(Capparis Mitchellii) and Native Lime (Citrus Glauca) Micropropagation

Further Investigation of Australian Native Orange (Capparis mitchellii) and Native Lime (Citrus glauca) Micropropagation

Syarifah Fadiya Hallaby

Submitted in fulfilment of the requirements for the degree of

Master of Science

Discipline of Plant Physiology, Horticulture and Viticulture School of Agriculture, Food and Wine Faculty of Science University of Adelaide

January 2012 Table of Contents

Table of Contents...... ii Abstract...... vi Declaration ...... viii Acknowledgement...... ix List of Figures ...... x List of Tables ...... xi List of Abbreviations ...... xii

CHAPTER ONE ...... 1 General Introduction and Literature Review...... 1 1.1 Introductory background ...... 1 1.2 Australian native food industry ...... 2 1.2.1 Capparis mitchellii and Citrus glauca as potential plants for the native food industry ...... 3 1.3 General description of Capparis mitchellii ...... 4 1.3.1 Classification and common names ...... 4 1.3.2 Distribution...... 5 1.3.3 Morphology ...... 6 1.3.4 Cultural value ...... 7 1.4 General description of Citrus glauca ...... 8 1.4.1 Classification and common names ...... 8 1.4.2 Distribution...... 10 1.4.3 Morphology ...... 11 1.4.4 Cultural value ...... 13 1.5 The limitation of production and cultivation ...... 13 1.6 Micropropagation as an alternative cultivation technique...... 15 1.6.1 Micropropagation Procedures ...... 15

1.6.1.1 Plant selection and preparation ...... 16 1.6.1.2 Establishing aseptic culture ...... 16 1.6.1.3 Production of suitable propagules ...... 20 1.6.1.4 Preparation for growth in the natural environment ...... 25 1.6.1.5 Transferring to ex vitro conditions ...... 28 1.7 Aim/Objective of the project ...... 29

CHAPTER TWO ...... 30 EXPLANT STERILISATION ...... 30 2.1 Introduction...... 30 2.2 Material and methods ...... 31 2.2.1 General preparation and sterilisation technique ...... 31 2.2.1.1 Equipment preparation ...... 31 2.2.1.2 Media preparation ...... 31 2.2.1.3 Explant preparation ...... 32 2.2.1.4 Incubation condition ...... 33 2.2.2 Effects of different sterilisation agents on explant surface contamination level ...... 34 2.2.3 Statistical analysis ...... 35 2.3 Results ...... 35 2.3.1 Capparis mitchellii ...... 35 2.3.2 Citrus glauca ...... 37 2.4 Discussion ...... 39

CHAPTER THREE ...... 42 Shoot Initiation ...... 42 3.1 Introduction...... 42 3.2 Material and Methods ...... 43 3.2.1 Preparation ...... 46 3.2.2 Effects of different basal media on shoot initiation ...... 46 3.2.3 Experimental design and statistical analyses ...... 47

iii

3.3 Results ...... 48 3.3.1 Capparis mitchellii ...... 48 3.3.2 Citrus glauca ...... 49 3.4 Discussion ...... 50

CHAPTER FOUR ...... 52 Shoot Multiplication and Elongation ...... 52 4.1 Introduction...... 52 4.2 Materials and Methods ...... 53 4.2.1 Preparation ...... 53 4.2.2 Effects of BA on shoot multiplication and elongation ...... 54 4.2.3 Experimental design and statistical analysis ...... 54 4.3 Results ...... 55 4.3.1 Capparis mitchellii ...... 55 4.3.2 Citrus glauca ...... 56 4.4 Discussion ...... 57

CHAPTER FIVE ...... 62 Rooting and Plant Establishment ...... 62 5.1 Introduction...... 62 5.2 Materials and Methods ...... 63 5.2.1 Preparation ...... 63 5.2.2 Effects of auxin level on root development ...... 64 5.2.3 Plant establishment ...... 65 5.2.4 Experimental design and statistical analyses ...... 65 5.3 Results ...... 66 5.3.1 Capparis mitchellii ...... 66 5.3.2 Citrus glauca ...... 67 5.4 Discussion ...... 68

CHAPTER SIX ...... 72 General Discussion ...... 72

Appendix. Supplier Information ...... 77 References ...... 78

Abstract

A protocol to micropropagate Capparis mitchellii and Citrus glauca through plant tissue culture techniques was investigated in this project. An efficient protocol will open a possibility to mass propagate both of these Australian native plants either for industrial or conservation purposes. The project was conducted using surface sterilisation, shoot initiation, shoot multiplication, rooting and acclimatisation experiments.

Microbial contamination level in this project was maintained at a low level by agitating the plant material for 4 hours in 5% (v/v) PPMTM solution prior to planting onto media supplemented with mancozeb. By this method, shoot initiation, multiplication, elongation and rooting experiments were made possible.

A range of different strength basal media and growth regulators was utilised to promote shoot and root induction from C. mitchellii and C. glauca nodal shoot explants. Shoot proliferation was observed in all treatments with the optimum rate obtained on MS media supplemented with 1 mg/L BA and ½MS media with 0.1 mg/L BA for C. mitchellii and C. glauca respectively. Healthy roots of C. mitchellii were promoted by exposing the proliferated shoots to high levels of NAA (100-200 mg/L) for 48 hrs. No roots were observed on C. glauca shoots.

Necrotic and secondary abscission zone became a major issue in this project. Prolonged use of antibiotics, imbalance of nutrients and exogenous growth regulators were

considered as the possible factors. Experimenting with nutrient level and growth regulators was necessary to improve shoot and root proliferation.

In conclusion, both C. mitchellii and C. glauca were propagated by tissue culture methods. This research has provided the first complete protocol to micropropagate C. mitchellii and basic information for micropropagating C. glauca through nodal shoot culture.

vii

Declaration

I, Syarifah Fadiya Hallaby, certify that this works contain no material which has been accepted for the award of any degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due references has been made in the text.

I give consent to this copy of my thesis, when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the

I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the Library catalogue and also through web search engine, unless permission has been granted by the University to restrict access for a period of time.

Signed :

Date :

viii

Acknowledgement

I would like to offer my sincerest gratitude to my supervisors Dr. Michelle

Wirthensohn, Dr. Phillip Ainsley and Dr. Manfred Jusaitis for their guidance, advice, support and patience, without them this thesis would never be completed.

I would also like to thank Dr. Chris Ford and Dr. Michelle Picard for their assistance and support during my candidature, Dr. Jennifer Gardner for access to the Waite

Arboretum and Stephen Forbes, Director of Botanic Garden of Adelaide, for full access to Botanic Garden of Adelaide facilities. Special thanks to fellow staff and students at

Botanic Garden of Adelaide Seed Conservation Centre, Jenny Guerin, Michael Thorpe,

Dan Duval, Thai Te, Emma Steggles, Rina Aleman and Nicole Dowling for their assistance and friendly support.

I express my deep appreciation to Human Resources Development Committee of Aceh

Government, Indonesia for awarding me a scholarship and to the University of

Adelaide for funding my research.

Finally, very special thanks for my family especially my parents for their love, support and understanding to let me study abroad. Thank you also to my friends in Adelaide, who I cannot mention one by one, for keeping me sane during my Master’s project.

List of Figures

Figure 1.1 Distribution of C. mitchellii in Australia (AVH, 2011a)...... 6

Figure 1.2 Morphology of C. mitchellii habit (A), leaves (B), flower (C), and fruit (D, Photo: P. Ainsley)...... 7

Figure 1.3 Distribution of C. glauca in Australia (AVH, 2011b)...... 11

Figure 1.4 Morphology of C. glauca habit (A), leaves (B), flower (C), and fruit (D)...... 12

Figure 2.1 Newly grown stems of C. mitchellii (A) and C. glauca (B) used for explants...... 33

Figure 2.2 Effects of different sterilisation treatments on contamination and initiated shoot level of Capparis mitchellii culture...... 37

Figure 2.4 Heavily (hdc) and slightly (sdc) discoloured tissue of C. glauca explants planted onto media with 0.2% PPMTM and miconazole, compared with healthy explants with minimum necrotic and growing shoots (sh) planted onto mancozeb and control media (no sterilisation agents)...... 39

Figure 5.1 Adventitious roots of C. mitchellii without (A) and with (B) callus formation...... 69

List of Tables

Table 1.1 Different surface sterilisation agents for disinfesting Capparis and Citrus explants...... 18

Table 1.2 Summary of the best treatments for shoot initiation and multiplication from published studies on Capparis and Citrus species micropropagation...... 23

Table 1.3 Summary of the best treatments for rooting from published studies of Capparis and Citrus species micropropagation...... 26

Table 2.1 Experimental design for shoot sterilisation experiment...... 34

Table 3.1 Effect of different basal media supplemented with 3mg/L BA on shoot initiation of Capparis mitchellii...... 48

Table 3.2 Effect of different basal media supplemented with 3mg/L BA on shoot initiation of Citrus glauca...... 49

Table 4.1 Effect of BA concentration on shoot multiplication and elongation of Capparis mitchellii...... 55

Table 4.2 Effect of BA concentration on shoot multiplication and elongation of Citrus glauca...... 57

Table 5.1 Effect of NAA on rooting of Capparis mitchellii...... 67

List of Abbreviations

ABA Abscisic acid;

ANFIL Australian Native Food Industry Limited

AVH Australia's Virtual Herbarium

B5 Gamborg Plant tissue culture medium

BA Benzyladenine

BAP 6-benzylaminopurine

CSIRO Commonwealth Scientific and Industrial Research Organisation

DKW Driver & Kuniyuki (1984) basal medium for walnut

GA3 Gibberellic acid

IAA Indole 3-acetic acid

IBA Indole 3-butyric acid;

NAA α-naphthalene acetic acid;

MS Murashige and Skoog basal medium

PGR Plant growth regulators pH Potential Hydrogen

PPMTM Plant Preservative MixtureTM

TDZ Thidiazuron

WPM Lloyd & McCown woody plant basal medium

ZR Zeatin riboside;

xii

Chapter One: General Introduction and Literature Review

CHAPTER ONE

General Introduction and Literature Review

1.1 Introductory background

Australia is well known for its high rate of biodiversity. Ten percent of the world’s biodiversity (plants, animal and birds) is native to Australia (Department of

Foreign Affairs and Trade, 2008). For thousands of years, numerous native fruits, nuts, berries, spices, herbs and vegetables have been widely used by Indigenous people. The textures, flavours, smells and colours of those native plants have been recently recognised for their marketable quality as well. At the end of the 1980s, the native food industry commercially started in Australia (Hele, 2001).

Capparis mitchellii (native orange) and Citrus glauca (native lime) are listed by

CSIRO as two of Australia’s economic plants (Lazarides & Hince, 1993). However, like other Australian economic native plants, most commercial stocks are still harvested from wild trees (Hele, 2001; Miers, 2004; Tworney et al., 2009). C. mitchellii is known to be difficult to propagate and to date there are no published reports about the commercial cultivation of this plant. On the other hand, the high value of C. glauca in the market elicited some serious attempts to cultivate it on an industrial scale by grafting (Tworney et al., 2009). However, in many cases conventional techniques are

Chapter One: General Introduction and Literature Review

time consuming, impractical, and occasionally fail to produce desirable outcomes

(Kumar & Sharma, 2005).

To expand cultivation options, micropropagation may be considered as an alternative. Micropropagation, also known as plant tissue culture, is a method to isolate protoplasts, cells, tissues, or organs and propagate them in a highly controlled environment, using nutrients and plant growth regulators (Hartmann et al., 2011). Until recently, the only research undertaken for C. mitchellii micropropagation focused on developing a sterilisation technique and selecting a basal medium for shoot induction and multiplication (Steggles, 2007). Similarly for C. glauca there was only one report published describing propagation by somatic embryogenesis (Jumin & Nito, 1996).

However, there are no detailed micropropagation protocols for either species. Thus there is a need for further research to optimise initiation, and establish multiplication/proliferation, rooting and acclimatisation methods for both C. mitchellii and C. glauca. This thesis set out to do just that.

1.2 Australian native food industry

Interest in Australian native food, both in the cultivation and wild harvesting areas, has rapidly increased, especially over this past decade. This has been influenced by the developing concept of Australian cuisine and understanding the value of native food based on Aboriginal and scientific knowledge. Furthermore, many native food

Chapter One: General Introduction and Literature Review

plants have been recognised for their marketable value as food resources either for national or international markets (Miers, 2004).

The native food industry is becoming a growth industry in Australia. At the beginning of this millennium, the estimated value of the native food industry was $10-

$16m per annum (Cherikoff, 2000; Hele, 2001). To ascertain whether this industry will be able to develop a sustainable market, the Australian Native Food Industry Limited

(ANFIL) was formed to act as the national body in 2006. This organisation is a non- profit organisation that represents the interests of the native food industry in Australia

(Rural Industries Research and Development Corporation, 2007; ANFIL, 2009).

1.2.1 Capparis mitchellii and Citrus glauca as potential plants for the native food

industry

Research has shown that native plants have the same or better nutritional characteristics as Western plants (Brand et al., 1983 cited in Ahmed & Johnson, 2000;

Konczak & Rural Industries Research and Development Corporation, 2009 ). Native fruits are rich in vitamins, antioxidants and minerals (Netzel et al., 2007; Konczak &

Rural Industries Research and Development Corporation, 2009). Some native plants are also recognized as anti-microbial agents (Forbes-Smith & Paton, 2002). Furthermore, other research has reported that by consuming native plants as their traditional diet,

Indigenous people are able to protect themselves from western diseases such as diabetes, obesity, and hypertension (O’Dea et al., 1984; Hodgson and Wahlqvist, 1992

Chapter One: General Introduction and Literature Review

both cited in Ahmed & Johnson, 2000). C. mitchellii (native orange) (Tworney et al.,

2009) and C. glauca (native lime) (Ahmed & Johnson, 2000) are two particular plants that have been recognized for some of those stated values.

1.3 General description of Capparis mitchellii

1.3.1 Classification and common names

Capparis mitchellii is a member of the family Capparaceae. Around 40-45 genera and nearly 700 species are classified within this family (Elliot & Jones, 1982;

Jessop & Toelken, 1986). The Capparaceae are known for diversity in their habit, fruits and flowers (Cronquist, 1981; Heywood, 1993; Mabberley, 1997 all cited in Hall et al.,

2002). These heterogeneous characters have triggered many taxonomic disagreements in this family (Hall et al., 2002). Species under the Capparaceae family are widely distributed in tropical and subtropical areas, while some species are even found in the arid zone (Jessop & Toelken, 1986). Five genera (one endemic) of this family can be found in Australia, i.e. Crateva, Capparis, Appophyllum, Cadaba, and Cleome. These 5 genera consist of 30 species, 20 of them endemic, 6 species are native and 4 species are naturalised (Hewson, 1982).

Capparis is the largest genus in the family Capparaceae. The phylogenic system of this genus varies depending on the characteristics used by the author to build the

Chapter One: General Introduction and Literature Review

system (Infantino et al., 2007). Seventeen species out of a total of 250 species in this genus are recorded as native or endemic in Australia. C. mitchellii is one of the endemic species (Hewson, 1982; Electronic Flora of South Australia, 2011a).

The scientific name of Capparis was derived from the Arabic word kabar, a

Persian name for Capparis spinosa which is commonly known as the Caper Tree. C. mitchellii was named after an explorer who collected it, Sir Thomas Mitchell

(Boomsma, 1972; Jessop & Toelken, 1986).

There are a few European colloquial names for C. mitchellii, including native or wild orange (Elliot & Jones, 1982; Hewson, 1982; Costermans, 2009), bumble and native pomegranate (Hewson, 1982). In Australian Indigenous language, this plant is known as Iga Tree (Jessop & Toelken, 1986; Tunbridge, 1988).

1.3.2 Distribution

Capparis mitchellii is widely distributed in Australia (Figure 1.1), and can be found in the Northern Territory, New South Wales, Queensland, South Australia and

Western Australia (AVH, 2011a). C. mitchellii is able to grow in a wide range of soil types including clay and limestone, making this plant suitable for garden culture, especially in dry areas (Elliot & Jones, 1982; Jessop & Toelken, 1986; Holliday, 1989;

Lazarides & Hince, 1993). Based on the Census of South Australian Vascular Plants, there are 65 records for these species in South Australia (Electronic Flora of South

Australia, 2011a).

Chapter One: General Introduction and Literature Review

Figure 1.1 Distribution of C. mitchellii in Australia (AVH, 2011a).

1.3.3 Morphology

Capparis mitchellii is a shrub or tree and has the ability to grow to 10m high

(Figure 1.2A). Leaves are 2-6 cm long by 1-3 cm wide with petioles. Sharp spines usually found on juvenile plants or new growth shoots on adult plants (Figure. 1.2B).

Inflorescences usually have a solitary flower or 2-4 flowers in a group. The flower is 3-

4 cm long and 5 cm across with white or creamy-yellow peduncles and numerous long stamens (Figure 1.2C). The fruit is a berry about 4-7 cm in diameter (Figure 1.2D). It has a pericarp with orange flesh inside the fruit that is filled with numerous seeds

(Elliot & Jones, 1982; Hewson, 1982; Jessop & Toelken, 1986; Lazarides & Hince,

Chapter One: General Introduction and Literature Review

1993; Harden, 2000). The fruits are edible and have been described as passion fruit like in taste with a kerosene aftertaste (Cribb & Cribb, 1987).

A. B.

Figure 1.2 Morphology of C. mitchellii habit (A), leaves (B), flower (C), and fruit (D, Photo: P. Ainsley).

1.3.4 Cultural value

Capparis mitchellii has been recognised for its economic, conservation and cultural value. C. mitchellii is characteristically drought resistant, making it suitable for use in Australian agro-forestry (Tworney et al., 2009). This plant is also an important

Chapter One: General Introduction and Literature Review

support for the arid ecosystem, where C. mitchellii acts as the host tree for the Caper

White Butterfly (Anaphaesis java spp. teutonia) and shade provider from heat

(Tunbridge, 1988; Harden, 2000). Furthermore, this plant also has potential for improving diets, due to its high level of vitamin C (Tworney et al., 2009). C. mitchellii is also listed as one of 21 native species which have been considered as valuable plants in the confectionery, flavouring and preservative industries (2004). In Indigenous culture, this plant not only forms part of a traditional diet, but also plays an important role in the dreaming (an Aboriginal term for religious belief about the Aboriginal Law

Creation) stories of Adnyamanthanha people of Northern Flinders Range, South

Australia (Tunbridge, 1988; Clarke, 2007). In general, C. mitchellii is a valuable

Australian native plant worthy of development and cultivation.

1.4 General description of Citrus glauca

1.4.1 Classification and common names

Citrus glauca belongs to the subfamily Aurantioideae of the Family Rutaceae.

This subfamily includes from 26 (Mabberley, 2008) to 33 (Swingle & Reece, 1967) genera, depending on the type of data used in determining the taxonomy of Citrus.

The phylogeny and taxonomy of the genus Citrus are complicated and controversial, mainly due to two factors. Firstly, Citrus species are able to cross with

Chapter One: General Introduction and Literature Review

other species in the same genus or related genera producing fertile and stable hybrids

(Mabberley, 1997; Nicolosi et al., 2000; de Araújo et al., 2003; Mabberley, 2004).

Secondly, citrus has a very long history of cultivation and distribution (Chapot, 1975 and Scora 1975 in de Araújo et al., 2003). Hence, there are several interpretations of

Citrus taxonomy. The two most widely used classifications are the Swingle system

(Swingle & Reece, 1967) and the Tanaka system (Tanaka, 1977 in Federici et al.,

1998).

Citrus glauca (Lindl.) was formerly known as Eremocitrus glauca (Lindl.)

Swingle (Elliot & Jones, 1984; Jessop & Toelken, 1986). However, Mabberley (2004) argued that the Citrus phylogeny constructed by Swingle was flawed because the understanding of the complex hybridisation history of commercial citrus was somehow overlooked. Thus, Mabberley (1998) proposed a new system for Citrus. In this proposed system, genera that were previously segregated from Citrus by Swingle, e.g.

Eremocitrus and Microcitrus, were restored to their former position as species of genus

Citrus. Further studies related to this issue have been unable to determine which version is more accurate, since some studies support the Mabberley system (de Araújo et al., 2003) while others support the Swingle system (Pang et al., 2007). Regardless of the arguments, Mabberley’s phylogeny system for Australian citrus was used in this thesis.

Citrus glauca was derived from the Latin words citron and glaucos, which mean citrus and greyish/bluish respectively, the latter referring to the greyish colour of its bark, and foliage (Boomsma, 1972). C. glauca is commonly known as Australian

Chapter One: General Introduction and Literature Review

desert lime (Swingle & Reece, 1967; Mabberley, 1998), wild lime, native lime, lime bush, desert lemon, native kumquat or desert kumquat (Boomsma, 1972; Elliot &

Jones, 1982; Cribb & Cribb, 1987; Low, 1991; Mabberley, 1998).

1.4.2 Distribution

The genus Citrus is generally recognised as being native to Southeast Asia,

China and Australia before it was widely distributed to other regions in the world

(Webber, 1967 and Calabrese, 1962 cited in Jessop & Toelken, 1986; Nicolosi et al.,

2000; Mabberley, 2004). Australia is acknowledged as the continent with the highest number of native Citrus species. Some of these native Citrus species are now valued as commodities for the bush food industry and as breeding lines for hybridisation with crop plants (Mabberley, 2004).

Citrus glauca is an Australian xerophyte native citrus (Swingle & Reece, 1967).

It is widely spread in drier forest (Cribb & Cribb, 1987), heavy clay soil (Elliot &

Jones, 1982; Low, 1991), semi arid or arid land region, and dry woodland (Elliot &

Jones, 1982). The distribution of this plant ranges from South-eastern Queensland,

South Australia and New South Wales (Swingle & Reece, 1967; Boomsma, 1972).

Based on the Australian Virtual Herbarium there are 283 records of C. glauca in

Queensland, New South Wales and South Australia (Figure 1.3) (AVH, 2011b).

Chapter One: General Introduction and Literature Review

Figure 1.3 Distribution of C. glauca in Australia (AVH, 2011b).

1.4.3 Morphology

Citrus glauca is a medium to tall shrub or small tree with numerous ascending branches (Figure 1.4A) (Boomsma, 1972). Thorns cover juvenile plants but are reduced or completely absent on adult plants (Swingle & Reece, 1967; Elliot & Jones, 1982;

Cribb & Cribb, 1987; Low, 1991). The leaves of C. glauca are grey green, thick and slender, 3-10 mm wide and several times longer with broader tips. They are also dotted with numerous oil glands and covered with fine greyish hair (Figure 1.4B) (Swingle &

Reece, 1967; Boomsma, 1972; Elliot & Jones, 1982; Jessop & Toelken, 1986; Cribb &

Cribb, 1987; Low, 1991). Fruits are small, 1-2 cm in diameter, rounded, and yellow- green or lemon coloured (Figure 1.4C) (Swingle & Reece, 1967; Boomsma, 1972;

Elliot & Jones, 1982; Jessop & Toelken, 1986; Cribb & Cribb, 1987; Low, 1991).

Chapter One: General Introduction and Literature Review

Seeds of C. glauca are mono-embryonic and protected by a rough testa (Swingle &

Reece, 1967). The sweet scented flowers of C. glauca bloom in early spring

(Boomsma, 1972; Low, 1991). The flower has 3-5 white-greenish petals, a short pistil, and 3 to 4 times as many stamens as petals (Figure 1.4D). Flowers may be single or form a group of 2-3 (Swingle & Reece, 1967; Boomsma, 1972; Elliot & Jones, 1982;

Jessop & Toelken, 1986; Cribb & Cribb, 1987; Low, 1991).

A. B.

Figure 1.4 Morphology of C. glauca habit (A), leaves (B), flower (C), and fruit (D).

Chapter One: General Introduction and Literature Review

1.4.4 Cultural value

Citrus glauca is listed as one of 14 important edible plants in the Australian native plant industry (Ahmed & Johnson, 2000). C. glauca has very good resistance to drought and pathogens, tolerance to salt, boron, hot-strong wind and the ability to survive in very hot (up to 45oC) and cold (-2 to -4oC) temperatures (Tworney et al.,

2009). These characteristics make this plant a good rootstock for grafting (Swingle &

Reece, 1967; Tworney et al., 2009). Its edible fruit is a perfect substitute for lemon or lime, having an even more intense flavour than either fruit, and higher levels of vitamin

C than orange (Tworney et al, 2009). It can be consumed raw or used as a food ingredient, e.g., drink, conserves, and marmalade (Cribb & Cribb, 1987; Low, 1991). C. glauca is also used in an Australian manufactured skin cleanser (Lawrence, 2002 cited in Mabberley, 2004). Recently, the retail selling price for this fruit reached $25-28/kg

(Tworney, et al., 2009).

1.5 The limitation of production and cultivation

One of the biggest problems in the Australian native food industry is the fact that most of the stocks in the markets, including C. glauca and C. mitchellii, are still harvested from wild trees. This practice could not only be a problem for fulfilling market demand, but also lead to ecological problems in Australia. Wild harvesting will reduce the reproduction and survival ability of harvested plants in the natural

Chapter One: General Introduction and Literature Review

population. It will also reduce food supplies for wild fauna. Moreover, there is a possibility that the presence of harvesters could impact other sensitive or threatened species around the harvested plants (CSIRO, 1996a cited in Berkinshaw, 1999).

In Australia, C. glauca is protected by law. Based on the National Parks &

Wildlife Act 1972, C. glauca is listed as a vulnerable species in South Australia, which means it is in danger of disappearing from the wild over the next 20-50 years

(Berkinshaw, 1999; Electronic Flora of South Australia, 2011b).

Thus, there is a need to develop propagation protocols for these plants for both economic and conservation purposes. Based on Tworney et al. (2009), both C. mitchellii and C. glauca are not easy to propagate. C. mitchellii can be difficult to propagate from seed but not from cuttings, while C. glauca propagates readily from seed but is difficult from cuttings. However, C. glauca fruit is often seedless. Swingle and Reece (1967) found grafting to be a good option for propagating C. glauca.

Nevertheless, in general conventional techniques are time consuming, impractical or unable to produce desirable outcomes (Kumar & Sharma, 2005). Thus, micropropagation was considered as an alternative option to cultivate these plants.

Chapter One: General Introduction and Literature Review

1.6 Micropropagation as an alternative cultivation technique

Micropropagation is a procedure for generating new plants using tissue culture based techniques (Kane, 2005). Tissue culture is a technique to cultivate plant organs

(embryos, shoots, roots and flowers) or tissues (cells, callus and protoplasts) in highly controlled environments (aseptic or in vitro) (George, 2008; Hartmann et al., 2011).

This propagating system is normally used to propagate plants that are slow or difficult to propagate by conventional techniques (Kane, 2005; Hartmann et al., 2011).

However, the expanded utilisation of this technique has also been very useful in producing disease or pathogen-free stocks (Flavell et al., 1986; Kane, 2005), rapid and industrial scale multiplication of a high quality plant, long term storage of germplasm, selecting mutants of either spontaneous or induced mutation, root initiation of recalcitrant plants, recovering hybrids from incompatible species and producing haploid plants (Taji et al., 2002).

1.6.1 Micropropagation Procedures

Hartmann et al. (2011) divides the micropropagation procedure into four stages

(I-IV). However, Debergh and Maene (1981) (cited in 2005; George & Debergh, 2008) recognise the importance of a preliminary stage to ensure that the plant tissues or organs are free of contamination before being planted onto sterile media. Therefore, they included another stage (stage 0) to the procedural steps. As a result, there are five stages (0-IV) in the micropropagation protocol (Kane, 2005; George & Debergh, 2008),

Chapter One: General Introduction and Literature Review

i.e. mother plant selection and preparation (stage 0), establishing aseptic cultures (stage

I), the production of suitable propagules (stage II), preparation for growth in the natural environment (stage III) and transfer to the natural environment (stage IV) (George &

Debergh, 2008). These stages will now be discussed in more detail.

1.6.1.1 Plant selection and preparation

The selection and preparation of the donor plant has a significant influence on the overall success of the micropropagation procedure. Debergh and Maene (1981)

(cited in Kane, 2005) state that the quality of explants, cultured material, and their ability to respond to treatments are highly influenced by the phytosanitary and physiological condition of the donor plant. George and Debergh (2008) suggested some steps to improve the donor plant quality, e.g. growing the intended plants in the greenhouse, treating them with growth regulators (if necessary) and using fungicides and/or pesticides to eliminate any potential disease and contamination issues.

1.6.1.2 Establishing aseptic culture

Establishing aseptic cultures consists of two steps: surface sterilisation and initiation. Surface sterilisation or disinfestation is described as the process to remove contaminants from the selected plant components (explants) (Hartmann et al., 2011).

Based on Taji et al. (2002) some of the aspects that need to be considered when selecting explants are age of the donor plant, physiological age of explants,

Chapter One: General Introduction and Literature Review

development stage of explants and size of explants. Selected and sterile explants are then planted onto media supplemented with various nutrients. Usually the establishment media will contain less (or no) growth regulators compared with the proliferation or multiplication media (Taji et al., 2002; Kane, 2005; Hartmann et al., 2011). Success of this stage will be achieved if adequate numbers of uncontaminated and healthy explants are produced (George & Debergh, 2008).

Methods for sterilising explants need to be carefully considered. An effective method will result in decreasing the level of contamination without damaging the explants (Hartmann et al., 2011).

Chapter One: General Introduction and Literature Review

Table 1.1 Different surface sterilisation agents for disinfesting Capparis and Citrus explants. Species Sterilisation Concentration Exposure References agents Time Capparis decidua HgCl2 0.1% 3 minutes (Tyagi & Kothari, 1997) 0.1% 3-4 minutes (Chahar et al., 2010) 0.1% 4-5 minutes (Deora & Shekhawat, 1995) 0.1% 10 minutes (Saxena et al., 2007)

mitchellii PPMTM 5% 18 hours. (Steggles, 2007)

spinosa NaOCl 0.2% 10 minutes (Chalak et al., 2003) 0.8% 5 minutes (Musallam & Duwayri, 2011) 1% 3 minutes (Al-Safadi & Elias, 2010)

Citrus aurantifolia NaOCl 1% 15 minutes (Al-Bahrany, 2002)

grandis 0.4% 25 minutes (Paudyal & Haq, 2000)

limon 1.2% 12 minutes (Kotsias & Roussos, 2001) 1.6% 20 minutes (Pérez-Tornero, Tallón & Porras, 2010) macroptera 4% 15 minutes (Miah, Islam & Hadiuzzaman, 2009)

sp and hybrid Ca(OCl)2 7% 20 minutes (Barlass & Skene, 1982)

Several chemical substances including calcium hypochlorite (Ca(OCl)2), mercuric chloride (HgCl2), sodium hypochlorite (NaOCl) and Plant Preservative

Mixture (PPMTM) have been used as surface sterilisation agents in micropropagation of

Capparis and Citrus species (Table 1.1). Generally, the utilisation of these agents

(except for PPMTM (Steggles, 2007)) were preceded with soaking explants in water with a few drops of liquid detergent (Tween 20 or 80) for a few minutes followed by a quick dip into 70% (v/v) ethanol (Deora & Shekhawat, 1995; Tyagi & Kothari, 1997;

Paudyal & Haq, 2000; Kotsias & Roussos, 2001; Al-Bahrany, 2002; Chalak et al.,

Chapter One: General Introduction and Literature Review

2003; Saxena et al., 2007; Al-Safadi & Elias, 2010; Chahar et al., 2010; Pérez-Tornero et al., 2010; Musallam & Duwayri, 2011). However, in some Citrus culture, rather than including Tween 20 in the pre-treatment solution, a few drops were directly incorporated into the sterilisation solution (Barlass & Skene, 1982; Miah, et al., 2009).

The main function of ethanol in the sterilisation procedure is to eliminate air bubbles and dissolve the epicuticular layer, while Tween functions to reduce the surface tension in order to increase the contact between explants and sterilisation agents (Pierik, 1997;

Dirr & Heuser, 2006).

Hypochlorite solutions (e.g. sodium hypochlorite (NaOCl) and calcium hypochlorite (Ca(OCl)2) are the most common chemical disinfesting agents in micropropagation studies. Many household bleach solutions contain 5.25% of NaOCl and are reported to be effective sterilisation agents at concentrations of 10-20% (v/v)

(Pierik, 1997; Dirr & Heuser, 2006; Hartmann et al., 2011) for many explants, including caper (Musallam & Duwayri, 2011), poinsettia (Castellanos et al., 2010) and some wetland monocots (Czakó et al., 2006). Ca(OCl)2 is less effective and enters the tissue more slowly than NaOCl. Hence, it is usually used at higher level (3.5-10%

(w/v)) and for sterilising NaOCl sensitive plants (Pierik, 1997).

Mercuric chloride (HgCl2) has stronger activity than NaOCl and Ca(OCl)2 and whilst historically has been commonly used today it is infrequently used. The mercury component makes it extremly toxic for plants, animals and humans. This solution needs to be carefully handled and properly disposed of (Pierik, 1997; Dirr & Heuser, 2006;

Hartmann et al., 2011). Chahar, et. al., (2010) soaked C. decidua explants in 70%

Chapter One: General Introduction and Literature Review

ethanol for 20-50 seconds followed by 0.1% (w/v) HgCl2 for 3-4 minutes, reporting that this approach eliminated contamination from explants. However, when the exposure time was increased (10 seconds for ethanol and 1 minute for HgCl2) this method was lethal.

Plant Preservative MixtureTM is a heat stable and wide spectrum anti microbial agent (Guri & Patel, 1998; Plant Cell Technology Inc., 2005). PPMTM had been reported to be significantly superior in controlling contamination on C. mitchellii explants when compared to NaOCl and Ca(OCl)2 (Steggles, 2007). Guri and Patel

(1998) claimed that PPMTM had no phytotoxic effects when incorporated into growing media at 0.05% (v/v) concentration. The suggested dosage for PPMTM is 0.05-0.2%

(v/v) in the media and 4-5% (v/v) in sterilisation solution (Plant Cell Technology Inc.,

2005). The capacity of PPMTM as an anti-microbial agent for C. mitchellii and C. glauca, in combination with an antifungal agent (e.g. mancozeb and miconazole), was studied in this thesis. Mancozeb was reported as an effective sterilisation agent in apple culture (Kolozsvári Nagy et al., 2005) while miconazole effectively controlled fungal contamination of 15 species from various families (Tynan et al., 1993).

1.6.1.3 Production of suitable propagules

This stage is mainly focused on producing outgrowths that are able to grow and develop into self sustaining plants (George & Debergh, 2008) and is also known as the multiplication stage (Taji et al., 2002). In this stage healthy explants from previous steps are planted into media supplemented with cytokinin to enhance microshoot

Chapter One: General Introduction and Literature Review

production and multiplication (Taji et al., 2002; Kane, 2005; Hartmann et al., 2011).

When micro shoots reach a suitable size, they are subcultured either to the same type of media (multiplication media) or moved to the next stage media (preparation for growth in the natural environment media) (George & Debergh, 2008; Hartmann et al., 2011).

The treatments required for shoot induction and multiplication vary between species (Pierik, 1997; Dirr & Heuser, 2006; Hartmann et al., 2011). In general, studies of Capparis and Citrus micropropagation have indicated that species from these genera favoured a high salt content media (i.e. Murashige and Skoog basal medium (MS))

(Murashige & Skoog, 1962) supplemented with various combinations of plant growth regulators (PGR) (Table 1.2).

Previous studies showed that MS basal medium was a suitable medium for shoot induction of Capparis and Citrus species. The percentage of responding explants and number of shoots developing in C. decidua cultures were highest in full strength

MS medium compared to reduced strengths of MS, B5 (Gamborg, Miller & Ojima,

1968) or WPM (Lloyd & McCown, 1980) (Deora & Shekhawat, 1995). However, studies of C. spinosa culture showed that even though there was no significant difference in the percentage response of explants between MS and modified MS (half strength nitrate and double calcium chloride and magnesium sulphate), the multiplication rate of explants on modified MS was superior to MS (Chalak et al.,

2003). Further research on the same species reported that modified MS and WPM media yielded significantly better results for shoot elongation than MS, where bud shoots showed only limited development (Musallam & Duwayri, 2011).

Chapter One: General Introduction and Literature Review

The incorporation of PGR into the media is important at this stage. PGR are chemical substances, natural or synthetic, with ability to influence plant growth and development (Gaspar et al., 1996; Gaba, 2005; McMahon et al., 2007). There are five types of commonly known PGR, including auxins, cytokinins, gibberellins, ethylene, and abscisic acid (Gaspar et al., 1996; Pierik, 1997; Nayak et al., 2002; Razdan, 2003;

Gaba, 2005; McMahon et al., 2007; Hartmann et al., 2011).

Studies of Capparis and Citrus micropropagation indicated that benzyladenine

(BA) (cytokinin) was the best PGR for shoot induction and multiplication. BA occasionally gave better results when combined with other PGRs, although this was not essential. Al Bahrany (2002) observed that the effect of NAA on shoot formation in C. aurantifolia was dependent on BA level in the medium. Paudyal & Haq (2000) also observed that auxin had no significant effect on C. grandis micropropagation. In C. decidua, NAA was only included in the initiation medium and not into the multiplication medium (Tyagi et al., 2010). These results confirm the ability of cytokinin to initiate cell division, promote axillary shoot formation and induce adventitious shoot differentiation in tissue culture (Pierik, 1997; Razdan, 2003).

Chapter One: General Introduction and Literature Review

Table 1.2 Summary of the best treatments for shoot initiation and multiplication from published studies on Capparis and Citrus species micropropagation.

Plant Growth Regulator Shoots per explant/somatic Species Explant Medium Cytokinin and Other Concentration Concentration Reference embryo Auxin PGR (mg/L) (mg/L) Capparis decidua N 20 MS BA 2.0 NAA 0.5 (Tyagi et al., 2010) N 7.2 MS BA 5.0 NAA 0.1 (Deora & Shekhawat, 1995) N, S 3-4 MS BA 5.0 - - (Chahar et al., 2010) S 8.5 MS BA 7 - - (Saxena et al., 2007)

C. mitchellii N 1.5 MS BA 5 IAA 0.05 (Steggles, 2007)

C. spinosa N 5.5 MS ZR+GA3 2+ 0.1 NAA 1 (Al-Safadi & Elias, 2010) Modified N 5.43 BA+GA 1.5+0.1 IBA 0.05 (Chalak et al., 2003) MS 3 N, S 45.7 MS TDZ 1 - - (Caglar et al., 2005) N, S 25-40/month MS BAP/Kinetin 0.5-2 - - (Safrazbekyan et al., 1990)

N 4.6 WPM BA/Kinetin +GA3 1.2/0.8+0.1 IBA 0.05 (Musallam & Duwayri, 2011) Citrus aurantifolia N 9 MS BA+ Kinetin 2.0+1 NAA 1 (Al-Bahrany, 2002) C. grandis S 5.2 MS BA 0.4 - - (Paudyal & Haq, 2000) C. jambiri N 83% MS BA 3 - - (Ali & Mirza, 2006) C. limon N 12-15 MS BA 0.05 - - (Rathore et al., 2004) N, S 2.5 DKW BA+ ABA 2+1 - - (Kotsias & Roussos, 2001) N Varied depend on cultivar DKW BA+GA3 2+2 - - (Pérez-Tornero et al., 2010) C. macroptera N, S 3.78 MS BA 1 - - (Miah et al., 2009) Abbreviation: N = nodal shoot; S = shoot tip; L = leaf segment; DKW = Driver Kuniyuki for Walnut; MS = Murashige and Skoog; WPM = Woody Plant Medium; BA = benzyladenine; GA3 = gibberellic acid; ZR = zeatin riboside; TDZ = thidiazuron; ABA = Abscisic acid; NAA = α-naphthalene acetic acid; IBA = Indole 3-butyric acid; IAA = Indole 3-acetic acid

Chapter One: General Introduction and Literature Review

Kinetin is a synthetic, adenine-derivative cytokinin. In micropropagating C. spinosa, kinetin was found to be the best treatment for shoot initiation and elongation.

However the number of shoots initiated and shoot length were not significantly different from that with BA (Safrazbekyan et al., 1990; Musallam & Duwayri, 2011).

The same results was also observed in Prosopsis sineraria, a desert tree, where both kinetin and BA at 5mg/L were the best treatments for bud breaking, shoot initiation and elongation when compared to the combination of cytokinin-auxin treatments (Kumar &

Singh, 2009). In C. aurantifolia, kinetin triggered the best response when it was combined with BA (Al-Bahrany, 2002).

Zeatin riboside (ZR) is a natural form of cytokinin (Gaspar et al., 1996). It was reported for its ability to stimulate production of protocorm like bodies (PLB) in

Cymbidium aloifolium (Orchidaceae) (Nayak et al., 2002) and shoot regeneration of C. spinosa (Al-Safadi & Elias, 2010).

Thidiazuron (TDZ) is usually described as synthetic chemical substance with cytokinin-type activity. Unlike the classic type of cytokinins which are adenine- derivative substances, TDZ is a derivative of diphenyl urea (Razdan, 2003).

Thidiazuron can be more active than an adenine-derived cytokinin. In micropropagation of Hydrastis canadensis, an endangered medicinal plant in Eastern North America,

TDZ was reported to be significantly more effective in inducing shoots than the combination of BA and NAA (He et al., 2007). TDZ was also required for regeneration of Artemisia judaica, an Egyptian medicinal plant (Liu et al., 2003) and Saintpaulia

Chapter One: General Introduction and Literature Review

ionantha, African Violet (Mithila et al., 2003). However, TDZ is often reported to inhibit elongation and cause hyperhydricity in micropropagation (Gaspar et al., 1996).

Gibberellic acid (GA3) and abscisic acid (ABA) are less commonly used in micropropagation studies (Razdan, 2003; Gaba, 2005). In some cases GA3 and ABA are required either to enhance or inhibit growth. Generally, GA3 is used to stimulate internode elongation while ABA is mainly known for its role in somatic embryo maturation (Razdan, 2003; Gaba, 2005).

1.6.1.4 Preparation for growth in the natural environment

The preparation for growth in the natural environment stage is simply known as root formation (Hartmann et al., 2011) or pre-transplant stage (Taji, et al., 2002; Kane,

2005). This stage is necessary to ensure that propagules are able to take water and nutrients from the medium to photosynthesis and produce their own carbohydrates rather than depending on the carbohydrates in the culture media. In the mean time, at this stage the propagules’ in vitro developed tissue may be modified to improve photosynthesis and reduce water loss in order to survive the ex vitro condition e.g. glass house condition (George & Debergh, 2008). The rooting procedure can be carried out in the in vitro or ex vitro environment (Kane, 2005; George & Debergh, 2008;

Hartmann et al., 2011). In the in vitro technique, microshoots are harvested and planted into root-inducing media that usually has a low level of cytokinin and a higher level of auxin. The basal media is usually reduced to half of the previous media concentration.

In the ex vitro techniques, microshoots are exposed to auxin and then inserted into soil-

Chapter One: General Introduction and Literature Review

less greenhouse rooting media. Some propagators tend to choose the ex vitro rooting technique rather than the in vitro technique due to the morphological difference between in vitro and ex vitro roots. Ex vitro roots usually have a better formed vascular system than in vitro developed roots which allows the ex vitro roots a better chance to survive when transferred to the normal environment (Hartmann et al., 2011).

Table 1.3 Summary of the best treatments for rooting from published studies of Capparis and Citrus species micropropagation. Rooting Concentration Exposure Species Media auxin References percentage (mg/L) Time Capparis decidua 37% MS IBA+NAA† 1.0+1.0 30 days (Chahar et al., 2010) 60-70% ½MS IBA 100.0 4 hours (Deora & Shekhawat, 1995) 86% MS IBA 1.0 nm (Saxena et al., 2007) spinosa 87% ½MS IAA 100.0 4 hours (Chalak et al., 2003) 80% ½MS IAA 5.0 6 weeks (Musallam & Duwayri, 2011) Citrus aurantifolia nm MS NAA or 2.0 10 weeks (Al-Bahrany, 2002) NAA+IBA 0.5+1.0 56% MS IAA 1.0 8 weeks (Al-Khayri & Al- Bahrany, 2001) grandis 77% ½MS NAA 1.0 nm (Paudyal & Haq, 2000) jambiri 70% MS NAA 0.5 nm (Ali & Mirza, 2006) limon 80% DKW IBA 1,000.0 5 seconds (Kotsias & Roussos, 2001) 94.8% DKW IBA 3.0 4 weeks (Pérez-Tornero, Tallón & Porras, 2010)

† pre-treated with 25 mg/L IBA in MS liquid for 6 days. Abbreviation: NAA = α-naphthalene acetic acid; IBA = Indole 3-butyric acid; IAA = Indole 3-acetic acid; nm: not mentioned

Chapter One: General Introduction and Literature Review

Capparis and Citrus species are capable of producing roots when planted onto media supplemented with auxins (IBA, NAA and/or IAA) (Table 1.3). Even though reducing salt level was recommended, Capparis and Citrus species seemed able to produce roots on both full and half strength media. Saxena et al. (2007) reported that when planting onto MS and ½MS media without any auxin C. decidua showed the same level of rooting percentage. Hence, auxin seemed to have more influence than the salt level in basal medium for promoting rooting in Capparis and Citrus.

Indole butyric acid, NAA and IAA have been reported as successfully initiating rooting in Capparis and Citrus species. The highest rooting percentage for woody plants such as C. decidua (Deora & Shekhawat, 1995; Saxena et al., 2007; Chahar et al., 2010) and Citrus (Paudyal & Haq, 2000; Kotsias & Roussos, 2001; Al-Bahrany,

2002; Ali & Mirza, 2006; Pérez-Tornero, Tallón & Porras, 2010) was usually obtained with IBA or NAA treatment. On the other hand, C. spinosa, which is usually described as a herbaceous plant, produced a high percentage of rooted plants when treated with

IAA (Chalak et al., 2003; Musallam & Duwayri, 2011). Generally, an increased level of auxin will be followed by an increased percentage of rooted plants. However when auxin levels are too high rooting percentages can be suppressed (Deora & Shekhawat,

1995; Paudyal & Haq, 2000; Al-Khayri & Al-Bahrany, 2001; Musallam & Duwayri,

2011). Therefore, in this thesis the capability of different levels and exposure times of

NAA and IBA in promoting rooting in C. mitchellii and C. glauca were evaluated.

Chapter One: General Introduction and Literature Review

1.6.1.5 Transferring to ex vitro conditions

The stage of transferring cultured plants from in vitro to ex vitro conditions may result in a significant loss of cultured plants (George & Debergh, 2008). Basically, at this stage the humidity around the cultured plants will be gradually reduced while inversely, light intensity will be gradually increased. Hence, plants will evenly adapt to the normal environment. Furthermore in this stage, cultured plants are also prepared for the transition from the heterotrophic to the photoautotrophic state (Kane, 2005; George

& Debergh, 2008; Hartmann et al., 2011). Earlier studies showed that the survival level of Capparis and Citrus varied between 40% (Chalak et al., 2003) to almost 100%

(Pérez-Tornero et al., 2010). The survival rate was also affected by the explant’s appearance and age. For C. spinosa, only shoots longer than 3 cm survived the acclimatisation stage (Chalak et al., 2003), while in C. macroptera culture, survival rate of older shoots was better than younger ones (Miah et al., 2009).

Even though theoretically almost all plants can be micropropagated by following these steps, the detailed procedures at each stage can be different for different species, especially for stages I-III (George & Debergh, 2008). Not all species will react the same way to a particular procedure, and hence, it is important to find the right protocol for each plant to ensure that optimal results are achieved.

Chapter One: General Introduction and Literature Review

1.7 Aim/Objective of the project

The aim of this research was to establish an efficient protocol to micropropagate

C. mitchellii and C. glauca. The protocol consisted of testing various sterilisation, shoot initiation, shoot multiplication, rooting and acclimatisation/hardening procedures for both these plants.

The specific objectives of this research were:

a. to identify the best sterilisation procedure for micropropagating C.

mitchellii and C. glauca

b. to identify the optimal basal media and PGRs for shoot initiation, shoot

multiplication-elongation and root production of C. mitchellii and C.

glauca explants, and

c. to develop acclimatisation methods for C. mitchellii and C. glauca.

Chapter Two: Explant Sterilisation

CHAPTER TWO

EXPLANT STERILISATION

2.1 Introduction

Micropropagation is a technique to propagate plants in axenic (aseptic or sterile) conditions (Hartmann et al., 2011). In this technique, selected tissues (explants) are planted onto a nutrient-rich medium, which promotes explant growth but also provides an ideal growth medium for microorganisms. Hence, explants need to be decontaminated before culturing, as micro-organisms can grow exponentially faster, resulting in explants being engulfed by the contaminants. Therefore, explant sterilisation procedures are crucial to ensure successful micropropagation (Bunn & Tan,

2002; Razdan, 2003; Iliev et al., 2010; Hartmann et al., 2011).

The ability of three sterilisation agents, PPMTM, mancozeb and miconazole, to control microbial contamination in C. mitchellii and C. glauca culture were investigated in this experiment. PPMTM is a heat-stable biocide claiming to effectively prevent or decrease the level of microbial contamination (Plant Cell Technology Inc., 2005) with no or minimum phytotoxic effect when utilised at recommended concentrations (Guri

& Patel, 1998; Niedz, 1998; Plant Cell Technology Inc., 2005). Mancozeb and

Chapter Two: Explant Sterilisation

miconazole are antifungal agents that reportedly decrease fungal contamination in plant tissue culture (Tynan et al., 1993; Kolozsvári Nagy, Sule & Sampaio, 2005).

2.2 Material and methods

2.2.1 General preparation and sterilisation technique

2.2.1.1 Equipment preparation

All equipment was sterilised before use. Heat resistant equipment was sterilised by dry or moist heat (Dodds & Roberts, 1995). Dry heat was used to sterilise stainless steel and glass. These items were wrapped in aluminium foil and placed in an oven at

180oC for a minimum of 3 hours. Moist heat was used for all autoclaveable materials.

In this method, items were wrapped in aluminium foil or put into a sealed autoclave bag and autoclaved for 20 minutes at 121oC. After autoclaving, paper products were briefly placed in an oven (150oC) to evaporate left over moisture. Reverse osmosis (RO) water was sterilized in Schott bottles with loose lids which were tightened after autoclaving.

2.2.1.2 Media preparation

Murashige & Skoog (1962) medium without any plant growth regulators (PGR) was used as basal medium (MS0). To prepare 1 L MS0, 4.43 g of Murashige & Skoog

Basal Medium with Vitamins and 20 g of sucrose were dissolved in 500 mL RO water

Chapter Two: Explant Sterilisation

in a 1 L beaker. The pH of the medium was adjusted to 5.7 using 1N sodium hydroxide

(NaOH) or hydrochloric acid (HCl). Gelling agent solution was prepared separately by adding 3.76 g gelrite to 500 mL warm RO water while stirring. Once fully dissolved, the gelling solution was added to the medium. The combined solution was heated to boiling point in a microwave oven and poured into a 1 L Schott bottle with a loose lid and autoclaved for 20 minutes at 121oC.

Sterilisation agents, PPMTM, mancozeb plus and miconazole, were added to media after autoclaving. To prepare 1 L of MS0 medium with 0.2% (v/v) PPMTM, 2 mL

PPMTM was added. However, mancozeb plus and miconazole were in powder form, thus both of these agents needed to be dissolved before they were added to the medium.

Mancozeb plus was dissolved by using sterile RO water while miconazole had to be dissolved with dimethyl sulfoxide (DMSO) in the fume hood. The amount of sterile RO water and DMSO used was kept as low as possible.

The process of adding the sterilisation agents to the medium was executed in a laminar flow hood to limit air contamination. The medium, once supplemented with a sterilisation agent, was dispensed into 30 mL sterile test tubes (10 mL per tube). After cooling, test tubes were refrigerated at 5±2oC until required.

2.2.1.3 Explant preparation

Newly grown nodal and apical shoots were used as explants in this experiment

(Figure 2.1). Shoots were harvested in September 2009 from a C. mitchellii tree located

Chapter Two: Explant Sterilisation

in the Botanic Gardens of Adelaide (BGA bed no. 4.1, G860746) and a C. glauca tree in the Waite Arboretum, Urrbrae (#163 (J11)). Both of these specimens were chosen for their abundance of healthy shoots. The harvested shoots were wrapped in damp paper and stored in a fridge at 5±2oC until required. The storage time was not more than 2 days.

A. B.

Figure 2.1 Newly grown stems of C. mitchellii (A) and C. glauca (B) used for explants.

Plant stems were cleaned of leaves and trimmed to approximately 1.5-2 cm lengths containing 1 or 2 nodal buds. These explants were rinsed under running tap water for 1 hour and transferred to sterilisation treatments.

2.2.1.4 Incubation condition

Cultures were maintained in a culture room at 25±2oC with 16 hour photoperiod, illuminated by cool white fluorescent light tubes.

Chapter Two: Explant Sterilisation

2.2.2 Effects of different sterilisation agents on explant surface contamination

level

Twelve treatments were studied in this experiment. The treatments combined pre-soaking with two levels of PPMTM with six media incorporating various levels of sterilisation agents (Table 2.1)

Table 2.1 Experimental design for shoot sterilisation experiment. Solid phase Liquid phase (MS medium incorporated with sterilisation agents) (MS medium) Sterilisation agents Concentration 2% (v/v) PPMTM None - PPMTM 0.2% (v/v) Miconazole 10 mg/L 20 mg/L Mancozeb 50 mg/L 100 mg/L 5% (v/v) PPMTM None - PPMTM 0.2% (v/v) Miconazole 10 mg/L 20 mg/L Mancozeb 50 mg/L 100 mg/L

Washed nodal sections were agitated in MS liquid media (pH was not adjusted) with 2 or 5% (v/v) PPMTM, for 4 hours. The liquid phase treatment was not necessarily conducted under aseptic conditions. After this, nodal shoots were planted, by inserting the shoots vertically without rinsing, directly onto the solid MS medium containing different levels of PPMTM, miconazole and mancozeb. This step was undertaken in a

Chapter Two: Explant Sterilisation

laminar flow hood to eliminate contamination. This experiment was monitored for 4 weeks. Each of twelve treatments (Table 2.1) was applied to 25 samples. Sample contamination was recorded after 4 weeks. Results were analysed and the best treatment (low contamination and healthy shoots) was then used as a sterilisation procedure to prepare explants for the shoot initiation experiment.

2.2.3 Statistical analysis

Data, (number of contaminants per treatment) were analysed using Statistical

Package for the Social Science software (SPSS Version 17 for Windows). The log linear analysis was used to evaluate significant differences between liquid and solid phases in controlling contamination level (Field, 2009).

2.3 Results

2.3.1 Capparis mitchellii

Two microbial contaminants (bacterial and fungal), were found during C. mitchellii establishment. Contaminants grew on both the explants and the medium. The three-way log linear analysis (Field, 2009) produced a final model that retained the solid phase × contamination frequency interaction with χ2(5)=28.533, P < 0.0001. This result indicated that the contamination frequency was only affected by the sterilisation

Chapter Two: Explant Sterilisation

agents in the solid media. There was no significant interaction between liquid phase treatment and contamination frequency (χ2(1)=1.887, P = 0.170) or between the liquid phase and solid phase treatment (χ2(5) = 0.199, P = 0.999). Further chi square tests of solid phase × contamination frequency confirmed the previous result (Fisher exact test value = 24.870, P < 0.0001). The contamination level in C. mitchellii cultures after 4 weeks was quite low. The highest contamination level observed was 36% in the treatment combining 2% (v/v) PPMTM and MS0 without any sterilisation agents and no contamination was observed on explants planted onto media with 0.2% (v/v) PPMTM

(Figure 2.2).

Chapter Two: Explant Sterilisation

80%

70%

60%

50%

40% level 30%

20% 10%

Shoot and Initiated ofContamination Percentage MS0 MS+0.2% MS+10mg/l MS+20mg/l MS+50mg/l MS+100mg/l PPM miconazole miconazole mancozeb mancozeb Solid Media Treatment

Liquid Phase 2% PPM - Contamination Level Liquid Phase 5% PPM - Contamination Level Liquid Phase 2% PPM - Initiated Shoot Level Liquid Phase 5% PPM - Initiated Shoot Level

Figure 2.2 Effects of different sterilisation treatments on contamination and initiated shoot level of Capparis mitchellii culture.

2.3.2 Citrus glauca

Fungi and bacteria were both found in C. glauca initiation cultures. The three- way log linear analysis test produced a final model that retained the liquid phase × contamination frequency and solid phase × contamination frequency. The liquid phase

× contamination frequency was significant, χ2 (1) = 9.713 P < 0.002. This interaction indicated that the contamination frequency was different between 2% (v/v) and 5%

Chapter Two: Explant Sterilisation

(v/v) PPMTM. The solid phase × contamination frequency interaction was also significant, χ2 (5) = 35.959, P < 0.0001, showing that the contamination frequency was significantly influenced by the sterilisation agents in media. The lowest level of contamination (4%) was observed in the media incorporated with 0.2% (v/v) PPMTM that had been pre-treated with either 2% or 5% (v/v) PPMTM (Figure 2.3). However, phytotoxic effects (discoloured tissues) were recorded for explants cultured on media supplemented with PPMTM and miconazole (Figure 2.4).

80%

70%

60%

50%

40%

30%

20%

10% Percentage of Contamination (%) ofContamination Percentage

0% MS0 MS+0.2% PPM MS+10mg/l MS+20mg/l MS+50mg/l MS+100mg/l miconazole miconazole mancozeb mancozeb Solid Media Treatment

Liquid Phase 2% PPM Liquid Phase 5% PPM

Figure 2.3 Effects of different sterilisation treatments on contamination level of Citrus glauca.

Chapter Two: Explant Sterilisation

hdc

sdc

2.4 Discussion

Bacteria and fungi were two types of microbial contaminants observed in this experiment. These microbes are common contaminating microorganisms in micropropagation (Bunn & Tan, 2002). The contaminants in this experiment started from the explants and spread to the media, indicating that the explants were the source of contamination.

Chapter Two: Explant Sterilisation

Data showed that 5% (v/v) PPMTM treatment was significantly more effective in controlling the contamination rate for C. glauca establishment than 2% (v/v) PPMTM

(Figure 2.3). Incorporating sterilisation agents into the solid medium produced a more significant reduction in contamination. PPMTM was more effective than miconazole and mancozeb. Adding PPMTM to the medium decreased the contamination levels to 0% for

C. mitchellii and 16% for C. glauca.

Phytotoxic activity was however observed on C. glauca cultures. Tissue became discoloured when cultured onto media supplemented with 0.2% (v/v) PPMTM and miconazole , indicating that this species was sensitive to these chemical substances.

Some previous studies reported PPMTM phytotoxicity in species including sweet orange

(Citrus sinensis) (Niedz, 1998 cited in Compton & Koch, 2001; Bunn & Tan, 2002), melon (Cucumis melo), petunia (Petunia hybrid) (Compton & Koch, 2001) and apple

(Kolozsvári Nagy et al., 2005). Miconazole was also reported to slightly reduce growth in some species including carnation, chrysanthemum, endive, lettuce, kumara, potato

(Tynan et al., 1993) and apple (Kolozsvári Nagy et al., 2005). Consequently, instead of

PPMTM, 100 mg/L mancozeb was chosen as the sterilisation agent for subsequent

Citrus glauca experiments. Results showed that 100 mg/L mancozeb in the medium was not only capable of inhibiting contamination, but also supported healthy shoot initiation from explants (Figure 2.4)

Plant Preservative Mixture did not have any phytotoxic effect on C. mitchellii tissue. However, even though statistically insignificant, further observation of the explant condition showed that media supplemented with 50 mg/L mancozeb in

Chapter Two: Explant Sterilisation

combination with 5% PPMTM as liquid treatment was able to support shoot induction better than other treatments (Figure 2.2). Therefore, 50 mg/L mancozeb was chosen as the sterilisation agent for subsequent C. mitchellii experiments.

Chapter Three: Shoot Initiation

CHAPTER THREE

Shoot Initiation

3.1 Introduction

In micropropagation, the shoot initiation phase aims to induce in vitro shoots from explants. In nodal shoot cultures, initiation is carried out by planting disinfected single or multiple nodes into cytokinin-enriched medium for at least 4 weeks (Iliev et al., 2010). Successful culture establishment is indicated by the production of stable micro shoots a few weeks after planting (George & Debergh, 2008; Hartmann et al.,

2011).

Direct shoot organogenesis of C. mitchellii from nodal shoots was previously reported on MS basal medium supplemented with 5.0 mg/L BA and 0.1 mg/L IAA

(Steggles, 2007). However, the study also found that the quality of micro shoots was difficult to maintain, indicating that the medium was not optimal for C. mitchellii establishment.

To the author’s knowledge, somatic embryogenesis is the only reported protocol for micropropagating C. glauca (Jumin & Nito, 1996). However, many citrus have been effectively propagated through nodal shoot culture (Paudyal & Haq, 2000; Kotsias &

Roussos, 2001; Al-Bahrany, 2002; Rathore et al., 2004; Ali & Mirza, 2006; Miah et al.,

Chapter Three: Shoot Initiation

2009). Therefore, it is feasible to assume that under the right conditions C. glauca would also be amenable to this approach.

Choosing an appropriate medium is essential to ensure successful culture.

Hence, the capability of different media to initiate in vitro shoots of C. mitchellii and C. glauca was studied in this chapter.

3.2 Material and Methods

The initiation experiment was the most challenging experiment to execute in the whole project. Various obstacles were found and needed to be solved in order to ensure that the initiation experiments were able to produce an acceptable number of in vitro shoots for multiplication and elongation experiments. Trials were performed over two spring seasons (2009 and 2010) before acceptable results were observed.

The initial proposal was to test full strength and half strength MS (Murashige &

Skoog, 1962) and WPM (Lloyd & McCown, 1980) basal media with and without BA supplementation. The level of BA tested was based on preliminary research (data not shown). The samples were sterilised using the optimal procedure from the sterilisation experiments. However, after 9 weeks most samples (especially in C. mitchellii) had to be discarded due to heavy fungal contamination. Harvesting time may contribute to this

Chapter Three: Shoot Initiation

issue as external contamination may vary depending on the environmental condition

(George, 2008). Furthermore, hyperhydricity was also observed on most in vitro shoots.

Hyperhydricity (vitrification) is an abnormal morphological and physiological condition of in vitro shoots characterised by glassy, translucent or water soaked appearance (Loreti & Pasqualetto, 1987; Pierik, 1997; Razdan, 2003; George & Davies,

2008; Ziv & Chen, 2008; Hartmann et al., 2011). Hyperhydric shoots usually lose their reproductive ability and show reduced survival when transferred to the external environment (Loreti & Pasqualetto, 1987; Taji, Kumar & Lakshmanan, 2002; George

& Davies, 2008). Hence, hyperhydricity is a very serious problem in plant tissue culture

(Loreti & Pasqualetto, 1987).

Hyperhydricity is triggered by high humidity, high organic and inorganic media components, high level of growth regulators, low light intensity and low level of gelling agent. Hyperhydricity can be reduced by venting cultures, raising the level of gelling agents, modifying the levels of growth regulators, organic and inorganic components, changing the source of gelling agent and adding antivitrification agents (Debergh,

1983; Ziv, 1991; Pierik, 1997; George & Davies, 2008).

In spring 2010, the media used for initiation experiments was modified in order to reduce hyperhydricity and fungal contamination. The modification included raising the sucrose level from 20 g/l to 30 g/l, using agar instead of gelrite as the gelling agent, using culture vessels with vented lids and increasing the mancozeb level in C. mitchellii culture media from 50 mg/L to 100 mg/L.

Chapter Three: Shoot Initiation

These modifications significantly reduced the level of hyperhydricity and fungal contamination. In contrast, the level of bacterial contamination increased. Eight weeks after planting, most explants were engulfed by bacteria. It has been previously reported that bacterial contamination can remain undetected for some time due to the media being used proving to support bacterial growth. However, if the conditions are optimised, bacteria will multiply rapidly (Cooke et al., 1992 and Leifert, 2000 cited inWojtania, Pulawska & Gabryszewska, 2005). The use of antibiotics was considered as a potential solution to bacterial contamination. Consequently, an additional experiment was conducted to study the sensitivity of plant tissue and microbial contamination to an antibiotic.

Due to time constraints, it was decided to examine only one type of antibiotic.

Streptomycin has reportedly controlled systemic infection with low phytotoxicity

(Razdan, 2003). In this experiment 4 levels of streptomycin (0 mg/L, 0.0625 mg/L,

0.125 mg/L and 0.250 mg/L) were incorporated into MS media containing 30 g/L sucrose, 7 g/L agar and 100 mg/L mancozeb. Each treatment was applied to 25 explants. Levels of contamination and tissue necrosis were recorded after 4 weeks. The lowest (16%) and highest (56%) level of contamination were observed on media with

0.250 mg/L and 0 mg/L streptomycin respectively. However the highest level of necrotic tissue (27%) was also observed on media with 0.250 mg/L streptomycin.

Considering the necessity to reduce the contamination level to ensure the continuation of this project, 0.250 mg/L streptomycin was included in all media for the remainder of these trials, as under this treatment more than 70% of cultures were healthy and free of contamination.

Chapter Three: Shoot Initiation

3.2.1 Preparation

Tools and materials were prepared according to the procedures described in

Section 2.2.1 with some modification to sterilisation procedures and media preparation as mentioned in section 3.2. In this experiment, sterilisation was carried out by soaking the explants in 5%PPMTM solution, followed by planting the explants, vertically and without rinsing, onto medium supplemented with 100 mg/L mancozeb and 0.250mg/L streptomycin. Agar was used as the gelling agent instead of gelrite. Agar is easily dissolved in hot water, so it was added directly to the medium without need to be separately dissolved in RO water. All media included 30 g/L sucrose, 7 g/L agar, 0.25 g/L streptomycin, 0.1 g/L mancozeb and BA (3 mg/L for C. mitchellii, or 0.3 mg/L for

C. glauca).

To prepare 100 mL of 1 mg/mL stock solution, 100 mg of growth regulator was dissolved with 2-5 mL solvent solution (1N HCl) and brought to volume by adding RO water. Stock solutions were refrigerated at 5±2oC until required.

3.2.2 Effects of different basal media on shoot initiation

In this experiment, full and half strength MS and WPM basal media were tested for microshoot initiation of C. mitchellii and C. glauca nodal explants. The planting process took 3 days. One hundred nodal shoots were planted each day across the 4 media treatments. In total 300 nodal shoots were cultured (75 nodal shoots per treatment).

Chapter Three: Shoot Initiation

Cultures were incubated in a culture room (as described in Section 2.2.1.4) and monitored weekly for any sign of contamination. Cultures with fungal contamination were discarded while cultures with bacterial contamination were kept as long as no signs of tissue deterioration were observed. Cultures were transferred onto fresh media every 3 weeks. Frequency of cultures with microshoots, number of microshoots per culture and maximum length of microshoots were recorded at the end of the experiment. The experiment was conducted over 9 weeks.

3.2.3 Experimental design and statistical analyses

A Randomised Block Design with 4 treatments (media), 3 blocks (planting days) and 25 explants per treatment per block was used in this experiment. Due to unequal sample number per treatment per block which was caused by contamination, the Gabriel test was chosen as a post hoc test as this test was specifically designed to analyse data with slightly unequal sample size (Field, 2009).

Frequency of cultures with microshoots was analysed by using crosstab model while number and length of microshoots were analysed using General Linear Model

Univariate Analysis in Statistical Package for the Social Science software (SPSS

Version 17 for Windows).

Chapter Three: Shoot Initiation

3.3 Results

3.3.1 Capparis mitchellii

Among the four media tested for C. mitchellii establishment (Table 3.1), there were no significant differences between WPM, MS and ½MS in promoting shoot initiation. However, the highest percentage (75.5%), number of microshoots per explant

(2.8) and the longest shoot length (3.3 mm) were observed on MS medium. Half strength WPM was the least suitable medium for C. mitchellii initiation, averaging only

13.3% explants producing 0.9 microshoots per explant with an average shoot length of

1.1 mm.

Table 3.1 Effect of different basal media supplemented with 3mg/L BA on shoot initiation of Capparis mitchellii. Number of Number of shoots Basal Medium explants with Shoot length (mm) per explant shoots (%) WPM 63.0±8.3 a* 2.3±0.3 ab 2.0±0.2 ab 1/2WPM 13.3±6.9 b 0.9±0.5 b 1.1±0.6 b MS 75.5±4.5 a 2.8±0.1 a 3.3±0.6 a 1/2MS 65.6±3.7 a 2.6±0.2 a 2.9±0.1 ab

F-Value (3, 6) 15.7 7.9 6.8 P-Value < 0.01 < 0.05 < 0.05 *Means with standard error within each column followed by the same letter(s) are not significantly different at P ≤ 0.05 according to Gabriel post hoc test.

Chapter Three: Shoot Initiation

3.3.2 Citrus glauca

Half strength MS was the best basal medium for inducing shoots of C. glauca

(Table 3.2). The percentage of explants with microshoots on ½MS was significantly higher than on other media (90% compared to 46-49%). The number of shoots per explant on ½MS media was not significantly different among all media tested. Half strength MS and WPM were the best media for shoot elongation. The least favourable result was consistently found on explants growing on full strength MS. Moreover, hyperhydricity was also more likely to be observed on full strength MS than on other media.

Table 3.2 Effect of different basal media supplemented with 3mg/L BA on shoot initiation of Citrus glauca. Number of Number of shoots Basal Medium explants with Shoot length (mm) per explant shoots (%) WPM 46.8±2.1 b* 4.9±1.0 a 1.8±0.2 a ½WPM 49.0±4.4 b 4.4±0.4 a 1.2±0.04 bc MS 48.3±7.2 b 4.4±0.1 a 1.2±0.1 c ½MS 89.7±2.0 a 6.5±0.6 a 1.7±0.2 ab

F-Value (3, 6) 20.1 3.4 11.5 P-Value < 0.01 > 0.05 < 0.01 *Means with standard error within each column followed by the same letter(s) are not significantly different at P ≤ 0.05 according to Gabriel post hoc test.

Chapter Three: Shoot Initiation

3.4 Discussion

Murashige and Skoog basal medium has been reported as an optimal medium for micropropagating many plants, including Capparis species. This medium has a high concentration of nutrients salts which are important for organogenesis and regeneration

(Razdan, 2003). However, non-significant results between MS and media with significantly lower nutrient concentration (WPM and ½MS) indicated that high nutrient content was not absolutely necessary for C. mitchellii to induce in vitro shoots.

Nevertheless, the poor growth performance of explants on ½WPM also suggested that the level of nutrient in WPM and ½MS was the minimum required for C. mitchellii growth.

In contrast, C. glauca explants growing on MS medium showed low levels of shoot growth and development. These results indicated a negative effect of high nutrient concentration on C. glauca establishment. The ability of C. glauca to grow on very low salt media, such as ½WPM, suggests that C. glauca has a lower requirement for nutrient salt than C. mitchellii.

Among the four media tested, the most significant difference is found in the ammonium level in the form of ammonium nitrate. The concentrations of ammonium nitrate in MS media, ½MS, WPM and ½WPM are 1,650 mg/L, 0.825 mg/L, 400 mg/L and 200 mg/L respectively (Sigma-BioScience, 1996). Ammonium is important for plants as the source of nitrogen, a component of proteins, DNA, RNA, ATP, NADPH,

NADH and other metabolites (McMahon et al., 2007). However, high levels of ammonium can be toxic to many higher plants (Britto & Kronzucker, 2002; George &

Chapter Three: Shoot Initiation

de Klerk, 2008) and is also one of the factors that induce hyperhydricity in plant tissue culture (George & de Klerk, 2008; Hartmann et al., 2011).

In conclusion, the nutrient requirement for successful establishment of these two species was species-dependent. Based on these results, MS and ½MS were selected for subsequent multiplication and elongation experiments for C. mitchellii and C. glauca respectively.

Chapter Four: Shoot Multiplication and Elongation

CHAPTER FOUR

Shoot Multiplication and Elongation

4.1 Introduction

Shoot multiplication is a stage where stabilised cultures are maintained and stimulated to multiply either by producing adventitious or axillary shoots (George,

1996; Dirr & Heuser, 2006; Hartmann et al., 2011). Axillary shoots develop from the axillary bud in the leaf axils (Pierik, 1997; Hartmann et al., 2011), while adventitious shoots originate from tissues that do not usually produce shoots (George, 1993;

Hartmann et al., 2011). The propagation ratio (the number of micro shoots per subculture) of adventitious shoots is usually greater than that of axillary shoots.

However, the possibility of somaclonal variation is also greater in adventitious shoots.

Hence, axillary shoots are generally more preferable when the main purpose is to produce true to type plants (Dirr & Heuser, 2006; Hartmann et al., 2011).

Capparis and Citrus species reportedly multiply rapidly when growth regulators are present (Table 1.2). Generally, shoot proliferation of these species can be promoted by the presence of cytokinins alone in the basal medium (Rathore et al., 2004; Chalak

& Elbitar, 2006; Rathore et al., 2007). Combining cytokinin with auxin may improve shoot number and quality in Capparis (Chahar et al., 2010; Tyagi et al., 2010;

Chapter Four: Shoot Multiplication and Elongation

Musallam & Duwayri, 2011) and Citrus (Al-Bahrany, 2002). However, in other studies shoot proliferation decreased after addition of auxin to the medium (Paudyal & Haq,

2000; Ali & Mirza, 2006).

Adding to the complexity is that the optimum concentrations of plant growth regulators for each stage of micropropagation often vary between species. Cytokinin levels suitable for triggering initiation and multiplication often inhibit elongation

(Saxena et al., 2007; Miah et al., 2009; Musallam & Duwayri, 2011).

An optimal level of cytokinin in the medium is essential to promote robust shoot proliferation and elongation. Thus, the effect of cytokinin level on C. mitchellii and C. glauca shoot multiplication and elongation was investigated in the following experiments.

4.2 Materials and Methods

4.2.1 Preparation

Tools and materials were prepared according to the procedures described in

Section 3.2.1. MS and ½MS media were used for culturing C. mitchellii and C. glauca respectively. Healthy shoots with growing microshoots from the initiation experiment

(Chapter 3) were used as explants for these experiments. In order to minimise carryover

Chapter Four: Shoot Multiplication and Elongation

effects from the previous initiation treatments, all samples were cultured onto their respective basal media without any plant growth regulators for 4 weeks.

4.2.2 Effects of BA on shoot multiplication and elongation

The effects of BA on shoot number and shoot length of C. mitchellii and C. glauca cultures were examined by planting nodal shoots onto media supplemented with different levels of BA. The BA levels were determined based on preliminary studies

(data not shown) which indicated that the BA requirement of C. mitchellii was higher than C. glauca. In the following experiment C. mitchellii was planted onto media supplemented with 0, 1, 3, and 5 mg/L of BA while C. glauca was planted onto 0, 0.1,

0.3, 0.5 and 0.7 mg/L of BA.

Cultures were incubated as described in Section 2.2.1.4. Explants were subcultured every 3 weeks without division. Number of microshoots per culture and maximum length of microshoots were recorded prior to each subculture and at the end of experiment (at the end of ninth week).

4.2.3 Experimental design and statistical analysis

A Randomised Block Design with BA level as treatments and 3 blocks with 10 to 15 explants per treatment per block was utilised in this experiment. Data recorded at the end of the ninth week were analysed using General Linear Model Univariate

Analysis in Statistical Package for the Social Science software (SPSS Version 17 for

Chapter Four: Shoot Multiplication and Elongation

Windows). Due to unequal sample size caused by contamination and necrosis in some samples, the Hochberg’s GT2 test was chosen as a post hoc test as this test is designed to analyse data when sample size is very different (Field, 2009).

4.3 Results

4.3.1 Capparis mitchellii

Shoot multiplication and elongation were both observed on explants planted on media supplemented with BA. In the absence of BA, only shoot elongation was observed. Moreover without BA in the media, deterioration was also observed on initiated microshoots, resulted in decreasing number of microshoots when compared to

Table 3.1 This result indicates that BA had a significant effect on C. mitchellii shoot proliferation (P<0.01) (Table 4.1).

Table 4.1 Effect of BA concentration on shoot multiplication and elongation of Capparis mitchellii. BA (mg/L) Number of Shoots Shoot length (mm) 0 2.9±0.1 a 4.6±0.4 a 1 6.3±1.0 b 7.6±2.6 a 3 5.0±0.7 b 5.3±0.5 a 5 6.1±0.4 b 4.3±0.7 a

F-Value (3, 6) 18.6 1.5 P-Value < 0.01 > 0.05 *Means with standard error within each column followed by the same letter(s) are not significantly different at P ≤ 0.05 according to Hochberg’s GT2 post hoc test.

Chapter Four: Shoot Multiplication and Elongation

Significant difference was not observed among BA treatments (1, 3 and 5 mg/L

BA) but all were significantly different from the control (P<0.01). The optimal rate of multiplication and elongation were observed when testing BA at 1 mg/L BA. Using this treatment, the average number of adventitious shoots per explant was 6.3. Shoot length varied from 4.3 to 7.6 mm across treatments but was not considered statistically different (P>0.05).

Ongoing observation of tissue appearance indicated that the quality (health) of

C. mitchellii shoots was in decline. Discolouration and necrosis were observed across all treatments during the experiment.

4.3.2 Citrus glauca

Significant variation in shoot number and length was observed between

0.1 mg/L BA and other treatments (0, 0.3, 0.5 and 0.7 mg/L BA) (P<0.01). The low number of microshoots (2.2 microshoots per explant) on 0.1 mg/L BA treatments was caused by shoot abscission rather than the inability of 0.1 mg/L BA in promoting shoot multiplication. On the other hand, the longest shoot length was found using 0.1 mg/L

BA (4.8 mm) and the shortest was observed on the control and media supplemented with 0.3-0.7 mg/L BA (Table 4.2).

Chapter Four: Shoot Multiplication and Elongation

Table 4.2 Effect of BA concentration on shoot multiplication and elongation of Citrus glauca. BA (mg/L) Number of Shoots* Shoot length (mm)* 0 3.7±0.3 ab 2.3±0.2 a 0.1 2.2±0.1 a 4.8±0.4 b 0.3 3.3±0.3 ab 3.1±0.2 a 0.5 4.5±0.4 b 3.1±0.1 a 0.7 4.2±0.2 b 2.3±0.3 a

F-Value (4, 8) 8.9 20.9 P-Value <0.01 <0.01 *Means with standard error within each column followed by the same letter(s) are not significantly different at P ≤ 0.01 according to Hochberg’s GT2 post hoc test.

Callus formation was observed at the base of some micro shoots, resulting in the separation of these micro shoots from the remainder of the cluster. These separated shoots usually deteriorated rapidly and died within a few days.

4.4 Discussion

Multiplication and elongation in C. mitchellii and C. glauca was affected by the concentration of BA in the medium. It was observed that new microshoots were rarely formed when BA was absent.

Adding 1-5 mg/L BA to C. mitchellii culture media significantly enhanced the ability of nodal shoots to proliferate and produce new axillary shoots. On the other hand, shoot length was suppressed in C. glauca cultures when the level of BA was higher than 0.1 mg/L. The suppressed elongation at higher levels of BA was also

Chapter Four: Shoot Multiplication and Elongation

observed in C. limon (Rathore et al, 2007) and C. aurantifolia (Al Bahrany, 2002).

Although cytokinins are necessary to stimulate cell division and promote shoot proliferation, caution is needed as high levels of cytokinin may inhibit elongation, increasing number of short shoots and inducing shoot hyperhydricity (Kadota & Niimi,

2003). While hyperhydricity is usually irreversible (Kevers et al., 2004), short shoots can be elongated by reducing the level of cytokinin in subculture media or by adding additional growth regulators to stimulate cell elongation e.g. auxin or gibberellic acid

(GA3). Suppressed elongation in C. decidua was reversed by adding NAA to the medium (Saxena et al., 2007) and GA3 were utilised to support elongation in C. spinosa

(Chalak & Elbitar, 2006; Musallam & Duwayri, 2011).

The declining quality of C. mitchellii shoots became a major issue in this experiment with some C. mitchellii cultures gradually yellowing a few weeks after planting. The decline of vigour was observed in all treatments with the worst quality observed in the control treatment. Subculturing and reducing the interval between subcultures did not improve the condition. It is possible that this issue was caused by an imbalance of nutrients in the medium (George, 1996). However, the inclusion of streptomycin into media to suppress contamination levels may also have been responsible. Phytotoxicity has been known to occur as a side effect of prolonged use of antibiotics in tissue culture (Leifert et al., 1992; Pierik, 1997; Chen & Yeh, 2007).

A secondary abscission zone induced by callus formation at the base and/or node of axillary shoots was observed in C. glauca cultures, causing shoots to shed

(Figure 4.1). Microshoot abscission was often found on long shoots rather than short

Chapter Four: Shoot Multiplication and Elongation

ones. Consequently abscission was more likely to be observed in the 0.1 mg/L BA treatment than in other treatments, as longer shoots were usually obtained in this treatment.

Shoot abscission is a common phenomenon in Citrus species (Plummer et al.,

1991). In Citrus sinensis, the abscission of leaves, flowers, fruit and shoots is part of its characteristic growth cycle (Hartmond et al., 2000). Several species of citrus have proved difficult to establish by tissue culture due to the production of secondary abscission zones, shoot abscission and extensive callus production (Plummer, 1987 cited in Plummer et al., 1991).

A B

Figure 4.1 Secondary abscission zones (arrows) at the base of axillary shoots (A) and leaves (B) in C. glauca nodal shoots culture.

Chapter Four: Shoot Multiplication and Elongation

Callus is an unorganized mass of undifferentiated and/or differentiated cells which actively multiply in response to wounding. In tissue culture, callus can be induced by growth regulators, especially auxin and/or cytokinin in media (Pierik,

1997).

Altman and Goren (1971) reported that callus formation was specifically stimulated at the abscission zone between the petiole and stem when bud explants of

Citrus sinensis were cultured on media supplemented with low levels (0.3-2.6 mg/L) of

ABA. The same result was also observed when C. sinensis bud explants were cultured on media supplemented with various combinations of ABA, IAA, and BA, except when

IAA or BA were alone in the medium. Another study with C. sinensis shoot cultures showed that a secondary abscission zone formed within internodes was highly influenced by ABA and NAA in the medium. Culturing shoots of this species on media supplemented with BA induced callus formation, but did not significantly affect abscission (Plummer et al., 1991). Instead, the secondary abscission zones in C. sinensis internodes were associated with ethylene production which was induced by

ABA and auxin in the medium (Sagee et al., 1980; Plummer et al., 1991). George

(1993) reported that the accumulation of ethylene in the culture vessel could lead to callus formation.

Abscisic acid and auxin were not added in C. glauca culture media and ventilated culture vessel lids were used to prevent ethylene accumulation in the present experiments. However, ABA and auxin can be formed naturally in C. glauca tissues and this may lead to ethylene production and subsequently induce callus formation. In

Chapter Four: Shoot Multiplication and Elongation

addition the ventilation of lids in these experiments might be insufficient to prevent ethylene accumulation.

In conclusion, shoot multiplication and elongation in C. mitchellii and C. glauca cultures was affected by BA level in the media. Adding 1 mg/L BA was sufficient to multiply and elongate axillary shoots of C. mitchellii from nodal shoot explants. The highest number of axillary shoots of C. glauca was observed on media supplemented with 0, 0.3, 0.5 and 0.7 mg/L BA whilst elongation was optimal on media supplemented with 0.1 mg/L BA.

Further studies of C. mitchellii and C. glauca micropropagation are needed to examine the effects of auxins and GA3 in combination with the levels of BA used in this study, to promote shoot elongation. Measurements of ethylene levels in culture vessels may be necessary to understand the phenomenon of secondary abscission zone formation in C. glauca.

Chapter Five: Rooting and Plant Establishment

CHAPTER FIVE

Rooting and Plant Establishment

5.1 Introduction

Plantlets (small plant with primary shoot and root) are obtained by transferring microshoots to rooting media to induce root production. Roots are required to prepare plantlets for growing in the natural environment outside the aseptic controlled conditions of the culture vessel.

Root production can be induced using either ex vitro or in vitro methods. The ex vitro methods treat the microshoots similar to conventional cuttings by directly inserting them into soilless rooting medium in high humidity to prevent excessive water-loss. Microshoots can also be treated with auxin by dipping into a powder or solution prior to planting if necessary. In in vitro methods, roots are usually obtained by planting the microshoots into salt-reduced media supplemented with auxins such as

IAA, IBA and NAA (George, 1996; Hartmann et al., 2011).

Ex vitro-produced roots usually have a stronger and better developed vascular system (McClelland et al., 1990), with more vigorous growth than in vitro roots, increasing the survival rate of ex vitro rooted plantlets in the natural environment (Dai et al., 2004). Ex vitro methods provide a good transition between the culture

Chapter Five: Rooting and Plant Establishment

environment and the natural one. These methods are more efficient and less expensive as compared to in vitro rooting methods (Martin, 2003; Husain & Anis, 2009;

Benmahioul et al., 2011).

On the other hand, in vitro methods are easier to control and manipulate

(George, 1996). Rooting percentage using in vitro methods is usually higher than when using ex vitro methods (Gonçalves et al., 1998; Dai et al., 2005). Many plants, including Capparis (Deora & Shekhawat, 1995; Chalak et al., 2003; Saxena et al.,

2007; Chahar et al., 2010; Musallam & Duwayri, 2011) and Citrus (Paudyal & Haq,

2000; Al-Khayri & Al-Bahrany, 2001; Al-Bahrany, 2002; Ali & Mirza, 2006; Pérez-

Tornero et al., 2010) species have been successfully rooted on culture media supplemented with auxin.

The capability of C. mitchellii and C. glauca microshoots to produce in vitro roots under the influence of auxin and to survive the establishment stage were examined in the following studies.

5.2 Materials and Methods

5.2.1 Preparation

Tools and materials were prepared as described in Section 3.2.1. Half strength

MS was used as the basal media in this experiment. Following 12 weeks exposure to

Chapter Five: Rooting and Plant Establishment

multiplication media, axillary shoots of C. mitchellii and C. glauca were still too short for rooting to be attempted. Due to the time constraints of this project, it was decided to use microshoots of C. mitchellii and C. glauca that had been established during year one of this project and regularly subcultured over the last 12 months with average shoot length range of 5 to 6 cm and 0.5 to 1 cm for C. mitchellii and C. glauca respectively.

5.2.2 Effects of auxin level on root development

The effect of auxin level on root development in C. mitchellii and C. glauca was examined by planting microshoots onto media containing different levels of auxin. It was initially proposed that NAA and IBA be used in this experiment. However, IBA was excluded due to the inability of C. mitchellii microshoots to develop roots when planted onto media supplemented with IBA in a preliminary experiment (data not shown). A preliminary experiment was not conducted with C. glauca due to the unavailability of microshoots. IBA was also excluded from the C. glauca experimental design due to the low number of microshoots being available for the main experiment.

Adventitious shoots approximately 10 mm long were excised and planted onto

1/2MS media supplemented with NAA at 0, 1, 3, 5, 100 and 200 mg/L. Microshoots that were planted onto 100 and 200 mg/L NAA were transferred to media without growth regulators after 48 hour (acute treatment), while microshoots in other treatments were kept on the same medium (chronic treatment) for the duration of the experiment.

All microshoots were incubated in the dark for three days before being exposed to 16 hr

Chapter Five: Rooting and Plant Establishment

photoperiod at 25±2oC. Cultures were monitored weekly to determine the percentage forming roots, root number and root length.

5.2.3 Plant establishment

Plantlets with roots longer than 5 mm were subjected to hardening and acclimatisation. Plantlets were carefully washed clean of media and soaked in mancozeb solution at 100 mg/L for 5 to 10 minutes. Cleaned plantlets were inserted into 250 mL culture jars which were 3/4 filled with a mixture of peat and perlite (1:4) and 6 or 7 beads of slow release fertilizer on the surface. Plantlets were then irrigated with 30 mL of RO water. Jars with loosely attached lids (to maintain humidity) were kept in a controlled environment (16 hr photoperiod, 25±2oC). Additional irrigation was given as necessary. Lids were removed after two weeks. A week later, plantlets were transferred to the glass house, a single plant per pot filled with previously described media, and misted twice daily with water. Plants were kept in the shade until considered ready for planting into soil.

5.2.4 Experimental design and statistical analyses

The experiment was set up as a Completely Randomised Block Design (CRBD) with 25 and 6 replications per treatment for C. mitchellii and C. glauca respectively.

Each replicate comprised a single micro-cutting.

Chapter Five: Rooting and Plant Establishment

Data were analysed using Crosstab model and General Linear Model Univariate

Analysis in Statistical Package for the Social Science software (SPSS Version 17 for

Windows). Tukey’s test was performed for mean comparison when possible (Field,

2009). Rate of rooting and callusing were analysed using the frequency form value

(number of samples with developed roots per treatment) instead of percentage.

5.3 Results

5.3.1 Capparis mitchellii

In the absence of auxin, C. mitchellii was able to produce adventitious roots, albeit at a very low level (4.0%, Table 5.1). Adding NAA to rooting media enhanced root formation and significantly affected root number and length (P<0.001). However there was no significant association between NAA level and rooting percentage

(Fisher’s Exact Test, P>0.05). Adventitious roots were first observed on microshoots five weeks after planting on media supplemented with NAA and eight weeks when

NAA was absent. The highest level of root formation was observed at 3, 100 and 200 mg/L NAA, while the highest root number (3.6) and length (7.6 mm) were obtained at

100 mg/L NAA. Callus formation was significantly associated with NAA (Fisher’s

Exact Test, P<0.001) and was only observed following chronic (ongoing) exposure of shoots to 3 or 5 mg/L NAA. No callus formation was observed using the acute (48 hr) exposure approach (Table 5.1).

Chapter Five: Rooting and Plant Establishment

Table 5.1 Effect of NAA on rooting of Capparis mitchellii. NAA (mg/L) % Rooting Number of roots* Root length (mm)* % Callusing 0 † 4 a 3.0 7.0 0 a 1 12 a 2.7±1.2 1.7±0.3 0 a 3 20 a 3.0±0.5 1.8±0.2 28 b 5 † 4 a 1.0 2.0 72 c 100 20 a 3.6±0.9 7.6±1.9 0 a 200 20 a 2.8±0.7 4.6±0.9 0 a

P-Value >0.05 < 0.001 < 0.001 <0.001 Percentage values within each column followed by the same letter(s) are not significantly different at P ≤ 0.05 according to ɀ-score (standardized residual). † Roots were only observed on one sample. *Post hoc tests were not performed because two treatments (0.0 and 5.0 mg/L BA) had only one sample with adventitious roots.

Plantlets were successfully planted onto plant establishment media but half of the plantlets were lost after a week in the glass house. Considering that the plantlets were pre-hardened in the incubator room without lids for more than 3 weeks, it was assumed that the glass house was still too dry for the plantlets to survive.

5.3.2 Citrus glauca

Adventitious root production was not achieved for C. glauca, and necrosis and a decline in shoot vigour were observed across all NAA treatments. Necrosis and decline also occurred in controls, but was slower to develop in the absence of NAA.

Chapter Five: Rooting and Plant Establishment

5.4 Discussion

In the current experiment the capability of C. mitchellii and C. glauca to develop in vitro roots in response to NAA was assessed. Whilst the maximum level of root production in C. mitchellii was only 20%, this result was higher than a previous study where rooting was only achieved on a single shoot after exposure to 1000 µM

(203 mg/L) IBA for 48 hours (Steggles, 2007). NAA has been reported to promote root production in other Capparaceae including Crateva nurvala (Sanayaima et al., 2006) and Cleome viscose (Anburaj et al., 2011). On the other hand, there was no root formation observed on C. glauca cultures in this study.

For C. mitchellii, exposing shoots to 100 mg/L NAA for 48 hours resulted in the higher rooting percentage, root number and root length without any callus formation

(Figure 5.1A) when compared to other treatments. Increasing the NAA level to

200 mg/L did not improve rooting percentage and both number and root length decreased (Table 5.1). Further anatomical and survival rate assessment is needed to examine root quality and ability to harden off.

Rooting was also obtained with prolonged application of low auxin levels

(chronic method). However, callusing was observed at NAA levels higher than 1 mg/L

(Figure 5.1B). Increasing the NAA level also increased the degree of callusing. Callus formation was undesirable at this stage, as roots developed from callus had an incomplete (or no) vascular connection to the shoots. Consequently, plantlets with this type of root rarely survived hardening (George, 1996).

Chapter Five: Rooting and Plant Establishment

A B

Figure 5.1 Adventitious roots of C. mitchellii without (A) and with (B) callus formation.

Auxin, although necessary for rhizogenesis, promoted callus and suppressed root and shoot growth if applied for prolonged periods (Woo & Wetzstein, 2008).

Suppressed root and shoot growth can be prevented by transferring shoots to media with reduced auxin or by pulse auxin exposure. C. decidua was successfully rooted (60-

70%) when exposed to 100 mg/L IBA for 4 hours (Deora & Shekhawat, 1995). On the other hand, this treatment was reported to significantly increase callus formation and shoot length of C. spinosa (Musallam & Duwayri, 2011). Rooting in C. spinosa was promoted (87%) by exposing shoots to 100 mg/L IAA for 4 hours (Chalak et al., 2003).

These results indicate that the effect of auxin on plant morphogenesis and organogenesis was genotype dependent

NAA was observed to have deleterious effects on C. glauca shoots in this study.

NAA was incapable of promoting root formation at the levels tested, but induced

Chapter Five: Rooting and Plant Establishment

necrosis and decreased the establishment rate of cultured plants. Similar deleterious effects of NAA were also observed on other species including Pinus roxburghii (Kalia et al., 2007) Arbutus unedo (Gomes et al., 2010) and Garcinia quaesita (Farzana et al.,

2010). Endogenous auxin levels may contribute to the variety of responses observed from different species to auxin treatment (Zhu et al., 2010). It is possible that the tested auxin levels were too high for C. glauca, which was found to be sensitive to a variety of other substances including sterilisation agents (Chapter 2), nutrients (Chapter 3) and other growth regulators (Chapter 4).

Nine plantlets of C. mitchellii were subjected to hardening and acclimatisation.

All plantlets were adapting very well to the incubator room condition (hardening stage).

No sign of deterioration was observed at this stage. However, half of the plantlets were lost a week after the plantlets were placed in the glass house (acclimatisation stage),

There was a possibility that the plantlets were exposed too soon to the glass house condition. Recovering the pots and removing it gradually to the glass house condition is suggested for the next study.

The rooting result of C. mitchellii and C. glauca in this experiment is similar to results for rooting of cuttings for these two species. Tworney et al. (2009) reported that

C. mitchellii can be propagated through cuttings while C. glauca has been known for its slow response to this technique. In general, C. glauca is propagated by being grafted onto a suitable rootstock (Swingle & Reece, 1967; Tworney et al., 2009).

This present experiment demonstrates the capability of C. mitchellii to produce in vitro roots without callus by exposing shoots to high levels of NAA for a short

Chapter Five: Rooting and Plant Establishment

period of time. Further study is necessary to further enhance root production in C. mitchellii and to determine the suitable type, level and time exposure of auxin for inducing roots on C. glauca shoots.

Chapter Six: General Discussion

CHAPTER SIX

General Discussion

Capparis mitchellii and C. glauca are both Australian native plants recognised as fruit commodities in the native food industry (Lazarides & Hince, 1993; Tworney et al., 2009).Wild harvesting is still the primary source for Australian native food (Hele,

2001; CSIRO, 2002; Miers, 2004; Tworney, et al., 2009). This practice is not only incapable of fulfilling the market demand but also can be a threat for Australian wildlife (Berkinshaw, 1999). Cultivating these plants is not only beneficial for the industry but also for conservation of these Australian native plants.

Both C. mitchellii and C. glauca can be propagated by conventional techniques with varying levels of difficulty (Tworney, et al., 2009). Previous studies to cultivate these plants by tissue culture techniques have resulted in methods to micropropagate C. glauca by somatic embryogenesis (Jumin & Nito, 1996) and initiate shoot and root proliferation from C. mitchellii nodal shoots (Steggles, 2007). However, further refinement is necessary to better understand micropropagation of these species. This project aimed to optimise the sterilisation, initiation, multiplication/proliferation, rooting and acclimatisation methods for C. mitchellii and C. glauca shoot cultures.

Chapter Six: General Discussion

Capparis mitchellii and C. glauca response to various sterilisation agents

(Chapter 2), basal media (Chapter 3), and plant growth regulators (Chapter 4 and 5) are observed and described in this project. The results obtained from these experiments confirmed that mass propagation of these plants by micropropagation through the nodal shoot culture method is possible.

Minimizing in situ contamination and damage to explants is an essential first step to achieve a healthy aseptic culture. Controlling the microbial contamination of field sourced woody plant material is not easy. Pre-soaking explant material in a wide spectrum anti-microbial solution (PPMTM) prior to planting onto medium supplemented with mancozeb proved to be the most preferable method due to the low level of contamination and phytotoxicity observed for both C. mitchellii and C. glauca.

Interestingly, mancozeb was also capable of promoting axillary shoot induction from C. mitchellii and C. glauca explants. Incorporating mancozeb into basal medium has been reported to effectively control fungal contamination without any phytotoxic effect in apple micropropagation (Kolozsvári Nagy et al., 2005). However, the capability of mancozeb to promote shoot proliferation has not been examined. Further study is needed to develop a better understanding of mancozeb’s effect to promote organogenesis in tissue culture.

Although PPMTM and mancozeb were able to reduce the contamination rate in the sterilisation experiment (Chapter 2), streptomycin was added to the culture medium to combat the increasing rate of bacterial contamination in initiation experiments

(Chapter 3). The contamination rate was manageable using streptomycin, but decline in

Chapter Six: General Discussion

vigour and necrosis were observed later on C. mitchellii (Chapter 4). Prolonged exposure of streptomycin was considered as one of the possible causes, as antibiotics not only inhibit bacterial growth but also may injure plant tissue (Leifert, Camotta &

Waites, 1992; Pierik, 1997; Chen & Yeh, 2007). As ongoing antibiotic is only required if the contamination is persistent (George, 1993; Bunn & Tan, 2002; Razdan, 2003), adding any antibiotic in future experiments should be avoided.

Generally, shoot initiation of woody species is promoted by planting sterilised explants onto basal medium with reduced or diluted levels of nutrients supplemented with growth regulators (Rathore et al., 2004). Nutrient and hormonal requirements differ between plants. Shoot initiation of C. mitchellii was observed in media with low levels of nutrients (½WPM), but shoot percentage, number of shoots and shoot length was optimized in MS medium. On the other hand, C. glauca tended to produce hyperhydric microshoots when planted onto MS medium. The highest initiation rate, shoot number and length of C. glauca were obtained in ½MS basal medium (Chapter

3).

Multiplication and elongation of C. mitchellii and C. glauca in this project was significantly influenced by BA in the medium (Chapter 4). Whilst increasing the BA level usually resulted in increased proliferation, elongation was usually suppressed.

This result was in agreement with previous studies of Capparis (Deora & Shekhawat,

1995; Musallam & Duwayri, 2011) and Citrus (Al-Bahrany, 2002). Addition of auxin and GA3 was found to stimulate shoot elongation in Capparis culture (Chalak &

Elbitar, 2006; Saxena et al., 2007). For Citrus, adding GA3 may improve shoot

Chapter Six: General Discussion

proliferation and elongation (Pérez-Tornero et al., 2010) but NAA may either stimulate, inhibit or have no effect on shoot multiplication and elongation depending on the cytokinin level in the media (Al-Bahrany, 2002). Furthermore, auxin reportedly promoted callus and led to the formation of secondary abscission zones on Citrus

(Plummer et al., 1991).

The tendency of C. glauca microshoots to develop secondary abscission zones was highlighted in this project. In previous studies, secondary abscission zones on

Citrus microshoots were promoted by ethylene which was induced by exogenous ABA and auxin (Altman & Goren, 1971; Altman & Goren, 1974; Sagee et al., 1980;

Plummer et al., 1991). A secondary abscission zone was rarely triggered by BA alone

(Altman & Goren, 1971; Altman & Goren, 1974; Plummer et al., 1991). However, exogenous BA can certainly influence the balance of endogenous growth regulators including ABA, auxins and ethylene. Further testing of both salt level in media and growth regulators is necessary to optimise microshoot quantity and quality for both species.

Rooting was promoted by exposing microshoots to a range of NAA levels

(Chapter 5). Although C. glauca microshoots failed to produce roots and became heavily necrotic, these treatments were capable of promoting roots on C. mitchellii microshoots. Generally, roots clean of callus were obtained when C. mitchellii shoots were exposed to high levels of auxin for short periods of time (pulse/acute treatment).

This method of auxin exposure also successfully promoted root formation in other

Capparis species (Deora & Shekhawat, 1995; Chalak et al., 2003; Chalak & Elbitar,

Chapter Six: General Discussion

2006). Despite the fact that auxin was capable of inducing rooting, over-exposure was found to inhibit root elongation and increase the potential for callus formation.

Lowering the NAA level and experimenting with other auxins is recommended for subsequent attempts at micropropagating C. glauca, and experimenting with timing of exposure to auxin should be studied to see if it can improve rooting percentage of C. mitchellii cultures.

In conclusion, significant improvements for micropropagating C. mitchellii and

C. glauca have been achieved through this project. A first complete protocol to propagate C. mitchellii by tissue culture is documented in this thesis. Although C. glauca proved more difficult to propagate, basic information on micropropagation of this species, including suitable basal media and growth regulator effects on shoot and root formation have been developed.

Appendix. Supplier Information

Product Name Acronym Manufacturer Addresses Product no. Batch/Lot no. /symbol /supplier 6-Benzylaminopurine BA Phytotechnology Shawnee Mission B800 08D80028A Laboratories KS-USA α-naphthaleneacetic acid NAA SIGMA St. Louis MO N-0375 23F-0232 USA Agar Phytotechnology Shawnee Mission A-111 03J1110D Laboratories KS-USA Gelrite, Gellan Gum Phytotechnology Shawnee Mission G469 04G469E Powder Laboratories KS-USA Murashige & Skoog’s MS Phytotechnology Shawnee Mission M-519 03D51911P Basal Medium with Laboratories KS-USA vitamins Mancozeb Plus Sulphur Richgro Garden Products Canning Vale WA Fungicide Australia Miconazole SIGMA St. Louis MO M-3519 51K1656 USA Lloyd & McCown Woody WPM Phytotechnology Shawnee Mission L-449 07F44938A Plant Basal Medium w/ Laboratories KS-USA Vitamin Plant Preservative PPMTM Phytotechnology Shawnee Mission P-820 10P82003A MixtureTM Laboratories KS-USA Streptomycin Sulphate Ducheva Biochemie Haarlem 3810-74-0 Netherland Tween 20 Polyoethylene ICN Biomedicals Inc Aurora Ohio, 103168 55238 sorbitan monolaurate USA Teflon membrane vent Flora Laboratories Menton, Victoria, spots Australia

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