UNIVERSITI PUTRA MALAYSIA

PHYSIOLOGICAL AND BIOCHEMICAL CAHNGES OF VITRO PROPAGATED BAHANA PLANTLETS INOCULATED WITH

RHIZOBACTERIA AND AGROBACTERIA

ZURAIDA AB. RAHMAN.

FBSB 2005 13

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF IN VITRO PROPAGATED BANANA PLANTLETS INOCULATED WITH

RHIZOBACTERIA AND AGROBACTERIA

BY

ZURAIDA AB. RAHMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Doctor of Philosophy

July 2005

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Doctor of Philosophy

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF IN VITRO PROPAGATED BANANA PLANTLETS INOCULATED WITH

RHIZOBACTERIA AND AGROBACTERIA

ZURAIDA AB. RAHMAN

July 2005

Chairman : Professor Maniah Mahmood, PhD

Faculty : Biotechnology and Biomolecular Sciences

A series of experiments were carried out to observe the effects of

rhizobacterial and agrobacterial inoculation, singly or combined on the total content,

concentration and distribution of the biochemical components (total soluble protein,

soluble nitrogen, proline, peroxidase activity, total soluble phenolic, nitrate

reductase activity, nitrate, chlorophyll), physiological characteristics (percentages of

growth, number of roots, fresh and dry weight of roots, maximum and total length of

roots) and mineral contents (N, P, K, Ca and Mg) of in vitro banana plantlets using

MS (Murashige and Skoog, 1962) basal medium. The effects of rhizobacterial

inoculation in modified MS medium containing sodium chloride (0.2%) and boron

(1 yM and 10 pM) on the biochemical components, physiological characteristics

and mineral content of the in vitro banana plantlets were also studied. Growth of

banana plantlets cultured in modified MS liquid medium supplemented with

different forms and concentrations of nitrogen or carbon sources and inoculated with

Bacillus sphaericus UPMBl 0 was estimated.

Results from the inoculation study using MS basal medium indicated that

inoculation with rhizobacteria (Azospirillum brasilense Sp7, Bacillus sphaericus

UPMBlO and Microbacterium oxydens UPMB11) or agrobacteria ( Agrobacterium

rhizogenes strains AR9402 and A4) showed positive response on growth of in vitro

banana plantlets compared to uninoculation after one month of culture. The

inoculation treatment also increased the number of root, fresh and dry weight of

roots and total length of root. At the same time, with inoculation the total content or

concentration of the respective biochemical activity as total soluble protein,

peroxidase, nitrate reductase, proline, nitrate, soluble nitrogen, phenolic and

chlorophyll of the host plants increased and varied according to the type of bacteria

used. Inoculation with these bacteria also enhanced the accumulation of N and P in

the plantlets. Co-inoculation with rhizobacteria (Azospirillum brasilense Sp7,

Bacillus sphaericus UPMB 1 0 and Microbacterium oxydens UPMB 1 1) and

agrobacteria (Agrobacterium rhizogenes strains AR9402 and A4) also showed

similar response as in single inoculation; UPMBlO+AR9402 treatment was the

most effective treatment. The presence of rhizobacteria in the medium supplemented

with 0.2% sodium chloride resulted in an improvement in growth and root biomass

compared to the control (uninoculated). This rhizobacterial inoculation also

produced an increase in protein, nitrate, soluble nitrogen and chlorophyll contents of

the plantlets cultured in MS modified medium containing 0.2% sodium chloride. The

descending order of effectiveness of the rhizobacteria in medium containing 0.2%

sodium chloride was:UPMB 1 1 NPMB 1 O>Sp7. Similar response was shown when

Bacillus sphericus UPMBlO was inoculated into medium containing boron at two

concentrations: 1 pM and 10 pM. An increase in percentage of growth (> 295%)

was shown when boron was applied into medium inoculated with Bacillus

sphericus UPMB10. Results fiom the experiment of modified MS medium

supplemented with different concentrations and forms of nitrogen also strongly

indicated that inoculation with Bacillus sphericus UPMBlO has the potential to

improve the in vitro plant growth especially in the absence of nitrogen. Inoculation

with Bacillus sphericus UPME310 showed significant increased plant growth in

treatment without nitrogen (- nitrogen) at 166% compared to un-inoculated only at

115%. Inoculation with Bacillus sphericus UPMBlO to enhance growth of in vitro

plantlets could partly replace the expensive chemical nitrogen requirement for the

plants. Bacillus sphericus UPMBlO seem to have the ability to increase growth of

plantlets in medium supplemented with asparagine, potassium nitrate and urea. The

descending order of effects of rhizobacterial inoculation on growth of plantlets

varied according to the following N-sources in the MS modified media: asparagine

(392%) > potassium nitrate (376%)> urea 291%. There was a negative response of

Bacillus sphericus UPMBlO inoculation in promoting growth of plantlets in media

containing KN03 (a range of 0 mM -300 mM) or (NH,+)2S04 (a range of 0 mM-

80mM) at all concentrations used. At 1.5% to 6.0% concentrations of sucrose,

inoculated plantlets with Bacillus sphericus UPMBlO showed increased growth

within a range of 250% to 304% compared to un-inoculated plantlets. It also indicate

that inoculation with Bacillus sphaericus UPMBlO into the media containing carbon

successfidly enhanced growth of in vitro plantlets. The descending order of effect of

UPMBlO inoculation on plant growth varied according to the following carbon

sources in the MS modified media: hctose (421%)> sucrose (356%)> glucose

(354%)> maltose (221%)> sorbitol (78%)> rnannitol(51%). Therefore, inoculation

with Bacillus sphaericus UPMBlO into the medium containing carbon sources

produced positive response on the host plant, an effect which is dependent on the

forms and concentrations of the carbon sources. The above finding provided

evidence that Azospiriflum brasilense Sp7, BacilIus sphaericus UPMB 10,

Microbacterium oxydens UPMBl 1, Agrobacterium rhizogenes strains AR9402 and

A4, singly or combined are potentially effective in promoting growth of in vitro

banana plantlets. Inoculation of rhizobacteria were showed beneficial to the plantlet

in saline conditions through increment of growth and improvement in rooting system

The effectiveness of inoculation is increased when associated with boron, nitrogen

or carbon into the medium. Thus these bacterial strains could be used as a

bioenhancer for growth of in vitro banana plantlets.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia Sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PERUBAHAN FISIOLOGI DAN BIOKIMIA KE ATAS ANAK PISANG IN VITRO YANG DIINOKULASI DENGAN RHIZOBACTERIA DAN

AGROBACTERIA

Oleh

ZURAIDA AB. RAHMAN

Julai 2005

Pengerusi : Profesor Maniah Mahmood, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Beberapa siri eksperimen telah dijalankan untuk memerhatikan kesan

inokulasi rhizobakteria dan agrobakteria secara tunggal atau digabungkan ke atas

jumlah kandungan, kepekatan dan pengagihan komponen-komponen biokimia

Cjurnlah protein terlarut, nitrogen terlarut, prolin, aktiviti peroxidase, jumlah fenolik

terlarut, aktiviti reduktase nitrat, nitrat, klorofil), ciri-ciri fisiologi (Peratus

pertumbuhan, jumlah akar, berat basah dan kering akar, jumlah dan panjang akar

maksimwn) dan kandungan mineral (N,P,K, Ca dan Mg) anak-anak pokok pisang in

vitro dengan menggunakan media basal MS. Kesan inoculasi rhizobakteria dalam

media MS (Murashige and Skoog, 1962) ubahsuai yang mengandungi natrium

klorida (0.2%) dm boron (1 pM dan 10 pM) ke atas kandungan biokimia, ciri-ciri

fisiologi dan kandungan mineral anak-anak pokok pisang in vitro juga dikaji.

Perturnbuhan anak-anak pokok pisang dalam media cecair ubahsuai yang

dibekakan dengan bentuk dan kepekatan nitrogen atau sumber karbon yang berbeza

serta diinokulasi dengan Bacillus sphaericus UPMB 1 0 juga telah dianggarkan.

vii

Hasil daripada kajian inokulasi yang menggunakan media basal MS

menunjukkan bahawa inokulasi dengan rhizobakteria (Azospirillum brasilense Sp7,

Bacillus sphaericus UPMBlO dan Microbacterium oxydens UPMBl1) atau

agrobakteria ( Agrobacterium rhizogenes strain AR9402 dan A4) menunjukkan

respon positif terhadap pertumbuhan anak-anak pokok pisang in vitro jika

dibandingkan dengan yang tidak diinokulasi selepas dikultur satu bulan. Rawatan

inokulasi juga meningkatkan bilangan, berat basah dan kering akar dan jumlah

panjang aka. Pada masa yang sama, melalui inokulasi jumlah kandungan protein

terlarut, peroxidase, reduktase nitrat, nitrat, prolin, nitrogen terlarut, fenolik dan

klorofil dari pokok perumah meningkat dan berbeza rnengikut jenis bakteria yang

digunakan. Inokulasi dengan bakteria ini juga merangsang pengumpulan kandungan

N dan P dalam anak-anak pokok. Gabungan inokulasi antara rhizobakteria

(Azospirillum brasilense Sp7, Bacillus sphaericus UPMB 1 0 dan Microbacterium

oxydens UPMB11) dan agrobakteria (Agrobacterium rhizogenes strain AR9402 dan

A4) menunjukkan respon yang sarna seperti inokulasi tunggal; di mana rawatan

UPMB1 O+AR94O2 ialah rawatan yang paling efektif. Kehadiran rhizobakteria di

dalam media yang dibekalkan dengan 0.2% natrium klorida menunjukkan

peningkatan dalam pertumbuhan dan biojisim akar berbanding dengan kawalan

(tidak diinokulasi). Inokulasi dengan rhizobakteria ini juga meningkatkan kandungan

protein terlarut, nitrat, nitrogen terlarut dan klorofil dalam anak-anak pokok yang

dikultur dalam media ubahsuai MS yang mengandungi 0.2% natrium klorida.

Keberkesanan dalam umtan menurun adalah: UPMB 1 l>UPMB 1 O>Sp7. Respon

yang sama juga dilihat apabila Bacillus sphaericus UPMBlO diinokulasi dalam

media yang mengandungi boron dalam dua kepekatan: 1 pM dan 10 pM.

Peningkatan peratus pertumbuhan (>295%) dilihat apabila boron digunakan dalam

media yang diinokulasi dengan Bacillus sphaericus UPMBlO. Hasil daripada ujikaji

media MS ubahsuai yang dibekalkan dengan kepekatan dan bentuk nitrogen yang

berbeza juga menunjukkan bahawa inokulasi dengan Bacillus sphaericus UPMBlO

mempunyai potensi untuk memperbaiki pertumbuhan pokok in vitro terutamanya

dalam ketidakhadiran nitrogen. Inokulasi dengan UPMBlO menunjukkan

peningkatan yang bererti terhadap pertumbuhan pokok dalam rawatan tanpa nitrogen

(-nitrogen) pada 166% berbanding dengan yang tidak diinokulasi pada hanya 1 15%.

Inokulasi dengan Bacillus sphaericus UPMBlO boleh merangsang pertumbuhan

anak-anak pokok in vitro di mana ia boleh menggantikan sebahagian daripada

keperluan bahan-bahan kimia nitrogen yang mahal untuk pokok. Bacillus sphaericus

UPMB 10 didapati mempunyai keupayaan untuk meningkatkan pertumbuhan anak-

anak pokok dalam media yang dibekalkan dengan asparagin, kalium nitrat dan urea.

Kesan inokulasi rhizobakteria terhadap pertumbuhan anak-an& pokok secara urutan

menurun mengikut sumber N- dalam media ubahsuai MS: asparagin> kalium

nitrat> urea. Terdapat respon negatif inokulasi Bacillus sphaericus UPMB 1 0 dalam

menggalakkan pertumbuhan anak-anak pokok dalam media yang mengandungi

KN03 (julat antara OrnM-30OmM) atau (NH&So4 (julat antara OmM-80 mM)

dalam semua kepekatan yang digunakan. Pembekalan sukrosa pada kepekatan 1.5%

hingga 6.0% dan diinokulasi dengan Bacillus sphaericus UPMB 10 menunjukkan

peningkatan pertumbuhan anak-anak pokok di antara julat 250-304% berbanding

dengan yang tidak diinokulasi. Ia juga menunjukkan bahawa inokulasi dengan

Bacillus sphaericus UPMB 1 0 dalam media yang mengandungi sumber karbon

dengan jayanya merangsang pertumbuhan a&-anak pokok in vitro. Kesan inokulasi

UPMBlO secara urutan menurun terhadap pertumbuhan pokok berbeza mengikut

sumber karbon di dalam media ubahsuai: fi-uktosa (421%)> sukrosa (356%)>

glukosa (354%)> maltosa (22 1 %)> sorbitol (78%)> mannitol (5 1 %). Maka,

inokulasi dengan Bacillus sphaericus UPMB 10 ke dalam media yang mengandungi

sumber karbon menghasilkan respon yang positif kepada pertumbuhan pokok

perurnah, di mana kesannya begantung kepada bentuk dan kepekatan sumber

karbon. Penemuan ini membuktikan bahawa Azospirillum brasilense Sp7, Bacillus

sphaericus UPMB 10, Microbacterium oxydens UPMB 1 1, Agrobacterium

rhizogenes strain AR9402 dan A4 secara tunggal atau digabungkan berpotensi dalam

menggalakkan pertumbuhan anak-anak pokok in vitro secara efektif Inokulasi

dengan rhiwbakteria juga memberi kebaikan kepada anak-an& pokok dalam

persekitaran garam melalui peningkatan pertumbuhan dan pengembangan sistem

pengakaran. Keberkesanan inokulasi meningkat apabila disatukan dengan boron,

nitrogen atau karbon dalam medium. Maka strain bakteria ini boleh digunakan

sebagai perangsang biologi ('bioenhancer') bagi perturnbuhan anak-anak pokok

pisang in vitro.

ACKNOWLEDGEMENTS

My sincere appreciation is extended to Professor Dr. Marziah Mahmood, the

Chairman of my Supervisory Committee, for her suggestions, advice support and

guidance throughout my study. I also would like to express my sincere appreciation

and a lot of thanks to the member of the Supervisory Committee, Professor Dr

Zulkifli Hj. Sharnsuddin, for his kind assistance and guidance throughout the

preparation of this thesis. I give thanks to Dr. Halimi Mohd. Saud, my member of

the Supervisory Committee and Dr Yunus B. Shukor for their advice and comments

during the planning and execution of this study. Appreciation is also to extend to my

Superior Officer at MPOB, Dr. Ahmad Tarmizi Hassim for his understanding

throughout the writing of this thesis.

A lot of thanks to colleagues at MMBPP 5, MPOB for their continuous moral

support, comment, advice and assistance throughout the study with a wish 'tetap

dalam ingatan'. Thanks are also extended to all my fiiends in Universiti Putra

Malaysia, especially those at Prof Maniah 's Laboratory, MARDI and MPOB.

I am deeply indebted to my beloved husband AHMAD B. ISMAlL and daughter

NUR KHADIJAH, my parents and my in-laws for their sacrifices and patience

during the course of my graduate study. May Allah guide us all.

I certify that an Examination Committee met on 1 3 ~ July 2005 to conduct the final examination of Zuraida Ab. Rahman on her Doctor of Philosophy thesis entitled "Physiological and Biochemical Changes of in vitro Propagated Banana Plantlets Inoculated with Rhizobacteria and Agrobacteria" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

JANNA ONG ABDULLAH, PhD . . Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (C hairrnan)

SIT1 KHALIJAH DAUD, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner)

ZAKARIA WAHAB, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

OTHMAN OMAR, PhD Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (External Examiner)

MAT ALI, PhD P- ~rofessorhe~u&ean School of Graduate Studies Universiti Putra Malaysia

Date: 25 OCT 2005

xii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows:

MARZIAH MAHMOOD, PhD Professor Faculty of Biotechnology and Biomolecula. Sciences Universiti Putra Malaysia. (Chairman)

ZULKIFLI HJ. SHAMSUDDM, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

HALIMI MOHD. SAUD, PhD Lecturer Faculty of Agricukure Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor /Dean School of Graduate Studies Universiti Putra Malaysia

Date: 17 NOV 2005

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

xiv

TABLE OF CONTENTS

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

1 INTRODUCTION

2 LITERATURE REVIEW 2.1 Importance of banana 2.2 Advantages of studies using in vitro system 2.3 Plant Growth-Promoting Rhizobacteria (PGPR)

2.3.1 Azospirillum sp. 2.3.2 Agrobacterium sp. 2.3.3 Benefits of co-inoculation

2.4 Biochemical and physiological changes in plants 2.5 Salinity stress 2.6 Benefits of boron 2.7 Nitrogen, carbon and pIant growth 2.8 Induction of phytohonnone 2.9 Associative nitrogen fixation in plant

3 GENERAL MATERIALS AND METHODS 3.1 Materials

3.1.1 Banana plant let culture 3.1.2 Bacterial strains

3.2 Media preparation 3.3 Culture conditions 3.4 Percentages of growth 3.5 Dry weight

. . . 111 ...

Vll l

X

xi xii XX

xxi xxvii

3.6 Biochemical studies 3 -6.1 Total soluble protein 3.6.2 Soluble nitrogen 3.6.3 Proline 3.6.4 Peroxidase activity 3.6.5 Total soluble phenolic 3.6.6 Nitrate reductase activity(NRA) 3.6.7 Nitrate 3 -6.8 Total chlorophyll

3.7 N,P,K,Ca and Mg content 3.8 Statistical Analysis

4 INFLUENCE OF RHIZOBACTERIAL AND AGROBACTERIAL INOCULATION ON SELECTED PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF BANANA PLANTLETS 4.1 Introduction 4.2 Materials and methods

4.2.1 Materials 4.2.1.1 Plant materials 4.2.1.2 Bacterial cultures

4.2.2 Methods 4.2.3 Statistical Analysis

4.3 Results and discussion 4.3.1 Growth and root biomass 4.3.2 Total soluble protein 4.3.3 Soluble nitrogen 4.3.4 Proline 4.3.5 Peroxidase activity 4.3.6 Phenolic compound 4.3.7 Nitrate reductase activity(NR4) 4.3.8 Nitrate 4.3.9 Total chlorophyll 4.3.1 0 N,P,K,Ca and Mg content

4.4 Conclusion

5 RESPONSE OF BANANA PLANTLETS TO RHIZOBACTERIAL INOCULATION UNDER SALT STRESS CONDITION 5.1 Introduction 5.2 Materials and methods

5.2.1 Materials 5 -2.1.1 Plant material 5.2.1 2 Bacterial cultures

5.2.2 Methods 5.2.3 Statistical analysis

xvi

Result and discussion 5.3.1 Growth and root biomass 5.3.2 Total soluble protein 5.3 -3 Soluble nitrogen 5.3.4 Proline 5.3.5 Peroxidase activity 5.3.6 Phenolic compound 5.3.7 Nitrate reductase (NR) activity 5.3.8 Nitrate 5.3.9 Total chlorophyll 5.3.10 N,P,K,Ca and Mg content Conclusion

6 INFLUENCE OF BORON IN GROWTH AND BIOCHEMICAL CHANGES IN PGPR INOCULATED BANANA PLANTLETS 6.1 Introduction 6.2 Materials and methods

6.2.1 Materials 6.2.1 -1 Plant material 6.2.1.2 Bacterial cultures

6.2.2 Methods 6.2.3 Statistical analysis Result and discussion 6.3.1 Growth and root biomass 6.3.2 Total soluble protein 6.3.3 Soluble nitrogen 6.3.4 Proline 6.3.5 Peroxidase activity 6.3.6 Phenolic compound 6.3.7 Nitrate reductase (NR)activity 6.3.8 Nitrate 6.3.9 Total chlorophyll 6.3.1 0 N,P,K,Ca and Mg content Conclusion

xvii

EFFECTIVENESS OF UPMBlO INOCULATION IN THE PRESENCE OF NITROGEN AND CARBON SOURCES ON GROWTH OF IiV VZTRO PLANTLETS 7.1 Introduction 7.2 Material and methods

7.2.1 Materials 6.2.1.1 PIant material 6.2.1.2 Bacterial culture Methods 7.2.2.1 Influence of NO? and N&+ 7.2.2.2 Ratio of NO<M&+ 7.2.2.3 Effect of various nitrogen sources 7.2.2.4 Effect of (NHs)2S04 and KN03

concentrations 7.2.2.5 Effect of sucrose concentrations and

light conditions 7.2.2.6 Effect of activated charcoal 7.2.2.7 Effect of various carbon sources

7.2.3 Statistical analysis Results and discussions 7.3.1 Influence of NO3- and NH.++ 7.3.2 Ratio of NO</NH~+ 7.3 -3 Effect of various nitrogen sources 7.3.4 Effect of (NH4)2S04 and KN03 concentrations 7.3.5 Effect of sucrose concentrations and light

conditions 7.3.6 Effect of activated charcoal 7.3.7 Effect of various carbon sources

7.4 Conclusion

8 GENERAL DISCUSSION AND CONCLUSION

REFERENCES APPENDICES BIODATA OF THE AUTHOR

xviii

LIST OF TABLES

Table

4.1 Effect of plant growth and root biomass of in vitro banana plantlets after one month culture in MS liquid medium and co-inoculated with rhizobacterial and agrobacterial.

Nutrients content(mg/gdw) of in vitro banana plantlets after one month culture in MS liquid medium co- inoculated with rhizobacterial and agrobacterial.

Nutrients content (mg/gdw) of in vitro banana plantlets cv.Berangan(a) and cv.Nocvaria(b) after one month culture in MS liquid medium containing sodium chlorite inoculated with rhizobacterial.

Effect of UPMBlO inoculation on root biomass of m vitro banana plantlets cv.Berangan culture in MS liquid medium supplemented with boron at two concentration, 1 pM and 10 pM.

Effect of UPMBlO inoculation on root biomass of in vitro banana plantlets cv.Novaria culture in MS liquid medium supplemented with boron at two concentration, 1 pM and 10 pM.

Nutrients content (mg/gdw) of in vitro banana plantlets cv.Berangan after one month culture in MS liquid medium containing boron ( I pM and 10 pM) and inoculated with UPMB 10.

Nutrients content (mg/gdw) of in vitro banana plantlets cv.Novaria after one month culture in MS liquid medium containing boron (I pM and 10 pM) and inoculated with UPMB 10.

Modification of NO3- and N&+ concentrations.

Ratios of N%-/N~' at five concentrations of nitrate and ammonium.

Effect ratio of NO</NH~' on plant growth of in vitro banana plantlets cv.Berangan after one month culture in modified MS liquid medium.

Page

4.15

LIST OF FIGURE

Page Figure

4.1A Growth of in vitro banana plantlets and root biomass after 4.6 one month culture in MS liquid medium and inoculated with rhizobacterial species: a (percentage of growth), b (number of roots), c (fresh weiht of roots), d (dry weight of roots), and e (maximum length of roots) and f (total length of roots).

Growth of in vitro banana plantlets after one month 4.7 culture in MS liquid medium inoculated with rhizobacterial

Growth and root biomass of in vitro banana plantlets after 4.1 1 one month culture in MS liquid medium inoculated with strains of Agrobacterium:a (percentage of growth), b (number of roots), c (fresh weiht of roots), d (dry weight of roots), and e (maximum length of roots) and f (total length of roots).

Growth of in vitro banana plantlets after one month 4.12 culture in MS liquid medium inoculated with agrobacterial.

Soluble protein content of in vitro banana plantlets after 4.18 one month culture in MS liquid medium inoculated with respective bacterial treatment

Soluble nitrogan content in part of banana plantlets after 4.21 one month inoculated with respective bacterial treatments: a (rhizobacteria), b (Agrobacterium) and c (co-inoculation of rhiiobacteria and Agrobacterium) in MS liquid medium.

Change of proline content of in vitro banana plantlets for 4.24 one month culture in MS liquid medium in the presence of respective bacterial treatment: a (rhiibacteria), b (Agrobactrium) and c (co-inoculation of rhizobacteria and Agrobacterium).

Change of peroxidase activity on in vitro banana plantlets 428 after one month culture in MS liquid medium inoculated with respective bacterial treatment: a (rhiuobacteria), b (Agrobacterium) and c (co-inoculation of rhiiobacteria and Agrobacterium).

xxi

Effect of bacterial: a (rhizobacteria, b ( Agrobacterium) 4.31 and c (co-inoculation of rhizobacteria and Agrobacterium) inoculation on changes of total phenolic compound of in vitro banana plantlets after one month culture in MS liquid medium.

Change of nitrate reductase activity of in vitro banam plantlets after one month culture in MS liquid medium inoculated with respective bacterial treatment: a (rhizobacteria), b (Agrobacteria) and c ( co-inoculation of rhizobacteria and Agrobacterium).

Effect of respective bacterial: a (rhizobacteria), b 4.39 (Agrobacterium )and c (co-inoculation of rhizobacteria and Agrobacterium) inoculation on nitrate content of in vitro banana plantlets for one month culture in MS liquid medium.

Chlorophyll content of in in vitro banana plantlets after one month culture in MS liquid medium inoculated with respective bacterial treatment: a ( rhizobacteria), b (Agrobacterium) and c) co-inoculation of rhizobacteria and Agrobacterium.

Effect of: a (*bacteria), b (Agrobacterium) and 4.47 c (co-inoculation of rhizobacteria and Agrobacterium) on nutrient content of in vitro banana plantlets after one month culture in MS liquid medium.

Growth (a) of in vitro banana plantlets and root biomass (b-f) cv. Berangan after one month c u h r e in MS liquid medium containing of NaCl inoculated with rhizobacterial.

5.1A-g Eff'ect of UPMBIO inoculation on growth of in vitro banana plantlets cv. Berangan culture in MS liquid medium containing 0.2% sodium chloride.

Growth (a) of in vitro banana plantlets and root biomass (b-f) cv. Novaria after one month culture in MS liquid medium containing of NaCl inoculated with rhizobacterial.

xxii

5.1- Effect of rhizobacterial inoculation on growth of in vitro banana plantlets cv. Novaria culture in MS liquid medium containing 0.2% sodium chloride A: Control, B: Sp7, C: UPMB 1 1, D: UPMB 10.

Total soluble protein content of in vitro banana plantlets 5.12 (a) cv. Berangan and (b) cv. Novaria after one month culture in MS liquid medium containing NaCl inoculated with rhizobacterial.

Soluble nitrogen content in part of ba- plantlets 5.15 (a) cv. Berangan and (b) cv. Novaria after one month culture in MS liquid medium containing NaCl inoculated with rhizobacterial.

Changes of proline content on in vitro banana plantlets (a) 5.17 cv.Berangan and (b) cv. Novaria after one month culture in MS liquid medium containing NaCl inoculated with rhizobacterial.

Changes of peroxidase activity on in vitro banana plantlets 5-20 (a) cv.Berangan and (b) cv.Novaria after one month culture in MS liquid medium containing NaCl inoculated with rhizobacterial.

Effect of rhiaobacteria inoculation on changes of phenolic 5-23 content of in vitro banana plantlets (a) cv-Berangan and (b) cv.Novaria after one month culture in MS liquid medium containing NaCl.

Changes of Nitrate reductase activity on in vitro banana 5-25 plantlets (a) cv. Berangan and (b) cv.Novaria after one month culture in MS liquid medium containing of NaCl and inoculated with rhizobactrial.

Effect of rhizobacteria inoculation on nitrate content of in vitro banana plantlets (a) cv.Berangan and (b) cv.Novaria 5.28

after one month culture in MS liquid medium containing NaCl.

Chlorophyll content of in vitro banana plantlets (a) cv.Berangan and (b) cv.Novaria after one month cultme in MS liquid medium containing NaCl inoculated with rhiiobacterial.

Growth of in vitro banana plantlets (cv.Berangan , cv. Novaria) after one month culture in MS liquid medium containing boron inoculated with UPMB 10.

6.1A Growth of in vitro banana plantlets cv.Berangan culture in MS liquid medium supplemented with two concentration of boron, 1 pM(A) and 10 pM(B) inoculated with rhiiobacterial species UPMB 1 0.

Total soluble protein content of m vitro banana plantlets 6.1 1 (B= cv. Berangan, N= cv. Novaria) after one month culture in MS liquid medium containing boron inoculated with UPMBl 0.

Soluble nitrogen content in part of banana plantlets (B=cv. 6.13 Berangan N= cv. Novaria) after one month inoculated with UPMBlO in MS liquid medium containing boron using in vitro system .

Changes of proline content on in vitro banana plantlets 6.15 (B= cv.Berangan, N= cv. Novaria ) after one month culture in MS liquid medium containing boron and inoculated with UPMB 10.

Changes of peroxidase activity of in vitro banana plantlets 6.17 (B= cv. Berangan, N= cv.Novaria) after one month culture in MS liquid medium containing boron and in inoculated with UPMB 10..

Effect of UPMBlO inoculation on changes of phenolic 6.20 content in in vitro banana plantlets (B= cv.Berangan, N= cv.Novaria) after one month culture in MS liquid medium containing boron.

Changes of Nitrate reductase activity on in vitro banana 6.23 plantlets (B= cv. Berangan, N= cv.Novaria) after one month culture in MS liquid medium containing boron and inoculated with UPMBlO..

Effect of UPMBlO inoculation on nitrate content of in 6.25 vitrobanana plantlets (B=cv.Berangan, N= cv.Novaria) after one month culture in MS liquid medium containing boron.

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Leaf chlorophyll content of in vitro banana plantlets 6.27 (B= cv.Berangan,N= cv.Novaria) after one month culture in MS liquid medium containing boron inoculated with UPMB 10.

Effect of UPMBlO inoculation on growth of in vitro 7.8 banana plantlets culture in the media containing different concentrations of N03- and NH~'

7.1A Growth of in vitro banana plantlet culture in MS liquid medium without nitrogen: A (-N control), B (- N+UPMB 10).

Effect ratio of NO</NI&' on growth of banana plantlets 7.15 cv.Berangan

Effect of UPMBlO inoculation on growth of in vitro 7.16 banana plantlets culture in the media containing different ratio of nitratelammonia (30 mM:lO mM).

Eff'ect of UPMBlO inoculation on growth of in vitro 7.28 banana plantlets culture in the media containing various forms of nitrogen

Effect of UPMBlO inoculation on growth of in vitro 7.21 banana plantlets culture in the media containing different concentrations of ammomium sulphate.

Effect of UPMBlO inoculation on growth of in vitro 7-23 banana plantlets culture in the media containing different concentrations of potassium nitrate.

Effect of UPMBlO inoculation on growth of in vitro 7-26 banana plantlets culture in the media containing different concentrations of sucrose and light conditions.

Effect of UPMBlO inoculation on growth of in vitro 7-31 banana plantlets culture in the media containing activated charcoal.

E f f i t of UPMBlO inoculation on growth of in vitro banana plantlets culture in MS liquid medium supplemented with activated charcoal.

Effect of UPMBlO inoculation on growth of in vitro 7.35 banana plantlets culture in the media containing various forms of carbon sources.

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LIST OF ABBREVIATIONS

BAP

MS

IAA

ABA

PGPR Plant Growth Promoting Rhizobacteria

PGRs Plant growth regulators

UPMBlO Bacillus sphaericus UPMB 10

UPMB11 Microbacterium oxydens UPMB11

s ~7 AzospiriIlum brasilense Sp7

NRA Nitrate reductase activity

C/N ratio Carbonlnitrogen ratio

NAA a-Napthalene acetic acid

6- Benzylaminopurine

Murashige and Skoog

3-Indoleacetic acid

Abscisic acid

NEU

WUE

ACC

Nitrogen use efficiency

Water use efficiency

1 -arninocyclopropane-1 carboxylate