In vitro regeneration of leatherleaf fern (Rumohra adiantiformis (G.Forst.) Ching).ABSTRACT.--Rumohra adiantiformis, also known as "leatherleaf fern", is an ornamental species that, because of its long display life, is widely used in floral arrangements. In this study, a new protocol for in vitro regeneration of the leatherleaf fern was established. For spore germination, two culture media (MS and Knop) were assessed with presence or absence of 1g [L.sup.-1] activated charcoal (AC) under different light and dark conditions. Frond, frond microcuttings, and prothallus explants were evaluated on Knop regeneration medium supplemented with 1g [L.sup.-1] AC, 0.5% agar, pH 5.0 in combination with 2,4 dichlorophenoxyacetic acid (2,4-D: 0.0, 0.1, 0.5 and 1.0 mg [L.sup.-1]) and 6-benzylamino purine (BA: 0.0, 0.1, 0.5 and 1.0 mg [L.sup.-1]). For rooting, four levels of [alpha]- naphthaleneacetic acid (NAA: 0.0, 0.01, 0.1 and 0.2 mg [L.sup.-1]) were tested. After 18 days of culture, spore germination rate was 100% on Knop medium with AC and 8 h light/16 h dark. After 120 days of culture, sporophytes 1.7 [+ or -] 0.4 cm in length developed on Knop medium, while those sporecultured on MS medium never produced sporophytes. From those germinated sporophytes, prothallus explants cultivated on Knop medium with AC and 0.5 mg [L.sup.-1] BA showed the highest regeneration rate with 235.7 gametophytes. The best sporophyte rooting response was obtained with 0.01 mg [L.sup.-1] NAA. Complete, regenerated sporophytes were obtained 183 days after culture initiation. By this procedure it would be possible to obtain up to 2 million sporophytes from one fertile frond. To determine the origin of the regenerated gametophytes, a histological analysis was performed with scanning electron microscopy (SEM). The analysis revealed that the gametophytes were regenerated from explant epidermal tissues on either the adaxial or abaxial surface.
KEY WORDS.--Leatherleaf fern, Rhumora, Sporophyte regeneration
Ferns are conventionally propagated both sexually and asexually. In asexual, or vegetative, propagation, new plants are produced from rhizomes, stolons, tubers, stipules, roots, buds, cuttings, and attached aerial stems (layering). Asexual fern propagation also includes apospory and apogamy (Kottackal et al., 2006), deviations from the "normal" life cycle in ferns. Apospory is the development of a gametophyte from an epidermal cell or cells of a sporophyte (Ambrozic-Dolinsek et al., 2002), while apogamy is the development of a sporophyte directly from a gametophyte without sexual fusion (Kottackal et al., 2006). In either direction, since only mitotic divisions are involved, the number of chromosomes remains the same (Foster and Gifford, 1974).
Rumohra adiantiformis (G.Forst.) Ching, also known as the "7-weeks-fern", "leatherleaf" or "samambaia-preta", is highly valued on the international florist greenery market because of its long post-harvest display life (D'Souza et al., 2006). Rumohra adiantiformis extractivism began in the 1970s as a major survival strategy for small-scale African and Brazilian farmers. It is a low-growing species indigenous to many parts of the Southern Hemisphere (Australia, South and Central America, Southern Africa and some Islands in the Indian Ocean). It is now cultivated commercially in American nurseries for the cut flower industry (Poole and Conover, 1978; Milton and Moll, 1988; Schumann and Mills, 1996). Most of the world's production of R. adiantiformis occurs in Florida where it is grown in controlled environments (Stamps et al., 1994; Stamps, 2004).
Commercial propagation of R. adiantiformis is done by rhizome division, but frequent replanting is necessary, making it difficult to satisfy the great demand for its leaves (Chert and Read, 1983; Strandberg, 2003). Improved regeneration procedures of in vitro culture are therefore desirable (Fernandez and Revilla, 2003). Little research on the in vitro culture of R. adiantiformis through rhizomes (Chen and Read, 1983; Amaki and Higuchi 1991) or spores (Drum and Randi, 2002; Drum and Randi, 2006) has been reported. To our knowledge, no attempt to increase the in vitro regeneration rate or to reduce the regeneration period has been reported. Therefore, the objectives of this study were to increase the regeneration rate and to reduce the time of regeneration of R. adiantiformis by in vitro culture.
MATERIALS AND METHODS
In vitro sorus germination and initial explants.--Mature Rumohra adiantiformis fronds bearing sori were used as the source of explants. For disinfection, fronds were washed under running tap water for 5 min, then the surface was sterilized sequentially, first in 70% (v/v) ethanol (30 sec), 0.1% sodium hypochloride solution with two drops of Tween 20[R] (10 min), and rinsed three times with sterile distilled water (3 min each). Five sori were dissected and placed in glass flasks containing 30 mL of Knop (Knop, 1865) or MS (Murashige and Skoog, 1962) medium, supplemented with 0 or 1 g [L.sup.-1] activated charcoal (AC) Hycel[R]. Each culture medium was adjusted to pH 5.0 with 1 N NaOH or HCl. Both media were gelled using 0.5% agar (Bioxon[R]) before autoclaving at 121[degrees]C and 1.2 kg [cm.sup.-2] for 20 min. The cultures were incubated at 25 [+ or -] 1[degrees]C for one month and maintained under a cycle of 16 h light and 8 h dark, with a light intensity of 39 [micro]mol [m.sup.-2] [s.sup.-1]. After spore germination and sporophyte regeneration, fronds (Fr) 20-30 mm long (Figure 1A) and frond microcuttings (MicFr) 10 mm long (Figure 1B) were used as initial explants for in vitro gametophyte regeneration. For a third explant, fronds bearing sori were cut into 2 mm pieces and placed in glass flasks containing sterilized distilled water. Subsequently, sori were centrifuged (Menendez et al., 2006) in Eppendorf tubes containing 70% ethanol for 1 min; 0.01% sodium hypochloride was then added, and the mixture was centrifuged for 3 min. Finally, the sori were washed three times with sterilized distilled water, and the obtained spores were placed on Whatman No. 42 filter paper (International Ltd Maidstone England) discs 1 cm in diameter and cultivated for one month on Knop medium supplemented with lg [L.sup.-1] AC under the same light conditions as described above. The resulting prothalli (Pr) (Figure 1 C) were used as the third type of explants for gametophyte regeneration.
[FIGURE 1 OMITTED]
Sporophyte regeneration rate and time reduction.--In order to increase the rate and reduce time of sporophyte regeneration, the effect of 2,4-dichlorophenoxyacetic acid (2,4-D Sigma[R]) (0.0, 0.1, 0.5 and 1.0 mg [L.sup.-1]) in combination with 6-benzylamino purine (BA Sigma[R]) (0.0, 0.1, 0.5 and 1.0 mg [L.sup.-1]) on Fr, MicFr and Pr was evaluated. Each group of explants was placed on Knop medium supplemented with 1 g [L.sup.-1] AC, 0.5% agar, at pH 5.0. All cultures were maintained under conditions of 16/8 h light/dark for three months at 25 [+ or -] 1[degrees]C. A total of 32 treatments were tested.
Rooting test--For rooting, regenerated sporophytes were transferred to MS medium (half-strength) supplemented with [alpha]-naphthaleneacetic acid (NAA Sigma[R]) (0.0, 0.01, 0.1 and 0.2 mg [L.sup.-1]), 3% sucrose and 1g [L.sup.-1] AC at pH 5.0. All media were gelled using 0.8% agar (Bioxon[R]). The cultures were incubated for one month at 25 [+ or -] 1[degrees]C under the same conditions described for the second stage.
Transfer to soil conditions and acclimatization.--Each well-rooted sporophyte was transferred to a pot containing cosmopeat[R] as substrate. The pots with transparent covers were maintained at a temperature of 25-28[degrees]C under a 16 h photoperiod. The covers were removed after 8 days.
Histological study.--In order to determine the origin of regenerated gametophytes from Fr, MicFr and Pr explant epidermal tissues on either the adaxial or abaxial surface, scanning electron microscopy (SEM) was used. For SEM observations, the selected explants were fixed in 2.5% glutaraldehyde for 12 h at 4[degrees]C then washed three times (30 min at 4[degrees]C) with Sorensen's 0.1 M phosphate buffer, pH 7.2. The explants were then dehydrated in a series of different grades of ethanol (30, 40, 50, 60, 70, 80, 90%) for 40 min and transferred to 100% ethanol (3 times, 3 min). After the samples were dehydrated in a critical-point dryer (Sandri-708A), the samples were sputter-coated with gold for 4 min in the ionizer (Ion Sputter JFC-1100, Jeol Fine Coat). All observations were documented on digital images using SEM (Jeol JSM 6390) at 15 Kv.
Statistical analysis.--Analysis of variance (ANOVA) and a test of least significant differences (LSD) were performed to assess germination rate, germination period, prothallus mass diameter, number of sporophytes, sporophyte height, number of regenerated sporophytes per explant (Fr, MicFr and Pr), length and number of roots, length and number of fronds, percentage of adaptation, and adaptation period, in a randomized-block design. Each flask contained five explants and was regarded as one block. Each test was replicated four times. All data were processed with the Statistical Analysis System V8.0 (SAS Institute, 1999).
RESULTS AND DISCUSION
In vitro sorus germination and initial explants.--In the present study, using the same MS medium, 70% spore germination was obtained both with and without AC, but 28 and 30 d after culture initiation, respectively, were necessary for sporophyte regeneration (Table 1). In contrast, a rate of 100% spore germination was obtained on Knop medium. When this medium was supplemented with AC, 18 d were required for germination, while 23 d were required without AC (Table 1). It was also noted that sporophytes formed only on gametophytes from spores cultured on Knop medium. Thus, it appears that medium salts and AC have an important effect on the rate and period of germination, as well as on sporophyte formation. The major difference between the two culture media is probably the amount of nitrogen. MS medium contains eight times the ammonium concentration contained in Knop medium.
It was reported in Nephrolepis exaltata (L.) Schott fern that 32% of spore germination took place within 28-30 d after culture initiation on MS media (Gonzalez et al., 2006). In Drynaria fortunei (Kunze) J.Sm. a spore germination rate of 15.3% was obtained after 7 d on MS medium (Chang et al., 2007). The influence of absolute and relative amounts of nitrate and ammonium on induction and differentiation of plant cell cultures has been reported for a number of in vitro systems (Ramage and William, 2002). Ammonium used as the sole source of nitrogen appears to have a negative effect on growth and morphogenesis (Walch-Liu et al., 2000). In addition, activated charcoal absorbs vitamins, cytokinins, auxins and inhibitory substances, thus altering the ratios of medium components and subsequently affecting plant regeneration (Fridborg et al., 1978; Ebert and Taylor, 1990; Druart and Wulf, 1993; Arzate et al., 2007).
All spore cultures germinated under light, but the spores cultured in the dark, on either Knop or MS medium and with or without AC, did not germinate even after 200 d of culture. However, when the cultures were kept under a dark to light cycle, all spores germinated after 15 d on Knop medium and after 18 d on MS medium. Weinberg and Bruce (2007) mentioned that the spore culture of Anemia phyllitidis (L.) Sw. needed light to induce its germination. Likewise, Chang et al. (2007) observed that spores of D. fortunei germinated only under light, indicating that light is one of the most important factors that affect events in the life cycle of a fern, functioning as a signal to awaken the dormant fern spore.
In the present study, after spore germination, a prothallus mass 1.4 cm in diameter was observed (Table 1, Figure 2A), but sporophytes were never induced on MS cultures, either with or without AC. In contrast, prothallus masses with developed sporophytes 1.6 cm tall (Figure 2B) were observed at 16 weeks on the Knop medium with AC (1.7 cm in height) or without AC (Table 1). Nevertheless, Brum and Randi (2006) reported that R. adiantiformis sporophyte induction took place after 19 weeks. This is probably consistent, as it was mentioned by Teng (1997) that the addition of AC to the culture media greatly improved efficiency of sporophyte regeneration.
[FIGURE 2 OMITTED]
Sporophyte regeneration rate and time reduction.--When Fr explants were cultured on Knop medium, masses of gametophytes were induced with 1 g [L.sup.-1] AC, 0.1 mg [L.sup.-1] 2,4-D and 0.1 mg [L.sup.-1] BA (Table 2, Figure 2C). In addition, the masses of gametophytes were observed on both sides (adaxial and abaxial) of Fr explants. Using MicFr explants, the best gametophyte regeneration response (13.5 gametophytes) was observed with lg [L.sup.-1] AC, 1.0 mg [L.sup.-1] 2,4-D and 0.1 mg [L.sup.-1] BA (Table 2, Figure 2D). On these explants, aposporous gametophytes developed. This physiological response is probably due to an effect caused by the exogenous growth regulators used. In contrast, when Pr explants were cultured with lg [L.sup.-1] AC and 0.5 mg [L.sup.-1] BA, 235.7, apogamous sporophytes developed (Table 2, Figure 2E). Pr explants produced 17 times more gametophytes than MicFr and 22 times more than Fr. Extrapolating this result, it would be possible to obtain up to 2 million regenerated sporophytes from one fertile R. adiantiformis frond. Moreover, Pr explants did not require the presence of 2,4-D, probably due to its high content of endogenous auxins and the regeneration capacity of the vegetative cells which were observed in those explants (Figure 2F). The differences in response to 2,4-D and BA of assayed explants obtained in the present study might depend on the type, age and the initial size of the explants.
The aposporously produced gametophytes were similar in appearance to gametophytes produced from spores. According to Bhojwani and Razdan (1983), the exogenous requirements for growth regulators depend on endogenous hormone levels in the plant system. Thus, the formation of adventitious buds on leaves without growth regulators may be a result of appropriate endogenous hormone levels (Camloha et al., 1994). These observations are in agreement with those of Ong and Ng (1998) on the fern Pyrrosia pilosellodes (L.) M. Price, in which regeneration of drought-stressed gametophytes was detected through the formation of unicellular protonemata on the surfaces of living cells; the unicellular protonemata continued normal development as if they were sporelings (gametophytes that develop following spore germination). These results show the beneficial effect of the combination auxin/citokinin in enhancing cell division (Camloha et al., 1994).
Chen and Read (1983) worked with rhizome tips of Rumohra adiantiformis on modified Prague's medium, using 2iP, kinetin and zeatin; they reported that the potential annual propagation rate from a single rhizome tip can be as high as 16,000,000 plantlets ready for potting and transfer to the greenhouse. Amaki and Higuchi (1992) reported that R. adiantiformis, in order to regenerate plants from segments of rhizome in MS medium, it was necessary to add 0.5 mg [L.sup.-1] NAA and to eliminate the use of BA.
Rooting test.--Regenerated sporophytes with an average of 7.3 fronds and 6.5 roots were obtained after 4 weeks. However, a better response was observed when 0.01 mg [L.sup.-1] NAA was added, resulting in 9.6 fronds and 8.2 roots in the same period. It was also observed that higher levels of NAA decreased number and length of both roots and fronds (Table 3, Figure 2G). These results are similar to those reported by Kottackal et al. (2006), who also increased the number of rhizoids in gametophytes of Pityrogramma calomelanos (L.) Link by adding NAA. On the other hand, Thakur et al. (1998) obtained 10.5 roots per regenerated Matteuccia struthiopteris plantlet in gelled half-strength MS medium supplemented with 1.0% AC after 8 weeks.
Transfer to soil conditions and acclimatization.--Acclimatization of 98% of the regenerated sporophytes with 10 cm long fronds was achieved after 15 days. Chang et al. (2007) reported that 5-month-old D. fortunei gametophytes were transferred to the greenhouse and juvenile sporophytes developed after 14 weeks. Chen and Read (1983) observed that preconditioning of intact rhizome tips of R. adiantiformis (4 weeks) to the air in the greenhouse is very important.
Histological study.--Induced aposporous gametophytes were observed on the adaxial surface of Fr (Figure 2C) and on the abaxial surface of MicFr (Figure 2D), while sporophyte regeneration by "apogamy" was observed on Pr explants (Figure 2E). Likewise, different stages of gametophyte regeneration were also observed in the vegetative cells (Figure 2F). Kottackal et al. (2006) in Pityrogramma calomelanos observed that, using crosier explants, gametophytes were induced from epidermal hair.
Conclusions.--The Knop medium supplemented with 1 g [L.sup.-1] AC under light was the best condition for spore germination and formation of sporophytes in Rumohra adiantiformis. The highest number of gametophytes (235.7) was obtained by culturing prothallus explants on Knop medium with 1 g [L.sup.-1] AC and 0.5 mg[L.sup.-1] BA, 183 d after culture initiation. The addition of 0.01 mg [L.sup.-1] NAA improved the rooting of regenerated sporophytes. The histological study revealed that gametophytes originated from vegetative cells on either the adaxial or abaxial surfaces of the assayed explants. Aposporous gametophytes were observed on Fr and MicFr explants and apogamous sporophytes on Pr explants. The high percentage (98%) of acclimatization and the relatively short time (183 d) of regeneration make this procedure applicable for obtaining up to 2 million completely regenerated sporophytes.
This research was supported by the Consejo National de Ciencia y Tecnologia (CONACYT). We thank to Hilda Araceli Zavaleta Mancera and Greta Hanako Rosas Saito of the Colegio de Postgraduados for their help in the histological analysis. We are also grateful to Jose Luis and Marie Ponce P., as well as to the enterprise "La Flor de Catemaco", for collecting plant material, and to the laboratory staff (Amaury M. Arzate F., Mariel Galindo T., Jose Luis Pina E., Rafael Mejia F., Guadalupe Gutierrez G., Laura Heredia B., Monica Bautista P. and Ariadna Rodriguez B.) and to Martha Eve Jimenez O. for her suggestions to the manuscript, as well as to Jose Guadalupe, Mariel Citli and Brisceida Guadalupe Munoz A.
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MARIA DEL CARMEN ROCIO AVILA-PEREZ Universidad Autonoma del Estado de Mexico, Facultad de Ciencias Agricolas, Carretera TolucaIxtlahuaca entronque al Cerrillo Km 15, Campus Universitario "El Cerrillo", El Cerrillo Piedras Blancas, Toluca, Estado de Mexico
LAURA WHITE-OLASCOAGA Universidad Autonoma del Estado de Mexico, Facultad de Ciencias, Centro de Investigacion en Recursos Bioticos. Carretera Toluca-Ixtlahuaca entronque al Cerrillo Km 15, Campus Universitario "El Cerrillo", E1 Cerrillo Piedras Blancas, Toluca, Estado de Mexico, tel. y Fax: (01722) 2965554 ext 120, e-mail: email@example.com
AMAURY M. ARZATE-FERNANDEZ *
Universidad Autonoma del Estado de Mexico, Facultad de Ciencias Agrfcolas, Carretera Toluca-Ixtlahuaca entronque al Cerrillo Km 15, Campus Universitario "El Cerrillo", El Cerrillo Piedras Blancas, Toluca, Estado de Mexico, tel. y Fax: (01722) 2965518 ext 144, e-mail: firstname.lastname@example.org
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TABLE 1. Effect of two culture media on spore germination rate and sporophyte regeneration of Rumohra adiantiformis. Values were obtained 30 d (for germination) and 120 d (for sporophyte) after culture initiation. Germination Germination Treatment (%) (days) Knop + 1 [gL.sup.-1] AC 100 18 [+ or -] 0.0 Knop + 0 [gL.sup.-1] AC 100 23 [+ or -] 0.0 MS + 1 [gL.sup.-1] AC 70 28 [+ or -] 0.0 MS + 0 [gL.sup.-1] AC 70 30 [+ or -] 0.0 Diameter of prothallus Number of Treatment mass (cm) sporophytes Knop + 1 [gL.sup.-1] AC 1.6 [+ or -] 0.2 5.9 [+ or -] 1.9 Knop + 0 [gL.sup.-1] AC 1.0 [+ or -] 0.3 3.1 [+ or -] 0.9 MS + 1 [gL.sup.-1] AC 1.4 [+ or -] 0.3 0.0 [+ or -] 0.0 MS + 0 [gL.sup.-1] AC 1.1 [+ or -] 0.4 0.0 [+ or -] 0.0 Height of sporophyte Treatment (cm) Knop + 1 [gL.sup.-1] AC 1.7 [+ or -] 0.4 Knop + 0 [gL.sup.-1] AC 0.7 [+ or -] 0.2 MS + 1 [gL.sup.-1] AC 0.0 [+ or -] 0.0 MS + 0 [gL.sup.-1] AC 0.0 [+ or -] 0.0 [+ or -] SD = Standard deviation MS = Murashige and Skoog medium Knop = Knop medium AC = Activated charcoal TABLE 2. Number of regenerated gametophytes from three kinds of explants in response to plant growth regulators and Knop medium with 1 [gL.sup.-1] active charcoal. Values were obtained 108 d after culture initiation and represent the means [+ or -] standard deviations (n [greater than or equal to] 50 cultured explants per treatment). Frond PGR: Microcutting 2, 4-D: BA Frond (Fr) (MicFr) (mgL-1) 0.0:0.0 1.5 [+ or -] 1.O f g h 4.5 [+ or -] 1.3 c d e 0.0:0.1 4.0 [+ or -] 1.8 c d 6.5 [+ or -] 1.2 c 0.0:0.5 2.0 [+ or -] 0.8 f g 0.0 [+ or -] 0.0 m h 0.0:1.0 3.5 [+ or -] 0.6 d e 6.2 [+ or -] 1.2 c d 0.1:0.0 1.2 [+ or -] 0.5 f g h i 4.2 [+ or -] 2.8 d e 0.1:0.1 10.5 [+ or -] 1.2 a 2.0 [+ or -] 1.4 f g h 0.1:0.5 1.0 [+ or -] 0.0 g h i 2.0 [+ or -] 1.6 f g h 0.1:1.0 0.2 [+ or -] 0.5h i 2.7 [+ or -] 1.2 e f g 0.5:0.0 5.0 [+ or -] 0.8 c 4.0 [+ or -] 1.8 e f 0.5:0.1 2.2 [+ or -] 0.9 f g 3.2 [+ or -] 1.7 e f g 0.5:0.5 1.0 [+ or -] 0.8 g h i 10.0 [+ or -] 1.8 b 0.5:1.0 7.2 [+ or -] 1.7 b 1.5 [+ or -] 1.0 g h 1.0:0.0 2.5 [+ or -] 1.2 e f 0.2 [+ or -] 0.5 h 1.0:0.1 0.5 [+ or -] 0.5 h i 13.5 [+ or -] 2.5 a 1.0:0.5 2.5 [+ or -] 1.2 e f 4.5 [+ or -] 1.2 c d e 1.0:1.0 0.0 [+ or -] 0.0 i 3.5 [+ or -] 1.2 e f g 2,4-D (A) 17.71 *** 8.22 *** BA (B) 19.47 *** 12.45 *** A X B 38.63 *** 25.78 *** Variation 35.92 36.75 Coefficient PGR: 2, 4-D: BA Prothallus (Pr) (mgL-1) 0.0:0.0 68.5 [+ or -] 6.4 h i 0.0:0.1 82.5 [+ or -] 9.8 g h 0.0:0.5 235.7 [+ or -] 15.3 a 0.0:1.0 122.0 [+ or -] 14.7 c d e 0.1:0.0 146.7 [+ or -] 15.6 b 0.1:0.1 135.5 [+ or -] 21.0 b c 0.1:0.5 91.7 [+ or -] 24.0 f g 0.1:1.0 61.5 [+ or -] 5.0 h i j 0.5:0.0 120.5 [+ or -] 10.8 c d e 0.5:0.1 45.2 [+ or -] 3.1 j 0.5:0.5 105.2 [+ or -] 7.3 e f 0.5:1.0 137.5 [+ or -] 19.8 b c 1.0:0.0 131.0 [+ or -] 25.3 b c d 1.0:0.1 111.7 [+ or -] 23.3 d e f 1.0:0.5 59.0 [+ or -] 7.4 i j 1.0:1.0 58.7 [+ or -] 9.0 i j 2,4-D (A) 16.22 *** BA (B) 15.07 *** A X B 52.93 *** Variation 14.36 Coefficient Means with same letters are not statistically different (LSD, 0.05). *** [less than or equal to] 0.001 TABLE 3. Effect of NAA on Rumohra adiantiformis root number, root length, frond number and frond length. Values were obtained at transplant and are means [+ or -] standard deviations (n [greater than or equal to] 50). Treatment NAA Root Root length (mg [L.sup.-1]) number (cm) 0 6.5 [+ or -] 1.9 c 0.5 [+ or -] 0.2 c 0.01 8.2 [+ or -] 0.8 a 0.7 [+ or -] 0.2 a 0.1 8.1 [+ or -] 2.4 a 0.5 [+ or -] 0.2 c 0.2 7.6 [+ or -] 1.5 b o.6 [+ or -] 0.1 b Treatment NAA Frond Frond length (mg [L.sup.-1]) number (cm) 0 7.3 [+ or -] 2.0 c 2.0 [+ or -] 0.4 a 0.01 9.6 [+ or -] 2.8 b 2.1 [+ or -] 0.6 a 0.1 10.1 [+ or -] 4.4 a 1.9 [+ or -] 0.5 b 0.2 9.5 [+ or -] 3.8 b 1.8 [+ or -] 0.4 b