Plant regeneration from leaf explants of the fern Platycerium bifurcatum in vitro

ScientiaHorticulturae, 56 ( 1 9 9 4 ) 2 5 7 - 2 6 6

257

Elsevier Science Publishers B.V., A m s t e r d a m

Plant regeneration from leaf explants of the fern Platycerium bifurcatum in vitro Marjana Camlohaa,*, Nada Gogalab, Janko Rode a ainstitute of Biology, University of Ljubljana, Karlovska 19, 61000 Ljubljana, Slovenia bDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Askerceva 12, 61000 Ljubljana, Slovenia (Accepted 27 July 1993)

Abstract Adventitious bud development was obtained on juvenile, in vitro grown leaves of Platycerium bifurcatum (Cav.) C.Chr. Entire leaves, less than 12 m m long, were cultured on Murashige and Skoog medium as modified by Hermen and Sheehan and supplemented with 0, 5 or 10 #M 6-benzylaminopurine (BA). Adventitious buds developed directly from leaf tissue without callus formation within 20 days. Bud organogenesis occurred on all tested media: it was found in greater numbers on media containing BA but in this media b u d growth was slower. In the majority of explants cultured for 40 days on BA-free media ten to 35 buds were produced, mainly on the abaxial surface. On media with added BA, buds often arose on both surfaces of the leaf. Bud induction was accompanied by formation of rhizoid-like structures. Spontaneous apospory was also observed. To induce rooting, shoots were transferred to media supplemented with 3 - 1 2 # M indole-3-butyric acid, indole-3-acetic acid or a-naphthaleneacetie acid. Optimal rooting was achieved in the presence of 6/tM IBA. The plantlets, successfully transferred to soil, appeared morphologically identical to the donor plants. Key words: Adventitious buds; Fern; Leaf explants; Plant regeneration; Platycerium bifurcatum Abbreviations: B A = 6-benzylaminopurine; B M = b a s a l medium; IAA=indole-3-acetic IBA = indole-3-butyric acid; NAA = a-naphthaleneacetic acid; RLS = rhizoid-like structures.

acid;

Introduction

The staghorn fern Platycerium bifurcatum (Cav.) C.Chr. (Polypodiaceae) is an epiphytic fern with typical forked fertile leaves. Among the genus Platycerium it represents one of the most widely cultivated greenhouse species (Tryon and Tryon, 1982). Asexual propagation occurs through shoot development from root buds (Hoshizaki and Price, 1990). Sexually, the fern propagates by spores, and this technique is most often used by horticulturists but * C o r r e s p o n d i n g author.

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it is generally slow (Thentz and Moncousin, 1984) and phytopathological problems are c o m m o n (Lane, 1981 ). In the last few decades the tissue culture m e t h o d has replaced the conventional methods of propagation in many ornamental species (Debergh et al., 1990), but there are only few reports on tissue culture ofP. bifurcatum (Thentz and Moncousin, 1984; Camloh and Gogala, 1991 ). The objective of the present study was the induction and regeneration of adventitious buds obtained on leaf explants, with special emphasis on the surface structures of the early stages of caulogenesis. Materials and methods Juvenile plants of P. bifurcatum (Cav.) C.Chr. were raised in aseptic culture from spores according to the m e t h o d of Camloh and Gogala (1992) however, sporophyte development occurred on solidified media and not on sterilised soil. Entire leaves, 8-12 m m in size, were detached from juvenile in vitro grown plants and placed flat on the surface of the m e d i u m so that the basal end and the abaxial surface were placed in contact with the medium.

Fig.1. An SEM micrograph of a leaf after 20 days on BA-freebud-induction medium showing adventitious buds, rhizoid-likestructures (RLS) and small unidentified structures (arrowed). Note presenceof some branched RLS (bar= 1 mm).

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Fig.2. An SEM micrograph of a leaf after 20 days on bud-induction medium supplemented with 5pM BA showing adventitious buds and many multiceUular scales. Table 1 Effects of BA on the regeneration of adventitous buds from leaves cultured on bud-induction medium and evaluated after 20, 30 and 40 days in culture. The percentage of regenerating leaves are presented. For each treatment, 20-25 leaves were taken Culture time (days)

20 30 40

BA concentration (#M) 0

5

10

88.9 94.1 100

76.5 92.9 100

75.0 93.3 100

The culture m e d i u m of Murashige and Skoog (1962), as modified by Henhen and Sheehan ( 1978 ), but without adenine sulphate contained 0.8% DifcoBacto agar and 3% sucrose. Media were adjusted to pH 5.7-5.8 before autoclaving. This basal m e d i u m (BA) was supplemented with 0, 5 or 10/tM BA to evolve a bud-induction media. The adventitious bud development on leaves was observed after 20, 30 and

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Fig.3. Leafcultured for 40 days on BA-freebud-inductionmedium.Explant is overgrownwith buds. Leavesof adventitiousbuds are clearlyvisible (bar= 1 mm). 40 days in culture using a stereomicroscope. The number of explants forming buds and the regeneration site on the leaves were determined, while the number of shoots was estimated. Surface structures developed on the explants were examined using a scanning electron microscope (SEM). Adventitious buds induced on growth-regulator free BM were excised after 8 weeks and elongated on the same medium for an additional 4 weeks. Elongated shoots, measuring 10-20 m m in length, were rooted on BM containing 2% sucrose, supplemented with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or ot-naphthaleneacetic acid (NAA) at 0, 3, 6, 9, and 12/tM, for 4 weeks. After another 2 weeks on growth-regulator free medium, the number and the length of roots and the number of rooted shoots were recorded. Cultures were maintained at 25 + 2 ° C under a 16 h photoperiod at 5.512.3 Wm -2 (Sylvania GroLux F40 T12 and fluorescent LV 20 lamps) and were transferred to fresh media every 4 weeks. Regenerated plantlets with well-developed roots were transferred to soil and maintained under high relative humidity for the first 2 weeks and then placed in a glasshouse. The Student's t-test was used to calculate the levels of statistical signifi-

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Fig.4. Leaf cultured for 40 days on bud-induction medium supplemented with lOaM of BA (bar= 1 mm). cance (p) for the length and n u m b e r of roots. For each treatment 15-20 ex' plants were used, and all experiments were repeated two or three times. For SEM observation explants were fixed in 2% glutaraldehyde (buffered in O.1M phosphate buffer, p H 7.0 ) for 3 h at 4 ° C. The leaves were then rinsed with phosphate buffer, dehydrated in a graded ethanol series and transferred to isopropanol. After critical-point drying (Bomar-9OOEX), samples were sputter-coated with gold and viewed with a SEM (Jeol JSM-840A). Results

In all treatments adventitious bud induction occurred within 20 days after the leaves were explanted on the m e d i u m (Fig. 1, Fig. 2). The percentage of leaves regenerating buds after 20 days in culture was lower on media containing BA, but after 30 and 40 days this effect disappeared. After 40 days in culture all explants regenerated buds (Table 1 ). On BA-free m e d i u m the majority of the explants produced buds on the abaxial surface, while on BA-con-

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Fig.5. Aposporousgametophytesdevelopingon a leaf culturedfor 40 dayson growth-regulator freebud-inductionmedium (bar= 1 mm). taining media buds often arose on both sides of the leaf (data not shown). The survival of explants in all treatments was above 90%. Little difference was found in responses to the two concentrations ofBA ( 5 and 10#M). In general, at lOaM BA the buds were smaller. However, at both concentrations the buds were smaller when compared with those on the BAfree medium (Fig. 3, Fig. 4). In the majority of the explants cultured for 40 days on BA-free medium, 10-35 buds developed (Fig. 3 ) and after 3 months in culture, up to 150 shoots were obtained from a single leaf. Development of adventitious buds was to a certain degree variable, b o t h within and among explants on the same medium. Bud differentiation was asynchronous. SEM observations showed that buds arose directly from leaf tissue without callus formation (Figs. 1 and 2). BA in the medium resulted in a higher frequency of multicellular scale development and bud organogenesis; however, the buds were smaller (Figs. 1 and 2). Beside buds, the formation of rhizoid-like structures (RLS) was observed on explants grown on all media. After 40 days in culture, RLS developed on

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Table 2 Effects of IBA, IAA and NAA on rooting o f shoots evaluated 6 weeks after the initiation of the experiment. Shoots were cultured on rooting medium for 4 weeks and then subcultured on growth-regulator free medium for 2 weeks. The number of roots per rooted shoot and the root length are given as mean + SE Auxin

Concentration (#M)

Rooted shoots (%)

Mean no. roots per explant

Mean root length (ram)

IBA

3 6 9 12 3 6 9 12 3 6 9 12

100 100 100 88.9 66.6 91.7 75.0 100 80.0 46.7 20.0 0 81.8

4.5 ___0.3 *.1 5.1 +0.8* 5.3 + 0.6** 5.4+0.9** 4.2+0.7 3,7+0.4 5.8 + 0.4*** 5.4+0.5*** 4.6 + 0.8 2.1 + 0.6 2.0 + 0.6 0 2.8+0.3

2.1 + 0.2*** 2.8+0.2*** 2.0 + 0.2*** 1.9+0.2"* 2.0+0.3*** 2.1 +0.2*** 2.7 + 0.2*** 2.4+0.2*** 3.0 ___0.2*** 2.4 + 0.3"** 1.3 + 0.2 0 1.0+0.1

IAA

NAA

Control

1The levels o f significant differences were calculated between control and other media. * P < 0.05; **P< 0.01; ***P< 0.001.

70-80% of the explants. The development of RLS coincides with bud induction. Some branched RLS were also observed (Fig. 1 ). During observations of bud regeneration, a few examples of spontaneous apospory were recorded occurring on the lamina. Aposporously produced gametophytes appeared similar to gametophytes produced from spores (Fig.5). Elongated shoots obtained on growth-regulator free bud-induction media were transferred to BM containing various concentrations of IBA, IAA or NAA for root induction and growth (Table 2 ). In all tested media RLS, callus and bud formation occurred at the base of the shoots. Although the highest number of roots per explant was achieved on the medium with 9#M IAA, the percentage of rooted shoots was relatively low. Therefore we assumed that 69gM IBA added to the medium gave best rooting results. However, we must point out that the amount of callus produced increased with increasing auxin concentration, which is also valid for the other auxins used. NAA in the medium resulted in the most vigorous callus growth. At higher concentrations root development on leaves of the shoots was observed. Plantlets with a well-developed root system were transferred to soil. The survival rate was very high. Regenerated plants looked normal both in culture and after transfer to soil. They did not show morphological abnormalities when compared with donor plants. The regeneration process, from primary explant to planflet appropriate for planting in soil, occurred within 18 weeks.

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Discussion

Adventitious bud regeneration is possible from juvenile leaves of P. bifurcatum without the involvement of an intermediary callus stage. Our results show that organogenesis could be induced without any growth regulator. Media used for inducing bud proliferation in ferns as well as in other plants generally include different growth regulators (Bhojwani and Razdan, 1983; Dykeman and Cumming, 1985; Ziv and Hadar, 1991 ). Apparently, P. bifurcatum is an exception. According to Bhojwani and Razdan ( 1983 ) the exogenous requirements for the growth regulators depend on endogenous hormone levels in the plant system. Thus, the formation of adventitious buds on the leaves of P. bifurcatum without growth regulators may be a result of appropriate endogenous hormone levels. Thentz and Moncousin (1984) used 8.9-13.3/zM BA for inducing adventitious buds on 1 cm 2 fragments of young P. bifurcatum leaves, while in our experiments there was no need for a growth regulator to obtain a satisfactory number of buds necessary for propagation. We found that medium containing BA resulted in a higher number of buds, but their elongation was slower in comparison with those on cytokinin-free medium, so that their separation was hindered. The differences in response to BA obtained in the present study in comparison with the results described by Thentz and Moncousin (1984) might depend on the age and the initial size of the explant. In the early stages of adventitious bud development on leaves, the formation of multicellular scales was observed. Richards et al. (1983) described similar scale development in the early stages of in vivo formation of root buds in P. bifurcatum. Small structures that are indicated by an arrow in Fig. 1 are similar to the initial stages of aposporous outgrowths obtained on Pteridium aquilinum leaves (Sheffield, 1984). However, it is possible that these structures are early developmental stages of adventitious buds. Histological and cytological analyses are necessary to define these structures exactly. The formation of RLS on the explants is an unusual phenomenon. They are probably rhizoids, but doubt exists because some of them are branched, which is very rare (E. Sheffield, personal communication, 1990). Thentz and Moncousin (1984) also observed rhizoid development on the leaf fragments of P. bifurcatum. It is noteworthy that Steil ( 1939 ) considers apospory in the broad sense to also include the production of only rhizoids on the explants. In a restricted sense, apospory is defined as regeneration of a more or less complete gametophyte from sporophytic tissue (Raghavan, 1989). Although the gametophytes observed on a few explants (Fig.5) could be defined as apospory in this restricted sense, further physiological and developmental studies are required to research the details of this interesting phenomenon, obtained for the first time in vitro in P. bifurcatum in this study.

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Among the rooting media tested the best rooting of elongated shoots was obtained on medium supplied with 6/tM IBA. However, the development of RLS, buds and callus at the base of the shoots undoubtedly slowed root formation and growth. According to the results obtained on media containing NAA, we may conclude that supraoptimal concentrations of NAA were used. In our recent research (Camloh and Gogala, 1991 ) we found that the dilution of BM resulted in better root development and growth. From some other reports on tissue culture of fern species, it is also evident that organogenesis is inhibited with standard levels of Murashige and Skoog inorganic nutrients (Loescher and Albrecht, 1979; Dykeman and Cumming, 1985 ). After rooting plantlets were successfully transplanted to soil and their morphology was found to be identical to the donor plants. The high percentage of regenerating explants, the large number of shoots produced per explant without an intermediary callus phase, good rooting of shoots, relatively simple in vitro requirements, and the uniformity of regenerated plants make this system applicable for mass propagation.

Acknowledgements We wish to thank Dr. E. Sheffield for comments on SEM micrographs, Dr. J. Zel for constructive comments on the manuscript, Dr. Z. Podlesek for his help with statistical analysis of the data, and N. Hojnik for technical assistance.

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