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Reproduction in selected New Zealand native ferns and 
their suitability for revegetation 

A thesis presented in partial fulfi lment of the requirements fo r the degree of 

Masters of Science in Plant Biology 

at Massey University 
Palmerston North, New Zeala nd 

Matthew Denton-G iles 

2006 



ABSTRACT 

The potential to use New Zealand native ferns for revegetation was assessed in 

laboratory, nursery and field experiments. Laboratory experiments indicated that the 

three native fern species, Blechnum novae-zelandiae, Cyathea medullaris and Dicksonia 

squarossa, had different maximum levels of spore germination. These differences also 

varied in response to seasonal changes in the environment. The effect of three soil 

conditioners on the germination of the same three species was minimal. Gametophytes 

appeared to be tolerant of low levels of maceration, as they were able to continue to 

grow and develop normally. Additional laboratory experiments indicated that B. novae­

zelandiae employs a mixed mating system, which utilizes an "antheridiogen" signal. 

The development of fern spores, laboratory propagated gametophytes and segmented 

rhizomes, was assessed in the nursery. Each experiment was applied with a 

hydroseeding mix of paper fibre, tackifier, fertilizer and water. Spore of B. novae­

zelandiae, C. medullaris and D. squarossa fai led to produce any long-lived 

gametophytes. The survival of laboratory propagated gametophytes of B. novae­

zelandiae, B. discolor and B. colensoi was low. However, a large proportion of 

surviving B. novae-zelandiae gametophytes produced sporophytes. B. novae-zelandiae 

rhizome segments produced healthy young ferns within 3 months of application . 

Field experiments were conducted on a sandstone/loess bank, 5 km east of Palmerston 

North. Aspects of the substrate were analysed including, pH, N, P and organic matter. 

The results indicated that the bank had a high soil pH, was deficient in several 

macronutrients and had no organic matter. Hydroseeding was applied using spore of 

the species B. novae-zelandiae, C. medullaris and D. squarossa. Hydroseeded spore 

fa iled to produce any visible gametophytes. Rhizome experiments using B. novae­

zelandiae and Microsorum pustulatum were also established. Low water availability 

resulted in poor rhizome establishment. 

The results suggest that there is great potential for utilizing native ferns in revegetation . 

Blechnum novae-zelandiae is the best species for revegetation in accordance to the 



results. Propagation via rhizome segmentation and gametophyte hydroseeding appear 

to be the most successful methods for establishing native ferns. 

This TIF project was carried out in conjunction with Rural Supply Technologies, 

Manaaki Whenua Landcare Research, Massey University and FoRST New Zealand. 

II 



ACKNOWLEDGEMENTS 

[ would like to thank all the fo llowing people and organizations for their help, support 

and encouragement over the past two years: 

An enormous thanks to my supervisor, John Clemens, whose encouragement and 

guidance kept me foc used on the job at hand. I especially thank him for making time to 

meet with me at regular interval s, to chat about my fe rns and their many wonders. A 

big thank you must al so go to Craig Ross who remained "on call " throughout the 

project. His helpful suggestions and vast experience in the field contributed greatly to 

the success of this research. 

A special thanks to Robert Coulson and Rural Supply Technologies Ltd, whose 

initiative and drive behind this project remained pos itive and enthusiastic. This project 

would not exist if it wasn' t fo r your prowess . Thank you to, Anne Parkinson, Eddie 

Charlton, Phillip Yalden, Bruce McCaskie and Pat Reedy, who all had a role in this 

project, whether it was unblocking hydroseeder pumps, organising meetings, or 

suppl ying me with tools fo r my fern growing attempts. 

[ would like to acknowledge the New Zealand Foundation for Research Science and 

Technology (FoRST), fo r providing me with a Technology in Industry Fellowship. [n 

addition, I would like to thank the providers of the J. P. Skipworth Scholarship, Coombs 

Memorial Bursary and Massey University Affinity Card Scholarship for their financial 

ass istance. 

On a more personal note I would like to thank my parents, Ken and Viv, for providing 

me with the means and encouragement to do what I enjoy most. I dedicate this work to 

you. 

"Nature will fo rever endeavour". 

Ill 



TABLE OF CONTENTS 

ABSTRACT 

ACKNOWLEDGEMENTS iii 

LIST OF TABLES viii 

LIST OF FIGURES viii 

1.0 IN TROD U CTI ON ------------------------------------------------------ 1 

1.1 New Zealand native ferns 1 

1.1.2 Distribution 1 

1.2 The homosporous fern life cycle 3 

1.2.1 Sexual mating systems and antheridiogen 5 

1.2.2 Asexual reproduction 7 

1.3 Spore germination and light 9 

1.4 Fern propagation 12 

1.4.1 ln vitro propagation 12 

1.5 Revegetation 14 

1.5.1 Hydroseeding 15 

1.5.2 Revegetation with native plants 18 

1.6 Project aims 20 

2.0 SPORE COLLECTION AND LABORATORY ANALYSES - 21 

2.1 Overview 21 

2.2 Materials and methods 22 

2.2.1 Spore collection 22 

2.2.2 Spore germination 24 

2.2.2.1 Media preparation 24 

2.2.2.2 Spore surface sterilization and sowing 24 

IV 



2.2.2.3 Assessing gennination 25 

2.2.2.4 Fluorescein diacetate spore viability test 27 

2.2.3 Sexual mating systems 28 

2.2.3.1 Sporophyte production and gametophyte sexuality 28 

2.2.3.2 Antherid iogen 29 

2.2.4 Gametophyte transplantation and maceration 30 

2.2.4.1 Transplantation of gametophytes 30 

2.2.4.2 Maceration of gametophytes 31 

2.2.5 Estimating spore quanti ty 32 

2.2.6 Statistical analyses 32 

2.3 Results 33 

2.3.1 Spore germination 33 

2.3 .1.1 Surface sterilization 33 

2.3.1.2 Spore germination in controlled, dark and natural conditions 34 

2.3.1.3 Seasonal variation in spore germination 38 

2.3 .1.4 The effect of three polyacrylamides on spore germination 40 

2.3.1.5 Fluorescein diacetate spore viability test 41 

2.3.2 Sexual mating systems 43 

2.3 .2.1 Sporophyte production and gametophyte sexuali ty 43 

2.3.2.2 Antheridiogen 45 

2.3.3 Gametophyte transplantation and maceration 49 

2.3.4 Estimating spore quanti ty 50 

2.4 Discussion 51 

2.4.1 Spore germination 51 

2.4.2 Sexual mating systems 56 

2.4.3 Gametophyte transplantation and maceration 60 

3.0 NURSERY EXPERIMENTS ----------------------------------------- 61 

3.1 Overview 61 

3.2 Materials and Methods 62 

3.2.1 Nursery site preparation and experimental design 62 

3.2.1.1 Nursery site preparation 62 

V 



3.2.1.2 Gametophyte propagation and establishment 63 

3.2.1.3 Rhizome establishment 66 

3.2.1.4 Spore establishment 66 

3.3.2.Statistical design and analyses 68 

3.3 Results 70 

3.3.1 Gametophyte experiments 70 

3 .3 .1.1 Gametophyte survival 70 

3.3.1.2 Sporophyte production on surviving gametophytes 72 

3.3.2 Rhizome experiments 74 

3.3.2.1 Shoot emergence and growth 74 

3.3.2.2 The rhizome root system 76 

3.3.3 Spore experiments 77 

3.3.3.1 Spore germination in the laboratory 77 

3.3.3.2 Gametophyte establishment in spore experiments 78 

3.3.4 Adventive plant species in the nursery 80 

3.4 Discussion 82 

3.4. l Gametophyte experiments 82 

3.4.2 Rhizome experiments 85 

3.4.3 Spore experiments 87 

4.0 FIELD EXPERIMENTS---------------------------------------------- 91 

4.1 Overview 91 

4.2 Materials and Methods 92 

4.2.1 Field experiment site preparation and analysis 92 

4.2.1.1 Site specifications 92 

4.2.1.2 Plot preparation 93 

4.2. I .3 Application 93 

4.2.1.4 Assessment 96 

4.2.2 Statistical design and analyses 96 

4.3 Results 97 

4.3.1 Pre-experimental analyses 97 

4.3.1.1 Soil/substrate analysis 97 

VI 



4.3 .1.2 Spore germination in the laboratory 97 

4.3.2 Hydroseeding fern spore in the field 99 

4.3 .2.1 Spore germination and gametophyte establishment 99 

4.3.2.2 Vegetation cover 99 

4.3.3 Fern rhizome experiment IO I 

4.3.3. 1 Rhizome establishment 101 

4.3.3.2 Rhizome viabi lity 101 

4.4 Discussion 103 

4.4.1 Site characteristics l 03 

4.4.2 Hydroseeding native fern spore 105 

4.4.3 Revegetation with rhizome segments 107 

5. 0 Genera I Discussion ----------------------------------------------------- 109 

5.1 Overview 109 

5.2 General Discussion and future work 110 

5.2.1 The success of Blechnum novae-zelandiae 110 

5.2.2 The development of commercial technology for native revegetation 112 

5.3 Conclusions 115 

References --------------------------------------------------------------------- 117 

Appendices -------------------------------------------------------------------- 12 5 

VII 



LIST OF TABLES 

Table 2.1 Blechnum novae-zelandiae gametophyte sexuality in the presence or 46 

absence of "antheridiogen". 

Table 2.2 Estimated quantity of dry spore in a l ml volume. 50 

Table 3.1 Pre-application gametophyte propagation information. 63 

Table 3.2 Gametophyte establishment and survival in nursery spore experiments. 78 

Table 4.1 Characteristics of the field site substrate compared to pasture soil. 98 

LIST OF FIGURES 

Fig. 1.1 The homosporous fern life cycle of Blechnum novae-zelandiae. 3 

Fig. 1.2 The phytochrome light-sensing system in fern spore. 11 

Fig. 2.1 Manawatu spore collection site map. 22 

Fig. 2.2 The effect of spore surface sterilization on spore germination. 33 

Fig. 2.3 Photoblastic spore germination. 35 

Fig. 2.4 Spore germination after a change in environment. 37 

Fig. 2.5 Seasonal spore viability using germination data. 39 

Fig. 2.6 The effect of various polyacrylamides on spore germination. 40 

Fig. 2.7 Fluorescein diacetate and determining spore viability. 42 

Fig. 2.8 In vitro propagation of ferns in the laboratory. 43 

Fig. 2.9 Blechnum novae-zelandiae archegonia and a single antheridium. 43 

Fig. 2.10 lntra-gametophytic selfing in B. novae-zelandiae. 44 

Fig. 2.11 Blechnum novae-zelandiae mating systems observed in vitro. 44 

Fig. 2.12 Dark spore germination in the presence/absence of "antheridiogen". 45 

Fig. 2.13 Gametophyte development in the presence/absence of "antheridiogen". 46 

Fig. 2.14 The effect of mature gametophytes on nearby developing spore. 48 

Fig. 2.15 Gametophyte transplantation and maceration. 49 

Fig. 2.16 Blechnum novae-zelandiae development after 66 days. 57 

Fig. 3.1 Nursery layout including the irrigation system and data logger. 63 

Fig. 3.2 The size distribution of B. novae-zelandiae, B. discolour and B. colensoi 65 

gametophytes used in the nursery gametophyte experiment. 

Vlll 



Fig. 3.3 Application of the rhizome experiment in the nursery 66 

Fig. 3.4 A diagrammatical representation of a single ½ m2 spore experiment plot. 67 

Fig. 3.5 The Newbury shade-house map showing experiments and individual plots. 69 

Fig. 3.6 Gametophyte survival in the nursery gametophyte experiments. 71 

Fig. 3.7 Sequential deve lopment of sporophytes from gametophytes in the nursery . 72 

Fig. 3.8 Mean number of gametophytes with sporophytes per replicate plot. 73 

Fig. 3.9 The proportion of surviving gameto phytes with sporo phytes. 73 

Fig. 3.10 Sequential development of B. novae-zelandiae shoots on chopped rhizome 74 

segments. 

Fig. 3.11 Viability of rhizome segments over a period of 27 weeks 75 

Fig. 3.12 Rhizome shoot length over a period of 27 weeks. 75 

Fig. 3.13 A diagrammatic representation of the position of roots, shoots and young 76 

fronds, on chopped rhi zome segments of different orientations. 

Fig. 3.14 The extent of the root system in B. novae-zelandiae rhizome segments. 76 

Fig. 3.15 Laboratory germination of spore used in nursery spore experiments. 77 

Fig. 3.16 Gametophytes isolated from nursery spore experiments. 79 

Fig. 3.17 Blechnum novae-zelandiae gametophytes showing increased necrosis at 79 

successive sampling dates. 

Fig. 3.18 Adventive plant species found in the nursery experiments. 80 

Fig. 3.19 Adventi ve fern species and gametophytes fou nd in nursery rhi zome plots. 81 

Fig. 3.20 A schematic representation of a B. novae-zelandiae rhi zome. 86 

Fig. 4.1 The field site, indicating loess and sandstone substrates. 92 

Fig. 4.2 The applicat ion of fern spore usi ng hydroseeding. 94 

Fig. 4.3 The application of B. novae-zelandiae and Microsorum pustulatum 95 

rhizome segments. 

Fig. 4.4 The failure of the initial hydroseeding spore application. 95 

Fig. 4.5 Laboratory germination of spore used in field spore experiments. 99 

Fig. 4.6 Vegetation on a hydroseeded region compared with a non-hydroseeded 100 

region . 

Fig. 4.7 Vegetation cover on different treatment plots, over 6 months. 101 

Fig. 4.8 Rhizome segment viability in the field, over 6 months. 102 

Fig. 4.9 A rock seam in the field site. 104 

Fig. 4.10 Changes in moss cover over the 6 month experimental period 105 

Fig. 4.11 Evidence of erosion on the field si te. 106 

Fig. 4.12 A rhizome segment exhibiting frond browning and cel l death in the field . 107 

IX 



INTRODUCTION 

1.1 New Zealand native ferns 

New Zealand is home to over 194 native fe rn species along with 18 fern allies. Of 

these, 89 (46%) are endemic and 105 (54%) are widespread (Brownsey, 2001). 

Although the number of species endemic to New Zealand is few compared to tropical 

countries, the abundance of native ferns growing in the wild and domestically is very 

high (Metcalf, 2003). Ferns make up a large part of the vegetation growing on the 

forest floor and vary morphologicall y, from large tree ferns to small water ferns . The 

variation in morphology of New Zealand ferns is a result of their adaptation to the 

different environments New Zealand presents. As a result New Zealand 's native ferns 

are distributed from cool alpine to warmer coastal regions. 

1.1.2 DISTRIBUTION 

The natural geographical regions of New Zealand consist of an array of environments 

from sub-tropical rainforest, to alpine regions, to desert. Each of these regions supports 

its own native fl ora (Dawson, 1993 ), and native ferns can be fo und growing in a wide 

range of these ecological niches. 

Native subtropical rainforest and subantarctic forests are home to the majority of native 

fern species. The native subtropical rainforest (dominated by Podocarps and 

Araucarians) is consistent with a warm, humid, wet environment, which is generally 

considered to be optimal for the growth of the majority of native fern species. The 

subantarctic fores t (dominated by Nothofagus sp.) provides slightly less optimal 

conditions than sub-tropical forest, and as a result a less di verse population of fern 

species is present. Within these fores ts it is common fo r different species of fem to 

inhabit areas such as stream banks, fallen trees, disturbed si tes, and clearings (Stewart et 

al. , 199 1 ). These areas often provide the ferns with the resources required for growth 

including sufficient water, low light levels and a suitable substrate. It has been 

suggested that species distribution is mainly affected by environmental gradients such 

as so il wetness (Norton, 1994). New Zealand native ferns are also distributed in more 



demanding locations, such as saline coastal environments (Blechnum blechnoides and 

B. durum) and alpine regions (Blechnum penna-marina and Polystichum cystosgia) 

(Chambers & Farrant, 1996; Brownsey & Smith-Dodsworth, 2000). The degree to 

which New Zealand's native fems can cope with extremes of heat and frost has 

previously been researched (Bannister & Smith, 1983; Bannister, 1984). Several native 

fem species common to the Otago and Fiordland regions of New Zealand were included 

in the research. Comparisons were made with northern hemisphere fems, and the 

results suggested that New Zealand fems are less well adapted to cope with extreme 

conditions, which is unsurprising given New Zealand's generally maritime climate. 

Research has also shown that New Zealand native fems form symbiotic relationships 

with mycorrhizal fungi. In Histiopteris incisa and Pteridium esculentum inoculation 

with mycorrhizal fungi caused substantial growth stimulation compared to controls 

(Cooper, 1975). 

Several native fem species are widely distributed across farmland and forestry. This 

observation is evidence of the ability of native fems to colonize land in competitive 

environments. Pteridium esculentum (bracken) and Paesia scaberula are examples of 

native fems that infest pastureland. Pteridium esculentum is also a major contributor to 

the under-storey vegetation of pine plantations (Hollinger, 1987). The native tree fems 

Dicksonia squarrosa, Cyathea medullaris and C. smithii are also significant members of 

the under-storey of older pine plantations (Ogden et al., 1997). In addition, Cyathea 

dealbata, C. medullaris and Dicksonia squarossa have been shown to predominate in 

bush margins (Young & Mitchell, 1994). Experimental evidence suggests that the 

gametophytes of several tree fem species preferentially grow at bush margins compared 

to the forest interior (Bernabe et al., 1999). This observation suggests that tree fems 

behave as pioneer species and have the ability to colonize deforested areas such as road 

cuttings. 

Statistical models have been designed to predict the distribution of native fem flora with 

respect to environmental conditions associated with geographic locations. Models are 

based on the concept that environments associated with preferential growing conditions 

would harbour common fem species in abundance. As a result, common species are 

generally modelled accurately, whereas rare species are not (Lehmann et al., 2002). 

2 



1.2 The homosporous fern life cycle 

A fern is typicall y defined as a non-flowering vascul ar pl ant that has chlorophyll and 

reproduces sexually by means of spore rather than seed (Brownsey & Smith­

Dodsworth, 2000) . A fe rn 's sexual life cycle consists of two separate free living 

generations called the gametophyte (gamete plant) and sporophyte (spore plant). The 

main di ffe rence between these two stages is that all gametophytes are haploid and all 

sporophytes are dipl oid. 

A homosporous fe rn is characterized by the growth of a single spore into a 

gametophyte, fo llowed by the initi ation of sexual organs, fe rtilization and the 

development of the sporophyte (F ig. I. I). 

-----t•• Fertilization 
Egg n - 2n 

Archegonia S;2 Sperm 

Antheridia r3 

Gametophyte (n) 

Germination 

Spore 

Meiosis 
2n ---• n 

Sporophyte (2n) 

I 
Sporangia 

Figure 1.1 The homosporous fem life cycle of Blee/mum novae-zelandiae (Kiokio), showing both the 

haploid gametophyte stage and the diploid sporophyte stage. 

3 



The sporophyte is the plant commonly observed growing in the bush or along roadsides. 

As it develops, spore is produced on the underside of fronds, in sporangia. The spore is 

shed and in the presence of water and red light, germinates and develops into the 

gametophyte. The gametophyte produces the sexual organs termed the antheridia (o) 

and archegonia (9). Sexual union occurs via fusion of the egg cell with a motile sperm 

cell. One important feature of this stage of the life cycle is that water is absolutely 

required as the sperm has to swim from the antheridia to the archegonia in order to 

initiate fertilization. The duration of the homosporous fern life cycle varies 

significantly between species of fern. The fastest growing ferns take only around 120 

days to complete one cycle, whereas slower growing species can take several years 

(Hoshizaki & Moran, 200 l ). The age at which a sporophyte becomes competent to 

produce spore has also been investigated in several ferns native to Japan (Sato, 1985). 

Fems growing on rocky banks, mossy rocks or tree trunks tended to reach a sexually 

competent stage earlier than those ferns growing on the forest floor. Therefore, the 

speed at which a fem produces spore is not only due to endogenous factors but also 

exogenous environmental factors. 

The homosporous fern life cycle is unique in plant development as it offers the ability to 

study various phenomena at both the developmentally simple haploid gametophyte 

stage, and at the more complex vascular sporophyte stage. Jts uniqueness has attracted 

interest in several scientific fields (Hickok et al., 1995). The water fern Ceratopteris 

richardii has been the preferred species in these studies because of its rapid life cycle 

and ease of culture (Hickok et al., 1987). Ceratopteris richardii produces large amounts 

of spore that can be mutagenized and screened. In addition, the ability to generate 

homozygote gametophytes from a single selfing event provides a powerful way of 

isolating mutations. The diploid sporophyte also provides a typical vascular plant 

system for developmental, genetic, biochemical or physiological research. The use of 

the fern C. richardii in early vascular plant research could perhaps be compared to the 

role of the angiosperm Arabidopsis thaliana in higher plant studies. 

4 



l.2.1 SEXUAL MATING SYSTEMS AND ANTHERIDIOGEN 

The sexual stage of the fem life cycle occurs in the gametophyte. Gametophytes can be 

unisexual , having either male antheridia or female archegonia. In some species 

gametophytes can al so be bisexual or hermaphroditic, hav ing both male and female 

organs. 

There are three basic sexual mating systems that fems use, depending on the sexual 

nature of the gametophyte. Bisexual gametophytes may se lf-fertili se, which is often 

termed intra-gametophytic selfing (Verma, 200 I). Thi s phenomenon involves the union 

of sperm and egg cell s from the same individual gametophyte. Most fems are diploid 

therefore intra-gametophytic se lfing often results in homozygous offspring. lt is al so 

poss ible that two gametophytes originating from the same sporophyte may initiate 

sexual union. This fo rm of selfing is termed inter-gametophytic selfing. A degree of 

genetic variation is achieved with inter-gametophytic selfing because spore from the 

same sporophyte has undergone meios is, and is therefore genetically diverse. The third 

poss ible mating system is termed inter-gametophytic cross ing, where gametophytes 

from different pl ants establish close to each other and sexual union is achieved. lnter­

gametophytic cross ing results in a predominantly heterozygous population, which is 

advantageous in terms of increasing genetic variation within a popul ation (Haufler & 

Welling, 1994). 

Some species of fe m have developed a pheromone system in order to promote inter­

gametophytic selfing and cross ing, and in turn, limit intra-gametophytic selfing. This 

pheromone is commonly known as "antheridiogen". Antheridiogens are produced by 

mature female fern gametophytes and can influence the sexual ontogeny of their 

neighbours (Haufler & Welling, 1994). The archegoni a of female or bisexual 

gametophytes release antheridiogen into the surrounding substrate. 

Antheridiogen stimul ates the germination of surrounding spore and, furthermore, the 

development of antheridia bearing, male gametophytes (Wen et al. , 1999). [n the 

absence of antheridiogen, spore develops into fe male or hermaphroditic gametophytes 

(Banks et al. , 1993). The concentration of the antheridiogen in the media determines its 

overall effectiveness to promote germination (Haufl er & Welling t 994). When the 

5 



concentration is high, more spores are induced to germinate and develop as male. At 

lower concentrations the amount of spore affected by antheridiogen is decreased. A key 

property of antheridiogen is its ab ility to substitute for the light requirement of fern 

spore and induce dark germination. Like seeds, spore remains dormant until certain 

factors promote its germination (Dyer, 1994 ). The soil spore bank is a biotic 

component of native plant communities and is especially important for regeneration and 

conservation of fem species (Ranai, 2003). The ability of antheridiogen to induce dark 

spore germination has implications for sexual reproduction via the utilization of spore 

banks. Spore buried in the soil can be induced to germinate and produce functional , 

antheridia bearing, male gametophytes. These male gametophytes are non­

chlorophyllous and produce sperm that is capable of swimming to the gametophyte 

from which the initial antheridiogen signal was produced (Haufler & Welling, 1994). 

Therefore, the antheridiogen signal promotes successful inter-gametophytic crossing 

between gametophytes. 

Antheridiogen has been proven to exist in a diverse range of fern species, including 

Blechnum spicant, Bommeria hispida, B. ehernbergiana, Ceratopteris richardii, 

Cryptogramma eris pa, Lygodium microphyllum, L. japonicum, L. reticulatum and 

Pteridium aquilinum (Banks et al. , 1993; Haufler & Welling 1994; Fernandez et al. , 

1997; Pajar6n et al. , 1999; Wen et al. , 1999; Kurumatani et al. , 2001 ; Lott et al. , 2003). 

It has been shown that some antheridiogens have similar structures to gibberellins and 

in fact are derivatives of this group of plant hormones (Kurumatani et al ., 2001 ). 

Initially, antheridiogens were thought to have a similar structure to gibberellins because 

their male induction response was able to be inhibited when gametophytes were treated 

with gibberellin biosynthetic inhibitors (Warne & Hickok, 1989). However, simple 

gibberellins (e.g. GA3) were unable to stimulate the male induction response typical of 

all antheridiogens, suggesting that antheridiogens were chemically divergent from 

gibberellins. Interestingly, abscisic acid (ABA) acted antagonistically to the 

antheridiogen male induction response, which is similar to its antagonistic role with 

gibberellins (Banks et al. , 1993; Wen et al. , 1999). This research laid the foundation 

for the identification of the chemical structure of antheridiogen. Gas chromatography­

mass spectrometry was used to successfully identify methyl esters of GA9 and GA 73 in 

media supporting the growth of the species Lygodium microphy llum and L. reticulatum 

6 



(Kurumatani et al. , 200 I). These compounds acted as functional antheridiogens and 

were able to induce male deve lopment. These two species were chosen because it was 

prev iously noted that they produced large amounts of antheridiogen. 

The ability to outcross is essential for maintain ing genetic variation within a population. 

However, in some instances outcross ing is di ffic ul t. When the abundance of 

gametophytes is very low, or the so il spore bank is either non-ex istent or fee ble, intra­

gametophytic selfing becomes a viable mating option. The ability to reproduce through 

intra-gametophytic se lfi ng promotes long-distance dispersal of a species (Lott et al. , 

2003). This fo rm of mating system allows for a single gametophyte to establish a new 

population. A species that uses intra-gametophytic se lfi ng can potenti all y radiate into 

areas devoid of a spore bank or res ident fe rn population. Research indicates that only 

some species are able to produce viable sporophytes this way (Soltis & Soltis, 1992). 

Most species prefer to outcross using an antheridiogen male-inducing system. It may be 

poss ible that a species has a mixed mating system and can use both outcross ing and 

selfi ng mechanisms, depending on environmental conditions . 

1.2.2 ASEXUAL REPRODUCTION 

Apart from the well characterized homosporous sexual li fe cycle, fe ms can also 

reproduce asexually by vegetati ve means or gametophytic apomixis. These 

characteristics are a variation on the bas ic fe m li fe cycle and in some cases are results of 

adaptation to environmental stress. 

Asexual reproduction leads to offspring that are genetically identi cal to the parent pl ant. 

Vegetative reproduction in fe rns can be executed either by the production of new ramets 

(shoots) through rhizomatous growth, fragmentation and re-establishment, or via the 

formation of viviparous bulbils in the frond margins (Koptur & Lee, 1993) . 

Asplenium bulbtferum (hen and chi cken fem) is a well known New Zealand native that 

reproduces vegetative ly via the production of bulb ils (Perrie et al. , 2005). Many native 

New Zealand fems, including Paesia scaberula and Pteridium esculentum, spread 

vegetatively via underground rhizomes. Once these fe rns establish new ramets (shoots), 

7 



rhizogenous connections to the parent can be terminated, thereby establishing an 

independent plant. 

Apomictic reproduction involves the development of the sporophyte from the 

gametophyte, without the fusion of gametes. This phenomenon has been observed in 

several fern species and has been studied in the model fern Ceratopteris richardii 

(Hickok et al., 1995). Interestingly, some fern species have completely lost the 

sporophytic stage of their sexual life cycle. Appalachian vittaria, which is native to the 

eastern United States, is an example of one of these species. For this fern, vegetative 

reproduction of the gametophyte generation has evolved as an adaptive advantage, as it 

has permitted the growth of this species beyond its normal geographic range (Farrar, 

1990). This phenomenon suggests that a ferns sexual reproductive mechanism is 

intolerant to an environment where water is unavailable. 

Research on the homos porous I ife cycle of ferns allows for the further understanding of 

how ferns develop and how they utilize variations in sexual reproduction successfully. 

In addition, some species have evolved variations on the basic sexual homosporous life 

cycle to allow them to reproduce successfully in unsatisfactory environmental 

conditions. 

8 



1.3 Spore germination and light 

A fern spore is an equivalent structure to a seed of a higher plant. Both structures give 

rise to a new generation and both require similar basic environmental parameters to 

stimulate germination. However, these two structures differ dramatically in size and 

ploidy level. Spore is dust-like, and individuals can only be observed clearly using a 

microscope, whereas seeds are generally much larger. In addition, all spores are haploid 

whereas the haploid generation in higher plants occurs prior to the formation of the 

diploid seed. Spores are periodically produced on mature sporophytes and released into 

the environment after a maturation period. 

The ability of a spore to retain its capacity to germinate has previously been used as a 

measure of viability (Perez-Garcia et al. , 1994). Spore is generall y at its most viable 

soon after it has been released from the parent sporophyte, although it has been shown 

that Platycerium b[/i1rcatum spore may require an after-ripening period (Camloh, l 999) . 

Green spores have high water content and an average Ii fe span of 7 weeks (Perez-Garcia 

et al. , 1994). This suggests that the environment in which these spores are deposited 

promotes conditions favourable for rapid germination. In comparison, non-green spores 

generally have low water content and can remain viable for a long period of time when 

subjected to the appropriate storage conditions. 

The conditions that favour high spore viability vary between different species. The 

conventional approach is to store spore in dry , cool conditions, similar to seed storage 

condit ions. However, research indicates that some non-green spores remain viable for 

at least two years when subjected to moist but dark conditions (Lindsay et al. , 1992; 

Perez-Garcia et al. , 1994). When taken in context, these findings seem plausible as 

spore stored in the soil is subjected to wet, dark conditions. Other research suggests that 

when spore is subjected to wet or dry conditions, there is little difference between the 

two treatments in terms of viability (S imabukuro et al. , 1998) . Therefore, the viability 

of fern spore varies enormously among fern species because of the phys iology of the 

spore and the conditions to which it is adapted. These factors have implications for the 

dynamics of spore banks in terms of the representation of different species in different 

environments. As stated previously, spore banks are thought to be important for 

9 



promoting outcrossing using antheridiogen. Therefore, the viability of spore in a spore 

bank influences the success of inter-gametophytic crossing in antheridiogen producing 

species. 

Fern spore viability has also previously been assessed using the fluorescein diacetate 

test (Large & Braggins, 1991 ). This test was previously developed for the evaluation of 

pollen viability (Heslop-Harrison & Heslop-Harrison, 1970). Fluorescein diacetate 

(FDA) is actively absorbed by viable spores and once it enters the cytoplasm the acetate 

group is cleaved by cytoplasmic esterases. This reaction forms a fluorescent compound. 

If the cytoplasm of the spore is dysfunctional, the esterases will also be dysfunctional 

and the fluorescent compound will not be formed. Therefore, viable and non-viable 

spore can theoretically be distinguished under a fluorescent microscope. 

In the laboratory, sterilizing spore may detrimentally affect its viability. It is standard 

procedure to sterilize spore prior to sowing. Sterilization kills any microbes, fungi or 

bacteria that may cause detrimental effects to the developing gametophyte. In addition, 

spore being kept in wet storage should also be sterilized in order to prevent 

contamination. Relatively recent data suggests that the sterilization process directly 

affects the viability of spore depending on the chemicals or techniques used 

(Simabukuro et al., 1998; Camloh, 1999). Some chemicals are very effective at 

preventing contamination, but in turn can dramatically affect the viability of the spore. 

The concentration of each chemical used should be optimized for preventing 

contamination but not reducing spore viability. 

After sterilization and sowing, spore is usually incubated at around 20 - 30°C, under 

low light conditions (20 µmo! m·2 s·1 
- 100 µmol m·2 s·1

) and at high humidity (Pangua 

et al., 1994; Camloh, 1999; Fernandez et al., 1999; Cox et al., 2003; Nondorf et al., 

2003; Pangua et al., 2003). The photoperiod reputedly used for incubation varies from 

constant light to 12 h darkness. Once imbibed a single flash of red light is enough to 

initiate the germination response in some species (Haupt, 1985). Fern spores are 

predominantly photoblastic and use the well characterized phytochrome light-sensing 

system to sense and respond to light (Tomizawa et al., 1983; Cooke et al., 1987; Haupt 

& Psaras, 1989). This system involves the stabilization of phytochrome in the presence 

of red light. When red light reaches the spore it causes one form of phytochrome (PFR) 

10 



to stabi lize. When enough PFR is stabilized, the germination response is initiated. 

However, far red light can cause reversion of this process and inhibit germination . The 

amount of red light required to initiate germination in spores varies between species. In 

addition, blue light can totally inhibit the phytochrome light-sensing system. This 

suggests that blue light influences factors downstream of the phytochrome mechanism 

in the spore germination induction pathway (Fig. 1.2) . 

Figure 1.2 A simplified, schematic diagram of the phytochrome light-sensing system in fem spore. Red 

light causes PFR to become active . If PFR remains acti ve for a certain period of time a biological 

Red light reaches spore 

~ Stabilization (PR- PFR) 

Phytochrome 
R (inactive) 

Reversion (PFR - PR) 

Phytochrome 
FR (active) 

Blue light reaches spore 

~ hibition downstream 

> Biological Response 
e.g. Germination 

Far Red light reaches spore 

response will be induced. If the spore perceives Far Red light PFR will be deactivated and reversion will 

occur. Reversion occurs instantly whereas the period of time it takes for PFR to stabilize and induce a 

biological response varies between spec ies from a single pulse ofred light to several hours. 

11 



1.4 Fern propagation 

New Zealand native ferns are successfully propagated commercially, with the tree ferns 

Cyathea dealbata, C. medullaris and Dicksonia squarrosa forming the bulk of 

production. New methods of propagation are being sought in order to increase 

production, as it can take at least a year for species to develop from spore to plants fit 

for sale. In vitro propagation of fern material has been researched extensively. In vitro 

propagation provides a method for growing species that are difficult to propagate using 

conventional methods. In addition, in vitro propagation can potentially be used to 

conserve endangered fern species. 

1.4.1 IN VITRO PROPAGATION 

In vitro propagation involves growing ferns on specialised growth media, usually under 

laboratory conditions. Scientists tend to use this approach because the technique is 

sterile and it is possible to manipulate growth conditions easily. This method of 

propagation has the potential to produce a large amount of fern material in a short 

period of time compared to conventional propagation methods. The preferred medium 

for in vitro propagation of spore is half strength Murashige and Skoog (MS) basal 

medium (Murashige & Skoog, 1962), supplemented with agar. 

In addition to spore, vegetative fem tissue can be grown on media containing 

appropriate concentrations of the plant hormones auxin and cytokinin (Fernandez et al., 

1999). Rapid generation of adventitious shoots can be accomplished relatively quickly 

compared to in vitro propagation of spore (Camloh et al., 1994; Ambrozic-Dolinsek et 

al., 1999). Various concentrations of sucrose, jasmonic acid and charcoal have all been 

shown to affect the rate of shoot formation. The addition of sucrose to the medium 

seems to give variable results. High concentrations of sucrose have been shown to 

inhibit development of the sporophyte, whereas jasmonic acid and charcoal promote 

growth (Pasha & Chakraborty, 1982; Ambrozic-Dolinsek et al., 1999; Camloh et al., 

1999). It has also been shown that addition of sucrose to the growth media can 

significantly enhance the growth rate of gametophytes (Goller & Rybczynski, 1995; 

Kuriyama et al., 2004). 

12 



Recent research has concentrated on the homogen ization of in vitro established 

gametophytes and sporophytes, and their regeneration capacity in MS liquid medium as 

well as more conventional media (Janssens & Sepelie, 1989; Fernandez et al. , 1993; 

Fernandez et al. , 1999). It has been suggested that for species with rapid life cycles, 

homogenization of gametophytes can be considered an excellent method for high yield 

propagation in a short amount of time. Species that are apomictic are also excellent 

candidates for homogenization. Homogenization of rhizomes has also proved to be 

successful, with an estimated 500 sporophytes being produced from 0.5g of vegetative 

tissue (Fernandez et al. , 1997). Another method of in vitro propagation of spore 

involves the use of an airlift fermenter. Spore of the ferns Pteridium aquilinum and 

Anemia phyllitidis were successfully cultured and subsequent growth and development 

was monitored (Sheffield et al. , l 997). Us ing this technique, both species produced 

more biomass than any other solid or liquid based culture system tested. Gametophytes 

grown in the airlift fermenter appeared characteristically similar to soil-grown plants, 

indicating development of the gametophyte was not impaired by this method of 

propagation. 

In vitro propagation has also been proposed as a useful method for propagating several 

endangered fern species . The two experiments reviewed implement different 

propagation techniques with varying success. In vitro propagation of spore was 

attempted for the conservation of Shizea dichotoma, which is native to Australia. 

However, poor germination rates hampered the ability to propagate the spore of this 

species (Cox et al. , 2003). In comparison, the South African tree fern Cyathea dregei 

has been successfully propagated via a method similar to the homogenization method 

described above, suggesting that vegetative propagation may be a more successful 

technique (Finnie & van Staden, 1987). The ability to propagate a particular fern 

mainly depends on the specific growth characteristics of that species . 

In vitro propagation techniques for the multiplication of fern tissue is an active field of 

research. The generation oflarge amounts of young ferns using this method has 

implications in commercial propagation, propagation of endangered fern species, and 

also in scientific experimentation. 

13 

• 



1.5 Revegetation 

The establishment of vegetative cover over an area ofland is the most successful way of 

controlling the phenomenon of erosion (Grace, 2000). Vegetation is able to hold the 

surface layer of soil together promoting stability and cohesiveness. Revegetation is 

particularly important in areas of land that are disturbed by civil engineering works. 

Erosion along roadsides affects the quality of the road and leads to large annual 

maintenance costs. It has been estimated, that in North America, forest roads account 

for as much as 90% of all sediment production from forested land (Grace, 2002). Since 

European deforestation in New Zealand began, sediment production has largely been 

determined by landslide events (Smale & McLeod, 1997; Glade, 2003). This research 

implies that land devoid of vegetation is prone to the processes of erosion and 

establishing vegetative cover is the best solution for this problem. In addition, 

establishing appropriate vegetation cover on substrates such as mine tailings and old 

quarry sites is the fastest way to rehabilitate these areas, which are usually infertile and 

sometimes highly toxic to plant growth. 

Disturbed land is naturally revegetated by a process known as succession. Succession is 

an ecological concept which states that there is a definable sequence of successive 

stages through which an ecosystem will pass. The speed at which succession occurs is 

influenced by certain factors associated with the specific area including temperature, 

rainfall, topography, slope, solar radiation and distance from intact forest (Leathwick & 

Rogers, 1996). Succession can be divided into two forms termed primary and secondary 

succession. Primary succession occurs in areas previously unoccupied by vegetation, 

whereas secondary succession occurs in areas where vegetation has been recently 

removed. In certain areas where environmental factors are unsuitable for rapid plant 

establishment and growth, succession occurs at a rate that is too slow to greatly reduce 

erosion. As a result, technology is continuously being developed that focuses on 

establishing vegetation in challenging environments as quickly and as cost effectively as 

possible. 

14 



1.5.l HYDROSEEDING 

Hydroseeding, also referred to as hydraulic seeding, is a common seeding practice used 

to sow areas of land. Hydroseeding involves the application of an aqueous slurry, 

commonly consisting of seed, fe rtilizer and tackifier, onto a target site via a high 

pressure hydraulic pump. The aqueous suspension is contained within a tank on the 

back of a slow moving tanker, and is progress ive ly sprayed over the target area at an 

appropriate consistency. The slurry is des igned to stick to the target site and promote 

germination and development of the incorporated seed. Hydroseeding has become 

particularly popular as a means of revegetating roadsides batters and verges. The main 

reason for this is that it is a cost effective way of es tab I ishing vegetation quickl y across 

large areas of land that are sometimes inaccessible to traditional seeding equipment. lt 

is prominently used for the reclamation of motorway embankments (Carr & Ballard, 

1980; Andres & Jorba, 2000). 

The ingredients included in the hydroseeding mix promote germi nation and 

development of the seed. Research has been carried out in order to identify which 

ingredients promote the best conditions for germination. However, results vary 

considerably as the spec ific environment into which the mix is introduced greatly 

influences the success of the seed. 

Seed is usually chosen to suit the condi tions of the site . Mainstream hydroseeding 

involves the sowing of mainly grasses and herbaceous legumes (Roberts & Bradshaw, 

1985). These plants are chosen on the bas is that they develop quickly, minimising the 

time it takes to successfull y establish a suitable vegetative cover, and are able to grow in 

conditions that are less than optimal (i.e. low water availabili ty, high light conditions). 

Legumes are particul arly important inclusions in the seed mix because soil s targeted for 

hydroseeding are usually devoid of organic material and are low in nitrogen (Roberts & 

Bradshaw, 1985). Legumes are known to form mutualisti c relationships with so il borne 

microbes that fix N into forms that are access ible to the pl ant. Initi ally, young seedlings 

have plenty ofN as the fe rtilizer included in the mix is still available in high 

concentrations. However, as the fe rtilizer is used by the plants and leached out of the 

soil , the N source becomes depleted. Legume species he lp to maintain the amount of 

15 



available nitrogen in the soil and, therefore, sustain the growth of the plants growing in 

the surrounding area. 

Several types of mulch are used in hydroseeding mixes including wood fibre, peat 

mulch, straw mulch and paper mulch. Research indicates that the mulch increases the 

chance of successful establishment of seedlings (Sheldon & Bradshaw, 1977). The 

mulch has water retaining capabilities and can act as a water source for the germinating 

seed. The seeds and seedlings are, therefore, less prone to drying out. In addition the 

mulch, in conjunction with tackifier, creates a protective coat over the top-soil 

immediately after application. This coat has the ability to resist erosion to a degree, and 

provides a physical barrier that protects the seed from predation and simply being 

washed away. The degradation of the mulch over time also adds to the organic matter 

in the soil, which can be important when hydroseeding on land that contains no top-soil, 

e.g. subsoil, roadside verges and mine tailings. 

ln the past, debate has arisen over the requirement of fertilizers in the mix. Experiments 

have shown that in some cases fertilizer can be phytotoxic and can reduce the 

germination rate of the seeds, when compared to non-fertilized plots (Sheldon & 

Bradshaw, 1977). These results were affected by the type of soil that was being used in 

the experiments. When fertilizer is applied to a porous, fine soil type such as sand, 

germination rate was negatively affected. It was thought that if water was not readily 

available, the toxicity of the fertilizer became a factor. In some cases fertilizer is 

absolutely essential (e.g. on sites that are deficient in several of the macronutrients). 

Fertilizer can provide the site with elements essential for normal growth and as 

discussed earlier, is also important in providing the initial N source commonly lacking 

in areas that are chosen for hydroseeding. In some cases it has been suggested that 

fertilizer could be applied later, when the seedlings have established. This would 

reduce possible toxicity effects of fertilizer on germination. An alternative is to use 

slow release fertilizer that is not physically available in such high concentrations during 

germination and provides nutrition for longer periods of time. 

The last component of the hydroseeding mix is often termed the tackifier or 

hydrocolloid. These chemicals are often polyacrylamides and act as soil conditioners 

and stabilizers. Properties of these chemicals include the ability to be dissolved in water, 

16 



to successfully enable germination and subsequent seedling growth, to be 

environmentally friendly , to limit erosion by creating a steadfast crust and to be cost 

effective. In the past it has been questioned whether these compounds were actually 

detrimental to seed development in some cases (Morrey et al. , 1983). More recently 

research contradicted this evidence suggesting that modern hydrocolloids have no 

adverse affects on germinating seeds (Merlin et al. , 1999). This paper conducted 

experiments to determine the effectiveness of these compounds on adhering seed to a 

sloped soil bank. It was shown that most of the compounds tested were able to induce 

high levels of adhesion of the seed to the so il. 

Each site is unique in both abiotic and biotic factors. In order to obtain the best possible 

result, an appropriate mix has to be prepared depending on the nature of the site. Factors 

that influence the success of the hydroseeding application include the roughness of the 

substrate, the density of the substrate, available nutrients, so il acidity, slope and solar 

radiation, as well as seasonal implications (Cano et al. , 2002). 

The soil substrate can be modified prior to application of the hydroseeding mix in order 

to alleviate some of the abiotic constraints. Scarification of the soil may be required 

when the soil is so compact that roots are unable to penetrate the surface . Scarification 

also increases the surface area of the face and provides a better substrate to which the 

hydroseeding mix can adhere. In addition, scarification creates microsites which 

promote successful plant establishment. 

Often the substrate chosen to be hydroseeded is deficient in several macronutrients . 

This deficiency can be compensated for by including a suitable fertilizer in the slurry. 

Acidic soils, such as those found at disused mine sites can be neutralised via the 

application oflarge amounts of lime (CaC03) . If this method is chosen, tests on the 

substrate have to be carried out in order to determine the correct amounts of lime to be 

applied (Bradshaw, 1997). The alternative is to choose certain species to hydroseed that 

are naturally adapted to acidic or toxic so il s. The fern Pteris vittata has been studied 

recently on its ability to grow on arsenic contaminated soi ls (Chen 2002; Visoottiviseth, 

2002) . This work has revealed that Pteris vittata is actually a hyperaccumulator of 

arsenic. As a result, a role for this fern has been proposed in the phytoremediation of 

arsenic containing soil, which can be prevalent at disused mine sites. 

17 



The gradient of the cut-slope is known to greatly affect hydroseeding success (Merlin et 

al., 1999). As discussed earlier, factors included in the mix, such as tackifier and 

mulch, can help to combat the effects of the slope on the establishment of the seed. 

Water availability and solar radiation are correlated with the seasons. Therefore, when 

planning the logistics associated with hydroseeding it is important to identify the most 

appropriate time of year to hydroseed. In New Zealand the summer months are usually 

the worst time to hydroseed as water availability is low and solar radiation is high. 

Seedlings sown on steep batters are unable to source sufficient amounts of water to cope 

with the higher temperatures. Therefore, the best time of the year is autumn, when 

temperatures are mild and water is more frequently available. 

Hydroseeding is a technology that requires varied applications for different sites. As 

more experiments are conducted and alterations to the basic method are implemented, 

the technology becomes more successful, as well as more flexible. An example of this 

flexibility is the development of hydroseeding applications for native species. 

1.5.2 REVEGETATION WITH NATIVE PLANTS 

The use of New Zealand native plants in revegetation offers a unique way to maintain 

areas of New Zealand's natural heritage. Instead of planting costly container plants, 

hydroseeding could potentially be used to establish native species in protected areas, 

reducing the immigration of exotic plants while maintaining the native plant flora. 

Road cuttings through native bush would be the main target for this treatment as 

legislation impedes the use of exotic species in revegetation projects within the 

boundaries of National Parks (National Parks Act, 1980). 

Hydroseeding with native species in New Zealand has produced mixed results (Simcock 

& Ross, 1997; Smale et al., 2005). Efforts have focused on using seeds of native 

shrubs. The most successful species at two different sites were Coprosma robusta 

(karamu) and Phormium cookianum (mountain flax). Factors thought to influence the 

outcome of these experiments included hare and rabbit predation and the colonization of 

plots by weedy foreign species. Interestingly, scarifying the surface of the batter did not 

improve the ability of seedlings to establish compared to smooth surfaces. Native 

18 



species tend to be slow to establish, therefore it is important to include in the mix an 

appropriate soi l stabilizer and in some cases, a nurse species . Nurse species have the 

ability to germinate and grow quickly without inhibiting the growth of the nati ve 

species. The nurse species prov ides initial protection against erosion until the native 

seed has germinated. 

In New Zealand hydroseeding has been used successfull y to establish native mosses on 

mine tailings (Ross et al. , 2003). Mosses are colonizers and have the abili ty to grow on 

infe rtile substrate. Over time moss co llects so il particles, building the beginnings of a 

fe rtile substrate into which seeds and spore can germinate and grow. 

Ferns are involved in the natural succession of plant growth on disturbed land 

(Kitayama et al. , 1995 ; Rivera et al. , 2000; Slocum, 2000). They generally establish 

after bryophytes, and are able to grow in relati ve ly infe rtile conditions. These 

characteristics promote their use on in fe rtile substrates, such as mine tai lings and 

subsoil. Ferns are also naturall y involved in secondary success ion. In mature stands of 

Pinus radiata , native tree fe rns are known to establish themselves naturally in the 

under-storey (Brockerhoff et al. , 2003) . New Zealand nati ve fe rns are fo und naturally 

on roadside slopes . Ferns such as Bfechnum novae-ze fandiae and Microsorum 

pustulatum are commonly found growing along roadsides in New Zealand (Isolde et al. , 

1995). These species of New Zealand nati ve fe rn are especially adapted to growing on 

banks. They characteristically have a creeping rhizome with a mat of roots growing 

into the substrate. As a result, these characteristics suggest that there is potential for the 

use of native fe rns in roadside revegetation. 

19 



1.6 Project aims 

20 

• To evaluate the potential use of several New Zealand native ferns for 

revegetation by conducting laboratory experiments focused on particular stages 

of their life cycles. 

• To assess the ability of native ferns to establish in the nursery and field, using 

various revegetation methods, including hydroseeding. 



SPORE COLLECTION AND LABORATORY ANALYSES 

2.1 Overview 

The experimental work in this thesis has been div ided into three separate chapters in 

order to separate laboratory experiments, nursery experiments and fie ld experiments. 

The work in all three chapters was conducted in accordance with appropriate 

experimental des ign, and the results were subjected to sui table statistical analys is. 

The first chapter includes the description of where, when and how fe rn spore was 

collected along with the design, results and discussion of several experiments conducted 

in the laboratory. Laboratory experiments were carried out in order to acquire 

info rmation on various aspects of the li fe cycle of several New Zealand native ferns. 

The germination of spore, collected mai nly from the Manawatu distri ct, was assessed 

under various conditions, using di ffe rent procedures. Aspects of the mating system of 

the species Blechnum novae-zelandiae are described for the first time. The robustness 

of gametophytes of several nati ve fe rn spec ies is also consi dered. The information 

provided by these laboratory experiments was used to consider the potential of several 

native fern species as candidates for revegetation, as we ll as to increase the knowledge 

base on several common New Zealand native fe rns. 

21 



2.2 Materials and Methods 

2.2.1 SPORE COLLECTION 

Spore was collected throughout the project for use in laboratory, nursery and field 

experiments. Most of the spore was collected in the Tararua Ranges, east of Palmerston 

North, New Zealand. Figure 2.1 shows the location of the main collection sites from 

which both spore and rhizomes were sourced. 

o_ tkm 

T,Hl AKA r,flJKU 

Man9aone Stream 

/ " < • 

i' 3 nAYANT 
It ILL 

I I 

I 

V 

Figure 2.1 Collection sites are marked by a red square. I. Moonshine Valley Road. 2. Massey Fernery. 

3. Greens Road. 4. Pahiatua Track West. 5. South Range Road. 6. Pahiatua Track East. 7. Harrison Hill 

Road. F. Location of Field site (purple square). Refer to herbarium for map references (via Appendix I). 

Species collected from the Manawatu region included Blechnum novae-zelandiae T. C. 

Chambers et P. A. Farrant (sites 2, 4, 5 & 7), Cyathea medullaris G. Forst (site 2 & 3), 

Dicksonia squarossa G. Forst (site 1), Asplenium bulbiferum G. Forst (site 6) and 

Microsorum pustulatum G. Forst (site not shown). 

Microsorum pustulatum was only required for the rhizome field experiments and was 

collected from Hall Block Road, near the Manawatu Gorge. Asplenium bulb(ferum 

spore was initially of poor quality, and therefore, this species was excluded from any of 

the experimental work. Only two collections took place outside of the Manawatu 

22 



di strict. Spore of Blechnum colensoi (Hook. f.) N. A. Wakef and Blechnum discolor G. 

Forst, was collected by Dr Craig Ross from the upper reaches of the Buller Gorge, in 

September 2004. A second coll ection of Blechnum discolor was made from the lower 

reaches of the Buller Gorge in February 2005 . 

The collection process involved harvesting fe rtile fronds of the required species and 

transporting them back to Massey University . Fertil e fronds were laid out onto fresh 

newspaper with the sori facing downwards. The fronds were left in this state for about 

7 days to allow them to dry . Once spore had been shed, it was separated from other 

debris that had collected on the newspaper, including sporangia, scales and hairs. The 

separation step involved gently shaking the newspaper at a downward angle. 

Sporangia, hairs and scales slid down the gradient fas ter than the spore, due to the 

greater mass of these structures. Once the separation process was compl ete the spore 

was tipped into envelopes and labelled with the species name, the date of collection, and 

the date of packaging. If spore was not required for use straight away it was kept in an 

unsealed paper envelope at 4°C. 

The ex istence of self-incompatibili ty mechanisms in the species of interest was 

unknown. Therefore, to protect against this compli cating factor, care was taken to make 

sure samples were taken from di fferent fe rns of the same species even when only a 

small sample was required. 

Voucher specimens were collected from each site. These specimens prov ide direct 

references to the species and populations used in the project. The samples were dried, 

pressed and labelled prior to inclusion in the Ell a Campbell Herbarium at Massey 

University. Each sample was given an MPN number for future reference (Appendix l). 

Unfortunately samples from the first Buller Gorge collection were unable to be included 

as suitable specimens were not collected at the time. 

The main species used in th is project were Blechnum novae-zelandiae, Cyathea 

medullaris and Dicksonia squarossa. All of these species were available in the 

Manawatu district. In addition, these three species are predominant spore producers. 

Spore was required throughout the project for various experi ments. 

23 



2.2.2 SPORE GERMINATION 

2.2.2.1 Media preparation 

The medium used in all experiments was Murashige and Skoog medium (MS) 

(Murashige & Skoog, 1962) supplemented with I% agar (Life Technologies, Scotland). 

This medium was decided upon due to its continual mention in the literature as a 

propagative medium for fern spore e.g. (Fernandez et al., 1993; 1999; Cox et al., 2003). 

Its specific effect on the species of interest was unknown as no scientific literature 

mentions the propagation of native New Zealand ferns on an artificial medium of this 

kind. The standard concentration of MS is 4.4 g L-1 of water. Throughout the text 

Murashige and Skoog medium will be abbreviated to½ MS, as this is the concentration 

used for spore germination and gametophyte development. Each batch of medium was 

prepared by adding appropriate amounts of MS, agar and water to a l L Schott bottle. 

The solution was then roughly mixed via swirling and placed in an autoclave at 121 °C 

andl 5 psi, for approx. I h. The solution was then kept warm in a 60°C water bath in 

order to keep the medium liquid prior to being poured into petri dishes. Pouring was 

carried out in a lamina flow cabinet in order to keep conditions sterile. Once cool, the 

Petri dishes were collected, labeled and stored in a refrigerator at 4°C until required. 

2.2.2.2 Spore surface sterilization and sowing 

It was important that spore was surface sterilized prior to sowing as contamination of 

plates with other microorganisms may have affected the germination process, as well as 

the growth and vigor of the gametophyte. Spore was initially removed from an 

envelope using a clean spatula and tapped into a sterile 1.5 ml micro-centrifuge tube. 

Around 0.05 ml - 0.1 ml of dry spore was a sufficient amount to sow ten plates. The 

next step required adding 1 ml of water to the spore to initiate imbibition and 

germination. The spore was then left to hydrate for at least 10 minutes. A solution of 

4.5 g L-1 of sodium hypochlorite, Tween20 (Sigma chemical Co, USA) and water was 

used to surface sterilize the spore. The Tween20 acts as a wetting agent as the spore 

tends to be hydrophobic if it has only recently been exposed to water. Tubes were then 

centrifuged at 2000rpm for 3 min in order to pellet the spore and separate it from the 

water. The supernatant was pipetted off and discarded, and I ml of fresh 

24 



hypochlorite/Tween20 solution was added. Dispersal of the spores in the solution was 

facilitated by swirling or shaking the tube vigorously. The spore was then allowed to 

soak for 3 min before the tubes were centrifuged once again. After centrifugation the 

supernatant was pipetted off, and 1 ml of autoclaved water was added to the pellet. In 

total three steril e water washes were conducted in order to remove any trace of the 

hypochlorite/Tween20 solution. Spore was sown onto fresh ½ MS, I% agar plates in a 

lamina fl ow cabinet. 0.1 ml of the sterile spore and water suspension was added to each 

plate. In addition, 2 ml of steril e distilled water was added to disperse the spore on the 

surface of the agar and fac ilitate germination. Plates were sealed with parafilm to reduce 

contamination by airborne micro-organisms. This process was successful at killing most 

microorganisms including paramecium, green algae and various fungi. In some cases 

contamination remained prevalent. However, due to the fac t that each experiment used 

many replicates, contaminated plates were able to be discarded if necessary without 

compromising stati sti cal analys is or the reliability of the results. 

The effect of this method of spore sterilization on the spore used in this project was 

tested. Germination rates were compared between non-sterilized spore and sterilized 

spore, subjected otherwise to the same conditions. 

2.2.2.3 Assessing germination 

The germination of spore was used as a key parameter to test the performance and 

viabili ty of spore collected throughout the project. Once spore was sterilized and sown 

it was usually transferred to a SANYO growth cabinet, model number MLR-350H. 

Plates were spread out on racks in order to maximise the amount of light available to 

them. The growth cabinet parameters were kept constant throughout the project. 

Constant white light was used at an intensity of ~ 40 - 60 µm ol m·2 s·1
, relative 

humidity was kept at 75% and temperature at 23°C ± 0.5°C. In some cases spore was 

kept under different conditions to those in the growth cabinet. 

Germination was recorded every two days after sowing. The number of replicates varied 

fo r each experiment. However, at least 3 replicates were used in order to validate the 

data. Germination was usually recorded for 20 days, or until germination was complete. 

Plates were positioned on a standard compound light microscope at 40x magnifi cation 

25 



and 50 spores were scored for each replicate . The emergence of the primary rhizoid 

indicated positive germination. In addition, spores with a broken exine and visible 

chloroplasts were also recorded as positive germinates if rhizoids could not be seen. 

For each treatment at least three replicates were examined. In some cases gametophytes 

grown from germination experiments were subsequently used in other experiments. 

One experiment analysed spore incubated in more "natural conditions" in the Massey 

University fernery and glass house . Light intensity, humidity and temperature in the 

Massey University fernery and glasshouse were measured using a Hobo™ data logger. 

Spore germination was assessed over a 20 day period and compared to a similar sample 

of spore grown in standard, growth cabinet conditions. In addition, dark sowing, using 

a green safe-light (0.0 1 µmot m-2 s-1
) , was utilized in order to observe the effects of 

darkness on spore germination. Dark sown spore was left for 10 days in the dark, 

whereupon its germination rate was measured at day 10. It was then incubated for 10 

more days in standard, growth cabinet conditions with germination being recorded 

every two days . 

Throughout 2005, spore from the species Blechnum novae-zelandiae, Cyathea 

medullaris and Dicksonia squarossa was collected from the collection sites shown (Fig. 

2.1) and sown as described above. Repeated collections were carried out at regular 

intervals in order to obtain information on the time of year that spore germination was at 

its highest in these species. It was perceived that this information would be valuable for 

planning future fem hydroseeding applications using these species. 

In another experiment germination was used to compare the effect of different 

polyacrylamides on spore of Blechnum novae-zelandiae, Cyathea medullaris and 

Dicksonia squarossa . Polyacrylamides are often used as soil conditioners and 

stabilizers, and are incorporated into the hydroseeding mix. The three polyacrylamides 

used in this experiment were Soilfix™, FLOBOND and Aquasorb. Soilfix™ and 

FLOBOND are simple soil conditioners and act to bind soil particles together. 

Aquasorb has an added property that allows it to absorb and store water as well as act as 

a soil conditioner. Spore was surface sterilised and sown onto standard ½ MS, 1 % agar 

plates. For each polyacrylamide two concentrations were tested. Plates were either 

subjected to a standard concentration of 1 g L- 1
, or a very high concentration of 10 g L- 1

• 

26 



For each treatment there were at least three replicates. Controls were included that only 

contained water without any polyacrylamide for each species. 

2.2.2.4 Fluorescein diacetate spore viability test 

The fluoresce in diacetate (F DA) test was adapted from a pollen viability test for testing 

the viability of fern spore (Shivanna & Rangaswamy, 1992). It had prev iously been 

suggested that this method could be used to ascertain the viability of spore of New 

Zeal and fe rn species (Large & Braggins, 199 1 ). Each time this test was carried out a 

fresh stock solution of FDA (S igma Chemical Co, USA) was prepared by adding 80 mg 

of FDA powder to 40 ml of acetone. Spore chosen fo r the test was freshly collected, 

and testing was carried out in conjunction with germination experiments. In addition 

"reduced viabili ty" spore was also prepared fo r use as a negative control by baking it in 

an oven at ~ 90°C for 2-3 h. Spore was imbibed overnight prior to incubation with 

FDA. Seven tubes were prepared fo r each spore sampl e. Five of these tubes contained 

various sucrose solutions (0. 1 M, 0.2M, 0.3M, 0.4M and 0.5M) in order to establish 

osmolarity effects of water on the spore. Several drops of FDA were added to each tube 

containing sucrose and one tube containing water. The seventh tube was a negative 

contro l and onl y contained water. This spore was expected to show onl y background 

fluorescence. These solutions were added to small samples of spore and the mixture was 

left to incubate for 1-2 hours. incubation was carried out on a slide or in a 

microcentrifuge tube. "Reduced viability" spore was also incubated with various 

solutions of FDA, sucrose and water. Samples were mounted on slides and viewed 

under an Olympus BXS I fluorescence mi croscope with a mounted digital camera. 

Viable spores were presumed to be able to change the fluorescein diacetate into a 

fluoresc ing compound, through the action of cytoplasmi c esterases. If the cytopl asm of 

a spore was not intact, the spore would not have any esterase activity and therefore 

would not fluoresce, rendering the spore non-viable. Photos of treated spore were taken 

and spore was scored in terms of its ability to fluoresce. 

27 



2.2.3 SEXUAL MATING SYSTEMS 

2.2.3.1 Sporophyte production and gametophyte sexuality 

The New Zealand native fern Blechnum novae-zelandiae was used in this experiment to 

study the sexual development of its gametophyte. Initially gametophytes were grown 

from spore in the growth cabinet in order to see if they were capable of producing 

sporophytes, and hence carry out the fertilization process in vivo, under laboratory 

conditions. Photos were taken using a hand-held Sony DSC-PS digital still camera 

(Fig. 2.8). Once it was discovered that this was possible an experiment was designed to 

test the ability of B. novae-zelandiae gametophytes to successfully carry out intra­

gametophytic selfing, inter-gametophytic selfing or inter-gametophytic crossing. Initial 

experiments, using only 4 replicates, were conducted looking primarily at intra­

gametophytic selfing. Blechnum novae-zelandiae spore was surface-sterilized, sown on 

standard growth media, sealed with parafilm and placed in a controlled environment as 

described previously. The spore germinated and produced small multicellular 

gametophytes. Four single, presexual gametophytes were isolated 40 days after sowing 

and transplanted onto four fresh 1/2MS, 1 % agar plates. In addition, 1 ml of sterile water 

was added to each of the four plates to facilitate growth and development. Isolated 

gametophytes were then left to develop for an additional 32 days before they were 

flooded with 10 ml of sterile water in order to facilitate fertilization. Each plate was left 

unsealed to allow the water to evaporate. Gametophytes were then periodically checked 

to see if they had produced sporophytes over the following 4 weeks. Photographs of 

antheridia/archegonia and sporophytes were taken with a Leica MZ 12 stereo 

microscope using dark-field microscopy. 

A more extensive experiment was also designed looking at all three possible mating 

systems i.e. intra-gametophytic selfing, inter-gametophytic selfing and inter­

gametophytic crossing. Spore from two separate populations of B. novae-zelandiae 

(Fig. 2.1, sites 4 and 7) were collected within two weeks of each other. These two 

populations were separated geographically, and therefore, it was anticipated that they 

would be slightly different genetically. The gametophytes of the Pahiatua Track West 

population were 50 days old when they were transplanted. Those of the Harrison Hill 

population were 3 7 days old. 18 replicates were set up for each treatment. The first 

28 



treatment used single, isolated gametophytes in order to test for intra-gametophytic 

selfing. The second treatment involved growing two gametophytes from the same plant 

in proximity to each other, to test for inter-gametophytic selfing. The third treatment 

involved growing two gametophytes from different plants in order to test for inter­

gametophytic crossing. The Pahiatua Track west gametophytes were used in all 

treatments . The Harrison Hill gametophytes were required for the third treatment only . 

Gametophytes were watered with sterile water periodically and left to develop. All 

plates were grown in the same conditions and positioned randomly in the growth 

cabinet. 72 days after setting up the experiment gametophytes were flooded with 5 ml 

of sterile water in order to facilitate ferti lization . Gametophytes were assessed every 

week after flooding for the production of sporophytes over a period of 11 weeks . 

2.2.3.2 Antheridiogen 

Antheridiogen experiments were conducted using the species Blechnum novae­

zelandiae, Cyathea medullaris and Dicksonia squarossa. The first experiment was 

intended to test for the presence of antheridiogen. This was done by designing an 

experiment that utilized the knowledge that antheridiogen is able to compensate for 

light, and cause the initiation of spore germination in the dark. Initially gametophytes of 

each species were grown on plates in batches of 50 gametophytes per plate. It was 

proposed that if these species utilized antheridiogen it would be exuded into the 

medium. After 87 days these gametophytes were removed from the plates and the 

medium was used as potential antheridiogen containing medium. Spore was sown onto 

both antheridiogen containing medium and normal medium in the dark using a green 

safe light. The normal medium was used as a dark control and would presumably result 

in non-germinated spore. In addition, spore was also sown in the light and kept in the 

growth cabinet at standard conditions. Spore was unable to be sterilized due to the fact 

that the equipment required for sterilization was only available in lighted areas. Dark 

sown spore was placed in envelopes and then sealed in a black plastic bag. Spore was 

left for 10 days. After the ten days treatment, dark, and light control plates, were 

examined for germination by counting I 00 spores on each replicate plate. All plates 

were then transferred to the growth cabinet. After 4 days they were examined again for 

any changes. After 22 days in the I ight the number of male gametophytes observed on 3 

rep I icates plates for each species and each treatment were recorded and compared. 

29 



The second experiment was designed to test the effect of a mature gametophyte on the 

ontogeny and development of surrounding spore. Presumably if a mature gametophyte 

produced antheridiogen, surrounding spore in close proximity to the gametophyte would 

predominantly develop as male gametophytes. 87 day old gametophytes of each species 

were isolated and transplanted onto fresh ½ MS, I% agar plates. Each plate contained a 

single gametophyte. Six replicates were set up for each species. Plates were watered 

with 2 ml of sterile water after transplantation. After 23 days of isolation, freshly 

collected spore was sterilized and sown around the gametophytes corresponding to that 

species. Control plates, missing mature gametophytes, were also sown. 46 days after 

sowing, the sexuality of gametophytes growing on both treatment and control plates was 

assessed. The sexuality of 50 gametophytes was determined at three different distances 

from the mature gametophyte, for each treatment plate. These distances were 0-1 cm, 

1-2 cm and 2-3 cm from the mature gametophyte. Samples were removed using a clean 

spatula and were mounted on a microscope slide. At each distance the first 50 

gametophytes, that were clearly distinguishable, were counted and sexed. Overall, the 

sexuality of 150 gametophytes was recorded for each plate, including controls. 

Photographs were taken for both antheridiogen experiments using an Olympus BX5 l 

compound microscope (Figs.2.12, 2.13 & 2.14). 

2.2.4 GAMETOPHYTE TRANSPLANTATION AND MACERATION 

2.2.4.1 Transplantation of gametophytes 

Gametophyte transplantation was required throughout the duration of this project in 

order to reduce competition between growing gametophytes and to provide fresh 

growing medium for gametophytes when old medium became exhausted. 

Transplantation was also carried out when setting up some experiments. The main 

challenge when transplanting gametophytes was to limit the amount of contamination 

from air and water borne microorganisms. Therefore, transplantation was carried out in 

a lamina flow cabinet. Gametophytes were uplifted using sterile toothpicks and were 

positioned on fresh plates. Lids were kept on plates at all times except when 

transporting gametophytes between them. Sterile water (2 ml) was added to each plate 

to facilitate growth and development of the gametophytes. 

30 



2.2.4.2 Maceration of gametophytes 

Experiments to assess the ability of gametophytes to withstand a low level of 

maceration were conducted using Blechnum novae-zelandiae, Cyathea medullaris and 

Dicksonia squarossa gametophytes. The degree of maceration was based on what may 

be expected to happen to these gametophytes in practi ce (i.e. when pumped through a 

hydroseeder). 

Spore samples from each species were sterili zed, sown and incubated in the standard 

growth cabinet conditions for 46 days . After this period of time the gametophytes were 

large enough to be transplanted (i. e. 5 mm in diameter). 300 gametophytes of each 

species were transplanted onto fresh ½ MS, I% agar plates (50 per plate). Two days 

after transplantation gametophytes were watered with I ml of steril e water per plate. 

Fourteen days after transpl antation a further 2 ml of steril e water was added to each 

pl ate. 4 1 days after transpl antation the gametophytes were suffi ciently large to 

macerate. 

Maceration was carried out in a lamina fl ow cabinet under steril e conditions. 

Gametophytes were macerated using a sterilized scalpel bl ade that had been soaked in 

70% ethanol and passed through a Bunsen fl ame. 25 pieces of chopped gametophyte 

were transplanted onto fresh medium. Five replicates pl ates of 25 gametophytes were 

transplanted for each species. In addition, 25 whole gametophytes were also 

transplanted onto fresh plates and replicated 5 times fo r each species. These whole 

gametophytes were controls. Survival of the chopped and whole gametophytes was 

assessed every 7 days fo r 6 weeks. Photos of the plates were also taken one day after 

maceration as well as 4 weeks after maceration using a hand-held Sony DSC-P8 digital 

still camera. 

3 1 



2.2.5 ESTlMATING SPORE QUANTITY 

A serial dilution was carried out in order to obtain an estimate of the amount of spore 

used in laboratory, nursery and field experiments. This estimate would also provide an 

indication of the amount of spore sown on each plate. 

Spore (0.1 ml), each of Blechnum novae-zelandiae, Cyathea medullaris and Dicksonia 

squarossa, was used in this dilution. The spore was diluted 1000-fold in order to obtain 

counts within the appropriate range of 30 to 300 spores per 0.1 ml of solution. This was 

done by initially adding l ml of water to the 0.1 ml of dry spore and thoroughly mixing 

the solution. Following this, 0.1 ml of the solution was added to 0.9 ml of water 

creating a IO-fold dilution. This I 0-fold dilution was repeated two more times in order 

to obtain a 1000-fold dilution. Throughout the serial dilution, solutions were thoroughly 

mixed in order to take uniform samples. Each dilution was carried out 3 times for each 

species and in addition 3 counts were made for each replicate. The amount of spore was 

counted for each replicate and an average was taken. The initial concentration of spore 

was then calculated for each species. 

2.2.6 ST A TISTICAL ANALYSES 

All germination curves were plotted using mean germination data at each time point, 

calculated from at least three replicates. In some figures the mean± the standard error 

was also included to show variability between replicates. ANOV A was used to 

determine the significance of several data sets including the effect of sterilization on 

spore germination (Fig. 2.2), the effect of polyacrylamide concentration on spore 

germination (Fig. 2.6 A, B & C) and the difference between two inductive environments 

(Fig. 2.4). All treatments were tested for significant difference at P < 0.05 unless 

otherwise stated. Replicate plates were positioned randomly when placed in growing 

conditions in order to reduce sampling bias, and possible environmental effects within 

the plate environment e.g. light, humidity, temperature. 

32 



2.3 Results 

2.3.1 SPORE GERMINATION 

2.3.1.1 Surface sterilization 

The germination of surface sterilized spore was compared with that of non-steril ized 

spore for the three species Bfechnum novae-zefandiae, Cyathea meduffaris and 

D icksonia squarossa (F ig. 2.2). The mean germination for each two day interval has 

been plotted. All species germinated within 6 days of sowing and nearly all treatments 

reached maximum germination after 6-1 0 days. Sterilized B. novae-zefandiae spore had 

an overall lower germination rate than that of non-sterili zed spore of the same species 

(5 0% and 70% respectively). However, analys is of the mean data, using ANO VA 

indicated there was no significant di ffe rence between the non-sterilized and sterilized 

spore germination data of B. novae-zefandiae. 

80 -

C 
60 · 0 

~ 
.!: 

T 
E T GI 

40 j Cl 1 ~ 

-+- B.nov Sterllzed 

B.nov Non-sterilized 

20 ., j_ 
0 - •---~--------------~---,----

-+- C. med Sterilized 

C. med Non-sterilized 

-+- D. squ Sterilized 

D. squ Non-sterllzed 
__ ..J 

0 2 4 6 8 10 12 14 16 18 20 

Days after sowing 

Figure 2.2 The effect of surface sterilization on spore gennination of three native New Zealand fem 

species, Blechnum novae-zelandiae (abbrev. B. nov), Cyathea medullaris (abbrev. C. med) and Dicksonia 

squarossa (abbrev. D. squ). 

33 



Cyathea medullaris and D. squarossa spore germination curves showed very little 

difference between sterilized and non-sterilized spore. This was supported by one-way 

ANOV A, which showed that there was no significant difference between the data of 

sterilized and non-sterilized spore of C. medullaris and D. squarossa. Cyathea 

medullaris and D. squarossa spore showed around 85% - 99% germination when the 

maximum germination potential was reached, regardless of sterilization. Standard error 

bars indicate that sterilized B. novae-zelandiae spore exhibited the most variation 

between replicates. These data suggest that the sterilization process described in this 

project did not significantly affect spore germination in these three fern species. 

2.3.1.2 Spore germination in controlled, dark and natural conditions 

Blechnum novae-zelandiae, Cyathea medullaris and Dicksonia squarossa spore was 

germinated on standard media, in controlled, dark, and natural conditions (Fig. 2.3). 

Under controlled, growth cabinet conditions, spore germinated between 4-6 days after 

sowing for all three species. Germination reached its maximum potential for all three 

species 10 days after sowing. This is indicated by a plateau visible on each curve. 

Blechnum novae-zelandiae spore germination plateaued at about 60%. Cyathea 

medullaris and D. squarossa spore germination plateaued at around 95%. These results 

are similar to the corresponding curves seen in Fig. 2.2. 

Spore sown in the dark did not germinate until it was transferred to the growth cabinet, 

10 days after sowing, whereupon germination commenced 4 to 6 days later. This 

indicated that B. novae-zelandiae, C. medul!aris and D. squarossa spore is photoblastic, 

i.e. requires light for germination. Germination plateaued at similar levels to the spore 

grown in the controlled environment, with one exception. Blechnum novae-zelandiae 

dark grown spore reached a higher maximum germination potential than spore grown in 

the controlled environment. 

Spore grown in natural conditions did not germinate after 20 days, except for a very low 

percentage ( < I%) of B. novae-zelandiae spores, from 18 20 days. 

34 



A 

-~ 

' l 
~ 0 

B 

C: 
0 

i 
C: 

-~ 
Cl 
~ 0 

C 

C: 
0 

i 
C: 

"i 
Cl 
~ 0 

100 

80 

60 

40 

20 

0 

100 

80 

60 

4 0 

20 

0 

100 

80 

60 

4 0 

20 

0 

0 2 

0 2 

0 2 

Controlled 

conditions 

4 

4 

4 

Blechnum novae-zelandiae 

T 
.1. 

6 

T 
j_ 

6 

T 

..1... 

6 

T T 
.1. l 

8 10 12 14 

Days after sowing 

Cyathea medullaris 

8 10 12 14 

Days after sowing 

Dicksonia squarossa 

8 10 12 14 

Days after sowing 

Dark ( 10 days) 

+ controlled conditions 

16 18 

16 18 

16 18 

Natural 

conditions 

20 

20 

20 

Figure 2.3 Germination of (A) B. novae-zelandiae, (8 ) C. medullaris and (C) D. squarossa in 

controlled, dark and natural conditions. 

35 



The spore used in each treatment was originally from the same collection, suggesting 

that even the non-germinated spore, grown in natural conditions, was potentially viable. 

Twenty-two days after sowing, this spore was transferred from natural to glasshouse 

conditions to test its ability to germinate. The changes in three environmental 

parameters (temperature, relative humidity and light intensity) compared with the 

germination of the three species, can be observed in Fig. 2.4. After four days in the 

glasshouse all three species germinated. Ten days after the change in conditions, all 

three curves plateaued, though germination reached a level substantially lower than that 

seen in controlled conditions (Fig. 2.3). 

A change in temperature can be observed 22 days after germination, corresponding to 

the change in conditions (Fig. 2.4A). When the temperature data for the two different 

conditions is compared, ANOVA indicates there is significant difference between the 

temperature of the natural conditions and the temperature of the glasshouse conditions 

(P < 0.0001 ). The mean temperature increased by 7.5°C corresponding with the onset 

of spore germination for each of the three species. 

A change in relative humidity was also observed (Fig. 2.4B). After plates had been 

transferred to the warmer glasshouse the relative humidity surrounding the plates 

decreased by 27.5%, an effect that was significantly different (P < 0.0001). These data 

suggest that a drop in humidity surrounding the incubation plates was associated with 

spore germination. However, the results shown here do not accurately measure the 

micro-environment to which the spore was exposed. The plates containing the spore 

were sealed with parafilm and, therefore, the humidity surrounding the plates (which is 

shown here) would only influence the humidity inside the plates indirectly, depending 

on how well sealed the plates were. It was observed that plates had dried out and lost 

most of their condensation by the conclusion of the experiment, indicating that water 

vapour was slowly escaping from the plates due to the lower humidity outside of the 

plates. 

There was no consistent difference in light intensity between the natural conditions and 

the glasshouse conditions (Fig. 2.4C). The mean light intensities for the two 

environments were not significantly different. 

36 



A 

B 

C 

Key 

Change in temperature 

25 100 

G' 20 80 
C: 

~ 0 

e 15 60 'i 
3 C: 

E 10 

1 
40 

.§ 
Q) Q) 
C. 

l Cl 
E 

l ~ Q) 5 20 0 

I-

0 0 
0 2 4 6 8 10 12 14 16 

I 
18 20 22 24 26 28 30 32 

Days after sowing 

Change in relative humidity 

100 - 100 

l 75 
~ 
'5 .E 

50 ::, 
~ 
Q) 

> 
i 25 w 

r - ,-

-
7 80 

C: 

I 
0 

60 i 
C: 

'§ 
Q) 

Cl 

20 
~ 

ix: 

0 - I I 0 
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 

0 
0 
~ 

400 
350 
300 

>- V)250 
~ .! 200 
C: "C 

2l ~ 150 
C: 0 

~ 100 
~ 50 

0 

Days after sowing 

Change in light intensity 

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 

Blechnum novae-zelandiae 

Dicksonia squarossa 

Days after sowing 

Cyathea medullaris 

Environmental parameter (i.e. temp 

humidity or light) 

100 

C: 
0 

60 i 
C: 

40 -~ 
Cl 

~ 20 

Figure 2.4 Spore germination of B. novae-zelandiae, C. 111edul/aris, D. quarossa in natural conditions (0-22 

days), and after transfer to glasshouse conditions (24 - 32 days). Changes in temperature (6A), relative 

humidity (68) and light intensity (6C) are shown . 

37 



2.3.1.3 Seasonal variation in spore germination 

The viability of spore of Blechnum novae-zelandiae, Cyathea medullaris and Dicksonia 

squarossa was assessed from April 2005 until September 2005, by measuring spore 

germination (Fig. 2.5). The early increase in spore germination seen in B. novae­

zelandiae and C. medullaris corresponded with the maturation of the immature spore 

through autumn and into winter. The onset of spore maturation in Dicksonia squarossa 

is not shown. The period of spore maturation varied between the three species. 

Blechnum novae-zelandiae spore matured over a 5 month period (April -August) and 

reaches its highest germination rate in late winter. Cyathea medullaris spore matured in 

mid April and maximum germination was reached several weeks alter. The maturation 

of D. squarossa spore was not clearly expressed. However, its maximum germination 

peaked in the late autumn, similar to the other tree fern species C. medullaris. All three 

species had highly viable spore in the late winter/early spring. 

Through field observations it was clear that by September the quantities of spore 

produced by each species, was in dramatic decline. The season's spore production was 

decreasing as indicated by the spent fronds. However, the small quantities of spore 

retained their ability to germinate. 

The variability within sample dates is shown by the standard error of the mean bars, 

located at each data point. Interestingly, for B. novae-zelandiae and D. squarossa the 

most variability within the samples was at the beginning of the season. Cyathea 

medullaris spore germination showed little variability at all stages. 

38 



A 
Blechnum novae-zelandiae 

C 100 0 
; 80 -113 
C 60 -
E 40 -... 
Q) 20 C) 

~ 0 0 
LC) LC) LC) LC) LC) LC) LC) LC) 

0 0 0 0 0 0 0 0 
0 0 0 0 0 0 0 0 
N N N N N N N N 

--- ~ --- co ;::: --- --- ---3 LC) co 0) 0) 
0 0 0 0 0 0 0 

--- --- LC) cri ~ a5 --- ;::: LC) 0 N 
C'? N ..- N 

Collection date 

B 
Cyathea medullaris 

C 100 0 ~ 

; 80 113 
C 60 
E 40 ... 
Q) 

20 C) 

~ 0 0 
LC) LC) LC) LC) LC) LC) LC) LC) 

0 0 0 0 0 0 0 0 
0 0 0 0 0 0 0 0 
N N N N N N N N 

--- --- --- co ;::: a5 cri cri 3 ,q- LC) 

0 0 0 0 0 0 0 
LC) --- --- --- ~ --- --- ;::: 0 LC) 0) co N 

C'? N N 

Collection date 

C 
Dicksonia squarossa 

C 100 0 

~ --; 80 ~ ---113 
C 60 
E 40 ... 
Q) 20 C) 

~ 0 0 
LC) LC) LC) LC) LC) LC) LC) LC) 

0 0 0 0 0 0 0 0 
0 0 0 0 0 0 0 0 
N N N N N N N N 

--- 3 LC) co ;::: a5 cri cri 3 0 0 0 0 0 0 
LC) 0 LC) cri ~ a5 N ;::: 

C'? N ..- N 

Collection date 

Figure 2.5 Mean germination rates of 8. novae-zelandiae (A), C. medullaris (8), D. squarossa (C) 

showing germination rates from Apri l 2005 to September 2005 Each time point shows the mean ± 

standard error after 12 days of gennination. 

39 



2.3.1.4 The effect of three polyacrylamides on spore germination 

The three chosen polyacrylamides; Soilfix™, FLOBOND and Aquasorb, did not 

influence the germination of B. novae-ze/andiae, C. medullaris or D. squarossa spore 

(Fig. 2.6A, B & C). Using ANOVA it was determined that the increase in concentration 

from lg L- 1 of polyacrylamide tol0g L-1 showed no significant change in germination 

compared to controls . The most variation between the three treatments was observed in 

B. novae-zelandiae spore. However, this variation is not statistically significant. 

80 

C: 
0 
'fi! 60 
C: 

-~ 
g, 40 

"#-

20 

SOILFIX 

--B. nov Cont. -- B.nov 1g/1 

- c .med Cont . --C.med 1g/l 

--• .squ Cont. D.squ 19/1 

0 +---~ ===~ 'L--~- -~----======;:::==:::;:=== 
0 2 4 6 8 10 12 14 

Days after sowing 

--B.nov 109/I 

C.med 109/I 

--• .squ 109/I 

16 18 20 

Figure 2.6A The effect ofSoilfix™ on the germination of B. novae-zelandiae, C .medullaris and D 

.squarossa spore. 

FLOBOND 
100 

80 

C: 
0 

60 'fi! 
C: 

-~ 
40 Cl 

~ 

--- B.nov Cont . --B.nov 1g/l - B.nov 10g/l 
20 

--c.medCont. --C.med 1g/1 C.med 10g/l 

--• .squ Cont . D.squ 1g/l - • .squ 10g/1 
0 

0 2 4 6 8 10 12 14 16 18 20 

Days after sowing 

Figure 2.6B The effect ofFLOBOND on the germination of B. novae-zelandiae, C .medullaris and D 

.squarossa spore. 

40 



AQUASORB 
100 · 

80 1 

C 
0 

60 ~ i 
C 

-~ 
40 l Cl 

~ 0 

-- B.novCont. --B.nov 19/1 B.nov 10g/l I 
20 , 

-- c .med Cont. C.med 1g/l Cmed 109/I 

-- D.squ Cont . D squ 1g/l D.squ 10g/I 
0 

0 2 4 6 8 10 12 14 16 18 20 

Days after sowing 

Fig 2.6c The effect of Aquasorb on the gennination of B. novae-zelandiae, C .medullaris and D .squarossa 

spore. 

2.3.l.5 Fluorescein diacetate spore viability test 

The results for the fluorescein diacetate (FDA) test were inconclusive. In some cases 

the test appeared to work successfull y. However, when repeated, vary ing results were 

obtained. Figure 2. 7 shows a sample of several of the photos taken using the 

fluorescence microscope. When considering the three species together, viable and 

"reduced viability" spore treated with water only appeared to have no fluorescence. 

Therefore, it was poss ible to ascertain the background fluorescence, which in the case of 

these photos was minimal. 

When FDA was added and left to incubate with the spore, several observations were 

made. Viable B. novae-zelandiae spore appeared to fl uoresce, with some spores 

fluorescing more brightly than others. However, "reduced viability" spore also had a 

low and comparable level of fluorescence. Viable C. medullaris spore did not fluoresce 

at all when exposed to FDA. However, one spore seen in the C. medullaris, FDA 

treated, "reduced viability" section, fluoresced brightly (F ig. 2. 7). FDA treated D. 

squarossa spore fl uoresced regardless of whether it was viable or "reduced viability" 

spore. Several attempts of the FDA test fai led to generate inte ll igible resul ts, producing 

photos of FDA treated spore that had absolutely no fl uorescence (data not shown). 

41 



Species Treatment "Viable" spore "Reduced viability" spore 

B. nov. Control (H20) 

FDA 

C. med. Control (H20) 

FDA 

D. squ. Control (H20) 

FDA 

Figure 2.7 Photos of viable and "reduced viability" spore treated with water (controls) and fluorescein 

diacetate (FDA). B. nov. = B. novae-zelandiae. C. med. = C. medullaris. D. squ. = D. squarossa. 

42 



2.3.2 SEXUAL MATING SYSTEMS 

2.3.2.1 Sporophyte production and gametophyte sexuality 

Blechnum novae-zelandiae was successfull y cultivated from spores, to sporophytes, 

under laboratory conditions (Fig. 2.8). The ability to produce sporophytes in the 

laboratory suggests that conditions were optimal for both antheridia and archegonia 

production. In addition, fertilization was able to be successfull y induced by flooding 

gametophytes with sterile water. Antheridia (o ) and archegonia (~ ) were identified 

using a compound microscope. In addition, the release of spermatozoids from a single 

antheridium was observed (Fig. 2.9). In one plate nearly the entire life cycle of B. 

no vae-zelandiae was observed in 66 days (i.e. from spore to sporophyte). 

, 

• 

• 
Ill . . 

Figure 2.8 The progression of laboratory grown Blechnu111 novae-zelandiae spore ( I00x mag.), to 

gametophytes, and finally sporophytes. 

A 

Figure 2.9 A. The arrow indicates the position of a cluster of B. novae-zelandiae archegonia next to the 

apical notch of the gametophyte (40x mag.). B. The arrow points to a single antheridium of the same 

species. Attached to the antheridium is a cluster of spenn ( I 00x mag ). 

43 



Initial experiments suggested that B. novae-zelandiae was capable of intra-gametophytic 

selfing. One out of four isolated, presexual gametophytes successfully selfed and 

produced visible sporophytic structures (Fig. 2.10). It appears that several fertilization 

events occurred at the same time, as three sporophytic structures can be observed 

growing from the gametophyte. 

A B 

Figure 2.10 Intra-gametophytic selfing. A. The arrows indicate the position of three sporophytes (!Ox 

mag.). 8 . A close up view of one sporophyte (20x mag.). 

In later experiments B. novae-zelandiae was found to be capable of both intra­

gametophytic selfing and inter-gametophytic crossing, when grown in standard 

laboratory conditions (Fig. 2.11 ). A low proportion of gametophytes subjected to inter­

gametophytic crossing, produced sporophytes . This type of mating was the most 

common. A lower proportion of gametophytes, subjected to intra-gametophytic selfing, 

produced sporophytes. Interestingly, gametophytes subjected to inter-gametophytic 

selfing did not produce any sporophytes. The data suggest a mixed mating system for B. 

novae-zelandiae. 

6-r--;:=========;--------------------, 

II) 

i :l: 4 
.c >, 
C. .c 
0 C. 
Q) ~ 
E o 2 
"' C. Cl Ill -0 

ci z 

0 

• lntra-garretophytic 
selfing 

• lnter-garretophytic 
crossing 

2 3 4 5 6 7 8 9 10 11 

Weeks after flooding 

Figure 2.11 A comparison of potential mating systems in B. novae-zelandiae. Inter-gametophytic selfing 

was not observed over the 11 week experimental period. 

44 



2.3.2.2 Antheridiogen 

The presence of antheridiogen was tested for in the species Bfechnum novae-zelandiae, 

Cyathea medullaris and Dicksonia squarossa . It was expected that spore grown in the 

dark on media that originally supported mature gametophytes, would germinate if 

antheridiogen was present in the medium. After IO days in the dark onl y Blechnum 

novae-zelandiae produced any germinants on the treatment plates (F ig. 2. 12). No spore 

germinated on the dark grown control , suggesting that the germination observed on the 

treatment plates was due to the presence of a substance previously excreted by the 

original gametophytes. The number of B. novae-ze fandiae spores that germinated on 

each treatment replicate plate varied from Oto 7%. The emerging rhizo ids observ