standardization of plant tissue culture and transformation protocols for

“STANDARDIZATION OF PLANT TISSUE

CULTURE AND TRANSFORMATION PROTOCOLS

FOR LINSEED (Linum usitatissimum L.)”

M.Sc. (Ag.) THESIS

by

PARITOSH

DEPARTMENT OF BIOTECHNOLOGY

COLLEGE OF AGRICULTURE

INDIRA GANDHI KRISHI VISHWAVIDYALAYA

RAIPUR (C.G.)

2009

“STANDARDIZATION OF PLANT TISSUE

CULTURE AND TRANSFORMATION PROTOCOLS

FOR LINSEED (Linum usitatissimum L.)”

Thesis

Submitted to the

Indira Gandhi Krishi Vishwavidyalaya, Raipur

by

PARITOSH

IN PARTIAL FULFILMENT OF THE

REQUIRMENTS FOR THE

DEGREE OF

Master of Science

in

Agriculture (Biotechnology)

ROLL No.9905 ID No.120107017

August, 2009

CERTIFICATE-I

This is to certify that the thesis entitled “Standardization of Plant Tissue

Culture and Transformation protocols for Linseed (Linum usitatissimum L.)”

submitted in partial fulfilment of the requirement for the degree of “MASTER OF

SCIENCE IN AGRICULTURE” of the Indira Gandhi Krishi Vishwavidyalaya,

Raipur, is a record of the bonafide research work carried out by Mr. PARITOSH

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

Student‟s Advisory Committee and the Director of Instructions.

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 have been duly acknowledged by him.

Date: Chairman Advisor Committee

THESIS APPROVED BY THE STUDENT’S ADVISORY COMMITTEE

Chairman : Dr. Girish Chandel ________________________

Member : Dr. D. K. Sharma ________________________

Member : Dr. Sanjay Sharma ________________________

Member : Dr. R. R. Saxena ________________________

Member : Dr. (Smt.) Zenu Jha ________________________

CERTIFICATE-II

This is to certify that the thesis entitled “Standardization of Plant Tissue

Culture and Transformation protocols for Linseed (Linum usitatissimum L.)”

submitted by Mr. PARITOSH to the Indira Gandhi Krishi Vishwavidyalaya, Raipur

in partial fulfilment of the requirements for the degree of M. Sc. (Ag.) in the

DEPARTMENT OF BIOTECHNOLOGY has been approved by the external

examiner and Student‟s Advisory Committee after oral examination.

EXTERNAL EXAMINER

Major Advisor ________________________

Head of the Department/ Section ________________________

Dean/ Dean Faculty ________________________

Director of Instructions ________________________

Acknowledgements

All the work presented in this thesis required the collaboration of a number of individuals. I

would like to express my sincere appreciation to all for their willingness and support.

This thesis would not have been possible without the kind support, trenchant critiques,

probing questions and remarkable patience of my thesis advisor: Dr. G. Chandel, associate Professor,

Department of Biotechnology, IGKV, Raipur. I am greatly indebted to his steadfast inspiration,

illuminating guidance, unfailing encouragement and pertinent suggestions in execution of this

research.

I extend a note of thanks to Dr. D. K. Sharma, Professor and Head, Department of

Biotechnology, IGKV, Raipur, Dr. Sanjay Sharma, Dr. (Smt.) Zenu Jha, Dr.R.R. Saxena, members of

my advisory committee for their consistent support and invaluable suggestions during my tenure of

research work. My sincere acknowledgements also to Dr. Raj Bhatnagar, Scientist, International

Center for Genetic Engineering (ICGEB), New Delhi for providing the valuable clones for this

research work.

I am especially thankful to all my seniors especially Shubha mam, Sudeshna mam and Neha

mam for their helping and coperative attitude. I can’t forget the moments shared with my friends,

Pramod, Ashish, Vikrant, Kaushalendra, Gulfishan, Taslima, Lovejot and Priyanka whose support

and helps always strengthened me.

I would especially like to thank Gulfishan, my lab mate, for walking beside me each step of

the way on this tough long journey.

I am also thankful to National Seed Project, IGKV, Raipur for providing valuable seed

source of LCK-9814, IA-32 and Kiran variety of linseed.

At last but obviously not the least I’m short of words to express my feelings for my beloved

parent and especially my elder brother Dr. Ashutosh, whose pampered support, care and constant

encouragement always gave wings to my ideas and made me to fly high.

Department of Biotechnology Paritosh

College of Agriculture, I.G.K.V., Raipur (C.G.).

Date:

CONTENTS

CHAPTERS PARTICULARS PAGE

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIALS AND METHODS

IV RESULTS AND DISCUSSION

V

SUMMARY, CONCLUSION AND

SUGGESTIONS FOR FUTURE

RESEARCH WORK

ABSTRACT

REFERENCES

LIST OF TABLES

TABLE No. TITLE PAGE No.

2.1 Classification of B. thuringiensis δ-endotoxin genes

and specificity.

3.1 General characteristics of three linseed varieties used

in the study.

3.2 Composition of different induction medium used in

study.

3.3 Composition of AB Media used for Agrobacterium

culture.

3.4 Solutions used in alkali lysis method of plasmid

DNA isolation.

3.5 PCR components with their quantity used for

screening the putative transformants of linseed.

3.6a Temperature profile used for the amplification of Bt

gene mcryIAc.

3.6b Temperature profile used for the amplification of Bt

gene mVIP.

4.1 Influence of genotype, explants type and hormone

conc. on mean callogenesis (%) of all the specified

linseed cultivar

4.2 Influence of genotype, explants type and hormone

conc. on mean shoot regeneration (%) of all the

specified linseed

4.3 Effect of medium composition and growth regulators

on number of shoots per hypocotyl derived callus of

three linseed (Linum usitatissimum L.) cultivars

4.4 Adventitious shoot regeneration of peeled and non-

peeled hypocotyl explants

4.5 Kanamycin sensitivity of explants cultured in basal

MSB5 media

4.6 Effect of OD and dipping time on transformation.

4.7 Effect of co-cultivation period on Agrobacterium

infected explant

LIST OF FIGURES

FIGURES TITLE BETWEEN

PAGES

2.1 Callus based shoot initiation from hypocotyl explant of

linseed.

3.1 Partial restriction map of mVIP gene and mcry1Ac gene.

4.1 Callogenesis and subsequent shoot induction in MSB5

media.

4.2 Influence of kinetin concentration on callusing and shoot

induction in LCK-9814 cultivar on MSB5 media.

4.3 3-4 inches long shoots ready for rhizogenesis.

4.4 In-vitro regenerated plants transferred into pots in green

house for acclimatization.

4.5 Development of morphogenetic calli inoculated on MSB5

along with kinetin.

4.6 Mortality of hypcotyl explants on diffenrt concentration

of Kanamycin.

4.7 Putative mVIP transformants in kiran cultivar of

linseed.

4.8 PCR screening of putative mVIP transformants.

LIST OF ABBREVATIONS

Abbreviations Details

l Microliter

2, 4-D 2, 4 – dichlorophenoxy acetic acid

AB Agrobacterium medium

ABA Abscisic acid

Agro-inoculum Agrobacterium inoculum

BAP Benzyleamino purine

bp Base pairs

Bt Baccillus thuriengiensis

CaMV Cauliflower Moisac virus

cry Crystal

CTAB Cetryl trimethyl ammonium bromide

cv. Cultivar

d Day

DMSO Dimethylsulfoxide

DNA Deoxyribose Nucleic acid

dNTP Deoxynucleotide triphosphate

EDTA Ethylene Diamine Trtra-acetic acid

g/L Gram per liter

GUS -glucuronidase

kb Kilo base

KIN Kinetin

L3 Lin and Zhang medium

LB Luria broth

LF Leaf folder

LS Linsmaier and Skoog medium

Mg Milligram

min Minute

mM Millimolar

MS Murashige and Skoog

MSM Murashige and Skoog Modified medium

mVIP Modified vegitative insecticidal protein

NAA α-Napthalene acetic acid

ng Nano gram

PCR Polymerase Chain Reaction

pH Log 1/(H+)

psi Pressure per square inch

rpm Revolution per minute

RT Room temperature

SDS Sodium dodecyl sulphate

T-DNA Target –DNA

CHAPTER I

INTRODUCTION

Linseed or flax (Linum usitatissimum L.) belongs to the family Linaceae, with more

than 200 species but, only one species– linseed (Linum usitatissimum L.) has a practical

and commercial use. Common flax is a plant with a long history of cultivation and

breeding in Lithuania (Bacelis, 2001), although it is attracting new interest in Europe.

The fiber flax cultivars are mostly grown in Lithuania, but recently the interest in oil

cultivars is also increasing. Despite of sizable acreage of linseed the contribution of crop

to oilseeds rarely goes beyond 2 percent. The average per hectare yield (328kg/ha) of

linseed in India is far lower than world average (852kg/ha) (DOR, 2007) due to various

constraints. One of the most important constraints is pest attack. Linseed is susceptible to

number of insect pests such as pod borer (Helicoverpa armigera), gall midge (Dasineura

oxycoccana), linseed caterpillar (Spodoptera litura), cut worm (Agrotis ipsilon) and

aphids (Aphis gossypii). According to latest report published by ministry of agriculture

2007 the productivity of linseed has decreased from 4.60 lacs hectare to 4.21 lacs

hectares and pest attack especially lepidopteran pests alone has 10% contribution in this

loss. Generally conventional breeding methods such as pedigree selection or bulk

breeding method to develop pest resistant linseed cultivars (Seiss et al., 1996).

The development of procedures for efficient regeneration of plants from cultured

cells, tissues and organs are a prerequisite for application of in-vitro culture techniques to

plant gene manipulation for crop germplasm enhancement (Zhang et al., 2004). The

capacity of cells to regenerate via different morphogenic programmes is a result of cell

dedifferentiation to become competent to the stimulus. This is then followed by induction

for the developmental programme and eventual development into the organ (Nhut et al.

2003). Regeneration methods frequently depend on the type of tissue used to initiate

cultures, with the generation or acquisition of starting material potentially becoming a

limiting factor (Koroch et al., 2003). Linseed improvement has not developed at the same

rate as in other crops (mainly cereals) in recent years. Tissue culture and use of transgenic

technology can speed up the novel breeding which can lead to linseed improvement and

even to incorporation of valuable and desirable traits into linseed cultivars (resistance to

fungal diseases, oil quality improvement and herbicide tolerance) through somatic

hybridization and somaclonal variation (Basiran et al., 1987) and genetic engineering

technology. Plant regeneration is a critical limitation to use of genetic transformation and

recovery of high number of transgenic plants (Dedicova et al., 2000). Therefore present

study entitled “Standardization of Plant Tissue Culture and Transformation

protocols for Linseed (Linum usitatissimum L.)” was undertaken first to standardize the

various factors viz. explant type, genotype and nutritional medium for efficient in-vitro

regeneration system in Linseed and further to develop the Agrobacterium mediated

transformation system using two Bt genes with following objectives:

1. Development of high frequency in vitro regeneration protocol for efficient

transformation in linseed.

2. Standardization of transformation protocol for Agrobacterium mediated / Biolistic

based technique.

3. Molecular screening of putative transformants.

CHAPTER II

REVIEW OF LITERATURE

Linseed is an important oilseed crop worldwide and is cultivated for its

innumerable utilities. It is a crop species widely adapted to warm and cool temperate

climates. The species has been used, for a long time as a source of industrial oil, for the

production of paints, varnishes inks and linoleum (Green and Marshall, 1984). Despite of

sizable acreage of linseed the contribution to oilseed rarely goes beyond 2 percent. The

average per hectare yield (328kg/ha) of linseed in India is far lower than world average

(852kg/ha) (DOR, 2007) due to various constraints. One of the most important

constraints is pest attack. Linseed is susceptible to number of insect pest. Pod borer

(Helicoverpa armigera), Gall midge (Dasineura oxycoccana), linseed caterpillar

(Spodoptera litura), cut worm (Agrotis ipsilon) and aphids (Aphis gossypii) are major

insect pest of linseed. There is huge loss in productivity in terms of quality and quantity.

According to latest report published by ministry of agriculture 2007, the productivity of

linseed has been decreased from 4.60 lac hectare to 4.21 lac hectares due to various

factors like water, disease, pest, temperature etc. But, pest attack is the most severely

contributing to this decrease. Thus, there is an urgent need to evolve superior cultivars

with genetic resistance to major lepidopteron pest such as Helicoverpa, linseed caterpillar

and cut worm.

2.1. Efforts in conventional breeding

Commonly, plant breeders use pedigree selection or bulk breeding method to

create pest resistance novel lines (Seiss et al., 1996). But is long time and tedious

process. A farmer largely relies on pesticides for pest control. An indiscriminate

application of pesticides during 1980 to 1990s has contributed a lot to the heavy outbreak

of Helicoverpa armigera (Ahmed et al., 2002). The use of chemicals, as a pest control

measure, is a two edged sword with both positive and negative impacts. Despite the

advantage of convenience, simplicity, effectiveness, flexibility and economics the

pesticide use has resulted in several problems such as insect pest resistance, resurgence

and outbreak of secondary pest, adverse effects on non-target organisms and other

externalities (Metcalf and Luckman, 1975). According to Ahmad (2002) there are more

than 500 insect species that have developed resistance to modern insecticides viz. H.

armigera has developed resistance to endosulfan, profenofos, thiodicarb, alpha

cypermethrin, zeta cypermethrin, delta methrin, lambda cyhalothrin, bifenthrin and

cyfluthrin (Ahmad et al., 2002).

To combat resistance pest species and to sustain agricultural productivity there is

need of novel mode of action, unrelated to traditional conventional methods. Tissue

culture technologies in linseed may be used seeking to provide cultivars with new and

useful characteristics (resistance to diseases, tolerance to herbicide and pest resistance)

through somatic hybridization and somaclonal variation (Marshall et al., 1962). The

biotechnological approaches like genetic engineering to improve the traits of important

crops strongly depends on an efficient recovery of plants through in vitro systems.

2.2. Tissue Culture in Linseed:

In plants Haberlandt gave the concept of tissue culture in 1902 that attempted to

cultivate isolated plant cells in- vitro on an artificial medium. The Linseed was cultured

as early as 1924 by Dieterich using embryo explant. However it was Laibach (1925) who

demonstrated the potential utility of culturing immature embryos to overcome hybrid

incompatibility. Sustainable literature available for in-vitro culture of linseed using

different explants. i.e. immature embryos (Laibach, 1925; Erdelska et al., 1973),

hypocotyls (Gamborg et al., 1974; Pretova and Williams, 1986), cotyledons (Rybczynski,

1975), stem (Murry et al., 1977), mature embryos (Kaul and Williams, 1987), leaf and

root (Zhan et al, 1989), petal (Sun and Dong, 1985), anther (Sun and Fu, 1981) proves

linseed to be excellent material for tissue culture. The capacity of linseed cells to grow as

callus, particularly in liquid suspension and callus formation from a single cell protoplast

offers immense scope to study the genetics, breeding and physiology of this species

(Gamborg et al., 1976). It is now possible to grow callus culture and differentiation into

complete plants rather easily from almost any part of linseed plant.

2.3. Callus mediated in-vitro regeneration:

Callus mediated plant regeneration is a stepwise process, starting from callus

induction, callus proliferation, morphogenesis and finally to plantlets. A callus consists of

an amorphous mass of loosely arranged thin walled parenchyma cells arising from the

proliferating cells of parent tissue. Frequently, as a result of wounding, a callus is formed

at the cut end of explant. The most important characteristics of callus, from the functional

point of view is that the abnormal growth has the potential to develop normal shoots,

roots and embryoids, that can form plants. The growth characteristic of a callus involves

a complex relationship between the explant used to initiate the callus, the composition of

medium and the environmental conditions during the incubation period (Dodds and

Roberts, 1985).

2.4. Factors affecting in-vitro culture

For efficient in vitro regeneration, it is necessary to understand the requirements

and factors important for the induction of callus and differentiation. Several factors that

influence the in vitro culture are nutrient media, genotype, size, type and age of explant,

culture conditions such as temperature and light intensity and culture techniques.

2.4.1. Culture media

Linseed explant responds to a range of callus induction and shoots regeneration

basal media i.e. macro and micronutrient composition. Rybczynski (1975) cultured

cotyledons on LS medium (Linsmaier and Skoog, 1965). They obtained callus culture

and plant regenerated from hypocotyls explants using Whites (1963) and MS media

(Murashige and Skoog, 1962), microelement and vitamins of B5 (Gamborg, 1975). Kaul

and Williams (1987) used Monnier basal medium for multiple shoot induction from the

hypocotyls of germinating seed. Nichtertein et al., (1991) obtained maximum shoots

regeneration from anther culture on modified N6 media.

Apart from salts present in the basal medium, several other supplements such as

sugar, vitamin, plant growth regulators, osmotica affects the in-vitro response. Many

workers have studied the effect of these medium supplements on in-vitro response of

linseed.

Bretagne et al., (1994) cultured hypocotyls, cotyledon and apical explants of

linseed on basal medium supplemented with the cytokinin, Thidiazuron, BAP, or Zeatin

alone or in combination with NAA, IAA or 2,4-D. Thidiazuron in combination with NAA

(0.01m) resulted in significantly higher shoot regeneration from hypocotyls segment as

compared with all other treatments.

Murry et al., (1977) derived callus from the stem explants of haploid and diploid

linseed and regenerated large number of shoots from callus supplemented with NAA and

BAP. Burbulis et al., (2005) used MS, B5 and MSB5 media for callus induction on

hypocotyl and shoot explant and found hypocotyl along with MSB5 has excellent

regeneration capacity.

2.4.2. Genotypes and explants

There are considerable evidences that variation exist among the different

genotypes for the response of in-vitro cell culture. It is widely considered that

morphogenesis is strongly affected by genetic and exogenous factors (Smith et al., 1990).

Nichtertein et al., (1991) observed significant differences among linseed

genotypes in capacity for root development. They studied the effect of genotypic factors

on shoot regeneration from anther culture of linseed and found to be significant.

Murry et al., (1977) reported a wide range of variations among genotype for

embryo culture response. Maximum in vitro response was obtained in genotype J-17

followed by J-23 and C-2 for mature embryo culture and C-2 followed by J-17 and J-23

for immature embryo culture. He reported these genotype exhibited higher callus

initiation, embroyogenic calli formation and plantlet regeneration.

Burbulis et al., (2007) reported that shoot number per explant was strongly

influenced by genotype, culture medium and different explants. Three varieties being

taken and under same conditions maximum shoots per explant was found in Szaphir

varieties followed by Lirina and Barbara respectively.

McHughen et al., (1991); Cunha et al., (1995) and Blinstrubiene et al., (2004) all

reported that almost any explant can be propagated but the hypocotyl is best in terms of

environmental suitability is concerned compare to stem, roots, cotyledon or others.

2.4.3. Culture conditions

Light and temperature are the two crucial factor in the tissue culture. Erdelska et

al., (1973) found that young embryos (7-9 days) needed light for embryogenic

development before germination could occur. The light condition used for linseed callus

culture ranges from complete darkness to continuous illumination. Zhan et al., (1989)

used 16 hours diffused light / 8 hours dark cycle for protoplast callus culture, whereas

McHughen (1989) used complete dark for hypocotyls callus culture.

Murry et al., (1977) observed the occurrence of high frequency shoot primodia at

300 C as compare to 25

0 C. However, callus formation was more vigorous at lower

temperature. In general 250-27

0 C temperature is widely used in linseed tissue culture.

For plant regeneration warm white fluorescent light (900-3000 Lux) with 16 to 20

hours photoperiod is generally used (Gamborg, 1976; Prêtova and Williams, 1986;

Kaul and Williams, 1987). The effect of light and darkness has profuse effect on

callusing and shoot initiation as per linseed regeneration is concerned. Thus, cultural

conditions should be standardized for optimum in- vitro regeneration.

2.4.4. Culture Techniques

Several culture techniques such as surface sterilization of explants, inoculation of

explants, sub culturing of callus etc affect the successful in vitro regeneration. Prêtova

and Williams (1986) used 1 % Sodium Hypochlorite solution to disinfect immature

embryo and seeds of linseed for 15 and 20 min. respectively followed by 3-4 rinsing.

Aseptic inoculation of explants and proper contact of explants with media is essential

for growth and development of culture. At particular stage it is necessary to

subculture the callus to a fresh medium.

Murry et al., (1977) reported that shoot regeneration from linseed callus was

influenced by the interval of time that explants were cultured on the medium

supplemented with growth regulators.

2.5. Transgenic research in linseed

The nuclear genome of linseed is small compare to many other plants (1.4 pg/2C

nucleus) with a chromosome number of 2n=30 (Bennet and Smith, 1976). In addition,

haploid plants can be generated readily from some linseed varieties. The property of

totipotency, small genome, availability of haploids and susceptibility to transformation by

Agrobacterium would recommend linseed as favorable system for mutagenesis studies by

random insertion of T-DNA.

2.6. Biotechnological approaches-Genetic Engineering

Numerous methods are there for the transformation i.e. Agrobacterium, gene gun,

electroporation, protoplast mediated but, Agrobacterium mediated transformation is most

common in plants. Agrobacterium tumefaciens is a gram-negative bacterium and soil

phytopathogen that genetically transforms host plants and causes crown gall tumors at

wound sites (Smith et al., 1990). The interaction of Agrobacterium and eukaryotic cells is

the only known mechanism for DNA transport between the different kingdoms in nature.

Agrobacterium tumefaciens-mediated transformation has been widely used for research

in plant molecular biology and for genetic improvement of crops since 1983. The

advantage of the method is the wide host-range of the bacterium, from microorganisms to

higher animals and plants, including crops such as soybean, cotton, rice, maize,

sugarcane, wheat, linseed, tobacco and many more. The other merits include integration

of the small copy number of T-DNA into plant chromosomes, and stable expression of

transferred genes. However, even with those superior attributes, it is still difficult to

achieve high reproducibility and consistency of transformation events that are

prerequisite for large scale transformation experiments in plant biology.

2.7. Agrobacterium mediated gene transfer

The Agrobacterium tumefaciens-mediated transformation method is commonly

used to create transgenic plants because it has several merits compared with direct gene

transfer methods such as particle bombardment, electroporation and silicon carbide

fibers. The advantages are (1) Stable gene expression because of the insertion of the

foreign gene into the host plant chromosome. (2) Low copy number of the transgene. (3)

Large size DNA segments can be transferred. Agronomically and horticulturally

important crops, flowers and trees have been genetically modified using this method (Ko

and Korban, 2004; Lopez et al., 2004).

A. tumefaciens is a gram negative soil bacterium which infects a wide range of

dicot plant species. It produces crown gall tumors by introducing a piece of DNA (T-

DNA) in to the host genome (Smith and Hood, 1995). It is the T-DNA delivery system,

which has been exploited for developing transformation techniques through the A.

tumefaciens approach. It is now known that for foreign gene transfer only the T-DNA

borders (25 bp repeats) and some flanking sequences are needed in cis position. Thus by

deleting T-DNA region and adding selectable markers, “disarmed” plasmid vectors can

be prepared and effectively used for transferring foreign genes without disturbing the

endogenous hormone balance of the host plant. Agrobacterium-mediated gene transfer

method in dicots is the most popular technique to obtain transgenic plants.

Basiran et al., (1987) used the Antares cultivar of linseed for Agrobacterium

mediated transformation. They used the C58C1 strain of Agrobacterium harbouring the

pGV3850:1103 plasmid along with npt gene for kanamycin selection. They used

hypocotyl explants for transformation by dipping it in Agrobacterium culture for 1-2

hours and obtained large number of transformed putative transformants.

McHughen et al., (1989) transformed the linseed hypocotyl explant with the C58

strain of Agrobacterium strain harbouring GV3850 plasmid. Plasmid contained

Acetolactate synthase (ALS) obtained from Arabidopsis conferred resistance to herbicide

chlorosulfuron. They grew the transgenic plant up to maturity and segregation pattern

was analyzed for novel gene of interest.

Basiran et al., (1992) did regeneration of transformed shoots via callus phase in

linseed by using C58C1 (pGV3850) strain of Agrobacterium tumefaciens and studied the

different factors affecting it.

Dong et al., (1991) used C58 strain of Agrobacterium having GV2260 plasmid

with GUS as marker. They regenerated the transformed plants of linseed and studied the

patterns of transformation intensity on flax hypocotyls inoculated with Agrobacteriun

tumefaciens.

Agrobacterium is an effective tool for plant genetic engineering, since a portion of

the plasmid DNA from Agrobacterium is incorporated into higher plant cells and results

in crown gall in the host plant (Chilton et al., 1977). Tumor induction is initiated by

bacterial recognition of monosaccharides and phenolic compounds secreted by the plant

wound site. Activated Agrobacterium transfers a particular gene segment, called transfer

DNA (T-DNA) from the Ti plasmid and T-DNA is stably integrated into the

chromosomal DNA in nucleus of the host plant; the genes for opine synthesis and tumor

inducing factors in T-DNA are transcribed in the infected cells. This expression of the

foreign gene in the host plant results in neoplastic growth of the tumors, providing

increased synthesis and secretion of opines for bacterial consumption (Nester et al.,

1984). Opine is the condensation product of an amino acid with a keto acid or sugar and

is a major carbon and nitrogen source for Agrobacterium growth. Agrobacterium is

classified based on the type of opine. Different Agrobacterium tumefaciens strains

produce different opine phenotypes of crown gall tumors, because a particular opine

expressed in the tumor is used for particular bacterial growth. Most common

Agrobacterium strains produce an octopine or nopaline form of opines (Hooykaas and

Beijersbergen, 1994). Octopine and nopaline are derivatives of arginine. Agropine was

found in octopine-type tumors, and it is derived from glutamate (Guyon et al., 1980).

Agrobacterium tumefaciens has been used for plant genetic engineering

extensively. Transgenic plants have been released commercially by several companies,

including Monsanto and Zeneca. Plants have been genetically engineered for the purpose

of developing resistance to herbicides, insects or viruses; tolerance to drought, salt or

cold and increasing yield (Birch, 1997). Transgenic tomatoes do not express the gene for

polygalacturonase, an enzyme that degrades pectin leading to softening of fruit tissues.

As a result, the tomatoes can accumulate flavor components for a longer period of time.

Cotton, potato and maize were genetically engineered for insect resistance and soybean,

canola and cotton were genetically developed for 11 herbicide resistance. The

Agrobacterium-mediated transformation method has not only been used for commercial

purposes, but also for basic biology research to study gene regulation or protein function

in transgenic plants (Mclntosh et al., 2004). Although it has been possible to make a

genetically modified plant in some plant species, it is still not easy to make transgenic

plants of all plant species. This may due to the poor transformation event caused by

improper environmental conditions during bacterial infection of the plant, poor plant

regeneration frequency and gene silencing due to position effects or transgene copy

number after stable integration into the plant chromosome. The Agrobacterium-mediated

transformation method still requires improvement in these aspects.

2.8. Agrobacterium-mediated plant transformation protocol development

Transformation efficiency can be increased by the manipulation of either the plant

or bacteria for enhancing competency of plant tissue and vir gene expression,

respectively (Henzi et al., 2000; Mondal et al., 2001; Lopez et al., 2004 and Chakrabarty

et al., 2002). Seedling age and pre-culturing of explants of linseed have been tested to

increase the transformation efficiency (Bretagne et al., 1994). These trials were

conducted to determine the best conditions for plant cell infection or increasing the

number of dividing plant cells before bacterial infection (Amoah et al., 2001;

Chakrabarty et al., 2002 and Mets et al., 1995). To increase the virulence of bacteria by

inducing the vir gene expression, temperature (Dillen et al., 1997), medium pH (Mondal

et al., 2001) and chemical inducers, such as acetosyringone (Som Leva et al., 2002) have

been tested. These factors likely enhance bacterial pili formation required for gene

transfer between bacteria, as well as between the bacteria and plants. Manipulation of

other factors, such as bacterial density, co-cultivation duration, have also increased

transformation efficiency in many experiments (Lopez et al., 2004). According to

previous experiments, inducing vir gene expression seems most important and effective

for increasing plant transformation efficiency, regardless of the type of plant being

studied. Although low temperature was very important to induce bacterial pili formation

by inducing the vir gene, optimization experiments were conducted at room temperature

or over 25°C for co-cultivation.

2.9. Factors to Increase Gene Expression and Transformation Efficiency

2.9.1. Agrobacterium concentration effect

It has been considered in several experiments that transformation efficiency might

be affected by bacterial growth phase and bacterial cell density. Different concentrations

of the Agrobacterium have been used by different research groups and for different plant

materials. In the standard protocol, cells are grown to the stationary phase (A600nm≈2-2.4),

pelleted and resuspended in inoculation medium to stationary or log or mid-log phase

(A600nm≈0.1-1.15). High concentrations of bacteria at the stationary phase have normally

been used for linseed transformation (McHughen et al., 1989 and Pretova et al., 1994),

and low concentrations of bacteria at the log or mid-log phase have been used for

broccoli (Mets et al., 1995), cabbage (Henzi et al., 2000), wheat (Cheng et al., 1997),

cottonwood (Han et al., 2000) and tobacco (Krugel et al., 2002). Clough and Bent (1998)

reported that different bacterial concentrations, ranging from 0.15 to 1.75 of A600nm,

resulted in different Arabidopsis transformation efficiencies Although throughout world

linseed transformation has been done with various concentration of Agrobacterium with

different type of cultivars. According to these results, it can be assumed that inoculum

density can make a difference in the transformation efficiency.

2.9.2. Co-cultivation duration effect

Co-cultivation for 2 to 7 days has been normally used in Agrobacterium-mediated

transformation under various co-cultivation temperatures (Cervera et al., 1998; Han et

al., 2000; Mondal et al., 2001 and SomLeva et al., 2002). Co-cultivation for 3 days

resulted in high transformation efficiency, and transformation efficiency reached a

maximum at day 5 in Citirange (Cervera et al., 1998). They reported that more than 5

days caused bacterial overgrowth and decreased the transformation efficiency. Many

transformation experiments in different plant species, such as tea (Camellia sinensis L.),

cauliflower and linseed showed that 2 to 3 days of co-cultivation resulted in high

transformation efficiency under room temperature co-cultivation conditions (Le et al.,

2001; Chakrabarty et al., 2002 and Lopez et al., 2004). Therefore, 2 to 3 days co-

cultivation has been routinely used in most transformation protocols, since longer co-

cultivation causes bacterial overgrowth that covers the explant and causes toxicity under

room temperature co-cultivation conditions. Cervera et al. (1998) reported that more than

5 days co-cultivation prevented callus formation and resulted in poor plant regeneration.

The co-cultivation duration recommended from published protocols for linseed

transformation varies from one research group to another. They are 2, 3 and 4 days at

26ºC, 24ºC, and room temperature co-cultivation conditions (Svab et al., 1995). Most

previous experiments indicated that 2 to 3 days were optimal at 25ºC co-cultivation

conditions, regardless of plant species. No experiments have been conducted yet to find

optimal co-cultivation duration lower than 25ºC. Thus, co-cultivation period is crucial for

getting transformed linseed shoot, because long co-cultivation leads to overgrowth of

bacteria and shorter co-cultivation period leads to unstable integration of T-DNA.

2.9.3. Effect of injured area

Linseed is easily regenerated in-vitro (Gamborg et al., 1976) and plant is

susceptible to Agrobacterium (Jordan et al., 1988). However, the regeneration efficiency

of transgenic plants is very low. Especially when hypocotyls explants are directly

cocultivated for 2-3 days with Agrobacterium, it is difficult to recover transformed shoots

(Jordan et al., 1988). Shoots regenerated from hypocotyls segments became non-

transgenic since they were grown from non transgenic cells protected by neighboring

transformed cells (Dong et al., 1993) Regenerants probably arise from non-transformed

cells that are thought to be protected from the selective agent by the surrounding

transformed cells, a phenomenon sometimes referred to as cross-protection. Because the

regeneration from hypocotyls segments of linseed occurs primarily from the epidermis,

while the transformed cells proliferate preferentially around the cut ends of explants, the

majority of regenerated shoots will come from epidermis tissue (Jordan and McHughen,

1988b). Thus, Yildiz et al., (2002) differentiated the effect of peeling on callus based

shooting and direct effect on efficiency of transformation. He peeled the epidermal layer

of hypocotyls explant, which increased the callus based shooting as compare to epidermis

based shooting. It increased tremendously the transformed shoots. Thus, peeled

hypocotyl explant can be used for getting high number of transformed shoots.

Fig.2.1.shows the differences between epidermis and callus based shoot initiation.

2.10. Use of Bacillus thuringiensis δ-endotoxins

"Bt" is short for Bacillus thuringiensis, a soil bacterium whose spores contain a

crystalline (Cry) protein. In the insect gut, the protein breaks down to release a toxin,

known as a delta-endotoxin. This toxin binds to and creates pores in the intestinal lining,

resulting in ion imbalance, paralysis of the digestive system and after a few days, insect

death. Different versions of the Cry genes, also known as "Bt genes", have been

identified. They are effective against different orders of insects or affect the insect gut in

slightly different ways. Bt cotton has been commercialized in India. Although others Bt

crop like rice, brinjal, potato, corn and soybean are in field trial stage. In foreign

countries corn, soybean, flax etc are available for commercial and personal use viz. Corn:

primarily for control of European corn borer, Cotton: for control of tobacco budworm and

cotton bollworm, Potato: for control of Colorado potato beetle. As per linseed

productivity is concerned, it is severely affected by different types of pest i.e. thrips, gall

midge, semilooper, pod borer etc. There are trails throughout the world for pest resistant

transgenic plants. In linseed different novel gene of interest has been transformed

successfully i.e. chlorosulfan resistant linseed by McHughen et al., (1989). The bacterium

B. thuringiensis was first discovered by scientist Ishiwata in Japan during 1902 in a

silkworm rearing unit and named it Bacillus scotto. In 1995, it was renamed as Bacillus

thuringiensis. In the early twentieth century, Berliner provided the first inkling that

microbes could control insect pests. Bacillus thuringiensis is a gram positive, sporulating

bacteria differing from Bacillus cereus only by the synthesis of several insecticidal

crystal proteins (ICPs). The ICPs produced by Bt are alpha-, beta- and gamma exotoxins

and beta-endotoxins are used in agriculture. The δ-endotoxin is the most extensively

studied toxin; its larvicidal specificity includes members of lepidopteran, dipterans and

coleopteran insects. In the natural form Bt has been used by farmers practicing organic

and other sustainable growing methods since 1950s as a spray to kill pests without

damaging non-targeted insects or other wildlife. For more than 40 years Bt proteins have

had a safe history as biopesticides preparations and are approved for organic farming.

Rats fed with very high doses of Bt proteins showed no detectable toxic effects whereas

synthetic pesticides, such as organophosphates and chlorinated biphenyles are toxic

(http:www.akademienunion.de). The growing realization of deleterious effects of

inorganic insecticides to the environment and human health spurred a renewed interest in

Bt in 1960 and viable biopesticide like Dipel, Thuricide etc. were introduced. The

inclusion produced by Bacillus thuringiensis consists of proteins exhibiting a highly

specific insecticidal activity. These observations led to the development of biopesticide

based on Bt for the control of certain insect species among the orders, Lepidoptera,

Diptera and Coleoptera (Beegla and Yamamoto, 1992). Fietelson et al., (1992) reported

that Bt was also found active against Hymenoptera, Homoptera, Orthoptera, Mallophaga

as well as against Nematodes, Mites and Protozoa.

Table 2.1. shows a summary of Bt gene and their specificity. Research efforts in

the past few years have led to the discovery of novel ICP which are produced by certain

isolates of B. thuringiensis. These proteins unlike well-characterized crystal proteins are

produced during vegetative growth of cells and are secreted in to the growth medium.

These results together with observed structural divergence of VIP with other

toxins make them an ideal candidate for deployment in insect management programs

together with the other category of Bt- toxins described earlier. Individually VIP has been

successfully expressed in monocots and dicots plants and efforts to pyramid VIP in the Bt

transgenic crops are underway in several laboratories (Ranjekar et al., 2003).

2.11. Mode of action

Most studies of histopathological and mode of action of Bt have been carried out

on lepidopteran larvae with toxin preparations derived from whole Bt crystals. These

investigations showed that mechanism of action of Bacillus thuringiensis cryI proteins

involve solubalization of the crystals in insect midgut receptors and insertion of the toxin

in to the apical membrane to create ion channels or pores. For most lepidopteron,

protoxins are solubilized under the alkaline conditions of the insect midgut proteases

(Tojo and Aizawa, 1983), to become activated toxins. Major proteases are trypsin like or

chymotrypsin like (Novillo et al., 1997). Activated cryI toxins have two known functions,

receptor binding and ion channel activity. Hoffman et al., (1988) have shown that the

activated toxin binds to specific receptors on apical brush border of the midgut

microvillae of susceptible insect binding being a two stage process involves reversible

and irreversible steps (Raja Mohan et al., 1995). Irreversible step involves a tight binding

between the toxin and the receptor, insertion into apical membrane of the columnar

epithelial cells follows the initial receptor mediated binding rendering the toxin

insensitive to proteases and monoclonal antibodies and induces ion channels or non-

specific pores in the target membranes. The formation of toxin-induced pores in the

columnar cell apical membrane allows efflux of cellular content/ions. The disruption of

gut integrity results in the death of insect from starvation or septicemia.

2.12. Selection and molecular characteristics of transgenic plant

Selection of transformed calli or plants is an important factor in plant transgenic

technology. A simple but efficient protocol is essential in selecting transformants from

which transgenic plants can be regenerated. A number of reporter genes Beta-

glucuronidase (GUS), luciferase (LUC), chloromphenical acetyl transferase (CAT) and

anthocyanins are being used in oilseed transformation in order to analyze gene

expression. The most commonly used reporter gene is GUS (Bauer et al., 1994). The

GUS gene was originally isolated from E. coli (Jefferson et al., 1987). The neomycin

phosphotransferase (nptII) confers resistance to kanamycin antibiotic (Uchimiya et al.,

1986).

In addition to transformation with selectable marker, the feasibility of

conformation with a non-selectable gene and a selectable marker had been used for the

production of transgenic linseed plants by Jain et al., (1999). Early selection and

screening of putative transformants for stable transformation is essential. Putative

transformants could easily and quickly be screened using polymerase chain reaction

(Potrykus, 1990). PCR helped in assessing the presence of foreign DNA sequence in

limited amount of putative transformed tissue. After identification of putative

transformants, rigorous proof of transformation is needed for the judgment of stable

transformation. Potrykus (1990) discussed different criteria necessary for confirmation of

stable transformation included proper positive and negative controls, correlation between

physical and genotype data (e.g. southern analysis and gene expression study by western

blot) comparison of predicted and actual results and data allowing discrimination

between false and true positives.

Different protocols have been developed to investigate the presence of introduced

foreign gene in the plant genome. The most important protocols are Polymerase Chain

reaction (PCR), DNA-DNA Hybridization (Dot blot and Southern blotting), Western

blotting and insect bioassay. PCR is quick and sensitive method in screening transgenic

plants. The polymerase chain reaction (PCR) has greatly simplified procedures for

cloning, modification of nucleic acid and efficient detection of specific DNA sequence in

individuals. PCR is a simple and powerful method invented and patented by Cetus

Corporation (Schafr et al., 1986). This method allows amplification of DNA, in-vitro,

through a succession of incubation steps at different temperatures. Typically, the double

stranded DNA is heat denatured primers are annealed at low temperature and extended at

an intermediate temperature. One step of these three consecutive steps is referred to as

cycle. PCR is based on the repetition of these cycles.

Now a days PCR is routinely used for confirming the presence of gene in

transgenic linseed plants. Very small amount of genomic DNA either in pure or crude

form is required for PCR. The stable integration of gene in plant genome could be

confirmed by dot blot hybridization or southern blotting. Southern blotting confirms the

right integration as well as the copy number of introduced gene in plants (Curtis et al.,

2000).

Table 2.1: Classification of Bt. δ-endotoxin gene and their specificity

cry gene Bt subspecies/

Bacterial strain

Target Insect Reference

Order Species

cryIAa HD1, Scotto,

entomocidus

L B. mori,

M. sexta

Schnepf et al., 1985;

Shibano et al., 1985;

Masson et al., 1989

cryIAb Kurustaki, HD1,

berkiner, NRD

12, ICI, IPL 7

L/D M. sexta,

Pieris brassicae,

Aedes agypti

Thorn et al., 1986; Geiser et

al., 1986; Hoftte et al., 1986

cryIAc HD 73, BTS 89A L H. virescens,

T. ni

Adang et al., 1985;

Dardenne et al., 1990

cryIB HD2 L P. brassicae Brizzard & Whieteley, 1988

cryIC entomocidus,

aizawai

L S. littoralis Sanchis et al., 1989; Honee

et al., 1988

cryICb Galleriae L S. exigua Kalman et al., 1993

cryID HD 68 L S. exigua,

M. sexta,

Hofte et al., 1990

cryIE Kenyae L S. littoralis Bosse et al., 1990

cryIF EG 6346 L Ostrinia nubilalis,

S. exigua

Chambers et al., 1991

cryIG galleriae,

DSIR 517

L Smulevitch et al., 1991;

Gleave et al., 1992

cryIX Galleria L Shevelev et al., 1993

cryIIA HD1, HD 263 L & D L. dispar,

A. aegypti

Widner & Whiteley, 1989;

Donovan et al., 1988

cryIIB HD 1 L L. dispar,

T. ni

Widner & Whiteley, 1989

cryIIC Shanghai S1 L M. sexta,

L. dispar

Wu et al., 1991

cryIIIA Tenebrionis,

San diego,

EG2158

C Leptinotarsa

decemlineta,

Phaedon

cochleriea

Sekar et al., 1987; Donovan

et al., 1988

cryIIIB Tolworthi C L.decemlineta Sick et al., 1990

cryIIIC C Diabrotica

undecimpunctata

Lambert et al., 1992

cryIIID Bt 1109P C Lambert et al., 1992

cryIVA Israelensis D A, aegypti,

Culex pipiens

Ward and Ellar, 1987

cryIVB Israelensis D A. aegypti Tungpradubkul et al., 1988

cryIVC Israelensis D A. aegypti Thorn et al., 1986

cryIVD Israelensis D A. aegypti,

C. pipiens

Donovan et al., 1988

cryV JHCC 4835 L/C O. nubilalis,

Diabrotica spp.

Tailor et al., 1992

VIP3 AaI AB 88 L/C A. ipsilon Estruch et al., 1996

VIP3 Aa2 AB 424 L/C S. frugiperda Warren., 1997

VIP3 Aa14 Bt. toworth L/C H. armigera

S. litura

E. vittela

P. brassicae

P. xylostella

Bhalla et al., 2005

Note: L. Lepidoptera; D. Diptera; C. Coleoptera

CHAPTER III

MATERIALS AND METHODS

The present study entitled “Standardization of Plant Tissue Culture and

Transformsation protocols for Linseed (Linum usitatissimum L.)” was carried out at

Plant Tissue Culture and Genetic Engineering Laboratory of the Department of

Biotechnology, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur

(C.G.).

Materials

3.1. Linseed varieties

In the present study, three Linseed varieties namely Kiran, Indira Alsi-32 and

LCK-9814 popularly grown in Central India were used. The details of Linseed varieties

used are mentioned in Table 3.1.

Table 3.1: General characteristics of the three Linseed varieties used in the study

Genotype Parantage Yr. of

release

Duration

(days)

Sailent feature

LCK-9814 Laxmi27×EC1387 1998 135-140 Powdery mildew and

rust resistant

IA-32 Kiran×Ayogi 2004 101-106 Moderately resistant to

powdery mildew

Kiran (Afg8×R1)×Afg8 1987 120-126 Budfly, wilt and rust

resistant

3.2. Gene Constructs

A strain of Agrobacterium tumefaciens, LBA-4404 strain harboring the plasmid

vector pBI-121 was used for transformation of linseed hypocotyls in Agrobacterium

mediated transformation system. Two Bt genes, mcry1Ac and mVIP were used to

develop pest resistant linseed and nptII as a reporter marker gene. These gene constructs

were obtained from International Center of Genetic Engineering and Biotechnology

(ICGEB), New Delhi through Material Transfer Agreement (MTA).

The partial construct map and gene sequence is shown in Figure 3.1. The plasmid

contains kanamycin resistant neomycin phosphotransferase (npt II) gene driven by CaMV

35S promoter for the selection of transformed plant.

3.3. Regeneration media used in linseed tissue culture

Three basal medias MS, B5 and MSB5 (MS major salt and B5 micro salt) were

prepared for in- vitro regeneration as listed in Table 3.2. All the Medias were added with

30g/l sucrose and pH adjusted to 5.7. Agar powder @ 8 g/l was added to media as gel

forming agent. Medias were sterilized by autoclaving at 1210C under 1.05 kg/cm

2

pressure for 20 min. Moderately warm media still in liquid state was poured in Petri-

plates under laminar hood.

3.4. Seed sterilization and inoculation

Mature seeds were surface sterilized for 1 min in 70% ethanol and then 5 min in 1%

HgCl2 followed by subsequent rinse with autoclaved distilled water. Seeds were

blotted dry on tissue paper and then inoculated on Petri plates in laminar hood.

3.5. Germination and selection of suaitable explant excision

Seeds were germinated in 5-7 days and two parts of seed i.e. hypocotyl and

cotyledon were selected as explant. The third explant, the leaf disc obtained from the seed

germinated in green house. The size of cotyledon and hypocotyl was in range of 0.5 –1

mm. For, normal regeneration of linseed we used MSB5 media as basic standard media

for linseed tissue culture.

3.6. Selection of hormone doses for shoot induction

In all analysis MSB5 was used as basic media for all the three varieties. For callus

induction four treatments of kinetin @250, 500, 750, 1000 μg/l. was used. Petri plates

containing different explants along with the different combination of hormones were

incubated at 260C under 16 hr light/8 hr dark photoperiod. The optimization of hormone

was done on the basis of embryogenic calli based shoot initiation coming out from the

explant.

3.7. Rooting

When the shootlets were near about 1-2 inches, which comes out from the cut end of

exlants, they are transferred to half strength of MSB5 with IBA as root inducing

hormone.

3.8Agrobacterium-mediated transformation

Transformation of the specified linseed varieties for the introduction of

recombinant construct carrying target gene (nptII, mcry1Ac or mVIP) has been done by

using Agrobacterium mediated transformation.

3.9. Preparation of Agrobacterium culture for co-cultivation

The Agrobacterium inoculum of strain LBA-4404 containing desired gene

construct from the stock stored at -20oC was grown on the minimal media containing

plates having 50mg/L kanamycin for 2 days at 28+1oC in the dark. Agrobacterium was

scrapped from freshly grown AB medium plates and inoculated the culture in 5 ml of

liquid AB media (Table 3.3) with respective antibiotic, kanamycin. Next day the same

overnight grown 5 ml media again inoculated in 50 ml freshly prepared AB medium

supplemented with kanamycin @50 mg/l on rotary shaker @150 rpm at 28oC for 2-3 hrs

till the inoculum density OD600 reaches 0.6. Inoculum density for the bacterial suspension

was measured at OD600 using Spectrophotometer.

Table 3.3: Composition of AB Media used for Agrobacterium culture

Stock Components Amount (g/ 500 ml)

Stock I K2HPO4

NaH2PO4

30.0

10.0

Stock II NH4Cl

MgSO4. 7H2O

KCl

CaCl2, 2H2O

FeSO4 7H2O

10.0

3.0

1.5

0.1

0.025

Stock III Glucose

Agar

5.0

15.0

3.10. Infection and co-cultivation of hypocotyl explant with A. tumefaciens

Hypocotyl segments cut from the germinated seeds of all the specified linseed

varieties were used as explant for Agrobacterium mediated transformation. To increase

the chances of transformation the epidermal layer of hypocotyls were peeled off and

precultured in MSB5 media along with kinetin for 5 days. These precultured peeled

hypocotyls were used as explant for Agrobacterium mediated transformation. The

hypocotyl explants were immersed in Agrobacterium inoculum for 2-4 min. Agro-

infected hypocotyls were blotted dry on sterile tissue paper and transferred on the same

medium as that for precultue under dark at 26+10C for 2-3 days. After co-cultivation for

three days the hypocotyls were shifted on fresh media containing 200 mg/l cefotaxime to

inhibit the over growth of Agrobacterium and 50 mg/l Kanamycin for selection at 28+1oC

in dark.

3.11. Selection of transformed hypocotyl explant

In Agrobacterium-mediated transformation system, infected hypocotyls were

transferred to selection medium after 3 days of co-cultivation. The selection cycle was

carried out for 4-5 weeks with subculturing after every 2 weeks. The transformed

shootlets coming out from cut ends of hypocotyl were separated and transferred onto

fresh selection medium for each cycle of selection.

3.12. Molecular screening of putative transgenic T0 plants by PCR analysis

3.12.1. Genomic DNA isolation from T0 plants

Genomic DNA was isolated by homogenizing 100-150 mg of leaf tissue and

extracting essentially according to CTAB method given by Saghai Maroof et al., (1984).

The stepwise protocol is given below:

~100 mg of tender shoot was grinded in 400 l 2X CTAB exaction buffer with a

glass rod on spot plate.

400 l more of 2X CTAB extraction buffer was added and mixed thoroughly, in ~

700 l of solution and then transferred, into 1.5 ml eppendorf tube.

Incubated at 65°C on water bath for 15 min and then cooled briefly and 700 l of

Chloroform Isoamyl Alcohol was added.

The contents were shaken by hands intermittently and kept at room temperature

for 15 min. Tubes were centrifuged at 13,000 rpm for 3 min.

600 l of upper aqueous phase was transferred into a new 1.5 ml eppendorf tube.

900 l of absolute ethanol was added and mixed gently and the tubes were kept

for 2 hrs at –20°C.

The sample was centrifuged for 3 min at 10,000 rpm, the supernatant was

decanted. The pellet was washed with 70 % ethanol and air-dried.

DNA pellet was air dried and then dissolved in 50 l of TE buffer

The extracted DNA was quantified by using Nanodrop spectrophotometer and

0.8% agarose gel electrophoresis. The DNA samples diluted to make the

concentration as 50 ng/µl to use as template for PCR analysis.

3.12.2. Quantification of Genomic DNA

Genomic DNA extracted from T0 plants was quantified with Nanodrop

spectrophotometer as well as 0.8% agarose electrophoresis. 3µl of DNA samples isolated

from each plant along with the standards of known quantity of DNA was loaded on 0.8%

agarose gel. The DNA was stained with ethidium bromide and observed under UV trans-

illuminator. The quantity of DNA was estimated by comparing with fluorescence of

standards. After the quantification DNA was diluted with sterile water such that the final

concentration of DNA was 50ng/µl.

3.12.3. Genomic DNA isolation Reagents and solutions

1M Tris- HCl (PH-8.0)

30.28g of Trizma base was dissolved in 200 ml of distilled water. The pH was set

to 8.3 using concentrated HCl. The solution was allowed to cool at room temperature

before making a final adjustment of pH. The final volume was adjusted to 250 ml with

distilled water and sterilized by autoclaving.

0.5M EDTA (pH-8.0)

186.1 g EDTA was dissolved in 800 ml distilled water stirred vigorously on a

magnetic stirrer and the pH adjusted to 8.0 with NaOH. The volume was made up to 1 L.

CTAB extraction Buffer

5 g CTAB, 20.35 g NaCl dissolved in 200 ml double distilled water later 25 ml 1

M tris HCl 10 ml 0.5 M EDTA were added and stirred vigorously on a magnetic stirrer.

Volume was made up to 250 ml and stored in room temperature and 20 l /20 ml 2-

Mercaptoethanol added into it prior to use.

Ethanol (70%)

70 ml of absolute ethanol was taken in measuring cylinder and volume was made

up to 100 ml using distilled water.

TE buffer (pH-8.0)

10 ml 1 M Tris HCl mixed with 2 ml 0.5 M EDTA the volume was adjusted by

using 988 ml of sterile double distill water.

PCR Reagents

• dNTPs: (dATP/dCTP/dGTP/dTTP)

100mM stock of each dNTP was diluted to 1mM of dNTP (i.e. 10 µl of each dNTP +

990 µl of sterile water).

• 2.5 M CaCl2

36.8 g of CaCl2 was dissolved in 50 ml of Autoclaved double distilled water.

• PCR Buffer (10X)

Components Concentration

1 M Tris (pH 8.3) 4.0 ml (100mM final Conc.)

1M KCl 10.0 ml (500 mM final Conc.)

1.5 mM MgCl2 2.0 ml

Gelatin 2.0 ml (1 mg/ml final Conc.)

Sterile water 4.0 ml

Total 20.0 ml

• 50X TAE buffer

Components Concentration

Trizma base 121.00 gm

Acetic acid (glacial) 28.55 ml

0.5M EDTA (pH 8.0) 100.00 ml

Water 50.45 ml

Total 300ml

3.13. Plasmid DNA isolation

Cloned plasmid vector pBI-121 was isolated from the Agrobacterium strain LBA-

4404 by using alkali lysis method given by Sambrook et al., (1989). The stepwise

protocol of alkali lysis method is described below:

Bacterial cells, LBA-4404 containing the desired clone (pBI-121) were grown

overnight at 28oC in LB medium containing suitable antibiotic (Kanamycin

@50 mg/l).

The cells were harvested by centrifuging 1.5 ml of culture at 12000 rpm for 7-

8 min at room temperature (RT).

The supernatant was discarded completely and left the tubes in an inverted

position on a tissue paper for 3-4 minutes to drain off AB completely.

50 µl of Lysozyme was added and the pellet was resuspended in 200 µl of

solution I by gentle vortexing or with the help of pipettman.

400 µl of freshly prepared solution II was added and mixed immediately by

gently inverting the tubes few times till cell suspension became clear and

incubated in ice for 5 min.

300 µl of chilled solution III was added (stored at -20oC) and mixed

thoroughly but gently by inverting the tubes several times till a white coarse

precipitation was visible then incubated on ice for 15 min. The composition of

Solution I, II and III are given in Table 3.4.

Centrifuged at 12000 rpm at 4oC for 20 min and transferred ~700µl of clear

supernatant to a fresh micro centrifuge tube. Centrifuged again at 12,000 rpm

at RT for 3 minutes and transferred 600 µl of clear supernatant to a fresh

micro centrifuge tube.

450µl of isopropanol was added and mixed well and incubated at RT for 10

min then pellet down plasmid DNA by centrifuging at 12,000 rpm for 5 min at

RT.

The supernatant was discarded carefully by decanting and left the tubes in an

inverted position on a tissue paper for few minutes to drain-off isopropanol

completely.

The pellet was washed by adding 800 µl of 70% ethanol to the micro

centrifuge tubes and then centrifuged at 12,000 rpm for 5 min at RT. The

supernatant was discarded carefully by decanting. Centrifuged once again at

above conditions for 1 min only and discarded the remaining ethanol with the

help of pipetteman.

The pellet was air dried for 5 min and dissolved the DNA pellet in 25-50 µl of

TE Buffer containing RNase A (50 µg/ml).

The DNA was resolved on 1% agarose gel by electrophoresis as well as

Nanodrop Spectrophotometer to check the quality and concentration of DNA.

Table.3.4: Solutions used in alkali lysis method of plasmid DNA isolation

Chemical Components Final Conc.

Solution I (pH 8.0) Glucose 50 mM

Tris (pH 8.0) 25 mM

EDTA (pH 8.0) 10 mM

Solution II SDS 1.0 %

NaOH 0.2 N

Solution III Ammonium acetate 7.5 M

TE Buffer Tris (PH 8.0) 1.0 M

EDTA (pH 8.0) 0.5 M

3.14. PCR analysis

The initial screening for the presence of transgene in regenerated plants was done

using PCR technique as per the methods described by Jain et al., (1999).Plant DNA

isolated from leaf tissue was used as template to obtain amplification product of mVIP

and mcryIAc genes for screening of putative transgenic plants. For integration of trans

gene, following gene specific primer pairs were used.

mcryIAc primer

Forward-5‟ATG GAT AAC CCA AAC ATT AAC-3‟

Reverse-5‟GTA CTC AGC CTC AAG AGT GGC-3‟

mVIP primer

Forward-5‟GTT GAC CAC TAG AGC TTT GC-3‟

Reverse-5‟CTT AAT AGA GAC ATC GTA G-3‟

Table.3.5: PCR components with their quantity used for screening the putative

transformants of linseed.

Components Concentration Quantity (µl)

1. Genomic DNA 10 ng/µl 2.0 µl

2. Taq Buffer 10 X 2.0 µl

3. dNTPs 0.5mM 2.0 µl

4. Primer F 50ng 1.0 µl

5. Primer R 50ng 1.0 µl

6.Taq Polymerase 1-2 U/µl 1.0 µl

7. Sterile Water - 11.0 µl

Total 20.0 µl

The 100 ng of genomic DNA isolated from putative transgenic linseed plants, the

negative control (non-transgenic/non-infected) plant and plasmid DNA which was used

for transformation were used as template for PCR reaction. The PCR reaction mixture

consisted of 10 ng of template DNA, 50ng/µl of each the primer, 0.5mM dNTPs, 10X

PCR buffer and 1-2 Unit of Taq DNA polymerase in final volume of 20µl (Table 3.5).

The PCR condition was performed in Thermal Cycler (MJ Research, PTC 100,

USA). The PCR conditions were set as per the transgene and primer combinations used

(Table 3.6a & 3.6b). PCR products were separated on 1% agarose gel (in 1X TAE

electrophoresis buffer) containing 0.5 µg/ml ethidium bromide. Separated products were

visualized under UV light and photographed using gel documentation system (Bio Rad,

USA) to examine the size of the amplification products. Based on the size of DNA band

and its comparative position with amplified product of a plasmid DNA, transgenic plants

were designated as positive.

Table 3.6a: Temperature profile used for the amplification of Bt gene, mcryIAc

Steps Temperature (oC) Duration

(min)

Cycles Activity

1

2

3

4

5

6

94

94

50

72

72

4

2.0

0.45

0.45

0.45

4.0

720

1

35 cycle

1

1

Denaturation

Denaturation

Annealing

Extension

Final extension

Storage

Table 3.6.b: Temperature profile used for the amplification of Bt gene, mVIP

Steps Temperature (oC) Duration

(min)

Cycles Activity

1

2

3

4

5

6

94

94

55

72

72

4

2.0

0.45

0.45

0.45

4.0

720

1

35 cycle

1

1

Denaturation

Denaturation

Annealing

Extension

Final extension

Storage

CHAPTER IV

RESULTS AND DISCUSSION

The present study was undertaken to standardized in–vitro regeneration and

transformation protocols for linseed (Linum usitatissimum L.). The study also aimed to

standardize the various factors affecting Agrobacterium mediated transformation of

linseed with two Bt genes, mcry1Ac and mVIP along with molecular characterization of

putative transformants for the development of borer (H. armigera) resistant linseed lines.

4.1. Development of in-vitro regeneration protocol for linseed (Linum usitatissimum)

The success of plant regeneration depends upon various factors such as

composition of culture media, explants, genotype of donor plants and cultural conditions.

Several researchers have stated that composition of the culture media (Pretova and

Williams, 1986; Kaul and Williams, 1987), explants source (Murry et al., 1977) and

genotypes (Zhan et al., 1989) are important parameters in determining the success of flax

or linseed plant regeneration.

4.1.1. Selection of explants

The type of explants has been identified as one of the major factors affecting the

tissue culture response in crop plants e.g. hypocotyl in case of linseed (McHughen et al.,

1989), shoot (Draper et al., 1987), cotyledon (Rybczynski et al., 1975). In the present

investigation hypocotyl and cotyledon from germinating seed and leaf discs of three

linseed varieties were taken as explant in order to analyze the source explants showing

best in-vitro response. All the three explants showed varied level of callogenesis and in

all tested genotypes the intensities of callus formation (Table.4.1) varied from 12.0 %

with leaf discs as expant (LCK-9814) to 92.0 % when hypocotyl was used as explant

(Kiran), in MSB5 media supplemented with Kinetin. It was observed that the response of

hypocotyl towards callogenesis was highest among all three explants irrespective of

genotype. Relatively higher frequency of callus formation was recorded in two linseed

variety Kiran and IA-32 under the same set of treatments in comparison to the cultivar

LCK-9814. The differences in callus formation from same explant tissue among the three

cultivars indicated that the genetic make up of a cultivar is also an important factor

affecting callus induction efficiency. The results are in agreement to findings of

Blinstrubiene et al., (2004) who have also reported differences in responsiveness among

linseed genotypes.

Variability in callogenesis and differences in growth regulators necessary for

callus production in each genotype may be attributed to the difference in levels of

endogenous hormones in these cultivars. On an average each of the three linseed

genotype produced callus in 10-15 days (Fig.4.1). Morphogenesis of tested linseed

cultivars was found to be determined by the genotype, explant and hormone

concentration (Table.4.2). As it was observed that hypocotyls showed maximum

frequency of callogenesis shoot induction was also found to be highest in hypocotyl as

compared to other two explants irrespective of the genotypes. It was also recorded that

organogenesis in terms of shoot induction was highest in hypocotyl explants of the

linseed variety LCK-9814 and lowest in Kiran cultivar. All the shoots were produced

spontaneously from the from 12-15 days old calli without sub-culturing onto fresh

medium. Mostly, leaf and cotyledon based callus did not showed any organogenic

structure and subsequently became necrotic which suggested that hypocotyl tissue have

either higher level or composition of endogenous hormones which leads to organogenesis

and formation of shoots. Similar observations were also recorded by Ferreria et al.,

(1993) in Antares cultivar of linseed. They reported more than 75% callusing in linseed

from three explants, hypocotyl, leaf and stem segments but the significant difference was

noticed in subsequent shooting among the calli derived from various explants. This

clearly indicated that shoot induction and subsequent plant regeneration in linseed is

explant dependent phenomena. Hypocotyl was found to be most suitable explant for

linseed in-vitro propagation, which is in confirmation to Draper et al., (1987); McHughen

et al., (1991) and Yildiz et al., (2002) and thus was used in all the further experiments.

The process of organogenesis appears to be complex, involving multiple internal

and external factors. The reinitiating of cell division, considered one of the key factor

during regeneration appear to be dependent on many factors but genotype, explant, media

and hormone concentration are most crucial ones (Burbulis et al., 2005). Our result

illustrated that genetic background is important factor determining in-vitro response for

both callogenesis and morphogenesis in linseed.

4.1.2. Effect of media and hormone concentration

There is marked effect of media and hormone concentration on morphogenesis of

linseed (Burbulis et al., 2005). To standardize the media and hormone supplementation,

linseed hypocotyls of all three specified cultivar were cultured under different culture

media (Table.3.2) and hormone composition. The hypocotyl explants from seven days

old germinated seeds were plated on MS, B5 and MSB5 (MS macro salt and B5 micro salt

with vitamin) supplemented with sucrose 30gm/l and 0.7% agar-agar.

Table.4.3: Effect of medium composition and growth regulators on number of

shoots per hypocotyl derived callus of three linseed (Linum usitatissimum L.)

cultivars. Data are mean± SE within two weeks of callus.

Nutrient media with Kinetin NUMBER OF SHOOTS/EXPLANT

LCK-9814 IA-32 KIRAN

MS Basal +Vitamin + 250 µg/l 1.99±0.16 1.91±0.11 0.98±0.09




B5 Basal +Vitamin + 250 µg/l 2.34±0.11 1.68±0.12 1.34±0.06




MSB5 Basal +Vitamin + 250 µg/l 3.84±0.22 2.78±0.17 1.89±0.11




Four treatments of hormone (kinetin) @250, 500, 750 and 1000 μg/l were used.

The media pH was 5.7±0.1, illumination-5000 lx, photoperiod-16 h and temperature

25±20 C. Effect of hormone and media interaction was marked and observation was

recorded (Table.4.3) in terms of mean number of shoots per hypocotyl explant of each

linseed cultivar. The number of shoots per explant was significantly affected by hormone

and culture media. The use of MSB5 medium resulted in significantly more shoots per

explant than any other medium and was observed for all three cultivars of linseed.

However, there were differences in shoot formation on MSB5 medium

supplemented with different concentration of growth regulators. Cultivars differed

significantly in number of shoots per explant.

Hypocotyl derived callus from cultivar LCK-9814 gave the best result, while the

cultivar Kiran had lowest organogenesis response. A regeneration rate of 3.84 shoots per

explant for LCK-9814 cultivar; 2.78 for IA-32 and 1.89 for Kiran cultivar was recorded

in MSB5 culture media supplemented with 250μg /l kinetin. It indicated that 250 μg/l

kinetin level produced the best regeneration frequency for all the three tested cultivars.

Different cultivars responded differently towards the same concentration of hormone

which clearly illustrated that morphogenesis or organogenesis is a genotype dependent

phenomenon (Fig.4.2). Our results were in accordance with the report that morphogenesis

is strongly affected by genetic and exogenous factors (Smith and Razdan, 1990) and

nutrient media has been often modified by adding different compositions of vitamins and

growth regulators depending on the plant species. The most widely used growth

regulators are cytokinins BAP, 2iP and kinetin (Bjowani and Razdan., 1990) and the

auxins IAA and NAA (Ferreria et al., 1993).

4.1.3. Plant regeneration

It was observed that linseed cultivars took seven to nine days for shoot initiation

from green callus and further five to seven days for shoot elongation. When the shootlets

were 1-2 inches long, they were removed from mother callus and transferred to the

rooting media for rhizogenesis. The rhizogenesis nutrient media have been modified by

the concentration of vitamins and growth regulator (Jain et al., 1999) and it has been

reported in flax that mostly IAA and NAA are more commonly used. As it is shown in

Fig.4.3.the shootlets about 3-4 inches were transferred onto rooting media supplemented

with 3% (w/v) sucrose, 0.1 µM IBA and 0.08 (w/v) agar-agar. After 10-15 days about

54% regenerated shootlets were transferred onto pots for acclimatization. Significant

means were revealed in the statistical analysis of variance for genotype explant

interaction in terms of shoot initiation at 1% level of significance.

4.2. Standardization of Agrobacterium mediated transformation protocol in linseed

Several agronomically important genes have been introduced into various crop plants

via Agrobacterium mediated transformation (McHughen et al., 1989). Callus

induction from the cut end of hypocotyl or other explants is primarily required for

high efficiency gene transfer using Agrobacterium tumefaciens as the Ti plasmids

transfers the gene of interest through cut end of the tissues. It has been reported that

transformation efficiency can be increased by the manipulation of either the plant or

bacteria for enhancing competency of plant tissue and vir gene expression,

respectively (Henzi et al., 2000; Mondal et al., 2001; Chakrabarty et al., 2002 and

Lopez et al., 2004). Age of the seedling and pre-culturing of linseed explants has

been assessed to increase the transformation efficiency by Bretagne et al., (1994) to

determine most suitable conditions for plant cell infection or increasing the number of

dividing plant cells before bacterial infection (Mets et al., 1995; Amoah et al., 2001

and Chakrabarty et al., 2002). These studies suggested that factors like increasing the

injury area, prolonged co-cultivation and optical density of Agrobacterium culture

significantly influence the efficiency of transformation. Here in following subsequent

headings, various factors influencing the Agrobacterium mediated transformation

had been analyzed.

4.2.1. Effect of peeling and pre-culturing on callus based shoot initiation

Processing of explants mainly high in vitro responsive hypocotyls by peeling the

epidermis layer and preculturing to produce calli from whole of the callus prior to

Agrobacterium infection resulted in enhanced production of transgenic plant (Yildiz et

al., 2002). To assess the effect of peeling and pre-culturing on hypocotyl derived callus,

two treatments were used which involved peeling of pre-cultured hypocotyls and culture

of hypocotyls without peeling. The mean response in terms of shoot induction and

number of shoots per explant were recorded and analyzed for both the treatments and the

findings are presented in Table.4.4

Table.4.4: Adventitious shoot regeneration of peeled and non-peeled hypocotyl

explants1

Callus formation

(%)

Hypocotyls producing

shoots (%) No. of shoots per

explant

Peeled hypocotyls 90 72.43 5.12

Non-peeled hypocotyl 78 52.67 2.96

t-value 1.321* 3.217* 1Each value is the mean of 3 replications each with 10 explants. Significantly different at the 0.01

The analysis of number of shoots per expant under different treatments indicated

that the callus formation was increased due to peeling and preculturing of hypocotyls as

well as increase in number of shootlets per explant was also recorded. A marked

difference in the pattern of callus formation had been observed in peeled precultured

hypocotyls (Fig.4.5)

The results are in agreement to the findings of Yildiz et al., (2002) who have

reported that increasing the injured area on hypocotyls explants of flax led to high

frequency callus based shoot regeneration which is more suitable for Agrobacterium

mediated transformation.

4.2.3. Selection of lethal dose of Kanamycin

Antibiotic resistance is the usual selectable marker of first choice in plant

transformation experiments (Fraley et al., 1986). The nptII gene provides resistance to

kanamycin and is used as selectable marker in many plant species including linseed

(Jordan et al., 1988 and Koronfel et al., 1994).

In view of the fact that selection of transformed calli is almost important before

shoot regeneration. Thus choice of selectable marker gene, selective agent and timing of

application is key step in the process of developing transgenic. The selectable marker

gene nptII encodes neomycin phosphotransferase enzyme which inactivates the antibiotic

Kanamycin and hence allows the growth of only transformed tissues. In order to

standardize the optimum dose of Kanamycin which ensure growth of only transformed

tissue but still not too toxic, the hypocotyls were incubated in media containing different

concentration of Kanamycin. A gradual decrease in survival of explants was observed in

hypocotyls explants cultured with increasing concentration of Kanamycin (Table.4.5.).

Table.4.5: Kanamycin sensitivity of explants cultured in basal MSB5 media

Kanamycin

Con. (mg/l)

No. of explants

cultured

No. of explants

survived

Lethality %

30 10 2 80

40 14 1 92

50 23 0 100

60 18 0 100

Kanamycin was found to be lethal at concentration of 50mg/l, as it completely

inhibited callusing but did not produced toxicity and survival of explants (Fig.4.6.); hence

50mg/l Kanamycin concentration was identified as optimum for selection of transformed

shoots. 50 mg/l kanamycin has been used for selection of transformed by different groups

(Jordan et al., 1988 and Jain et al., 1999).

4.2.4. Agrobacterium inoculums density and dipping time

In order to standardize the Agrobacterium inoculum density and dipping time for

efficient transformation, different concentrations of Agrobacterium strain with different

dipping period were tested. Table.4.6. shows the effect of Agro culture density and

dipping time for both the clones. Out of total 52 explants treated, only two putative

transformants were produced on selection medium in kiran cutivar tretaed with mVIP

clone. These putative transformants were found at OD600 ≈ 0.6 of Agro culture along with

dipping time of 4 minutes. Similarly, significant effect of Agrobacterium inoculum

density and dipping time has been found by different groups for recovery of successful

transgenic production. Our results are in accordance with the findings of Chakrabarty et

al., (2002) who reported that Agrobactrium density and dipping time is critical in

transofming plants.

4.2.5. Effect of co-cultivation duration on Agrobacterium infected explants

Co-cultivation for 2 to 7 days has been normally used in Agrobacterium mediated

transformation for various crops. (Han et al., 2000 and Mondal et al., 2000). They

reported that more than 5 days caused bacterial overgrowth and decreased the

transformation efficiency

Co-cultivation period is the time taken by the Agrobacterium to insert its T-DNA

in the plant genome and considered as a very critical step in the successful transformation

as reported by Jain et al., (1999). Lesser time hinders the proper insertion of T-DNA into

plant genome as well as prolonged co-cultivation leads to bacterial overgrowth

(McHughen et al., 1991). Thus after removing explants from Agrobacterium culture and

treated explants were dried between filter paper and cultured onto co-cultivation medium.

Table.4.7: Effect of co-cultivation period on Agrobacterium infected explant

Days No. of Agrobacterium

infected explants

No. of clean

explants

No. of shoot

regenerated on

selection medium

2 26 26 18

3 22 17 12

4 24 8 2

Out of three co-cultivation period tested, 2-3 days is suitable for regeneration of

shoots on selection medium. This clearly indicates that three days of co-cultivation is

optimum for transfer of T DNA. Other workers have also reported three days co-

cultivation for proper integration of T-DNA in wheat (Holme et al., 2006).

4.2.6. Molecular screening of putative transformants

Gene specific primers designed from the coding region of both the genes were

used for molecular screening of putative transformants. Two shoots grown on

selection mediums as shown in Fig.4.7. were analyzed for the response of mVIP gene

by PCR. The two regenerants of linseed cultivar kiran were found positive for the

mVIP gene as the PCR analysis yielded 900 bp amplicon (Fig.4.8).

CHAPTER V

SUMMARY, CONCLUSIONS AND SUGGESTIONS FOR FUTURE

RESEARCH WORK

Linseed (Linum usitatissimum L.) is an annual dicot belonging to family Linaceae

and is the only species in its family that is cultivated commercially (Burbulis et al.,

2005). Generally conventional breeding methods such as pedigree selection, bulk

breeding etc have been used to develop pest resistant linseed cultivars (Seiss et al., 1996).

Linseed is susceptible to number of insect pests such as pod borer (Helicoverpa

armigera), gall midge (Dasineura oxycoccana), linseed caterpillar (Spodoptera litura),

cut worm (Agrotis ipsilon) and aphids (Aphis gossypii). Ministry of Agriculture (2007)

reported that pest attack is the most severely contributing reason for decrease in its

productivity from 4.60 lakhs hectare to 4.21 lakhs hectares among all factors including

scarcity of water, disease, pest, temperature etc. The indiscriminate application of

pesticides for control of lepidopteron insects during 1980 to 1990s has created high

selection pressure and heavy outbreak of H. armigera (Ahmed et al., 2002). Thus, there

is an urgent need to evolve superior cultivars with genetic resistance to major

lepidopteron pest such as Helicoverpa, linseed caterpillar and cut worm.

Biotechnological tools and techniques are being used to improve a large number

of crop species (Bretagne et al., 1994) and genetic engineering is the most commonly

used technique for incorporation of novel traits from diverse organisms. The development

of procedures for efficient regeneration of plants from cultured cells, tissues and organs

are a prerequisite for application of in-vitro culture techniques to plant gene manipulation

for crop germplasm enhancement (Zhang et al. 2004). In the present study was

undertaken to ass4ess the various factors affecting in-vitro regeneration response and

Agrobacterium mediated transformation using two Bt genes (mcry1Ac and mVIP) in

three linseed cultivars.

Effect of three factors viz. genotype, type of explant and vitamin along with

hormone concentration on callus induction and morphogenesis of linseed varieties

(LCK9814, IA-32 and Kiran) were studied. We have successfully examined the effect of

the above stated factors and developed a high efficiency plant regeneration protocol for

linseed. All the three explants showed varied level of callogenesis and in all tested

genotypes the intensities of callus formation varied from 12.0 % with leaf discs as expant

(LCK-9814) to 92.0 % when hypocotyl was used as explant (Kiran), in MSB5 media

supplemented with Kinetin. It was observed that the response of hypocotyls towards

callogenesis was highest among all three explants irrespective of genotype. The

differences in callus formation from same explant tissue among the three cultivars

indicated that the genetic make up of a cultivar is also an important factor affecting callus

induction efficiency. It was also recorded that organogenesis in terms of shoot induction

was highest in hypocotyl explants of the linseed variety LCK-9814 and lowest in Kiran

cultivar. The number of shoots per explant was significantly affected by hormone and

culture media as highest number of shoots per explant (3.84/expalnt) was recorded in

MSB5 medium supplemented with 250µg/l Kinetin.

In a pilot experiment the concentration of Kanamycin required to kill non-

transformed cells were examined. Out of four Kanamycin concentrations tested (30, 40,

50 and 60 mg/l), hypocotyls explant showed 100% mortality after 7 days of inoculation

on selection medium containing 50mg/l Kanamycin. This concentration of Kanamycin

was used for the selection of transformed calli and shoots from Agrobacterium infected

explants.

We also investigated the Agrobacterium culure density, dipping time and pre-

culture of explants for Agrobacterium mediated transformation in Linseed. The Agro-

culture density (0.4 and 0.6), dipping time (2.0 and 4.0 min) and explant tissues

(hypocotyls, cotyledon and leaf discs) were found to be critical for the recovery of

transformants. It was also observed that peeling of epidermal layer of hypocotyl led to

increase in number of shootlets per explant from 2.96 to 5.12. Co-cultivation period is the

period taken by Agrobacterium for transferring its T-DNA. Because lesser time hinders

the proper insertion of T-DNA into plant genome as well as prolonged co-cultivation

leads to bacterial overgrowth. Thus, in our linseed cultivar 3 days of co-cultivation period

was found to be optimum co-cultivation period for successful transformation. Out of 52

hypocotyls infected only two regenerants produced on selection medium (MSB5 + 250

µg/l kinetin + 50 mg/l Kanamycin) were found positive for mVIP gene by PCR analysis.

Conclusions:

The in-vitro studies revealed significant effect of genotype, explant tissues,

vitamins and kinetin concentration on the callus induction and morphogenesis of

Linseed.

The response of hypocotyl towards callogenesis was highest among all three

explants irrespective of genotype. Out of the three explants tested the hypocotyl

showed highest average number (3.84/exapnt) of shoots per explant for all the

three linseed cultivars under study.

Organogenesis in terms of shoot induction was highest in hypocotyl explants of

the linseed variety LCK-9814 and lowest in Kiran cultivar.

The number of shoots per explant was significantly affected by hormone and

culture media as higher number of shoots per explant were recorded in MSB5

medium supplemented with 250 µg/l kinetin in all the tested cultivars.

50 mg/l Kanamycin was found to be leathal for the killing of hypocotyl explants

and used for selection.

The Agro-culture density (0.6 OD), dipping time (4.0 mins) and explant tissues

(hypocotyl) were found to be critical for the recovery of transformants.

It was also observed that peeling of epidermal layer of hypocotyls enhances the

number of shootlets per explant from 2.96 to 5.12.

Three days of co-cultivation period was found to be optimum for the transfer of

T- DNA into target tissues and subsequently recovery of transformants.

Two regenerants were found positive for mVIP gene by PCR analysis from total

52 regenerants developed on selection medium.

The development of high efficiency in-vitro plant regeneration system and

Agrobacterium mediated transformation protocol provides an alternative for

genetic manipulation of linseed for biotic and abiotic stresses.

Suggestions for future work:

The tissue culture protocol developed in this study for three linseed cultivars must

be tested for more popular linseed varieties.

The Agrobacterium mediated transformation protocols should used to develop

transgenic with agronomically important genes.

The PCR positive Bt transgenic plants must further analyzed by Southern

analysis for confirmation of gene integration.

Standardization of Plant Tissue Culture and Transformation protocols

for Linseed (Linum usitatissimum L.)

by

Paritosh

ABSTRACT

Linseed (Linum usitatissimum L.) is one of major oilseed crops popularly grown

in Canada, Luthania, Argentina and India. Commonly, pedigree selection or bulk

breeding methods are used by linseed breeders to develop novel lines but have achieved

limited success in genetic improvement of the crop. The application of biotechnology

particularly tissue culture and genetic engineering have been helpful in accelerating

breeding programs or improving the efficiency of selection, as demonstrated in several

crops including linseed (Seiss et al., 1996). The present study was carried out to

standardize the in-vitro regeneration of three linseed cultivars and also to assess the

various factors affecting Agrobacterium mediated transformation in linseed. Two novel

Bt genes, mVIP and mcry1Ac cloned in the background of binary vector pBI-121 were

used in the study.

The regeneration capacity of the three linseed cultivars, „LCK-9814, IA-32 and

Kiran‟ was analyzed for adventitious shoot organogenesis and it was observed that

number of shoots per explants were significantly affected by genotype, culture media and

application of growth regulators. The intensity of morphogenesis was found to be

affected not only by the use of exogenous growth regulators but also by the type of

expant and genotype. The hypocotyls from all tested genotypes were more responsive in

shoot induction compared to that of cotyledons and leaf discs. The highest average

number of shoots per explants (3.84) was recorded from hypocotyls derived calli of

cultivar LCK-9814 on culture medium MSB5 + 250 µg/l kinetin.

Further various factors viz. peeling of epidermis from hypocotyl explant, optical

density, dipping time and co-cultivation period affecting Agrobacterium mediated gene

transfer were also standardized. The Agro-culture density (0.6 OD), dipping time (4.0

mins) and explant tissues (hypocotyl) were found to be critical for the recovery of

transformants. Out of 52 hypocotyls infected only two regenerants produced on selection

medium (MSB5 + 250 µg/l kinetin + 50 mg/l Kanamycin) were found positive for mVIP

gene by PCR analysis. The high efficiency hypocotyl derived in-vitro plant regeneration

and Agrobacterium mediated transformation protocols developed in the present study

opens new avenues for the improvement of linseed for various agronomically important

traits using modern biotechnological tools.

Date: (Dr. G. Chandel) Place: Major Advisor

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Table 3.2: Composition of different callus induction medium used in study

Note: a- Murashige and Skoog media, b- Gamborg media; c-MSB5 media

Source Component Concentration (mg/L)

MS a

B5 b

MSB5c

MS I

NH4NO3

KNO3

CaCl2.2H2O

MgSO4 .7H2O

KH2PO4

(NH4)2 SO4

NaH2PO4. H2O

1650

1900

440

370

170

-

-

-

3000

150

500

-

134

150

1650

1900

440

370

170

-

-

MS II

KI

H3BO3

MnSO4.4H2O

ZnSO4. 7H2O

Na2MoO4.2H2O

CuSO4.5H2O

CoCl2.6H2O

MnSO4.H2O

0.83

6.20

22.30

8.60

0.25

0.025

0.025

-

0.75

3.00

-

2.00

0.25

0.025

0.025

10.00

0.75

3.00

-

2.00

0.25

0.025

0.025

10.00

MS III FeSO4.7H2O

Na2EDTA

37.30

27.80

37.30

27.80

37.30

27.80

MSIV Nicotnoic acid

Pyridoxine HCl

Thiamine HCl

Glycine

Inositol

0.5

0.5

0.1

2.00

100

1.00

1.00

10.00

-

100

1.00

1.00

10.00

-

100

Hormones Kinetin 250µg/l 250µg/l 250µg/l

Sucrose 30.0 20 20

Agar agar 7.0 7.0 7.0

pH -5.6-5.8 5.8 5.8 5.8

Table.4.1: Influence of genotype, explants type and hormone conc. on mean callogenesis (%) of all the specified linseed cultivar

Nutrient media LCK-9814 INDIRA ALSI-32 KIRAN

Hypocotyl Cotyledon Leaf Hypocotyl Cotyledon Leaf Hypocotyl Cotyledon Leaf

MSB5 +0 µg/l Kinetin 26 17 12 22 27 16 31 47 17

MSB5+250 µg/l Kin. 63 76 14 47 81 22 60 76 18

MSB5+500 µg/l Kin. 58 81 23 76 77 34 74 80 25

MSB5+750 µg/l Kin. 44 73 29 82 80 27 86 72 21

MSB5+1000 µg/l Kin. 27 59 17 90 86 22 92 71 32

Table.4.2: Influence of genotype, explants type and hormone conc. on mean shoot regeneration (%) of all the specified linseed

Nutrient media

LCK-9814 INDIRA ALSI-32 KIRAN

Hypocotyl Cotyledon Leaf Hypocotyl Cotyledon Leaf Hypocotyl Cotyledon Leaf

MSB5+ 0 µg/l Kin 23 0 0 19 0 0 4 0 1

MSB5+250 µg/l Kin 87 19 9 32 10 2 24 14 7

MSB5+500 µg/l Kin 72 12 4 49 7 6 47 22 9

MSB5+750 µg/l Kin 56 23 3 67 14 3 59 17 14

MSB5+1000 µg/l Kin 39 14 1 79 8 7 67 19 11

Table.4.6a: Effect of Agrobacterium inoculum density and dipping time (2 min.) on transformation efficacy

Clone OD


No.of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative (%)

transformant

No.of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative (%)

transformant

No.of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative(%)

transformant

mVIP (0.4) 47 0 0 53 0 0 43 0 0

mVIP (0.6) 32 0 0 10 0 0 22 0 0

mcry1Ac (0.4) 21 0 0 11 0 0 31 0 0

mcry1Ac (0.6) 57 0 0 44 0 0 45 0 0

Table.4.6b: Effect of Agrobacterium inoculum density and dipping time (4 min.) on transformation efficacy

Clone OD


No. of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative (%)

transformant

No.of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative (%)

transformant

No .of

explants

inoculated

No. of

survived

explants

after

selection

Percentage

putative(%)

transformant

mVIP (0.4) 41 0 0 43 0 0 39 0 0

mVIP (0.6) 34 0 0 18 0 0 52 2 3.84

mcry1Ac (0.4) 31 0 0 21 0 0 31 0 0

mcry1Ac (0.6) 37 0 0 34 0 0 35 0 0

Fig.2.1:Callus based shoot initiation from hypocotyl explant of linseed

a, b- Callus formation (cf) from cut ends of hypocotyl explant;

epidrmis based shooting (ebs).

c, d- Callus based shooting (cbs) from peeled hypocotyl.

Figure 3.1:Partial restriction map of mVIP gene and mcry1Ac gene

Partial restriction map of mcry1Ac gene

T-Border (L)

Poly A

Xho I

NPT II

Xho I Bst

CaMV35S

Promoter

T-Border (R)

EcoR I

Bgl II

Nco IHind III

cry1Ac

Sac1

1.8 kb 0.27 kb0.8 kb

Nos TerminatorCaMV35S

Partial restriction map of mVIP gene

(a)

(b)

T-Border (L)

Poly A

Xho

I

NPT II

Bst

CaMV35S

Promoter

EcoR I

Bgl II

Nco IHind III

mVIP

Sac1

1.2 kb 0.27 kb0.8 kb

Nos TerminatorCaMV35S

Xho IT-Border (R)

a b

c d

Fig.4.1: Callogenesis and subsequent shoot induction in MSB5 media.

a-leaf explant, b-cotyledon explant, c, d- peeled hypocotyl explant

b

d

a b

c

Fig.4.2:Influence of kinetin concentration on callusing and shoot induction in

LCK-9814 cultivar on MSB5 media.

Four treatment of hormone 250, 500, 750 & 1000 μg/l was used.250 μg/l kinetin

was found to be optimum for callusing & number of shoots per explant in LCK-

9814 cultivar of linseed.

b

Fig.4.3: 3-4 inches long shoots ready for rhizogenesisFig.4.4: In-vitro regenerated plants transferred

into pots in green house for acclimatization

a

c

b

d

Fig.4.5:Development of morphogenetic calli inoculated on MSB5 along with kinetin.

a- Peeled hypocotyls, increase the chances of transformation because transformation

occurs in callus based shootlets not in epidermis based shooting.

b- Precultured peeled hypocotyls.

c- Morphogenesis and calli developed after 10 days of culture.

d- Induction of shoots, arrow shoes the shoot initiation.

C d

50mg/l

40mg/l

Fig.4.6:Mortality of hypocotyl explants on different concentration of Kanamycin.

a- Hypocotyl explant culture on control medium.

b- Hypocotyl explants cultured on Kan. 50 medium showing 100% mortality.

c- 15 dyas old shootlets cultured on control medium.

d- 15 dyas old shootlets cultured on kan. 50 medium showing killing of cells.

dc

ba

a

b

Fig.4.7:Putative mVIP transformants in kiran cultivar of linseed.

a- Arrow shows the putative transformed shootlet along with dead

non-transformed shootlets and hypocotyls in kan. selection.

b- Arrow shows putative transformed calli in kan. selection along

with non-transformed dead shootlets in kan. selection.

M PC NC1 1 2 NC2

M PC NC1 NC2 1 NC3 2

Figure 4.8: PCR screening of putative mVIP transformants

M- Lambda Hind III DNA marker2 (125-23130 bp)

PC- Positive control (Plasmid DNA) of mVIP.

NC1 – Control linseed plant negative for mVIP.

NC2 – without template DNA i.e., Water

Lane 1–transformants positive for mVIP gene.

NC3– Control linseed plant negative for mVIP.

Lane 2– transformants positive for mVIP gene

900 bp

standardization of plant tissue culture and transformation protocols for

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