early developmental stages of homalothecium sericeum (hew...

American International Journal of Biology June 2015, Vol. 3, No. 1, pp. 1-18

ISSN: 2334-2323 (Print), 2334-2331 (Online) Copyright © The Author(s). All Rights Reserved.

Published by American Research Institute for Policy Development DOI: 10.15640/aijb.v3n1a1

URL: http://dx.doi.org/10.15640/aijb.v3n1a1

Early Developmental Stages of Homalothecium sericeum (Hew.) Schimp (Brachytheciaceae) under in vitro Conditions

Bengi Erdağ1, Münire Nihan Bağdatli2, İlknur Kuzu, Yelda Emek

Abstract

In this study, spore germination features, protonemal and gametophytic development stages of Homalothecium sericeum (Hew.) Schimp. had been investigated. Effects of plant growth regulators on colony size and number of gametophyte per colony were also studied. Spore germination is in exosporic type and dependent upon on light. The highest germination percentage is obtained in Distilled water exposed to 24 ± 2 °C and 16:8 photoperiod conditions. It is followed by 1/8 MS, ¼ MS, ½ MS and full strength MS media. Inspite of observing the highest germination percentage in Distilled water, this was not used in the further experiments because of weak chloronemal development and no colonization has been observed. Among the MS media in four different strength, the best colonial growth was obtained in full strength MS medium. In terms of the level of colony growth, the MS medium was followed by the ½ MS medium. The ¼ and 1/8 MS media inhibited the forward growth of protonemata. Sporelling type of H. sericeum is Bryum type due to spore germination and protonemal form with long cylindiric cells. Response to cytokinins in colonial growth and gametophyte differentiation was observed as abnormal bud formation. Auxine type and concentration effected colony size and number of gametophyte per colony. At the and of sixth months, maximum colony size and maximum number of gametophyte per colony has been held in MS medium containing 1mg/L IAA.

Keywords: Homalothecium sericeum, in vitro, spore germination, protonema, PGRs, gametophytes

1Department of Biology, Faculty of Arts & Sciences, Adnan Menderes University, 09010, Aydın, Turkey. Phone number: +90 256 2128498 / 1712, Fax number: +90 256 2135379, Email: [email protected], 21Department of Biology, Faculty of Arts & Sciences, Adnan Menderes University, 09010, Aydın, Turkey.

2 American International Journal of Biology, Vol. 3(1), June 2015 1. Introduction

Bryophytes s. str. (mosses) are evolutionarily one of the oldest groups of land plants. However, despite representing the second largest group of land plants after angiosperms (15000-25000 species; Hallinback and Hodgetts, 2000; Gradstein et al., 2001), the amount of information regarding the biology and early life stages of bryophytes is still limited in comparison to vascular plants. Furthermore, this lack of information has persisted even though the results of basic and applied physiological, genetic, morphogenetic, ecological and evolutionary experiments are comparatively easier to observe in bryophytes (Sabovljevic et al., 2003).

Most bryophytes are clonal organisms. Bryophytes produce new colonies

either through spores, fragments and/or asexual propagules. Many researchers have reported that reproduction through vegetative diaspores is the most effective among these propagational ways (Keever, 1957; Mishler and Newton, 1988). In order to colonize new sites, spores are the effective way because they provide sufficient genetic diversity. Owing to their small size, spores can be easily carried and dispersed over long distances by the wind (Van Zanten and Pocs, 1981).

Spore germination is the first and most important stage of development

during colonization. The development observed following the germination of the spore (or, in other words, during the development of the sporeling) reflects the basic characteristics of the gametophyte, and is far more specific than the other developmental processes (Nehira, 1983). However, studying spore germination and the development of the protonema in situ is relatively difficult (Wiklund and Rydın, 2004). In vitro culture conditions thus present a more practical alternative. Furthermore, moss provide excellent and very convenient material for in vitro culture, as they are widespread, easy to manipulate experimentally, occupy little space, and are cheapter to use compared with vascular plants (Bijelović et al., 2004). Numerous researchers have conducted axenic culture studies on different species of mosses under controlled conditions (Basile and Basile, 1988; Kowalczyk et al., 1997; Sabovljević et al., 2003; Sabovljević et al., 2009). However, as is the case with higher plants, the development observed under axenic conditions tends to be species-specific, and different species may display different responses under the same in vitro conditions (Bijelović et al., 2004; Sabovljević et al., 2005).

Erdağ, Bağdatli & Emek 3

Homalothecium sericeum (Hedw.) Schimp. is a species of the Brachytheciaceae family. It is widely distributed in Eurasia. As is the case with many moss species, there is no direct information regarding the early life stages of H. sericeum. In fact, information regarding these stages are mostly obtained through generalizations based on the biology of model moss species such as Funaria sp. Polytrichum sp. Similar to many other species of bryophytes, H. sericeum possesses a relatively simple morphology. In addition, owing to its wide distribution and substrate tolerance, it also represents a potential test organism for studying plant physiology and developmental biology. However, for H. sericeum to be effectively used as a test organism, it is first necessary to obtain information regarding this species’ early development stages.

This study represents the first study on the early developmental stages of H.

sericeum under in vitro conditions. In our study trials, we investigated the spore germination characteristics and the stages of protonema and gametophyte development of the Homalothecium sericeum species under in vitro conditions, and evaluated the effect of different plant growth regulators on colony size and the number of gametophytes per colony.

2. Material and Methods 2.1. Plant Material, Sterilization and in vitro Spore Germination

Full developed H. sericeum plants were collected from mountain of Subice-

Beşparmak province AYDIN in March 2013. The voucher specimens are kept in the herbarium of Adnan Menderes Üniversity (AYDN 3403).

The cultures were initiated from mature spores, from ripped and unopened

capsules. Fresh, unopened capsules were surface sterilized by dipping in 1 % NaOCl for 6 minutes, and thoroughly rinsed three times in sterile distilled water.

To form spore suspensions, sterilized spore capsules were opened with the aid

of sterile forceps and dissection needle in laminar air flow cabinet and spores were released on eppendorf tubes containing 1 ml distilled water. Spore suspensions (approximately 10 µL) were taken with a micropipette and transferred to sterile petri dishes 90 mm in diameter containing 25 mL nutrient medium.

4 American International Journal of Biology, Vol. 3(1), June 2015

Distilled water (DW) and Murashige and Skoog (MS) (Murashige and Skoog, 1962) media in four different strengths (MS, ½ MS, ¼ MS, 1/8 MS) were used to test the effect medium compositions on the germination of H. sericeum spores. All the media were enriched 3% (w/v) sucrose. In all experiments, the pH of media was adjusted to 5.8 with 1M NaOH or HCl, and 0.8% agar-agar (w/v) (Sigma) was added before autoclaving at 105 kPa for 15 min at 121° C.

Cultures were maintained at 24 ± 2 °C with 16:8 photoperiod, with

illumination provided by cool white fluorescent lamps at 20 µmol m-2s-1. The same experiments were conducted in dark conditions and the effects of the dark on germination were investigated. 2.2. Protonema Structure, Protonemal growth and Gametophyte Formation

Protonemal colony pieces of approximately 2 mm diameter were collected

from protonemal colonies 2 months after they were first cultured. The protonemal colony pieces were then transferred to media consisting of full MS, ½ MS, ¼ MS and 1/8 MS in order to determine the effects of MS media with different concentrations. The media giving the best growth was selected based on colony diameter. In addition, to investigate the effect on exogenously-added plant growth regulators on colony size and the number of gametophytes per colony, colonies were transferred to full strength MS media with auxins [Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) and 2,4-D (2,4-Dichlorophenoxyacetic acid); 0.1, 1 and 2 mg/L] and cytokinins [Benzyl adenine (BA), Kinetin (Kin) 0.5, 1 and 2 mg/L, and Thidiazuron (TDZ) 0.0001, 0.001 and 0.05 mg/L]. The media without plant growth regulators was used as control.

All cultures were incubated in sterile 90 mm petri dishes containing 25 ml of

media. Incubation was performed for 6 months at 24±2 °C in a climate chamber with a 16/8 photoperiod (light intensity approximately 20 µmol m-2s-1). Subculturing was performed at 4-week intervals.


2.3. Data Collection and Statistical Analysis The spore germination trials were performed with 2 petri dishes for each type

of media and in 3 repeats. Starting from day 10 of incubation, the spore germination characteristics and the spore germination percentages in different media were recorded at 3-day intervals. In this context; randomly selected 100 spores were observed under light microscope and germ tube formation with or without few celled protonemata were considered as “germination”. Microscopic photographs were taken using an Olympus E 330 digital photograph machine attached to an Olympus CX 31 optical microscope and Leica EC3 digital photograph machine attached to Leica S8APO stereomicroscope. Photographs were taken in situ (in their media) or on a slide (a part dissected from colony).

Protonemal colony size and the number of gametophytes that formed per

colony were determined at the end of month 3 and 6. Observations regarding growth and development were recorded using an Olympus E 330 digital photograph machine attached to an Olympus CX 31 optical microscope, or a Leica EC3 digital photograph machine attached to a Leica S8APO stereomicroscope. The samples were photographed either in their media, or by separating them from their media and placing them on a lamella.

The data obtained from the trials were analyzed with the One-Way ANOVA

test by using the SPSS statistical package program (SPSS for WINDOWS, standard version, release16.0). Mean values in the data were compared using the Duncan Multiple Range Test, and p-values less than 0.05 were considered as statistically significant. 3. Results

Homalothecium sericeum spores which were taken from surface-sterilized capsule

(sterilization was 100% successful) are isosporic and spherical with 10-20 micrometers diameter (Figure 1a). In the media under constant dark conditions, the spores were swollen, but had failed to continue to the following stages of germination. In all media under photoperiod conditions, spore germination was observed. Spore germination of the H. sericeum spores was exosporic.


Three to four days after the beginning of culture, the spores had turned bright green and had started to swell (Figure 1b). Approximately ten days later, the walls of the swollen spores had split and a germ tube had formed (Figure 1c, Figure 1d, Figure 1e). In terms of germination polarity, unipolar and bipolar spores were common but tri and tetrapolar spores were also oserved with increasing rarity (Figure 1f, Figure 1g, Figure 1h). In the early stages of development, numerous primary chloronemal branches had formed on the main chloronema filament; these primary branches were then followed by secondary branches (Figure 1i and Figure 1j).

Figure 1. Germination Stages of H. sericeum Spores Cultured in MS Media.

a. Matured spore b. Swollen spore c. Ruptured spore d. Germ tube formation e. Unipolar spore germination and two celled protonema f. Bipolar spore germination g. Three polar spore germination h.Tetra polar spore germination i. Primary chloronemal branches developed on main chloronemal filament j. Chloronemal filament showing secondary branching . Scale bar: 20 µm

The spores of the H. sericeum species displayed different germination

percentages in different media (Figure 2). About 16 days after the beginning of culture, the highest germination percentage was observed among spores in the distilled water medium. It was respectively followed by spores in the 1/8 MS, 1/4 MS, 1/2 MS and MS media. The media used in the experiments varied considerably from one another with respect to their mineral nutrient contents. The highest mineral nutrient content was found in the MS medium.


Distilled water, on the other hand, had no mineral nutrients. A decrease was observed in the germination percentages in parallel with the increase in the mineral nutrient content of their media.

Figure 2. Germination Percentages of H. sericeum Spores in Different in vitro Media. The Error Bar Showed in the Graph is İndicating Standard Error

Although the highest germination percentage during the experiments was

observed in the distilled water medium, this medium was not used in the following stages of the experiment since it lead to no branching, and resulted in weak chloronemal development and no colonization. However, six months after the beginning of the protonema culture in this medium, gemma-like structures formation was observed on the tip or mid-sections of the protonemal (chloronema) branches (Figure 3a and Figure 3b). At the beginning, the gemmae-like structures appeared to have numerous chloroplasts and to be formed on spherical single cells. Afterwards, these gemmae-like structures acquired a composite form composed of 4-6 cells (Figure 3c).


Figure 3: Gemma Formation in Distilled Water A. Uni-Celled Gemma Developing at Apex of Protonemal Filaments B. Uni-Celled Gemma

Developing at The Middle of Protonemal Filaments C. Composite Gemma There was no difference in primary chloronematal branching and

development in different strength media spore. Chloronemal filaments had long cylindiric cells, vertical septated with plenty sphaerical chloroplast. In the early stages of development, numerous primary chloronemal branches had formed on the main chloronema filament; these primary branches were then followed by secondary branches. Most of the chloronemal branches had developed horizontally, while some had developed vertically. About one month after the beginning of culture, the terminal cells of the chloronema began to turn transparent, and started to show differentiation in the caulonema. The caulonema filaments had lighter colors and oval-shaped chloroplasts, along with cells whose septals were characteristically bent (Figure 4). Caulonemal filaments showed horizontal growth in the culture media, and formed numerous secondary chloronemal filaments. About 35-40 days after the beginning of culture, the cultures displayed the heterotrichous protonemal stage in the form of chloronema and caulonema. Sporelling type of H. sericeum is Bryum type because of spore germination and firstly appearing globose-subglobose cells followed by long cylindric cells. This sporelling type is characterized by filamentous form of protonema having long cylindric cells and contains only chloronema or chloronema with caulonema together (Nehira, 1983). In our experiments, both chloronema and caulonema were observed.


Figure 4. Chloronemal and Caulonemal Filaments Developing in MS Medium. Asteriks İndicate an Oblique Cross wall Characteristic of Caulonemal Cells

The 2 mm diameter protonemal colony pieces obtained from the MS media

with four different strengths demonstrated different levels of colony growth depending on the type/concentration of media. The 1/4 MS and 1/8 MS media inhibited the further growth of protonemata. After three months of incubation, the best colony growth (assessed based on colony diameters) was observed in the MS medium. In terms of the level of colony growth, the MS medium was followed by the 1/2 MS medium.

During the experiments investigating the effect of exogenous plant growth

regulators on colony size and gametophore differentiation; it was observed that the response to cytokinins application was callus formation (data not shown). Furthermore, in MS medium with 1 mg/L BA, abnormal or moruloid buds were induced (Figure 5). These buds did not result in gametophore differentiation.


The type and concentration of auxins being applied had a discernible effect on colony size and the number of gametophytes forming per colony (Table 1).

Figure 5. Abnormal and Moruloid Buds Developed in MS Medium Containing 1 mg/L BA


Table 1: Effects of auxines on colony size and number of gametophyte per colony in cultures after 3 and 6 months of incubation. The data obtained from the trials were analyzed with the One-Way ANOVA test by using the SPSS statistical package program (SPSS for WINDOWS, standard version, release16.0). Mean values in the data were compared using the Duncan Multiple Range Test, and p-values less than 0.05 were considered as statistically significant

At the end of the 3rd month, the maximum colony size was observed in the

MS medium with 1 mg/L IAA, while the lowest values were obtained in the MS medium with 0.1 and 2 mg/L IAA. The maximum number of gametophytes per colony was obtained in the MS control medium without any growth regulator. The statistical results indicated that data from other media could be considered within the same group, and no significant difference was observed.

By the end of the 6th month of the study, the number of gametophytes

increased in parallel to the colony diameter. The maximum colony size and the maximum number of gametophytes per colony was observed in the MS medium with 1 mg/L IAA (Table 1 and Figure 6). Gametophyte development shows similar its with the development in the natural conditions (Figure 7a, Figure 7b, Figure 7c). During gametophyte differentiation, the development of rhizoidal filaments was observed. Rhizoids were formed on caulonemata at the base of the buds that degenerated. They are smaller than chloronema and caulonema, and lack any chloroplasts. The transverse walls of the rhizoids are slanting relative to the long axis of the filaments.


Figure 6. Effect of auxines on colony size in cultures having MS medium on 6 months of incubation. The mean colony diameter in millimeters ± SEM for at

least four colonies is shown below each picture


Figure 7. Gametophyte development in MS medium containing 1 mg/L IAA a. A young gametophyte bud (Arrows show leaf primordia) b. Further gametophyte development and rhizoids c. Mature gametophytes

4.Discussion Under dark conditions, H. sericeum spores only completed the water intake

stage of germination, and were unable to continue into the other stages of germination.

There is no general consensus on how spore germination should be identified.

Certain researchers consider swollen spores as “germinated” (Bauer and Mohr, 1959; Mogensen 1978). However, Glime (2007) describes that swelling during spore germination is a passive process, and that it cannot be used as an indication of germination. From physiological perspective, the rupturing of the spore wall and the formation of the germ tube are active processes that can be considered as indications that spore "germination" has taken place. Valanne (1966) considers the presence of a carbon source (e.g. sucrose) in the in vitro media as a factor that promotes germination under dark. Our experiments demonstrated that, even when sucrose was present in the media as a source of carbon, the spores were not able to germinate under dark conditions. Similarly, Physcomitrella patens was also unable to germinate under dark despite the addition of an exogenous carbon source to the media (Cove et al., 1978; Schild, 1981).

Among mosses, the requirement of light for spore germination is species-

dependent (Meyer, 1948). According to the results of our experiments, germination of H. sericeum spores is light-dependent.


The blocking of germination processes until adequate light conditions are present appears to be an important survival strategy (Silva et al., 2010). This prevents the spore from germinating somewhere where it cannot receive light (e.g. underground) (Glime, 2007).

The low germination percentage observed in high mineral content media

appears to be associated with the negative osmotic potential caused by high mineral concentrations. Although bryophytes are generally desiccation-tolerant organism, spore germination is a critical stage that requires water (Wiklund and Rydın, 2004). Silva et al. (2009) previously reported that the specific water potential at which seeds and spores germinate is related to the water profile of the habitats in which the relevant species are naturally found.

Although mineral-free distilled water resulted in the highest germination

percentage, it also inhibited protonemal development and caused gemma formation. In unsuitable environments, it is common for bryophytes, and especially for mosses, to form asexual propagules such as gemmae (Whitehouse, 1973, 1980; Side and Whitehouse, 1987; Duckett and Ligrone, 1992; Goode et al., 1993a, b, 1994). Such structures are observed both in nature and under in vitro culture conditions. Asexual propagules such as gemmae, bulbils and tubers represent a survival strategy against unsuitable conditions (Duckett and Pressel, 2003; Preston, 2004; Pressel et al., 2005). In addition to the desiccation-tolerance they exhibit under conditions where water is scarce, asexual propagules such as gemmae allow bryophytes to continue propagating and living under unsuitable environmental conditions for extended periods of time (Oliver et al., 2000a, b; Proctor, 2001). In our experiments, gemma formation by H. sericeum as a survival strategy under the unsuitable conditions presented by mineral-free distilled water partly explains the considerable spread of H. sericeum that was observed in almost every type of substrate.

Germination is a physiological process that can take place in nutrient-poor

environments, such as distilled water (Meyer, 1948; Mogensen, 1978; Olesen and Mogensen, 1978). However, nutrients are necessary and important for protonema development (Nishida, 1978; Nehira, 1983; 1988). On the other hand, high macronutrient concentrations result in poor growth in most moss species (Awasthi et al., 2012). A preference for diluted culture solutions can be seen in some studies (Voth, 1943; Awasthi et al., 2010a, 2010b).


In our experiments, the best colony growth was observed in the full strength MS medium; the second-best level of growth was observed in the 1/2 MS medium. The 1/4 MS and 1/8 MS media, on the other hand, had an inhibiting effect on the further growth of colonies. Colony growth and spore germination results were negatively correlated. These results indicate that spore germination and protonemal development are processes that are independent from one another.

Cytokinins have the effect of promoting bud formation in certain moss

species (Bopp, 1968; Valadon and Mummery, 1971). However, in our experiments, the response to cytokinin application was callus formation and abnormal bud development. In a study on the regulation of gemma and bud formation Bryum capillare Hedw. with cytokinins, Sarla (1989) reported abnormal bud development in BA-containing media. A large number of gametophytes developed in the control medium, while cytokinin-containing media did not result in gametophore differentiation. This observation regarding gametophytes is possibly related to the spontaneous and endogenous synthesis of cytokinin in caulonemal cells. The concentration of exogenous cytokinin and the amount of endogenous cytokinin might have reached a level sufficient for inhibiting gametophore differentiation.

The relationship between colony growth and gametophore differentiation

varies from one species of moss to another. In certain moss species, the protonema must first reach a “critical size” before gametophyte formation can take place. In other species, the age of the protonema is more important than its size (Chopra and Kumra, 1988). By the end of the 6th month of the study, the number of gametophytes increased in parallel to the colony diameter. However, comparisons with data from the 3rd month suggest that gametophyte differentiation is associated more with colony age than colony size.

According to the data from the experiments investigating the effect of

exogenous auxins on colony size and the number of gametophytes per colony, it was observed that the type and concentration of auxins being applied had a discernible effect on colony size and the number of gametophytes forming per colony. Bıjelovıc et al. (2004) had previously reported a relationship between protonemal (colonial) growth and the formation of gametophytes, and that this relationship is affected by the concentration of auxins.


In this study, we investigated firstly the spore germination characteristics and the stages of protonema and gametophyte development of the H. sericeum species under in vitro conditions, and evaluated the effect of different plant growth regulators on colony size and the number of gametophytes per colony. We hope these data will contribute to our knowledge on moss biology, especially on physiology. Acknowledgments

This study was supported by the Scientific Research Project Fund of Adnan

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early developmental stages of homalothecium sericeum (hew...

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