Interaction between sucrose and pH during in vitro culture of Nephrolepis biserrata (Sw.) Schott (Pteridophyta)

Interação da sacarose e níveis de pH durante o cultivo in vitro de Nephrolepis biserrata (Sw.) Schott (Pteridophyta)

Interaction between sucrose and pH during in vitro culture of Nephrolepis biserrata (Sw.) Schott (Pteridophyta)

Interação da sacarose e níveis de pH durante o cultivo in vitro de Nephrolepis biserrata (Sw.) Schott (Pteridophyta)

Sandra Tereza Ambrósio; Natoniel Franklin de Melo ^*

Embrapa Semi-Árido, C. Postal 23, CEP 56302-970, Petrolina, PE, Brasil

ABSTRACT

The present work reports the effect of different pH and sucrose concentrations on in vitro propagation of Nephrolepis biserrata. Fronds aseptically obtained from stolon segment culture were cultivated in MS semi-solid medium supplemented with 0, 15, 30, 45 and 60g.L^-1 sucrose and pH adjusted to 3, 5, 7 and 9, in a factorial design. Frond number and length, pinnae number, raquis length and diameter, fresh and dry matter weight were measured. Inhibition of shoot and leaf regeneration was observed in all the pH treatments in the absence of sucrose. On the other hand, when sucrose was added to the medium, the shoot number increased, reaching the maximum average values of 51.25 and 38.25 shoot per explant at pH 5 and 7, respectively. Sucrose concentrations from 15 to 45g.L^-1 increased leaf length and diameter and the pH 9 did not affect the dry matter weight, and was still not adequate for development of new fronds. Young sporophytes were successfully acclimated.

Key words: ferns, ornamental plants, tissue culture

RESUMO

O presente trabalho relata o efeito de diferentes valores de pH e concentrações de sacarose na propagação in vitro de Nephrolepis biserrata. Frondes obtidas assepticamente a partir do cultivo de estolões foram inoculadas em meio semi-sólido MS, suplementado com 0, 15, 30, 45 e 60g.L^-1 de sacarose e pH ajustado para 3, 5, 7 e 9, em arranjo fatorial. Foram avaliados: número e comprimento das frondes, número de pinas, comprimento e diâmetro da raque e biomassa fresca e seca da parte aérea. Em geral, houve inibição da regeneração de brotos e folhas em todos os tratamentos de pH na ausência de sacarose. Por outro lado, a presença de sacarose no meio induziu 51,25 e 38,25 brotos por explante no pH 5 e 7, respectivamente. Concentrações entre 15 e 45g.L^-1 de sacarose aumentaram o comprimento e o diâmetro das folhas e o pH 9 não afetou o peso foliar seco, sendo inadequado para o desenvolvimento de novas frondes. Os esporófitos jovens foram submetidos, com sucesso, ao processo de aclimatação.

Palavras-chave: pteridófitas, plantas ornamentais, cultura de tecidos

Introduction

In the sporophytic phase, pteridophytes commonly present large and complex fronds, with elaborate leaves, which, besides facilitating spore dispersion and supporting asexual reproduction for the spore presence, also provide excellent ornamental qualities (Jones 1987). Therefore, the pteridophytes fronds are known worldwide due to their ornamental value, and several studies have been conducted for optimizing in vitro conditions, not only to contribute for the propagation of ornamental species in commercial scale, but also to understand the species biology (Fernández et al. 1999). Among the pteridophytes species used as ornamental foliages in commercial and residential environments, we can find those of the genus Nephrolepis (Morton 1958; Zimmerman & Jones 1991). It is a highly distinctive genus characterized by abundant stolons, disarticulate, inequilateral and usually entire pinnae, hydathodes in the upper surface of the pinnae and spores with a relative thin and tuberculate-rugose perispore (Tryon & Tryon 1982).

Several difficulties imposed by specificity of fern life cycle interfere on in vitro cultivation protocol establishment (Morini 2000). According to Stokes (1984), in vitro propagation of pteridophytes species can require prolonged periods of time, which sometimes might extend to several years, so that the frond and plant size can reach the uniformity demanded by the market of ornamental plants. During in vitro regeneration of ferns species, some factors affect the multiplication efficiency and many aspects have been examined, such as explant donor source (Atmane et al. 2000; Whittier & Braggins 2000), nutrient type (Padhya & Mehta 1982; Fernández et al. 1999) and physical culture conditions (Lin & Yang 1999). Sporophyte fragments have been used as explants source, which are usually the most practical and viable form of micropropagation of ornamental species in commercial scale (Bertrand et al. 1999).

Since the 1970's, in vitro multiplication protocols of Nephrolepis species have been reported. Loescher & Albrecht (1979) observed the effect of environmental, nutritional and hormonal conditions on in vitro organogenesis of N. exaltata, var. bostoniensis, using juvenile segments of stolon. Lê (1983) reported the influence of sucrose concentrations on sporophyte propagation of N. exaltata (L.) Schott using gametophytic tissue as explant source, while Pasqual et al. (1994) propagated the same fern by shoots, in order to study in vitro multiplication under the effect of sucrose and mineral salt association. Higuchi et al. (1987) described the effect of growth regulators and sucrose on histological differentiation and fresh matter weight of N. cordifolia Prsel. The association of these variables with pH is important because it could influence the multiplication efficiency, establishment and quality of the propagated sporophytes (Handreck 1992; Koedam et al. 1992; Pevalek-Kozlina 1996).

In the present work four pH treatments and five levels of sucrose concentrations were studied, in order to evaluated in vitro induction, regeneration and establishment of N. biserrata sporophytes.

Material and methods

The material studied was obtained from cultivated plants and identified as Nephrolepis biserrata (Sw.) Schott (Davalliaceae/Filicopsida) (Tryon & Tryon 1982). The plants were maintained in the Experimental Glasshouse of Embrapa Semi-Árido, Petrolina-PE, for sanitary treatment.

Initially, young sporophytes cultivated in a glasshouse, in vases with substrate (organic matter and vermiculite 1:1), were treated with a solution containing Benomyl (0.5%), Nuvacron (1mg.L^-1) and Extravon (1mg.L^-1) solution once a week. Stolon segments were collected, washed in water containing 1-3 drops of commercial detergent, and re-washed in running distilled water. The disinfection process consisted of immersion in 70% alcohol (v/v) for 1-2 minutes, followed by 20 minutes of immersion in a 2% sodium hypochloride solution and rinsed in sterile distilled water.

A hundred stolon segments were cut into pieces of 2cm length, placed in MS semi-solid medium (Murashige & Skoog 1962) supplemented with 30g.L^-1 sucrose, with pH adjusted to 5.9. Juvenile sporophytes were obtained after 90 days. Single shoots initiated from juvenile sporophytes were used as explants for the experimental assays, as previously described (Byrne & Caponetti 1992).

Under sterile condition, explants were transferred to MS semi-solid medium supplemented with factorial combinations of sucrose at 0, 15, 30, 45 and 60g.L^-1 and pH adjusted to 3, 5, 7 and 9. Each treatment consisted of four replicates and was maintained in a growth chamber at 25 ± 1ºC and 16 h light photoperiod. All media were solidified with 7g.L^-1 agar and autoclaved at 120ºC, 1kg.cm^-2 pressure, for 20 minutes. After autoclaving, the final pH shifted downwards around 0.2.

After 45 days of cultivation, frond and pinnae numbers, frond length, rachis diameter, and fresh and dry matter weight of aerial parts were evaluated. The Duncan test (p < 0.05) was used for evaluating the data obtained with different sucrose concentrations and pH levels after ANOVA assay (Statsoft Inc 1997). Plants from in vitro culture were transferred to plastic boxes (veg-box), using substrate composed by vermiculite, sand and organic matter in the ratio 1:1:1, previously sterilized. The acclimatization was carried out in a glasshouse at an average temperature of 27±2ºC, 85% relative humidity and 250-560µmol.m^-2.s^-1 light intensity.

Results and discussion

The sporophyte sanitary state was considered a critical factor for in vitro cultivation. Explants with endophytic microorganisms displayed growth and differentiation into primary leaves, but the development was interrupted around the 20^th day after inoculation. This kind of explant was not viable for establishment or regeneration experiments. However, new shoots from health explants were obtained 10 to 15 days after inoculation. There was 80% of shoot regeneration and 20% showed both necrosis and contamination. According to Morini (2000), the main problem for establishment of in vitro propagation protocols in pteridophytes is the presence of microorganism in the plants, hindering the disinfection process and affecting their survival.

Frond and pinnae numbers and fresh matter weight were significantly affected by the interaction between sucrose concentration and pH (Table 1). Regarding frond and pinnae numbers, satisfactory results were obtained with the increase of sucrose concentration in all pH treatments. The increase of sucrose concentration significantly improved frond production. Maximum values of 51.25 and 38.25 fronds per explant were obtained, with 60g.L^-1 sucrose at pH 5 and 7, respectively. Sucrose at 45g.L^-1 and pH 5 also increased the fresh matter weight. On the contrary, an increase in sucrose concentration reduced the frond induction in N. exaltata (Pasqual et al. 1994). It was also reported that sucrose concentrations higher than 50g.L^-1 caused a decrease in the formation of new prothallias colonies in this species (Lê 1983).

Length, diameter and dry matter weight of aerial parts were not significantly affected by the interaction between sucrose concentration and pH. In this case, the Figure 1 shows data analyzed separately, because pH and sucrose levels in ANOVA were significant. For simplicity, however, we have presented the graphs with levels pooled (Figure 1). Responses were obtained at sucrose concentration of 30 and 45g.L^-1 regarding frond length, rachis diameter and dry matter weight. The absence or higher sucrose concentration (60g.L^-1) significantly reduced these parameters. Data suggested inhibition of plant development at pH 9, in which reduction of frond length and rachis diameter was also observed. Similar response was observed for dry matter weight, although statistical differences were not observed among pH levels 3, 5 and 9. Thus, pronounced growth responses were obtained at pH 5 and 7. This intense growth is possibly due to increase in cell division with subsequent frond production, as reported by Pevalek-Kozlina (1996) in Platycerium bifurcatum. In this species, neutral or acid pH stimulates in vitro frond production. Under natural conditions, pH also appears to have a selective effect in the establishment of many ornamental species (Symonds et al. 2001). Some pteridophytes have been reported to prefer alkaline soils (Handreck 1992), whereas others are acidiphilous (Koedam et al. 1992) or yet generalists (Ranal 1995). Under in vitro conditions, the medium pH may have significant effects on growth and differentiation of cells, although there are many reports showing that cells in culture can significantly alter the pH of the external medium through differential uptake of nutrients and/or through H⁺ fluxes (Minocha 1987).

Some factors, such as limited photosynthetic activity due to in vitro conditions, may explain the significant decrease of regeneration rate under sucrose absence. However, high sucrose level in the culture medium might result in chlorophyll degradation and reflect on the frond development (Fernández et al. 1999). Despite of in vitro plant regeneration can be successful without sucrose, its presence usually promotes a significant increase of new regenerated structures, since in the induction phase, the carbon source accumulated in the plant tissue is enough to start the development (Lalonde et al. 1999). Meanwhile, during the development phase, large and available carbon amounts are necessary for structural growth, differentiation and development. In general, sucrose in a medium can serve as an osmotic agent and as a carbon and energy source. Many cultures, especially embryo and some shoot cultures, require a medium with high osmotic potential (Tremblay & Tremblay 1995).

In the present study, all in vitro regenerated plants did not show any observable morphological abnormalities. During acclimatization process, the regenerated sporophytes produced new fronds quite well distributed in the apex, and show a more intense green color when compared to the original sporophyte source. In conclusion, the results described here provided efficient approaches for in vitro multiplication of N. biserrata.

Acknowledgments

The authors are very thankful to Maria Tereza Ataíde, for her technical assistance; to Adriana Mayumi Yano-Melo, for the statistical analyses and help in the manuscript; to CNPq (Proc. 300392/96-6) and Embrapa Semi-Árido, for their financial support.

Received: April 15, 2003. Accepted: April 24, 2004

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Publication Dates

• Publication in this collection
  28 Mar 2005
• Date of issue
  Dec 2004

History

• Accepted
  24 Apr 2004
• Received
  15 Apr 2003