in vitro regeneration of shorea parvifolia dyer ssp ... vitro regeneration of shorea parvifolia dyer...

IN VITRO REGENERATION OF SHOREA PARVIFOLIA DYER SSP PARVIFOU A

Kho Pei Ee

Bachelor of Science with Honours (Resource Biotechnology)

2005

Pusat Khidmt Maklumllt Alcadcmilc UNIVERSITI MALAYSIA SAftAWAK

IN VITRO REGENERATION OF SHOREA PARVIFOLIA DYER SSP PARVIFOLIA

KHOPEIEE

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor Science with Honours

(Biotechnology Resource)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2005

ACKNOWLEDGEMENTS

I would like to express my sincere appreciation and deepest gratitude to my obliging

supervisor Dr Ho Wei Seng for his continual guidance in completing my final year

project thesis Besides I also want to express my truthful gratitude to my co-supervisor

Assoc Prof Dr Sim Soon Liang for her guidance on tissue culture technique Special

thank to Assoc Prof Dr Kasing Apun for her kindly assistance in completing the Drug

sensitivity test Not forgetting all of the staff at Forest Seed Bank (FSC) Semenggok

who well-maintained S parvifolia Dyer parvifolia seedlings I am also truly grateful to

Mr Lau Ee Ting (Msc) Chong Yee Ling (Msc) postgraduates of Plant Tissue Culture

and microbiology laboratory for their kindly assistance Lastly thanks to my beloved

parents Then JipKhong and all of my friends (Tan Siew Khim Chia Sze Wooi Tan Sia

Hong and Loh Pihk Foong) for being supportive and understanding

ABSTRACT

Shorea parvifolia Dyer ssp parvifolia or meranti sarang punai is a woody species from Dipterocarpaceae family It is economically important and its timber as well as nonshytimber products have long been recognized in Southeast Asian and other regions The present study was carried out to establish the micropropagation protocols for use in largeshyscale propagation and the development of an in vitro regeneration system for the use in genetic manipulation of S parvifolia Dyer parvifolia Lamina internodal nodal and shoot tips explants from healthy 2-year-old seedlings of S parvifolia Dyer parvifolia were surface sterilized in 70 ethanol prior to treatment of 10 (vv) clorox with I mIll Tween 20 for IS minutes Internodal nodal and shoot tip explants were inoculated on Y2 MS (without vitamins and sucrose) soft agar medium incorporated with IS mIll PPM 10 mlll tetracycline and 3g1 activated charcoal Meanwhile lamina explants were inoculated on the same formulation of medium except reducing the PPM to 10mlll After three days the explants were subcultured to Y2 MS soft agar medium which incorporated with S mill PPM and S mlll tetracycline BAP was found effective for shoot growth of this species There was about 80 of shoot tip explants sprouted new shoots on Y MS soft agar medium incorporated with Imlll PPM and BAP either at OSmgll or 2Smgll Meanwhile only SO of nodal explants sprouted axillary buds on Y MS soft agar medium supplemented with lmlll PPM and OSmgll BAP Most of the nodal explants failed to react with 10mgli and 2Smgll BAP Yellowish and greenish granulated callus were successfully induced from cut edges of internodal explants cultured on Y MS soft agar medium supplemented with I mill PPM Kinetin at 02mgll along with O2mgl NAA However lamina and internodal explants failed to induce callus formation on Y MS soft agar medium supplemented with different concentration of2 4-D and picioram

Key words Shorea parvifolia Dyer ssp parvifolia in vitro regeneration callus induction shoot growth

11

ABSTRAK

Shorea parvifolia Dyer ssp parvifolia alau meranti sarang punai merupakan spesies berkayu yang diklasifikasikan dalam famili Dipterocarpaceae Spesies ini memainkan peranan penling dalam ekanomi Negara Kayu balak dan produk jenis bukan kayu spesies ini amal popular di Asia tenggara dan wilayah yang lain Kajian ini dilaksanakan dengan lujuan menubuhkan protokol micropropagasi yang digunakan dalam propagasi secara besar-besaran serla berusaha untuk membangunkan in vilro sislem regeneragti bagi kegunaan dalam manipulasi genetik S parvifolia Dyer parvifolia Kombinasi klorox 10 (vA) yang dilambahkan dengan 1mll l Tween 20 selama 15 minil dengan pendedahan kepada 70 elanol lelah digunakan unuk menseril permukaan eksplan daun balang nodus dan hujung pucuk dari anak pokok S parvifolia Dyer parvifolia yang berumur dua tahun Eksplan batang nodus dan hujung plcuk dikulur dalam Y MS separa pejal media (Iiada vitamin dan sukrosa) yang diambahkan dengan 15 mill P Pvi 10 mli lieiracyciine dan 3gl1 arang yang diaktifkan Manakala eksplan daunjlga dikullur dalam media yang sama kecuali kepekatan PPM telah dikurangkan kepada 1OmIl l Selepas hari ketiga kesemua eksplan di subkultur ke Y MS separa pejal media yang dilambahkan dengan 5 mill PPM dan 5 mill tetracycline SAP memberi kesan yang baik dalam p erlumbuhan pucuk bag spesies ini Kira-kira 80 eksplan hujung pucuk berjaya mengeluarkan pucuk baru apabila dikultur dalam Y MS separa pejal media yang dilambahkan dengan 1mlll PP1vf dan SAP pada kepekatan 05mgll alau 25mgll Manakala hanya 50 eksplan nudus beljaya mengeluarkan tunas sisi pada Y MS gtepara pejal media yang dilambahkan dengan 1mlll PPM dan 05mgIISA Kebanyakkan eksplannodus lidak berinleraksi baik dalam kehadiran rOmg1 dan 25mgl l BAP Buliran kalus yang berwarna hijau kekuningan telah berjaya diinduksi dari kawasan palangan eksplan batang yang dikullurkan di Y MS separa pejal media yang diambahkan dengan 1ml1 PPM 02mgl kinetin dan O2mg1 NAA Manakala kebanyakan eksplan daun dan balang gagal membenluk kalus dalam MS separa pejal media yang dilambahkan dengan 2 4-D dan piciaram yang berlainan kepekalan

Kala kunci Shorea parvifolia Dyer ssp parvifolia in vilro regenerasi induksi kalus perlumbuhan pucuk

III

~1 r

ru_ Khidmat Makluml Akademik UNIVERSITI M LAySIA SARAWAK

TABLE OF CO TENTS

ACKNOWLEDGEMENTS ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIO S

CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW

Shorea parvifolia Dye r parvifolia 21 1 212 21 3 2 14 21 S 2 16

Taxo nomic characteristics Vernacular names Distribution and habitat General morphology Fl owering and fruitin g Economic s ignificance

Micropropagation stud y on woody species Plant tissue Culture and micropropagati on Ju venility of trees Condition of plant material Surface sterili zation Contamination Browning effect of explants

MA TERIALS AND METHODS

Plant materi al and explants source Surface sterili zation of ex plants materi a l 32 1 322 323 324 325 326 Inducti on

Experiment I Experiment 2 Experi men t 3a and 3b Experiment 4a and 4b Experi ment Sa and 5b Dru g Sensiti vity Test (DST)

of multiple s hoots fo rmation through direct organogenesIs

33 I Induction of multiple shoots by BAP

Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV

332 Induction of multiple shoots by BA P and 24 NAA

34 Indu ction of callus formation through indirect organogenesIs 341 Induction of callus by 2 4-D and picloram 24 342 Induction of callus by NAA and Kinetin 25

CHAPTER IV RESULTS

41 Surface ste rilization of exp lants 411 Experiment 1 26 412 Experiment 2 26 413 Data analysis 29 4 14 Experiment 3a and 3b 30 415 Experiment 4a and 4b 31 416 Experiment Sa and 5b 32 417 Drug Sensitivity Test 33

42 Induct ion of multiple shoots formation through direct organogenesis 421 Induction of multiple shoots by BA 35 422 Induction of multiple shoots by BA and NAA 38

43 Induction of callus formation through indirect organogenes Is 431 Induction of callus by 2 4-D and Picloram 39 432 Induction of callus by combinations of NAA 40

and Kinetin

CHARTER V DISCUSSlON

51 Surface sterilization 41 511 Type of contaminants 512 Factors affecting failure of surface 43

steril ization 52 Optimized protocol for surface sterilization of S parvijalia 44

Dyer parvifolia 53 Organogenesis 48

CHAPTER VI CONCLUSIONS AND RECOMMENDATIONS 5 L

REFERENCES 53

v

APPENDICES

APPENDIX A APPENDIX B APPENDIX C APPENDIX 0 APPENOlX E

APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M

Preparation of 1 liter MS medium 60 Preparation of I liter MS + 1 ml PPM medium 61 Preparation of I liter MS + I ml PPM + 2 4-0 medium 62 Preparation of Iliter MS soft agar medium 63 Number of contaminated damaged and axenic lamina 64 explants after sterilization with 40 clorox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic internodal 65 explants after sterilization with 40 c1orox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic nodal 66 explants after sterilization with 40 clorox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic lamina 67 explants after sterilization with 45 c1orox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic lamina 68 explants after sterilization with 40 clorox for 15 20 25 and 30 minutes recorded from day 1 to 7 after initiation of culture Number of contaminated damaged and axenic lamina 69 explants after sterilization with 35 clorox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic internodal 70 explants after sterilization with 45 clorox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic internodal 71 explants after sterilization with 40 c1orox for 15 20 25 and 30 minutes recorded from day 1 to 7 after initiation of culture Number of contaminated damaged and axenic internodal 72 explants after sterilization with 35 c1orox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture

VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U

Number of contaminated damaged and axenic nodal 73 explants after sterilization with 45 clorox for 152025 and 30 minutes recorded from day 1 to 7 after initiation of cu Iture Number of contaminated damaged and axenic nodal 74 explants after sterilization with 40 clorox for J 5 20 25 and 30 minutes recorded from day 1 to 7 after initiation of cu Iture Number of contaminated damaged and axenic nodal 75 explants after sterilization with 35 clorox for 15 20 25 and 30 minutes recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic explants on 76 MS medium with 1 mill PPM recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic explants on 77 MS medium with 1 mIll PPM recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic explants on 78 MS medium with I mill PPM medium recorded from day I to 7 after initiation of culture Number of contaminated damaged and axenic explants on 79 MS medium with I mill PPM recorded from day 1 to 7 after initiation of culture Number of contaminated damaged and axenic explants on 80 Y2 MS soft agar medium with 5 mill PPM 5mlll TET and 3 gil AC recorded from day I to 7 after initiation of culture

VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page

Number of axenic lamina explants after sterilized in three different concentrations of clorox at four different timing recorded during day 7 after initiation of culture

28

Number of axenic internodal explants after sterilized in three different concentrations of clorox at four different timing recorded during day 7 after initiation of culture

28

Number of axenic nodal explants after sterilized in three different concentrations of clorox at four different timing recorded during day 7 after initiation of culture

28

Factorial analysis of variance of clorox concentration exposure time and interaction of clorox and exposure time (duration) for axenic lamina internodal and nodal explants

29

Number of contaminated damaged and axenic explants on Y MS soft agar medium with 5 mill PPM 5ml1 TET and 3 gil AC recorded during day 7 after initiation of culture

32

The diameter of inhibition zone (mm) and growth of bacterial colonies on medium

33

Effects of BAP on mUltiple shoot induction of S parvifolia Dyer parvifolia

35

Effects of 2 4-D and Picloram parvifolia Dyer parvifolia

on callus induction of S 39

Vlll

= f

LIST OF FIGURES

Figure No Page

2 1 Mature S parvifolia Dyer parvifolia tree 7

22 (a) The bark of S parvifolia is greyish brown outside and reddish pink or orange inside

(b) The ovoid nuts of S parvifolia are enclosed by 3 longer and 2 shorter calyx lobes

(c) The leaves ofS parvifolia are broadly ovate

8

41 (a) (b) (c) Lamina internodal and nodal explants contaminated by whitish or brownish fungi

(d) Lamina explants turned brown and damaged by highest concentration of clorox

(e) Browning of nodal explants (f) Glutinous spots on the surface of stem explants after three

weeks of initial inoculation

27

42 (a) (b) (c) (d) Swollen internodes explants 30

43 (a) The axenic lamina internodal nodal and shoot tip explants after culturing on v MS soft agar medium supplemented with Imlll PPM 75 mIll tetracycline OSmgl and 2Smgl 2 4-D medium

(b) One of the shoot tip explants show cell enlargement on the nodes

(c) (d) (e) (f) Most of the internodes explants remained greenish and swollen as a result of cell growth

31

44 (a) Glutinous spots on the surface of most internodes explants after three weeks of inoculation

(b) (c) Bacteria colonies on Nutrient agar (NA) after cultured for overnight

(d) Microscopic gram negative bacteria colonies after Gram Stain technique

(e) (f) Antibiotic discs were placed on the Mueller-hinton agar swabbed with bacteria broth cultured overnight in fresh LB liquid medium

(g) (h) Clear zone of resistance ring can be observed after incubated overnight

34

IX

45 (al) Axenic shoot tips explants after 2 weeks cultured on Y MS 36 soft agar medium supplemented with I mill PPM and O5mgll BAP

(a2) The shoot tip explants sprouted two new shoots and leaf primordia after 5 weeks

(a3) Elongation of new shoots and the shoot tip explants turned brown after 17 weeks

(b) 2-weeks old axenic explants were sliced into CI and dl (CI) Development of some greenish shoots after 5 weeks

inoculated on Y MS soft agar medium supplemented with Imlll PPM and OSmgl BAP

(C2) New shoots and leaf primordia turned brown after 17 weeks of initial cUlturing

(d ) Greenish shoots developed from explants on weeks s (d2) Emergence and elongation of new shoots after 17 weeks

46 (al) Axenic shoot tips explants after 2 weeks cultured on Y MS 37 soft agar medium supplemented with I mill PPM and 2Smgll BAP

(a2) New greenish shoot was sprouted from the explants after 5 weeks of initial inoculation

(a3) Development of two new shoots from explants and it turned brown after 17 weeks

(b l) Greenish shoots were sprouted from explants after 2 weeks inoculated on Y MS soft agar medium supplemented with I mill PPM and 2Smgl BAP

(b2) New shoots and leaf primordia turned brown after 17 weeks of initial inoculation

(c) After 17 weeks cultured on Y MS soft agar medium supplemented with Imlll PPM and 2S mgll BAP leaf primordia and explants turned brown

47 (al) Axenic nodal explants after 5 weeks cultured on Y MS soft 38 agar medium supplemented with OSmgll BAP

(a2) Swollen yellowish axillary buds were sprouted from the explants after 7 weeks of initial inoculation

(a3) Emergence of more axillary buds from explants after 17 weeks of initial inoculation

(b) (c) (d) Yellowish axillary buds were sprouted from three individual explants after 17 weeks inoculated on soft agar Y MS supplemented with OSmgl BAP

x

48 (al) Axenic internodal explants after 7 weeks cultured on h MS 40 soft agar medium supplemented with Imil PPM combinations of 02mgll NAA and 02mg Kinetin

(a2) Yellowish and light greenish callus were sprouted out from the cut edges of explants after 17 weeks of initial inoculation

(b l) Axenic internodal explants after 7 weeks cultured on Y2 MS soft agar medium supplemented with I mIll PPM combinations of 02mgl NAA and 02mgll Kinetin The bark of explants was subsequently peeled off

(b2) Greenish callus were developed from the wounded area of explants after 17 weeks of initial culturing

(c) (d) Swollen internodal explants after 7 weeks of initial culturing and subsequently contaminated by endogenous bacteria

Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D

LIST OF ABBREVIA nONS

Abscisic acid

Activated charcoal

6-benzyl amino purine

Indole-3-acetic acid

Indole-3-butyric acid

Diameter at breast height

6- furfurylamino purine

Murashige and Skoog medium (1962)

Naphthalene acetic acid

Sodium hypochlorite

PicJoram 4 Amino-256-trichloropicolinic acid

Plant preservative mixture

Polyvinylpyrrolidone

Tetracycline

2 4-dichlorophenoxy acetic acids

Xll

CHAPTER I

INTRODUCTION

Dipterocarpaceae is the most well known trees family in the tropical forest This family

consists of 580 species in 15 genera is of palaeotropical distribution but is found chiefly

in Indomalaysia They are trees which often dominate humid tropical forests The

dipterocarps also constitute important timbers for domestic needs in the seasonal

evergreen forests of Asia As well as being valuable timber trees several species yield a

useful balsam or resin dammar camphor butter fat and tannin when tapped

Shorea is the largest and economically most important genus in the family

Dipterocarpaceae (Symington 1943) The genus Shorea consists of 188 species and is

widely distributed from Sri Lanka India Myanmar through Indochina and Malaysia The

genus has been divided into 10 sections based on the appendages to the connectives the

foml of the anthers and the number of pollen sacs (Ashton 1982)

Dipterocarpaceae is economically important and its timber as well as non-timber

products have long been recognized in Southeast Asian and other region~ In Malaysia

the chief export timbers are mainly produced by Shorea sp However many dipterocarp

species previously the durable heavy hardwoods were valued but growth rates are too

slow such as Neobalanocarpus sp and some Shorea sp The Dipterocarpaceae are

valuable source of hardwood timber production in South and Southeast Asia and thus

have been heavily logged Natural regeneration of some species in logged forest is poor

thus stocks are being depleted and species diversity is being reduced (Abdulhadi et ai

1981 )

Shorea parvijolia Dyer parvijolia is a tree species of the rain forest in South and

Southeast Asia It is most frequent in Mixed Dipterocarps Forest on a wide range of soils

and becoming abundant in regenerated forest (Anderson 1975) The flowering behaviour

of S parvijolia is notoriously unpredictable There is always some flowering of this

Shorea every year however the amount of flowering and consequent seed production is

very small (Ng 1977) The dipterocarp seeds are produced sporadically and exhibit loss

of viability after four-days of maturity due to the reduction of moisture content below

37 and become nonviable on the 8th day Thus seeds cannot be preserved or stored for

long periods as they lack dormancy Besides this the recalcitrant seeds also intolerant to

desiccation (Appanah and Cossalter 1994 Krishnapillay 1994 Roberts 1973)

Furthermore only limited success has been achieved with conventional vegetative

propagation Thus conventional forestry practices are difficult to implement Therefore

concerted efforts must be made to evolve methods for the vegetative propagation of

forest trees for use in reforestation genetics conservation and cloning of superior

genotypes for the purpose of forest plantations and tree improvement programmes

Mature trees demonstrating superior genotypes are ideal for mass propagation

However it is usually more difficult to establish shoot culture from mature trees than

from juvenile plants (Bonga 1987 Hackett 1987) The practicable method of

propagation for many trees is through seeds Seed raised plants show wide variations in

2

the field due to heterozygous of the parents naturally The conventional method of

selection breeding and progeny testing of timber trees is very slow and difficult due to

long life cycle of the trees (30-50 years) which may add to the problem of contamination

in vivo by the symbiotic association of microorganisms In order to overcome this

problem pruning hedging air layering budding and grafting can be used to induce

rejuvenation

Increasing demands for forest products are expected to rise sharply over the

coming decade and storages have been forecasted by the end of this century (Keays

1974) Thus there is an urgent need for large number of genetically improved planting

materials for timber production One of the most important aspects of a tree improvement

program is the production of genetically superior genotypes Traditional propagation of

elite tree through rooting of cuttings layering budding etc is very poor or in many

cases not possible Thus vegetative propagation plays an important role in the genetic

improvement of timber trees Selection of tree with desire characteristics and planting the

clonal material of the selection trees is the simplest approach It can be carried out

immediately by identifying desired trees or plus tree among the trees in the existing

population Tissue culture technique is the most obvious choice for mass propagation of

plus trees Besides genetic engineering will rapidly accelerate plant breeding and offer

new ways to increase forest productivity wood quality and other breeding and other

desirable properties such as resistance to pests pathogens stress and herbicides

3

During the last decade significant progress has been made in the propagation of

forest trees through in vitro culture technology In vitro culture techniques can rapidly

increase the number of individuals of endangered species with reproductive problems and

or extremely reduced populations due to deforestation (Iriondo amp Perez 1990) These

techniques could be useful in in vitro regeneration of S parvifolia Dyer parvifolia Thus

the objectives of this study are

1 To establish the micropropagation protocols for use in large scale propagation of

S parvifolia Dyer parvifolia

2 To attempt the development of an in vitro regeneration system for the use in

genetic manipulation of S parvifolia Dyer parvifolia

4

Pusat Khidmt Maidumat Akademik UNIVEftSm MALAYSIA SARAW K

CHAPTER II

LITERATURE REVIEW

21 Shorea parvifolia Dyer

211 Taxonomic classification (Wong 1988 Newman et 01 1996)

Division Spermatophyta

Class Angiosperma

Sub-class Dicotyledonne

Group Thalam iflorae

Order Guttiferales

Family Dipterocarpaceae

Genus Shorea

Section Mut ica

Species Shorea parvifolia Dyer

Sub-species Shorea parvifolia Dyer ssp parvifolia

Shorea parvifolia sp Velutinata PS Asthon

212 Vernacular names

S parvifolia has various common names it is locally known as meranti sarang punai

(Brunei Sarawak) seraya punai (Sabah) awang belah dangar burau dangar siak kontoi

kontoi burong lampung lampung nasi lampung tembag merangan merangan batu

merangan nasi perawan lop pooga bahaya ponga payur ponga pipit sawang puteh

5

bull ~ f

(Kalimantan) (Newman e al 1996) In Thailand S parvifolia commonly known as Saya

lueng or Saya lang khao (Pooma and Newman 2001)

213 Distribution aDd habitat

Shorea parvifolia Dyer (Figure 21) is a woody plant species of the tropical rain forest in

South and Southeast Asia This species are widespread throughout Peninsular Thailand

Peninsular Malaysia (type locality) Borneo Singapore and Sumatra from lowlands (sea

level) to upper hills (800 m altitude) In Malaysia S parvifolia distributed throughout

Sarawak Peninsular Malaysia except Perl is Northern Kedah and Langkawi In Thailand

it is widely scattered in evergreen forest in southernmost areas It is probably the

commonest dipterocarp in the Malaysian region (Ashton 1982) Meranti sarang punai is

most frequent in Mixed Dipterocarps Forest on a wide range of soils and becoming

abundant in regenerated forest (Anderson 1975)

S parvifolia grow well in the humid forest especially on the sandstone clay soil

yellow clay soil yellow sandy clay soil sandy loam and yellow sand The preferred

habitat of this emergent plant is usually along or near river running fresh water gentle

slope steep slope ridge areas where the soil has higher water retention ability (Ashton

1968) It is absent in the dry hilly or mountainous areas

6

Figure 21 Mature S parvi[oiia Dyer parvi[olia tree

214 General morphology

S parvifolia are large to very large trees which attain 65-70 m tall and over 200cm in

diameter at breast height (dbh) with short and sharp buttress up to 4 m high with straight

gradually tapering cylindrical boles which are without branches up to 10-42 m high

(Figure 21) The Barks of this species are smooth or variously fissured flaking in

longitudinal flakes or scaling usually from below up It is usually thick greyish brown

outside and reddish pink or orange inside (Figure 22a)

The leaves are broadly ovate 4-9 x 3-5 cm midrib depressed above secondary

nerves 10-13 pairs with few intennediate nerves and became reddish brown when it dries

(Figure 22b) The indumentum is short fine tu fted hairs on buds stipules and panicles

7

sparse on leaves beneath Domatia of S parvifolia are pubescent or scale-like and

frequently one or two pairs at base Inflorescences are terminal or axillary panicles the

flowers irregularly crowded scented and cream to pinkish color The flower buds are

ovoid and peta ls fa lcate oblong with white tinged pink or pinkish-red at base Stamens

are glabrous connec tives filiform curved downwards exceeding the anth ers globose

(Smitinand e l al 1980)

The fruits are a nut borne On short stalk containing one seed The nut are ovo id to

oblong and pubescent (15 x 1 cm) Fruiting calyx saccate calyx bases enclosing half of

the nut it has 3 longer calyx lobes (55-7 x 1-15 cm) and 2 short lobes (2-25 x 03- 05

cm) embrace the lower portion ofthe nut (Figure 22c)

plusmn~r~J

~j-

shybull

u

Figure 22 (a) The bark of S parvifoli11 is greyish brown outside and reddish pink or orange inside

(b) The ovoid nuts of S parvifoli11 are enclosed by 3 longer and 2 shorter calyx lobes

(c) The leaves ofS parvifolia are broad ly ovate

I I tG b

8

215 Flowering and Fruiting

It was reported by Appanah and Chan (1981) Ashton (1982) and Bawa (I 994) that this

species are pollinated by a wide variety of animal and insects The amount of fruits bears

will depends on the maturity of the tree and its crown structure Flowering usually occurs

in September to October (Appanah and Chan 1981) and the fruit ripen in the following

January and February The yield may continue to late March and April in very favourable

years The flowering behaviour of S parvifolia is notoriously unpredictable However its

have normally a pattern of mass fruiting only once in every 2 to 5 years The

phenomenon is called gregarious or general flowering Mass fruiting seems to be

synchronized with droughts which tend to be associated with El Nifto event periodic

water stress and the accumulation of carbohydrates during the dry season in August to

October (Burgess 1972)

216 Economic significance

Meranti sarang punai is an important source of quality wood Light red meranti timber

has great advantage for not having siliceous content It provides for its good workability

to come up with various wood works and products From its wide range of applications it

is particularly important for producing plywood veneer (face and core) hardboard

particleboard and furniture It is used for heavy construction works even under exposed

conditions but treatment is a requirement for it to last longer

9

Barks of this meranti produce in large quantities opaque yellow low graded

dammar used locally for torches plasters varnish and lacquer materials and as animal

and vegetable preservative solution (chloroform) Its trade name is daging dammar

batu or dammar meranti sarang punai Barks are also used as house panelling walls

stripped into slabs basket bins and in talll1ing material Nuts of red meranti yield fats

used in the manufacture of chocolate cosmetics soaps and candles Fruits are boiled and

eaten by the local people

22 Micropropagatioo study 00 woody species

Successful micropropagation of woody plants is relatively a recent phenomenon Several

woody species such as poplars wild cherry eucalypts red wood radiate pine and teak

are at present commercially micropropagated (Thorpe 1988 Bajaj 1997) Vegetative

propagation both by conventional and in vitro tissue culture methods for some woody

plants like Shorea species have been carried out but For example S pinanga S

compressa S leprosula S palembanica S ovalis S selanica S stenoptera and

Dryobalanops aromatica To-date there is no report on such work on S parvifolia has

been carried out and published

Dick and Aminah (1994) reported that a total of 75 species 10 the

Dipterocarpaceae had been successfully propagated a third of which belong to the genus

Shorea Smits and Struycken (1983) induced callus and roots on leaf explants of Shorea

curtisii whereas nodal explants of S obtuse sprouted axillary shoots but complete

plantlets were not obtained Scott et al (1987) produced complete plantlets of Shorea

10

Pusat Khidmt Maklumllt Alcadcmilc UNIVERSITI MALAYSIA SAftAWAK

IN VITRO REGENERATION OF SHOREA PARVIFOLIA DYER SSP PARVIFOLIA

KHOPEIEE

This project is submitted in partial fulfillment of the requirements for the degree of Bachelor Science with Honours

(Biotechnology Resource)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2005

ACKNOWLEDGEMENTS












ABSTRACT



11

ABSTRAK



III

~1 r


TABLE OF CO TENTS


CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW









Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

ACKNOWLEDGEMENTS












ABSTRACT



11

ABSTRAK



III

~1 r


TABLE OF CO TENTS


CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW









Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

ABSTRACT



11

ABSTRAK



III

~1 r


TABLE OF CO TENTS


CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW









Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

ABSTRAK



III

~1 r


TABLE OF CO TENTS


CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW









Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10


TABLE OF CO TENTS


CHAPTER I

CHAPTER II

2 I

22 23 24 25 26 27 28

CHAPTER III

3 1 32

33

INTRODUCTION

LITERATURE REVIEW









Page

III

IV

VII I

IX

XII

5 5 6 7 9 9 10 1 I 13 14 14 16 17

19

19 20 21 21 22 22

23

IV



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10



CHAPTER IV RESULTS




and Kinetin






REFERENCES 53

v

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

APPENDICES


APPENOlX F

APPENDIX G

APPENDIX H

APPENDIX 1

APPENDIX]

APPENDIX K

APPENDIXL

APPENDIX M


VI

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

APPENDIXN

APPENDIX 0

APPENDIX P

APPENDIXQ

APPENDIX R

APPENDIX S

APPENDIX T

APPENDIX U


VII

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

LIST OF TABLES

Table No

41

42

43

44

45

46

47

48

Page


28


28


28


29


32


33


35



Vlll

= f

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

LIST OF FIGURES

Figure No Page





8





27





31






34

IX


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10


















x






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10






Xl

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

ABA

AC

BAP

IAA

IBA

dbh

Kinetin

MS

NAA

NaOCI

Pic

PPMtrade

PVP

TET

24-D


Abscisic acid

Activated charcoal








Sodium hypochlorite




Tetracycline


Xll

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10

CHAPTER I

INTRODUCTION




















1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10


1981 )




















2






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10






rejuvenation















3











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10











4


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10


CHAPTER II

LITERATURE REVIEW




Class Angiosperma



Order Guttiferales


Genus Shorea

Section Mut ica









5

bull ~ f


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10


















6












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10












7











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10











plusmn~r~J

~j-

shybull

u




I I tG b

8




















9






















10




















9






















10






















10

in vitro regeneration of shorea parvifolia dyer ssp ... vitro regeneration of shorea parvifolia dyer...

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