F. Initial plant groWth regulator (PGR) treatments of ancymidol, paclobutrazol or uniconazole were applied as a soil drench or foliar spray on 1 Feb. 1996 when plants were ca. 4 inches tall. Subsequent PGR applications were made at floral bud emergence (FBE) on 28 Feb. or at floral bud stretch (FBS) on 6 Mar. Plants drenched with ancymidol at 0.25, 0.375, or 0.5 mg ai/pot were 18.3, 17.8, and 16.3 inches tall, compared to 22.2-inch tall plants drenched with water. Plants drenched with paclobutrazol at 2, 3, or 4 mg ai were 18.0, 16.3, and 14.3 inch tall, while plants drenched with uniconazole at 0.05, 0.07, 0.09, or 0.11 mg ai were 19.0, 16.8, 16.1, and 14.5 inches tall, respectively. Optimum plant height would be 18 inches. Multiple applications of ancymidol had no effect on plant height. Paclobutrazol at 2.0 mg/pot at FBE yielded shorter plants than those plants given a single 2.0 mg/pot drench but had no effect at FBS. All second applications of uniconazole produced plants with shorter inflorescence than

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