Vegetative Propagation of Selected Nepenthes Species by Stem Cuttings

VEGETATIVE PROPAGATION OF SELECTED NEPENTHES SPECIES BY STEM CUTTINGS

Nurul Syahira Binti Karim

Bachelor of Science with Honours (Plant Resource Science and Management) 2016

VEGETATIVE PROPAGATION OF SELECTED NEPENTHES SPECIES BY STEM CUTTINGS

NURUL SYAHIRA BINTI KARIM

(43623)

The project is submitted in partial fulfilment of the requirement for the degree of Bachelor of

Science with Honours

(Plant Resource Science and Management)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2016

APPROVAL SHEET

Name of Candidate : Nurul Syahira binti Karim

Title of Dissertation : Vegetative Propagation of Selected Nepenthes Species by Stem Cuttings

………………………………

(Prof. Dr. Hamsawi bin Sani)

Supervisor

…………………………......

(Dr. Freddy Yeo Kuok San)

Coordinator

Plant Resource Science and Management Department of Plant Science and Environmental Ecology Faculty of Resource Science and Technology Universiti Malaysia Sarawak

DECLARATION

I am Nurul Syahira binti Karim, the final year student of Plant Resource Science and

Management hereby declare that this thesis is my own work with the guidance of my supervisor, Professor Dr. Hamsawi bin Sani. No portion of the work referred to this dissertation has been submitted in support of an application for another degree of qualification of this or any other university or institution of higher learning.

______

Nurul Syahira binti Karim (43623)

Plant Resource Science and Management Department of Plant Science and Environmental Ecology Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ACKNOWLEDGEMENT

Alhamdulillah, for His abundant grace towards me, I managed to finish my final year project within the time provided. Here, I want to express my gratitude to all those people that always lend their hands and always support me to accomplish my research works.

First of all, it difficult to overstate my gratitude to my supervisor, Prof. Dr. Hamsawi bin

Sani for his advices, guidance, motivation and support from the beginning until the end of this project. Thanks for always cared, support and taught me a lot of new things through the accomplishment of this project. Thanks for being such a patience lecturer for me and treat me like a daughter. I also would like to thank my examiner Dr. Rebicca Edward for her comments and advices.

For my parents, Karim bin Abu Sha and Haniza binti Adnan, thank you for great sacrifice, endless prayers, blessing and support to reach me at this level of higher education. They are such a good listener of me, always being supportive especially when I’m in the breaking point from the beginning until now. Their love and care gave me motivation to complete my project and thesis writing.

My very big thanks to my special person and my adviser because not getting tired to give me inspiration and motivation to accomplish my research works. Thank you also to all my fypmates, coursemates and friends who help me a lot and being such a good friend of me.

Lastly, my gratitude to my true self for not giving up even there are many times I want to give up. I always said to myself “You can do it”. And at the end, I managed to complete my Final Year Project and writing thesis. Thanks to myself and everyone.

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TABLE OF CONTENTS

APPROVAL SHEET i

DECLARATION ii

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS iv

LIST OF ABBREVIATIONS vii

LIST OF FIGURES viii

LIST OF PLATES ix

LIST OF TABLES x

ABSTRACT 1

1.0 INTRODUCTION

1.1 Research background 2

1.2 Problem statements 5

1.3 Objectives 5

2.0 LITERATURE REVIEW

2.1 Botanical description

2.1.1 Nepenthes ampullaria 6

2.1.2 Nepenthes mirabilis 7

2.1.3 Nepenthes gracilis 8

2.2 Economic importance 9

2.3 Vegetative propagation 9

2.4 Vegetative propagation of Nepenthes sp. 10

2.5 Factor affecting rooting of cuttings

2.5.1 Effect of types of cutting 11

2.5.2 Effect of cutting position 11

2.5.3 Effect of age of cutting 12

2.5.4 Effect of leaf retention 13

2.5.5 Effect of Plant Growth Regulator 14

2.5.6 Effect of Rooting Media 15

3.0 MATERIAL AND METHODS

3.1 Study site 16

3.2 Stock plant preparation 17

3.3 Rooting Media preparation 17

3.4 Preparation of Plant Growth Regulator 18

3.5 Cutting preparation 18

3.6 Experimental design 20

3.7 Experimental layout 21

3.8 Data analysis 22

4.0 RESULTS

4.1 Rooting success 23

4.2 Number of root formed 30

4.3 Length of root 33

4.4 Root dry weight 35

4.5 Number of cuttings produce shoot 37

4.5.1 Number of shoots formed 37

4.6 Shoot dry weight 38

4.7 Root to shoot ratio 40

5.0 DISCUSSIONS

5.0 Rooting success of Nepenthes sp. between treatments 42

5.1 Effect of rooting media to the rooting of cuttings 43

5.2 Effect of leaf retention to the rooting of cuttings 45

5.3 Effect of Plant Growth Regulators (PGR) to the rooting of cuttings 46

5.4 Effect moisture surrounding to the rooting of cuttings 47

5.5 Effect of environmental factors to the rooting of cuttings 48

6.0 CONCLUSION AND RECOMMENDATIONS 49

7.0 REFERENCES 51

8.0 APPENDIXES

Appendix 1 54

Appendix 2 55

Appendix 3 56

Appendix 4 57

Appendix 5 58

LIST OF ABBREVIATIONS

ANOVA Analysis of Variance

IBA Indole-butyric acid

PGR Plant Growth Regulator

PPM Parts per million sp. Species

% Percentage

P P-value

χ2 Chi-square

UNIMAS Universiti Malaysia Sarawak

RDW Root Dry Weight

SDW Shoot Dry Weight

RSR Root to Shoot Ratio

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LIST OF FIGURES

Figure 1 : Distribution of Nepenthes sp. (Honda, n.d.) 3

Figure 2 : Image of pitcher Nepenthes ampullaria (Clarke, 1997) 6

Figure 3 : Image of pitcher Nepenthes mirabilis (Clarke, 1997) 7

Figure 4 : Image of pitcher Nepenthes gracilis (Clarke, 1997) 8

Figure 5 : The percentage of rooting success for 24

Nepenthes ampullaria

Figure 6 : The percentage of rooting success for 26

Nepenthes mirabilis

Figure 7 : The percentage of rooting success for 28

Nepenthes gracilis

Figure 8 : Mean Number of Roots Per Cuttings of N.ampullaria 31

Figure 9 : Mean Number of Roots Per Cuttings of N.mirabilis 31

Figure 10 : Mean Number of Roots Per Cuttings of N.gracilis 32

Figure 11 : Mean of Root Dry Weight 36

Figure 12 : Mean of Shoot Dry Weight 39

Figure 13 : Mean of Root to Shoot Ratio 41

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LIST OF PLATES

Plate 1 : Concrete bed covered with transparent plastic sheet and equipped 16

with a simple misting system.

Plate 2 : Container box containing 5 ml of tap water 17

LIST OF TABLES

Table 1 : Experimental design for this experiment 20

Table 2 : The experimental layout for each species 21

Table 3 : Comparison of Chi-square between treatments for rooting on 23

N.ampullaria

Table 4 : Comparison of Chi-square between treatments for rooting on 25

N.mirabilis

Table 5 : Comparison of Chi-square between treatments for rooting on 27

N.gracilis.

Table 6 : Rooting percentage of three different Nepenthes sp. 29

Table 7 : Mean number of roots produced by three different 30

Nepenthes sp.

Table 8 : Length of roots (cm) in cuttings of three different 33

Nepenthes sp. with respect to different rooting media

and leaf retention.

Table 9 : Overall comparison of length of roots (cm) shown by different 34

rooting media and leaf retention

Table 10 : Root dry weight of three different Nepenthes sp. 35

Table 11 : Overall comparison root dry weight (g) shown by different 36

rooting media and leaf retention

Table 12 : No of shoots produced by different species of Nepenthes sp. 37

with respect to different rooting media and present of leaf retention

Table 13 : Shoot dry weight of three different Nepenthes sp. 38

Table 14 : Overall comparison shoot dry weight (g) shown by 39

different rooting media and leaf retention

Table 15 : Root to shoot ratio of three different Nepenthes sp. 40

Table 16 : Overall comparison root to shoot ratio (g) shown by 41

different rooting media and leaf retention

Table 17 : Raw data for overall results (exclude died cuttings) of 54

species 1(N.ampullaria)

Table 18 : Raw data for overall results (exclude died cuttings) of 55

species 2(N.mirabilis)

Table 19 : Raw data for overall results (exclude died cuttings) of 56

species 3(N.gracilis)

Table 20 : Raw data for overall rooting success for three species 57

Vegetative Propagation of Selected Nepenthes Species by Stem Cuttings

Nurul Syahira Binti Karim Department of Plant Science and Environmental Ecology Faculty of Resources Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Pitcher plant has a high potential and beneficial as ornamental and medicinal plants. However, only a few studies have been carried out. Due to the clearing of lands and natural forests, the population of pitcher plant is nearly extinct. Hence, in order to save the extinction of Nepenthes sp., the conventional propagation by rooting of stem cuttings is done because low percentage of seed germination showed by Nepenthes sp. The present study focused on N.ampullaria, N.gracilis and N. mirabilis. There are five treatments altogether have been tested which include three different rooting media with present or without of leaf retention (Sand with leaf, sand with no leaf, cocopeat with leaf, cocopeat with no leaf, water with leaf and water with no leaf).To enhance rooting, the cutting were treated with commercial hormone, SADEX 1 for sand media and cocopeat media while for water media were treated with 600 ppm of IBA hormone. The results showed cuttings N.gracilis has the highest percentage rootings success (13.33%) compared to N.ampullaria and N.mirabilis (11.67%). Overall rooting success was recorded by cuttings with leaf rooted in sand (21.7%) followed by leaf rooted in cocopeat (6.7%). While for cuttings without leaf rooted in sand (5.8%) and in cocopeat (2.5%). There is no rooting success for rooting media water. Leaf retention on cuttings proved to be a beneficial factor to enhance the root and shoot development of Nepenthes compared to those cuttings without leaf.

Keywords: Pitcher plants, conventional propagation, vegetative propagation, rooting of stem cutting ABSTRAK

Periuk kera sangat berpotensi tinggi dan bermanfaat sebagai tanaman hiasan dan tanaman perubatan. Walau bagaimanapun, hanya beberapa kajian telah dijalankan. Berikutan pembersihan tanah dan hutan semula jadi, populasi periuk kera hampir pupus. Oleh itu, dalam usaha untuk menyelamatkan kepupusan species Nepenthes, kaedah pembiakan konvensional dengan perarakan keratan batang dilakukan kerana peratusan percambahan biji benih yang ditunjukkan oleh species Nepenthes adalah rendah. Kajian ini memberi tumpuan kepada N.ampullaria, N.gracilis dan N.mirabilis. Terdapat lima jenis rawatan yang telah diuji termasuk tiga jenis media perakaran yang berbeza dengan pengekalan daun atau tanpa pengekalan daun (Pasir dan daun, pasir tanpa daun, serat sekam kelapa dan daun, serat sekam kelapa tanpa daun). Untuk menggalakkan perakaran, keratan batang telah dirawat dengan hormon komersial, SADEX 1 bagi media pasir dan media serat sekam kelapa manakala bagi media air telah dirawat dengan 600 ppm hormon IBA. Hasil kajian telah menunjukkan N.gracilis mempunyai peratusan perakaran yang paling tinggi (13.3%) berbanding N.ampullaria dan N.mirabilis (11.67%). Secara keseluruhannya, kejayaan perakaran yang dicatatkan oleh keratan batang berdaun yang berakar dalam pasir (21.7%) diiukti oleh keratan batang berdaun yang berakar dalam cocopeat (6.7%). Sementara untuk keratan batang tanpa daun yang berakar dalam cocopeat (2.5%). Tiada peratusan kejayaan bagi perakaran menggunakan media perakaran air. Pengekalan daun pada keratan batang telah dibuktikan menjadi satu faktor yang baik untuk menggalakkan perngeluaran akar dan pucuk untuk Nepenthes berbanding dengan keratan batang tanpa daun.

Kata kunci: Periuk kera, pembiakan konvensional,perakaran keratan batang,

CHAPTER ONE

INTRODUCTION

1.1 Background study

Nepenthes sp. is a carnivorous plant belongs to the family Napenthaceae. Nepenthes sp. is also known as a pitcher plant, ‘periuk-kera’ (monkey’s cooking pot) (Phillipps & Lamb,

1988). According to the Clarke (1997), Nepenthes sp. is called as pitcher plant because it is producing jug- shaped leaves or leaf extension which functions are to attract, trap and digest animals or insect in order to get nutrient beneficial.

According to Clarke (1997), the world’s largest center of distribution of pitcher plant is

Borneo Island with 31 recorded species. Peninsular Malaysia is also recognized as one of the centre of distribution of Nepenthes species in the world (Adam & Hamid, 2007). This species is known to be the largest genus, Nepenthes (Nepenthaceae). This genus is distributed from northern Australia throughout Southern China. Figure 1 (Honda, n.d.).

The distribution of Nepenthes is focus on the tropical area of the world. The species is growing from sea level up to 3400 m above sea level. The species is more likely for the exposed habitats and on nutrient poor soils (Adam et al. 1991). Besides, the species also grows in heath forest (kerangas), in lowland dipterocarp forest and on the edge of freshwater swamps

(Adam & Wilcock, 1990).

Figure 1 Distribution of Nepenthes sp. (Honda, n.d.)

Pitcher plant is divided into two discrete groups that are based on their habitat. For the species which grow from sea level up to 1000 m are known as lowland species. While for the species which grow above 1000 m above sea level are known as highland species (Clarke, 1997).

According Sani et al. (2000), several parts of the Nepenthes sp. was used to treat some ailments such as stomach-ache, dysentery and diarrhea by the Sarawak, Brunei and

Kalimantan natives. Moreover, they are not only known for their medicinal value but also recognised as their unique features which they are used as ornamental plant. Their varieties in colour, shape and size of the pitchers can be an attractive features of landscape.

Then, pitcher plant also has been recorded the medically important species which is Aedes albopictus (Beaver, 1979). It is a nepenthexene species that from its breeding sites. Nepenthes mirabilis can be used to treat gonorrhea (Razafindraib et al. 2014).

Nepenthes sp. is known to have a very low percentage of germination by seeds because of the small size of seeds (Sani et al. 2000). The best alternative method for planting the stock plant of Nepenthes sp. is by vegetative propagation. The study of vegetative propagation for

Nepenthes comes in two ways: in vitro seed germination and vegetative propagation by stem cuttings. The best vegetative propagation of Nepenthes sp. is vegetative propagation by stem cuttings. This is based on the previous study made by Sani et al. (2000) and Asun (2015).

Thus, an experiment about vegetative propagation of selected Nepenthes sp. by stem cuttings will be carried out.

Vegetative propagation is the most effective method to produce a true-to-type plant

(Krisnawati et al. 2011). This is because the plant which produced through vegetative propagation will maintain the same genes as their mother plant. Usually, vegetative propagation is used when there is low percentage in germination for some species of plant.

Hence, it is also can acts as an alternative way to some species which is hard to produce seed.

Thus, vegetative propagation is been applied to ensure the continuity of the species.

Furthermore, the labour cost is low and the technique for vegetative propagation is simple compare to the other techniques such as tissue culture.

1.2 PROBLEM STATEMENT

Nepenthes sp. or known as pitcher plant is difficult to propagate by seed. This is because

Nepenthes sp. has small seeds and shows low percentage germination by seeds (Sani et al.

2000). Moreover, the population of pitcher plant is nearly extinct due to the clearing of the land and natural forests. Hence, the vegetative propagation through rooting of stem cuttings is as an alternative method to conserve the Nepenthes sp. The conservation of Nepenthes sp. is very important because it has high potential as ornamental plants and medicinal plants.

However, there is still lack of studies to be done on the propagation of Nepenthes sp.

1.3 OBJECTIVES

The objectives of this study were as follow:

1) To investigate the rooting propensity of Nepenthes ampullaria, Nepenthes mirabilis and

Nepenthes gracilis.

2) To determine the effect of leaf retention on rooting propensity.

3) To determine the effect of different rooting media on rooting of cutting.

CHAPTER TWO

LITERITURE REVIEW

2.1 Botanical description

Nepenthes sp. comprises 70 species and widely found over to the Madagascar, Southeast Asia to the Western Pacific. There is about 31 species had been recorded in the Borneo Island

(Clarke, 1997). This study would focused on the three selected species that can be found in

Borneo Island which is Nepenthes ampullaria, Nepenthes mirabilis and Nepenthes gracilis.

2.1.1 Nepenthes ampullaria:

Figure 2 Image of pitcher Nepenthes ampullaria (Clarke, 1997).

N. ampullaria has a cylindrical stem which climbing up to 15 m high. Leaves are sub-acute to rounded apex. The pitcher is usually urceolate with two wings from top to bottom. The lower pitchers are arised from the rossett shoots with reducing lamina. Then, the inner surface is covered with the glandular. Hence, the upper pitcher is infundiular with less of wings. The

6 mouth structure is avate and horizontal (Clarke, 1997). Usually the female inflorescence is shorter than male inflorescence. N.ampullaria is distributed to “Borneo, Peninsular Malaysia,

Sumatra and New Guinera”(Adam et al. 1991). It is commonly found at the habitat with low altitude such as kerangas forest, secondary forest and within the gaps of lowland primary forest (Adam et al. 1991).

2.1.2 Nepenthes mirabilis:

Figure 3 Image of pitcher Nepenthes mirabilis (Clarke, 1997). N.mirabilis has cylindrical stem with less than 10 m length and less than 10 mm thick. Leaves have petiolate when the plant is matured. The picthers are same like the N.gracilis whereas has bulbous at the base. The lower pitcher has flattened peristome with glandular in the inner surface. The upper pitcher is cylindrical shape with two wings from top to bottom. The inner surface at the bulbous part is covered with the glandular region. The structure of the mouth is horizontal to oblique. This species is usually found in damp habitats, swampy areas that are below 100 m (Clarke, 1997). N.mirabilis can survive more in the swampy region because of its roots are permanently under water (Phillipps & Lamb, 1988). N.mirabilis is distributed to

“Borneo, Peninsular Malaysia, Java, Sumatra, Thailand, Indo-China, Southern China,

Moluccas, Philippines, Celebes, New Guinea and Australia” (Adam et al. 1991).

2.1.3 Nepenthes gracilis:

Figure 4 Image of pitcher Nepenthes gracilis (Clarke, 1997).

N. gracilis has triangular stem with less than 6 m in length and less than 5 mm thick. Leaves are coriaceous type and sessile (Clarke, 1997). According to the Phillipps and Lamb (1988)

N.gracilis has green pitcher rarely over 15 cm in length. The pitcher is bulbous at the base with less than 10 cm high and less than 3 cm wide. The mouth is circular shape and peristome is not highly developed. Peristome is almost green and reddish brown. The inner pitcher is white and rarely light purple and brown colour. The upper pitcher is lack of wings (Clarke,

1997). The distribution of N. gracilis is widely to the Borneo, Sumatra and Peninsular

Malaysia. This species is commonly found in peat swamp forest, heath forest, at the side of the paddy fields and the lowland evergreen rain forest (Adam et al. 1991).

2.2 Economic importance

The pitcher plant has high potential uses as an ornamental plant and medicinal plant. In Egypt, the pitcher plant is planted as source of medicinal which mixed with wine and bring the feeling of happiness to human (Rosso, 2010).

Besides, pitcher plants are also used as ornamental plants which cultivated by tissue culture.

This is because they have attraction and easily grown as landscape plants (Plachno, 2007).

2.3 Vegetative propagation

Vegetative propagation by stem cuttings is an alternative method to increase the planting for species that have hard germination seed or for clonal propagation (Noor Aini Shukor & Liew,

1994). Besides, vegetative propagation is asexual propagation that duplicated the genotype of the stock plant through mitotic cell division (Hartmann & Kester, 1983). Vegetative propagation also is the type of reproduction method which produced true-to-type plant

(Macdonald, 2006). Vegetative propagation of forest tree is very useful as the cloning and replicate of the stock plants. It is very important to the plant that cannot germinate directly through their vegetative reproduction part or produce small amount due to the sterility, genetic incompatibility or flowering season (Barden et al. 1987).

There are a few types of vegetative propagation such as cutting, runners, layering, grafting and tissue culture. There is study on Garcinia lucida Vesque (Clusiaceae) about vegetative propagation by stem cuttings in non-mists propagators and through graftings by Takoutsing et al. (2014). The result showed that Garcinia lucida Vesque can undergo successfully in vegetative propagation through cuttings and graftings.

In propagation by cuttings, Hartmann and Kester (1983) stated that a portion of the vegetative parts of the plants like modified stem (rhizome, tubers, corms and bulbs), root or leaf is cut from the stock plant or known as parent plant to produce new progeny.

In conclusion, there are few advantages that can gain due to the purpose of doing vegetative propagation. This method is very cheap, rapid, simple and does not require any additional knowledge or specific techniques (Hartmann & Kester, 1983).

2.4 Vegetative propagation of Nepenthes sp.

There are two methods for vegetative propagation on Nepenthes sp.: in-vitro propagation and stem cuttings (Brittnacher, n.d.).

However, the best vegetative propagation of Nepenthes sp. is vegetative propagation by stem cutting. This is based on the previous study made by Sani et al. (2000) and Asun (2015). Sani et al. (2000) reported that Nepenthes sp could be propagated vegetatively using stem cuttings and tissue culture technique. While Asun (2015) reported that Nepenthes sp. could be propagated vegetatively with the use of commercial prepared rooting hormone.

2.5 Factor affecting rooting of cuttings

2.5.1 Effect of types of cuttings

Cuttings were made up from the various body part of the plant, for example stems, modified stem (rhizomes, tubers, corms and bulbs), leaves and roots. They will be classified according to the part of the body plant that they are taken like stem cuttings, hardwood, deciduous, narrow-leaved evergreen, semi hardwood, softwood, herbaceous, leaf cuttings, leaf-bud cuttings and root cuttings. The cuttings that are selected must be free from diseases and pests

(excellent health), moderately vigorous and of known identity (Hartmann & Kester, 1983).

2.5.2 Effect of cutting position

The position of the stem where cuttings are taken will influence the rooting success.

According to the experiment effect of cutting position on Gonystylus bancanus by Aini et al.

(2010) the top position of the cutting recorded the highest survival percentage, 90.7% then followed by 86.0% and 74.7% for middle and bottom. This is because the top position gives a good response because present of juvenile tissue that actively differentiating more than in the middle and bottom position (Tchoundjeu & Leaky, 2001). In vegetative propagation, the top cuttings give the higher percentage of rooting of cutting and produce more number of roots than bottom cutting (Araya, 2005).

Aini et al. (2010) stated that bottom position of cuttings produced more root as compared to the top and middle positions. This is because bottom position has more storage part for nutrient such as carbohydrates. Hence, the carbohydrates can enhance the rooting ability of the cuttings. In conclusion, the bottom position of cuttings will give the higher success of rooting more than the others position.