In Vitro Shoot Induction of Kaempferia parviflora Wall. Ex. Baker

MULTIPLE

IN VITRO

SHOOT INDUCTION OF

KAEMPFERIA

PARVIFLORA

WALL. EX. BAKER

VITHO ALVENO A24070078

AGRONOMY AND HORTICULTURE DEPARTMENT

FACULTY OF AGRICULTURE

BOGOR AGRICULTURAL UNIVERSITY

2012

ABSTRACT

VITHO ALVENO. Multiple In Vitro Shoot Induction of Kaempferia parviflora Wall. Ex. Baker. (Supervised by NURUL KHUMAIDA and SINTHO WAHYUNING ARDIE)

This research aimed to find the best MS salt concentration and cytokinin (BAP) concentration for K. parviflora rapid multiplication by using in vitro buds as explants. The experiment was done at Plant Tissue Culture Laboratory of Agronomy and Horticulture Department, Faculty of Agriculture, Bogor Agricultural University from January to November 2011.

This research began by sterilizing shoots of K. parviflora rhizomes received from PT. Ogawa Indonesia to produce axenic culture. After about 8 weeks of growth, young shoots began to sprout. The young shoots of K. parviflora axenic culture were then cut and cultured individually on the treatment medium, which consist of two different MS salt concentrations (MS and ½ MS

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and five different 6-benzylaminopurine (BAP) concentrations (0, 1.25, 2.5, 3.75, and 5 ppm) respectively.

Explants started to grow after 2 weeks of culture. Both MS salt concentration and BAP concentration treatments did not give a significant effect on the first shoot induction time, but ½ MS medium supplemented with 5 ppm BAP could induce first shoot faster than other treatments, which is within an average of 2.5 weeks after cultured (WAC). At early observation (2 to 4 WAC), medium enriched with 3.75 ppm BAP induced more shoot than the other treatments, but at the end, control medium (MS0) induced more shoot than other

treatments. Starting from 2 WAC, the explants cultured on 0 ppm BAP medium grow more rapidly than the other treatments, but after 10 WAC, they grow slower. All treatments have the same trend of leaf growth. At 2 to 8 WAC, the number of leaves is rising rapidly, but at the 9 WAC, it began to slow down.

Generally, MS salt concentration showed significant different result on early development of leaf (2 and 3 WAC). BAP concentration treatments showed significant different result for number of shoots at the end of observation (9 to 12 WAC) and explants height at early to mid observation (2 to 8 WAC), but did not

affect number of leaves. Interaction between MS salt concentration and BAP concentration was observed at the end of observation (7, 8, 11, and 12WAC) for number of shoot, at early observation for explants height (2 to 6 WAC) and number of leaves (2 and 3 WAC).

There were total 100 explants cultured on 10 different medium compositions, but by the end of observation, fungus and bacteria contaminated 33 explants. Among 67 surviving explants, 5 explants or 7.42% of surviving explants

didn‟t regenerate shoot by the end of observation (12 WAP). There is no callus developed and all surviving plantlets have developed roots.

MULTIPLE

IN VITRO

SHOOT INDUCTION OF

KAEMPFERIA

PARVIFLORA

WALL. EX. BAKER

This bachelor thesis was submitted to the Faculty of Agriculture as a requirement for achieving the Bachelor of Agricultural Science

Vitho Alveno A24070078

AGRONOMY AND HORTICULTURE DEPARTMENT

FACULTY OF AGRICULTURE

BOGOR AGRICULTURAL UNIVERSITY

2012

BIOGRAPHY

The author was born in Jakarta at March 29, 1989 as the first of two sons of Hendra Gunawan and Livy Winata Gunawan.

The author began his formal education in Bogor Budi Mulia elementary school and graduated at 2001. The author continued his education in Budi Mulia junior high schools and graduated at 2004. After that, the authors continued his education in Senior High School I Bogor.

After graduated from senior high school, the author enrolled as an undergraduate student at Agronomy and Horticulture Department, Bogor Agricultural University at 2007 through USMI.

During his enrollment at Bogor Agricultural University, the author has joined as committee member in “Festival Tanaman XXXI” at 2010 and National Seminar of PERHORTI at 2011. The author have also worked as field assistant of Indoflowers Nursery at 2011 and practice assistant in “Plant Propagation” and

FOREWORD

Praise be to Allah SWT who has provided guidance and strength so that the author could finishes this bachelor thesis “Multiple in vitro shoot induction of Kaempferia parviflora Wall. Ex. Baker”. This bachelor thesis was submitted as one of the condition to achieve the Bachelor of Agricultural Science degree.

The author has received many help when doing the research and composing this bachelor thesis, therefore the author would like thank:

1. Dr. Ir. Nurul Khumaida and Dr. Sintho Wahyuning Ardie, SP., MSi. as supervisors who have provided guidance and assistance during the research and composing of this bachelor thesis.

2. Dr. Dewi Sukma, SP., MSi. as examiner who have advised this bachelor thesis so it could be better.

3. Dr. Ir. Eko Sulityono, MSi as academic counselor who has guided and gave many advises to the author.

4. His late beloved mother, who has given a lot of love, though she could not watch and accompany the author grew up.

5. His father and brother, who have given a lot of prayers, advices and supports.

6. Mrs. Siti Kholifah and Mrs. Juariah, AGH Plant Tissue Culture Laboratory staff, who helped author during his research.

7. All friends from AGH 44 family for the supports given to author.

8. To all parties who have provided assistance during the preparation of this thesis, the authors deliver so many thanks.

Bogor, December 2012 Author

CONTENTS

LIST OF TABLES ... iii

LIST OF FIGURES ... iv

LIST OF APPENDIX ... v

INTRODUCTION ... 1

Background ... 1

Objectives ... 3

Hypothesis ... 3

LITERATURE REVIEWS ... 4

Kaempferia galanga ... 4

Kaempferia parviflora ... 4

Plant Tissue Culture ... 6

Basal medium ... 6

Plant Growth Regulator (PGR) ... 8

Cytokinin ... 9

Bud Culture ... 10

Secondary Metabolites Production ... 11

Micropropagation of Zingiberaceae ... 12

MATERIAL AND METHODS... 14

Place and Time ... 14

Materials and Equipments ... 14

Statistical Analysis ... 14

Methods ... 15

Observation ... 16

RESULT AND DISCUSSION ... 17

General Condition ... 17

Shoot Induction of K. parviflora ... 20

K. parviflora Plantlets Height ... 24

K. parviflora Number of Leaves ... 27

CONCLUSION AND SUGGESTION ... 30

Conclusion ... 30

Suggestion ... 30

REFERENCE ... 31

LIST OF TABLES

Table 1. Analysis of variance recapitulation for first shoot induction time, number of shoot, explants height, and number of leaf observation for K. parviflora ... 19 Table 2. Effect of MS salt concentration and concentration of BAP on shoot

induction rate ... 20 Table 3. Interation effect of MS salt strength and BAP concentration on

number of K. parviflora shoot ... 23 Table 4. Interaction effect of MS salt concentration and BAP concentration

on K. parviflora plantlets height ... 26 Table 5. Effect of MS salt concentration on K. parviflora number of leaf ... 27 Table 6. Interaction effect of MS salt concentration and BAP concentration

LIST OF FIGURES

Figure 1. Morphology of K. parviflora ... 6

Figure 2. Structure of native and synthetic cytonkinins ... 10

Figure 3. Explants used. ... 14

Figure 4. ContaminatedK. parviflora plantlets ... 17

Figure 5. Performance of K. parviflora explants ... 18

Figure 6. Various BAP concentration effect on number of K. parviflora in vitro shoots. ... 21

Figure 7. Fitted line plot for number of K. parviflora in vitro shoot at 12 WAC ... 21

Figure 8. Representation of K. parviflora explants ... 22

Figure 9. Shoot regeneration rate of K. parviflora plantlets ... 24

Figure 10. Various BAP concentration effect on K.parviflora plantlets height .. 25

Figure 11. Fitted line plot for K. parviflora plantlets height at 6 WAC. ... 25

Figure 12. Effect of MS salt concentration on number of K. parviflora leaves... 27

LIST OF APPENDIX

Appendix 1. Murashige and Skoog Basal Medium Composition ... 35

Appendix 2. Analysis of Variance for Plantlets Height ... 36

Appendix 3. Analysis of Variance for Number of Shoots ... 38

INTRODUCTION

Background

Health is an important thing for human life. The use of herbal plant

products is increasing along with improved human‟s awareness on the concept of

a harmonious relationship between human and nature. Moreover, with the development of “back to nature” thinking, many people start to consume food and medicines that are made from natural ingredients. One result is the development of medicinal plant industry.

Kaempferia parviflora is a kind of galingale (aromatic ginger), which belongs to Zingiberaceae family. This plant originally came from northern part of Thailand. Most of the Thai use K. parviflora as seasoning for their traditional food, traditional medicine, and anti-inflammatory agent. Several research about Kaempferia parviflora extract bioactivity showed that it has, among others anti malarial and anti microbial effect (Yenjai et al., 2004), antioxidant effect on fermented K. parviflora rhizomes (Vichitpan et al., 2004), antidepressant effect on aged rats (Wattanathorn et al., 2007), anti inflammatory effect trough the inhibition of NO and PGE2 release (Tewtrakul and Subhadirasakul, 2008), and

inhibitory effect on Helicobacter pylori activity (Chaichanawongsaroj et al., 2010). Although K. parviflora has been used to treat inflammatory related

diseases, but it hasn‟t been commercially cultivated in a large scale.

Indonesia itself has a very large dependence toward imported medicine and conventional medicine ingredients. Development of Indonesian traditional medicine (herbal medicine), which majority made of medicinal plants, has an important role in the development of Indonesian drugs industry and community health services. Approximately 85% of the raw materials for traditional medicine industry were obtained from nature without special cultivation efforts. Besides the uncertain quantity issues, the quality of these materials are also less reliable (Indonesian Ministry of Agriculture, 2007).

The growing medicinal plant industry, requires a sustainable supply of raw materials, so it requires a stable availability of good quality plant materials. To fulfill that demands, medicinal plant cultivation needs to be improved, and to do

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that, planting material availability needs to be maintained. A germ free and true-to-type plant material will be continuously needed in large amount. Conventional propagation of K. parviflora by splitting rhizomes will not sufficient for a large scale cultivation in the future. A whole year was needed to produce rhizomes for planting materials (ICS Unido, 2009). Pathogen infestation and dormancy also posed as potential problem for conventional propagation technique. Another propagation technique will be needed and plant tissue culture holds promise for rapid multiplication.

Plant tissue culture is a plant propagation technique by isolating cell, tissue, or organ of a plant under an aseptic condition until it grows into a perfect plant. Plant tissue culture offers many advantages over conventional techniques. The main advantage is that it could offer a rapid multiplication in short time. Another advantage is with special technique, a germ-free plant could be grown, so it could be used as mother plant. Generally, there are two pathways that were widely used in tissue culture technique, which are organogenesis and embryogenesis (Gunawan, 1988).

Organogenesis can be defined as a process of plant cell or tissue forming various organs. This process provides the basis for asexual plant propagation largely from non-meristematic somatic tissues. Plant tissues have the ability to dedifferentiate from their current structural and functional state and to begin a new developmental path towards other endpoints. In vitro plant propagation used this flexibility as a common approach by regenerating multiple shoot meristems followed by root meristem induction.

Embryogenesis can be defined as the process of embryo development, Embryo itself is the earliest stage of an organism before it develops any structures or organs. Usually, in higher plants, embryos are a product of gametic fusion (zygotes), but as mentioned above, plant cells are unique, thus a morphologically and functionally correct non-zygotic embryos can also arise from widely disparate cell and tissues type at different points of plant life cycle. According to Altman and Loberant (1998), there are two developmental sequences leading to organogenesis and embryogenesis in tissue culture. They differ in the presence or absence of a callus stage, but it‟s important mainly because it relates to the genetic

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stability. A callus stage and meristem cell derived from callus usually lead to genetic aberrations than direct regeneration.

In this tissue culture technique, a part of a plant (explants) was grown on a special basal medium. Those basal medium composed of mineral nutrients (macro and micro), plant growth regulator, vitamins, amino acid, carbohydrate source (sucrose), solidifying agent (agar), distilled water, and additional organic materials. Several researchers have developed mediums for different use, like MS (Murashige and Skog), WPM (Woody Plant Medium) for woody plants, Gamborg B5 medium, and other basal mediums. There are a lot of basal mediums, but Murashige and Skoog medium is the first and most common medium for plant propagation.

The whole medium composition will determine the success rate of plant propagation trough tissue culture, especially mineral nutrients and plant growth regulators (PGR). PGR like auxin and cytokinins are used to regulate plant growth for various needs. PGR is widely used in plant tissue culture technique, but different plant varieties may respond a plant growth regulator in different ways. The effects of PGR are generally specific for explant type and plant species; therefore, a study was needed to determine the best medium composition for rapid multiplication.

Objectives

The aims of this research were to study the effect of different MS salt concentration and cytokinin (BAP) concentration to K. parviflora growth and shoot multiplication.

Hypothesis

1. There is an optimum MS salt concentration for in vitro shoot multiplication of K. parviflora.

2. There is an optimum BAP concentration for in vitro shoot multiplication of K. parviflora.

3. There is an interaction between MS salt concentration and BAP

concentration, which optimally induce multiple in vitro shoot of K. parviflora.

LITERATURE REVIEWS

Kaempferia galanga

Plants like ginger, turmeric, and galingale have been used as traditional medicine and seasonings. Galingale or kencur (Kaempferia galanga), is a member of the Zingiberaceae family. It is widely used in Southeastern Asia as food seasoning and is also widely cultivated in Southeast Asia. Rajendra et al (2011) reported that K. galanga rhizome extract showed the presence of sterols, triterpenoids, alkanoids, saponins, flavonoids, carbohydrates, and proteins.

This plant is used as herb in Indonesian cuisines, and especially in Javanese and Balinese cuisines. Beras kencur, which combines dried K. galanga powder with rice flour, is a particularly popular traditional herbal drink (jamu) used to treat rheumatism and abdominal pain.

Kaempferia parviflora

Kaempferia parviflora is a kind of galingale, which belongs to Zingiberaceae family. This plant is indigenous to the northeastern part of Thailand

and known as „Krachai Dum‟ or „Black Galingale‟ due its purplish black

rhizomes. Most of the Thai use K. parviflora as seasoning for their traditional food and medicine for various disease, especially inflammatory related diseases. The Thai also believed that K. parviflora have an aphrodisiac effect, though it

hasn‟t been scientifically proved. Although it has been used widely, but its

cultivation hasn‟t been done in a large scale. Some research showed various

bioactivities from K. parviflora rhizome extract among others: antioxidant effect from fermented K. parviflora rhizomes (Vichitphan et al., 2007), antidepressant on aged male rats (Wattanathorn et al., 2007), anti-inflammatory effect (Tewtrakul et al., 2008), and inhibitory effect on Helicobacter pylori activity (Chaichanawongsaroj et al., 2010). Yenjai et al. (2004) reported that K. parvilora rhizomes also showed an anti malarial and mild bactericidal effect.

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Botanically, Kaempferia parviflora belongs to: Kingdom : Plantae

Sub-kingdom : Phanerogamae Divisi : Spermatophyta Subdivisi : Angiospermae Kelas : Monocotyledonae Ordo : Scitaminales Family : Zingiberaceae Genus : Kaempferia

Species : Kaempferia parviflora source : ICS Unido (2009)

K. parviflora has a purplish-black rhizome (Figure 1b), straight oval leaves with 1-10 cm leaf stem, and purplish flower with white colored stem (Figure 1a). Evi (2012) showed that K. parviflora morphology is affected by environmental conditions. Shading condition affected adaxial leaf color but not the abaxial leaf color. Plants grown under natural shading and 55% artificial shading had greener adaxial leaf color than those grown under full sun condition.

K. parviflora is best grown on highland about 500-700 m above sea level (asl). Kaempferia parviflora is grown very well in a good aerated soil under mild sunlight. Old rhizomes aged 11-12 months, germ free should be kept in dry and cool place for 1-3 months before growing. Fertilizer formula 15-15-15 about 25-30 kg/ha is recommended. Harvesting time for the best crop is at 8-9 months after planting (ICS Unido, 2009). According Evi‟s reasearch (2012) for K. parviflora early vegetative growth, it was best planted at 240 m asl with natural shading. Zulfa (2012) reported that K. parviflora planted with 15 L ha-1 of bio-fertilizer and 50% of chemical fertilizer application, had the best respond on late vegetative growth. Zulfa also reported that Bio-fertilizer application could reduce the usage of chemical fertilizer by 50%. Bio-fertilizer application could also relatively increase yield and suppress disease severity of root-knot caused by nematode in the field.

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Figure 1. Morphology of K. parviflora, the whole plant (a) and K. parviflora rhizome (b) (source: Evi, 2012)

Plant Tissue Culture

Plant tissue culture is a propagation technique on an aseptic condition using a small part of plant (explants) until it grows into a perfect plant (Hartmann and Kester, 1978). Plant tissue culture was based on cellular totipotency theory, which is every cell has an ability to grow and differentiated into a new plant, even after a cell has undergone final differentiation in the plant body (Caponetti et al, 2005). According to Fowler (1983), the first and main advantage of tissue culture technique is this technique does not affected by various environmental factors, like climate, season, nor soil condition. Plant tissue culture could also be done on both large and small scale, because it requires only small area and the plants produced with this technique are uniform and pathogen free.

Basal medium

Basal medium on tissue culture is an important factor on plant tissue culture. Basal medium itself is an aseptic medium, which will provide every mineral nutrient needed by plantlet to grow well. The success rate of plant propagation trough tissue culture was generally affected by medium composition. On this technique, macro nutrient and micro nutrient has a very large effect on its success rate, therefore, various basal medium has been developed. Those mediums usually contained similar chemical materials; the only difference was its amount.

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Generally, the main composition for basal medium is inorganic salts, plant growth regulator, vitamins, amino acids, carbohydrate source, solidifying agent (agar), distilled water, and additional organic materials (Dodds and Roberts, 1995). Inorganic salts are minerals which needed by plant to grow well (Hoagland, 1972). Usually, mineral nutrients were grouped into two groups, which is macro nutrient and micro nutrient. Macro nutrients are nutrients that are needed in large amount, such as N, P, K, Ca, Mg, and S, while micro nutrients are nutrients that are needed in small amount, such as Fe, Mn, B, Cu, Zn, I, and Co.

The most used vitamins in tissue culture medium are: thiamine (vitamin B1), nicotinic acid (niacin), and pyridoxine (vitamin B6). Thiamine is an essential vitamin in tissue culture, because thiamine affects cell growth and differentiation. Vitamin C, like citric acid and ascorbic acid, also sometimes used as antioxidant to prevent or reduce browning explants.

Sucrose or sugar used as carbohydrate source in basal medium, because generally inoculated explants are still heterotroph, therefore, the explants needed enough carbohydrate for energy source. According to Chawla (2002), sucrose and glucose are a better carbohydrate source compared to fructose, lactose, maltose, galactose, raffinose.

In tissue culture technique, there are two kind of basal medium, which is solid medium and liquid medium. Solid medium has been used widely, since the use of liquid medium has some limitation, which is cultured explants could be drowned in the medium and die. For preventing that to happen, the medium have to be solidified (Bhojwani and Razdan, 1983). The most popular medium solidifying agent is agar. Agar is a mixture of polysaccharide which obtained from a species of algae (Chawla, 2002). Beside agar, other medium solidifying agent which can be used are Alginate or Gelrite.

Explants grown with tissue culture technique, usually has a narrow pH toleration with optimum point between 5.0-6.0. Plants growth will be limited under acidic condition (pH< 4.5) or alcalic condition (pH> 7.0) (Bhojwani and Razdan, 1983).

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Plant Growth Regulator (PGR)

Plant growth regulator (PGR) is an organic compound, which naturally formed on plant that will affect growth and development of a plant (Chawla, 2002). PGR distributed across plant tissues and organs, and will influence plant growth and development. In tissue culture technique, PGR is an important part of basal medium. There are five main groups of PGR, which is auxin, cytokinin, gibberellin, abscisic acid, and ethylene, but only auxin, cytokinin and gibberellin group were widely used in plant tissue culture.

Auxin has the most basic role for plant growth, which is trough its influence to cell division, growth and differentiation. Auxin was found on every plant tissue, but highest concentration of auxin was found on meristematic tissues, which is the young part of a plant. Indole-3-acetic acid or IAA, was the first successfully isolated natural auxin, but because IAA easily damaged, its use in plant tissue culture was limited. Some synthetic auxins, like 2,4-dichlorophenoxyacetic acid (2,4-D), 3-indole butric acid (IBA), and 1-napthalenacetic acid (NAA) were more widely used in medium composition. Auxin has many function on plant tissue culture, depends on its concentration and the plant tissue, but generally, auxin will promote cell growth and division on cambium and also promote callusing and root growth (Moore, 1979).

Second important PGR group is cytokinin, which will promote cell differentiation. That cell differentiation will be useful for in vitro shoot regeneration and callusing. Generally, a high concentration of cytokinin will inhibit root growth, but break apical shoot dormancy. The first natural cytokinin

ever founded was adenine, which one of the purine‟s (6-aminopurin) derivate and one component for nucleid acid. Adenine was also the most natural cytokinin found on plant tissue. Beside adenine, there are some compounds which has the same, but stronger effect as adenine, such as kinetin, benzyladenin (BA), and tetrahydropyranylbenzyladenine (PBA). Those three compound were synthetic cytokinins and will never be naturally formed on a plant (Moore, 1979).

Auxin and cytokinin were the most common PGR used in plant tissue culture. According to Scott (2008), auxin and cytokinin concentration will affect explants growth and differentiation. A high auxin concentration combined with

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low cytokinin concentration will promote root growth, while low auxin concentration and high cytokinin concentration will promote shoot growth. When both PGR have the same concentration, then it will promote callus growth.

The third PGR group is gibberellin, which was found by Kurosawa, a Japanese researcher on 1920 when he researched about Gibberella fujikuroi (Fusarium moniliforme) (Warreing and Phillips, 1981). Kurosawa found that the fungus release a compound to the infected plant that promote leaf and stem elongation. That compound was then successfully isolated and named gibberellic acid. That compound was not only found on Gibberella fujikuroi, but also on some high level plant. There are total 57 compound which have the same chemical and biological activity with gibberellic acid that isolated from Gibberella fujikuroi and named GA1-GA58. GA3 was the gibberellicacid isolated

from Gibberella fujikuroi. Generally, gibberellin promote cell elongation.

Cytokinin

Cytokinins were discovered in the search for factors that stimulate plant cell to divide. The first cytokinin ever discovered was kinetin, which was identified from autoclaved DNA, but zeatin was the first natural cytokinin ever discovered, which was extracted from immature endosperm of maize. The molecular structure of zeatin was similar to that of kinetin. Both molecules are adenine and aminopurine derivates. All naturally occurring cytokinins also have an adenine ring structure with a 5 carbon isopentenyl side chain from N6 of the adenine molecule (Figure 2). Beside zeatin, there are some compounds which has the same, but stronger effect as zeatin, such as isopentenyl-adenine (ip) and dihydrozeatin (DZ). Some synthethic compounds, such as benzyladenin (BA), and tetrahydropyranylbenzyladenine (PBA) can mimic cytokinin action or for some other compounds, antagonize it.

Generally, cytokinin is defined as PGR group, which have biological activities similar to those of trans-zeatin (Taiz and Zieger, 2006). These activities include, induce cell division in callus cell in the presence of auxins, promote bud or root formation from callus cultures in the appropriate molar ratios to auxins, delay leaves senescence, and promote expansion of dicot cotyledons.

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When a high concentration of cytokinin applied to plant cells, root growth will be inhibited, but break apical shoot dormancy (Taiz and Zieger, 2006). It is possibly because when a high level cytokinin was applied to plant cell, the level of free auxins are reduced and vice versa (Srivastava, 2001).

Figure 2. Structure of native and synthetic cytonkinins. (source: Smith, 1977)

Bud Culture

Bud culture was one of micropropagation technique by inducing axillary shoot from apical buds and lateral buds (Kane, 2005). This technique was based

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on cytokinin induced primordial growth of a shoot, which will produce small shoots. Through bud culture, million explants could be produced from just one inoculated shoot, although the shoot regeneration speed was mainly affected by plant varieties. According to Chawla (2002), there are two methods of bud culture, which are single node culture and axillar bud.

In single node method, a single node was isolated along with bud and then grown on basal medium. After it grows, the node could be rooted and then acclimated to the field. In this method, cytokinin was not used and its propagation speed is very dependent on the explants. The more explants used, the faster propagation could be done.

The second method is axillary buds. In this method, inoculated buds were grown on a high cytokinin medium. The high cytokinin concentration will break apical dominance and promote axillar buds growth. Those axillary buds were then cut into a few nodes to be used as explants or rooted and then acclimated to the field.

Secondary Metabolites Production

Secondary metabolites itself are compounds with a restricted occurrence in taxonomy groups, that are not necessary for cell (organism) to live, but play a role in the interaction of the cell (organism) with its ecosystem (Verpoorte, 2000). Plant secondary metabolites play an important role from economical point since it was widely used for drugs, fragrances, flavors, and natural dyes. As a medicinal plant K. parviflora was also cultivated for its secondary metabolites.

Cell cultures offered a hope for secondary metabolite production through cell suspensions. With plant cell suspension, plant secondary metabolites could be produced on all year-round continuously without seasonal restrain. Kurz and Constabel (1991) stated that cell suspension is an alternative method to produce secondary metabolite, especially for wild and scarce plant species. Dodds and Roberts (1995) added that although cell suspension was promising, the secondary metabolites produced often instable and some cell lines also loses its ability to synthesize the desired compound after prolonged culture.

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Micropropagation of Zingiberaceae

As a family, that has a lot of members with medicinal uses, research on zingiberaceae family member propagation has been done repeatedly. Some of them are Curcuma soloensis (Zhang et al., 2011), Etlingera elatior (Yunus et al., 2011), Hedychium spicatum (Giri and Tamta, 2011), Kaempferia galanga (Geetha et al., 1997; Chirangini et al., 2005; and Parida et al., 2010). Kaempferia rotunda (Geetha et al., 1997 and Chirangini et al., 2005) Zingiber montanum (Hamirah et al., 2010), and Zingiber serumbet (Faridah et al., 2011).

Zhang et al (2011) reported that Thidiazhuron (TDZ) promote shoot much better than BA on C. soloensis. Explants cultured on medium enriched with BA could only produce shoot up to 5 shoots/explant, but when TDZ was present, explant could produce shoot up to 18.7 shoots/explant (2.5 μM). When combined with BA, NAA could also promote shoot regeneration for C. soloensis. When TDZ was used, addition of NAA to the medium did not resulted significant improvement. These was achieve within 30 days.

Yunus et al (2011) reported that E. elatior explants cultured on medium enriched with BAP produced more shoot than explants cultured on medium enriched with kinetin or 2-ip. The quality of shoots and overall growth response were also better at all level of BAP treatment compared to other cytokinins. The highest number of shoot within 12 weeks was observed at medium enriched with 0.7 ppm BAP, which was 4.59 shoots/explant.

Giri and Tamta (2011) reported that H. spicatum responded very well to TDZ. Just as C. soloensis, when TDZ was present, number of shoots/explant increased about 200% compared to BAP and kinetin. The maximum number of

shoots was achieved by explants cultured on medium enriched with 1 μM TDZ,

which is 3.86 shoots/explant.

K. galanga has been cultivated in large scale and research about its micropropagation has also been done repeatedly. Some of those researches are done by Geetha et al (1997), Chirangini et al (2005), and Parida et al (2010). Geetha et al reported that the best medium for shoot induction was MS medium supplemented with 1 ppm BAP and 0.5 ppm NAA (8 shoots/explants), while Parida et al reported that MS medium supplemented with 1 ppm BA and 0.5 ppm

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IAA is the best medium for K. galanga shoot multiplication (11.5 shoots/explants). On the other hand, Chirangini reported that when BAP and IAA or NAA were combined, it did not promote shoot growth, but when BAP was used separately, it promoted better shoot growth up to 8.75 shoots/explants. This is probably happened because Chirangini et al subcultured the plantlets every 4 weeks. Chitra et al (2005) also reported that the plantlets established on field without in vitro developed rhizome did not dorm rhizome even after 7 months after transplantation. Similar respond might occur in K. parviflora.

In contrast with K. galanga, K. parviflora hasn‟t been cultivated in large scale; therefore not much research about K. parvflora micropropagation has been done. One of this research was done by Dheeranupattana et al (2003) about K. parviflora in vitro shoot induction. On that research, best shoot induction was observed on MS basal medium supplemented with 0.5 ppm NAA and 3 ppm BA, which is 2,4 shoot/explants within 4 weeks with sterilized K. parviflora shoots as explants.

MATERIAL AND METHODS

Place and Time

This experiment was done at Plant Tissue Culture Laboratory of Agronomy and Horticulture Department, Faculty of Agriculture, Bogor Agricultural University from January to November 2011.

Materials and Equipments

The explants used in this study were in vitro shoots from axenic culture of K. parviflora (Figure 3a). The axenic culture itself were initiated by sterilizing young shoots of K. parviflora (Figure 3b), which were received from PT. Ogawa Indonesia. The other materials used in this study are MS basal medium stock solution, sucrose, sterile distillate water, solidifying agent (agar), alcohol, spirits, plastic, and iodine (10%). The equipments used in this study are 300 ml culture bottles, laminar air flow cabinet, autoclave, baker glass, pipette, measuring glass, scissors, scalpels, pincers, analytical balance, and camera.

Figure 3. Explants used; (a) young shoot of K. parviflora; (b) axenic culture of K. parviflora cultured for 8 weeks.

Statistical Analysis

This study was prepared using Two Factors Completely Randomized Design. The first factor is MS salt concentration that consist of two concentrations, which are mediums made using normal MS medium nutrient solution concentration and mediums made using only half of normal MS medium nutrient solution concentration (Appendix 1). The second factor was BAP

B A

15

concentrations that consist of five concentrations, which are 0 ppm, 1.25 ppm, 2.5 ppm, 3.75 ppm, and 5 ppm. Therefore, this experiment consisted of 10 combinations of treatments, which are MS + 0 ppm BAP, MS + 1.25 ppm BAP, MS + 2.5 ppm BAP, MS + 3.75 ppm BAP, MS + 5 ppm BAP, ½ MS + 0 ppm BAP, ½ MS + 1.25 ppm BAP, ½ MS + 2.5 ppm BAP, ½ MS + 3.75 ppm BAP, and ½ MS + 5 ppm BAP. Each treatment was repeated 10 times, so there are 100 bottles of culture. On each bottle contained a explant, which totaled 100 explants.

The bottle‟s placements on culture rack were randomized in each replication.

Additive linear model used was as follows:

Yijk = µ + τi + αj+ ταk+ εij i = 1, 2…r and j= 1, 2…r

Yij = observation on ith MS salt concentration, jth BAP concentration µ = mean

τi. = ith MS salt concentration effect

αj = jth BAP concentration effect

ταk = kth MS salt concentration and BAP concentration interaction effect

εij = random effect of ith MS salt concentration, jth BAP concentration.

Observation data will then be subjected to analysis of variance (ANOVA) using SAS 9.2 program. If the treatments gave significant result, the data will be subjected to Duncan Multiple Range Test at α=5%.

Methods Equipment preparation

All the equipments used in this study were washed and autoclaved on 121oC and 17.5 psi of pressure for one hour. All autoclaved equipments were then placed in an oven to keep them sterile.

Medium Preparation

This experiment used two different MS salt concentration, (MS and ½ MS

)

and five different 6-benzylaminopurine (BAP) concentration (0, 1.25, 2.5, 3.75, and 5 ppm) respectively, which resulted 10 different medium composition. For preparing 1 L of medium, the medium were prepared by adding MS stock solution and BAP into distilled water half of the medium volume and dissolving

16

sucrose (3%). The medium pH was adjusted to 5.8 to 6 before adding agar and autoclaved on 121oC and 17.5 psi of pressure for 20 minutes. Autoclaved medium were then stored in the culture room for a week.

Explants Preparation

Explants preparation began by sterilizing shoots from K. parviflora rhizomes to produce axenic culture. The shoots were washed on running water and treated with fungicide for one night. The shoots were then surface sterilized with 20% hypochlorite followed by 5% hypochlorite for 10 minutes in laminar air flow cabinet and then rinsed with sterilized distilled water. The shoots were then planted to Murashige and Skoog medium without plant growth regulator (MS0). After about 8 weeks of growth, young shoots began to sprout. The young shoots of K. parviflora axenic culture were cut and then cultured individually on the treatment medium. The mother plants were separated and cultured into MS0

medium as stock. The explants were then placed at culture room under 1200 Lux light intensity for 24 hours with an average of 22oC temperature.

Observation

Observations were done every weeks starting from the 1 week after cultured (WAC) to 12WAC. Observed variables include:

1. The time when the first shoot appeared (week) 2. Number of shoots per plantlets

3. Explants height

4. Number of leaves per plantlets

5. The number and percent of contaminated culture 6. The number and percent of callused culture 7. The number and percent of explants rooted

For plantlet height, observations were done from outside culture bottles by measuring plantlet height starting from medium surface to the end of the longest leaf.

RESULT AND DISCUSSION

General Condition

This research began by sterilizing shoots from K. parviflora rhizomes to produce axenic culture. The K. parviflora rhizomes were received from PT. Ogawa Indonesia. After about 8 weeks of growth, young shoots began to sprout. The young shoots of K. parviflora axenic culture were cut and then cultured individually on the treatment medium. The explants were then placed in culture room with 22o C average temperature under 1200 Lux light intensity for 24 hours. There are total 100 explants cultured in 10 different medium compositions, but by the end of observation, fungus and bacteria contaminated 33 explants. Among 67 surviving explants, 5 explants or 7.42% of surviving explants didn‟t regenerate shoot by the end of observation (12 WAC). There is no callused culture and all surviving explants have developed roots. Contaminated explants are presented in Figure 4.

Figure 4. Kaempferia parviflora plantlets contaminated by bacteria (a) and fungi (b). Arrow indicates bacteria and fungi colonies.

Explants inoculated in medium with additional 2.5 ppm BAP showed different appearance and leaf color compared to the other treatment. Explants inoculated in both MS + 2.5 ppm BAP and ½ MS + 2.5 ppm BAP formed vitrous shoot (Figure 5). Explants cultured on medium enriched with 2.5 ppm BAP also produce less shoot than other treatment.

Vitrous plant itself is a developmental anomaly, which cause deficiency in lignification. It is suspected to be caused by ethylene, but Phan (1990) reported that vitreous explants were observed on apple explants inoculated in medium

18

enriched with BAP. On the other hand, ethylene did not cause vitrous explants, unless BAP was present. It is possibly caused by excessive cell-division promotion by cytokinins.

Figure 5. Performance of K. parviflora explants cultured on (a) ½ MS +2.5 ppm BAP, (b) MS + 2.5 ppm BAP, and (c) ½ MS + 1.25 ppm BAP

Generally, MS salt concentration did not give significantly different result except on early development of leaf (2 and 3 WAC). BAP concentration treatments give significantly different result for number of shoots at the end of observation (9 to 12 WAC) and explants height at early to mid observation (2 to 8 WAC), but did not affect number of leaf. Interaction of MS salt concentration and BAP concentration was observed on every observation. MS salt concentration and BAP concentration interaction was observed at the end of observation (7, 8, 11, and 12WAC) for number of shoot, at early observation for explants height (2 to 6 WAC) and number of leaf (2 and 3 WAC).

Although generally ½ medium produce lower result than MS medium, but medium composition did not gave a significantly different result (Table 1). Half MS medium was composed using only half of Murashige and Skoog medium macro and micro nutrient (Appendix 1), thus ½ MS medium could reduce the cost.

19

Table 1. Analysis of variance recapitulation for first shoot induction time, number

of shoot, explants height, and number of leaf observation for K. parviflora

Variable Age

(WAC)

MS salt concentration

BAP

Concentration Interaction Coeff. Var

First Shoot

Induction Time ns ns ns 62.88 (28.12)

Number of Shoots

2 ns ns ns 250.52 (33.53)

3 ns ns ns 123.13 (40.40)

4 ns ns ns 122.59 (41.43)

5 ns ns ns 122.20 (41.05)

6 ns ns * (ns) 96.26 (35.84)

7 ns * (ns) ** (*) 90.32 (35.76)

8 ns * (ns) ** (*) 90.32 (35.76)

9 ns ** (*) * (ns) 81.47 (34.44)

10 ns ** (*) * (ns) 74.40 (31.75)

11 ns ** ** 59.14 (25.24)

12 ns ** ** (*) 55.02 (24.70)

Plantlet Height 1 ns ns ns 24.03

2 ns * ** 31.15

3 ns ** ** 29.91

4 ns ** ** 28.94

5 ns ** ** 30.7

6 ns ** ** 28.25

7 ns ** ns 32.57

8 ns ** ns 31.5

9 ns ns ns 30.7

10 ns ns ns 29.51

11 ns ns ns 28.62

12 ns ns ns 28.06

Number of Leaves

2 ns ns ns 31.72

3 * ns * 42.09

4 ** ns * 40.23

5 ns ns ns 39.47

6 ns ns ns 33.23

7 ns ns ns 34.91

8 ns ns ns 34.91

9 * ns ns 29.53

10 ns ns ns 28.84

11 ns ns ns 26.61

12 ns ns ns 26.23

Note : WAC = Week(s) After Cultured; ns = not significantly different; * = significantly

different at α=5%; ** = significantly different at α=1%. Values inside brackets are

20

Shoot Induction of K. parviflora

Analysis of variance showed that MS salt concentration treatment did not show significantly different results on the number of shoots/explant. This is consistently observed at 2 to 12 WAC, as shown on Table 1 that both MS salt concentration did not differ significantly. However, BAP concentration treatment showed significantly different results at 9 and 10 WAC and very significantly different results at 11 and 12 WAC. Interaction between MS salt concentration and BAP concentration that affects the number of shoots was observed at 7, 8, 11, and 12 WAC.

Effect of MS salt concentration and BAP concentration on first shoot induction time is presented in Table 2. Generally, explants starts to grow new buds at 3 and 4 WAC. Both MS salt concentration and BAP concentration do not give a significantly different effect on the speed of shoot regeneration.

Table 2. Effect of MS salt concentration and concentration of BAP on shoot induction rate

Treatment

Percentage of culture forming shoot (%)

Average first shoot induction time

(WAC) MS salt

concentration BAP (ppm)

MS

0 100 4.00

1.25 87.5 6.85

2.5 100 7.12

3.75 78.8 3.87

5. 75 4.00

½ MS

0 100 6.00

1.25 100 5.00

2.5 83.3 5.50

3.75 100 4.87

5 100 2.50

At the beginning of observation (2 to 4 WAC), there are no BAP treatment that gave better result for number of shoots, but generally, 3.75 ppm BAP treatment induce more shoot (Figure 6). At 7 WAC, explants cultured on medium without BAP started to produce more shoot than other BAP treatments. At the end of observation, 0 ppm BAP treatment produced more shoot (3.16 shoots/explant) than other treatments, while the lowest number of shoots was observed at 2.5 ppm BAP concentration with average 1.64 shoots/explant.

21

Figure 6. Various BAP concentration effect on number of K. parviflora in vitro shoots.

Regression analysis showed that BAP concentration has negative quadratic response to K. parviflora shoot with regression equation as follows: Y = 0.127x2 - 0.846x + 3.153 and R² value = 0.127 (Figure 7). The low R2 value (0.127) showed that only 12.7 % of the whole variance could be explained by this equation. The number of shoot will decrease as BAP concentration raised to 3.331 ppm and then number of shoot will start to increase again. Similar response was also reported by Hamirah et al (2010) on Zingiber montanum (red ginger) when the medium was enriched with 2-ip.

Figure 7. Fitted line plot for number of K. parviflorain vitro shoot at 12 WAC Regression analysis did show that BAP concentration has quadratic response with concave graph, but several researchers reported that additional cytokinin would promote shoot induction up to certain point for some Zingiberaceae family member. Some of them are K. galanga (Geetha et al., 1997; Chitrha et al., 2004; and Parida et al., 2010), K. rotunda (Geetha et al., 1997),

0 0.5 1 1.5 2 2.5 3 3.5

0 2 4 6 8 10 12

N um be r o f S h o o ts/ expl a n t

Week(s) After Cultured

0 ppm 1.25 ppm 2.5 ppm 3.75 ppm 5 ppm 0 1 2 3 4 5 6 7

0 1.25 2.5 3.75 5

N um b er o f S h o o t(s ) BAP Concentration

22

Z. serumbet (Faridha et al., 2011), Z. montanum (Hamirah et al., 2010). Cytokinins can promote cell division and cause release of shoot apical dominance, thereby stimulating growth of lateral buds and resulting in multiple shoot formation (Gaba, 2005).

Additional BAP did not promote shoot regeneration on K. parviflora, but cytokinins also affected other plant function, like cell division and cell development. Figure 8 shows that explants cultured on BAP enriched medium looks more vigorous than explants cultured on medium without BAP. Explants cultured on medium without BAP looks thinner compared to those cultured on medium enriched with BAP.

Figure 8. Representation of K. parviflora explants cultured on: a) MS medium; b) MS + 3.75 ppm BAP; c) MS + 5 ppm BAP; d) ½ MS medium e) ½ MS + 3.75 ppm BAP; f) ½ MS + 5 ppm BAP.

Interaction of MS salt strength and BAP concentration was observed on 7th, 8th, 11th and 12th week after planting. At the beginning of observation (2nd to 6th week after planting), the best medium was MS medium + 5 ppm BAP which produced 1.57 shoots/explants at 6th week. Starting from 7th week to 12th week, the highest number of shoot was observed on MS +0 ppm BAP medium (Table 3), which produce an average of 5.25 shoots/explants at 12th week. Duncan Multiple

a b c

23

Range Test also showed that MS + 0 ppm BAP was the best medium for K. parviflora shoot regeneration.

Table 3. Interation effect of MS salt strength and BAP concentration on number of K. parviflora shoot

MS salt concentration

BAP (ppm)

Age (WAC)

7 8 11 12

MS

0 3.16 (1.80) a 3.16 (1.80) a 4.20 (2.11) a 5.25 (2.39) a

1.25 0.37 (0.90) c 0.37 (0.90) c 1.75 (1.45) b 2.37 (1.63) b

2.75 0.50 (0.96) bc 0.50 (0.96) bc 1.00 (1.22) b 1.50 (1.40) b

3.75 1.62 (1.34) abc 1.62 (1.34) abc 2.00 (1.48) b 2.12 (1.53) b

5 1.20 (1.21) bc 1.20 (1.21) bc 1.00 (1.17) b 1.33 (1.26) b

1/2 MS

0 1.22 (1.25) bc 1.22 (1.25) bc 2.00 (1.57) b 2.12 (1.60) b

1.25 2.00 (1.49) ab 2.00 (1.49) ab 2.28 (1.62) b 2.28 (1.62) b

2.75 1.00 (1.15) bc 1.00 (1.15) bc 1.66 (1.41) b 1.83 (1.45) b

3.75 1.16 (1.25) bc 1.16 (1.25) bc 1.60 (1.43) b 1.80 (1.50) b

5 1.71 (1.46) abc 1.71 (1.46) abc 2.14 (1.59) b 2.28 (1.62) b

Note : Values inside bracket were transformed values using 2 �+ 1_{; means followed by same} letter in the same collumn did not differ significantly based on Duncan Multiple Range Test

at α=5%

Table 3 also showed that during the mid to end of observation MS salt concentration have more effect on number of shoots compared to BAP concentration. It is possibly caused by no subculture was done during this research.

In this research, the highest number of shoot within 4 weeks was observed on MS supplemented with 3.75 ppm BAP, which produced only 1.77 shoot(s)/explants. In other study, Dheeranupattana et al. (2003) showed that best medium for K. parviflora shoot induction was MS basal medium supplemented with 0.5 ppm NAA and 3 ppm BA, which is 2.4 shoot(s)/explants within 4 weeks. Compared to this study, the number of shoot induced within 4 weeks in Dheeranupattana et al. study was higher. This is probably caused by the absent of auxin in the mediums used in this study, as stated by Geetha et al (1997) for K. galanga and K. rotunda. When BAP was used separately, both K. galanga and K. rotunda only produced 2-3 shoots/explants, but when BAP was combined with NAA, K. galanga could produce up to 10 shoots/explants and K. rotunda could produce up to 7 shoots/explants. Parida et al (2010) also reported similar respond on K. galanga when low concentration BAP (1 ppm) was combined with IAA

24

(0.5 ppm), but not when higher concentration of BAP was used. Kaempferia parviflora might respond in similar way.

Figure 9. Shoot regeneration rate of K. parviflora plantlets

Figure 9 showed that short-term rapid propagation of K. parviflora could use MS medium supplemented with 3.75 ppm BAP for 3 - 4 weeks. Although it

doesn‟t produce shoot as much as explants cultured on MS0 medium at the end of

observation (Table 3), its shoot regeneration rate was very high on early observation (about 0.4 shoot/explant/week at 4 WAC), which for some cases, better than MS0 medium that reach similar rate only at 7 WAC. Long-term

propagation could use MS0 medium, since it was easier and certainly cheaper to

make.

K. parviflora Plantlets Height

Analysis of variance showed that MS salt concentration treatment did not give significantly different results on the plantlets height. This is consistently observed from 1 to 12 WAC, as shown on Table 1. BAP concentration treatment showed significantly different results at 2 WAC and very significantly different results starting from 3 week to 8 WAC. Interaction between MS salt concentration and BAP concentration that affects the plantlets height was observed at 2 to 6 WAC. At the end of observation, no treatment showed significantly different result. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

2 3 4 5 6 7 8 9 10 11 12

S h o o t( s) /w ee k /e xp la n ts

Week(s) After Cultured

MS + 0 MS + 1.25 MS + 2.5 MS + 3.75 MS + 5 1/2 MS + 0 1/2 MS + 1.25 1/2 MS + 2.5 1/2 MS + 3.75 1/2 MS + 5

25

Explants cultured on BAP free medium grow faster than the other treatments, as shown on Figure 10. Starting from 2 WAC, the explants cultured on 0 ppm BAP medium grow more rapidly than the other treatments, but after 10 WAC, they grow slower. On the other hand, explants cultured on medium enriched with BAP keep growing steadily. It is possible that the growth speed declining on BAP free medium was because the explants have reached its maximum growth in bottle. It is obvious that BAP did not induce K. parviflora plantlets to grow taller.

Figure 10. Various BAP concentration effect on K.parviflora plantlets height Regression analysis on explants height at 6 WAC showed that BAP concentration had quadratic response with concave graph and regression equation as follows: y = 0.303x2 - 1.958x + 8.284 and R² value = 0.240 (Figure 11). Just as number of shoots, regression analysis on explants height showed low R² value, which is 0.240. It means that only 24% of the whole variance could be explained by this equation. The quadratic response with concave graph also provides hope that an increased BAP concentration above 5 ppm can increase plantlets height growth. The same applies to number of shoots.

Figure 11. Fitted line plot for K. parviflora plantlets height at 6 WAC. 0 2 4 6 8 10 12

0 2 4 6 8 10 12

P la n tl et h ei gh t (c m )

Week(s) After Cultured

0 ppm 1.25 ppm 2.5 ppm 3.75 ppm 5 ppm

y = 0.303x2_{- 1.958x + 8.284} R² = 0.240

0 5 10 15

0 1.25 2.5 3.75 5

P la n tl et s H egh t (c m )

26

Interaction between MS salt concentration and BAP concentration for Kaempferia parviflora plantlets height was observed from 2 WAC to 6 WAC. From the beginning of observation, explants cultured on ½ MS medium + 0 ppm BAP grow faster than other treatments, especially at 1 to 3 WAC. As shown on Table 4, explants cultured on ½ MS medium without BAP grow faster than other treatments. Duncan Multiple Range Test also showed that ½ MS medium with no BAP was the best medium for plantlets height growth for the first four weeks. This is possibly caused by nutrient concentration on medium. ½ MS medium composed using only half of macro and micro nutrients of MS medium, therefore osmosis would be easier and the explants would grow faster. However, starting from 6 WAC, explants cultured on MS + 0 ppm BAP and MS + 1.25 ppm BAP medium started to grow faster. This is also suspected caused by medium concentration. At 6 WAC, some of the nutrients in medium have been absorbed by the explants, thus decreasing medium concentration, allowing easier osmosis. Table 4. Interaction effect of MS salt concentration and BAP concentration on

K. parviflora plantlets height MS salt

concentration

BAP concentration (ppm)

Age (WAC)

2 3 4 5 6

MS

Plantlet height (cm)

0 3.01 bc 4.35 bc 5.13 bc 5.68 bc 7.38 ab

1.25 3.57 bc 4.87 b 5.67 b 6.71 ab 7.67 ab

2.75 3.75 b 3.96 bc 4.50 bc 5.22 bc 5.87 bcd

3.75 3.50 bc 4.53 bc 5.07 bc 5.61 bc 6.60 bc

5 3.13 bc 4.27 bc 4.23 bc 4.46 c 5.15 cd

1/2 MS

0 5.33 a 7.61 a 8.02 a 8.42 a 9.21 a

1.25 2.68 bc 3.28 c 3.77 c 4.38 c 4.73 cd

2.75 2.81 bc 3.24 c 3.66 c 4.05 c 4.20 d

3.75 2.48 c 3.45 bc 3.58 c 3.93 c 4.81 cd

5 3.02 bc 3.90 bc 4.37 bc 4.98 bc 6.32 bc

Note : Means followed by same letter in the same collumn did not differ significantly based on

Duncan Multiple Range Test at α=5%

Based on Table 4, ½ MS without BAP could be recommended for a rapid growth of K. parviflora plantlets. ½ MS + 0 ppm BAP medium could not promote K.parviflora shoot growth, but it promote growth faster than other treatments. That means, ½ MS + 0 ppm BAP medium was best used to promote plantlets growth before acclimatized and planted to the field.

27

Number of Leaves

Some explants strated to grow leaf at 2 WAC, and at 3 WAC, all leaves have fully emerged. Analysis of variance showed that MS salt concentration gave a very significantly different result for number of leaves only at 4 WAC and a significantly different result on the 3 and 9 WAC (Table 1). Generally, explants cultured on MS medium produce more leaf than on ½ MS medium throughout the observation (Table 5).

Table 5. Effect of MS salt concentration on K. parviflora number of leaves.

MS salt concentration

Age (WAC)

3 4 5 6 7 8 9 10 11 12

MS 1.79 a 1.88 a 2.25 2.67 3.00 3.00 3.60 a 3.59 3.71 3.77 1/2 MS 1.42 b 1.45 b 2.15 2.51 2.71 2.71 3.08 b 3.08 3.24 3.48

Note : Means followed by same letter in the same row did not differ significantly at α=5% (Duncan Multiple Range Test).

Analysis of variance showed that MS salt concentration gave a very significantly different result for number of leaves only at 4 WAC and a significantly different result on the 3 and 9 WAC (Table 1). Figure 12 showed MS salt strength effect on K. parviflora number of leaves at 3, 4, and 9 WAC. It is clear that salt concentration affect K. parviflora ability to produce leaf at early stage of growth. At 12WAC, MS medium produce more leaf than ½ MS medium, which is 3.77 leaf/explants whereas ½ MS medium only produce 3.48 leaf/explants.

Figure 12. Effect of MS salt concentration on number of K. parviflora leaves. 0 1 2 3 4 5

3 4 9

N ur o f L ea v es /e xp la n ts

Week(s) After Cultured

MS 1/2 MS

28

BAP treatment did not show significantly different result throughout the observation (Table 1). The highest number of leaf was observed on explant cultured on medium enriched with 3.75 ppm BAP, while BAP free treatment produce the lowest number of leaf. Regression analysis showed that BAP concentration has quadratic response on number of leaf with equation as follows : y = -0.052x2 + 0.347x + 3.316 and R2 value = R² = 0.070. The same respond also observed on torch ginger (Etlingera elatior) by Yunus et al (2011). In addition to that, explants inoculated on medium enriched with BAP, would grow larger leaves (Figure 8).

Figure 13. Fitted line plot for number of K. parviflora leaves at 12 WAC.

Interaction between MS salt concentration and BAP concentration was observed at 3 and 4 WAC. At 12 WAC, the highest number of leaves was observed on MS + 1.25 ppm BAP and MS + 3.75 ppm BAP with average 4.12 leaf/explants. Although ½ MS + 0 ppm BAP did not induce higher number of leaves, but it did not differ significantly to other treatments (Table 6). Since ½ MS + 0 ppm BAP also promote faster K. parviflora plantlets height growth, it could be said that ½ MS + 0 ppm BAP was the best medium for K. parviflora vegetative growth.

y = -0.052x2_{+ 0.347x + 3.316}

R² = 0.070 0

1 2 3 4 5 6

0 1.25 2.5 3.75 5

N

um

b

er

o

f

le

av

es

29

Table 6. Interaction effect of MS salt concentration and BAP concentration on K. parviflora number of leaves from 3 to 12 WAC

MS BAP WAC

Salt (ppm) 3 4 5 6 7 8 9 10 11 12

MS

0 1.85 abc 2.00 ab 2.50 2.67 2.83 2.83 3.40 3.40 3.40 3.25

1.25 1.87 abc 2.25 a 2.62 3.12 3.25 3.25 4.00 4.00 4.12 4.12

2.75 1.50 abc 1.50 abc 1.87 2.25 3.00 3.00 3.25 3.25 3.25 3.37

3.75 2.22 a 2.25 a 2.50 2.87 3.12 3.12 3.75 3.75 4.00 4.12

5 1.42 bc 1.33 bc 1.66 2.33 2.60 2.60 3.50 3.33 3.67 3.67

1/2 MS

0 2.00 ab 2.00 ab 2.77 3.00 3.11 3.11 3.22 3.12 3.37 3.37

1.25 1.10 c 1.10 c 1.80 2.40 2.50 2.50 2.75 2.75 2.71 2.85

2.75 1.55 abc 1.62 abc 2.28 2.85 2.85 2.85 3.42 3.33 3.50 3.83

3.75 1.14 c 1.16 c 2.00 2.00 2.33 2.33 3.00 3.40 3.60 3.80

5 1.28 bc 1.28 bc 1.85 2.14 2.71 2.71 3.00 3.00 3.14 3.71

Note : Means followed by same letter in the same collumn did not differ significantly based on

Duncan Multiple Range Test at α=5%

Short-term rapid propagation of K. parviflora could use MS medium supplemented with 3.75 ppm BAP for 3 - 4 weeks. One drawback of this method was relatively expensive. For massive propagation, the produced shoot should be sub-cultured to the same medium for more shoot production. That means, MS medium supplemented with 3.75 ppm BAP will be required in monthly base. For this case, MS0 medium could be used as an alternative. It produced more shoot,

but at a slower rate (about 0.4 shoot/explant/week at 7 WAC). Long-term propagation should use MS0 medium, since it was easier and certainly cheaper to

make.

Based on Table 4 and Table 6, ½ MS without BAP could be recommended for a rapid growth of K. parviflora plantlets. ½ MS + 0 ppm BAP medium could not promote K. parviflora shoot growth, but it promote faster growth compared to the other treatments, and although ½ MS + 0 ppm BAP did not induce higher number of leaves, but it did not differ significantly with other treatments. That means ½ MS + 0 ppm BAP medium was best used to promote plantlets growth before acclimatized and planted to the field.

CONCLUSION AND SUGGESTION

Conclusion

Based on this research, short-term rapid propagation of K. parviflora could use MS medium supplemented with 3.75 ppm BAP for 3 - 4 weeks. Although it

doesn‟t produce shoot as much as explants cultured on MS0 medium at the end of observation, its shoot regeneration rate was very high on early observation (about 0.4 shoots/week at 4 WAC), which for some cases, better than MS0 medium that

reach similar rate at 7 WAC. Long-term propagation could use MS0 medium,

since it was easier and certainly cheaper to make. The produced shoot could then be sub-cultured to the same medium for more shoot production or to ½ MS medium for rapid growth before acclimatized and then planted on the field.

Suggestion

Further study should use a wider range of BAP (higher than 5 ppm) or other cytokinins (kinetin, 2ip) and combined with auxins. Further study should also use medium enriched with organic source like coconut water. Research on somatic embryogenesis should also be done.

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Appendix 1. Murashige and Skoog Basal Medium Composition Stock

Solution Materials

Concentration in Stock Solution (mg/L)

MS ½ MS

Volume of stock solution (ml)* Final concentrati on (mg/L) Volume of stock solution (ml)* Final concentrat ion (mg/L) Macronutrients

A NH4NO3 82.500 20 1,650.000 10 825.0000

B KNO3 95.000 20 1,900.000 10 850.0000

C CaCl2.2H2O 44.000 10 440.000 5 220.0000

D MgSO4.7H2O 37.000 ₁₀ 370.000 ₅ 185.0000

KH2PO4 17.000 170.000 85.0000

Micronutrients

E FeSO4.7H2O 5.560 5 27.800 2.5 13.9000

Na2EDTA 7.460 37.300 18.6500

F MnSO4.H2O 3.380 5 16.900 2.5 8.4500

ZnSO4.7H2O 1.720 8.600 4.3000

H3BO3 1.240 6.200 3.1000

KI 0.166 0.830 0.4150

Na2MoO4.2H2O 0.050 0.250 0.1250

CoCl.6H2O 0.005 0.025 0.0125

CuSO4.5H2O 0.005 0.025 0.0125

Organics

Vitamins Glycine 0.200 10 2.000 10 2.0000

Nicotinic acid 0.050 0.500 0.5000

Pyridoxine.HCl 0.050 0.500 0.5000

Thiamine.HCl 0.010 0.100 0.1000

Myo-inositol 100.000 1 100.000 1 100.0000

Sucrose 30 g 30 g

Agar 7 g 7 g

Source : Gunawan (1988);

36

Appendix 2. Analysis of Variance for Plantlets Height

df MS

2 WAC

MS Salt Concentration 1 0.27

BAP Concentration 4 0.18

Medium * BAP 4 0.6

Error 83 1.07

Total 92

3 WAC

MS Salt Concentration 1 0.2

BAP Concentration 4 13.94

Medium * BAP 4 14.95

Error 71 1.7

Total 80

4 WAC

MS Salt Concentration 1 1.07

BAP Concentration 4 15.03

Medium * BAP 4 13.67

Error 66 1.98

Total 75

5 WAC

MS Salt Concentration 1 2.69

BAP Concentration 4 14.87

Medium * BAP 4 15.61

Error 65 2.78

Total 74

6 WAC

MS Salt Concentration 1 8.41

BAP Concentration 4 22.39

Medium * BAP 4 16.03

Error 65 3.12

Total 74

7 WAC

MS Salt Concentration 1 13.54

BAP Concentration 4 23.38

Medium * BAP 4 12.68

Error 64 6.1

Total 73

37

Appendix 2. Analysis of Variance for Plantlets Height (continued)

df MS

8 WAC

MS Salt Concentration 1 13.07

BAP Concentration 4 25.59

Medium * BAP 4 12.45

Error 64 6.28

Total 73

9 WAC

MS Salt Concentration 1 15.11

BAP Concentration 4 22.42

Medium * BAP 4 15.62

Error 60 7.3

Total 69

10 WAC

MS Salt Concentration 1 13.39

BAP Concentration 4 16.91

Medium * BAP 4 11.56

Error 56 7.53

Total 65

11 WAC

MS Salt Concentration 1 9.62

BAP Concentration 4 13.94

Medium * BAP 4 11.27

Error 55 7.78

Total 64

12 WAC

MS Salt Concentration 1 2.31

BAP Concentration 4 11.36

Medium * BAP 4 10.53

Error 55 8.23

Total 64

38

Appendix 3. Analysis of Variance for Number of Shoots

df MS

2 WAC

MS Salt Concentration 1 0.16

BAP Concentration 4 0.08

Medium * BAP 4 0.08

Error 83 0.07

Total 92

3 WAC

MS Salt Concentration 1 0.06

BAP Concentration 4 0.29

Medium * BAP 4 0.27

Error 71 0.19

Total 80

4 WAC

MS Salt Concentration 1 0.02

BAP Concentration 4 0.18

Medium * BAP 4 0.29

Error 66 0.21

Total 75

5 WAC

MS Salt Concentration 1 0.06

BAP Concentration 4 0.42

Medium * BAP 4 0.47

Error 65 0.24

Total 74

6 WAC

MS Salt Concentration 1 0.18

BAP Concentration 4 0.12

Medium * BAP 4 0.4

Error 65 0.18

Total 74

7 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.44

Medium * BAP 4 0.7

Error 64 0.2

Total 73

39

Appendix 3. Analysis of Variance for Number of Shoots (continued)

df MS

8 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.6

Medium * BAP 4 0.74

Error 64 0.27

Total 73

9 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.67

Medium * BAP 4 0.43

Error 60 0.21

Total 69

10 WAC

MS Salt Concentration 1 0.006

BAP Concentration 4 0.52

Medium * BAP 4 0.38

Error 56 0.36

Total 65

11 WAC

MS Salt Concentration 1 0.02

BAP Concentration 4 0.51

Medium * BAP 4 0.37

Error 55 0.14

Total 64

12 WAC

MS Salt Concentration 1 0.09

BAP Concentration 4 0.59

Medium * BAP 4 0.45

Error 55 0.15

Total 64

40

Appendix 4. Analysis of Variance for Number of Leaves

df MS

2 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.08

Medium * BAP 4 0.2

Error 83 0.09

Total 92

3 WAC

MS Salt Concentration 1 2.71

BAP Concentration 4 0.88

Medium * BAP 4 1.17

Error 71 0.45

Total 80

4 WAC

MS Salt Concentration 1 3.64

BAP Concentration 4 0.98

Medium * BAP 4 1.53

Error 66 0.44

Total 75

5 WAC

MS Salt Concentration 1 0.17

BAP Concentration 4 1.54

Medium * BAP 4 1.15

Error 65 0.75

Total 74

6 WAC

MS Salt Concentration 1 0.44

BAP Concentration 4 0.86

Medium * BAP 4 1.55

Error 65 0.73

Total 74

7 WAC

MS Salt Concentration 1 1.46

BAP Concentration 4 0.23

Medium * BAP 4 0.88

Error 64 0.99

Total 73

41

Appendix 4. Analysis of Variance for Number of Leaves (continued)

df MS

8 WAC

MS Salt Concentration 1 1.46

BAP Concentration 4 0.23

Medium * BAP 4 0.88

Error 64 0.99

Total 73

9 WAC

MS Salt Concentration 1 4.8

BAP Concentration 4 0.03

Medium * BAP 4 1.12

Error 60 0.96

Total 69

10 WAC

MS Salt Concentration 1 4.21

BAP Concentration 4 0.24

Medium * BAP 4 0.92

Error 56 0.92

Total 65

11 WAC

MS Salt Concentration 1 3.68

BAP Concentration 4 0.43

Medium * BAP 4 1.42

Error 55 0.85

Total 64

12 WAC

MS Salt Concentration 1 1.33

BAP Concentration 4 0.66

Medium * BAP 4 1.54

Error 55 0.9

Total 64

Appendix 2. Analysis of Variance for Plantlets Height

df MS

2 WAC

MS Salt Concentration 1 0.27

BAP Concentration 4 0.18

Medium * BAP 4 0.6

Error 83 1.07

Total 92

3 WAC

MS Salt Concentration 1 0.2

BAP Concentration 4 13.94

Medium * BAP 4 14.95

Error 71 1.7

Total 80

4 WAC

MS Salt Concentration 1 1.07

BAP Concentration 4 15.03

Medium * BAP 4 13.67

Error 66 1.98

Total 75

5 WAC

MS Salt Concentration 1 2.69

BAP Concentration 4 14.87

Medium * BAP 4 15.61

Error 65 2.78

Total 74

6 WAC

MS Salt Concentration 1 8.41

BAP Concentration 4 22.39

Medium * BAP 4 16.03

Error 65 3.12

Total 74

7 WAC

MS Salt Concentration 1 13.54

BAP Concentration 4 23.38

Medium * BAP 4 12.68

Error 64 6.1

Total 73

Appendix 2. Analysis of Variance for Plantlets Height (continued)

df MS

8 WAC

MS Salt Concentration 1 13.07

BAP Concentration 4 25.59

Medium * BAP 4 12.45

Error 64 6.28

Total 73

9 WAC

MS Salt Concentration 1 15.11

BAP Concentration 4 22.42

Medium * BAP 4 15.62

Error 60 7.3

Total 69

10 WAC

MS Salt Concentration 1 13.39

BAP Concentration 4 16.91

Medium * BAP 4 11.56

Error 56 7.53

Total 65

11 WAC

MS Salt Concentration 1 9.62

BAP Concentration 4 13.94

Medium * BAP 4 11.27

Error 55 7.78

Total 64

12 WAC

MS Salt Concentration 1 2.31

BAP Concentration 4 11.36

Medium * BAP 4 10.53

Error 55 8.23

Total 64

Appendix 3. Analysis of Variance for Number of Shoots

df MS

2 WAC

MS Salt Concentration 1 0.16

BAP Concentration 4 0.08

Medium * BAP 4 0.08

Error 83 0.07

Total 92

3 WAC

MS Salt Concentration 1 0.06

BAP Concentration 4 0.29

Medium * BAP 4 0.27

Error 71 0.19

Total 80

4 WAC

MS Salt Concentration 1 0.02

BAP Concentration 4 0.18

Medium * BAP 4 0.29

Error 66 0.21

Total 75

5 WAC

MS Salt Concentration 1 0.06

BAP Concentration 4 0.42

Medium * BAP 4 0.47

Error 65 0.24

Total 74

6 WAC

MS Salt Concentration 1 0.18

BAP Concentration 4 0.12

Medium * BAP 4 0.4

Error 65 0.18

Total 74

7 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.44

Medium * BAP 4 0.7

Error 64 0.2

Total 73

Appendix 3. Analysis of Variance for Number of Shoots (continued)

df MS

8 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.6

Medium * BAP 4 0.74

Error 64 0.27

Total 73

9 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.67

Medium * BAP 4 0.43

Error 60 0.21

Total 69

10 WAC

MS Salt Concentration 1 0.006

BAP Concentration 4 0.52

Medium * BAP 4 0.38

Error 56 0.36

Total 65

11 WAC

MS Salt Concentration 1 0.02

BAP Concentration 4 0.51

Medium * BAP 4 0.37

Error 55 0.14

Total 64

12 WAC

MS Salt Concentration 1 0.09

BAP Concentration 4 0.59

Medium * BAP 4 0.45

Error 55 0.15

Total 64

Appendix 4. Analysis of Variance for Number of Leaves

df MS

2 WAC

MS Salt Concentration 1 0.1

BAP Concentration 4 0.08

Medium * BAP 4 0.2

Error 83 0.09

Total 92

3 WAC

MS Salt Concentration 1 2.71

BAP Concentration 4 0.88

Medium * BAP 4 1.17

Error 71 0.45

Total 80

4 WAC

MS Salt Concentration 1 3.64

BAP Concentration 4 0.98

Medium * BAP 4 1.53

Error 66 0.44

Total 75

5 WAC

MS Salt Concentration 1 0.17

BAP Concentration 4 1.54

Medium * BAP 4 1.15

Error 65 0.75

Total 74

6 WAC

MS Salt Concentration 1 0.44

BAP Concentration 4 0.86

Medium * BAP 4 1.55

Error 65 0.73

Total 74

7 WAC

MS Salt Concentration 1 1.46

BAP Concentration 4 0.23

Medium * BAP 4 0.88

Error 64 0.99

Total 73

Appendix 4. Analysis of Variance for Number of Leaves (continued)

df MS

8 WAC

MS Salt Concentration 1 1.46

BAP Concentration 4 0.23

Medium * BAP 4 0.88

Error 64 0.99

Total 73

9 WAC

MS Salt Concentration 1 4.8

BAP Concentration 4 0.03

Medium * BAP 4 1.12

Error 60 0.96

Total 69

10 WAC

MS Salt Concentration 1 4.21

BAP Concentration 4 0.24

Medium * BAP 4 0.92

Error 56 0.92

Total 65

11 WAC

MS Salt Concentration 1 3.68

BAP Concentration 4 0.43

Medium * BAP 4 1.42

Error 55 0.85

Total 64

12 WAC

MS Salt Concentration 1 1.33

BAP Concentration 4 0.66

Medium * BAP 4 1.54

Error 55 0.9

Total 64