Studies on Agrobacterium-mediatedTransformation in Oat (Avena sativa L.)

THESIS

Submitted to the

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur

In partial fulfilment of the requirement forthe degree of

MASTER OF SCIENCE

In

AGRICULTURE(MOLECULAR BIOLOGY AND BIOTECHNOLOGY)

By

NAGESH RAOSAHEB DATTGONDE

Biotechnology centreJawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (MP)

2013

CERTIFICATE- I

This is to certify that the thesis entitled, “Studies on Agrobacterium-mediated Transformation in Oat (Avena sativa L.)” submitted in partial

fulfillment of the requirement for the degree of MASTER OF SCIENCE INAGRICULTURE (Molecular Biology and Biotechnology) of Jawaharlal

Nehru Krishi Vishwa Vidyalaya, Jabalpur is a record of the bonafide research

work carried out by Mr. NAGESH RAOSAHEB DATTGONDE under my

guidance and supervision. The subject of the thesis has been approved by the

Student’s Advisory Committee and the Director of Instruction.

No part of the thesis has been submitted for any other degree or

diploma (Certificate awarded etc.) or has been published / published part has

been fully acknowledged. All the assistance and help received during the

course of the investigation has been acknowledged by him.

(Dr. S. Tiwari)Chairman of Advisory Committee

THESIS APPROVED BY THE STUDENT’S ADVISORY COMMITTEE

Chairman: (Dr. S. Tiwari) ……………………………………………..

Member: (Dr. L.P.S. Rajput) ……………………………………………..

Member: (Dr. A. K. Naidu) ……………………………………………..

CERTIFICATE-II

This is to certify that the thesis entitled, “Studies on Agrobacterium-mediated Transformation in Oat (Avena sativa L.)” submitted by Mr.NAGESH RAOSAHEB DATTGONDE to the Jawaharlal Nehru Krishi Vishwa

Vidyalaya, Jabalpur, in partial fulfillment of the requirement for the degree of

MASTER OF SCIENCE in AGRICULTURE (Molecular Biology andBiotechnology), JNKVV, Jabalpur, after evaluation has been approved by the

Student’s Advisory Committee and the External Examiner and by the student’s

Advisory Committee after an oral examination on the same.

Place : Jabalpur (Dr. S. Tiwari)Date: ………… Chairman of Advisory Committee

MEMBER OF THE STUDENT’S ADVISORY COMMITTEE

Chairman : Dr. S. Tiwari ……………………

Member :

Member :

Dr. L.P.S. Rajput

Dr. A. K. Naidu

……………………

……………………

Director, Biotechnology centre : Dr. S. Tiwari .……………………

Director of Instructions : Dr. P.K. Mishra ..……………………

ACKNOWLEDGEMENT

Firstly I would express my sincere gratititude to almighty God, who gave me this

opportunity to giving my heartfelt thank to all the dedicated people who gave me support

and kind co-operation, encouragement during my studies and research work.

In presenting this text, I feel highly privileged to the chairman of my advisory

committee Dr. S. Tiwari, Director of Biotechnology Centre, JNKVV, Jabalpur for his

invaluable counsel, keen interest and constant encouragement during the course of the

study and preparation of thesis work.

I am deeply obliged to all the members of my advisory committee Dr. L.P.S.

Rajput, Principal Scientist, Biotechnology Centre and Dr. A. K. Naidu for their valuable

guidance and timely suggestions during the course of investigation. I am deeply obliged

and express my sincere gratitude to Mrs. Keerti Tantwai, Biotechnology Centre, whose

constant encouragement and valuable suggestions is unforgettable.

I would like to mention and express my special thanks to Dr. Iti Gontia-Mishra,

Mr. Niraj Tripathi and Mr. Sunil Kumar for helping me with tissue culture and molecular

work.

I also owe an everlasting debt of gratititudes to all the members of Biotechnology

Centre Ms. Shaly Sasidharan, Ms. Ritu Sharma and Mr. Sandip Rangdale for their advice

and help during the tenure.

If I forget to mention here about, my senior, Mr. Swapnil Sapre, Ms. Sapana

Varandani and Mr. Vijay Prakash Bansal; they always stands behind me like a pillar. I

would express my gratititude and heart full feeling to them and no words to giving them

thank.

I express my sincere thanks to my friends Yogesh Patil, Kachare Satish, Rupesh

Kulkarni, Roshani Sawalakhe, Shrikant Karale, Vaibhav Gaikwad, Chetan Bondre, Amol

Ganore, Kunal Bhalerao, Krishna Ambhure, Shantanu Zayale, Jagdeep Bilolikar and

Husain Basha for their support, best wishes and encouragement.

At this inexplicable moment of joy, I deem it a proud privilege to recall all the

cooperation and the contribution of my dear juniors Pankaj, Deva, Yogesh, Vishwajeet,

Vishwavijay and Sumit.

I would like to express my heartfelt gratitude to my grandfather Shri Ramrao L.

Delmade and grandmother Late Chandrabhagabai R. Delmade who always gave me a

helping hand, in any condition stand behind me and gave constant encouragement with a

smile of love and affection.

I would like to express my heartfelt gratitude to my parents Shri Raosaheb N.

Dattgonde and Smt. Laxmibai R. Dattgonde who always gave me a helping hand, in any

condition stand behind me and gave constant encouragement with a smile of love and

affection.

I have no words to thank my brothers Ambadas R. Dattgonde and sister Utkarsha

whose love and cheerful presence, joy and energy filled in my life with the joy of success

and prosperity.

Finally, I would like to thanks all those who directly and indirectly support me in

my life.

Place : Jabalpur

Date : July 2013 (Nagesh Raosaheb Dattgonde)

LIST OF CONTENTS

SI. Title Page

1 Introduction 1-4

2 Review of Literature 5-12

3 Materials and Methods 13-35

4 Results 36-48

5 Discussion 49-56

6Summery, Conclusions and Suggestions for further

work57-59

References 60-64

Appendices I-III

Vita

LIST OF TABLES

Number Title Page

3.1 List of oat genotypes used in the present studyalong with their pedigree

13

3.2 General composition and stock solutions of MS(Murashige and Skoog) basal medium

15

3.3 General composition and stock solutions of B5 basal(Gamborg) medium

16

3.4 Preparation and storage of growth regulator stocksolutions

18

3.5 Preparation of MS medium from stock solutions 19

3.6 Concentrations of plant growth regulators fortifiedwith MS culture media for preliminary experiments

21

3.7 Composition of Co-cultivation (CCM) 26

3.8 Shoot Elongation (SEM) and Rooting Medium (RM) 28

3.9 Composition of GUS staining solution 29

3.10 Composition of DNA Extraction buffer 30

3.11 PCR programme for different primers used in thestudy

32

3.12 List of primers and their features 34

3.13 The time course of the transformation procedure 35

4.1 Observation on in vitro regeneration of Oat cultivarJO-1

37

Number Title Page

4.2 Percent survival and regeneration in oat explants in

the presence of different concentrations of

antibiotics in regeneration (MS02B0.1N) medium

40

4.3 Effect of different treatment on transformation

efficiency (TE %) of embryo explants based on

survival on hygromycin containing media

42

4.4 Effect of different treatment on transformation

efficiency (TE %) of embryo explants based on

criteria of GUS assay

42

4.5 Effect of different treatment on transformation

efficiency (TE %) of embryo explants based on

criteria of PCR

43

4.6 Effect of different treatment on transformation

efficiency (TE %) of leaf base explants based on

hygromycin survival

44

4.7 Effect of different treatment on transformation

efficiency (TE %) of leaf base explants based on

criteria GUS assay

44

4.8 Effect of different treatment on transformation

efficiency (TE %) of leaf base explants based on

criteria PCR putative transgenic explants

45

4.9 Overall effects of co-cultivation period and different

treatments on transformation efficiency (TE %)46

List of Figures

SI. Titles Page

1Flow diagram of Agrobacterium-mediated transformation in

oat using different explants56

LIST OF PLATES

Number TitlePage

(In between)

1. A. Oat spiklets 36-37

B. Oat spiklets with immature seed 36-37

C. Oat plants with immature seeds spiklets 36-37

D. Oat mature seeds 36-37

E-F. Germination of oat seeds on MS media without

growth regulators (E: 2 days and F: 5 days after

culture)

36-37

2. 2. Culture of isolated oat embryos 37-38

A-B. Freshly isolated embryos 37-38

C-D. Callus initiation 37-38

E-F. Direct shoot and root proliferation from isolated

oat embryos

37-38

3. 3. Different stages of cultured leaf base of oat 38-39

A-B. Freshly cultured leaf base 38-39

C-D. Callus initiation 38-39

E-F. Direct shoot and root proliferation from cultured

leaf base of oat

38-39

4. 4. In vitro morphogenesis in oat 39-40

A-C. In vitro regeneration from cultured embryos 39-40

D-F. In vitro morphogenesis in cultured oat leaf base 39-40

Number TitlePage

(in between)

5. 5. Explants of oat showing GUS expression 46-47

A. Non transformed callus 46-47

B. Transformed callus 46-47

C. Shoot with root 46-47

D. Leaf 46-47

E. Morphogenic calli 46-47

F. Callus 46-47

G-H. Shoot with root 46-47

I. Leaf venation showing GUS expression 46-47

6. A. Diagrammatic representation of the binary vector

pCAMBIA 1305.1 containing 35S CaMV

promoter from Cauliflower mosaic virus and poly

A terminator, hptII Hygromycin phospho

transferse II gene, (NOS) Nopaline synthase

terminator, Kanamycin resistance gene for

bacterial selection

47-48

B. Culture of Agrobacterium tumefaciens strain

GV3101 harboring binary vector pCAMBIA

1305.1 carrying reporter gene uidA (β-

glucuronidase, GUS) and plant selectable

marker gene hptII under the CaMV 35S

promoter

47-48

C. Plasmid DNA of Agrobacterium tumefaciens

strain GV3101 harboring binary vector pCAMBIA

1305.1

47-48

Number TitlePage

(in between)

7. 7. Molecular analysis of PCR putative transgenic plant 47-48

A. PCR amplification of hptII gene from control plant and

putative transformants, Lane M-1kb DNA ladder,

Lane P-Positive control, Lane N-Nontransformed

control plant, Lane 1,2,3 and 5 putative transformants

47-48

B. PCR amplification of virD2 gene for Agrobacterium

DNA contamination free transgenic selection. M-

Marker, P-Positive control as a Agrobacterium DNA

amplified on 338bp, N-Negative control and 1,5

Positive, 2-4 Negative transgenic

47-48

C. PCR amplification of CaMV 35S promoter gene from

control plant and putative transformants, Lane M-1kb

DNA ladder, Lane P-Positive control, Lane N-

Nontransormed control plant, Lane 1-2 and 3-5

putative transformants

47-48

8. A-C. Plants regeneration with root formation 48-49

D. Regenerated plants ready for hardening 48-49

List of Abbreviations

Sl. Abbreviations Stands for

1 MS Murashige and Skoog’s medium

2 B5 Gamborg’s medium

3 BAP or BA Benzyl amino purine or 6-Benzyladenine

4 IBA Indole-3-butyric acid

5 IAA Indole-3-acetic acid

6 2,4-D 2,4-Dichlorophenoxyacetic acid

7 GA3 Gibberalic Acid

8 NAA Naphthalene acetic acid

9 bp Base pair

10 CTAB Cetyl Tri methyl Ammonium Bromide

11 EDTA Ethylene Diamine Tetra Acetate

12 mg Milli Gram

13 g Gram

14 µg Micro gram

15 ng Nano gram

16 L Liter

17 ml Milli Liter

18 µl Micro Liter

19 mm Milli Meter

20 M Molar

21 mM Milli Molar

22 µM Micro Molar

23 % Percentage

24 PCR Polymerase Chain Reaction

25 O.D. Optical density

26 rpm Revolutions per minute

27 TAE Tris base Acetic acid Glacial EDTA

28oC Degree centigrade

29 cv Cultivar

30 RH Relative humidity

31 h Hour

32 min Minute

33 DNA Deoxy-ribose Nucleic Acid

34 SDS Sodium Dodacile sulphate

35 LB Luria Bertaini

36 VAAT Vacuum Assisted Agrobacterium-mediated

transformation

37 VIAAT Vacuum Infiltration Assisted Agrobacterium-mediated

transformation

38 SAAT Sonication Assisted Agrobacterium-mediated

transformation

39 SVIAAT Sonication and Vacuum infiltration Assisted

Agrobacterium-mediated transformation

40 Vol. Volume

1

INTRODUCTION

Cultivated oat (Avena sativa L.) is an important agronomic cereal crop.

The oat crop is primarily produced for animal feed and human food, but recent

research has elevated its potential dietary value for human consumption. Oat

ranks sixth in world cereal production following wheat, maize, rice, barley and

sorghum (Choubey et al., 1996). It is important winter forage in many parts of the

world and is grown as a multipurpose crop for grain, forage or as a rotation crop.

It has excellent growth habit, quick recovery after cutting and good quality fodder.

It is a palatable, succulent and nutritious fodder crop with excellent protein

quality. Oat requires a long, cool season for its growth; therefore, it is

successfully grown in the plains and hilly areas of the country. Currently, oat

remains an important grain and forage crop in many parts of the world and grown

on 13.2 million hectares with a grain production of 26.2 million metric tons in

2003. The Russian federation is the largest producer followed by Canada and the

USA. Land area devoted to oat has fallen substantially in the past several

decades with oat being placed by higher value crops, such as soybean in USA.

In India, this crop occupies maximum area in Uttar Pradesh (34%) followed by

Punjab (20%), Bihar (16%), Haryana (9%) and Madhya Pradesh (6%) (Choubey

et al., 1996).

In India, oat productivity is quite low as compared to other cereal crops. It

suffers heavy yield losses due to several biotic and abiotic stresses. Biotic

stresses including diseases such as crown rust (caused by Puccinia coronata

f.sp. avenae), stem rust (caused by Puccinia graminis f.sp. avenae), powdery

mildew, septoria leaf blight, victoria blight, bacterial blights, soil-borne viruses

and nematodes etc. have been the primary disease problems in the major oat-

producing areas around the world. Abiotic stresses comprises extreme

temperatures, drought, high salinity, cold and water logging which often result in

significant losses to the yield of oat crop.

Crop and plant improvement is a major area of commercial interest. A

great deal of efforts has been made towards the development of new cultivars of

2

oat with improved disease, pest and herbicide tolerance. Genetic improvement of

commercially important oat cultivars through classical breeding is laborious and

time-consuming, expensive and sometimes even unsuccessful.

The pre-requisite for any genetic engineering process is the availability of

an efficient in vitro regeneration system from cell and callus cultures. The

transformation system is requires, efficient system for embryogenic callus

induction and shoot regeneration have been considered to be the basic step in

production of stable transgenic plants. Various tissue culture techniques are

being applied for varietal development of cereal crops including oat in different

countries (Kim et al., 2004). Among these techniques, anther culture, protoplast

fusion, leaf culture, root culture and dehusked seed culture are important in oat

an ancillary techniques for the formation of novel oat varieties.

The new gears of biotechnology such as genetic transformation has

enabled us to insert any gene for any quality character such as delayed

flowering, early maturity and increment in girth etc. as well as against biotic and

abiotic stresses. A remarkable progress has been made in the development of

gene transfer technology (Somers et al., 1992), which ultimately has resulted in

the production of large number of transgenic plants both in dicots and monocots.

Genetic modification is an important experimental tool that can be used to

analyze and understand the mechanisms responsible for the expression of

transgenes or endogenous genes and to create plants with the desired

characteristics. For genetic transformation, two basic methods are available i.e.

biolistic and Agrobacterium-mediated transformation (Gasparis et al., 2008).

Potential benefits from these transgenic plants include higher yield and enhanced

nutritional value reduction in pesticide and fertilizer use.

Transgenic oat plants have been obtained using particle bombardment

methods for gene transfer (Pawlowski and Somers, 1998). DNA integration

patterns in transformed plant tissue obtained via particle bombardment tend to be

highly variable and multiple or fragment copies of introduced DNAs are common,

especially when older cultures are targeted. Cho et al., (2003) studied the

3

expression of green fluorescent protein (gfp) and its inheritance in transgenic oat

plants transformed with a synthetic green fluorescent protein gene driven by a

rice actin promoter where proliferating SMCs were bombarded with a mixture of

plasmids containing the sgfp (S65T) gene and one of three selectable marker

genes, phosphinothricin acetyltransferase (bar), hygromycin phosphotransferase

(hpt) and neomycin phosphotransferase (nptII).

Till date there is only a single report available on Agrobacterium mediated

transformation of oat by Gasparis et al. (2008). Two different types of explants:

immature embryos and leaf base segments were used for transformation.

Immature embryos are composed of highly totipotent, meristematic cells,

whereas leaf base segment consist of differentiated cells, which have to undergo

dedifferentiation before somatic embryo development and plant regeneration and

these difference in these explants were employed to test cell-competence to

Agrobacterium-mediated transformation and transgene expression.

A new and potentially more efficient method, Sonication Assisted

Agrobacterium-mediated Transformation (SAAT) (Trick and Finer, 1997) was

developed for delivery of Agrobacterium to plant target tissues. SAAT is a very

easy, low cost method to substantially enhance the efficiency of Agrobacterium-

mediated transformation of low or non-susceptible plant species. The strength of

this method is that the cavitation caused by sonication results in thousands of

micro-wounds on and below the surface of the plant tissue. This wounding

pattern permits Agrobacterium to travel deeper and more efficiently throughout

the tissue than conventional microscopic wounding, increasing the probability of

infecting plant cells. In some another studies the Vacuum Assisted

Agrobacterium-mediated Transformation (VAAT) (Lin et al., 2009), and some

with application of both the methods (SAAT and VAAT) (Amoah et al., 2001)

used to increase the transformation efficiency by increasing the infiltration of

Agrobacterium in the plant cell through these wounds.

Since there are few reports of Agrobacterium-mediated transformation

studies in oat, this was taken as the basis to take up the present investigation to

4

generate an efficient protocol for Agrobacterium-mediated transformation of oat

using various explants. Also check the transformation efficiency by using

Sonication and Vacuum Agrobacterium-mediated assisted transformation

separately and by combining both the methods.

In the light of above facts, the present experiments were envisaged to

fulfill following objectives:

1. To study the response of different explants of oat to Agrobacterium -

mediated transformation.

2. To standardize an efficient protocol for Agrobacterium - mediated

transformation in oat.

3. To validate the genetically transformed plants.

5

REVIEW OF LITERATURE

Oat is an important cereal crop produced mainly for animal feed and

human food. Presently, it has also being known for its potential dietary value for

human consumption. There are several coordinated public breeding programs

focusing on the genetic enhancement of oat. Despite these efforts, a yield and

quality losses due to stress, pathogens and insects continue to occur. The

continued development of biotechnological approaches based on genetic study

can manipulate oat which would aid in oat quality enhancement efforts (Carlson

et al., 2007).

2.1 In vitro plant regenerationThe establishment of in vitro regeneration systems plays a significant role

in the biotechnological improvement of cereals. While in the major cereals like

maize, rice, wheat and barley, impressive progress has been achieved towards

developing efficient plant regeneration systems from different tissues and organs,

but in oats only a few work have been reported. However, since the first

publication on successful plant regeneration from oat callus (Lorz et al., 1976),

plant tissue culture methods have advanced considerably.

Plant tissue culture technology is playing a vital role in basic and applied

studies, including crop improvement. In modern agriculture, about 150 plant

species are extensively cultivated. Many of these are reaching the limits of their

improvement by traditional methods. The application of tissue culture technology,

as a central tool or as an adjunct to other methods, including recombinant DNA

techniques, is an initial step towards plant modification and improvement for

agriculture, horticulture and forestry. The initial development in the field of plant

tissue culture i.e. the ability to recover plants, not only from micropropagated

meristematic tissues, but from in vitro cultures of protoplasts, cells,

undifferentiated plant tissues (callus), pollen, ovules, embryos, cotyledons and

other explants tissues, take quite a long time for its development hence, for any

tissue culture system sterilization and maintaining aseptic condition is the pre-

6

requisite step. The most tedious parts of in vitro techniques are sterilizing plant

materials and media and maintaining aseptic conditions. Obtaining sterile plant

material is difficult and despite precautions taken, 95% of cultures will end up

contaminated if the explants are not disinfected in a proper manner. Because

living materials cannot be exposed to extreme heat and retain their biological

capabilities, plant organs and tissues are sterilized by treatment with a

disinfecting solution.

According to the protocol by Chen et al. (1995) the seeds were surface

sterilized with 70% ethanol for two minutes, followed by two washes with sterile

distilled water, and then treating with 5% sodium hypochlorite solution for 10 min

followed by five washes with sterile distilled water. The sterilized seeds were

germinated on solid MS medium in tissue culture jars (70 x 75mm) at 27°C in the

dark for one day. The germinated seeds were grown in the dark at 25°C in

incubator or in a growth cabinet at 25°C with 16h day illumination.

The best callus induction in oats was achieved when immature embryos

were used as explants. However, Chen et al. (1995) reported that immature

embryos are not a convenient source of material for transformation studies

because they require mature plants for their production, require specialized

growth environments and may be restricted to a short season. Therefore, it is an

urgent need for development a reliable alternative regeneration system in oats.

Although mature embryos produce lower callus yields, their availability

throughout the year makes them an excellent explants source (Birsin et al.,

2001).

An efficient in vitro regeneration system for oat using leaf bases as

explants has been developed by Chen et al. (1995). Leaf segments isolated from

seedlings grown for 2 to 5 days in the dark which were subjected to an initial

screening for callus formation. Callus was induced from cultured leaf base

segments on MS medium containing 2mg l-1 2,4-D. The frequency of callus

formation in leaf explants was strongly dependent on the position of the segment

taken from seedling and age of the seedling. The callus was induced only from

7

the basal segments of leaves and the frequency of callus induction of the first

segment from the leaf base was higher than that of the subsequent segments

from the leaf base. Callus tissue first became visible on the leaf explants within a

week on callus medium. Some compact and opaque callus developed within soft

callus masses one week later. The four to five week old calli were transferred to

a shooting medium. Shoots of 3mm long were excised from the callus and

transferred to fresh shooting medium. Seven days later, when most of the shoots

had grown 15 to 20mm long, they were transferred to a rooting medium. Roots

developed within 5 days. The plants with shoots and well developed roots were

transplanted to pots containing soil. Whole plants were able to grow in soil to

normal mature plants within ten weeks.

Chen et al. (1995) developed an efficient short term regeneration system

using seedling derived from oat (Avena sativa). Callus derived from the leaf base

showed a higher response of plant regeneration than callus initiated from

mesocotyls and more mature parts of the leaves. A correlation between the

nuclear DNA content of the donor material, was analyzed with flow cytometry and

its ability to form callus was observed. Somatic embryogenesis was developed

from callus derived from tissue close to the apical meristern. Plant regeneration

media with various concentrations of auxins were examined. Callus from three

different cultivars showed a similar regeneration potential with an optimal

regeneration frequency of 60%. About 2 months after inoculation, regenerated

plantlets were obtained which were further transferred to green house for

cultivation.

Gless et al. (1997) developed a reliable and efficient protocol for the

regeneration of fertile plants derived from leaf base segments of young in-vitro-

grown oat seedlings. Callus induction and shoot regeneration were achieved

when the basal region of young seedlings was cultured on auxin-containing

medium. Callus induction efficiencies as well as regeneration frequencies were

correlated with the developmental stages and the genotype of the explants.

8

Nuutila et al. (2002) developed a protocol for regeneration where the

nitrogen composition, sugar and auxin concentrations of callus induction medium

were optimized in order to improve the regeneration of green plants from two

elite oat cultivars, Aslak and Veli. For both the cultivars, the production of

plantlets was doubled by optimization. The results obtained also clearly

demonstrated that cultivars of the same species may differ drastically in their

requirements for essential media components. Veli required higher total amounts

of nitrogen (67.8mM) than Aslak (44.9mM) but less maltose and 2,4-

dichlorophenoxyacetic acid (28g l-1 and 0.6mg l-1) than Aslak (38g l-1 and 2mg l-

1).

Kim et al. (2002) reported that mature embryos of five oat genotypes were

cultured to develop an efficient method of callus induction and plant regeneration.

Murashige and Skoog (MS) and N6 media supplemented with 2,4-

dichlorophenoxyacetic acid (2,4-D) and kinetin was used for callus induction.

Significant callus induction was observed among the combinations of various

plant growth regulators. Callus induction showed highest efficiency in medium

containing 3mg l-1 of 2,4-D. The highest frequency of callus induction was

obtained in Gwiri37. For plant regeneration calli induced from mature embryos

were transferred onto MS and N6 media supplemented with combinations of 6-

benzyladenine (BA) and napthaleneacetic acid (NAA) for 5 weeks. Highest

percentage of plant regeneration showed in MS medium containing 0.2mg l-1 of

NAA and 1mg l-1 of BA. The callus initiation medium affected the subsequent

plant regeneration. Treatment with 3mg l-1 of 2,4-D and 3mg l-1 of kinetin in callus

induction media showed high frequency for plant regeneration. Regenerated

shoots were treated with indole 3-butyric acid induced root formation.

Kim et al. (2004) reported plant regeneration of Korean oat using mature

embryo and leaf base segment as explant. MS media supplemented with 2,4-

dichlorophenoxyacetic acid, kinetin and picloram was used for callus initiation

from mature embryos and leaf base segments. 3mg l-1of 2,4-D and 3mg l-1 of

picloram when used in callus induction medium showed highest frequency for

plant regeneration from mature embryos. Leaf base segments were transferred

9

to callus induction medium and incubated at 25°C in 16/8 hour light/ dark cycle

for 3 weeks. Callus induction from leaf base segments of Malgwiri variety showed

highest efficiency in medium containing 3mg l-1 of 2,4-D and 1mg l-1 of kinetin

(91.8%). In case of Samhangwiri, the combinations of phytohormones did not

show significant difference. Regeneration from leaf base segments showed

highest frequency in shoot medium containing 1mg l-1 of antiauxin, tri-

iodobenzoic acid (TIBA) and 1mg l-1 of 6-benzyladenine (BA). Calli induced from

leaf base segments of Samhangwiri and Malgwiri in media containing 3mg l-1 of

2,4-D and 3mg l-1 of picloram showed better regeneration. Thus callus induction

medium is an important factor for plant regeneration.

Hao et al. (2006) developed an efficient protocol where enhanced somatic

embryogenesis and plant regeneration was obtained using young leaf bases of

naked oat (Avena nuda) as explants by including salicylic acid (SA) and carrot

embryogenic callus extracts (CECE) in media. Five and four-fold improvement

was achieved in somatic embryogenesis and plant regeneration, on the

corresponding media supplemented with 0.5mM SA and CECE as compared to

control, respectively. Some physiological and biochemical changes were

assayed in both embryogenic callus (EC) and non-embryogenic callus (NEC) and

results indicated that superoxide dismutase activity was stimulated and catalases

and ascorbate peroxidase activities were inhibited, while the O2 (superoxide

anion) content was reduced and the hydrogen peroxide level was promoted in

EC compared with NEC. Reduced malondialdehyde content and relative

electrolyte leakage were also detected in EC.

2.2 Agrobacterium-mediated transformationGene transfer is the introduction of genetic information from any living

organism into a new host that can help provide a solution to certain constraints

that limit crop production or quality. Such crops are genetically modified (GM) or

transgenic. Transgenic crops that have been commercialized include maize,

soya, cotton and canola. Two popular strategies for gene transfer to plants are

the Agrobacterium method and direct DNA introduction by micro-particle

bombardment. The efficient production of transgenic plants requires stringent

10

selection procedures supported by a selectable marker gene that confers

resistance to agents such as antibiotics or herbicides. Several such selection

systems have recently been described for grain legumes, based on the marker

genes neomycin phosphotransferase II (nptII), hygromycin phosphotransferase

(hph, aph IV or hyg) ( Popelka et al., 2004).

Kuai et al. (2001) produced fertile transgenic plants of oat (Avena sativa

L.var. Melys) followed by microprojectile bombardment of primary embryogenic

calli from immature embryos with two plasmids containing the bar gene or the β-

glucuronidase (uidA) gene, after selection with glufosinate ammonium. Eleven

plants were regenerated from phosphinothricin resistant callus, with three of the

eleven plants containing either intact or rearranged copies. No plants co-

transformed with the non-selected uidA gene were detected. Stable transmission

and expression of the bar gene in the T1 inbred progenies occurred in a

Mendelian manner in one line, which contained an intact bar gene, and in all six

T2 lines tested from this transformant.

Cho et al. (1999) developed a highly efficient and reproducible

transformation system for oat (Avena sativa L. cv. GAF/Park-1) using

microprojectile bombardment of highly regenerative tissues derived from mature

seeds. Callus was induced under dim light conditions on medium containing 2,4-

D, BAP and high cupric sulfate. Highly regenerative tissues, generated from

embryogenic callus, were used as a transformation target. From 327 individual

explants bombarded with the β-glucuronidase gene and a hygromycin

phosphotransferase gene, 84 independent transgenic events were obtained after

an 8-12 weeks selection period on hygromycin. All events were regenerative,

giving an effective transformation frequency of 26%; co-expression of GUS

activity occurred in 70% of the independent events. Presence of the foreign

genes in DNA from leaf samples of T0 and T1 plants was confirmed by PCR

amplification and/or DNA blot hybridization. Fertility of the plants from the

transgenic lines was 63% (24/38) and the transgene(s) was stably transmitted to

T1 and T2 progeny.

11

Gasparis et al. (2008) reports on the successful Agrobaterium-mediated

transformation of oat. Three cultivars, two types of explants and three

combinations of strain/vectors, were successfully used for transformation of other

cereals. Transgenic plants were obtained from the immature embryos of cv.

Bajka, Slawko and Akt and leaf base explants from Bajka after transformation

with A. tumefaciens strain LBA4404(pTOK233). The highest transformation rate

(12.3%) was obtained for immature embryos of cv. Bajka.

2.2.1 Sonication assisted Agrobacterium-mediated transformationTrick and Finer (1997) described a new and efficient Agrobacterium-based

transformation technology that overcomes the barriers and enhances DNA

transfer in such diverse plant groups as dicots, monocots and gymnosperms.

They used sonication-assisted Agrobacterium-mediated transformation (SAAT),

involves subjecting the plant tissue to brief periods of ultrasound in the presence

of Agrobacterium. It was observed that SAAT increases transient transformation

efficiency in several different plant tissues including leaf tissue, immature

cotyledons, somatic and zygotic embryos, roots, stems, shoot apices,

embryogenic suspension cells and whole seedlings. A 100 to 1400-fold increase

in transient β-glucuronidase expression has been demonstrated in various

tissues of soybean, Ohio buckeye, cowpea, white spruce, wheat and maize.

Stable transformation of both soybean and Ohio buckeye has been obtained

using SAAT of embryogenic suspension culture tissues.

SAAT is an easy, low cost method to substantially enhance the efficiency

of Agrobacterium-mediated transformation of low or non-susceptible plant

species. These method was also used in different crops, as like cowpeas (Pathak

and Hamzah, 2008), flax (Beranova et al., 2008), lily (Kim et al., 2007), soybean

(Santarém et al., Trick and Finer, 1998), (Meurer et al., 1998), sunflower (Weber

et al., 2003). The strength of this method is that the cavitation caused by

sonication results in thousands of micro-wounds on and below the surface of the

plant tissue. This wounding pattern permits Agrobacterium to travel deeper and

more completely throughout the tissue than conventional microscopic wounding,

increasing the probability of infecting plant cells.

12

2.2.2 Vacuum assisted Agrobacterium-mediated transformationLin et al. (2009) used the mature embryos of soaked seeds were pierced

by a needle and soaked in the Agrobacterium inoculum under vacuum infiltration.

They worked on herbicide or antibiotic analysis and molecular analysis were

conducted on T0 plants. The results showed that although the efficiency of

transformation was about 6.0%, it was easier to transform indica rice using this

method and the transformation process was significantly shortened. The success

of transformation was further confirmed by the genetic and molecular analyses of

T1 transformants. This method was also used in different crops like Arabidopsis

(Ye et al., 1999), soybean (Mariashibu et al., 2013), pakchoi (Xu et al., 2008),

alfalfa, radish, pakchoi and petunia (Grabowska and Filipecki, 2004).

2.2.3 Sonication and Vacuum assisted Agrobacterium-mediatedtransformation

Amoah et al. (2001) reported an efficient transformation of inflorescence

tissue from ‘Baldus’ which is a commercial wheat variety, using the

agrobacterium strain AGLI harbouring the binary vector pAL 156. The effect of

different factors used for transformation as like duration of preculture, vacuum

infiltration, sonication treatment and Agrobacterium cell density was studies for

the expression of uidA gene.

The reports for use of sonication and vacuum assisted Agrobacterium-

mediated transformation are known for kidney bean (Liu et al., 2005) and radish

(Park et al., 2005) citrus (Oliveira et al., 2009), and banana (Subramanyam et al.,

2011), which showed tremendous increase in transformation efficiency.

13

MATERIALS AND METHODS

In the present investigation, experiments related to “Studies on

Agrobacterium-mediated transformation in oat (Avena sativa L.)” were carried

out in the Plant Tissue Culture and Transgenic Laboratory, Biotechnology

Centre, Jawaharlal Nehru Krishi Vishwavidyalaya, Jabalpur. Details of the

protocols are given below. List of oat genotypes used in the present study

along with their pedigreeare given in Table 3.1.

3.1 Experimental material3.1.1 Explants material

The commercial cultivar of Oat ‘JO-1’ was selected for the “Studies on

Agrobacterium-mediated transformation in Oat (Avena sativa L.)” on the basis

of good regeneration capacity ‘JO-1’ variety was selected for study

(Varandani, 2011) among other five selected genotype. Seeds of oat varieties

JO-1, JHO-822, JHO-851, OS-6 and Kent were obtained from the ‘All India

Coordinated Forage Research Project (ICAR), Department of Agronomy,

JNKVV, Jabalpur’. Various explants viz. leaf base, mature embryo of oat were

obtained from in vitro germinated seeds (Plate 1). Oat genotypes used in the

present study along with their pedigree are shown in Table 3.1.

Table 3.1 List of oat genotypes used in the present study along withtheir pedigree.

Sl. Genotype Pedigree

1. JO-1 Cross Kent × UPO 50

2. OS-6 Cross Hfo-10 × Hfo 55-P2-2

3. Kent Introduction from USA

4. JHO-822 Cross 4268 × Indio

5. JHO-851 Selection from Japanese introduced materialHIUGAKAIRYOKURO)

14

3.1.2 Agrobacterium cultureFor genetic transformation studies, Agrobacterium tumefaciens strain

GV 3101 containing binary plasmid vector pCAMBIA 1305.1 (11846bp) was

obtained from National Research Centre on Plant Biotechnology (NRCPB),

IARI, New Delhi. This vector comprise of gus (β-glucuronidase) gene with

catalytic intron under control of CaMV35S promoter and nos terminator,

and hptII (Hygromycin phosphotransferase II) gene as plant selectable

marker under the control of CaMV35S promoter and poly A terminator

(Plate 6A).

3.1.3 Glasswares, plasticwares, Chemicals and reagentsAll the glassware’s and plastic wares used in the study were procured

from Borosil and Tarsons respectively. All the chemicals used in the present

investigation were of plant tissue culture and molecular biology grade

procured from reputed companies and suppliers.

3.2 Methods3.2.1 In vitro plant regeneration

For the development of transgenic oat, efficient and reproducible plant

regeneration system from explants cultures is a prerequisite.

3.2.1.1 ExplantsThe explants viz. mature embryos were excised from mature seeds

(Plate 2), while leaf bases were excised from 6 days old seedlings (Plate

3) respectively and cultured to initiate in vitro cultures and transformation

via Agrobacterium tumefecians.

3.2.1.2 Culture MediaMurashige and Skoog (MS) medium (Murashige and Skoog, 1962)

and Gamborg’s B5 medium (Gamborg et al., 1968) fortified with different

combination of auxins and cytokinin were used for plant tissue culture. A

general composition of basal MS and B5media are given in the Table 3.2

and 3.3.

15

Table 3.2 General composition and stock solutions of MS (Murashigeand Skoog) basal medium.

ConstituentsAmount (mg l-1)

present inoriginalmedium

Amount to betaken for stocksolutions (g l-1)

Inorganic compounds

a. Macronutrients (1X) (40X)

KNO3 (Potassium nitrate) 1900 76

NH4NO3 (Ammonium nitrate) 1650 66

CaCl2 .2H2O (Calcium chloride) 440 17.6

MgSO4.7H2O (Magnesium sulphate) 370 14.8

KH2PO4 (Potassium phosphate) 170 6.8

b. Micronutrients (200X)

MnSO4.4H2O (Manganese sulphate) 22.3 4.46

ZnSO4.7H2O (Zinc sulphate) 8.6 1.72

H3BO3 (Boric acid) 6.2 1.24

KI (Potassium iodide) 0.83 0.17

Na2MoO4.H2O (Molybdic acid) 0.25 0.05

CoCl2 (Cobalt chloride) 0.025 0.005

CuSO4.2H2O (Copper sulphate) 0.025 0.005

c. Iron stock (200X)

Disodium EDTA (Na2- EDTA) 37.25 7.45

Ferrous sulphate (FeSO4) 27.85 5.57

Organic compounds

d. Vitamins (1000X)

Nicotinic acid 0.50 0.5

Pyridoxine HCl 0.50 0.5

Thiamine HCl 0.10 0.1

e. Amino acid (1000X)

Glycine 2 2

16

Table 3.3 General composition and stock solutions of B5 basal(Gamborg) medium.

ConstituentsAmount (mg l-1)

present inoriginal medium

Amount to betaken for stocksolutions (g l-1)

a. Macronutrients (1X) (40X)KNO3 (Potassium nitrate) 2500 100.00

CaCl2.H2O (Calcium chloride) 150 6.00

MgSO4.7H2O (Magnesium

sulphate) 250 10.00

(NH4)2SO4 (Ammonium sulphate) 134 5.36

NaH2PO4.H2O (Sodium

phosphate) 150 6.00

b. Micronutrients (200X)H3BO3 (Boric acid) 300 60.0

MnSO4.H2O (Manganese

sulphate) 1000 200.0

ZnSO4.7H2O (Zinc sulphate) 200 40.0

KI (Potassium iodide) 75 15.0

Na2MoO4.2H2O (Molybdic acid) 25 5.0

CuSO4 (Cupric sulphate) 2.5 0.5

CoCl2.6H2O (Cobalt chloride) 2.5 0.5

c. Iron stock (200X)Disodium EDTA (Na- EDTA) 37.25 7.45

Ferrous sulphate (FeSO4) 27.85 5.57

d. Vitamins (100X)Myo-inositol 100 10.0

Nicotinic acid 1 0.1

Pyridoxine-HCl 1 0.1

Thiamine-HCl 10 1.0

3.2.1.3 Preparation of stock solutions3.2.1.3.1 Stock solution of macronutrients

To prepare 1L of macronutrients stock solution, the salts mentioned

in Tables 3.2 and 3.3 were dissolved one after another in 600ml of double

17

distilled water and then the volume was made to 1L after filtering, this

solution was stored in refrigerator at 4°C.

3.2.1.3.2 Stock solution of micronutrientsTo make 1L of this stock solution, the salts were dissolved

sequentially as shown in Table 3.2 and 3.3, in 800ml of distilled water and

final volume was adjusted to 1L.

3.2.1.3.3 Stock solution of ironNa2-EDTA (7.45g) was dissolved in boiling water. Thereafter, 5.57g

of FeSO4 was added gradually. The solution was stirred for at least 1h in

hot condition until the colour of the solution changes to golden yellow.

Finally the volume was made up to 1L and stored in refrigerator, in an

amber coloured bottle.

3.2.1.3.4 Stock solution of vitaminsTo make 50ml of vitamin stock solution, 25mg of nicotinic acid was

first dissolved in 25ml of boiling distilled water and after cooling, other two

vitamins pyridoxine-HCl (25mg) and thiamine-HCl (5mg) were added. The

final volume was made to 50ml. This solution was stored in refrigerator at

0°C for a maximum period of 10 days.

3.2.1.3.5 Stock solution of amino acidGlycine (100mg) was dissolved in 50ml of distilled water and stored

at 0°C for a maximum period of 15 days.

3.2.1.3.6 Stock solution of growth regulatorsGrowth regulators were not directly dissolved in water. Firstly it was

dissolved in water-miscible solvents and finally water was added to make

up to the desired volume. Auxins, NAA and IBA (100mg) were initially

dissolved into 3 to 5ml of absolute ethanol and then volume was made up

to 100ml by adding ultra pure water. Cytokinin, BAP (100mg) was first

dissolved in to 3 to 5ml of 1N NaOH and then, final volume was made up

to 100ml by adding ultrapure water.

18

Table 3.4 Preparation and storage of growth regulator stocksolutions.

Growth regulators

Solution preparation

Solvent DiluentsPowderstorage

Liquidstorage

Sterilization

2,4-Dichlorophenoxy-

acetic acidEthanol Water RT 2-8°C CA

Indole-3-acetic acid

Free acid (IAA)1N NaOH Water 0°C 0°C CA/F

Indole-3-acetic acid

Sodium salt1N NaOH Water 2-8°C 0°C CA/F

α-Naphthalene acetic

acid Free acid (NAA)1N NaOH Water RT 2-8°C CA

6-Benzylaminopurine

(BAP)1N NaOH Water RT 2-8°C CA/F

Kinetin 1N NaOH Water -0°C -0°C CA/F

CA=Co-autoclavable, F=Filter sterilization.

To obtain the final working concentration of 1mgl-1 of plant growth

regulator in culture medium, 1ml of the stock solution was added to 1L

medium. It was calculated by using following formula to meet the

requirement as given in the Table 3.4.

Vol. of stock solution=Desired growth regulators concentration × Medium Vol.

Stock solution concentration

3.2.1.4 Preparation of culture mediaFor in vitro regeneration of oat, MS basal medium was prepared

from stock solutions as per Table 3.5.

19

Table 3.5 Preparation of MS medium from stock solutions.

Components MS Medium (1l)

Macronutrients (ml l-1) 25

Micronutrients (ml l-1) 5

Iron stock (ml l-1) 5

Vitamin stock (ml l-1) 1

Amino acids (ml l-1) 1

Growth regulators (ml l-1) as required

Myo-inositol (mg l-1) 100

Sucrose (g l-1) 30

Agar (g l-1) 8

Desired concentration of auxin and cytokinin were added from stock

solutions according to the culture media combinations used (Table 3.4).

Unless specified otherwise, all fortified MS media supplemented with micro

and macro nutrients, vitamins, 30g l-1 sucrose and 8g l-1 agar and the pH of

the medium was adjusted to 5.8 with addition of 1N NaOH or 1N HCl and

final volume was made to 1L (Table 3.2). Culture media were sterilized in

one litre aliquots by autoclaving at 121oC and 15 psi stream pressure for 20

min before pouring into pre-sterilized 100x17mm glass Petri dishes (30-

35ml/Petri dish) under aseptic condition on laminar air flow cabinet.

3.2.2 Sterilization and germination of seedsInitially oat seeds were washed thoroughly with tap water in order to

remove dust and other particles followed by washing with distilled water with

2-3 drops of tween-20 for 20 min. The seeds were rinsed with distilled water

8-9 times. Further sterilization was carried out inside the laminar air flow

chamber. Seeds were treated with 70% ethanol for 2 min followed by 0.5%

HgCl2 solution for 5 min. Sterilized seeds were washed thoroughly with

autoclaved distilled water for 4-5 times to overcome the poisonous effect of

20

HgCl2. Finally, seeds were soaked in sterile water for 1-2 min prior to

inoculation in to MS medium without any growth regulator.

3.2.3 Mature embryo as explantsSeeds were sterilized as described in section 3.2.2. The mature

embryos were removed from the imbibed seeds and placed, scutellum-up, on

MS medium with different combinations of growth regulators as mentioned in

Table 3.6 (Plate 2). Plates were incubated at 25°C for 15 days in dark. For the

regeneration, embryogenic part of the callus was cut into small pieces

approximately 2-3mm in size and inoculated on MS regeneration media

(MS2B.1N) and incubated for 5 weeks at 25°C in a 16h light and 8h dark

photoperiod. Small shoots (2-3cm) were sub-cultured on growth regulator-free

root regeneration medium. Plants were hardened under laboratory conditions

before they were transferred to the greenhouse.

3.2.4 Leaf base as explantsSeeds were sterilized as described in section 3.2.2. They were

germinated under light conditions on MS medium. Leaf-base segments were

taken from 4-6 day-old seedlings. The leaves were separated from coleoptiles

and sequentially cut into 1mm transverse sections, starting from the original

leaf base with a scalpel in a sterile petri- dish, a sheet of millimetre paper was

placed underneath to allow accurate sizing during dissection. Segments 1-6

starting from base (1mm each) were compared for their embryogenic callus

induction efficiency (Plate 3). Only those calli, containing distinct embryogenic

development visualized under the stereomicroscope were considered to be

embryogenic. Other types of calli (e.g. watery, translucent callus) were

discarded. On the basis of this comparison, only segments of 1-3mm were

further used for callus development. After about 4-5 weeks, calli were

transferred to shoot induction medium (for regeneration) with different

combinations of growth regulators (Table 3.6) and cultured in light at 25°C.

Shoots were sub-cultured on growth regulator-free root regeneration medium.

Plants were kept under laboratory conditions for 10-15 days before they were

transferred to the greenhouse for hardening.

21

Table 3.6 Concentrations of plant growth regulators fortified with MSculture media for preliminary experiments.

Sl. MediaGrowth regulators (mg l-1)

2,4-D IAA NAA BA IBA

1 MS2D 2.0 - - - -

2 MS6D 6.0 - - - -

3 MS10D 10.0 - - - -

4 MS50D 50.0 - - - -

5 MS2B.1N - - 0.1 2.0 -

6 MS5B.1N - - 0.1 5.0 -

7 MS1B1N - - 1.0 1.0 -

8 MS2IBA - - - - 2.0

9 MS3IBA - - - - 3.0

10 MS3IAA - 3.0 - - -

3.3 Culture conditionsPetri dishes were incubated at 25 ± 2°C under dark condition for one

week. Later they were subjected to a photoperiod regime of 12h at 1200 lux

luminance provided by photosynthetically active radiation (PAR) lamps.

3.4 Regeneration of plantsAfter 45 days, observations were recorded for all explants cultures and

calli were transferred to MS medium without growth regulators for maturation.

For the plant regeneration, the calli were subsequently transferred into the

regeneration medium consisting of MS basal medium supplemented with

different concentrations of plant growth regulators in varying combinations

(Table 3.6), 30g l-1 sucrose and 8g l-1 agar powder. Where root formation was

not obtained on regeneration medium, plantlets were subsequently sub

cultured to rooting medium, (MS basal medium supplemented with 1.1mg l-1

BAP, 0.1mg l-1 NAA, 15g l-1 sucrose and 8g l-1 agar).

22

3.5 Hardening of regenerated plantsThe roots of plantlets were rinsed with sterile lukewarm water to wash-

off the adhering agar (Plate 8). The plantlets were transferred to root trainers

filled with sterilized mixture of sand, soil and FYM in 1:1:1 ratio. Transplanted

root trainers were transferred to a glass house under 30 ± 2oC and 60 ± 5%

RH for 2-3 weeks for acclimatization.

3.6 Experimental designThe factorial completely randomized design (CRD) was used to study

the effect of culture medium and genotypes and their interactions on callus

induction, formation of morphogenic and embryogenic calli, organogenesis

and plant regeneration of oat from cultured explants. 90 explants each were

cultured for each separate treatment for both explants and each treatment

was performed in two replications and computation of the effect of all factors

was carried out separately.

3.7 Observations recordedFor all explants cultures, observations were recorded at 3 stages;

Stage 1-after 35 days of initial culturing; after 28 days from reculturing of calli

on regeneration media; Stage 3-when the complete plants were obtained. All

observations were based on initial culture media, irrespective of regeneration

medium or rooting medium.

3.7.1 Number of callus forming explants per 90 explants platedCultured explants on different media were recorded for callus formation

after 30 days.

3.7.2 Number of embryogenic calli per 90 explants platedProliferated calli from cultured explants were classified into live and

dead calli. A count was made for live calli identified by their phenotypic

appearance. Such calli were semitransparent, glossy or swelled structures,

partially shooting or rooting on white friable callus observed and sometimes

green calli with shoots were observed (Plate 4).

3.7.3 Number of organogenic calli per 90 explants platedProliferated calli from cultured explants were classified into live and

dead calli during observations. A count was made for calli identified by their

23

phenotypic appearance. Such calli were white to yellowish green in colour

with dense and glossy appearance and regenerating shootlets and/or roots as

well as shoots with roots. Calli regenerated from cultured explants, showing

shoot formation were referred as shoot forming calli (Plate 4).

3.7.4 Number of plants regenerated per 90 explantsComplete plantlets (shoots + roots) regenerated were recorded as

regenerated plantlets.

3.8 Statistical analysis of dataThe data were analyzed in factorial completely randomized design

(CRD) to find out the significance of different culture media combination,

genotypic effects and their interactions with two replications. The analysis of

data was carried out as per the method suggested by Snedecor and Cochran

(1989) to study the single as well as interactive effects.

3.9 Genetic transformation of Oat3.9.1 Maintenance of Agrobacterium cultures

A. tumefaciens GV3101 was cultured on Luria Bertanni agar plates

(LA). The bacterial culture was subcultured within two to three weeks for

maintenance. To prepare 1 litre of LB medium; Tryptone (10g), Yeast

extract (5g) and NaCl (5g) were dissolved together in 900 ml ultra pure

water; pH was adjusted to 7.5; volume was made up to 1L, added with

agar (15g) followed by autoclaving at 121ºC and 15 psi for 20 min. LB

broth was prepared without agar. The medium was supplemented with

antibiotics viz. kanamycin (50μgml-1) and rifampicin (50μgml-1) (Plate 6B).

3.9.2 Plasmid isolation (Miniprep method)1. From all the 10 cultures, 1ml culture each was taken in Eppendorf tube

and centrifuged at 5500 rpm for 10 min.

2. The supernatant was discarded and 100µl freshly prepared sol. I was

added to the tube, vortexed vigorously till the pellet gets dissolved.

3. 200µl of sol. II was added in the tube and mixed gently by inverting 5-7

times and incubated at room temperature for 5 min.

4. 150µl of chilled sol. III was added to each tube and mixed by inverting

15-17 times and incubated on ice for 15 min.

24

5. Tubes were centrifuged at 12000 rpm for 20 min. After centrifugation,

supernatant was taken out in a fresh Eppendorf tube and equal volume

of chilled isopropanol was added to each tube and mixed by inversion.

6. Centrifuged at 12000 rpm for 20 min. Supernatant was discarded and

700µl of 70% ethanol was added in each tube and mixed well by tubes

horizontally.

7. Centrifuged at 12000 rpm for 10 min, discarded ethanol by pouring and

dried pellet properly in vacuum drier.

8. 20-25µl of sterilized double distilled water was added in each tube and

pellet was dissolved properly by tapping the tubes.

3.9.3 Plasmid PCRPCR was carried out using plasmid samples as template in individual

reaction. (Working concentration)

10X PCR buffer 2µl 1X

25mM MgCl2 2µl 2.5mM

Plasmid DNA 2µl 50ng

dNTPs mix 0.5µl 200µM

Forward primer 0.5µl 20pM

Reverse primer 0.5µl 20pM

Taq polymerase (Fermentas) 0.5µl 1 Unit

Nuclease free water 12µl For volume making

Total 20µl

Reaction conditions included one cycle of initial denaturation at 94ºC

for 5 min followed by 35 cycles each of denaturation at 94 ºC for 1 min,

annealing at 48ºC for 1 min and DNA replication at 72ºC for 2 min with a final

extension step at 72ºC for 10 min. Finally hold at 4ºC.

25

3.9.4 Colony PCR of positive Agrobacterium culture harbouringpCAMBIA constructs

Agrobacterium culture grown on LA plate containing rifampicin

(50µgml-1) and kanamycin (50µgml-1) was confirmed for the presence of the

construct through colony PCR. A single bacterial colony was picked up and

dissolved in 2µl nuclease free water which was further used as template. The

PCR reaction and thermal conditions were same as that of above mentioned

plasmid PCR except an initial denaturation step for 10 min at 94ºC.

3.9.5 Screening of explants for antibiotic sensitivityThe explants were inoculated on MS media with different

concentrations of hygromycin for the assessment of antibiotic sensitivity.

Hygromycin was used in concentrations of 5mg l-1, 10mg l-1, 25mg l-1 and

50mg l-1 with MS basal medium as control.

3.9.6 Pre-culture of explantsIn order to determine the influence of pre-culture of explants on

transformation efficiency, callus developed from mature embryos and leaf

base were inoculated in shoot induction medium and incubated at 25 ± 2°C

with 12h photoperiod at a light intensity of 1200 lux for 12, 18, 24, and 48h. A

control was maintained in similar way without pre-culturing the explants. Each

experiment comprised of 100 immature explants in replicates of three.

3.9.7 Co-cultivation of Oat explants with AgrobacteriumA. tumefaciens was inoculated in LB containing antibiotics rifampicin

(50mgl-1) and kanamycin (50mg l-1). The Agrobacterium colony was picked

from LA plate with the help of sterile inoculation loop and inoculated in LB.

The culture was incubated in dark at 28°C for 16h with shaking at 200rpm.

Agrobacterium cultures in LB were centrifuged at 5,000 rpm for 5 min

at 28°C. Supernatant was discarded and pellet was dissolved in 20 ml of

liquid co-cultivation medium [(containing B5 Major & Minor Salts, Ferrous-

NaEDTA, Sucrose, 2-[N-morpholino] ethanesulfonic acid (MES), B5 Vitamins,

Acetosyringone (3', 5'- Dimethoxy-4-hydroxyacetophenone), GA3 (Gibberellic

acid), BAP, L-Cysteine, Na-thiosulphate and DTT); (Table 3.7)].

26

Acetosyringone was dissolved in ddH2O first and then added to the mixture

for further sterilization.

Table 3.7 Composition of Co-cultivation (CCM).

Components Co-cultivation medium (1l)

B5 Major Salts (40X) 2.5ml

B5 Minor Salts (200X) 0.5ml

Ferrous-NaEDTA (200X) 0.5ml

Sucrose (3%) 30g

2-[N-morpholino]ethanesulfonic acid (MES) 3.9g

Agar (plate only) 8g

B5 Vitamins (100X) 10ml

Acetosyringone (100mM) 1ml

GA3 (Gibberellic acid) mgml-1 1ml

BAP (6-benzyl-aminopurine) (1.67mg ml-1) -

L-cysteine (3.3 mM) 400mg

Na-thiosulphate(1.0mM) 248mg

Dithiothreitol (DTT, 1mM) 154mg

3.9.8 Sonication assisted Agrobacterium-mediated transformation(SAAT)

For SAAT, plant tissue was sonicated in a bath sonicator (HF-

frequency: 35 KHz) in the presence of Agrobacterium culture. The explants

were kept in liquid co-cultivation medium and subjected to sonication in 20ml

Agrobacterium suspension (in co-cultivation medium) for 0-30s followed by

soaking on sterile Whatman filter paper to remove excess co-cultivation

medium. Ten treated calli developed from embryo and leaf base were placed

on top of solid co-cultivation medium. Plates were then sealed with ParafilmTM

and incubated at 25±2ºC for 3-4 days in dark.

27

3.9.9 Vacuum Infiltration assisted Agrobacterium-mediatedtransformation (VIAAT)

During co-cultivation, explants submerged in co-cultivation medium

with bacteria, were subjected to various time periods of vacuum infiltration viz.

10 and 15min at different vacuum pressure ranging from 15 to 30 inch of Hg.

3.9.10 Sonication and vacuum infiltration assisted Agrobacterium-mediated transformation (SVIAAT)The conditions optimized in the methods SAAT and VIAAT were

applied in combination to achieve maximum transformation efficiency. This

method was referred to as Sonication and vacuum infiltration assisted

Agrobacterium-mediated transformation (SVAAT) of oat.

3.9.11 Selection and plant regenerationCalli were washed with cefotaxime (250mg l-1) containing MS Liquid

medium to remove the excess Agrobacterium culture. Calli were kept in

cefotaxime solution for fifteen minutes and blot dried on sterile filter paper and

cultivated on MS medium supplemented with hygromycin 20mg l-1 and

cefotaxime 500mg l-1. Only calli which were able to regenerate on MS medium

supplemented with hygromycin 20mg l-1 were considered as putative

transformed plants.

After 8 days of culturing transformed plants survived and regenerated

on hygromycin containing medium and appeared greenish in colour and the

non-transformed plants does not showed any growth and were light yellowish

to brown necrosis. Hence they were removed and only green and healthy

plants were allowed to regeneration.

3.9.12 Statistical analysisA range of parameters influencing transformation was evaluated using

fifty explants for each experiment. Each experiment was repeated at least

three times. All the parameters were evaluated and optimized on the basis of

GUS activity in treated explants or the number of regenerating explants. The

data were analyzed using one-way analysis of variance (ANOVA) in a

completely randomized design (CRD). For each experiment, the treatment

means and least significant difference (LSD) were determined (α= 0.05). LSD

28

was used to separate the means for significant effect at significance level P<

0.05. All statistical analysis was performed using web based statistical

package WASP2 (http://icargoa.res.in).

Table 3.8 Shoot Elongation (SEM) and Rooting Medium (RM).

ComponentsShoot

ElongationMedium (1L)

RootingMedium (1l)

MS Major Salts (40X) 25ml 25ml

MS Minor Salts (200X) 5ml 5ml

MSIII Iron Stock (200X) 5ml 5ml

Sucrose (3%) 30g 30g

2-[N-morpholino] ethanesulfonic acid

(MES)

0.6g 0.6g

Purified Agar 8g 8g

B5 Vitamins (100X) 10ml 10ml

L-Aspartic acid 50mg 50mg

GA3, (1mg ml-1) 1ml 0.3ml

BAP (1mg ml-1) 2ml -

NAA (1mg ml-1) 0.1ml 0.0 or 0.1 ml

Cefotaxime, (250mg ml-1) 1ml 1ml

Carbenicillin, (100mg ml-1) 1ml 1ml

pH 5.6 5.6

3.9.13 GUS Histochemical assayTransient GUS expression in explants was histochemically assayed

after 3 to 5 days of co-cultivation with A. tumefaciens GV3101 containing

pCAMBIA 1305.1 vector by staining the explants in a buffer containing X-

GLUC (Table 3.9). Briefly, 10 explants with shoots were incubated in X-GLUC

29

overnight at room temperature in dark and washed with ethanol for removing

chlorophyll content. Experiment was performed in triplicate. The developing

callus, shoots and the putative transgenic plantlets regenerated through

transformation experiments were also analyzed through histochemical assay.

GUS histochemical assay was performed according to the method described

by Jefferson (1987). Histochemical localization of GUS activity was examined

under a Nikon DXM 1200F stereomicroscope (Plate 5).

Table 3.9 Composition of GUS staining solution.

SI. ConstituentsConcentration

For 1ml final volumeStock required

1 Distilled water - - 830μl

2 Sodium phosphate pH 7.0 1M 0.1M 100μl

3 EDTA 0.5M 10mM 20μl

4 Triton 100X 10% 0.1% 10μl

5 Potassium ferri-cyanide 50mM 1mM 20μl

6 X- Gluc 0.1M 2mM 20μl

3.9.14 Molecular analysis through Polymerase Chain Reaction (PCR):

3.9.14.1 DNA Isolation

The technique of DNA isolation rely upon the fact that nucleic acid (NA)

would form suitable complex with detergent cetyltrimethylammonium bromide

(CTAB) under high salt concentration and when the concentration reaches

0.4M NaCl the CTAB-NA complex would precipitate. Genomic DNA was

isolated using method adopted by Saghai-Maroof et al. (1984) with suitable

minor modifications. DNA Extraction buffer [100mM Tris-HCl (pH 8.0),

20mM EDTA (pH 8.0), 1.4M NaCl, 2% CTAB and 0.2% β-

mercaptoethanol] was made without β-mercaptoethanol on a magnetic stirrer

to avoid foaming. β-mercaptoethanol was added to the cooled solution at

room temperature.

30

Table 3.10 Composition of DNA Extraction buffer.

Reagents Concentration

Tris-HCl (pH 8.0) 100mM

EDTA (pH 8.0) 20mM

NaCl 1.4M

CTAB 2%

β-mercaptoethanol 0.2%

Sample (2g) was homogenized in liquid nitrogen using a pre-chilled

pestle and mortar. The fine powder was transferred to a 50ml Oakridge

tube and 10ml of DNA extraction buffer (preheated to 65ºC) was added

and mixed thoroughly. The samples were incubated in a water bath at

65ºC for 1h with intermittent mixing every 10min to ensure complete and

even extraction. The samples were then removed from water bath and

cooled to room temperature. Samples were then centrifuged for 15min at

10,000rpm at room temperature. Supernatant was transferred to a fresh

tube. Then an equal (to supernatant) volume of chloroform: isoamyl

alcohol (24:1) v/v was added and mixed thoroughly but gently for about

5min. The mixture was then centrifuged for 15min at 10,000rpm at room

temperature. Supernatant was transferred to a fresh tube and equal (to

supernatant) volume of chilled isopropanol was added and mixed gently

by inverting tubes and kept for 10min undisturbed. The DNA precipitate

was then spooled out using 1ml cut tips and transferred to a 1.5ml

microcentrifuge tube. DNA was again centrifuged at 10,000rpm for 10min.

The supernatant was discarded and pellet was washed with 70% ethanol.

The pellet was dried at room temperature and dissolved in 200µl of TE

buffer for further use.

3.9.14.2 DNA purificationThe purification of DNA was carried out in order to remove the

impurities like RNA, proteins and polysaccharides. These are considered as

inhibitors in DNA amplification during PCR.

31

5µl of RNaseA (5mg ml-1) was added to DNA, mixed well and

incubated at 37ºC for 30min. This was followed by the addition of equal

volumes of chloroform: isoamyl alcohol (24:1) v/v and mixed vigorously.

The above mixture was centrifuged at 14,000 rpm for 10min. Supernatant

was transferred to a fresh microcentrifuge tube and 1/10 volume of 3M

sodium acetate (pH 5.4) was added followed by further addition of two

volumes of pre-chilled ethanol and mixed gently for DNA precipitation. The

precipitated DNA was centrifuged at 12,000rpm for 5min to obtain pellet.

The pellet was dried at room temperature to completely remove ethanol

and then dissolved in 100µl of TE buffer and stored at -20ºC for further

use.

3.9.14.3 Quantification of DNAIsolated DNA was quantified by measuring the absorbance at 260nm

and 280nm on a UV-spectrophotometer. 50µgml-1 concentration of double

stranded DNA shows an absorbance of 1 at 260nm. Concentration of DNA

samples was calculated using formula: (O.D.260nm x 50µg DNA/ml x

Dilution factor)/1000.

3.9.14.4 Dilution of DNAThe quantified DNA was diluted according to the DNA quantity needed

in each sample for PCR amplification using sterile nuclease free water.

Dilutions were carried out according to the following formula:

Dilution =Required concentration of DNA (ng/µl) x Total volume required (µl)

Available concentration of DNA (ng/µl)

3.9.14.5 PCR amplification with gene specific primersPCR reaction was prepared with following concentrations: 10X Taq

buffer with MgCl2, 100µM dNTPs, 10pmol primers (forward and reverse),

1UTaq DNA polymerase and 25-100ng of template DNA.

3.9.14.6 PCR conditionsPCR conditions were standardized considering different parameters

viz. initial denaturation, denaturation, annealing, extension and final extension

using Thermo Hybrid (Px2) PCR Machine. PCR thermal and reaction profiles

32

(Table 3.12) were optimized for amplification purpose by using specific

primers.

3.9.14.7 Gel electrophoresis of PCR productDNA molecules obtained throughout the study were visualized on

agarose gel. Agarose gel electrophoresis was carried out according to

Maniatis et al. (1989). According to the purpose, different concentrations (0.8-

1%) of gel solutions were prepared in 0.5X TBE buffer. For 0.8% agarose gel,

0.8g of agarose was melted completely in 100ml of 0.5X TBE buffer by

heating. Then the solution was left to cool around 50°C and 50µl of ethidium

bromide (10mg ml-1) added to the gel solution. The gel was poured into the

electrophoresis tray having a comb, which will form the wells for the sample

loading. The gel was left at room temperature until it was solidified and

electrophoresis tank was filled with 0.5X TBE buffer. The samples were

prepared by mixing the samples with 6X loading buffer to the final

concentration of 1X and loaded into the wells, along with DNA size markers

(λ-phage DNA digested with PstI), in a separate well. Then the tank was

connected to a power supply and run under constant voltage of 50-60V in

agarose gel electrophoresis apparatus (BioRad, USA). The gel was visualized

under UV transilluminator and photographed.

Table 3.11 PCR programme for different primers used in the study

No. ofcycles Steps

Temperatures (ºC) and durations

hptII caMV 35S virD2

1 Initialdenaturation

94 ºC for 5min

94 ºC for 5min

94 ºC for 10min

35

Denaturation 94 ºC for 1min

94 ºC for 1min

94 ºC for 1min

Annealing 52 ºC for 1min

52 ºC for 1min

50 ºC for 1min

Primerextension

72 ºC for 2min

72 ºC for 2min

72 ºC for 2min

1 Final extension 72 ºC for 10min

72 ºC for 10min

72 ºC for 10min

33

3.10 Time course of transformation

A general time course of the transformation procedure followed in the

present investigation has been presented in Table 3.13.

34

Table 3.12 List of primers and their features.

S.N.PrimerCode

Sequence

(5’-3’)

No. ofNucleotides

GCContent

(%)

Tm(°C)

AnnealingTemp. (°C)

Expectedfragment size

(bp)

1Hpt F TCGTCCATCACAGTTTGCC 19 52.6 55.4

52 499Hpt R AAAAGCCTGAACTCACCGC 19 52.6 56.0

2CamVF GCTCCTACAAATGCCATCA 19 47.3 52.6

52 522CamVR GATAGTGGGATTGTGCGTCA 20 50.0 54.8

3Vir D2A ATGCCCGATCGAGCTCAAGT 20 55.0 58.9

50 338Vir D2E CTGACCCAAACATCTCGGCTGCCCA 25 60.0 65.1

35

Table 3.13 The time course of the transformation procedure.

Steps Description Materials Time period

1. Explant preparation (100 explants) Oat mature Embryos calli and Leaf base 30 to 45 Days

2. Agrobacterium inoculation Liquid co-cultivation medium, overnight bacterial culture (12-16 hr),sonicator for sonication, vacuum pump with desiccator for vacuuminfiltration

~ 20 min

3. Co-cultivation Co-cultivation medium in petri dishes (No antibiotics) 3 Days

4. Washing of explants Sterile water added with 400 mg l-1cefotaxime 1 min each

5. Shoot regeneration Regeneration medium supplemented with hygromycin in petri dishes 5-6 weeks

6. Rooting of shoots Rooting medium supplemented with hygromycin in bottle jars 2-3 weeks

7. Gus analysis (between steps 3-4) X-Gluc, assay buffer, vacuum pump with desiccators for vacuuminfiltration, 70 % ethanol

2 days

8. PCR analysis Primers, Taq polymerase and PCR buffer, Agarose powder, TBEbuffer

1 day

9. Hardening of regenerated plants Pots covered with a plastic bag with sterile Soil:Sand:Vermiculite(1:1:1)

2-3 weeks

Total time to obtain fully hardened transgenic plants 12-16 weeks

11. Green house growth Pots with potting mix, irrigation-100 ml/pot/day Up to maturity

36

RESULTS

The present investigation was carried out with an objective to achieve

Agrobacterium-mediated transformation in oat. For transformation of oat, mature

seeds were sterilized, germinated and used for explants generation. For

transformation, Agrobacterium tumefaciens GV3101 carrying the binary vector

pCAMBIA 1305.1 was used, which contain a reporter gene (gus) and after

transformation, further experiments were carried out for confirmation of

transformation. The putative transformants were selected on media

supplemented with antibiotics and these were further validated through molecular

analysis. The results obtained are presented below.

4.1 In vitro morphogenesis studiesFor the in vitro morphogenesis studies in oat two separate experiments

were conducted with mature embryo and leaf base explants. Explants of five

genotypes viz. JO-1, OS-6, KENT, JHO-822 and JHO-851 were cultured on

different combination of MS media. The media were selected on the basis of

preliminary experiments conducted to screen suitable plant growth regulators

and their combinations for in vitro response. The basal MS medium was fortified

with different combinations of BAP, NAA, IAA, IBA and 2,4-D in varying

concentrations. During present investigation, observations were recorded for

callus induction, embryogenic callus and organogenic callus formation and plant

regeneration abilities.

4.1.1 Morphogenesis in cultured explantsTwo explants, mature embryo and leaf base were cultured on MS medium

fortified with different concentrations and combinations of plant growth regulators.

Leaf base explants were obtained by germination of oat seeds on MS basal

medium for 6 days (Plate 1). The first response of cultured explants was

visualized after one week and mostly independent of culture media combinations

and accessions. During the second week, explants became swollen and no

callus proliferation was evident. The callus initiation started from the upper

37

portion of explants usually not in contact with the culture medium. After 28-35

days of culture, callus initiating explants were counted.

After callus induction from the explants, callus tissue developed distinct

characteristics such as dense, rough, soft and sometimes glossy. These

distinctness reflected diversity in in vitro developmental potentials with the

different culture medium regimes.

In vitro morphogenesis, the way in which a callus forms a new plant in

vitro, was variable. During the present investigation plant regeneration from the

explants cultures appeared to be direct as well as via callus phase. Culture

media played an important role in the formation of morphogenic callus (Table

4.1).

Table 4.1 Observation on in vitro regeneration of Oat cultivar JO-1.

Explants tobe taken

No. of callusformation/ 90

explants

No. explantshaving shoot

formation

No. of explantshaving rootformation

Embryo 62 48 43

Leaf base 75 53 39

4.1.2 Mature embryo culture

Mature embryos were cultured on different combinations of MS medium.

After, callus induction, embryogenic calli and organogenic calli formation were

observed on different combination of MS medium.

4.1.2.1 Callus inductionThe callus induction from mature embryo cultures varied from 90-97%.

Maximum callus induction was evident from JO-1 (97.0%) followed by OS-6

(90.0%). In terms of the culture media response to in vitro culture, the

performance of culture media MS6D with 6mg l-1 2,4-D (97.0%) was found to be

the best in terms of callus initiation (Plate 2).

38

4.1.2.2 Embryogenic callus initiationThe mean embryogenic callus formation from mature embryo cultures

varied from 86.5-96%. Among five accessions, maximum embryogenic calli

formation was observed for JO-1 (96.0%). In terms of the culture media response

to in vitro culture, the performance of culture media MS6D (96.0%) followed by

MS10D (86.5%) was found to be most responding for embryogenic callus

initiation (Plate 4).

4.1.2.3 Organogenic callus formationThe mean organogenic callus formation from mature embryo cultures

varied from 2.45-13.3%. Maximum organogenic calli formation was observed for

cv. JO-1 (17%). (Plate 2)

4.1.3 Leaf base cultureLeaf base explants were cultured on three different combinations of MS

medium. Callus induction, embryogenic calli and organogenic calli formation

were observed from all the accessions and on all combinations of MS medium;

however their frequency varied among the different media combinations and

accessions. Leaf bases were observed to swell 5-10 days after plating. Callus

formation was observed after 20-25 days of plating. Callus proliferation started

from the cut edges of the leaf. After callus induction, initiated callus tissue

developed distinct phenotypes viz. wet, rough, hard dense and glossy, reflecting

different developmental potentials. After 40-45 days of inoculation, calli could be

distinguished on the basis of their phenotypic appearances. Compact, light green

coloured calli with few or many dark green bead like structures and sometimes

partially covered with thin layer of white loose callus were recognized as

embryogenic calli.

4.1.3.1 Callus inductionThe mean callus induction frequencies from leaf base cultures varied from

1.5%-89.0%. Maximum callus induction was evident from JO-1 (89.0%) and

minimum by OS-6 (1.5%). Among different culture media, MS6D (98.0%), was

found the best (Plate 3).

39

4.1.3.2 Embryogenic callus initiationThe overall formation of embryogenic calli varied from 2.27% to 88.1%.

Most embryogenic callus were generated from JO-1 (95.5%) followed by OS-6

(Plate 3).

4.1.3.3 Organogenic callus formationThe mean organogenic callus formation from leaf base cultures varied

from 2.67% to 12.82%. Higher organogenic calli formation was observed from

JO-1 (12.82%), and least by OS-6 (2.67%). (Plate 4)

4.2 Agrobacterium-mediated transformation of OatExplants each of oat mature embryo calli and leaf base calli were infected

with Agrobacterium suspended in co-cultivation liquid medium and then the calli

were transferred on to the co-cultivation solid medium and kept in dark for 1-3

days to observe transformation efficiency.

It was observed that the co-cultivated calli which were kept for incubation

for two days showed best transformation efficiency than one day and three day

incubation periods.

4.2.1 Assessment of antibiotic sensitivity of explantsThe sensitivity of explants to the antibiotic hygromycin was established

prior to actual transformation experiments in order to determine the effective

concentration for selection of transformants. The explants were cultured on MS

medium containing 2mgl-1 BAP, 0.1mgl-1 IAA and supplemented with either of five

different concentrations of hygromycin (10, 15, 20, 25 and 30mg l-1) to test

hygromycin sensitivity with a control in each and observed for growth up to at

least 6 weeks. In the absence of antibiotics, the explants regenerated normally

and produced calli and shoots. The regeneration capacity of explants was

restricted even at 25mg l-1 concentrations hygromycin respectively, resulting into

very slow growth and a maximum of 2 per cent of explants showed regeneration

of adventitious shoots after three weeks of culture. While, explants cultured on

medium with 30mg l-1 Hygromycin caused total inhibition of shoot regeneration

within 2 weeks and finally resulting into bleaching of explants (Table 4.2).

40

However, explants cultured on higher concentrations of antibiotic (hygromycin,

30-40mg l-1) became necrotic and dried up within one week. Hence, antibiotic

concentrations on and above of 20mg l-1 of hygromycin were used for selection of

transformants in subsequent transformation experiments.

Table 4.2: Percent survival and regeneration in oat explants in the presenceof different concentrations of antibiotics in regeneration(MS02B0.1N) medium.

Cultivars Explants Hygromycin concentration (mg l-1)

Control 10 15 20 25

JO-1Embryos calli 68 14 6 0 0

Leaf base calli 82 17 9 0 0

4.2.2 Culture of co-cultivated explantsExplants after co-cultivation with Agrobacterium strains, were placed on

regeneration medium with antibiotics cefotaxime (250mg l-1) and hygromycin

(20mg l-1). The regenerated explants were subsequently subcultured on

regeneration medium. This culture strategy greatly stimulated the in vitro

regeneration of transformed callus with embryogenic and organogenic calli.

It was observed that embryogenic and organogenic transformed calli

showed maximum regeneration on MS medium fortified with 2mg l-1 BAP and

0.1mg l-1 NAA.

4.2.3 Optimization of plant transformation conditions4.2.3.1 Bacterial inoculum density and inoculation duration

Exposure of embryo calli and leaf base calli explants to an undiluted

culture (OD600 = 0.5) of Agrobacterium tumefaciens GV3101 resulted in severe

necrosis of the explants. Diluted culture (1:2 dilutions) reduced necrosis to a

great extent. The GUS response varied significantly among the treatments. The

maximum GUS response was obtained with 1: 2 dilution for 20 min. Therefore,

41

subsequent experiments were carried out for 20 min inoculation using 1:2 dilution

of Agrobacterium culture (OD600 = 0.5).

4.2.3.2 Effect of co-cultivation duration on transformationFollowing transformation, explants were co-cultivated on semisolid co-

cultivation medium at 25±2ºC for different durations to test its effect on

transformation frequency in oat explants. Co-cultivation durations up to 2-4 days

showed better results in oat transformation (Table 4.9). Experiment failed

completely when duration of co-cultivation was increased to 5 days giving lowest

transformation (4%) with high mortality of explants. A 3-day co-cultivation period

was the best option over 2 or 4 days resulting into highest transient

transformation efficiency of 40%.

4.2.3.3 Effect of different treatment on transformation of embryo explantsThe embryo explants tested for transformation in oat. For transformation

of embryo different treatments as like sonication, vacuum infiltration was used.

On the basis of hygromycin survival, T5 (73.33) was found to be statistically

superior over all other treatments and minimum survival was found in T9 (47.78)

with 2.32 at 5% CD (Table 4.3). In addition, the T5 (52.96) was also found to be

statistically superior over all other treatments for transformation efficiency with

GUS assay in embryo explants (Table 4.4) however, leaf base explants exhibited

less transformation efficiency. The minimum transformation efficiency with GUS

assay was observed in T3 (32.59). During transgenic selection based on PCR, T5

(40.74) proved to be superior over all other treatment used for transformation

(Table 4.5) and minimum value was observed in T3 (26.67). Finally, in case of

embryo highest transformation efficiency with hygromycin survival, GUS assay

and PCR were 73.33%, 52.96% and 40.74% respectively.

42

Table 4.3 Effect of different treatment on transformation efficiency (TE %)of embryo explants based on survival on hygromycin containingmedia.

Embryos Transformationtreatment

Co-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 52.5972 T2 55.1996 T3 51.11

Vacuum Infiltration48 T4 65.9372 T5 73.3396 T6 60.37

Sonication+

Vacuum Infiltration

48 T7 52.2272 T8 57.4196 T9 47.78

Control - T10 4.44SEM ± 0.78, CD 1% 3.17, CD 5% 2.32

Table 4.4 Effect of different treatment on transformation efficiency (TE %)of embryo explants based on criteria of GUS assay.

EmbryosTransformation

treatmentCo-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 35.5672 T2 37.7896 T3 32.59

Vacuum Infiltration48 T4 50.0072 T5 52.9696 T6 46.67

Sonication+

Vacuum Infiltration

48 T7 46.3072 T8 46.6796 T9 42.22

Control - T10 0.0SEM ± 0.74, CD 1% 3.11, CD 5% 2.28

43

Table 4.5 Effect of different treatment on transformation efficiency (TE %)of embryo explants based on criteria of PCR.

Embryos Transformationtreatment

Co-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 27.7872 T2 29.2696 T3 26.67

Vacuum Infiltration48 T4 38.1572 T5 40.7496 T6 37.78

Sonication+

Vacuum Infiltration

48 T7 31.8572 T8 36.3096 T9 33.70

Control - T10 0.0SEM ± 0.81, CD 1% 3.28,CD 5% 2.40

4.2.3.4 Effect of different treatment on transformation of Leaf base explantsThe leaf base explants also tested for transformation in oat. For

transformation of leaf base also the different treatments as like sonication,

vacuum infiltration were used. On the basis of hygromycin survival, T5 (70.0) was

found to be statistically superior over all other treatments and minimum survival

was found in T9 (46.3) with 2.5 at 5 % CD (Table 4.6). In addition, the T5 (51.85)

was also found to be statistically superior over all other treatments for

transformation efficiency with GUS assay in leaf-base explants (Table 4.7)

however, embryo explants exhibited more transformation efficiency. The

minimum transformation efficiency with GUS assay was observed in T3 (33.33).

During transgenic selection based on PCR, T5 (37.04) proved to be superior over

all other treatment used for transformation (Table 4.8) and minimum value was

observed in T3 (28.89). With leaf base, highest transformation efficiency with

hygromycin survival, GUS assay and PCR were 70.0%, 51.85% and 37.04%

respectively.

44

Table 4.6 Effect of different treatment on transformation efficiency (TE %)of leaf base explants based on hygromycin survival.

Leaf base Transformationtreatment

Co-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 50.0072 T2 52.2296 T3 48.89

Vacuum Infiltration48 T4 64.4472 T5 70.0096 T6 58.89

Sonication+

Vacuum Infiltration

48 T7 50.0072 T8 54.4496 T9 46.30

Control - T10 3.70SEM ± 0.85, CD 1% 3.41, CD 5% 2.50

Table 4.7 Effect of different treatment on transformation efficiency (TE %)of leaf base explants based on criteria GUS assay.

Leaf base Transformationtreatment

Co-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 34.8172 T2 37.7896 T3 33.33

Vacuum Infiltration48 T4 48.8972 T5 51.8596 T6 45.56

Sonication+

Vacuum Infiltration

48 T7 41.1172 T8 43.7096 T9 41.11

Control - T10 0.0SEM ± 0.82, CD 1% 3.31, CD 5% 2.42

45

Table 4.8 Effect of different treatment on transformation efficiency (TE %)of leaf base explants based on criteria PCR putative transgenicexplants.

Leaf baseTransformation

treatmentCo-cultivationperiod (Hours) Treatment Transformation

efficiency (%)

Sonication48 T1 31.1172 T2 30.0096 T3 28.89

Vacuum Infiltration48 T4 36.6772 T5 37.0496 T6 33.33

Sonication+

Vacuum Infiltration

48 T7 33.3372 T8 34.4496 T9 32.22

Control - T10 0.0SEM ± 0.919, CD 1% 3.692, CD 5% 2.719

4.2.3.5 Overall effects of different treatment on both oat explantstransformation

When observed transformation data of both explants combinedly analysed

on basis of hygromycin survival, T9 (73.33) was found to be statistically superior

over all other treatments and minimum survival was found in T18 (46.30) with 2.34

at 5% CD. In addition, the T9 was also found to be statistically superior over all

other treatments for with 52.96% transformation efficiency in GUS assay of leaf-

base explants. The minimum transformation efficiency with GUS assay was

observed in T5 (32.59). During transgenic selection based on PCR, T9 proved to

be superior over all other treatment used for transformation with transformation

efficiency of 40.74% (Table 4.9).

4.3 GUS assay in transformed calli and shootsAfter selection on media containing hygromycin, the putative transformed

calli and shoots were assayed for transient GUS expression. Blue stained leaf

46

Table 4.9 Overall effects of co-cultivation period and different treatments on transformation efficiency (TE %).

Transformationtreatment

Co-cultivationperiod (Hours)

Explants tobe taken Treatment

Transformation efficiency (%) based on

Hygromycin resistanttransgenic explants

GUS putativetransgenic explants

PCR putativetransgenic explants

Sonication

48Embryo T1 52.59 35.56 27.78

Leaf base T2 50.00 34.81 31.11

72Embryo T3 55.19 37.78 29.26

Leaf base T4 52.22 37.78 30.00

96Embryo T5 51.11 32.59 26.67

Leaf base T6 48.89 33.33 28.89

VacuumInfiltration

48Embryo T7 65.93 50.00 38.15

Leaf base T8 64.44 48.89 36.67

72Embryo T9 73.33 52.96 40.74

Leaf base T10 70.00 51.85 37.04

96Embryo T11 60.37 46.67 37.78

Leaf base T12 58.89 45.56 33.33

Sonication +Vacuum

Infiltration

48Embryo T13 52.22 46.30 31.85

Leaf base T14 50.00 41.11 33.33

72Embryo T15 57.41 46.67 36.30

Leaf base T16 54.44 43.70 34.44

96Embryo T17 47.78 42.22 33.70

Leaf base T18 46.30 41.11 32.22

Control -Embryo T19 4.44 0.0 0.00

Leaf base T20 3.70 0.0 0.00SEM ± 0.820CD 1% 3.132CD 5% 2.343

SEM ± 0.799CD 1% 3.055CD 5% 2.285

SEM ± 0.869CD 1% 3.329CD 5% 2.483

47

venation and spots could be clearly visualized on transformed calli and shoots

while untransformed calli and shoots were completely bleached and were devoid

of any stained spots/area (Plate 5).

4.4 Molecular analysis of putative transformants of oat

After selection on antibiotic containing media, the putative transformants

plantlets were analyzed at molecular level for confirmation of transformation.

4.4.1 Colony PCR using Agrobacterium colony as template for positivecontrolAgrobacterium tumefaciens strain GV 3101 containing binary plasmid

vector pCAMBIA 1305.1 were grown in LB medium (Plate 6A, 6B). Single colony

of the Agrobacterium strains was used in colony PCR to test the product sizes as

well as for its further use as a positive control. Plasmid DNA was isolated from

Agrobacterium colonies and further used as positive control (Plate 6C). When

Agrobacterium strain GV3101 harbouring pCAMBIA 1305.1 vector was used as

template, while hptII primers led to a product size of 499bp as amplified upon gel

electrophoresis of colony PCR product (Plate 7A).

4.4.2 Absence of Agrobacterium contamination in transformed plantsGenomic DNA from putatively transformed plants was subjected to PCR

using virD2 gene specific forward and reverse primers which led to the

amplification of 338bp fragment specific to the Agrobacterium chromosomal

DNA. Based on the results of the analysis thirteen plants were found with an

amplified fragment of 338bp i.e. virD2 gene specific fragment, indicating that the

plants were contaminated with Agrobacterium (Plate 7B), rest of the putative

transgenic plants were devoid of any such contamination.

4.4.3 Analysis of plants transformed by pCAMBIA 1305.1 vector.The genomic DNA from different samples of putative transformants were

analyzed for amplification of fragment of hptII gene using gene specific primer.

Negative control (non-transformed plants) and the positive control (plasmid

isolated form Agrobacterium strain pCAMBIA 1305) were also used for

48

amplification of this gene. Some putative transformants gave positive results with

a product size of approximately 499bp with gene specific primer of hptII gene and

products of 522bp size in case of CaMV35S promoter specific primers (Plate

7C).

Molecular analysis employing polymerase chain reaction clearly indicated

the integration of the transgenes from the T-DNA region of Agrobacterium to the

oat host genome. Finally the transformation efficiency observed approximately

40% in used explants of oat.

4.5 RootingThe transformed explants further separated and subculture on rooting

medium (1B0.1I) (Plate 8). After sufficient rooting the transgenic plantlets should

be hardened under green house condition. Finally they were transfer to soil.

4.5 HardeningAfter successful transfer to soil, the hygromycin resistant putative

transgenic plants were subjected to molecular analysis to validate the genetic

transformation. The genomic DNA was used as template for PCR analysis with

gene specific primer sets. Among all the hygromycin resistant plants, some were

found with Agrobacterial contamination based on the amplification of virD2 gene

specific amplicons of 338bp. Remaining plants were tested by PCR using hptII

gene specific primer set. Remaining putative transformants gave positive results

with a product size of approximately 499bp with gene specific primers of hptII

gene. Same amplification products were also visualized in positive control using

A. tumefaciens GV3101 single colony as a template while, they were absent in

negative control. DNA from the non-transformed plantlets did not show any

amplified product.

49

DISCUSSION

Totipotency and genetic engineering of somatic cells is the basis for

transformation and in vitro propagation which is being extensively used for

obtaining a large number of genetically identical transformed plantlets. These

plantlets would serve as an excellent source for tolerance to various biotic and

abiotic stresses. The abiotic stresses and biotic stresses are the major

constraints in oat production. So for the development of transgenic plants; callus

induction, genetic transformation and plant regeneration is a critical step.

Despite many years of research, tissue culture of oat is still not easy and

varies in each and every genotype. Selecting a suitable subculture medium to

improve the quality of calli might be a key step for the success of transformation.

Production of embryogenic calli with high regeneration ability is prerequisite for

highly efficient transformation of oat (Somers et al., 1992; Torbert et al., 1995).

During present investigation plant regeneration appeared to be a stepwise

process, starting from callus induction, callus proliferation, morphogenesis i.e.

embryogenesis and/or organogenesis followed by plantlet regeneration (Plate 2,

3, 4). Plants from explant cultures followed one of the two pathways: direct

organogenesis and somatic embryogenesis. Organogenesis was accomplished

by the de novo organization of gamogenesis (only shoot) or rhizogenesis (only

root). Direct somatic embryogenesis or formation of embryoids in callus cultures

was obtained without complete plantlet regeneration after transformation.

Multiple shoots proliferation was obtained from callus after three to five weeks of

co-cultivation culture of mature embryo and leaf base.

The transformed explants were selected on the basis of antibiotic provided

in the media (hygromycin 20mg l-1) and then individual shoots were aseptically

excised and GUS stained for validating transformation (Plate 5). Major factors,

which produced considerable variation in the pattern of development in culture,

were cultivars and media combinations. Genotypic differences are usually related

to variation in endogenous growth regulator levels (Seraj et al., 1997). Different

50

explants of single genotype do not responded identically in cultures, most likely

due to varying gradients of endogenous growth regulators. Single explants

collected from same source behaved differently in culture depending upon size

and location on donor plant. Response of explants from well-nourished plants

was different from those of nutrient deficient plants. Even in our single

experiment, with similar explants and media, culture responses varied

considerably.

During present investigation, three types of media were used for callus

culture establishment. A basal MS medium supplemented with auxins, a growth

regulator free basal MS medium for germination of oat seeds and, the third MS

medium supplemented with low auxin and higher cytokinin for regeneration of

plantlets from embryogenic calli. The use of in vitro grown seedling as starting

materials for tissue culture experiments has several advantages. Sterile plant

material of constant quality can be easily provided within a short time and

specialized and usually extensive growth conditions are not necessary. Results

exhibit that the embryogenic callus induction largely depends upon the nature of

initial culture medium i.e. the first medium, where as other two media supported

the germination of seeds and for regeneration of plantlets, respectively.

The presence of auxin in the medium is generally essential for callus

induction. Tissue or callus maintained continuously in an auxin containing

medium. The callus initiated and multiplied on a medium rich in auxin that

induced differentiation of localized group of meristematic cells called

embryogenic clumps. Developed embryogenic calli were transferred to media

devoid of auxin, or with the reduced levels of auxin and high level of cytokinin.

When transferred to a medium with low auxin and higher cytokinin, the

embryogenic callus showed shoot proliferation.

For mature embryo and leaf base culture, during course of preliminary

investigations three auxins (2,4-D, IAA and IBA) and a cytokinin (BAP) in

different concentration and combinations were used for culture establishment.

Results clearly indicated varying response of growth regulators. Some

51

combinations led to organogenesis, some followed the process of

embryogenesis from cultured explants (Plate 4).

In the present study, mature embryos were placed on ten different

modifications of MS medium (Table 3.6) to evaluate callus initiation. Medium

MS6D induced maximum number of calli (97%) from mature embryo and from

leaf base calli too. In the same cultural conditions medium MS1B1N have shown

less callus initiation (0.75%). It was also observed that incubating in dark,

increased the percentage of calli initiation in genotype JO-1 even up to 50% as

compared to light incubation. Calli obtained in this condition were generally

cream in colour and turned green after transferring to light. Calli turned brown if,

they were not subcultured within three weeks. Almost similar results were

recorded in case of both mature embryo and leaf base explants. Callus induction

from mature embryo of JO-1 (97%) showed high efficiency in medium containing

6mg l-1 of 2,4-D. However low level of 2,4-D i.e. 3mg l-1 in combination with 3mg l-

1 picloram gave similar results (Kim et al., 2004). This may be due to synergistic

action of picloram with auxin 2,4-D.

Plant regeneration efficiency was examined in MS medium containing

2mg l-1 BAP and 0.1mg l-1 NAA. Percentage of plant regeneration from mature

and immature embryos of JO-1 and OS-6 showed high in calli induced from

medium containing 6mg l-1 2,4-D. However, Kim and Lee (2002) reported that

treatment with 3mg l-1 of 2,4-D and 1mg-l-1 of kinetin in callus medium showed

high frequency for plant regeneration in oat cultivars. This result supports that

plant regeneration was influenced by the callus initiation medium (Kim and Lee,

2002). The regeneration efficiencies of JO-1 and OS-6 were 97% and 96% in

medium containing 6mg l-1 2,4-D increased the frequencies of callus induction.

While medium containing MS2B.1N showed high frequency of multiple shoot

proliferation.

During present investigation, growth regulator 6 mg l-1 2,4-D induced

callus in higher frequencies in mature embryo culture (91%) as compared to leaf

base culture (89.9%) among varying concentrations tested ranging from 2.0 to

52

50.0 mg l-1. Kim et al. (2002) have also shown that low level of 2,4-D i.e. 3mg l-1

in combination with 3mg l-1 picloram is quite effective for callus initiation. In

addition, combination of both auxin and cytokinin supplied in culture medium

gave rise to multiple shoot induction from meristematic zones.

From leaf base the embryogenic calli were initiated in medium MS2D

(95.5%), MS3I (65.0%), MS3IBA (62.0%) and MS2IBA (60.0%). Maximum

organogenesis was found on medium MS2B.1N (13.69 %) and minimum on

medium MS3IAA (1.5%) from leaf base culture. However, calli initiated on an

auxin alone failed to initiate embryogenic calli and this lacunae was overcome by

adding a cytokinin in the media.

Leaf base segments have been proved to be a very suitable target for the

production of transgenic oat plant due to their easy availability, the short culture

period and their high regeneration potential (Gless et al., 1998). During the

present investigation for the leaf-base culture, the leaf length was a vital

parameter for obtaining maximum in vitro response. Leaf-base isolated from 6

day old seedlings responded better as compared to younger or older seedlings.

In another experiment on oat by Chen et al. (1995) maximum calli could be

obtained from 3-day-old seedlings. The gradient of morphogenic competence of

leaf-base and apex has been reported for many other cereal species, viz. rice

(Werincke et al., 1981), wheat (Wernicke and Milkovits, 1984), rye (Linacero and

Vazquez, 1986), barley (Becher et al., 1992) and oat (Chen et al., 1995).

During present investigation in a preliminary experiment, among 5

cultivars tested for their in vitro potential, JO-1 showed best results for callus

induction, embryogenic calli formation and organogenic calli formation from

mature embryo and leaf base. In another study on oat, Kim et al. (2004) also

documented genotypic differences for callus induction and regeneration. The

efficiency of in vitro regeneration system being genotype dependent has also

been reported by other groups (Varandani, 2011; Birsin and ozgen, 2001; Chen

et al., 1995). Although strong genotypic effects are still general characteristics of

the in vitro response in cereal tissue culture, studies by Karp and Lazzeri (1992)

53

suggested that with improved techniques, some of these genotypic variations can

be overcome.

The results of present study demonstrated that under experimental

conditions, in vitro responses of explants i.e. mature embryo and leaf base

cultures were dependent on genotype and media combinations. It was also

noticed that mature embryo initiated callus comparatively earlier than the leaf

base. Presence of higher endogenous auxins in mature embryo explants as

compared to leaf base may be the possible reason for an early induction of

callus. One more possible reason for the occurrence of such phenomenon may

be that mature embryo contains meristemoids that are known for higher and

faster regeneration potential. For the regeneration from calli, medium containing

higher level of cytokinin and low level of auxin such as MS2B.1N showed the

best result for both the explants. For regeneration capabilities, both the explants

responded differently. During present investigation maximum plantlets

regeneration was observed from mature embryo followed by leaf base.

Due to the remarkable progress made in the development of gene

transfer technology (Somers et al., 1992), which ultimately resulted in the

production of large number of transgenic plants both in dicots and monocots.

Genetic modification is an important experimental tool that can be used to

analyze and understand the mechanisms responsible for the expression of

transgenes or endogenous genes, and to create plants with the desired

characteristics. The two basic methods of genetic transformation—biolistic and

Agrobacterium-mediated transformation are the methods available (Gasparis et

al., 2008). Potential benefits from these transgenic plants include higher yield,

enhanced nutritional value reduction in pesticide and fertilizer use. Transgenic

oat plants have been obtained using particle bombardment methods of gene

transfer (Pawlowski and Somers, 1998). DNA integration patterns in transformed

plant tissue obtained via particle bombardment tend to be highly variable and

multiple or fragment copies of introduced DNAs are common, especially when

older cultures are targeted. Cho et al. (2003) studied the expression of green

fluorescent protein (GFP) and its inheritance in transgenic oat plants transformed

54

with a synthetic green fluorescent protein gene driven by a rice actin promoter

where proliferating SMCs were bombarded with a mixture of plasmids containing

the sgfp (S65T) gene and one of three selectable marker genes, phosphinothricin

acetyltransferase (bar), hygromycin phosphotransferase (hpt) and neomycin

phosphotransferase (nptII). Cho et al. (1999) developed highly efficient and

reproducible transformation system for oat using microprojectile bombardment of

highly regenerative tissues derived from mature seeds. Highly regenerative

tissues were generated from embryogenic callus which were used as a

transformation target and transformation was achieved successfully with

transformation efficiency of 26%. Hence till now, not many reports were available

on transformation of oat via Agrobacterium-mediated transformation and only

one report was available developed by Gasparis et al. (2008), where they

developed procedures of oat regeneration from two different types of explants:

immature embryos and leaf base segments. Immature embryos are composed of

highly totipotent, meristematic cells, whereas leaf base segment consist of

differentiated cells, which have to undergo dedifferentiation before somatic

embryo development and plant regeneration and this difference encouraged to

test cell-competence to Agrobacterium-mediated transformation and transgene

expression.

The transformation efficiency may be dependent on the factor incubation

period, sonication, vacuum infiltration treatments and concentration of

acetosyringone. The transformation efficiency at four days incubation period

(96h) with vacuum infiltration of calli was higher, but growth of Agrobacterium

was higher. In order to decrease the excess growth of Agrobacterium, addition of

appropriate antibiotic is an essential requirement. Hence, 3 days (72h) incubated

calli culture was better preferred as they showed lesser growth of Agrobacterium,

but sufficient to cause infection as compared to four days (96h) incubated callus

(Table 4.9).

During the present investigation for primary antibiotic selection of

transformed explants we used MS media containing hygromycin (20mg l-1) and

fortified with 2mg l-1 BAP and 0.1mg l-1NAA. In embryos and leaf base explant,

55

transformation efficiency obtained was 73.33 and 70.00% respectively. Validation

of the transformed plant was done by the GUS histochemical assay. As per other

plant, transformed oat also showed blue coloured spots in multiple shoot

regenerated explants and calli. But especially in regenerated oat plant the GUS

expression was showed in leaf venation as blue colour. The callus that would be

transformed showed partial or complete transformation by expressing blue colour

which confirming transformation. The GUS reporter system utilizes a bacterial

gene from Escherichia coli (uidA) coding for a β-glucuronidase (GUS) and

consists in placing this gene in the Ti-plasmid, which is transferred to plant cells

following infection. When the plant tissue is assayed, transformation events were

indicated by blue spots, which is a result of the enzymatic cleaving of an artificial

substrate to give a blue product. In present investigation it was observed, in case

of embryo and leaf base transformations on basis of GUS assay the

transformation efficiency were 52.96% and 51.85% respectively (Plate 5). (Table

4.3 and 4.7)

In the molecular analysis of putative transformants amplified hptII specific

primer were further used for the amplification of hygromycin phosphotransferase

resistant gene. The PCR product showed the presence of amplified bands of the

hptII gene specific primers at 499bp and CaMV 35S promoter region specific

primer at 522bp. This confirmed the transformation however, absence of this

band/amplification at 499 bp possibility of transformation was rejected (Plate 7A).

In this manner putative transformants were validated with the PCR technique.

Presence of agobacterial contamination in transformants was checked by virD2

gene specific primer which amplified a 338bp fragment. Overall, highest

transformation efficiency (40.74%) in oat was observed with the treatment of

vacuum infiltration with 72h incubation in dark during co-cultivation of embryo

explants with Agrobacterium.

An overall protocol for efficient transformation of oat using Agrobacterium-

mediated transformation has been developed which is present in Fig 1.

56

Sterilization of oat seeds

Inoculation in callus induction (MS6D) medium

Infection with Agrobacterium along with vacuum treatment

Co-cultivation of explants for 72h under dark condition

Washing with Cefotaxime solution (250mg l-1)

Selection of putative transformants on regeneration media(MS2B0.1N+Hygromycin 20mg l-1+ Cefotaxime 250mg l-1) for 3 cycles of 10 days

Regeneration of explants

Selection of GUS putative transformants

Detection of transgene through gene specific PCR

Rooting of selected putative transformants on rooting medium (MS)

Hardening

Fig 1. Flow diagram of Agrobacterium-mediated transformation in oat usingdifferent explants

Embryo as explantsLeaf base as explants

Inoculation of seed on MSmedia for germination

Excise the Leaf base from 6days old seedlings aseptically

Soak the seeds overnight indistilled water

Excise the swollen embryounder microscope aseptically

57

SUMMARY, CONCLUSIONS AND SUGGESTIONS FORFURTHER WORK

6.1 Summary

Oat is one of the most important cereal crops worldwide. In spite of its

nutritional importance, its area of cultivation has been low, with virtually no

increase. Conventional breeding has resulted in several important improvements

in this crop and recent advances in biotechnology, such as plant tissue culture

and genetic transformation can significantly contribute to better sustainability of

this important food and fodder crop.

Currently, use of in vitro technology for improvement of oat is rapid,

reliable and sustainable option. However, so far this crop is very difficult to

manipulate in vitro due to its very sensitive response during culturing practices.

Therefore, very extensive and broader approach was applied in this study in

selection of explants, media and other physio-chemical parameters.

For the in vitro regeneration studies in oat from different explants, mature

embryo and leaf base were cultured on MS medium supplemented with different

concentrations and combinations of auxins and cytokinin. In vitro regeneration in

oat was studied by culturing explants on MS medium supplemented with BAP

(0.1-5.0mg l-1) in combination with NAA (1-2mg l-1), IAA (1-3mg l-1), IBA (2-3mg l-

1) and 2,4-D (2-50mg l-1). After 40-45 days of culture, the calli were transferred

into shooting medium. The pH of the entire medium was adjusted to 5.8 prior to

autoclaving. All cultures were incubated at 25±2°C under PAR light and a 12/12

hrs light/dark photoperiod regime.

Culture responses were accounted based on callus induction,

embryogenic calli formation and organogenesis. Among different explants, leaf

base demonstrated best response for regeneration. In mature embryo callus

induction was observed on MS medium supplemented with 2,4-D at 6.0mg l-1,

whereas in case of leaf base, callus induction was observed maximum on MS

medium supplemented with 2,4-D at 2.0mg l-1.

58

In case of embryogenic calli MS medium supplemented with 2,4-D (6mg l-

1) was found to be best and in organogenesis MS medium supplemented with

BAP (0.5-2mg l-1) and NAA (0.1-2mg l-1) was found to be better. Among different

genotypes JO-1 produced maximum number of induced calli, embryogenic calli

as well as organogenic calli.

Regeneration from callus cultures is a prerequisite for the application of

modern methods of in vitro culture for crop improvement. In this study, in vitro

regeneration in oat using mature embryo and leaf base as explants with different

treatments of Agrobacterium-mediated transformation have been described.

Experiments were conducted to examine callus induction from leaf base, mature

embryo and then transforming the calli with Agrobacterium by using different

treatments. The co-cultivation treatment were supplemented with sonication,

vacuum infiltration and both in combination used for transformation with different

incubation period at dark condition of 48h, 72h and 96h (Table 4.9). Among

different transformation treatments, it was found to be the vacuum treatment with

72h dark incubation period observed good results of transformation over other

treatments.

After explants selection on hygromycin containing media, transformed

explants and calli were again validation through GUS staining followed by the

molecular analysis employing polymerase chain reaction. These validations

clearly indicated the integration of the transgenes from the T-DNA region of

Agrobacterium to the oat host genome. However, the transformation efficiency

was less, as approximately only 24 per cent of the explants and calli of oat

exhibited transgenic presence. On the other hand, vacuum infiltration technique

during co-cultivation increased the transformation efficiency up to 40.74 per cent.

6.2 ConclusionOn the basis of results obtained from different explants, mature embryo

and leaf base was preferred over other explants. It can be concluded from the

experiments, that callus induction takes place when basal medium is

supplemented with higher levels of 2,4-D. Whereas shoot differentiation occurs

59

on basal medium supplemented with higher concentrations of BAP with low level

of NAA. Among genotypes, JO-1 was found to be most responsive for in vitro

regeneration hence, was used for further transformation related experiments.

Finally based on data obtained, it was concluded that the mature embryo

calli become infected with Agrobacterium tumefaciens and were transformed with

co-cultivation with vacuum treatments. Higher transformation efficiency at

different transgenic selection criteria was observed when transformed calli were

kept in incubation in dark for 72h followed by an antibiotic wash to remove

Agrobacterium contamination. Finally, based on PCR analysis putative

transgenic calli and/or plants were selected with a highest transformation

efficiency of 40.74 per cent.

6.3 Suggestion for further work

1. In vitro responsive genotypes may be used in inter-specific and

inter-generic hybridization programmes, where in vitro embryo

rescue technique will be required to obtain hybrids.

2. Embryogenic callus culture from mature embryos can be used to

obtain embryogenic cell suspension cultures and for totipotent

protoplast isolation. Suspension cultures can be used for in vitro

selection at cell level, and protoplast for somatic hybridization,

cybridization and genetic transformation purposes.

3. Regeneration protocol developed during this study from embryo/ leaf

bases should be used for development of transgenic lines with

different transgene transformed through this technique.

4. Transformation of the local cultivars of oat with transgenes like

phytase to increase its nutritive values for feed purposes.

5. Development of biotic and abiotic stress resistant varieties of oat

through transformation.

60

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APPENDICES

ANOVA Table 4.3 Effect of different treatment on transformation efficiency(TE %) of embryo explants based on survival onhygromycin containing media.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F prob

Treatments 9 7384.833 820.530 439.574 5.585

Error 20 37.333 1.866 - -

Total 29 - - - -

SEM ± 0.789, CD 1% 3.177, CD 5% 2.32

ANOVA Table 4.4 Effect of different treatment on transformation efficiency(TE %) of embryo explants based on criteria of GUS assay.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F

probTreatments 9 4827.866 536.426 298.014 2.633

Error 20 36.000 1.800 - -

Total 29 - - - -

SEM ± 0.744, CD 1% 3.115, CD 5% 2.28

ANOVA Table 4.5 Effect of different treatment on transformation efficiency(TE %) of embryo explants based on criteria of PCR.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F prob

Treatments 9 2952.800 328.088 164.044 9.536

Error 20 40.000 2.000 - -

Total 29 - - - -

SEM ± 0.816, CD 1% 3.281, CD 5% 2.40

ANOVA Table 4.6 Effect of different treatment on transformation efficiency(TE %) of leaf base explants based on hygromycinsurvival.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sumof squares F cal F prob

Treatments 9 6975.366 775.047 357.711 4.317

Error 20 43.333 2.166 - -

Total 29 - - - -

SEM ± 0.850, CD 1% 3.412, CD 5% 2.50

ANOVA Table 4.7 Effect of different treatment on transformation efficiency(TE %) of leaf base explants based on criteria GUS assay.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F prob

Treatments 9 4337.500 481.944 237.028 2.538

Error 20 40.666 2.033 - -

Total 29 - - - -

SEM ± 0.823, CD 1% 3.313, CD 5% 2.426

ANOVA Table 4.8 Effect of different treatment on transformation efficiency(TE %) of leaf base explants based on criteria PCRputative transgenic explants.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F

probTreatments 9 2533.200 281.466 111.102 4.316

Error 20 50.666 2.533 - -

Total 29 - - - -

SEM ± 0.919, CD 1% 3.692, CD 5% 2.719

ANOVA Table 4.9 Over all effects of co-cultivation period and differenttreatments on transformation based on criteria ofhygromycin containing media.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sumof squares

F cal F prob

Treatments 19 14416.266 758.758 376.241 4.358

Error 40 80.666 2.016 - -

Total 59 - - - -

SEM ± 0.820, CD 1% 3.132, CD 5%= 2.343

ANOVA Table 4.9 Over all effects of co-cultivation period and differenttreatments on transformation based on criteria GUSassay.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sum ofsquares F cal F

probTreatments 19 9183.516 483.349 252.179 1.207

Error 40 76.666 1.916 - -

Total 59 - - - -

SEM ± 0.799, CD 1% 3.055, CD 5% 2.285

ANOVA Table 4.9 Over all effects of co-cultivation period and differenttreatments on transformation based on criteria PCR.

Source ofvariation

Degrees offreedom

Sum ofsquares

Mean sumof squares F cal F prob

Treatments 19 5489.266 288.907 127.457 8.116

Error 40 90.666 2.266 - -

Total 59 - - - -

SEM ± 0.869, CD 1% 3.329, CD 5% 2.483

VITA

The author of this thesis Mr. Nagesh Raosaheb Dattgonde

S/O Shri. Raosaheb Nagoji Dattgonde was born on 21 May 1988 at

Barepurwadi, Tahsil Vasamat, District Hingoli of Maharashtra.

He completed his primary school education in Malegaon, District

Nanded; S.S.C. and H.S.C. from Ahmadpur, District Latur (M.H.)

under Latur board, Latur.

He joined College of Agriculture, Naigaon (Bz.) affiliated to

MAU, Parbhani (M.H.) in the year 2005 and successfully

completed B.Sc. (Agriculture) degree in the year 2009 with an

O.G.P.A. of 7.85.

Subsequent to graduation, he joined M.Sc. in Agriculture

(Molecular biology and Biotechnology) at Biotechnology Centre,

J.N.K.V.V. Jabalpur (M.P.) in the year 2010. In partial fulfillment

of master degree he was allotted a research problem entitled

“Studies on Agrobacterium-mediated Transformation in Oat

(Avena sativa L.)”.

Nagesh Raosaheb Dattgonde

At BarepurwadiPost/Tahsil. VasamatDist. Hingoli (M.H.)Mobile No.: +918600683423

Email ID: nrd2010@gmail.com,nageshdattgonde1@gmail.com