GENETIC TRANSFORMATION OF GROUNDNUT FOR
RESISTANCE TO TIKKA DISEASE
MAHMOOD UL HASSAN
95-arid-52
Department of Plant Breeding and Genetics Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2013
GENETIC TRANSFORMATION OF GROUNDNUT FOR
RESISTANCE TO TIKKA DISEASE
by
MAHMOOD UL HASSAN
(95-arid-52)
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Plant Breeding and Genetics
Department of Plant Breeding and Genetics Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2013
ii
CERTIFICATION
I hereby undertake that this research is an original one and no part of this thesis falls under plagiarism. If found otherwise, at any stage, I will be responsible for the consequences.
Student’s Name: Mahmood ul Hassan Signature: ______
Registration No: _____95-arid-52_____ Date: ______
Certified that the contents and form of thesis entitled “Genetic Transformation of Groundnut for Resistance to Tikka Disease” submitted by Mr. Mahmood ul
Hassan have been found satisfactory for the requirement of the degree.
Supervisor: ______(Dr. Zahid Akram)
Co-Supervisor: ______(Dr. Yusuf Zafar)
Member: ______(Dr. Ghulam Shabbir)
Member: ______(Dr. Tariq Mukhtar)
Chairperson: ______
Dean: ______
Director, Advanced Studies: ______
iii
"In the Name of Allah, the most Beneficent, the most Merciful"
iv
DEDICATED TO UNFATHOMABLE LOVE OF MY PARENTS
v
CONTENTS Page List of Tables xi
List of Figures xiii
List of Abbreviations xvi
Acknowledgements xviii
ABSTRACT 1
1 GENERAL INTRODUCTION 3
2 IN VITRO REGENERATION FROM COTYLEDONS 7
2.1 INTRODUCTION 7
2.2 REVIEW OF LITERATURE 9
2.3 MATERIALS AND METHODS 12
2.3.1 Explant Preparation 12
2.3.2 Data analysis 14
2.4 RESULTS AND DISCUSSION 14
2.4.1 Number of Responding Explants (%) 14
2.4.2 No. of shoots/responding explants (%) 15
2.4.3 Rooting percentage 15
3 STANDARDIZATION OF IN VITRO CULTURE SYSTEM FROM 26
LEAF DISCS
3.1 INTRODUCTION 26
3.2 REVIEW OF LITERATURE 27
3.3 MATERIALS AND METHODS 30
3.3.1 Explant preparation 30
vi
3.3.2 Medium and Culture Conditions 31
3.3.3 Data analysis 33
3.4 RESULTS AND DISCUSSION 33
3.4.1 Number of Responding Explants (%) 33
3.4.2 No. of shoots/responding explants 34
3.4.3 Rooting percentage 34
4 STANDARDIZATION OF IN VITRO REGENERATION VIA CALLUS 41
INDUCTION
4.1 INTRODUCTION 41
4.2 REVIEW OF LITERATURE 42
4.3 MATERIALS AND METHODS 45
4.3.1 Explant Preparation 45
4.3.2 Culture Medium and Conditions 46
4.3.3 Data Analysis 46
4.4 RESULTS AND DISCUSSION 47
4.4.1 Number of Responding Explants (%) 47
4.4.2 Number of Differentiated Embryos/ Responding Explant 48
4.4.3 Germination Percentage of Embryos 48
5 AGROBACTERIUM MEDIATED TRANSFORMATION 55
5.1 INTRODUCTION 55
5.2 REVIEW OF LITERATURE 56
5.3 MATERIALS AND METHODS 72
5.3.1 Explant Preparation 72
5.3.2 Bacterial Strain and Vector 73
vii
5.3.3 Determination of Lethal Dose of Hygromycin for In Vitro 73
Grown Shoots
5.3.4 Selection and Rooting of Putative Transformed Shoots 74
5.3.5 PCR Analysis of Putative Transgenic Plants 74
5.3.6 Gel Electrophoresis 75
5.3.7 Southern Blot 75
5.3.8 Determination of Lethal Dose of Hygromycin for Field Grown 77
T1 Plants
5.3.9 Selection of T1 Plants 77
5.3.10 Pathogenicity Test 77
5.3.11 RT-PCR Analysis 78
5.4 RESULTS AND DISCUSSION 78
5.4.1 Effect of Co-Cultivation Period and Acetosyringone 78
Concentration
5.4.1.1 Co-cultivation Period 78
5.4.1.2 Acetosyringone 79
5.4.2 Determination Of Lethal Dose Of Hygromycin For In Vitro 80
Grown Shoots
5.4.3 PCR analysis of Putative Transgenic Plants 80
5.4.4 Southern Blot 80
5.4.5 T1 Generation 80
5.4.6 Determination of Lethal Dose of Hygromycin for Field Grown 85
Plants
5.4.7 Selection of T1 Plants 85
viii
5.4.8 Pathogenicity Test for Intact Plants 85
5.4.9 RT-PCR Analysis 85
6 SILICON CARBIDE WHISKER MEDIATED TRANSFORMATION 91
6.1 INTRODUCTION 91
6.2 REVIEW OF LITERATURE 92
6.3 MATERIALS AND METHODS 96
6.3.1 Production of Embryogenic Callus 96
6.3.2 Preparation of Silicon Carbide Whiskers and DNA Delivery 96
6.3.3 Selection for Stable Transformants 96
6.3.4 PCR analysis 97
6.3.5 Selection of T1 Plants 97
6.3.6 Pathogenicity test for intact plants 97
6.3.7 RT-PCR analysis 98
6.4 RESULTS AND DISCUSSION 98
6.4.1 T0 generation 100
6.4.2 T1 generation 100
6.4.3 Pathogenicity test 100
6.4.4 RT-PCR analysis 104
7 TRANSFORMATION USING GENE GUN 106
7.1 INTRODUCTION 106
7.2 REVIEW OF LITEATURE 108
7.3 MATERIALS AND METHODS 111
7.3.1 Production of Embryogenic Callus 111
7.3.2 Preparation of Micro-carriers 112
ix
7.3.3 Coating DNA onto Micro Particles 112
7.3.4 Selection of Transformants 113
7.3.5 PCR analysis 113
7.3.6 Selection of T1 Plants 114
7.3.7 Pathogenicity test for intact plants 114
7.3.8 RT-PCR analysis 114
7.4 RESULTS AND DISCUSSION 115
7.4.1 T0 generation 115
7.4.2 T1 generation 117
7.4.3 Pathogenicity test 117
7.4.4 RT-PCR analysis 120
8 GENERAL DISCUSSION 124
SUMMARY 135
LITERATURE CITED 139
x
LIST OF TABLES
Table No. Page
2.1 Treatments (Hormone Combination Used) 16
2.2 Hormone concentrations for root induction 16
2.3 Analysis of variance for number responding explants (%) 16
2.4 Analysis of variance for No. of shoots/responding explants (%). 17
2.5 Analysis of variance for rooting percentage 19
2.6 Duncan's Multiple Range Test for ranking of treatments with 20
respect to rooting percentage
2.7 Duncan's Multiple Range Test for ranking of varieties with rooted 20
plants/explant (%
3.1 Treatments (Hormone Combination Used) 32
3.2 Analysis of variance for number responding explants (%) 35
3.3 Analysis of variance for No. of shoots/responding explants 37
3.4 Analysis of variance for rooting percentage 38
3.5 Duncan's Multiple Range Test for ranking of treatments with 38
respect to rooting percentage
3.6 Duncan's Multiple Range Test for ranking of varieties with rooting 38
percentage.
4.1 Analysis of variance for number responding explants (%) 49
4.2 Analysis of variance for No. of embryos/responding explants 50
4.3 Analysis of variance for germination percentage 52
4.4 Duncan's Multiple Range Test for ranking of treatments with 52
respect to germination percentage
xi
4.5 Duncan's Multiple Range Test for ranking of varieties with respect 52
to germination percentage
5.1 Quantities of different reagents in PCR mixture 76
5.2 Analysis of variance for factors affecting transformation efficiency 81
5.3 Mean values of transformation efficiency at different co- 81
cultivation periods ranked by Duncun’s Multiple Range Test
5.4 Mean values of transformation efficiency at different 82
acetosyringone concentrations ranked by Duncun’s Multiple Range
Test
5.5 Table of means for Infection frequency (IF, number of lesions/cm2 85
leaf area), incubation period (IP, number of days from inoculation
to appearance of first lesion), lesion diameter (LD), leaf area
damage (LAD) and disease score of transgenic and control plant
5.6 Modified 9-point scale used for screening groundnut genotypes for 90
resistance to late leaf spot disease
6.1 Analysis of variance for factors affecting transformation efficiency 99
7.1 Analysis of variance for factors affecting transformation efficiency 116
7.2 Mean values of transformation efficiency at different distances as 116
ranked by Duncan's Multiple Range Test
7.3 Mean values of transformation efficiency at different particle size 116
as ranked by Duncan's Multiple Range Test
xii
LIST OF FIGURES
Fig. No Page
2.1 Duncan's Multiple Range Test for ranking of interaction means with 17
respect to number of responding explants (%).
2.2 Duncan's Multiple Range Test for ranking of interaction means with 19
respect to No. of shoots/responding explants (%)
2.3 Bunch of spontaneous roots emerged from point of contact with 20
solid
surface due to mechanical stress
2.4 Grafting in groundnut 23
2.5 Different steps in peanut tissue culture 24
3.1 Duncan's Multiple Range Test for ranking of interaction means with 35
respect to number of responding explants (%)
3.2 Duncan's Multiple Range Test for ranking of interaction means with 37
respect to No. of shoots/responding explants
3.3 Different steps of shoot induction from leaf discs 40
4.1 Duncan's Multiple Range Test for ranking of interaction means with 49
respect to number of responding explants (%)
4.2 Duncan's Multiple Range Test for ranking of interaction means with 50
respect to No. of embryos/responding explants
4.3 Different steps in peanut somatic embryogensis 54
5.1 Diagram for vector containing RCG-3 under EN4 promoter 76
5.2 Effect of acetosyringone and length of co-cultivation period on 82
production of hygromycin resistant shoots (%)
xiii
5.3 Amplification of RCG 3 gene fragment by PCR in 4 surviving T0 83
plants.
5.4 Southern blot analysis of four surviving T0 plants 83
5.5 Cut twigs of plants on different concentrations of hygromycin 84
5.6 Amplification of RCG 3 gene fragment by PCR in T1 plants 84
5.7 Pathogenicity test of intact plants for susceptibility to late leaf spot 87
disease
5.8 Means for Infection frequency, incubation period, lesion diameter, 87
leaf area damage and disease score of transgenic and control plants
5.9 RT-PCR for RCG-3 gene in T1 plants. 88
6.1 Effect of callus age and whisker concentration on production of 99
hygromycin resistant shoots (%)
6.2 Emergence of hygromcine resistant shoots in selection medium 101
6.3 Amplification of RCG 3 gene fragment by PCR in 3 surviving T0 101
plants.
6.4 Southern blot analysis of three PCR positive plants 102
6.5 Amplification of RCG 3 gene fragment by PCR in T1 plants. 102
6.6 Pathogenicity test of intact plants for susceptibility to late leaf spot 103
disease
6.7 Means for Infection frequency, incubation period, lesion diameter, 103
leaf area damage and disease score of transgenic and control plants
6.8 Six PCR positive plants from progeny of T0-3 plant were subjected 103
to RT-PCR analysis
7.1 Emergence of hygromcine resistant shoots in selection medium 118
xiv
7.2 Amplification of RCG 3 gene fragment by PCR in 3 surviving T0 118
plants.
7.3 Southern blot analysis of three PCR positive plants 119
7.4 Amplification of RCG 3 gene fragment by PCR in T1 plants. 119
7.5 Pathogenicity test of intact plants for susceptibility to late leaf spot 122
disease
7.6 Means for Infection frequency, incubation period, lesion diameter, 123
leaf area damage and disease score of transgenic and control plants
7.7 Seven PCR positive plants were subjected to RT-PCR analysis 123
xv
LIST OF ABBREVIATIONS
Abbreviation Stands for
ANOVA Analysis Of Variance
ATP Adenosine Triphosphate
ATPase Adenosine Triphosphatase
BAP 6-Benzyl Amino Purine bp Basepair
CaCl2 Calcium Chloride
CaMV Cauliflower Mosaic Virus cDNA Complementary Dna
CTAB Cetyl Trimethylammonium Bromide
DNA Deoxy Ribonucleic Acid
EC Embryogenic Callus
EDTA Ethylenediaminetetraacetic Acid
GFP Green Fluorescent Protein
GUS Glucuronidase
IAA Indole Acetic Acid kb Kilobase kDa Kilodalton
Kn Kanamycin
LB Luria-Bertani mRNA Messenger Rna
MS Murashige And Skoog
NAA Naphthalene Acetic Acid
xvi
NaCl Sodium Chloride
NaOCl Sodium Hypochlorite
NaOH Sodium Hydroxide
OD Optical Density
PAGE Polyacrylamide Gel Electrophoresis
PCR Polymerase Chain Reaction
PEG Polyethylene Glycol
PEG Polyethylene Glycol
PGRs Plant Growth Regulators
PR Pathogenesis Related
QTL Quantitative Trait Locus
RAPD Random Amplification Of Polymorphic Dna
RFLP Restriction Fragment Length Polymorphism
RNA Ribonucleic Acid rpm Revolutions Per Minute
RT-PCR Reverse Transcriptase Pcr
SDS Sodium Dodecyl Sulphate
TAE Tris-Acetate Edta
TBE Tris-Borate Edta
TDZ Thidiazuron
UV Ultraviolet v/v Volume Per Volume w/v Weight Per Volume
xvii
ACKNOWLEDGEMENTS
Praise is due to Allah whose worth cannot be described by speakers, whose bounties cannot be counted by calculators and whose claim (to obedience) cannot be satisfied by those who attempt to do so, whom the height of intellectual courage cannot appreciate, and the divings of understanding cannot reach; He for whose description no limit has been laid down, no eulogy exists, no time is ordained and no duration is fixed. He brought forth creation through His Omnipotence, dispersed winds through His Compassion, and made firm the shaking earth with rocks.
Salutations upon the Holy Prophet Muhammad (Sallallah-o-Alaih-
Wassalam), Sun of the sky of secrets, the made-visible of the Luminescence, the
Majestic Centre around which all revolves, the Beautiful Axis of the firmament, the city of knowledge, who has guided his followers to seek knowledge from cradle to grave.
The words are inadequate to express my deepest sense of appreciation and devotion to my worthy Supervisor Dr. Zahid Akram, Associate Professor,
Department of Plant Breeding and Genetics, Faculty of Crop and Food Sciences, Pir
Mehr Ali Shah Arid Agriculture University, Rawalpindi. I am extremely grateful to his scholastic and sympathetic attitude, inspiring guidance and enlightened supervision in the accomplishment of resereach work and this manuscript.
xviii
I wish to extend my thanks to Dr.Yusuf Zafar, ex-Project Director, National
Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad for his obligation, well wishes, generous assistance, timely advice, useful critiques and encouragement during the course of my research work and presentation of this manuscript.
Completing this work would have been all the more difficult were the support and help not provided by Dr. Ghulam Shabbir, Assistant Porfessor,
Department of Plant Breeding and Genetics and Dr. Tariq Mukhtar, Associate
Professor, Department of Plant Pathology, Pir Mehr Ali Shah Arid Agriculture
University, Rawalpindi.
I feel pleasure to record my deep sense of gratitude to Prof. Dr M. Kausar
Nawaz Shah, Chairman Department of Plant Breeding and Genetics department
PMAS-Arid Agriculture University Rawalpindi, Pakistan for his constant encouragement throughout this endeavor.
I offer deepest thanks to Mrs. Farhat Nazir, Scientific officer NIGAB,
NARC, Islamabad for enthusiastic guidance and support in designing and execution of this research.
xix
I am especially thankful to Dr. Ghulam Muhammad Ali, Project Director, and Dr. Shaukat Ali, Senior Scientific Officer, NIGAB, NARC, Islamabad for provision of gene construct (RCG3) and laboratry facilities for part of this research.
I offer my cordial gratitude and genuine credit to my friends Dr. Saad Imran
Malik, Matloob Javed, Dr. Umar Rashid, Dr. Javed Iqbal, Dr. Mubashar Hussain, Dr.
Muhammad Ahmad, Dr. Abdul Qayyum, Shahid Riaz Malik and Sarfraz Ahmad for their well wishes and prayers. I am obliged by the cooperation of the staff of the department, especially Hafiz Wajid Imran and Amir Nadeem. I am also indebted to my students Shagufta Naseem, Muhammad Tayyab and Muhammad Yasir for their help in my research work.
I cannot overlook to articulate my admiration and unfathomable sagacity of appreciation from the core of my heart to my loving brothers, sisters, my beloved wife and children, father and mother in law who experienced all the ups and downs of my endeavor with patience and always remembered me in their prayers. At this stage,
I cannot forget my caring and loving brother, Hafeez ur Rehman (Late), who always wished and dreamt for my success in higher education but could not see his dream coming true. May Allah bless his soul with eternal peace.
Financial assistance by Higher Eductioan Commission, Islamabad, under indigenous scholarship programme is highly appreciated.
(Mahmood ul Hassan)
xx
ABSTRACT
Groundnut is grown worldwide as an important cash crop and its seeds are a major source of premium quality vegetable oil and protein. Yield loss up to 70 per cent has been reported due to reduced photosynthesis resulting from increase in necrotic leaf area and defoliation after severe attack of leaf spot disease. The best way to control this foliar disease is to grow resistant cultivars, but higher levels of resistance are not available in groundnut cultivars. Genetic transformation is the best alternative in such circumstances and availability of efficient regeneration and gene delivery systems is a prerequisite for this technique. Therefore during present study, regeneration protocols were standardized by culturing three explants of each of four commercial varieties on
MS medium supplemented with different hormone combinations, subsequently followed by transformation of best responding variety with chitinase gene.
78.33 per cent cotyledonary explants responded to shoot induction with 5.14 shoots/explant in Golden variety on MS medium containing 0.1 mg/l NAA and 4 mg/l
BAP. Roots were induced successfully (61.30 per cent) on MS medium fortified with
1.0 mg/l NAA. Direct shoots were induced from 52.67 per cent leaf disc explants.
Somatic embryos were obtained (98.33 per cent) from epicotyl explants on MS medium having 8mg/l picloram. 14.18per cent transformation efficiency through
Agrobacterium, 6.88per cent by silicon carbide whisker and 6.59per cent by gene gun method has been achieved. This is first known report of Silicon carbide whisker mediated transformation in peanut in world. The transgene (RCG3) over expressed well and showed significant resistance to the Tikka disease.
1
2
In present study it has been proven that rice chitinase gene (RCG-3) confers strong resistance against leaf spot disease of peanut. The stable peanut lines produced will be helpful to evolve cultivars with built-in solution to control fungal diseases, specially the leaf spot. The standardized protocols of regeneration and transformation will be used for incorporation of other desirable genes in this crop. Chapter 1
GENERAL INTRODUCTION
The peanut (Arachis hypogaea), is the one of world’s most important legume and oilseed crop, native to South America and grown all over the tropical and temperate regions of the world. It is grown on 20.84 million hectares area worldwide with the production of 36.35 million metric tons and yields 1.74 metric tons per hectare. Presently peanut is being grown in 108 countries in the world however its production is more concentrated in Asia and Africa, where the crop is mostly grown by farmers with small holdings under rainfed conditions with limited inputs (ICRISAT,
2007). China and India are the largest producers while Israel ranks at top in yield, that is, 6325 kg/ha followed by USA and Egypt having yield of 3508 and 3234 kg/ha respectively (FAO, 2011) .
Groundnut seed has high oil (43-55per cent), protein (25-30 per cent) and carbohydrate (21per cent) contents. It is a rich source of fiber, niacin, vitamin E, folate, magnesium, phosphorus (Ahmad et al., 2007). Like other vegetable oils, it does not contain toxic compounds and linolenic acid, which causes oxidative rancidity. The oil is also used to make margarines and mayonnaise (Sanders et al., 2003). Moreover this oil is very much stable and is preferred for deep-frying in industries, since it has a smoke point of 229.4°C compared to the 193.5°C of extra virgin olive oil (Deane,
2004).The seed meal is used in confectionary industry and as a protein concentrate in livestock feed (Khalil and Jan, 2005).
3
4
In Pakistan first planting on a commercial scale began in year 1949 with cultivation on 400 hectares area in Rawalpindi division. Its production in Pakistan is
67800 tons on an area of 82900 hectares on an average yield of 817.9 kilograms per hectare Out of total, 84per cent of peanut is produced in potohar tract of Punjab, 13per cent in khyber pakhtun khwa and 3 per cent in Sindh (Anonymous, 2011). Therefore, it is the most important crop of rainfed area of Pakistan and is livelihood of resource-poor farmers. Oil extraction is not a usual practice here; rather it is consumed as roasted kernels and in confectionary items (Asif et al., 2004).
Main reasons of low yield are high infestation of weeds and leaf spot disease commonly known as “Tikka” disease. Early and late leaf spot diseases are caused by
Cercospora arachidicola and Cercosporadium personatum respectively. Symptoms of early leaf spot include roughly circular lesions, dark brown on the upper leaflet surface, slightly lighter on the lower surface and surrounded by a yellow halo. Late leaf spot has similar symptoms however, lesions are darker brown and without a definite chlorotic yellow halo. On the lower side of the leaflets, lesions are almost black, in contrast to the lighter coloured lesions of ELS. In case of severe intensity lesions may combine together causing defoliation. Lesions can also develop on stems, petioles and pegs
(Woodroof, 1933; Jenkins, 1938; Van Wyk and Cilliers, 2000).
Severity of disease varies with localities and seasons and yield reductions of 20 to 70per cent have been reported (Venkataraman and Kazi, 1979; Subrahmanyam et al.,
1992; Godoy et al., 2001). Yield and quality can be affected by leaf spot and in particular by the reduced photosynthesis resulting from increase in necrotic leaf area
5
and defoliation after severe infection. Weakened pegs by leaf spot and reduced ability of diseased plants to maintain healthy pegs contributes to loss of yield (Subero, 1992).
In Pakistan, 94per cent disease incidence and 50per cent yield loss has been reported (Ijaz, 2011). Like most developing countries, farmers in Pakistan can hardly afford fungicide applications to protect the crop from fungal diseases mainly due to economic reasons. The best way to control these foliar diseases is to grow resistant cultivars, but higher levels of resistance are not available in groundnut cultivars
(Subrahmanyam et al., 1995).
Groundnut is genetically less diverse crop with respect to fungal disease resistance, particularly, leaf spot, limiting the scope of marker assisted selection and
QTL mapping (Ozudogru et al., 2013). However, genes for resistance against certain diseases and pests are present in some wild relatives of groundnut but inter-specific crosses have limited success due to crossing barriers (Stalker and Moss, 1987; Prasad and Gowda, 2006; Tiwari and Tuli, 2008). Few traits have been tagged in peanut using molecular markers; most of them have been identified in interspecific introgression populations. Four low density genetic maps have been published by utilizing variation among Arachis species, but no maps have been reported to date from cultivated x cultivated species crosses (Selvaraj et al., 2009). Markers for resistance to root-knot nematode (Garcia et al. 1995; Burow et al. 1996; Choi et al. 1999), the aphids
(Herselman et al. 2004) and Aspergillus (Yong et al. 2005) have been identified.
Pandey et al. (2012) also maintained that peanut still lacks availability of linked markers for important traits. First study on QTL mapping for leaf spot resistance in
6
cultivated peanut has been reported just recently by Wang et al (2013) with very low phenotypic variance.
Genetic transformation, coupled with conventional breeding procedures, will provide a gateway to develop disease resistant groundnut varieties. Standardization of a robust and genotype-independent tissue culture system is a pre-requisite for successful transformation system in any crop, and groundnut is no exemption. To produce a large number of transgenic plants, it is necessary to indentify a large number of cells that are capable of receiving foreign genes and can develop into fertile plants.
Groundnut shows low response to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants and insufficient availability of explants (Livingstone and Birch,
1999).
The present study therefore was conducted with the following objectives:
1. To standardize an efficient in vitro culture system in local groundnut
varieties.
2. To standardize Agrobacterium, gene gun and silicon carbide whisker
mediated transformation methods using chitinase gene.
3. To assess expression of transgene at molecular and field/green house levels.
Chapter 2
IN VITRO REGENERATION FROM COTYLEDONS
2.1 INTRODUCTION
Groundnut (Arachis hypogaea L.) is an important amphidiploid species belonging to legumionous family “Fabaceae”. It has been originated in southern
Bolivia or northern Argentina (Gregory et al.,, 1980) is cultivated for food and oil around the world. Production is concentrated in Asia and Africa, where the crop is mostly grown by farmers with small holdings under rainfed conditions with limited inputs (ICRISAT, 2007). The groundnut seed contains 40-50 per cent oil, 20-30 per cent proteins and is an excellent source of B vitamins. The oil is of premium quality and due to high protein contents, meal can be used as animal feed (Pardee, 2002).
Groundnut is the most important crop of rainfed area of Pakistan and is livelihood of resource-poor farmers. It is grown on an area of 81000 hectares with an average yield of 996 kg/ha (Govt. of Pak., 2008). In Pakistan, groundnut is consumed as roasted kernels and in confectionary items (Asif et al.,, 2004).
Poor yields of peanut in developing countries is mainly due to the biotic and abiotic stresses such as unreliable rainfall, drought, unavailability of high yielding varieties, damage by pest and diseases, poor cultural practices, and limited use of inputs
(Nageshwara and Nigam, 2001). Fungal and viral pathogens are the most common diseases of groundnut worldwide (Yang et al.,, 1998). Early and late leaf spot diseases
7
8
caused by Cercospora arachidicola and Cercosporadium personatum respectively can devastate a peanut crop by reducing yield upto 70 per cent (Subrahmanyam et al.,, 1984 and Subrahmanyam et al.,, 1992). In most developing countries, groundnut is grown mainly by resource-poor farmers who can hardly afford chemical protection. The existence of genetic variation for disease resistance has been noted among commercial groundnut cultivars (Branch, 1996), yet the development of resistant cultivars is considered as the most practical and cost effective means of controlling diseases.
Many traits of groundnut including yield and disease resistance have been improved by conventional breeding. However, due to its limited applicability, many of the important traits including disease, insect and herbicide resistance have yet to be improved. To address the specific problems in groundnut, it is imperative to optimize the transformation method which in turn requires a rapid, efficient, robust and reproducible regeneration protocol. Large number of transgenic plants can be produced only if there are large number of cells, capable to uptake foreign DNA and subsequent regeneration to full-fledged plants.
In most of tissue culture experiments of groundnut, regeneration is accomplished through somatic embryogenesis from different explants including leaf discs (Chengalrayan et al., 1994), cotyledons (Kim et al., 2004), embryos (Hazra et al.,1989), mature and dry seeds (Baker et al., 1995) cotyledons with embryos, epicotyls, leaflets (Cucco and Jaume, 2000) and mature epicotyl (Little et al., 2000).
However, regeneration by organogenesis has also been reported using leaves, cotyledons, cotyledonary node, hypocotyl, epicotyl, and zygotic embryos with very low
9
shoot frequencies (McKently et al., 1990, Eapen and George, 1993), Present study was designed to simplify the regeneration system in local cultivars by reducing the steps of tissue culture producing more shoots from readily available seed explants round the year. This method proved to be cost and time effective as there was less contamination and direct multiple shoot induction was accomplished in short span of time.
2.2 REVIEW OF LITERATURE
Evidence of first cultivation of groundnut are traced near rivers of the Parana and Paraguay. At the time of European invasion, groundnut was taken to rest of the world including Asia and African (Acquahh, 2007). The plant has compound leaves with two pairs of opposite leaflets each having length of 1-7 cm and width of 1-3 cm.
Like other legumes the peanut flowers have one standard, two keel and two wing petals. Diameter of fully opened flower is 2 to 4 cm with yellow colour. Cultivated groundnut is tetraploid and is normally self-pollinated. After pollination, the fruit pedicle elongates in the form of a peg, penetrating the fruit into the soil where it develops to maturity. Pod is constricted and is 3-7 cm long having 1 to 5 seeds.
A. hypogaea has two subspecies viz hypogea and fastigiata, which are distinguished by branching pattern and distribution of vegetative and reproductive axes.
Subspecies fastigiata has four viz. fastigiata, peruviana vulgaris, and aequatoriana whereas subspecies hypogaea has two varieties viz hypogaea and hirsuta. The botanical name has been derived from the Greek word arachis meaning ‘legume’ and hypogaea meaning ‘below ground’, indicating the formation of pods inside the soil
(Pattee and Stalker, 1995. Arachis hypogaea L. (2n = 40) is an allotetraploid species
10
with monophyletic origin having comparatively little genetic diversity (Pattee and
Young, 1982). Polyploidy usually creates severe genetic bottlenecks, contributing to the genetic vulnerability of major crops (Company et al.,, 1982). Groundnut is best grown in well drained sandy loam soils in the areas with 500 to 1000 mm rainfall.
Regeneration in peanut has been reported from mature and immature embryo axes (Baker et al., 1995; Brar et al., 1994) cotyledons (Ozias-Akins, 1989; Atreya et al., 1984) and leaves (Livingstone and Birch, 1995; Baker and Wetzstein, 1992).
Regeneration from protoplasts has also been reported in Arachis paraguariensis but in
A. hypogea this technique was unsuccessful (Li et al., 1993). In general, most of the reported regeneration protocols are not reproducible, less efficient and genotype dependent. Moreover, most of the reported methods are commercially less important cultivars.
There is a considerable advancement in understanding of disease resistance mechanism in peanut; hence, the techniques of genetic transformation developed in other crops can be applied in this crop once regeneration protocol is established.
Introduction of specific genes usually requires tissue-specific expression instead of expression in all body parts. The isolation of promoters is important for understanding the regulation of plant gene expression. Evaluation of promoter activity, that is, when, where and how much a gene is expressed, provides information about behaviour of transgenic plant when regulated by such a promoter sequence. Smith and Beliaev
(1995) isolated peanut pod - and seed coat -specific genes in order to isolate their promoters. These promoters can then be used to express transgenes specifically in the
11
seed coat and pod. The application of scientific advances to the improvement of peanut will broaden its genetic base and will assist future peanut breeding programs and the peanut industry.
Robinson et al., (2011) described a protocol for in vitro regeneration of groundnut through cotyledon explants. Only 10 per cent of the explants were contaminated by using non-succulent dry mature seeds to start the in vitro culture.
Atreya et al., (1984) regenerated plants from cotyledon segments of peanut on
MS medium supplemented with NAA and/or BA. On MS medium, 2 mg of BA/l poduced maximum shooting while 1 mg/l of NAA induced the maximum frequency of rooting compared to any other level of NAA tested. Combination of NAA and BA induced regeneration of whole plants directly. Shoot induction was more concentrated from the portion of cotyledon near the embryo axes.
Pestana et al., (1999) studied the effect of different temperatures on in vitro regeneration response of groundnut on MS medium supplemented with BA using cotyledonary explants. Leaf explants were cultivated in the presence of the same growth regulator at different temperatures viz. 25, 28, and 35 ° C. Best shoot induction was obtained at 35° C and 95 per cent shoots developed roots at this temperature.
Burns et al., (2012) developed a direct regeneration protocol for six American cultivars of peanut using two types of cotyledonary explants. They cultured cotyledons
12
with and without a small portion of embryo axis on MS medium supplemented with different concentrations (10-640 μM) of BAP for four weeks. On a 1-4 shoot induction scale, explant with a small portion of embryo axis gave 1.8 shooting response while that without axis gave 1.6. Shoot bud induction ranged from 7.1 to 24.6 per cent among different genotypes while visual rating ranged between from 1.7 to 2.1. The researchers recommended this method of regeneration for Agrobacterium transformation of peanut as transgenic plants could be obtained in just four months.
Vernma et al., (2009) developed a direct regeneration system using cotyledonary node explant of groundnut on MS medium supplemented with 1 to 50 mg/l of BAP. Maximum shooting was observed on 15mg/l of BAP while higher concentrations had inhibitory effects. Flowering was also observed in some varieties when NAA was added to induction medium at the concentration of 1 mg/l. Shoots were shifted to root induction medium consisting of MS medium supplemented with 1 mg/l
NAA. The rooted plants were successfully transferred to pots containing 3 parts of soil,
1 part of sand and 1 part of farmyard manure.
2.3 MATERIALS AND METHODS
2.3.1 Explant Preparation and Culture
Seeds of four groundnut cultivars viz. BARD-92, BARI-2000, BARD-479
Golden and were provided by Barani Agriculture Research Institute, Chakwal,
Pakistan. Freshly de-shelled seed were sterilized by treating with 70 per cent (v/v) ethanol for one minute and then with 20 per cent clorox (commercial bleach) for ten minutes. After rinsing with sterile double distilled water, seeds were soaked in same
13
water for three hours at room temperature. Each cotyledon was halved longitudinally after removing the seed testa and embryo. In this way four explants were obtained from one seed.
The explants were cultured on MS basal salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al.,, 1968), 30g/L sucrose and supplemented with 12 different hormone combinations (Table 2.1). 8g/L agar was added after adjusting the pH at 5.8 at
25 ◦C. The de-embryonated explants were placed with their cut edges in contact with medium for 3 weeks at 25± 2 ◦C with a 16/8 hours photoperiod. The experiment was conducted in completely randomized design and was repeated in three batches, each batch consisting of 10 explants for each treatment. The data on number of shoots/explants, gain of chlorophyll by explants, callus percentage and increase in explant size were recorded after two weeks of culture.
The regenerated shoots were excised and incubated on the same medium on which shoots were induced. On attaining a height of 5.0 cm, these shoots were shifted to rooting medium containing different concentrations of NAA (Table 2.2).
The rooting medium consisted of MS salts, B5 vitamins, 30g/L sucrose and was supplemented with 2mg/L NAA. The rooted plants were transplanted in pots containing peat moss kept in greenhouse. For the first week the pots were covered with polythene sheet to maximize the humidity. The above data for number of shoots
14
induced per explant and rooting efficiency were recorded for the four genotypes used in the experiment.
2.3.2 Data Analysis
The experimental data regarding number of explants showing shoot induction
(responding explants), number of shoots/responding explant and rooting percentage
(number of shoots showing root development) were subjected to analysis of variance
(ANOVA). Significance of means was computed by Duncan’s Multiple Range Test (at
P=0.05) using M-STATC software.
2.4 RESULTS AND DISCUSSION
To see whether various treatments and varieties make some significant difference or not, analysis of variance (ANOVA) is performed. If treatments and varieties make significant differences, Duncan’s Multiple Range Test is used for ranking of treatments and varieties.
2.4.1 Number of Responding Explants (%)
Number of explants showing shoot development has been referred as responding explants. Shoot induction, the basic objective of the experiment, was observed after three weeks of the inoculation. Analysis of variance (Table 2.3) revealed that treatments, varieties and their interactions have highly significant impact on number of explants responding for shoot induction. Duncan’s Multiple Range Test for interactions means showed that highest mean number of responding explant (78.33 per
15
cent) was obtained at T3 in Golden variety while lowest (11.67 per cent) was achieved in BARD-92 at T9 (Figure 2.1). Javed (2009) conducted experiment on Golden and
BARI-2000 varieties using different combinations of BAP and NAA and obtained comparative less number of shoots. Hormone type and combinations, especially inclusion of BAP in current study had better effects on shoot induction.
2.4.2 Number of Shoots/Responding Explants (%)
Analysis of variance (Table 2.4) revealed that treatments, varieties and their interactions had highly significant impact on number of shoots/responding explants ( per cent). Duncan’s Multiple Range Test for interaction means showed that highest mean number of shoots/responding explants (5.14 per cent) were obtained in Golden variety at Treatment No. 3 while lowest (1.10 per cent) at treatment No. 9 in BARD-
479 variety(Figure 2.2). It was also observed that if the shoot producing explant is left in petri plate for a longer period, spontaneous rooting started (Figure 2.3).
On the other hand no root induction was observed if same explant is left in jars.
The most probable reason for this phenomenon is the mechanical or physical stress in jar due to limited space available.
2.4.3 Rooting Percentage
The prime objective of the study was to get maximum number of rooted plants/explant as this will be an ultimate output in any tissue culture based transformation process. Analysis of variance showed that treatments and varieties
16
Table 2.1: Hormone combinations used for shoot induction
Treatment (T) No. Hormonal combinations NAA mg L-1 BAP mg L-1
1 0.1 0
2 0.1 2 3 0.1 4 4 0.1 6 5 0.5 0 6 0.5 2
7 0.5 4 8 0.5 6 9 1 0 10 1 2 11 1 4
12 1 6
Table 2.2: Hormone combinations used for root induction
Treatment (T) No. Concentration of NAA (mg L-1) 1 0.5 2 1.0 3 1.5
Table 2.3: Analysis of variance for number of responding explants (%)
Source of Variance DF Sum of Squares Mean Square F Value Treatments 11 3361.458 1120.486 126.54** Varieties 3 19786.458 1798.769 203.155** Treatment x variety 33 1893.583 55.745 6.2959** interaction Error 96 850.000 8.854 Total 143 25837.500 ** Highly significant
17
BARI‐2000 Golden BARD‐479 BARD‐92 A ‐ 78.33 B ‐ (%)
C ‐ C ‐ 60.00 D 55.00 D ‐ ‐ explant DE DE DE
51.67 ‐ ‐ ‐ EF ‐ EFG EFG EFG ‐ ‐ ‐ 45.00 FG 44.17 FGH ‐ GH ‐ GHI GHI GHI GHI
41.67 41.67 41.67 ‐ ‐ ‐ ‐ HIJ HIJ 38.33 ‐ ‐ IJK IJK IJK IJK IJK 36.67 36.67 36.67 ‐ ‐ ‐ ‐ ‐
34.17 HIJ JKL JKL 33.33 KL ‐ ‐ ‐ 32.50 KL KL KL KL Responding ‐ 31.67 31.67 31.67 31.67
‐ ‐ ‐ ‐ LM LM ‐ ‐ LMN LMN LMN 28.33 28.33 LMNO LMNO LMNO ‐ ‐ ‐ of ‐ ‐ ‐
26.67 26.67 26.67 26.67 26.67 MNOP MNOP ‐ NOP ‐ ‐ 23.33 23.33 23.33 OP 22.50 P ‐ 21.67 21.67 21.67 21.67 ‐ 20.00 20.00 18.33 18.33 18.33 17.50 17.50 17.50 15.83 15.00 14.17 Number 12.50 11.67
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Treatments
Figure 2.1: Duncan's Multiple Range Test for ranking of interaction means with respect to number of responding explants (%) (Values sharing same letters do not differ significantly at 5 per cent level of probability)
Table 2.4: Analysis of variance for No. of shoots/responding explants (%).
Source of Variance DF Sum of Squares Mean Square F Value Treatments 11 20.222 6.741 275.20** Varieties 3 60.556 5.505 224.7595** Treatment x variety 33 9.192 0.279 11.37** interaction Error 96 2.351 0.024 Total 143 92.321 ** Highly significant
18
had highly significant impact on this parameter (Table 2.5). 61.30 per cent shoots developed roots at 1.0 mg/liter NAA while only 12.21 per cent shoots developed roots at 0.5 mg/liter NAA.
However, at 1.5 mg/liter NAA, moderate (54.36 per cent) root development was achieved (Table 2.6). As far as varieties are concerned, Golden responded best to root induction (45.176 per cent) while lowest root induction was observed in BARD-92
(Table 2.7). The shoots which did not produce roots were used for grafting on healthy root stock with 58 per cent success. A wedge shaped 5cm long scion was inserted in a
T-shaped cut made in 20 days old seedling grown in 25 x 25 cm earthen pot (Figure
2.4). The joint was wrapped with parafilm and pot was covered with polythene bag which was removed gradually in 7 days. The plants reached to maturity but produced very small number of seeds as they mainly remained in the form of a single shoot.
Tiwari and Tuli (2012) also reported grafting in peanut. The whole procedure of experiment has been shown pictographically in Figure 2.5.
Plant growth regulators have been regarded as important parameter in determining the success of regeneration system. A deviation from an optimum level of hormone will have a significant impact. The combination of cytokinin and auxin, particularly the concentration of cytokinin, has been described as more critical to affect the regeneration. As for as treatments (hormone combinations) are concerned in our experimentation, T3 (BAP 4 mg/L and NAA 0.1mg/L) was the best treatment for shoot induction as it produced78.33 per cent shoots (Figure 2.1).
19
BARI‐2000 Golden BARD‐479 BARD‐92 A ‐ 5.14 (%)
B B ‐ ‐ explant
3.72 C 3.69 C ‐ ‐ 3.17 D 3.12 ‐ DE DEF ‐ ‐ DEFG ‐ EFG EFG EFG EFG EFGH ‐ EFGH ‐ ‐ ‐ FGH GH ‐ ‐ ‐ 2.64 ‐ 2.50 HI 2.48 HI ‐ ‐ 2.40 2.31 2.31 IJ 2.29 IJK 2.28 IJKL 2.23 IJKL 2.23 ‐ ‐ ‐ 2.19 2.17 JKLM ‐ JKLMN JKLMNO ‐ shoots/responding JKLMNO JKLMNO JKLMNO ‐ ‐ JKLMNOP
‐ KLMNOP KLMNOPQ ‐ ‐ LMNOP 1.96 LMNOP 1.95 ‐ LMNOP 69 LMNOPQR ‐ ‐
‐ ‐ MNOPQR MNOPQR MNOPQR MNOPQR ‐ ‐ NOPQR OPQR of ‐ ‐ ‐ ‐ 1.78
PQR PQR ‐ 1.75 ‐ QR 1.70 QR 1. ‐ ‐ ‐ 1.64 R ‐ R 1.61 1.59 ‐ 1.55 ‐ 1.54 1.54 1.50 1.47 1.46 1.43 1.43 1.42 1.40 1.34 1.34 1.34 1.34 1.30 1.29 1.23 1.23 1.19 1.18 1.11 1.10 Number
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Treatments
Figure 2.2: Duncan's Multiple Range Test for ranking of interaction means with respect to No. of shoots/responding explants (%). Values sharing same letters do not differ significantly at 5 per cent level of probability
Table 2.5: Analysis of variance for rooting percentage
Source of Variance Degree of Sum of Mean Square F Value Freedom Squares Treatments 2 16935.789 8467.895 632.282** Varieties 3 383.992 127.997 9.5573** Treatment x variety 6 135.619 22.603 1.6877 interaction Error 24 321.422 13.393 Total 35 ** Highly significant
20
Table 2.6: Duncan's Multiple Range Test for ranking of treatments with respect to rooting percentage
Treatment No. Mean Ranking 2 61.30 A 3 54.36 B 1 12.21 C Values sharing same letters do not differ significantly at 5 per cent level of probability
Table 2.7: Duncan's Multiple Range Test for ranking of varieties with rooted plants/explant (%)
Variety Mean Ranking Golden 45.176 A BARD-479 44.463 A BARI-2000 43.830 A BARD-92 37.028 B Values sharing same letters do not differ significantly at 5 per cent level of probability
Figure 2.3: Bunch of spontaneous roots emerged from point of contact with solid surface due to mechanical stress
21
The use of de-embryonated cotyledons as explant was in agreement with Swathi et al., (2006) who suggested that if cotyledon is cut vertically and its adaxial side is in direct contact with medium, its regeneration efficiency is improved.
It is evident from the results that in general by increasing BAP concentration in medium, number of shoots/explants increased upto 4 mg/L and then it dropped. While on other hand the callus formation was reduced by increasing BAP concentration.
Explant enlargement and chlorophyll accumulation remained almost unaffected with change in hormone combination. Golden produced highest shoots and rooted plants followed by BARD-479. However in chlorophyll gain and explant enlargement both the bold seeded varities viz GOLDEN and BARI-2000 performed better than the small seeded varieties viz BARD-479 and BARD-92.
McKently et al., (1995) emphasized the use of zygotic embryo axes as explants but cotyledons have several comparative benefits like robustness, time saving and cost effectiveness. Venkatachalam et al (2000) also used cotyledons as explants to produce fertile plants via embryogenesis which involves much more tissue culture (too many media, culture conditions and time) as compared to direct regeneration from cotyledons as done in present study. This explant is easy to use as sterilization is simple and does not need too much care. Moreover it also saves time and sources needed for in vitro regeneration which is a pre-requisite for protocols involving epicotyls or leaves as explants. Golden is a commercial cultivar released for Potowar region of Pakistan. The successful establishment of regeneration of this variety will have a number of benefits in future.
22
Use of de-embryonated cotyledon as explant makes this protocol more attractive for Agrobacterium tumefacien transformation. Traits like drought tolerance, disease and herbicide resistance would be transferred through genetic engineering into
Golden variety using our standardized protocol. By incorporating these traits, the researcher would be able to enhance the productivity of the crop.
23
A
Red seed testa of Golden variety
Off-white seed testa of B Chakori variety
Figure 2.4: Grafting in groundnut
A: Scion of Golden variety has been grafted on root stock of Chakori variety
B: Grafted plant after harvesting: the seed testa in Golden (scion) is red while that in
Chakori (root stock) is off-white
24
A B
C D
E F
25
G H
I
Figure 2.5: Pictographic summary of steps involved in peanut tissue culture
A: Explant sterilization
B, C: Explant preparation
D: Inoculation of explant on medium
E: Increase in size, callusing and gaining of chlorophyll
F: Multiple shoot formation from one of explants
G: Root induction
H: Mature peanut plant in pot in green house
I: Plant after harvesting
Chapter 3
STANDARDIZATION OF IN VITRO CULTURE SYSTEM
FROM LEAF DISCS
3.1 INTRODUCTION
Peanut is a crop of immense importance as it is the source of dietary proteins, high quality vegetable oil and peanut butter. Genetic improvement of crops has been expedited by modern techniques like genetic transformation. New pest- and disease-resistant varieties of crops have been created and the nutritional value has been enhanced in other varieties (Jauhar, 2001; Vega et al., 1999). For success of any genetic improvement programme, a robust and reproducible plant regeneration system is a basic requirement (Jahne et al., 1995).
In vitro regeneration in plants is accomplished either through embryogenesis or organogenesis. Complete procedure of organogenesis is less understood. The effects of cultivar, explant and plant growth promoters on somatic embryogenesis and organogenesis have been evaluated in many plant species. In model plants like tobacco, leaf discs are the most commonly used explants for
Agrobacterium mediated and biolistic transformation (Horsch et al., 1985; Tomes et al., 1990). Mroginski et al., (1981) produced plants of six varieties of groundnut through organogenesis from immature leaf culture. Many researchers have expanded this technique to include various genotypes of peanut (Cheng et al.,
1992; McKently et al., 1991; Seitz et al., 1987; Utomo et al., 1996). Similar work has been reported in other species of Arachis genus (Mansur et al., 1993; McKently
26
27
et al., 1991; Pittman et al., 1983). Regeneration through organogenesis has also been achieved using mature leaf discs in different species of Arachis (Burtnik and
Mroginski 1985; Rey et al., 2000). In vitro tissue culture response is governed by combination of internal hormones naturally present inside the explant tissue and those present in the medium (Evans et al., 1981). Different species of Arachis have been regenerated by various combinations of 2,4-D or NAA and KIN or BA
(Mroginski and Kartha 1984). However, some researchers have used thidiazuron in regeneration experiments of cultivated peanut (Akasaka et al., 2000; Gill and
Ozias-Akins 1999). Thidiazuron has potent activity as a cytokinin in the induction of shoot organogenesis in several plant species (Murthy et al., 1998; Mithila et al.,
2001). While regeneration through organogenesis has been achieved from leaf discs in peanut (McKently et al., 1991). It was necessary to standardise this technique for the Pakistani cultivars for experiments on transformation. In order to induce the proliferation of meristematic tissues and convert the explants into complete plants, a precise combination of hormones is required. Present study was designed to optimise a reproducible and high-efficiency regeneration system for peanuts using young leaves in medium supplemented with Naphthaleneacetic acid and thidiazuron.
3.2 REVIEW OF LITERATURE
The recalcitrance of peanuts to tissue regeneration and genetic transformation impedes the development of genetically modified approaches for pest and disease control. Several exogenous genes have been introduced into peanuts by particle bombardment (Chu et al., 2008) or Agrobacterium-mediated
28
transformation (Bhatnagar et al., 2010), but these genetic transformation approaches are time-consuming and labour-intensive, and a vast amount of explants are needed due to the low frequency of regeneration in peanuts. The successful exploitation of in vitro techniques in peanuts depends on the establishment of efficient regeneration systems. Leaflets are the most widely used explants in peanut tissue culture.
Bele et al (2012) achieved direct shoot induction in 36.69 per cent of explants by inoculating leaf discs explants on MS medium supplemented with TDZ in Santalum album.
Several other types of explants, such as cotyledonary nodes (Srinivasan et al., 2010), epicotyls, hypocotyls (Marion et al., 2008), axillary meristems (Singh and Hazra, 2009) and cotyledons (Bhatnagar et al., 2010; Tiwari and Tuli, 2008), have also been used in peanut regeneration systems. Although, great efforts have been made to enhance the frequency of regeneration in peanuts, it was still difficult to obtain a sufficient number of explants in a short period of time. It even takes 4 to
6 months for explants to regenerate and recover from selection (Bhatnagar et al.,
2010).
McKently et al. (1991) evaluated plant development via organogenesis from in vitro cultured immature leaf tissue of the cultivated peanut and a perennial peanut species, A. glabrata Benth. Leaflets, 5 mm in length from peanut seedlings and 5 to 10 mm in length from perennial peanut plants, were cultured in vitro on
29
MS medium supplemented with 1 mg /L Naphthaleneacetic acid and four concentrations (1, 3, 5, and 10 mg/ L) of 6-benzylaminopurine . Bud regeneration occurred from the adaxial surface of the cultivated peanut explants on all BA concentrations, with the largest quantity produced on 5 mg/ L. 84 per cent of the cultures with bud tissue continued growth and differentiation into shoots within
120 d. These shoots developed roots within 30 d of transfer to basal medium supplemented with 1 mg/L NAA. Plantlets transferred to soil and placed in a greenhouse developed successfully, matured, and set seed. Phenotypic variation was not observed. A wide range of cultivated peanut genotypes was evaluated for organogenic responsiveness. The perennial peanut leaf explants callused and produced shoot meristems within 50 d of culture. Ten percent of the meristems continued growth and development into whole plants.
Akasaka et al., (2000) developed shoot buds from leaf discs explants of groundnut on MS medium supplemented with 1 mg/l NAA and various cytokinins such as benzyladenine, isopentenyladenine, kinetin, chloropyridylphenylurea, thidiazuron, zeatin in various combinitions. Among all the cytokinins tested, thidiazuron proved to be the best to induce shoot buds. But longer exposure to TDZ caused abnormality in buds and they were not able to convert into plants.
Disorganized vascular bundles and absence of shoot apical meristem was observed in histological studies. However, if explants are exposed to 10 mg/l TDZ only for 7 days before transferring them to hormone free medium, 35 per cent buds developed into shoots. All the shoots developed roots at medium supplemented with 1 mg/l
NAA.
30
Fontana et al., (2009) cultured leaf discs of three accessions of Arachis villosa on basic MS medium supplemented with different combinations of indole-
3-butyric acid, α-naphthalenacetic acid, kinetin, 6-benzylaminopurine and thidiazuron. Among all the combinitions, 13.62 μM TDZ and 4.44 μM BAP was found best. In absence of TDZ, induced buds were elongated only. The shoots developed excellent root nework on MS medium supplemented with 0.54 μM
NAA. Ponsamuel et al., (1998) cultured plumule explant of cultivated groundnut initially on MS medium supplemented with 2, 4-D and Kinetin and got 30-40 buds per explants. Later on, these explants were shifted to MS medium containing BAP and NAA and 4 shoots were obtained per explant in first 25 days. They continuously harvested shoots from the same explants at the rate of 5 shoots per month for seven months by addition of brassin in this medium.
Geng et al., (2011) obtained 41 per cent regeneration frequency by culturing leaf disc explants of Arachis hypogaea on MS medium containing
0.5mg/l of each of NAA and TDZ. The induced shoots were successfully elongated, adventitious roots were induced, plants were acclimatized and reproduced normally. This procedure was highly efficient and is feasible for the genetic transformation of cultivated groundnut.
3.3 MATERIALS AND METHODS
3.3.1 Explant Preparation
Seeds of four groundnut cultivars viz. BARD-92, BARI-2000, BARD-479
Golden were provided by Barani Agriculture Research Institute, Chakwal,
31
Pakistan. Freshly de-shelled seeds were sterilized by treating with 70 per cent (v/v) ethanol for one minute and then with 20 per cent clorox for ten minutes. After rinsing with sterile water thrice, few seeds of each variety were inoculated in simple solidified autoclaved MS medium for germination. Leaf discs of 0.5 cm diameter containing mid rib were cut from leaves of 10 days old seedlings under sterile conditions.
3.3.2 Medium and Culture Conditions
The explants were cultured on MS salts (Murashige and Skoog 1962), B5 vitamins
(Gamborg et al., 1968), 30g/L sucrose and augmented with 12 different hormone combinations (Table 3.1). 8g/L agar was added after adjusting the pH at 5.8 at 25
◦C. The leaf disc explants were placed with their abaxial side in contact with medium at 25± 2 ◦C with 16 hours light duration. Completely randomized design was used for this experiment which consisted of three batches each having 30 explants. The data on number of responding explants and shoots/explant were recorded after four weeks of culture.
After four weeks the responding discs were shifted to fresh MS medium containing 8 mg/l BAP and 0.5 mg IAA. On attaining a height of 5.0 cm, these shoots were cut and shifted to rooting medium.
The rooting medium consisted of half MS salts with vitamins, 15g/L sucrose and three different concentrations of NAA viz. 0.5, 1.0 and 1.2mg/L. The rooted plants were transplanted in pots containing coconut husk compost. Pots were covered with polythene sheet to maximize the humidity for first seven days.
32
Table 3.1: Hormone combinations used to induce shoot buds
Treatment (T) Hormonal combinations
NAA mg L-1 TDZ
1 0.1 0.1
2 0.1 0.5
3 0.1 1
4 0.1 1.5
5 0.5 0.1
6 0.5 0.5
7 0.5 1
8 0.5 1.5
9 1 0.1
10 1 0.5
11 1 1
12 1 1.5
33
3.3.3 Data Analysis
Analysis of variance (ANOVA) was performed using M-STATC software.
Means of parameters showing significant variations were ranked by Duncan’s
Multiple Range Test.
3.4 RESULTS AND DISCUSSION
To see whether various treatments and varieties make some significant difference or not, analysis of variance (ANOVA) is performed. If treatments and varieties make significant differences, Duncan’s Multiple Range Test is used for ranking of treatments and varieties.
3.4.1 Number of Responding Explants (%)
Number of explants showing shoot development has been referred as responding explants. After ten days of culture, it was observed that leaf disc was enlarged and thickened and callus was observed from cut surface (Figure 3.3).
Shoot induction, the basic objective of the experiment, was observed after four weeks of the inoculation from cut ends of midrib. These finding were in agreement with Eapen and George, (1993) and Geng et al., (2011). Analysis of variance
(Table 3.2) revealed that treatments, varieties and their interactions had highly significant impact on number of explants responding for shoot induction. Duncan’s
Multiple Range Test for interaction means showed that highest mean number of responding explants (52.67 per cent) were obtained at Treatment No. 6 in Golden
34
variety while lowest (1.00 per cent) number of explants responded at treatment No.
1 and 9 in BARD-92(Figure 3.1).
Akasaka et al., (2000) reported 34.7 per cent response rate while Geng et al
(2011) observed that 40.9 per cent explants responded to shoot induction on a medium containing thidiazuron. Difference in hormone combinations and genotypes might have contributed to this difference.
3.4.2 Number of Shoots/Responding Explants
Analysis of variance (Table 3.3) revealed that treatments, varieties and their interactions have highly significant impact on number of shoots/responding explants.
Duncan’s Multiple Range Test showed that highest (5.69) mean number of shoots/responding explants were obtained at Treatment No. 6 in Golden variety while lowest value (1.00) was observed in Treatment No. 1 and 9 in BARI-2000 and BARD-479 respectively (Figure 3.2). Eapen and George(1993) observed 7.0 shoots/responding explant on average while Geng et al, (2011) reported 5.0 shoots
/responding explant.
3.4.3 Rooting Percentage
The prime objective of the study was to get maximum number of rooted plants/explant as this will be an ultimate goal in any tissue culture based
35
Table 3.2: Analysis of variance for number responding explants (%)
Source of Variance DF Sum of Squares Mean Square F Value Treatments 11 1153.299 384.433 45.19** Varieties 3 53343.576 4849.416 570.0538** Treatment x variety 33 1532.118 46.428 5.4576** interaction Error 96 816.667 8.507 Total 143 56845.660 ** Highly significant
BARI‐2000 Golden BARD‐479 BARD‐92 A ‐ 52.67 B ‐ B ‐ 45.00 43.67 C ‐ D ‐ 36.00 explants(%)
E 33.00 ‐ F 27.33 ‐ F ‐ G ‐ GH responding ‐
GHI 21.50 20.33 ‐‐ HIJ HIJ HIJ of GH
‐ ‐ ‐ ‐ IJ ‐ IJK IJK IJ 17.00 ‐ ‐ JKL JKL ‐ 16 16.00 ‐ ‐ JKL JKL ‐ ‐ 13.20 KLM KLM KLM KLM KLM 17.33 13.00 13.20 ‐ ‐ ‐ ‐ ‐ LM LM LM LM LM LM LM LM LM 11.1 18.25 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ M M 11.00 11.00 ‐ ‐ Number 8.25 10.00 10.00 7.00 8.20KLM 8.20 8.20 8.00 8.20 8.20 7.00 7.00 7.10 7.00 7.00 8.25 7.00 7.00 7.00 N N N N N N N 7.00 6.00 ‐ ‐ ‐ ‐ ‐ ‐ ‐ N ‐ 1.20 1.10 1.0 1.00 1.00 1.00 1.00 1.00
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Treatments
Figure 3.1: Duncan's Multiple Range Test for ranking of interaction means with respect to number of responding explants (%). Values sharing same letters do not differ significantly at 5 per cent level of probability
36
transformation process. Analysis of variance showed that treatments and varieties had highly significant impact on this parameter (Table 3.4).
56.07 per cent shoots developed roots at 1.5 mg/liter NAA while only 12.69 per cent shoots developed roots at 0.5 mg/liter NAA. However root development achieved (55.82 per cent) at 1.0 mg/liter NAA was non-significantly less than top value (Table 3.5). Golden variety responded best to root induction (44.06 per cent) while lowest (36.03 per cent) root induction was observed in BARD-92 (Table
3.6). Verma et al (2009) used 0.5, 1.0, 1.5, and 2.0 mg/l NAA for root inductions but observed root induction only at 1.0 mg/l NAA in four commercial Indian
Peanut varieties. In our study 1.0 and 1.5 mg/l NAA gave excellent result, however, root induction was observed at all hormone levels used.
Venkatachalam et al., (1996) regenerated plantlets from leaf discs of peanut through organogensis using combination of BAP, Kinetin and NAA while Sarkar and Isam (2000) used only BAP and kinetin. However, results of current study coincide with those of many researchers who emphasized the use of TDZ in regeneration of plantlets from leaf explants of different Arachis species; A. hypogaea (Kanyand et al., 1994, 1997), A. correntina (Mroginski et al., 2004), A. stenosperma (Vijaya Laxmi and Giri, 2003) and A. villosa (Fontana et al., 2009).
A very low concentration of TDZ (0.01 to 1.0 mg/l) is used in most of tissue culture experiments and its concentration is of immense importance in determining the pathway of plant growth (Akasaka et al., 2000).
37
Table 3.3: Analysis of variance for No. of shoots/responding explants
Source of Variance DF Sum of Squares Mean Square F Value
Treatments 11 7.839 2.613 43.1522**
Varieties 3 171.550 15.595 257.5540**
Treatment x variety 33 21.013 0.637 10.5161** interaction
Error 96 5.813 0.061
Total 143
** Highly significant
A
‐ BARI‐2000 Golden B ‐ 5.69 5.11 explant
C ‐ CD FG CD ‐ ‐ ‐ GHIJKL ‐ E 3.39 ‐ 3.23 3.23 EF EF 3.17 ‐ ‐ FG FG 2.89 FG FGH ‐ ‐ FGHI ‐ ‐ 2.72 GHIJ ‐ GHIJK ‐ GHIJKL GHIJKLM GHIJKLM ‐ 2.52 2.50 ‐ GHIJKLMN ‐ ‐ ‐ HIJKLMNO HIJKLMNO IJKLMNO IJKLMNO IJKLMNO IJKLMNO ‐ ‐ JKLMNO ‐ 2.23 2.22 ‐ ‐ ‐ KLMNO ‐ 2.14 LMNO 2.13 LMNO LMNO MNO MNO 2.08 ‐ NO NO NO ‐ NO NO ‐ ‐ 2.00 ‐ ‐ O O O ‐ ‐ ‐ O ‐ 1.92 O O ‐ 1.88 ‐ 1.87 JKLMNO ‐ ‐ ‐ 1.83 ‐ ‐ 1.77 ‐ P P Shoots/responding 1.67 1.67 1.65 ‐ ‐ 1.61
1.60 1.60 1.55 1.45 1.43 1.40 1.40 1.39 1.39 1.34 1.34 1.33 1.33 of 1.30 1.28 1.25 1.25
1.23 1.20 1.20 1.15 1.00 1.00 Number
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 Treatments
Figure 3.2: Duncan's Multiple Range Test for ranking of interaction means with respect to No. of shoots/responding explants. Values sharing same letters do not differ significantly at 5 per cent level of probability
38
Table 3.4: Analysis of variance for rooting percentage
Source of Degree of Sum of Mean Square F Value Variance Freedom Squares Treatments 3 369.971 123.324 10.1936** Varieties 2 14970.914 7485.457 618.7266** Treatment x variety 6 128.617 21.436 1.7718 interaction Error 24 290.356 12.098 Total 35 15759.857 ** Highly significant
Table 3.5: Duncan's Multiple Range Test for ranking of treatments with
respect to rooting percentage
Treatment Mean Ranking 1.5 mg/l NAA 56.07 A 1.0 mg/l NAA 55.82 A 0.5 mg/l NAA 12.69 B Values sharing same letters do not differ significantly at 5 per cent level of probability
Table 3.6: Duncan's Multiple Range Test for ranking of varieties with rooting
percentage.
Variety Mean Ranking Golden 44.06 A BARD-479 43.19 A BARI-2000 42.83 A BARD-92 36.03 B Values sharing same letters do not differ significantly at 5 per cent level of probability
39
It has been found that low concentration of TDZ induces multiple shoots while slightly higher concentration switches the pattern to callus induction in
Cajanus cajan (Singh et al., 2003).
The shoot bud elongated well when shifted to medium containing BAP but lacking TDZ. These results are in close agreement with those of Gill and Ozias-
Akins (1999), Pelah et al., (2002), Ahmad et al.,( 2006) and Fontana et al., (2009).
Most of cytokinins used in plant tissue culture like BAP, Zeatin and kinetin contain adenine in their structural formula while TDZ is a non-adenine type highly active cytokinin which has a wide range of effects on cultures including induction of multiple buds and inhibition of their elongation (Mok et al., 1987; Huetteman and
Preece, 1993). This problem is overcome by shifting of buds to a medium containing adenine-type cytokinens (Fontana et al., 2009).
40
B A
C D
Figure 3.3: Different steps of shoot induction from leaf discs
A. Increase in size, thickening and callus induction from cut surface of leaf disc after
10 days of culture.
B. Development of shoot buds
C. Development of roots
D. Plantlet growing in coconut husk peat
Chapter 4
STANDARDIZATION OF IN VITRO REGENERATION VIA
CALLUS INDUCTION
4.1 INTRODUCTION
Development and standardization of an efficient and robust in vitro regeneration system is a pre-requisite for a successful transformation through
Agrobacterium or micro projectile bombardment. Transformation of somatic embryos is more desirable as a single cell is transformed and subsequently converted into genetically stable, non-chimeric plant. Many researchers have developed regeneration systems for embryogenic cultures from immature and mature seed explants of diverse groundnut genotypes (Saxena et al., 1992; Baker et al., 1995). Immature seeds have the demerits of seasonal availability and difficulty in accurate assessment of proper developmental stage. On the other hand, dry mature seeds are available round the year and have no variation regarding developmental stage (Baker et al., 1995; McKently et al, 1995). Induction of somatic embryogenesis and organogenesis have been reported in groundnut using different plant growth hormones including auxins (Ozias-Akins, 1989; Baker and
Wetzstein, 1994; McKently et al, 1995) and thidiazuron (TDZ) (Gill and Saxena,
1992; Kanyand et al., 1994; Li et al., 1994). Embryogenic cultures of groundnut have been reproducibly produced from diverse genotypes by using picloram
(Ozias-Akins et al., 1992) or from a small number of genotypes with 2,4-D
(Durham and Parrott, 1992). Multiple subcultures of embryogenic callus on
41
42
picloram were used to produce the first transgenic groundnut plant (Ozias-Akins et al., 1993).
Conventional plant breeding methods have achieved limited success in genetic improvement of groundnut due to the narrow genetic base. Optimization of protocols for in vitro plant regeneration is a basic prerequisite for genetic improvement of plants using biotechnological techniques (Rey et al., 2000).
While the use of explants from immature embryos for somatic embryogenesis and regeneration is very common, several intrinsic complications are faced in using such materials in groundnut. Identification of proper developmental stage of explant and its maintenance, particularly in off season is not less than a challenge. Moreover, under-ground organs are prone to high contamination rates. The developmental stage of immature embryo is estimated from morphology of pod shell which is not always precise depending upon multiple factors like weather conditions, soil moisture, structure and texture. The optimization of protocol for somatic embryogenesis using epicotyls from mature seeds would eliminate many of these problems. A simple and cost effective system for highly efficient somatic embryogenesis using harvested, dry, stored seeds has been elaborated in this chapter.
4.2 REVIEW OF LITERATURE
In vitro regeneration can occur either through organogenesis or somatic embryogenesis. In somatic embryogenesis, the culture has merit such as single cell origin which results in low frequency of chimeras and high number of regenerants
43
(Rey et al., 2000, Sato et al., 1993). Somatic embryogenesis has been reported from immature embryo axis (Eapen and George, 1993; Baker et al., 1994), leaf discs (McKently, 1991; Gill and Saxena, 1992; Baker and Wetzstein, 1998) mature and immature cotyledons (Gill and Saxena, 1992; Wetstein and Baker, 1993), hypocotyls (Venkatachalam et al.,1997) and epicotyls (Little et al., 2000). Most of the protocols developed for regeneration of groundnut have low frequencies of plant recovery ranging from 18-38 per cent (Ozais-Akins, 1989; Reddy and Reddy,
1993; Wetzstein and Baker, 1993).
Somatic embryogenesis in groundnut has been standardized using diverse explants including leaf discs (Baker and Wetzstein 1992), immature cotyledons
(Baker and Wetzstein 1995), immature embryo axes (Eapen et al., 1993, Roja Rani and Padmaja 2005), mature embryo axes (McKently, 1991), and immature embryos
(Sellars et al., 1990).
The ability of formation and regeneration of embryogenic callus is genotype dependent, and higher efficiencies have generally been achieved using immature embryos at specific developmental stages as explants (Baker and Wetzstein 1992;
Durham and Parrott 1992; Ozias-Akins and Anderson, 1992). Livingstone and
Birch (1999) elaborated that for continuous supply of embryogenic callus for transformation experiments, round the year availability of explants should be ensured. Immature embryos have only seasonal availability and they have more risk of contamination. A possible remedy to this problem is long-term maintenance of cultures but it will give rise to infertility and genetic abnormalities (Wang et al.,
1998).
44
Chengalrayan et al.,(1994) produced embryogenic callus from the leaflets of mature embryo axes and established conditions for conversion of 25 per cent of somatic embryos to plants. Baker et al., (1995) used embryo axes from mature seeds of 14 cultivars of groundnut and obtained high quality embryogenic callus at a frequency of 0.83 somatic embryos per cultured axis, with a regeneration frequency of 15 per cent.
Efficiency of the developed protocols is low, probably because of morphological abnormalities in the apical meristem of the embryos as observed in the cultivar JL-24 (Chengalrayan et al., 2001). The malformation in somatic embryos lead to low frequency of embryo conversion rate in medium lacking growth hormones. To overcome this problem various time, labour and resource consuming procedure are adopted which also increase the risk of contamination
(Joshi et al., 2005). That is why shortening of duration from initiation of culture to production of transgenic plant is an important objective for Agrobacterium mediated and biolistic methods of transformation (Joshi et al., 2005). In recent years TDZ is being used to convert abnormal culture to embryos (Chengalrayan et al., 1997; Joshi et al., 2008). In another study it was observed that auxin concentration in induction medium has no effect on rate of embryo conversion
(Wetzstein and Baker 1993). In most of the experiments for somatic embrogenseis
2,4-D is routinely used but its effects on morphology and performance of somatic embryos has not been clearly elaborated.
Induction of somatic embryos is not the ultimate goal, but conversion of these embryos to plants is the real measure of a successful culture system. Low
45
conversion rate usually hinders the success of a protocol. Many reseachers have triumphed to overcome this problem in groundnut (Ozias-Akins et al., 1992;
Chengalrayan et al., 1997). Hazra et al., (1989) proposed a protocol for callus induction and subsequent embryo conversion from immature zygotic embryos.
Chengalrayan et al., (1997) on the other hand developed somatic embryogenesis system from embryonic leaflets of mature dry seeds on MS medium supplemented with 2,4-D. Embryo conversion was however achieved on hormone free medium.
The embryo developed radicals but shooting was not observed. When these embryos were shifted to the medium supplemented with TDZ, high shooting frequency was observed. Induction and conversion of somatic embryos in groundnut on medium supplemented with TDZ have been reported by many scientists (Saxena et al., 1992; Gill and Saxena, 1992; Murthy et al., 1995; Victor et al., 1999)
4.3 MATERIALS AND METHODS
4.3.1 Explant Preparation
Seeds of four groundnut cultivars viz. BARD-92, BARI-2000, BARD-479
Golden were provided by Barani Agriculture Research Institute, Chakwal,
Pakistan. Freshly de-shelled seed were sterilized by treating with 70 per cent (v/v) ethanol for one minute and then with 20 per cent clorox (commercial bleach) for ten minutes. After rinsing with sterile double distilled water, seeds were soaked in same water for three hours at room temperature, seed testa was removed surgically, the cotyledons were opened and embryos were separated with full care. About one third portion of embryos (epicotyl) was cut with surgical blade and used as explant.
46
4.3.2 Culture Medium and Conditions
The explants were cultured on callus induction medium (CIM) consisting of
MS macro and micro salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al., 1968), 30g/L sucrose and supplemented with picloram (2, 4, 6, 8 and 10 mg/l).
8g/L agar was added after adjusting the pH at 5.8 at 25 ◦C. the explants were inoculated on medium for 3 weeks at 25± 2 ◦C with a photoperiod of 16/8 hours.
The experiment was conducted in completely randomized design and was repeated in three batches, each batch consisting of 20 explants for each treatment. After three weeks the callus was transferred to embryo conversion medium (ECM) consisting of MS salt, B5 Vitamins, 30g/l Sucrose different concentrations of 2,4-D
(0.1, 0.5, 1.0, 1.5, 2.0 mg/l) and cultured under the same conditions as mentioned above.
The embryos thus developed were shifted to embryo germination medium
(EGM) consisting of MS salt, B5 Vitamins, 30g/l Sucrose different concentrations of BAP (0.1, 0.5, 1.0, 1.5, 2.0 mg/l).
The data on number of explants showing callus, number of embryos and number of germinated embryos were recorded.
4.3.3 Data Analysis
Analysis of variance (ANOVA) was performed using M-STATC software.
Means of parameters showing significant variations were ranked by Duncan’s
Multiple Range Test.
47
4.4 RESULTS AND DISCUSSION
To see whether various treatments and varieties make some significant difference or not, analysis of variance (ANOVA) is performed. If treatments and varieties make significant differences, Duncan’s Multiple Range Test is used for ranking of treatments and varieties.
4.4.1 Number of Responding Explants (%)
Number of explants showing friable, granular embryogenic callus has been referred as responding explants. After ten days of culture, it was observed that plumule was enlarged and callus formation started (Figure 4.3). Analysis of variance (Table 4.1) revealed that treatments, varieties and their interactions have highly significant impact on number of responding explants. Duncan’s Multiple
Range Test for interactions showed that highest mean number of responding explants (98.33 per cent) were obtained at Treatment No. 4 ( 8 mg/l picloram) in
Golden variety while lowest number of explants (33.33 per cent) responded at
Treatment No. 1 in BARI-2000 variety (Figure 4.1).
Importance of picloram in induction of embryogenic callus has been reported in Arachis hypogaea (Little et al., 2000), Arachis pintoi (Rey et al., 2000) and Arachis glabrata (Vidoz et al., 2004).
Percentage of responding explants increased with increasing concentration of picloram till 8mg/l and then reduced slightly. This finding is in agreement with
48
that of McKently (1990) but differ significantly from that of Cucco and Jaume
(2000) who got non-embryogenic callus on all concentrations of picloram.
Vidoz et al., (2006) used 5, 10, 15 and 20mg/l picloram and got maximum
(56 per cent) embryo conversion at 15 mg/l picloram concentration in Arachis correntina. This indicated the difference in genetic makeup responsible for concentration of endogenous hormone level of germplasm used.
4.4.2 Number of Differentiated Embryos/ Responding Explant (%)
Analysis of variance (Table 4.2) revealed that treatments, varieties and their interactions have highly significant impact on number of differentiated embryos/responding explants. Duncan’s Multiple Range Test for interaction means showed that highest (19.33) mean number of embryo/responding explants was obtained at Treatment No. 4 while lowest value (5.33) was observed in Treatment
No. 1 in Golden variety, each (Figure 4.2).
Our findings differ from that of Vidoz et al., (2006) who reported better embryo conversion on hormone free MS medium in Arachis correntina.
Chengalrayan et al., (1997) also used cytokinins like BAP and TDZ in embryo conversion medium in Arachis hypogea whereas in present study auxin produced excellent results.
4.4.3 Germination Percentage of Embryos
The prime objective of the study was to get maximum number of fully grown plants/explant as this will be an ultimate goal in any tissue culture based
49
Table 4.1: Analysis of variance for number responding explants (%)
Source of Variance DF Sum of Squares Mean Square F Value
Treatments 4 14005.833 3501.458 182.6848**
Varieties 3 2891.250 963.750 50.2826**
Treatment x variety 12 2160.833 180.069 9.3949** interaction
Error 40 766.667 19.167
Total 59 19824.583
** Highly significant
BARI‐2000 Golden A BARD‐479 BARD‐92 ‐ B ‐ (%)
C 98.33 ‐ D 90.00 DE DE ‐ ‐ ‐ EF F ‐ F F 78.33 ‐ ‐ ‐ FG explants ‐
70.00 GH 68.33 68.33 H ‐ ‐ HI HI 61.67 ‐ ‐ 60.00 IJ IJ 58.33 58.33 IJ IJ ‐ ‐ 55.00 ‐ ‐ J ‐ 48.33 46.67 43.33 43.33 38.33 38.33 36.67 36.67 responding 33.33
of
Number
T1 T2 T3 T4 T5 (2 mg/l) (4 mg/l) (6 mg/l) (8 mg/l) (10 mg/l) Treatments (Picloram Concentration)
Figure 4.1: Duncan's Multiple Range Test for ranking of interaction means with respect to number of responding explants (%). Values sharing same letters do not differ significantly at 5 per cent level of probability
50
Table 4.2: Analysis of variance for No. of differentiated embryos/responding explants
Source of Variance DF Sum of Squares Mean Square F Value Treatments 4 362.267 90.567 51.264** Varieties 3 98.183 32.728 18.5252** Treatment x variety 12 139.067 11.589 6.5597** interaction Error 40 70.667 1.767 Total 59 670.183 ** Highly significant
BARI‐2000 Golden BARD‐479 BARD‐92 A ‐ 19.33 explant
B ‐ C ‐ 14.67 CD ‐ DE DE ‐ ‐ DEF 12.33 EFG DEF ‐ ‐ ‐ 11.33 EFG EFG ‐ EFG EFG ‐ ‐ ‐ FGH FGH 10.00 10.00 9.67 ‐ ‐ GH GH GH GH 8.00 9.33 ‐ ‐ ‐ ‐ 8.67 8.33 H 8.00 8.00 ‐ 7.33 7.33 6.67 6.67 6.67 6.67 embryos/responding
5.33 of
Number T1 T2 T3 T4 T5 (0.1 mg/l) (0.5 mg/l) (1.0 mg/l) (1.5 mg/l) (2.0 mg/l) Treatments ( 2,4‐D conentration)
Figure 4.2: Duncan's Multiple Range Test for ranking of interaction means with respect to No. of embryos/responding explants. Values sharing same letters do not differ significantly at 5 per cent level of probability
51
transformation process. Analysis of variance showed that treatments and varieties had highly significant impact on this parameter (Table 4.3).
74.72 per cent embryos germinated at Treatment No.1 while only 71.09 per cent embryos germinated at Treatment number 5 (Table 4.4). Golden variety responded best to embryo germination (74.142 per cent) while lowest (71.374 per cent) root induction was observed in BARD-92 (Table 4.5). Linvingstone and
Birch (1999) reported that embryo germination was 3 per cent on hormone free MS medium but it increased to 30 per cent when cytokinins were used in the medium.
Previously Chengalrayan et al., (1994) also reported similar results.
In present study embryo germination is significantly high (74.72 per cent) on a very low concentration (0.1mg/l) of BAP. Venkatachalam et al., (1997) also found that higher concentration of BAP along with very low concentration of NAA trigger embryo germination.
In most of the experiments combination of two, three, four or even five plant growth regulators were used (Venkatachalam et al., 1999; Vidoz et al., 2006;
Robinson et al., 2011) but in present study only single hormone was used in each step to avoid complexity. Moreover, time duration of each step was kept minimum to avoid somaclonal variation which is a problem in most of peanut cultures
(Ozias-Akins and Gill, 2001; Vidoz et al., 2006)
52
Table 4.3: Analysis of variance for germination percentage
Source of Variance Degree of Sum of Mean Square F Value Freedom Squares Treatments 4 110.887 27.722 0.7409** Varieties 3 77.072 25.691 0.6866** Treatment x variety 12 487.806 40.651 1.0865 interaction Error 40 1496.601 37.415 Total 59 2172.367 ** Highly significant
Table 4.4: Duncan's Multiple Range Test for ranking of treatments with respect to germination percentage. Values sharing same letters do not differ significantly at 5 per cent level of probability
Treatment Mean Ranking 1 (0.1 mg/l BAP) 74.72222 A 3 (1.0 mg/l BAP) 74.03 A 4 (1.5 mg/l BAP) 72.44 B 2 (0.5 mg/l BAP) 71.81 B 5 (2.0 mg/l BAP) 71.09 C
Table 4.5: Duncan's Multiple Range Test for ranking of varieties with respect to germination percentage. Values sharing same letters do not differ significantly at 5 per cent level of probability
Variety Mean Ranking Golden 74.142 A BARD-92 73.68 A BARI-2000 72.074 B BARD-479 71.374 B
53
A B
C D
F E
54
H G
Figure 4.3: Different steps in peanut somatic embryogensis
A, B: Increase in explant size and callus development
C, D and E: Malformation of embryos if kept on induction medium
F: Normal embryo development on ECM
G: Embryo germination on EGM
H: Root development
Chapter 5
AGROBACTERIUM MEDIATED TRANSFORMATION
5.1 INTRODUCTION
Groundnut is one of the important cash crops, especially in the rainfed areas of Pakistan. Genetic improvement in this crop by conventional breeding is not as rapid as needed to meet the demands of increasing population. Groundnut has a very narrow genetic base (Kochert 1996). It is believed that a single hybridisation event between Arachis duranensis and A. ipaensis, both diploid species, gave rise to what we recognise as peanut today. There is little in the way of resistance genes to fungal or viral diseases available to breeders. Furthermore, introgression of desirable traits, if available, from wild Arachis relatives is difficult, especially as peanut is a self-pollinating species. Although many yield related traits have been genetically improved by conventional breeding methods, many traits are yet to be improved. These include resistance to insect pests as well as to diseases caused by nematodes, viruses, bacteria and fungi especially those causing leaf spot and producing carcinogenic aflatoxin (Porter et al., 1990). Other beneficial traits would include tolerance to water stress, uniform fruit maturity, and enhanced nutritional quality by modification of the amino acid and lipid composition.
Genetic transformation is the best choice for improvement of many important traits which are not being improved by conventional methods.
Establishment of a suitable gene delivery system and a protocol for subsequent recovery of plants is a basic requirement for recalcitrant peanut varieties.
55
56
Genetic transformation of groundnut has been reported previously but recovery of viable reproductive plants is very limited because of very low transformation efficiencies. The success of Agrobactenum-mediated transformation appears to be heavily influenced by the peanut cultivar, the strain of A. tumefaciens used, the plasmid that the bacterium carries, as well as the co-cultivation conditions and the efficiency of the regeneration system.
Most reports describing Agrobacterium-mediated transformation of peanut have used kanamycin as the selection agent (Eapen and George, 1994). However, many scientists found hygromycin B to be a more suitable selective agent.
Present study was designed to simplify the transformation and subsequent regeneration system in local cultivars by reducing the steps of tissue culture producing more transgenic shoots from readily available seed explants round the year. This method proved to be cost effective and time saving as there was less contamination and direct multiple shoot induction on selection medium was accomplished in short span of time.
5.2 REVIEW OF LITERATURE
Several diseases including early and late leaf spots stem rot and rust attack peanut and cause yield reduction of up to 70 per cent (Subrahmanyam et al., 1992).
Weeds are another major constraint for limited peanut production as yield loss of 30-80 per cent due to weeds is reported (Gill et al., 1986). Weeds compete
57
with crops for light, nutrients, water and carbon dioxide and major loss (25-45 per cent) occurs in early stages of crop development (Reddy, 1984).
Early leaf spot (ELS) is caused by the fungus Cercospora arachidicola
Hori. Conidiophores on groundnut leaves produce conidia, which are dispensed by splashing rain, wind, insects and mechanical dissemination and can germinate within two weeks to repeat the process (Porter et al., 1990; Subrahmanyam et al.,
1992). Conidia germinate to form germ tubes, which penetrate into opened stomata and epidermal cells. The mycelium is initially intercellular but on the death of host cells it becomes intracellular (Gibbons, 1966; Porter et al., 1990). Stomata produce viable conidia after storage for 12 months at 20 to 30°C and 75 to 81 per cent relative humidity (Alabi, 1986).
Climate, micro-environments and method of irrigation affect disease severity. Optimum temperatures of 25-31°C, high humidity and late rainy season favour sporulation (Venkataraman and Kazi, 1979; Subrahmanyam et al., 1992).
Asci and ascospores are formed by the pathogen in the perfect stage
(Mycosphaerella arachidicola) during over-wintering on crop residue and together with mycelial fragments can also be potential sources of initial inoculum in coming spring (Hemmingway, 1957).
The pathogen perpetuates from season to season on plant debris and volunteer peanut plants, building up an inoculum reservoir for the following season
(Subrahmanyam et al., 1992). Rao et al. (1993a) reported that the conidia,
58
ascospores and mycelium could only survive for 30-60 days on underground peanut debris and for one year on the debris stored indoors.
It produces roughly circular lesions, dark brown on the upper leaflet surface, slightly lighter on the lower surface and surrounded by a yellow halo. They may combine together in cases of severe intensity causing defoliation. Lesions can also develop on stems, petioles and pegs (Woodroof, 1933; Jenkins, 1938; Van
Wyk and Cilliers, 2000).
Severity of disease varies with localities and seasons and yield reductions of
20 to 100 per cent have been reported (Venkataraman and Kazi, 1979;
Subrahmanyam et al., 1992). Yield and quality can be affected by ELS and in particular by the reduced photosynthesis resulting from increase in necrotic leaf area and defoliation after severe infection. Yield can also be reduced when the pegs are weakened by ELS and by the reduced ability of diseased plants to maintain healthy pegs (Subero, 1992).
Thiram and Mancozeb are two major fungicides for seed treatment for control of ELS.Chlorothalonil and Tebuconazole have been used for foliar spray at a 14-, 21-, 28-day schedule and a control (Grichar et al., 1998). The best results were obtained by 14-day schedule as there was significant increase (43 per cent) in yield. Cole (1981) observed that if chlorothalonil, mancozeb, vinclozolin and chlorothalonil are applied one by one after 21 days, ELS infection is reduced to much extent, yield is improved, percentage of pods left in the ground after harvest
59
and number of decayed pods were reduced. It was also shown that combination of
Mancozeb and benomyl was more effective than chlorothalonil . However web blotch increase rapidly where ELS was controlled by fungicides. Subrahmanyam and Hildebrand (1997) reported that yield increase was achieved in fungicide treated groundnut only if good rainfall or irrigation was available, whereas in water stress conditions, fungicide application was ineffective.
Disease may be effectively controlled by accurate application of fungicides but, these may cost a considerable financial burden on farmer. Moreover sometimes it is not possible for farmers to spray the crop in time as was in the case of outbreak of ELS in North Carolina after Hurricane Floyed in 1999 (Isleib et al.,
1999).
A computerized weather based advisory system have been used in USA to help farmers in determining the appropriate time for fungicide spray which have resulted in significant increases in the yield of groundnut and net profit ( Horne et al. 2005).
Intensive use of fungicides may result in development of resistance in fungal pathogens in few years. C. arachidicola had been reported to develop resistance to benzimidazole in France (Rao et al., 1993b).
Fungal diseases can be effectively controlled by fungicides but this is not an economically viable solution as subsistence farmers cannot afford this (Weeks et
60
al., 2000). Green and Wynne (1986) proved that all components of resistance in greenhouse conditions were significantly correlated to those in field conditions.
Hence evaluation of lines for partial resistance to ELS can be done in greenhouse to develop resistant for field conditions.
Tuggle et al. (1999) identified 43 strains of C. arachidicola in groundnut fields and found significant variation in their virulence. They suggested that resistance to ELS can be affected by the virulence of the strain. It is necessary to conduct trials for several year on multiple locations to evaluate the durability of resistance.
Subrahmanyam et al. (1995) screened 1508 lines, 743 advanced lines and
4177 early generation breeding lines and 126 interspecific hybrids for resistance to
ELS. Only 80 germplasm lines, 46 breeding lines and four interspecific hybrids showed an adequate level of resistance. Rao et al. (1993b) studied the response of four genotypes to eight C. arachidicola strains. The genotypes showed a variable response to all eight isolates with respect to different components of resistance. It is, therefore, important that the different pathotypes present in a production area be taken into consideration in resistance breeding programmes. In many cases, it has been observed that a line showing resistance to disease in one locality proved to be susceptible in another locality (Chandra et al., 1995).
Continuous cultivation of groundnut years after years in the same field increases inoculum and spread of disease, while rotation with different crops like
61
bahiagrass, cotton, sorghum and corn decreases the incidence to much extent
(Brenneman et al., 1995). Burying the crop residues by deep ploughing also helps in disease control because spore forming ability of fungus is suppressed. Moreover incidence of epidemics are less in fields with reduced tillage as compared to conventionally tilled fields provided no previous crop residue was present (Weeks et al., 2000; Brenneman and Culbreath, 2005).
Monfort et al. (2004) also suggested that four fungicide sprays combined with reduced tillage are enough to control ELS instead of seven sprays with conventional tillage. In this way cost of production may significantly be reduced keeping in view the price of fungicides and labour. Some herbicides like lactofen are reported to reduce fungal attacks while others like 2,4-D have opposite effects
(Baysinger et al., 1999).
Kokalis-Burelle et al. (1992) reported positive results after treatment of leaves with chitin and the bacteria Bacillus cereus. Knudsen et al. (1987) obtained more effective control using Pseudomonas cepacia. Verticillium lecanii has been reported as a parasite on several groundnut pathogens in India, including C. arachidicola (Subrahmanyam et al., 1990). The hyperparasitic fungus Dicyma pulvanata feeds on leaf spot fungi but this fungus has not been tested yet for the control of ELS in field trials (Brenneman and Culbreath, 2005).
Kishore et al., (2005) reported that Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 reduced lesion frequency by 60 per
62
cent improved increased the pod yields by 62 and 75 per cent, respectively, compared with the control.
Late leaf spot (LLS) is caused by the fungus Cercosporidium personatum.
This fungus over-winters on the crop residues and debris. Conidiophores are mostly hypophyllous, arising in more or less distinctly concentric reddish-brown tufts, generally with hyaline tips. Conidia are usually cylindrical, pale brown with somewhat attenuated tips and one or more septates.
Increase in temperature and humidity in spring causes an increase in fungal activity. The optimum range for growth and sporulation for C.personatum is 25-
30°C. Light is a requisite for sporulation. Temperature required for germination is slightly less than that of C. arachidicola (Pattee and Young, 1982). Conidia, produced by conidiophores, on groundnut residue in the soil and off-season groundnut plants, serve as the principal source of initial inoculum. Intercellular haustoria are produced at temperatures from 25-31°C and lesions develop within
10-14 days. The lesion forming cycle starts all over again and the conidia are dispersed by insects, farm implements (Pattee and Young, 1982), splashing water
(from overhead irrigation or rain) and wind (Smith and Crosby, 1973; Horne et al.,
1976; Hagan, 1998; Subrahmanyam, et al., 1992). In spring ascospores (Jenkins,
1938), chlamydospores and mycelial fragments (Hemmingway, 1957) are also potential sources of initial inoculum produced on crop residue that over-wintered in the soil (Pattee and Young, 1982; Porter et al., 1990). The fungus leads its life
63
through different seasons on volunteer groundnut plants and plant debris, building up an inoculum for the coming season (Subrahmanyam et al., 1992).
The lesions are similar to those of ELS to much extent. However, these are darker brown and without a definite chlorotic yellow halo. On the lower side of the leaflets, lesions are almost black, in contrast to the lighter coloured lesions of ELS.
LLS generally occur later in the season and is often misinterpreted with other leaf spots. C. personatum produced cellulolytic and pectolytic enzymes that altered the starch, sugar and amino acid content of leaf tissue, resulting in reduced leaf efficiency and premature abscission (Pattee and Young, 1982). An active phytotoxin, Cercosporin, was also isolated from C. personatum. Mohapatra (1982) showed that diseased leaves contained higher quantities of reducing sugars than healthy ones.
In a study conducted by Pattee and Young (1982), leaf spot reduced the leaf area index by 80 per cent, the carbon dioxide uptake by 85 per cent and the canopy carbon exchange rate by 93 per cent. Photosynthesis of diseased canopies was reduced not only by defoliation but also by inefficient fixation of carbon dioxide by diseased attached leaves. Horne et al. (1976) reported that haustoria produced by the Late Leaf Spot fungus penetrate leaf cells which in turn increase respiration.
The severity of the disease varies with amount and pattern of rainfall and method of irrigation. If plant residues are left in the field and there is no proper crop rotation, the incidence of disease is increased (Swanevelder, 1998).
64
Although number of pods is reduced, the major loss is by shedding of pods due to disease attack on pegs. Ghuge et al. (1980) observed that decrease in disease incidence resulted in an increase in the dry matter, a higher number of mature pods, heavier nuts (as expressed in 100-kernel weight) and enhanced pod yield. In a variety having yield potential of 4400 kg suffered a loss of 57kg per each percent of defoliation by leaf spot (Backman and Crawford, 1984).
In an experiment, chlorothalonil, benomyl, mancozeb, fentin hydroxide, tiophanate methyl and benomyl/mancozeb-combination were proved to very effective in controlling LLS (Pauer et al., 1983). Hagan et al. (2005) in a study showed that tebuconazole alone or in combination with chlorothalonil can be used as curative as well as protective measure against LLS while chlorothalonil is only protective fungicide. Waliyar et al., (2000) reported that if fungicides were applied at 40, 55, 70 and 85 days after sowing date, a yield increase of 1.5 to 3 tonnes was achieved.
Gorbet et al. (1990) applied fungicides on 14 genotypes of peanut and found that all the lines gave higher yield when sprayed on 14th day as compared to unsprayed plots.
Culbreath et al. (2002) studied the phenomenon of development of resistance to fungicides in fugal strains. They reported that due to this fact new chemical like sterol biosynthesis inhibitor are being registered for control of fungal diseases in peanut.
65
Hagan (1998) conducted an experiment to evaluate the efficacy of flusilazole to control LLS. The flusilazole enters the lipid layer on leaf surface and shows its effectiveness in three hours especially in wet weather. Different electronic weather stations which could measure relative humidity and temperature have been devised to advise proper fungicide with appropriate time and quantity just by pressing a key (Bailey and Matyac, 1985; Jacobi et al., 1995).
Cost of production will be reduced to a great extent if resistant cultivars are planted because less fungicides, equipment and labour will be required (Johnson and Beute, 1986). Nigam and Dwivedi (2000) reported that resistance to LLS was correlated with undesirable pod and seed characteristics, low partitioning and late maturity.
Chiteka et al. (1988) conducted identical experiments in the field and greenhouse to identify the LLS resistant lines of peanut. The performance of genotypes in the greenhouse was significantly correlated with their performance in the field conditions for latent period, lesion diameter and sporulation. Resistant genotypes can be selected on the basis of sporulation level only because other components are associated with this.
Luo et al. (2005) identified 56 genes for resistance to LLS using micro array and RT-PCR and proposed to develop specific gene probes for marker- assisted selection in breeding programmes. Anderson et al. (2000) developed highly resistant line by crossing peanut with A. durenensis. This line was used in
66
breeding programmes to incorporate resistance genes into well adaped high yielding varieties. The segregating lines were used for molecular marker studies.
Shokes and Gorbet (1990b) used LLS resistant, partially resistant and a susceptible variety in their experiment in which three grades of disease control were applied; high intensity control, low intensity control and no-disease control.
They found that yield loss in no disease control programme was 60.3 per cent while
17.0-24.6 per cent was recorded for the resistant and partially resistant lines. Seed weight was the lowest with the no disease management programme and greatest with the maximal management programme. Seed weight of the susceptible cultivar gave the largest response to LLS control. Hagan (1998) reported that plant appearance scores generally resulted in the best separation of all genotypes particularly under the no disease control programme.
Deep ploughing, disinfection of equipment, elimination of volunteer plants, burning or proper removal of crop residues helps to stop increase in inoculum for next crop. Irrigation during cool weather increase fungus growth so it should be avoided (Swanevelder, 1998; Hagan, 1998; Phipps, 2000).
It has been observed that resistance to leaf spots could not be induced by using strains of rhizobacteria and chemical elicitors in peanut contrary to many crops including rice, potato, tomato, vegetables, pome fruit, mango, citrus, grape, banana, peppers and tobacco. However, in one of two experimental tests, foliar
67
sprays with DL-β-amino-n-butyric acid (an elicitor of localised acquired resistance) resulted in less LLS infection (Zhang et al., 2001).
Chitinase is considerd to be the best antifungal agent because of its ability to degrade chitin, a chain homopolymer of N-acetylglucosamine (GlcNAc) connected by β-1, 4 glucosidic linkages, which is essential component of cell wall of most of fungi. Chitin is also a major component of exoskeleton of insects and at the time of molting chitinase is produced to digest the exoskeleton. Degradation of chitin at inappropriate time may prove lethal or insect or can disarm them.
Pace of genetic improvement in peanut through conventionl breeding methods is limited because of low frequency of successful crosses and linkage of desirable traits with undesirable ones (Pattee and Stalker, 1995). There is an urgent need to improve peanut cultivars especially for disease resistance. As there is a limited availability of resistance sources in the gene pool of peanut and inter- specific crosses have very little success, tools of genetic engineering can be employed to improve the crop.
Genetic transformation involves development of a robust and reproducible regeneration system, preparation of construct and its integration into an appropriate vector, optimization of transformation methods, selection and multiplication of transformants, morphological and molecular analysis of transgenics, and assessment of these plants for their effectiveness against biotic and abiotic stresses along with biosafety measures (Birch, 1997).
68
Kumar and Kirti (1996) analyzed the interaction of cercospora with wild
Arachis diogio at molecular level by differential expression of genes and found that an 886 kb long AdCyp transcripts encoding 172 amino acid long polypeptide was accumulated during cercospora attack. Thus gene for this transcript can be identified and used as a marker in peanut breeding and biotechnology.
Cantonwine et al. (2007) suggested that strip tillage in peanut sowing delays onset of early leaf spot due to lesser initial infections from soil borne stroma. Using cDNA microarray technique, Luo et al. (2005) identified 56 genes which were expressed in ELS resistant peanut variety as a response to Cercospora personatum.
In a study conducted by Kishore et al. (2005) late leaf spot was controlled biologically upto 60 per cent by foliar spray of peanut associated bacteria Bacillus circulans, GRS 243 and Serratia marcescens GPS 5. Chitinases purified from culture of these bacteria effectively inhibited the germination of cercospora in vitro.
A non tissue culture based approach was applied by Rohini and Rao (2009) to introduce chitinase genes driven by CaMV35S into peanut through
Agrobacterium strain LBA 4404. Integration of transgene into host DNA was confirmed by Southern Blot and plants proved to be resistant to Cercospora arachidicola to varying degrees.
69
Anuradha et al. (2008) isolated a defensin gene from mustard having 90 per cent resemblance to previously known defensins. This gene when transferred to peanut expressed well and conferred resistance to leaf spot disease in field trials.
The good thing is that nutritional value of genetically modified peanut with anti- fungal and anti-pest transgenes is not affected.
Chu et al. (2008) successfully produced transgenic peanut in which allergen
Ara h 2 and Ara h 6 were silenced and binding of human lgE with these allergens were reduced significantly. Traits like seed weight, germination percentage and fungal susceptibility remained unaffected.
Sobolev et al. (2007) found that peanut resists the attack of early/late leaf spot by synthesis of stilbene phytoalexins. The quality of these phytoalexins in different genotypes can be used as markers in peanut breeding.
Lei et al. (2008) identified a SCAR marker AFs-412 for resistance to a flavours infection. This marker can help in identification of resistant lines during peanut breeding and hence reducing the aflotoxin contamination which is a major problem of peanut industry all over the world.
Crown gall causing Agrobacterium tumefaciens is a natural genetic engineer which transfers its tumor inducing (Ti) genes to host plant’s DNA
(Chilton, 1983). Tumor inducing genes present in T-DNA part of plasmid can be replaced with desired genes like those conferring disease/pest resistance. This
70
genetically modified Agrobacterium is then allowed to infect plant cell or a wounded tissue which can regenerate into a full grown plant.
Eapen and George (1994) co-cultivated primary leaf explants with
Agrobacterium strain LBA4404 containing the GUS and NPTII reporter genes and cultured on regeneration medium supplemented with kanamycin to select transformed shoots. Transformants were subsequently rooted and plantlets were transferred to soil. Stable insertion and expression of the transgenes were confirmed by kanamycin/GUS assay and Southern blot.
Cheng et al. (1997) studied the inheritance of transgenes in sexually derived progenies of transformed peanut and found 3:1 expression in some plants while in others it was 100 per cent expression of the GUS gene in T2 plants, indicating homozygous and heterozygous T1 plants. PCR analysis confirmed the results of
GUS assay. It is evident from results that transgenes transferred to peanut through
Agrobacterium can be inherited through Mendelian manner.
Egnin et al. (1998) calculated the transformation frequency of GUS gene driven by CaMV 35S promoter by counting the number of patches expressing
GUS activity on peanut leaf and epicotyl explants cultured on different hormonal combinations. They concluded that high concentration of 2, 4-D (1.0 or 2.5 mg/liter) and low BAP (0.25 or 0.5 mg/liter) resulted in higher transformation efficiency than thidiazuron-containing or hormone-free medium. The improved protocol was used to achieve transformation frequencies ranging from 12- 36 per
71
cent for leaf and 15-42 per cent for epicotyls. These results were endorsed by PCR and Southern blot analysis.
Livingstone and Birch (1999) reported a highly efficient transformation method in groundnut. This method comprised of three steps: particle bombardment into embryogenic callus derived from mature seeds, escape-free (not stepwise) selection for hygromycine resistance and brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media. This resulted in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produced three to six independent transformants per bombardment of 10 cm2 embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment.
Transgene copy number ranged from one to twenty with multiple integration sites.
Rohini and Rao (2000) conducted some transformation experiments using
Agrobacterium strain LBA 4404 containing binary vector pKIWI105 which carried the GUS and NPT-II genes. GUS assay and PCR analysis confirmed that 3.3 per cent of the plants were transfomed. Molecular analysis of T0, T1 and T2 generation had shown successful insertion, expression and inheritance of foreign genes in groundnut.
Luo et al. (2005) identified genes for resistance to LLS in groundnut using micro array and real-time polymerase chain reaction (PCR). They detected 56 genes in several functional categories. Seventeen of the 20 most effective genes
72
were selected for validation and they proposed to develop characterized gene probes for marker-assisted selection in breeding programmes.
Zheng et al. (2005) manipulated the vitamin and salt combination of the medium and concluded that glutathione, sodium selenite and DL- alpha -tocopherol increased the regeneration frequency and transformation efficiency of groundnut by
Agrobacterium tumefaciens. They pointed out that ascorbic acid is not suitable antioxidant in MS medium due to the stimulation of oxidation in the presence of iron.
Yogranjan et al. (2006) reported that shoot differentiation via callusing was attained on MS medium supplemented with 2 mg/lit BA, 0.1 mg/lit Indole Acetic
Acid, and 150 mg/lit adenine sulphate, whereas direct shoot differentiation was achieved on medium containing 0.1 mg/lit TDZ. They also found that a strong
GUS expression was detected in the putative transgenic shoots of groundnut by the histochemical assay. Transformation was confirmed by the presence of 0.70 kb amplified fragments for NPT-II and 1.40 kb for uidA genes by PCR analysis.
5.3 MATERIALS AND METHODS
5.3.1 Explant Preparation
Only the Golden variety was used in transformation experiments as it had shown best results in tissue culture experiments. Freshly shelled seeds were treated with 70 per cent ethanol for one minute and then with 20 per cent clorox for ten
73
minutes. The seeds were rinsed with distilled autoclaved water for five times and then soaked in the same water for two hours.
The seed testa was removed, cotyledons were separated, radical and plumule were removed and each cotyledon was cut longitudinally into two halves.
In this way four explants were obtained from each seed. Ten explants were used in each treatment with three replications.
5.3.2 Bacterial Strain and Vector
The Agrobacterium strain LBA-4404 harbouring pB1333 binary vector was employed for transformation. The plasmid carried rice chitinase (RCG-3) and hygromycin phosphotransferase (hpt) genes driven by EN4 and CaMV 35S promoters respectively. Kanaymycine gene was present outside tDNA for bacterial selection (Figure 5.1)
Five ml of Agrobacterium culture (OD=0.5), grown overnight at 280C in
LB medium (pH=7.2) was placed in sterile glass plates and explants were submersed in it for 2 minutes. The explants were removed from culture, blotted on sterile filter paper and inoculated in petri plates containing shoot induction medium
(SIM) as described in chapter 2. The plates were sealed with parafilm and incubated at 28 oC for 48, 72, and 96 hours in dark. Control experiment, lacking co-cultivation was also conducted in parallel.
5.3.3 Determination of Lethal Dose of Hygromycin for In Vitro Grown Shoots
Shoots from control explants were shifted to SIM containing different concentrations (15, 20, 25, 30, 35 mg/l) of hygromycin in five replicates to determine the lethal dose.
74
5.3.4 Selection and Rooting of Putative Transformed Shoots
At the end of co-cultivation period, explants were rinsed thrice with sterile cefotexime solution (250 mg/ml) followed by washing with sterile water. The explants were then inoculated on SIM containing cefotexime at the rate of 250 mg/ml and kept at 25ºC with 16/8 hours photoperiod for three weeks. Shoots were separated from explants with sharp surgical blade and placed on selective SIM
(SIM with 25mg/ml hygromycin) for two weeks. Approximately 5 cm long shoots were shifted to root induction medium (RIM) as explained in chapter 2. Fully rooted plants were shifted to coconut husk compost in small cups, covered with transparent polythene bags to maintain humidity and kept in green house. The bags were removed gradually to avoid sudden desiccation shock to plants. After three weeks the plants were shifted to bigger pots (30 x 30 cm) where they flowered and produced seeds normally.
5.3.5 PCR Analysis of Putative Transgenic Plants
DNA from putative (T0) and control peanut plants was isolated by CTAB method. For PCR analysis of the RCG-3 in the genome of transformants, forward primer 5’-CATATCAAGCATGAGGTGTA-3’ and reverse primer 5’-
CAACAACGATTTT GCTATAA-3’ were employed to amplify a 696 bp fragment. DNA was denatured at 94ºC for 3 minutes for first time and for 30 seconds in each cycle. Annealing temperature was 520C for 30 seconds while extension was done for 45 seconds at 720C. After the completion of 40 cycles, final extension was given at 720C for twenty minutes.
75
Following PCR profile was used for amplification
PCR Steps Time Number of Cycles
Denaturation at 94 oC 3 min 1
Denaturation at 94 oC , 30 sec
Annealing at 52 oC 30 sec 40
Extension at 72 oC 45 sec
Final Extension at 72 oC 20 min 1
Standby temperature 4 oC
Lid temperature 105 oC
Eppendorf Thermal cycler (96 well) was used for amplification.
5.3.6 Gel Electrophoresis
PCR product was electrophoresed on 1 per cent agarose gel for 30 minutes at 100 volts and viewed under UV light by using Biorad gel documentation system.
5.3.7 Southern Blot
For southern blot analysis, 10µg DNA of four transgenic (T0) plants was digested with Hind III endonuclease, electrophoresed on a 0.8 per cent agarose gel and blotted on a nylon membrane. Non-radioactively labelled (Fermentas Biotin
DecaLabel DNA labeling kit) 696 bp PCR-amplified chitinase fragment was used as probe.
76
Ca.3.8 Kb
EcoRI Hind III SacI SacI EcoRI
RB LB P 35S HPT TCaMV PEN4 RCG3 T NOS
Kmr pB1333-EN4-RCG3 ca.1.4 Kb ca.1.1 Kb
Figure 5.1: Diagram for vector containing RCG-3 under EN4 promoter
Table 5.1: Quantities of different reagents in PCR mixture.
Reagents Quantity(µL)
Buffer (10X) 5
MgCl2 (25 mM) 4
Dntp (10 mM) 1
Taq polymerase (5u µL-1) .5
Primer forward 1.0
Primer reverse 1.0
Template 5
Deionized double distilled water. 32.5
Total Volume 50
77
5.3.8 Determination of Lethal Dose of Hygromycin for Field Grown T1 Plants
Twigs of 25 days old plants were detached and their cut ends were submerged in Hoagland solution containing different concentration of hygromycin
(50, 100,150 and 200 mg/l) in five replicates to determine the lethal dose.
5.3.9 Selection of T1 plants
Lateral twigs of 25 days old T1 plants, growing in field, were detached and their cut end was submerged in Hoagland solution containing 150mg/lit hygromycin (previously determined lethal dose for twigs of field growing plants) in test tubes. Plants showing necrosis and wilting were discarded while those retaining normal green colour and vigour were selected and shifted into bigger (30 x 30 cm) earthen pots. Presence of transgenes was confirmed by PCR analysis in these plants.
5.3.10 Pathogenicity Test
Ten hygromycin resistant T1 plants were subjected to pathogenicity test.
Spores of Cercosporadium personatum were collected in distilled autoclaved water by thoroughly washing a large number of infected leaves. Concentration of spores was measured by haemocytometer and was adjusted to the concentration of 2 x 105 spores/ ml. Resistance of control and transgenic plants was evaluated by spraying of spores on leaves till run off. 100 per cent humidity was maintained by using humidifier in greenhouse. Leaf area was measured by leaf area meter and necrotic area ( per cent) was calculated by multiplying average size of lesion with number of lesions divided by leaf area.
78
5.3.11 RT-PCR Analysis
RNA from control and confirmed 10 transgenic (T1) plants was extracted using TRI reagent® (Sigma-Aldrrich). cDNA was synthesized using Omniscript®
Reverse Transcription Kit of Qiagen company following manufacturer’s instructions. Total reaction volume was 20µl having 2µg RNA, 2µl 10 x RT
Buffer, 2µl dNTP Mix, 2µl Oligo-dT primer (10 μM), 1µl RNase inhibitor (10 units/μl) and 1µl Reverse Transcriptase. PCR was performed using the primers mentioned above in section 5.3.5 and product was separated on 1 per cent agarose gel.
5.4 RESULTS AND DISCUSSION
5.4.1 Effect of Co-cultivation Period and Acetosyringone Concentration
Analysis of variance showed that variation in co-cultivation period and acetosyringone concentration affected transformation efficiency highly significantly (Table 5.2).
5.4.1.1 Co-cultivation Period
It is evident from Table 5.3 that co-cultivation period of three days (72 hours) is optimum and it gives maximum transformation efficiency of 12.51 per cent. Longer co-cultivation results in overgrowth of Agrobacterium which is not controlled by antibiotic, hence decreasing the recovery of transformants (8.90 per cent). Shorter co-cultivation also produced significantly low number of transgenic shoots (3.55 per cent).
79
Co-cultivation period of two days was found best in many legumes such as pigeonpea (Shrivastava et al., 2001), chickpea (Kar et al., 1996; Husnain et al.
1997) and mashbean (Karthikeyan et al., 1996). Barik et al, (2005), however, obtained maximum transformants on 4 days of co-cultivation in grasspea. Plant species, explant tissue, Agrobacterium strain and nature of medium might be responsible for this variation.
5.4.1.2 Acetosyringone
Duncan’s Multiple Range Test demonstrated (Table 5.4) that addition of acetosyringone at a concentration of 100 mg/l was highly beneficial as transformation efficiency was raised to 10.08 per cent in comparison to 5.18 per cent in control. Further increase in Acetosyringone concentration decreased transformation efficiency slightly (9.70 per cent).
To further refine the picture, the interaction means has been shown graphically in Figure 5.2. Maximum transformation efficiency (18.14) was achieved when explants were co-cultivated for 72 hours at 100 mg/l acetosyringone concentration while no transformant was recovered when explants were co- cultivated for 48 hours at acetosyringone free medium.
Acetosyringone is mostly added in co-cultivation medium in case of monocots; however it is also beneficial in dicots. Becker et al. (1994) recommended 100µM acetosyringone for maximum transformation efficiency in
Phaseolus vulgaris while Clercq et al. (2002) observed that 200 µM concentration of AS was optimum in Phaseolus acutifolius transformation.
80
5.4.2 Determination of Lethal Dose of Hygromycin for In Vitro Grown Shoots
At hygromycin concentration of 25 mg/l and above, growth of explants stopped and they started turning brown. At 20 mg/l the growth was affected but there was no browning or necrosis. Below this concentration the shoots showed normal growth pattern. Hence 25 mg/l hygromycin was used for selection of transformed shoots.
5.4.3 PCR Analysis of Putative Transgenic Plants
DNA from six putative transgenic plants surviving till maturity and control plants was isolated and subjected to PCR analysis for presence of RCG3 gene. The band of required size 696 bp was observed in all the 6 plants and positive control while no such band was observed in control plant (Figure 5.3).
5.4.4 Southern blot
Southern blot analysis of four surviving plants showed three copies of transgene in plant No. T0-1, two copies in plant No. T0-2 and T0-4. Only plant No.
T0-3 has single copy, so its progeny was selected for further studies (Figure 5.4).
5.4.5 T1 generation
As To plants were developed through tissue culture, they were comparatively weak and could produce a small number of seeds. Only 16 seeds were obtained from T0-3 plant which were shelled and sown in 30 x 30 cm pots in next sowing season with normal cultural practices.
81
Table 5.2: Analysis of variance for factors affecting transformation efficiency.
Source of Variance Degree of Sum of Mean Square F Value
Freedom Squares
Co-cultivation Period 2 366.153 183.077 221.75**
Acetosyringone 2 133.523 66.76 80.86**
Interaction 4 8.106 2.026 2.45 NS
Error 18 14.861 0.826
Total 26 522.643
Table 5.3: Mean values of transformation efficiency at different co-cultivation periods ranked by Duncan’s Multiple Range Test.
Co-cultivation Period Mean No. of hygromycin Ranking
(Hrs) resistant shoots
72 12.51 A
96 8.90 B
48 3.55 C
Values followed by same alphabet do not differ significantly
82
Table 5.4: Mean values of transformation efficiency at different acetosyringone concentrations ranked by Duncan’s Multiple Range Test.
Acetosyringone Conc. Mean No. of hygromycin Ranking
(mg/l) resistant shoots
100 10.08 A
200 9.704 A
0 5.187 B
Values followed by same alphabet do not differ significantly
Figure 5.2: Effect of acetosyringone and length of co-cultivation period on production of hygromycin resistant shoots ( per cent)
83
Figure 5.3: Amplification of RCG 3 gene fragment by PCR in 4 surviving T0 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant;
Lanes 1-4: transgenic plants (T0-1 to T0-4)
T0-1 T0-2 T0-3 T0-4 -
+
Figure 5.4. Southern blot analysis of four surviving T0 plants
84
Figure 5.5. Cut twigs of plants on different concentrations of hygromycin.
Figure 5.6. Amplification of RCG 3 gene fragment by PCR in T1 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant; Lanes 1-
16: T1 plants
85
5.4.6 Determination of Lethal Dose of hygromycin for field grown plants
At hygromycin concentration of 25 mg/l and above, growth of explants stopped and they started turning brown. Below this concentration the shoots showed normal growth pattern. Hence 150 mg/l was used as a lethal dose for field grown plants (Figure 5.5).
5.4.7 Selection of T1 plants
Out of sixteen, ten hygromycin resistant transgenic plants were selected by detached cut twig method and presence of transgene was confirmed by PCR
(Figure 5.6). The inheritance pattern was not in exact conformity with Mendelian law; however final conclusion cannot be drawn on the basis of such a small sample.
5.4.8 Pathogenicity Test for Intact Plants
Ten randomly selected T1 plants along with a control plant were subjected to leaf spot inoculum spray on lower and upper surface of leaves (Figure 5.7 ). One upper most leaf, comprising of three leaflets, was tagged from three main branches of each plant for disease evaluation. It is clear from Table 5.5 and Figure 5.7 that infection frequency (IF, number of lesions per cm2 of leaf area), incubation period
(IP), lesion diameter (LD), leaf area damage (LAD) and disease score are significantly high in control plant as compared to transgenic plants. This indicates the practical importance of transgenic to control the leaf spot disease.
5.4.9 RT-PCR Analysis
RT-PCR of 10 T1 plants showed bands in all the plants, brightness and thickness of bands, however, differed significantly (Figure 5.5). In plant number
86
T1-3, T1-4 and T1-10 the bands were much brighter and thicker indicating higher number of mRNA transcripts which in turn gav higher expression of chitinase enzyme. In control plant there was no such band while in plant number T1-2 and
T1-8 the bands were comparatively less bright indicating less mRNA copies, hence lower expression.
A transgenic production system can be called best if it is fast, produces abundant non-chimeric plant with least tissue culture steps and avoids costly and lobours media preparations (Tiwari and Tuli, 2012). In most of tissue culture based transformation methods whole plant is regenerated from a single transformed cell and differentiated tissues are not used as recipients of foreign DNA (Higgins et al.,
2004).
The current study has further strengthened the evidence that transgenic plants can be successfully produced using differentiated tissues like cotyledons.
Some researchers obtained stable transgenic peanut plants from fully differentiated explants like epicotyls (Egnin et al., 1998; Qiusheng et al., 2004) and cotyledons
(Tiwari et al., 2007; Tiwari and Tuli, 2008; Tiwari and Tuli, 2012).
It has been observed by many researchers that Kanamycin does not kill non-transformed cells completely, enabling pseudo positive or chimeric plants to
87
Figure 5.7: Pathogenicity test of intact plants for susceptibility to late leaf spot
disease: transgenic (right) and control (left) plant
45
40
35
30 Incubation Period 25 Infection Frequency Lesion Diameter 20 Leaf Area Damage (%) Disease Score 15
10
5
0 1 2 3 4 5 6 7 8 9 0 ol - 1 tr T1 T1- T1- T1- T1- T1- T1- T1- T1- n T1- o C
Figure 5.8: Means for Infection frequency, incubation period, lesion diameter,
leaf area damage and disease score of transgenic and control plants.
88
Table 5.5: Table of means for Infection frequency (IF, number of lesions/cm2 leaf area), incubation period (IP, number of days from inoculation to appearance of first lesion), lesion diameter (LD), leaf area damage (LAD) and disease score of transgenic and control plant (each value is mean of three leaves each having three leaflets, however disease score was on whole plant basis)
Plant No. IP IF LD LAD SCORE
T1-1 10.67 0.48 2.2 1.84 2
T1-2 9.67 0.38 2.04 2.83 2
T1-3 12.33 0.47 2.14 1.76 2
T1-4 13.33 0.6 2.45 1.67 2
T1-5 12.67 0.6 1.83 1.56 2
T1-6 10.67 0.54 2.27 2.21 2
T1-7 12.33 0.35 1.63 0.75 1
T1-8 10.11 0.28 2.07 2.5 2
T1-9 12.67 0.68 2.1 2.37 2
T1-10 12.33 0.29 2.38 1.38 2 Control 7.33 1.29 6.22 39.12 6
Figure 5.9: RT-PCR for RCG-3 gene in T1 plants. Lanes 1-10 represent the plant numbers T1-1 to T1-10 respectively while C indicated control plant. The brightness and thickness of band has been compared with bands of housekeeping gene actin from peanut
89
be selected (Cheng et al., 1996; Sharma and Anjaiah, 2000; Dodo et al., 2008). On the other hand hygromycin not only eliminates the non-transformed cells completely, but also increases the potential for multiple shoot induction (Tiwari and Tuli, 2012). Moreover, probability of inducing somaclonal variation is reduced by the short duration cycle of transformation and regeneration (Olhoft et al., 2003).
Resistance against different fungal diseases has been achieved by over expression of rice chitinase in different plant species including bread wheat (Chen et al., 1998), rice (Lin et al., 1995), strawberry (Asao et al., 1997), grapevine
(Yamamoto et al., 2000), pigeon pea (Kumar et al., 2004) and taro (He et al.,
2008).
In present study it has been proven that rice chitinase gene (RCG-3) confers strong resistance against leaf spot disease of peanut. The stable peanut lines hence produced will be helpful to evolve cultivars with built-in solution to control fungal diseases, especially the leaf spot.
90
Table 5.6: Modified 9-point scale used for screening groundnut genotypes for resistance to late leaf spot disease
Disease Disease Description Damaged area score of leaf (%)
1 No disease 0 2 Lesions present largely on lower leaves; no defoliation 1-5 3 Lesions present largely on lower leaves, very few on middle 6-10 leaves; defoliation of some leaf lets evident on lower leaves 4 Lesions present on lower and middle leaves but severe on 11-20 lower leaves; defoliation of some leaflets evident on lower leaves 5 Lesions present on lower and middle leaves, over 50 % of 21-30 defoliation of lower leaves 6 Severe; eosins on lower and middle leaves; lesions present but 31-40 less severe on top leaves; extensive defoliation of lower eaves; some defoliation on middle leaves 7 Lesions on all leaves but less severe on top leaves; defoliation 41-60 of all lower and middle leaves 8 Defoliation of all lower and middle leaves; severe lesions on 61-80 top leaves evident. 9 Almost all leaves defoliated, leaving bare stem; some leaflets 81-100 may remain, but show severe leaf spot
Chapter 6
SILICON CARBIDE WHISKER MEDIATED
TRANSFORMATION
6.1 INTRODUCTION
Plant cell wall is commonly found as the non-living barrier in the ways of
DNA delivery technologies being attempted for plant genetic engineering. In case of biological systems, the cell wall is dissolved by cell wall degrading enzymes secreted by donor host for contact of donor cell with recipient cell allowing exchange of biological materials along with net DNA delivery into recipient cells.
But this limitation cannot be overcome in monocots which are non-host for prokaryotic mediated deliveries. The cell wall problem in monocots was ruled out by use of protoplasts, biolistic particles penetration opening the way for DNA entry into plants whether monocots or dicots. Protoplasts are an excellent target for genetic transformation, but regeneration of protoplasts to plants is problematic.
Genetic transformation by gene gun is also getting increasing attention but it requires sophisticated, expensive equipments, expertise and consumables.
Microinjection method also requires highly skilled workforce. Electroporation method has wider application range but transformation efficiency is very low. In current circumstances, Silicon Carbide Whiskers are very favorable means of direct gene delivery as it is very simple and does not require much technical equipment and skill.
Studies show that Silicon carbide whiskers have high tensile strength and elastic modulus alongwith having resistance to shock and degradation (Choi et al.,
91
92
1997). Silicon carbide whiskers are inherently hard and are cleaved easily producing sharp cutting edges (Greenwood and Earnshaw, 1984). These whiskers are obtained by the thermal reduction of silica, which can also be obtained from rice husks (Mutsuddy, 1990). Silicon Carbide whiskers are used in industries to sharpen cutting tools and in the production of amalgamated materials. Silicon carbide and other whiskers from different sources have been utilized in the transformation of monocot and dicot plant species embryo and cell suspension cultures. The mechanism for whisker-mediated transformation is based on micro puncturing of cells for DNA delivery. Scanning electron micrograph by Kaeppler et al., (1990) suggested that a Silicon Carbide whisker pierced the cell wall of Zea mayz. The surface of Silicon Carbide whiskers is negatively charged contrary to asbestos fibers (Appel et al., 1988). As DNA is also negatively charged, so it cannot bind to whiskers for ultimate delivery into the host cell. Kaeppler et al.,
(1990, 1992) also ruled out the benefit of premixing the DNA with whiskers. It means that whiskers only puncture the cell during vortex process paving the way for DNA entry.
This study was conducted to develop a rapid, simple, economical and less laborious protocol for transformation of local peanut with higher efficiency using silicon carbide whiskers.
6.2 REVIEW OF LITERATURE
Cell wall is a big barrier in transfer of DNA from one cell to another during genetic transformation of plant cells. In some plants, cell wall is degraded by host
93
cells during Agrobacterium mediated transformation but this is not true in all cases making the process less efficient. To overcome this hurdle protoplasts are used for transformation but regeneration in protoplasts is again a problem. Therefore, gene gun or biolistic method of transformation is getting much popularity in plants where transformation and regeneration is problematic.
Silicon carbide whisker mediated transformation is probably the simplest known method of genetic transformation. It involves vortexing of target tissues along with plasmid (harboring gene of interest) in presence of silicon carbide.
Silicon carbide is micro-needle shaped hardest man-made material used as an abrasive and is component of saw blades (Dunwell, 2011).
Studies reveal that silicon carbide whiskers have high tensile strengths, elasticity and resistance to degradation (Choi et al., 1997). Moreover they have high intrinsic strength and their easy cleavage produces sharp cutting edges needed to penetrate into nucleus of cells (Greenwood and Earnshaw, 1984).
In industries these whiskers are used as abrasive material to manufacture and sharpen the cutting tools. Silicon carbide whiskers have been utilized in transformation of tobacco and maize (Kaelper et al., 1992), rice (Nagatani et al.,1993), wheat (Omirulleha et al., 1996) ryegrass ( Dalton et al.,1998), soybean (
Khallafalla et al., 2006) and cotton ( Asad et al., 2008).
94
Electron microscope scan has shown that whisker penetrate into nucleus of maize thus perforating the cell to pave way for delivery of DNA (Kaeppler et al.,
1990). Like DNA, whisker surface is also negatively charged, hence DNA is not attached to whisker, however, the perforation caused by whiskers facilitates DNA movement to nucleus. Somatic embroygenic callus is a pre-requisite for silicon carbide whisker mediated transformation. In Gene gun or PEG methods, callus cells are used as target tissues but in case of whisker mediated transformation cell suspension is used making the quantity of DNA quite adjustable. Moreover, other direct transformation methods deliver multiple copies of transgene making the process complicated, but in case of silicon carbide whisker mediated transformation usually a single copy is delivered (Asad et al., 2008).
Transformation efficiency varies with types of whiskers or even manufacturing process of same whiskers. Different forms of silicon carbide whiskers like AA, TW, Alfa Aesar and Sc-9 have been utilized in transformation.
In maize transformation, SC-9 has shown five times higher efficiency than other forms used.
Mixing method and duration are very important in achieving high transformation efficiency. Vortex mixing is the most common and initial method for mixing callus and whiskers (Asano et al., 1990; Dunahay et al., 1993; Kaeppler et al., 1990; 92). Increased quantity of whiskers, fast, vigorous and prolonged shaking increases the transformation efficiency but survival of cells and their regeneration is adversely affected (Frame et al., 1994; Petolino et al., 2000;
95
Mizuno et al., 2004). So, a balance of whisker quantity, mixing speed and survival rate is needed for better efficiency.
Before mixing with plasmid and whiskers, callus is given some treatment to increase the transformation efficiency. Maize callus incubated for 30 minutes with
1 ml of N6 medium having 0.5 M sorbitol and mannitol increased the transformation efficiency 3-5 times as compared to non-treated callus (Vain et al.,
1993). Moreover, duration of intervals for subculture also affected the transformation efficiency in cotton (Asad et al., 2008). In Agrostis alba plants best results were obtained when transformation was done after 6 days of subculture
(Asano et al., 1991) while in cotton 14 days were best(Asad et al., 2008)
Kindle (1990) observed only 10% cell survived if callus of clamydomonas is vortexted for 3 minutes with glass beads and PEG while cell survival increased to 80%, if vortexed for same duration with silicon carbide whiskers. He recovered
10-100 transformants from107 cells using silicon carbide whiskers.
Transformation of dry wheat embryos by Serik et al (1996) through silicon carbide whiskers negated the old concept that only suspension culture could be used for transformation using this technique.
Matsushita et al., (1999) conducted two experiments for transformation of rice through silicon carbide. Firstly, they vortexed scuteller tissues of embryos along with pAct1-F plasmid harbouring GUS gene and silicon carbide in liquid medium. They got 302 GUS spots in 250mg sample. In second experiment, they
96
utilized two plasmids; pAct1-F having GUS gene and pDM302 having bar gene
(which confers resistance to Bialaphos). 873 embryos were used in the experiment and 57 calli were proved to be bialaphos resistant, some of which were also GUS positive. They also reported that vortexing duration of 30 minutes was optimum.
6.3 MATERIALS AND METHODS
6.3.1 Production of Embryogenic Callus
Embryogenic callus was produced by inoculating the epicotyl explants of the best responsive variety, Golden, on callus induction medium (CIM) as described in Chapter 4.
6.3.2 Preparation of Silicon Carbide Whiskers and DNA Delivery
Different quantities of silicon carbide whiskers (100, 200 and 300 mg) were added to a pre-weighed 50 ml plastic tube and covered with aluminum foil and autoclaved at 121°C for 15 minutes. 5 ml of separately autoclaved callus induction medium and 5µg of plasmid (containing RCG-3 gene as mentioned in chapter 6) were added into this tube and vortexed for two minutes. 2 g of embryogenic callus
(10, 20, and 30 days old) was added into this tube and vortexed again for 2 minutes. Control experiment lacked plasmid DNA.
6.3.3 Selection for Stable Transformants
Small clumps of callus treated with silicon carbide whiskers were shifted to petri plates containing callus induction medium with 30mg/l hygromycin (lethal dose). Surviving calli were sub cultured on fresh medium after every two weeks.
97
Callus was regenerated into plantlets through somatic embryogenesis as described in Chapter 4.
6.3.4. PCR Analysis
DNA from putative (T0) and control peanut plants was isolated by CTAB method. For PCR analysis of the RCG-3 in the genome of transformants, same primers and PCR profile was used as described in section 5.3.5.
6.3.5 Selection of T1 Plants
Lateral twigs of 25 days old T1 plants were detached and their cut end was submerged in Hoagland solution containing 150mg/lit hygromycin in test tubes having cotton plugs on their mouths. Plants showing decolouration, necrosis and wilting were discarded while those retaining normal green colour and vigour were selected and shifted into bigger (25 x 25 cm) earthen pots.
6.3.6. Pathogenicity test for intact plants
Six intact plants from which the twigs were cut for above mentioned test were subjected to pathogenicity test. Spores of Cercosporadium personatum were collected in distilled autoclaved water by thoroughly washing a large number of infected leaves. Concentration of spores was measured by haemocytometer and was adjusted to the concentration of 2 x 105 spores/ ml. Resistance of control and transgenic plants was evaluated by spraying of spores on leaves till run off. 100% humidity was maintained by using humidifier in green house. Leaf area was measured by leaf meter and necrotic area (%) was calculated by multiplying average size of lesion with number of lesions divided by leaf area.
98
6.3.7. RT-PCR analysis
RNA from control and confirmed 5 transgenic (T2) plants was extracted using TRI reagent® (Sigma-Aldrrich). cDNA was synthesized using Omniscript®
Reverse Transcription Kit of Qiagen company following manufacturer instructions.
Total reaction volume was 20µl having 2µg RNA, 2µl 10 x RT Buffer, 2µl dNTP
Mix, 2µl Oligo-dT primer (10 μM), 1µl RNase inhibitor (10 units/μl) and 1µl
Reverse Transcriptase. PCR was performed using the primers mentioned above in section “PCR analysis” and product was separated on 1% agarose gel.
6.4 RESULTS AND DISCUSSION
Analysis of variance showed that callus age, whisker quantity and their interactions had highly significant effect on transformation efficiency (Table 6.1).
Highest transformation efficiency (6.88%) was achieved when 200 mg of whisker was used for 20 days old callus while lowest (2.00%) was observed at 100 mg whisker on 30 days old callus (Figure 6.1). Callus cells must be at an optimum turgidity at the time of treatment, so that whiskers can pierce into them to delivered the desired foreign DNA (Asad et al, 2008).
Callus cell at the age of 10 days are fully turgid and hence they are more prone to excessive damage by piercing of whiskers (Petolino et al 2000). On the other hand 30 days old callus cells lose much of their turgidity and are least wounded by whiskers decreasing the transformation efficiency. Callus cells at the age of 20 days seem to be at an optimum turgidity for delivery of foreign DNA in this case giving maximum transformation efficiency.
99
Table 6.1: Analysis of variance for factors affecting transformation efficiency.
Source of Degree of Sum of Squares Mean Square F Value
Variance Freedom
Callus age 2 15.5 7.75 20.92**
Whisker 2 10.66 5.33 14.4** quantity
Interaction 4 11.66 2.917 7.87**
Error 18 6.667 0.37
Total 26 44.5
8.000 7.000
shoots 6.000
5.000 4.000 3.000 transgenic 2.000 of 1.000 0.000
Number 100 mg 200 mg 300 mg 100 mg 200 mg 300 mg 100 mg 200 mg 300 mg Whisker Whisker Whisker Whisker Whisker Whisker Whisker Whisker Whisker 10 days 20 days 30 days Whisker concentration and callus age
Figure 6.1: Effect of callus age and whisker concentration on production of hygromycin resistant shoots (%)
100
6.4.1 T0 generation
Embryos showing normal growth in selective medium (Figure 6.2) were subcultured on the same medium to confirm their resistance to hygromycin. Out of
4 hygromycin resistant plants obtained, required band of 696 bp was observed in three plants in PCR analysis confirming the presence of RCG3 gene (Figure 6.3).
These plants were shifted to coconut husk compost, where they reached maturity and produced seeds normally.
Southern blot analysis showed a single copy of gene in T0-3 plant only while two copies were observed in remaining two plants (Figure 6.4)
6.4.2 T1 generation
Among the progeny as of T0-3 plant, 6 were hygromycin resistant while 4 were sensitive. PCR analysis for presence of RCG-3 gene revealed the required band of 696bp in the hygromycin resistant plants, while no band in sensitive plants was observed (Figure 6.5).
6.4.3 Pathogenicity Test
The upper most leaf, comprising of three leaflets, was tagged from three main branches of each plant for disease evaluation. It is clear from Figure 6.7 that incubation frequency (IF, number of lesions per cm2 of leaf area), incubation period (IP), lesion diameter (LD), leaf area damage (LAD) and disease score are significantly high in control plant as compared to transgenic plants. This indicates the practical importance of transgenic to control the leaf spot disease.
101
Figure 6.2: Emergence of hygromcine resistant shoots in selection medium
L + - 1 2 3
Figure 6.3: Amplification of RCG 3 gene fragment by PCR in 3 surviving T0 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant;
Lanes 1-3: transgenic plants (T0-1 to T0-3)
102
Figure 6.4: Southern blot analysis of three PCR positive plants
Figure 6.5: Amplification of RCG 3 gene fragment by PCR in T1 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant; Lanes 1-
11: T1 plants
103
Figure 6.6: Pathogenicity test of intact plants for susceptibility to late leaf spot
disease: control (above) and transgenic (below) plant
Figure 6.7: Means for Infection frequency, incubation period, lesion diameter,
leaf area damage and disease score of transgenic and control plants
C 1 2 3 4 5
EN4‐RCG3
Actin
104
Figure 6.8: Six PCR positive plants from progeny of T0-3 plant were subjected to RT-PCR analysis
6.4.4. RT-PCR analysis
RT-PCR of 6 PCR positive T1 plants from the progeny of T0-1 plant showed bands in all the plants, brightness and thickness of bands, however, differed significantly. In plant number 1, 3 and 6 the bands were much brighter and thicker indicating higher number of mRNA transcripts which in turn gives higher expression of chitinase enzyme (Figure 6.8). In control plant there was no such band while in plant number 2, 4 and 5 the band was comparatively less bright indicating less mRNA copies, hence lower expression.
Silicon carbide whisker mediated transformation is probably the simplest known method of genetic transformation. It involves vortexing of target tissues along with plasmid (harbouring gene of interest) in presence of silicon carbide.
Silicon carbide is micro-needle shaped hardest man-made material used as an abrasive and is component of saw blades (Dunwell, 2011)
Studies reveal that silicon carbide whiskers have high tensile strengths, elasticity and resistance to degradation (Choi et al., 1997). Moreover they have high intrinsic strength and their easy cleavage produces sharp cutting edges needed to penetrate into cell walls and ultimately nucleus of cells (Greenwood and
Earnshaw, 1984).
105
Electron microscope scan has shown that whisker penetrate into nucleus of maize thus perforating the cell to pave way for delivery of DNA (Kaeppler et al.,
1990). Like DNA, whisker surface is also negatively charged hence DNA is not attached to whisker, however, the perforation caused by whiskers facilitates DNA movement to nucleus.
Increased quantity of whiskers and prolonged shaking increases the transformation efficiency but survival of cells and their regeneration is adversely affected. So a balance of whisker quantity, mixing speed and survival rate is needed for better efficiency (Frame et al., 1994; Petolino et al., 2000; Mizuno et al., 2004).
Moreover, callus age affected the transformation efficiency greatly in different crops. In Agrostis alba plants best results were obtained when transformation was done after 6 days of subculture (Asano et al., 1991) while in cotton 14 days were best(Asad et al., 2008). Matsushita et al. (1999) conducted two experiments for transformation of rice through silicon carbide. Firstly they vortexed scuteller tissues of embryos along with pAct1-F plasmid harbouring GUS gene and silicon carbide in liquid medium. They got 302 GUS spots in 250mg sample. In second experiment they utilized two plasmids; pAct1-F having GUS gene and pDM302 having bar gene (which confers resistance to Bialaphos). 873 embryos were used in the experiment and 57 calli were proved to be bialaphos resistant, some of which were also GUS positive.
106
No report of any previous work on transformation of peanut through silicon carbide whiskers was found in literature for comparison of this study.
Chapter 7
TRANSFORMATION USING GENE GUN
7.1 INTRODUCTION
Foreign genes can be delivered indirectly through Agrobacterium or directly via microprojectile bombardment. Microprojectile bombardment involves the coating of gold or tungsten particles with DNA and accelerating them at high velocity into target plant tissue. Some particles will penetrate into the nuclei of some of the cells, where the DNA may integrate into the plant's genome. Cells that survive the impact and are able to regenerate can give rise to whole transgenic plants.
The choice between using microprojectile bombardment or Agrobacterium as the means by which to deliver DNA is determined by several factors including the laboratory facilities and technical skills available, the species and/or cultivar to be transformed, and the regeneration system. There is a general belief that
Agrobacterium-mediated transformation is preferable to microprojectile bombardment for gene delivery. This belief is based on several assumptions, which are becoming less compelling due to recent new information. Agrobacterium- mediated transformation is thought to result in fewer integrated transgene copies, thus reducing the risk of transgene rearrangements and gene silencing. Further,
Agrobacterium is considered a 'cleaner' approach, since the T-DNA is thought to be the only DNA which is transferred. It is now known that DNA from outside the T-
DNA region can also be transferred into the plant's genome (Smith, 1998).
106
107
Microprojectile bombardment can result in multiple transgene copies. However, we have observed in many cases that the copy number is equivalent to that observed using Agrobacterium. The transgene copy number and hence the chances of gene rearrangements can be reduced by careful optimisation of bombardment conditions.
Thus microprojectile bombardment is an appropriate means by which we can genetically modify important crop species, including peanut.
The use of microprojectile bombardment as a means of developing transformed peanut plants was first reported by Ozias-Akins et al. (1993). This method was developed using immature peanut seeds as the source of explants.
Bombardment of 1-2 year old embryogenic callus derived from immature embryos and cotyledons resulted in 1 % of bombarded callus pieces producing stable, transformed lines. It was not reported if the plants were fertile. However, a more recent report indicated that the efficiency has been improved 25-fold and that plants are capable of producing pods (Ozias-Akins et al., 1996). There have been other reports of microprojectile bombardment of peanut tissue. For example. Livingstone and Birch (1995) and Clemente et al. (1992) reported bombarding leaflets from mature embryos. However, neither group reported the regeneration of transformed plants using this target tissue. Brar et al. (1994), on the other hand, reported transformation and regeneration of fertile peanut plants using the bombardment system on exposed meristems from mature seeds of the elite cultivars Florigiant and Florunner. Since transformed lines were identified via histochemical staining for GUS activity, this method would be likely to result in chimeric plants.
108
The current study was conducted to optimize the conditions for gene gun mediated transformation of local peanut variety with best callus induction and regeneration ability.
7.2 REVIEW OF LITERATURE
In this method gold or tungsten microparticles coated DNA having genes of interest are bombarded at very high speed on plant cells. Some of these particles penetrate in cells upto cytoplasm and some penetrate into nucleus, out of which only a few get integrated into host’s DNA. Some of the cells having integrated transgene can survive and regenerate to make a whole independent plant.
Ozias-Akins et al. (1993) bombarded the callus obtained from immature peanut seeds and got transgenic plants but with a little success (1%) but the inheritance of gene to next generation was not reported. However, in a recent work the transformation efficiency was improved to 25% and inheritance of transgene was also confirmed (Ozias-Akins et al., 1996). Mostly the callus is bombarded but some researchers like also reported bombardment of other plant tissus (Livingstone and Birch, 1995; and Clemente et al., 1992). Brar et al. (1994) reported bombardment of meristem of mature seeds of peanut and transformation was confirmed by GUS activity but it is suspected that resulting plants will be chimeric.
Sanford and coworkers (1993) developed a novel transformation method in which metal particles coated with plasmids harboring desired genes are bombarded on target plant tissue with high velocity. The device used is usually called gene gun
109
and method is also referred as biolistic method. Several higher plants, yeast, chlamydomonas Escherichia coli, Agrobacterium and animals have been successfully transformed by this method (Christou, 1994 ; Sanford 1993).
The biolistic method have several merits lik exclusion of T-DNA borders, no genotype dependency, widest range of host organisms, least tissue culture and somaclonal variation (Rasmussen et al., 1994)
Gene gun of biolistic method of transformation has been utilized in many species (Sanford et al., 1993). Legumes such as soybean, common bean and peanut, showing partial recalcitrance to Agrobacterium mediated transformation have been transformed by this method (Russell et al., 1993; Aragfio et al., 1996).
Lacorte et al., 1997 observed that efficiency of transformation in peanut through particle bombardment method was negatively affected by amount of DNA and particles upto a threshold level. Ikea et a., (2003) reported biolistic transformation in cowpea using embryonic axes as explant but transmission of transgene to next generation was not stable. Ivo et al. (2008), however, obtained transgenic plants of cowpea by particle bombardment method which transmitted the transgene to next generation stably in Mendelian manner.
Thu et al. (2003) obtained transgenic plants of pigeon pea by bombarding the 5 days old seedling with particles coated with plasmids harboring uidA and nptII genes. Seedlings were cultured on callus induction medium containing
110
kanamycin. Transgenic plants thus obtained were analyzed and inheritance to next generations was confirmed.
Singsit et al. (1997) produced BT peanut by bombarding callus obtained from immature cotyledons with particles coated with vectors containing Bt cryIA(c) and hph gene. Bioassay showed 100% mortality rate of lesser cornstalk borers and 66% reduction in larvae weight.
Taylor et al. (1993) compared the effects of two vectors pBARGUS and pAHC25 on transformation efficiency of wheat, sugarcane and maize using particle bombardment method. Transformation efficiency in terms of number of blue spots was 3-50 times greater with pAHC25 than pBARGUS in different species. GUS activity as assayed by flourometer showed an increase of 2.5 times in maize while
30 times in wheat with pAHC25 versus pBARGUS.
Effect of promoter and tissue type on transformation efficiency was studied by Gallo-Meagher and Irvine (1993) in sugarcane using gene gun method. They found that GUS foci and activity was higher in young expanding leaves as compared to older leaves. Comparison of promoters showed that transformation efficiency was highest with maize ubiquitin as compared to pEmu, rice actin 1, and
CaMV 35S promoters.
Chowdhury et al. (1997) transformed embryogenic callus and young leaves of palm by microprojectile bombardment with five constructs harboring different constructs and observed that the expression of the GUS gene driven by Emu or
111
Ubil promoters was significantly higher than those of the Actl, 35S, and Adhl promoters.
Ozias-Akins et al. (1993) produced the first transgenic peanut plants by particle bombardment of 1-2 years old embryogenic callus. They did not reported about the fertility of plants, but the problem of fertility in transgenic soybean obtained by particle bombardment method has been reported earlier (Finer and
McMullen, 1991).
Clemente et al. (1992) bombarded leaflets of peanut seedlings and observed
GUS activity in the callus initiated from these leaflets. But this callus could not be regenerated to differentiated tissues and hence no transgenic plant was obtained. As mature tissues like leaflets are easily available, this technique could be quite helpful if regeneration efficiency is improved.
Ismail (1999) successfully transformed Vicia faba using 35S promoter and
GUS and Bar genes as reporters through particle bombardment method. Southern blot and PCR analysis confirmed the integration of genes and inheritance was also confirmed by analysis of T1 generation.
7.3 MATERIALS AND METHODS
7.3.1 Production of Embryogenic Callus
Embryogenic callus was produced by inoculating the epicotyl explants of the best responsive variety, Golden, on callus induction medium (CIM) as described in Chapter 4.
112
7.3.2 Preparation of Micro-carriers
In a 1.5ml microfuge tube, weighed out 3 mg of 0.6 μm gold particles for 6 bombardments. Added 1ml of freshly prepared of 70 % ethanol in this tube, mixed on platform and vortexed for 2-3 minutes. The mixture was incubated for 15 minutes on ice. By a short spin of 5 seconds, micro particles were pelleted and supernatant was discarded. Repeated the following steps three times.
a. Added 1ml of sterile water.
b. Vortexed for 1 minute.
c. Allowed the particles to settle down for one minute.
d. Pelleted the micro particles by spinning for two seconds and discarded the
liquid.
Added 50 μl of sterile water to bring the micro particle concentration to 60 mg/ml.
7.3.3 Coating DNA onto Micro Particles
Vortexed the micro carriers prepared in sterile water (60 mg/ml) for 5 minutes on a Platform and removed 50 μl (3 mg) of micro carriers to a 1.5 ml tube.
While vortexing vigorously, added in order:
a. 20ul spermidine (0.1 M)
b. 50 ul CaCl2 (2.5 M) was added drop by drop on vortex and continued for
2-3 minutes.
After centrifugaton for 20 seconds, micro carriers were pelleted and supernatant was discarded. Pellet was washed with140 μl of 70 % ethanol followed by same volume of pure ethanol. Finally the pellet was resuspended in 48 μl of pure ethanol from which 6ul was transferred to each of macro carriers. The micro
113
carriers were spread uniformly over the central 1 cm of the macro carrier using the pipette tip and were dehydrated immediately.
Rupture disks, macro carriers and stopping disk was fixed on the instrument. A distance of 6 cm from macro carrier to target callus was kept and helium pressure was built upto 1100 psi at which rupture disk was burst and DNA coated micro carriers were shot on callus. The experiment was replicated thrice along with control experiment lacking plasmid DNA was also conducted in parallel.
The same procedure was performed after changing the size of gold particle
(1.0 μm and 1.6 μm) and target distances (9cm and 12 cm).
7.3.4 Selection of Transformants
After bombardment callus clumps were shifted to petri plates containing callus induction medium (as explained in chapter 5) with 30mg/l hygromycin.
Surviving calli were sub cultured on fresh medium after every two weeks. Callus was regenerated into plantlets through somatic embryogenesis.
7.3.5. PCR analysis
DNA from putative (T0) and control peanut plants was isolated by CTAB method. For PCR analysis of the RCG-3 in the genome of transformants, forward primer 5’-CATATCAAGCATGAGGTGTA-3’ and reverse primer 5’-
CAACAACGATTTT GCTATAA-3’ were employed to amplify a 696 bp
114
fragment. DNA was denatured at 94ºC for 3 minutes for first time and for 30 seconds in each cycle. PCR profile was same as mentioned in chapter 5.
7.3.6. Selection of T1 Plants
Lateral twigs of 25 days old T1 plants were detached and their cut end was submerged in Hoagland solution containing 150mg/lit hygromycin in test tubes having cotton plugs on their mouths. Plants showing decolouration, necrosis and wilting were discarded while those retaining normal green colour and vigour were selected and shifted into bigger (25 x 25 cm) earthen pots.
7.3.7. Pathogenicity Test for Intact Plants
Six intact plants from which the twigs were cut for above mentioned test were subjected to pathogenicity test. Spores of Cercosporadium personatum were collected in distilled autoclaved water by thoroughly washing a large number of infected leaves. Concentration of spores was measured by haemocytometer and was adjusted to the concentration of 2 x 105 spores/ ml. Resistance of control and transgenic plants was evaluated by spraying of spores on leaves till run off. 100% humidity was maintained by using humidifier in green house. Leaf area was measured by leaf meter and necrotic area (%) was calculated by multiplying average size of lesion with number of lesions divided by leaf area.
7.3.8. RT-PCR analysis
RNA from control and confirmed 5 transgenic (T2) plants was extracted using TRI reagent® (Sigma-Aldrrich). cDNA was synthesized using Omniscript®
115
Reverse Transcription Kit of Qiagen company following manufacturer instructions.
Total reaction volume was 20µl having 2µg RNA, 2µl 10x Buffer RT, 2µl dNTP
Mix (5 mM each dNTP) , 2µl Oligo-dT primer (10 μM), 1µl RNase inhibitor (10 units/μl) and 1µl Reverse Transcriptase. PCR was performed using the primers mentioned above in section “PCR analysis” and product was separated on 1% agarose gel.
7.4 Results and Discussion
Analysis of variance showed that target distance and particle size had highly significant while interaction has non-significant effect on transformation efficiency (Table 7.1). Duncan Multple Range Test for Target distance showed that
6 cm distance was the best as it gave highest transformation efficiency (6.59%) while least efficiecny (3.61%) was observed at 12 cm (Table 7.2).
As for as effect of particle size is concerned, 0.6 μm proved to be the best in achieving maximum transformation efficiency (7.073) while minimum transformation efficiency of 3.611% was achieved when particles of 1.6 μm were used (Table 7.3).
7.4.1. T0 generation
Embryos showing normal growth in selective medium (Figure 7.1) were subcultured on the same medium to confirm their resistance to hygromycin. Out of
4 hygromycin resistant plants obtained, required band of 696 bp was observed in three plants in PCR analysis confirming the presence of RCG3 gene (Figure 7.2).
116
Table 7.1: Analysis of variance for factors affecting transformation efficiency.
Source of Degree of Sum of Squares Mean Square F Value Variance Freedom
Target distance 2 44.277 22.139 20.92**
Particle size 2 68.647 34.324 14.4**
Interaction 4 5.108 1.277 7.87
Error 18 5.75 0.319
Total 26 123.783
Table 7.2: Mean values of transformation efficiency at different distances as
ranked by Duncan's Multiple Range Test
Target distance (cm) Mean Ranking 6 6.592 A 9 4.259 B 12 3.610 C Values followed by same alphabet do not differ significantly
Table 7.3: Mean values of transformation efficiency at different particle size
as ranked by Duncan's Multiple Range Test
Particle size (μm) Mean Ranking 0.6 7.073 A 1.0 3.777 B 1.6 3.611 B Values followed by same alphabet do not differ significantly
117
These plants were shifted to coconut husk compost; where they reached to maturity and produced seeds normally.
Southern blot analysis showed single copy of gene in T0-2 plant only while four and two copies were observed in T0-1 and T0-3 plants respectively (Figure
7.3). Multiple copy insertion by biolistic transformation method has been reported in peanut by many researchers (Singsit et al., 1997; Livinigstone et al., 2005 and
Niu et al., 2009).
7.4.2. T1 Generation
Among the progeny of T0-2 plant, 7 were hygromycin resistant while 3 were sensitive. PCR analysis for presence of RCG-3 gene revealed the required band of 696bp in the hygromycin resistant plants while no band in sensitive plants was observed (Figure 7.4)
7.4.3. Pathogenicity Test
Seven confirmed transgenic T1 plants, named from T1-1 to T1-7, along with a control plant were subjected to leaf spot inoculum spray on lower and upper surface of leaves (Figure 7.5). One upper most leaf, comprising of three leaflets, was tagged from three main branches of each plant for disease evaluation. It is clear from Figure 7.6 that incubation frequency (IF, number of lesions per cm2 of leaf area), incubation period (IP), lesion diameter (LD), leaf area damage (LAD) and disease score are significantly high in control plant as compared to transgenic plants.
118
Figure 7.1: Emergence of hygromycine resistant shoots in selection medium
Figure 7.2: Amplification of RCG 3 gene fragment by PCR in 3 surviving T0 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant;
Lanes 1-3: transgenic plants (T0-1 to T0-3).
119
T0-1 T0-2 T0-3 - +
Figure 7.3: Southern blot analysis of three PCR positive plants
Figure 7.4: Amplification of RCG 3 gene fragment by PCR in T1 plants. L: 1 kb ladder; (+): Plasmid containing RCG3 gene; (-): control plant; Lanes 1-
11: T1 plants
8.2.4. RT-PCR Analysis
120
RT-PCR of 7 PCR positive T1 plants showed bands in all the plants, brightness and thickness of bands, however, differed significantly. In plant number
3, 4, 5 and 7 the bands were much brighter and thicker indicating higher number of mRNA transcripts which in turn gave higher expression of chitinase enzyme
(Figure 7.7). In control plant there was no such band while in plant number 1, 2 and
6 the band was comparatively less bright indicating less mRNA copies, hence lower expression.
The biolistic method have several merits like no genotype dependency, widest range of host organisms, least tissue culture and somaclonal variation
(Rasmussen et al., 1994). Genetic transformation through gene gun is accomplished by piercing of cells by microparticles and insertion of the transgene into the genome of host cell followed by subsequent growth of transformed cells into plantlet and finally the expression of transgene (Russell et al., 1992).
Transformation efficiency and subsequent expression is affected by several factors including particle size, helium gas pressure, macro and micro carrier travel distance and vacuum pressure. In current investigations, a distance of 6 cm from stopping screen to target tissue proved to be the best, however, Zuraida et al.
(2010) reported that a distance of 9 cm is best in rice.
Size of the particle can affect the speed, the quantity of attached DNA and damage area of tissue which affects cell survival (Janna et al., 2006). Particle size of 0.6 to 1.6 μm is usually used for transformation of plants (Tian and Seguin,
121
2004). Smaller particles have less force to penetrate into the tissue while larger particles cause excessive damage to the cells. In present study, 1.0 μm sized particles gave the best results as compared to 0.6 and 1.6 μm. Similar results have been achieved in corn (Klein et al., 1988), tobacco (Russell et al., 1992), peanut
(Clemente et al., 1992) and rice (Zuraida et al., 2010). Folling and Olsen (2002), however, reported that a size of 0.6 μm was best in wheat as the larger particles caused extreme damage to tissues. Therefore, the appropriate size for different species and tissues must be investigated for maximum efficiency.
122
Figure 7.5: Pathogenicity test of intact plants for susceptibility to late leaf spot
disease: transgenic (above) and control (below) plant
Figure 7.6. Means for infection frequency, incubation period, lesion diameter,
leaf area damage and disease score of transgenic and control plants
123
Figure 7.7: Seven PCR positive plants were subjected to RT-PCR analysis
Chapter 8
GENERAL DISCUSSION
Groundnut (Arachis hypogaea L.) has been originated in South America and is cultivated on over 50 million acres in almost hundred tropical, subtropical and temperate countries, with an annual seed yield of 28 million tonnes. Cultivated groundnut has been most probably derived from a single hybridization event between Arachis ipaensis and Arachis duranensis (Kochert, 1996). Having a narrow genetic base, it does not possess considerable level of resistance to several major fungal diseases including Cercospora, a major destructive disease which could totally devastate the crop. Use of fungicides is neither cost effective, nor environment friendly. The best way to control this foliar disease is to grow resistant cultivars, but higher levels of resistance are not available in groundnut cultivars.
Thus, there is a strong incentive for genetic engineering, but groundnut proved to be recalcitrant to transformation. Moreover, availability of efficient regeneration and gene delivery system is a prerequisite for this technique. Such techniques have been developed for local genotypes of different areas of the world with limited success. The present studies therefore were conducted to standardize an efficient in vitro culture and transformation system in Pakistani groundnut varieties.
Direct regeneration from cotyledonary and leaf disc explants while indirect regeneration through somatic embryogenesis was optimised. The best responding variety in regeneration experiments was used to standardize Agrobacterium, silicon carbide whisker and biolistic methods of transformation using rice chitinase (RCG-
3) gene.
124
125
High frequency shoot induction (78.33%) was induced from cotyledonary explants of a widely grown variety, Golden, on MS medium supplemented with 0.1 mg/l NAA and 4 mg/l BAP. These shoots were rooted successfully (61.30%) on
MS medium supplemented with 1.0 mg/l NAA.
The result indicated that by increasing BAP concentration in medium, number of shoots/explants increased upto 0.5 mg/L and then it dropped. While on other hand the callus formation was reduced by increasing BAP concentration.
Explant enlargement and chlorophyll accumulation remained almost unaffected with change in hormone combination. Golden produced highest shoots and rooted plants which were followed by BARD-479. However, in chlorophyll gain and explant enlargement both the bold seeded varieties viz GOLDEN and BARI-2000 performed better than the small seeded varieties viz BARD-479 and BARD-92.
Cotyledons have several comparative benefits like robustness, time saving and cost effectiveness. Venkatachalam et al (2000) and also used cotyledons as explants to produce fertile plants via embryogenesis, while Swathi et al. (2006) used cotyledons for direct regeneration. The cotyledonary explants are easy to use as sterilization is simple and does not need too much care. Moreover it also saves time and sources needed for in vitro germination which is a pre-requisite for protocols involving epicotyls or leaves as explants.
Twelve different combinations of TDZ and IAA were used to induce direct regeneration from leaf discs of all the four varieties. Highest number of responding
126
explants (52.67%) with highest (5.65) number of shoot buds per responding explant was achieved at a combination of 0.5 mg/l BAP and 0.5 mg/l TDZ in
Golden variety. 56.07% shoots developed roots at 1.5mg/l NAA.
Venkatachalam et al. (1996) regenerated plantlets from leaf discs of peanut through organogensis using combination of BAP, Kinetin and NAA while Sarkar and Isam (2000) used only BAP and kinetin. However, results of current study coincide with those of many researchers who emphasized the use of TDZ in regeneration of plantlet from leaf explants of different Arachis species; A. hypogaea (Kanyand et al., 1994, 1997), A. correntina (Mroginski et al., 2004), A. stenosperma (Vijaya Laxmi and Giri, 2003) and A. villosa (Fontana et al. 2009).
The shoot bud elongated well when shifted to medium containing BAP but lacking TDZ. These results are in close agreement with those of Gill and Ozias-
Akins (1999), Ahmad et al.( 2006) and Fontana et al. (2009). Most of the species of Arachis are recalcitrant to regeneration through organogenesis except Arachis pintoi (Rey et al., 2000) . Mroginski et al (2004) tried different combinitions BAP,
2,4-D, NAA, KIN and PIC to initiate organogenesis from leaf discs of Arachis correntina but failed. However they successfully produced shoots from leaf disc by using TDZ. Most of cytokinins used in plant tissue culture like BAP, Zeatin and kinetin contain adenine in their structural formula while TDZ is a non-adenine type highly active cytokinin which has a wide range of effects on cultures including induction of multiple buds and inhibition of their elongation. This problem is overcome by shifting of buds to a medium containing adenine-type cytokinens
(Fontana et al., 2009).
127
Epicotyl explants of all the four varieties were inoculated on callus induction medium (CIM) consisting of MS medium supplemented with 2, 4, 6, 8 and 10 mg/l picloram. Maximum (98.3%) number of explants responded to embryogenic callus induction on 8mg/l picloram. After three weeks the callus was transferred to embryo conversion medium (ECM) consisting of MS salt, B5
Vitamins, 30g/l Sucrose different concentrations of 2,4-D (0.1, 0.5, 1.0, 1.5, 2.0 mg/l) and cultured under the same conditions as mentioned above. Maximum 19.33 embryos/explant were observed in Golden variety on ECM containing 1.5 mg/l 2,
4-D. The embryos thus developed were shifted to embryo germination medium
(EGM) consisting of MS salt, B5 Vitamins, 30g/l Sucrose different concentrations of BAP (0.1, 0.5, 1.0, 1.5, 2.0 mg/l). Maximum (74.72%) embryo germination was observed at 0.1mg/l BAP in Golden variety.
In present study embryo germination is significantly high (74.72%) on a very low concentration (0.1mg/l) of BAP. Venkatachalam et al. (1997) also found that higher concentration of BAP along with very low concentration of NAA trigger embryo germination.
In present study epicotyls from mature seeds have been used as explants.
Thus these explants are readily available round the year and there is no need to grow the seeds in greenhouse or in vitro to obtain explants as is in the case of leaves or immature embryos. Moreover rate of contamination is low in spite of minimal sterilization. As radical portion produces non-embryogenic callus, only epicotyl was used in this study instead of whole embryo axis. Cutting the epicotyl
128
portion produced a wound which could provide a good infection site for
Agrobacterium in genetic transformation is intended.
In most of the experiments combination of two, three, four or even five plant growth regulators were used (Venkatachalam et al., 1999; Vidoz et al., 2006;
Robinson et al. 2011) but in present study only single hormone was used in each step to avoid complexity. Moreover, time duration of each step was kept to minimum to avoid somaclonal variation which is a problem in most of peanut cultures (Ozias-Akins and Gill, 2001; Vidoz et al., 2006).
Binary vector pB1333 harbouring rice chitinase (RCG-3) and hygromycin phosphotransferase (hpt) genes driven by EN4 and CaMV 35S promoters, respectively was employed for all the three methods of transformation.
Kanamycine gene was present outside tDNA for bacterial selection. Effect of co- cultivation period and acetosyringone concentration was evaluated in transformation through Agrobacterium using direct regeneration system from cotyledonary explant. Co-cultivation period of three days (72 hours) and acetosyringone concentration of 100 mg/l gave maximum transformation efficiency of 14.18%. The experiment, right from start to harvesting of T0 plants, was completed in 211 days.
A transgenic production system can be called best if it is fast, produces abundant non-chimeric plant with least tissue culture steps and avoids costly and lobours media preparations (Tiwari and Tuli, 2012).
129
In most of tissue culture based transformation methods whole plant is regenerated from a single transformed cell and differentiated tissues are not used as recipients of foreign DNA (Higgins et al., 2004).
The current study has further strengthened the evidence that transgenic plants can be successfully produced using differentiated tissues like cotyledons.
Some researchers obtained stable transgenic peanut plants from fully differentiated explants like epicotyls (Egnin et al., 1998) and cotyledons (Tiwari et al., 2007;
Tiwari and Tuli, 2008; Tiwari and Tuli, 2012).
It has been observed by many researchers that Kanamycin does not kill non-transformed cells completely, enabling pseudo positive or chimeric plants to be selected (Cheng et al., 1996; Sharma and Anjaiah, 2000; Dodo et al., 2008). On the other hand hygromycin not only eliminates the non-transformed cells completely, but also increases the potential for multiple shoot induction (Tiwari and Tuli, 2012). Moreover, probability of inducing somaclonal variation is reduced by the short duration cycle of transformation and regeneration (Olhoft et al., 2003).
Resistance against different fungal diseases has been achieved by over expression of rice chitinase in different plant species including bread wheat (Chen et al., 1998), rice (Lin et al., 1995), strawberry (Asao et al., 1997), grapevine
(Yamamoto et al., 2000), pigeon pea (Kumar et al., 2004).
130
In present study it has been proven that rice chitinase gene (RCG-3) confers strong resistance against leaf spot disease of peanut. The stable peanut lines hence produced will be helpful to evolve cultivars with built-in solution to control fungal diseases, especially the leaf spot.
Silicon carbide whisker mediated transformation was optimized by varying the callus age (10, 20 and 30 days old) and whisker quantity (100, 200 and 300 mg). Highest transformation efficiency (6.88%) was achieved when 200 mg of whisker was used for 20 days old callus. This is first known report of Silicon carbide whisker mediated transformation in peanut. This experiment took 265 days from inoculation of epicotyls to harvesting of T0 plants.
Silicon carbide whisker mediated transformation is probably the simplest known method of genetic transformation. It involves vortexing of target tissues along with plasmid (harbouring gene of interest) in presence of silicon carbide.
Silicon carbide is micro-needle shaped hardest man-made material used as an abrasive and is component of saw blades (Dunwell, 2011).
Studies reveal that silicon carbide whiskers have high tensile strengths, elasticity and resistance to degradation (Choi et al., 1997). Moreover they have high intrinsic strength and their easy cleavage produces sharp cutting edges needed to penetrate into cell walls and ultimately nucleus of cells (Greenwood and
Earnshaw, 1984).
131
Electron microscope scan has shown that whisker penetrate into nucleus of maize thus perforating the cell to pave way for delivery of DNA (Kaeppler et al.
1990). Like DNA, whisker surface is also negatively charged hence DNA is not attached to whisker; however, the perforation caused by whiskers facilitates DNA movement to nucleus.
Previously it was thought that only suspension culture can be the target for silicon carbide mediated transformation but Serik et al. (1996) successfully demonstrated the transformation of dry wheat embryos by these whiskers
Moreover, callus age affected the transformation efficiency greatly in different crops. In Agrostis alba plants best results were obtained when transformation was done after 6 days of subculture (Asano et al., 1991) while in cotton 14 days were best (Asad et al., 2008). Matsushita et al (1999) conducted two experiments for transformation of rice through silicon carbide. Firstly they vortexed scuteller tissues of embryos along with pAct1-F plasmid harbouring GUS gene and silicon carbide in liquid medium. They got 302 GUS spots in 250mg sample. In second experiment they utilized two plasmids; pAct1-F having GUS gene and pDM302 having bar gene (which confers resistance to Bialaphos). 873 embryos were used in the experiment and 57 calli were proved to be Bialaphos resistant, some of which were also GUS positive.
No report of any previous work on transformation of peanut through silicon carbide whiskers was found in literature for comparison of this study.
132
Genetic transformation through gene gun is accomplished by piercing of cells by microparticles and insertion of the transgene into the genome of host cell followed by subsequent growth of transformed cells into plantlet and finally the expression of transgene (Russell et al., 1992).The biolistic method have several merits like no genotype dependency, widest range of host organisms, least tissue culture and somaclonal variation (Rasmussen et al 1994).
In present study embryogenic callus was produced by the method mentioned above and bombarded gold particles coated with vector harbouring chitinase gene using PDS 1000/He Gene Gun by Bio-Rad. Effect of gold particle size and micro carrier travel distance on transformation efficiency through gene gun method was evaluated. Particle size of 0.6 μm proved to be the best in achieveing maximum transformation efficiency (7.073) while 6 cm distance was the best as it gave the highest transformation efficiency (6.59%). A time period of
237 days was required to complete this experiment from inoculation of epicotyls to harvesting of T0 plants. Transformation efficiency and subsequent expression is affected by several factors including particle size, helium gas pressure, macro and micro carrier travel distance and vacuum pressure. In current investigations, a distance of 6 cm from stopping screen to target tissue proved to be the best, however, Zuraida et al., (2010) reported that a distance of 9 cm is the best in rice.
Size of the particle can affect the speed, the quantity of attached DNA and damage area of tissue which affects cell survival (Janna et al., 2006). Particle size of 0.6 to 1.6 μm is usually used for transformation of plants (Tian and Seguin,
133
2004). Smaller particles have less force to penetrate into the tissue while larger particles cause excessive damage to the cells. In present study, 1.0 μm sized particles gave the best results as compared to 0.6 and 1.6 μm. Similar results have been achieved in corn (Klein et al., 1988), tobacco (Russell et al., 1992), peanut
(Clemente et al., 1992) and rice (Zuraida et al. 2010). Folling and Olsen (2002), however, reported that a size of 0.6 μm was the best in wheat as the larger particles caused extreme damage to tissues. Therefore, the appropriate size for different species and tissues must be investigated for maximum efficiency.
PCR, RT-PCR and Southern blot analyses were performed on T0 generations in all the transformation experiments to confirm insertion, expression and copy number of the transgene, respectivly. Progeny of the T0 plant having single copy number of transgene was subjected to pathogenicity test for leaf spot disease and transgenic plants proved to be resistant to the disease.
Transformation through Agrobacterium using cotyledonary explant proved to be the best method as it involved minimum tissue culture, least media preparation and above all it was time saving with exceptionally high comparative transformation efficiency. The explants used in this method are readily availabe and need minimum sterilization. Moreover, probability of inducing somaclonal variation is reduced by the short duration cycle of transformation and regeneration.
In present study it has been proven that rice chitinase gene (RCG-3) confers strong resistance against leaf spot disease of peanut. The stable peanut lines hence produced will be helpful to evolve cultivars with built-in solution to control fungal
134
diseases, especially the leaf spot. The standardized protocols of regeneration and transformation will be used for incorporation of other desirable genes in this crop.
SUMMARY
Groundnut (Arachis hypogaea L.) is the one of world’s most important legume and oilseed crop, native to South America and grown all over the tropical and temperate regions of the world. Leaf spot, commonly known as Tikka disease, caused by fungus Cercospora have been a major destructive disease of groundnut and could cause a yield loss of up to 70 %. The best way to control this foliar disease is to grow resistant cultivars, but higher levels of resistance are not available in groundnut cultivars. Genetic transformation is the best alternative in such circumstances and availability of efficient regeneration and gene delivery systems is a prerequisite for this technique. The present study therefore was conducted to standardize an efficient in vitro culture and transformation system in local groundnut varieties.
The experiment involved standardization of three regeneration methods viz. direct regeneration from cotyledonary explants, direct regeneration from leaf discs and regeneration via callus induction using epicotyl explants.
The best responding variety in regeneration experiments was used to standardize Agrobacterium, silicon carbide whisker and biolistic methods of transformation using rice chitinase (RCG-3) gene.
Excellent direct shooting (78.33%) was induced from cotyledonary explants of local variety, Golden, on MS medium supplemented with 0.1 mg/l NAA and 4 mg/l BAP. These shoots were rooted successfully (61.30%) on MS medium
135
136
supplemented with 1.0 mg/l NAA. Rooting was also achieved successfully by physical stress method. Grafting also proved to be a good alternative of rooting.
Regeneration from different explants has been achieved but cotyledons have several comparative benefits like robustness, time saving and cost effectiveness.
Twelve different combinations of TDZ and IAA were used to induce direct regeneration from leaf discs of all the four varieties. Highest number of responding explants (52.67%) with highest (5.65) number of shoot buds per responding explant was achieved at a combination of 0.5 mg/l BAP and 0.5 mg/l TDZ in Golden variety. 56.07% shoots developed roots at 1.5mg/l NAA.
Epicotyl explants of all the four varieties were inoculated on callus induction medium (CIM) consisting of MS medium supplemented with 2, 4, 6, 8 and 10 mg/l picloram. Maximum (98.3%) number of explants responded to embryogenic callus induction on 8mg/l picloram. After three weeks the callus was transferred to embryo conversion medium (ECM) consisting of MS salt, B5 Vitamins, 30g/l sucrose supplemented different concentrations of 2,4-D (0.1, 0.5, 1.0, 1.5, 2.0 mg/l) and cultured under the same conditions as mentioned above. Maximum 19.33 embryos/explant were observed in Golden variety on ECM containing 1.5 mg/l 2,4-
D. The embryos thus developed were shifted to embryo germination medium (EGM) consisting of MS salt, B5 Vitamins, 30g/l sucrose and different concentrations of
BAP (0.1, 0.5, 1.0, 1.5, 2.0 mg/l). Maximum (74.72%) embryo germination was observed at 0.1mg/l BAP in Golden variety.
137
The Agrobacterium strain LBA-4404 harbouring pB1333 binary vector was employed for all the three methods of transformation. The plasmid carried rice chitinase (RCG-3) and hygromycin phosphotransferase (hpt) genes driven by EN4 and CaMV 35S promoters, respectively. Kanamcine gene was present outside tDNA for bacterial selection. Effect of co-cultivation period and acetosyringone concentration was evaluated in transformation through Agrobacterium using direct regeneration system from cotyledonary explant. Co-cultivation period of three days
(72 hours) and acetosyringone concentration of 100 mg/l gave maximum transformation efficiency of 14.18%. The experiment, right from start to harvesting of T0 plants, was completed in 211 days.
Silicon carbide whisker mediated transformation was optimized by varying the callus age (10, 20 and 30 days old) and whisker quantity (100, 200 and 300 mg).
Highest transformation efficiency (6.88%) was achieved when 200 mg of whisker was used for 20 days old callus. This is first known report of Silicon carbide whisker mediated transformation in peanut. This experiment took 265 days from inoculation of epicotyls to harvesting of T0 plants.
Effect of gold particle size and micro carrier travel distance on transformation efficiency through gene gun method was evaluated for peanut embryogenic callus. As far as effect of particle size is concerned, 0.6 μm proved to be the best in achieving maximum transformation efficiency (7.073) while 6 cm distance was the best as it gave highest transformation efficiecy (6.59%). A time
138
period of 237 days was required to complete this experiment from inoculation of epicotyls to harvesting of T0 plants.
PCR, RT-PCR and Southern blot analyses were performed on T0 generations in all the transformation experiments to confirm insertion, expression and copy number of the transgene, respectively. Progeny of the T0 plant having single copy number of transgene was subjected to pathogenicity test for leaf spot disease and transgenic plants proved to be resistant to the disease.
Transformation through Agrobacterium using cotyledonary explant proved to be the best method as it involved minimum tissue culture, least media preparation and above all it was time saving with exceptionally high comparative transformation efficiency. The explants used in this method are readily availabe and need minimum sterilization. Moreover, probability of inducing somaclonal variation is reduced by the short duration cycle of transformation and regeneration.
In present study it has been proven that rice chitinase gene (RCG-3) confers strong resistance against leaf spot disease of peanut. The stable peanut lines hence produced will be helpful to evolve cultivars with built-in solution to control fungal diseases, especially the leaf spot. The standardized protocols of regeneration and transformation will be used for incorporation of other desirable genes in this crop.
LITERATURE CITED
Ahmad, N. and M. Rahim. 2007. Evaluation of promising groundnut (Arachis
hypogaea L.) varieties for yield and other characters. J. Agric. Res., 45 (3):
185-189.
Akasaka, Y., Daimon H. and M. Mii. 2000. Improved plant regeneration from
cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure
to thidiazuron. Pl. Sci., 156: 169-175.
Alabi, R. O. 1986. Dispersal and survival of conidia of Cercospora arachidicola
Hori. Archiv fur Phytopathologie und Pflanzenschutz. 22: 33-39.
Anderson, W. F., T. Stalker, H. Wood and K. Moore. 2000. Identification of
marker genes associated with late leaf spot resistance. APRES. 32: 22.
Anderson, W. P. 1983. Weed Science Principles. 2nd edition, West Pub. Co. St.
Paul. Minn., USA: 33-42.
Anonymous. 2004. Annual Report of the Project Co-ordinator, All India Co-
ordinated Cotton Improvement Project, CICR, Coimbatore, pp. 1-21.
Anthony, T. T., H. B. Stephen, V. K. Igor, E. M. M. Ignacio, K. V. Wayne, A. B.
Laura, S. Heungsop, J. C. Tzyy, K. Hiroaki, R. D. Gary, W. Detlef and J. H.
139
140
Maria. 2000. Transformation of Medicago truncatula via infiltration of
seedlings or flowering plants with Agrobacterium. The Pl. J., 22 (6): 531-
541.
Anuradha, S. T., K. Divya, S. K. Jami and P. B. Kirti. 2008. Transgenic tobacco
and peanut plants expressing a mustard defensin show resistance to fungal
pathogens. Plant Cell Rep., 27 (17): 77-86.
Anuradha, S. T., S. K. Jami, R. S. Datta and P. B. Kirthi. 2006. Genetic
transformation of peanut (Arachis hypogaea L.) using cotyledonary node as
explant and a promoter less gus::nptII fusion gene based vector. J. Bio. Sci.,
31 (2): 235-246.
Aragfio, F. J. L., L. M. G. Barros, A. C. M. Brasileiro, S. G. Ribeiro, F. D. Smith,
J. S. Sanford, J. C. Faria and E. L. Rech. 1996. Inheritance of foreign genes
in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle
bombardment. Theor. Appl. Genet., 93: 142-150.
Asad, S., Z. Mukhtar, F. Nazir, A. J. Hashmi, S. Mansoor, Y. Zafar and M. Arshad.
2008. Silicon carbide whisker-mediated embryogenic callus transformation
of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants.
Mol. Biotech., 40: 161-169.
141
Asano, Y., Y. Otsuki and M. Ugaki. 1991. Electroporation-mediated and silicon
carbide whisker-mediated DNA delivery in Agrostis alba L. (Redtop). Pl.
Sci., 9: 247–252
Asao, H., Y. Nishizawa, S. Arai, T. Sato, M. Hirai, K. Yoshida, A. Shinmyo and T.
Hibi. 1997: Enhanced resistance against a fungal pathogen Sphaerotheca
humuli in transgenic strawberry expressing a rice chitinase gene. Pl.
Biotechnol. 14: 145-149.
Atreya, C. D ., R. J. Papa and N. C. Subrahmanyam. 1984. In vitro regeneration of
peanut (Arachis hypogaea L.) plantlets from embryo axes and cotyledon
segments Plant Science Letters, 34 (3): 379-383.
Backman, P. A. and M. A. Crawford. 1984. Relationship between yield loss and
severity of early and late leaf spot diseases of peanut. The Am. Phytopath.
Soc., 74 (9): 1101-1103.
Bailey, J. E. and C. A. Matyac. 1985. Advances in the deployment of the peanut
leafspot advisory in North Carolina using an electronic weather station.
Proc. Am. Peanut Res. Educ. Soc., 17: 48.
Baker, C. M. and H. Y. Wetzstein. 1992. Somatic embryogenesis and plant
regeneration from leaflets of peanut, Arachis hypogaea. Plant Cell Rep., 11:
71–75.
142
Baker, C. M., R. E. Durham, J. A. Burns, W. A. Parrott and H. Y. Wetzstein.
1995.High frequency somatic embryogenesis in peanut. (Arachis hypogaea
L.) using mature dry seed. Plant Cell Rep., 15: 38–42.
Bechtold, N., J. Ellis and G. Pelletier. 1993. In planta Agrobacterium-mediated
gene transfer by infiltration of adult Arabidopsis thaliana plants. Comptes
Rendus Academie Sciences, Paris, Series 3, Life Sciences, 316: 1194-1199.
Bele, D., M.K. Tripathi, G. Tiwari, B.S. Baghel and S. Tiwari. 2012. Microcloning
of sandalwood (Santalum album L.) from cultured leaf discs. J. Agric. Tech.
8(2): 571-583.
Bhatnagar, M. P., J. Devi, D. S. Reddy, M. Lavanya, V. Vadez, R. Serrajs S. K.
Yamaguch and K. K. Sharma. 2007. Stress induced expression of At
DREB1A in transgenic peanut (Arachis hypogaea L.) increases
transcription efficiency under water - limiting conditions. Plant Cell Rep.,
26: 2071-2082.
Bhatnagar. M., K. Prasad., P. Bhatnagar-Mathur., M. L. Narasu., F. Waliyar and K.
K. Sharma. 2010. An efficient method for the production of marker free
transgenic plants of peanut (Arachis hypogaea L.). Plant Cell Rep., 29 (5):
495-502.
143
Birch, R. G 1997. Plant transformation: Problems and strategies for practical
application.Ann. Rev. Plant Physiol. Plant Mol. Biol., 48: 297-326.
Bradford, M. M. 1976. A dye binding assay for protein. Ann. Biochem., 72: 248-
254.
Brenneman, T. B. and A. K. Culbreath. 2005. The integrated pest management
system. http://nespal.cpes.peachnet.edu/pa
Brenneman, T. B., D. R. Sumner., R. E. Baird, G. W. Sutton and N. A. Minton.
1995. Suppression of foliar and soil borne peanut diseases in bahiagrass
rotations. Phytopath., 85: 948-952.
Burns. S. P., M. Gallo and B. L. Tillman. 2012. Expansion of a direct shoot
organogenesis system in peanut (Arachis hypogaea L.) to include US
cultivars. In vitro Cell. Dev. Biol. Plant., 48(1): 58-66
Burow, M.D., C. E Simpson, A. H. Paterson and J. L. Starr. 1996. Identification of
peanut (Arachis hypogaea L.) RAPD markers diagnostic of rootknot
nematode (Meloidogyne arenaria (Neal) Chitwood) resistance. Mol. Breed.,
2(4):369-379.
144
Burtnik. O. J., L. A. Mroginski. 1985. Regeneracio´n de plantas de Arachispintoi
(Leguminosae) porcultivo in vitro de tejidosfoliares.Ole´agineux 40: 609–
611.
Cantonwine, E. G., A. K. Culbreath and K. L. Stevenson. 2007. Characterization of
early leaf spot suppression by strip tillage in peanut. Phytopath., 97: 187-
194.
Chandra, S., B. K. Sarma, J. Singh and D. K. Pandrey. 1995. Screening and
stability analysis of peanut (Arachis hypogaea) for resistance to early leaf
spot (Cercospora arachidicola). Ind. J. Agri. Sci., 65: 876-879.
Chang, S. S., S. K. Park, B. C. Kim, B. J. Kang, D. U. Kim and H. G. Nam. 1994.
Stable genetic transformation of Arabidopsis thaliana by Agrobacterium
inoculation in planta. The Plant J., 5: 551-558.
Chee, P. P. and J. L. Slighton.1995. Transformation of soybean (Glycine max) via
Agrobacterium tumefaciens and analysis of transformed plants. In :
Agrobacterium Protocols : Methods in Molecular Biology, Eds. K. M. A.
Gartland, M. R. Davey, Humana Press, Totowa, New Jersey, 44: 101-119.
Chen, W. B., X. Xu, G. H. Liang, S. Muthukrishnan, P. D. Chen, D. J. Liu and B.
S. Gill. 1998. Introduction and constitutive expression of a rice chitinase
145
gene in bread wheat using biolistic bombardment and bar gene as a
selectable marker. Theor. Appl. Genet. 97: 1296-1306.
Chen, W. S., C. C. Chiu., H. Y. Liu., T. L. Lee., J. T. Chen., C. C. Lin., Y. J. Wu
and H. Y. Chang. 1998. Gene transfer via pollen-tube pathway for anti-
fusarium wilt in watermelon. Biochem. Mol. Biol. Int., 46: 1201-1209.
Cheng, M., R. L. Jarret, Z. Li and J. W. Demski. 1997. Expression and inheritance
of foreign genes in transgenic peanut plants generated by Agrobacterium-
mediated transformation. Plant Cell Rep., 16: 541–544.
Cheng. M., D. C. H. Hsi and G. C. Phillips. 1992. In vitro regeneration of
Valencia-type peanut (Arachis hypogaea L) from cultured petioles and
other seedling explant. Peanut Sci., 19:82–87.
Chengalrayan, K., S. Hazra and M. Gallo-Meagher. 2001. Histological analysis of
somatic embryogenesis and organogenesis induced from mature zygotic
embryo derived leaflets of peanut (Arachis hypogaea L.). Pl. Sci., 161:415–
442.
Chengalrayan, K., S. Sathaye and S. Hazra. 1994. Somatic embryogenesis from
mature embryo-derived leaflets of peanut (Arachis hypogaea L.). Plant Cell
Rep., 13: 578–581.
146
Chengalrayan, K., V. B. Mhaske and S. Hazra. 1997. High-frequency conversion of
abnormal peanut somatic embryos. Plant Cell Rep., 16(11):783-786.
Chiteka, Z. A., D. W. Gorbet, F. M. Shokes, T. A. Kucharek and D. A. Knauft.
1988. Components of resistance to late leaf spot in peanut. I. Levels and
variability –implications for selection. Peanut Sci., 15: 76-81.
Choi, G. J. 1997. Silicon carbide fibers from copolymers of commercial
polycarbosilane and silazane. J. Ind. Eng. Chem., 3: 223-228.
Choi, K., M.D. Burow, G. Church, G.Burow, A.H. Paterson, C.E. Simpson and J.L.
Starr. 1999. Genetics and mechanism of resistance to Meloidogyne arenaria
in peanut germplasm. J. Nematology, 31(3): 283-290.
Chowdhury, M. K. U., G. K. A. Parveez and N. M. Saleh. 1997. Evaluation of five
promoters for use in transformation of oil palm (Elaeis guineensis Jacq).
Plant Cell Rep., 16: 277-281.
Chowrira, G. M., V. Akella and P. F. Lurquin. 1995. Electroporation-mediated
gene transfer into intact nodal meristems in planta : generating transgenic
plants without in vitro tissue culture. Mol. Biotech., 3: 17-23.
147
Christou, P. 1994. Application to plants. Agricultural Biotechnology and Crop
Improvement in Particle Bombardment Technology for Gene Transfer
(Yang, N. and Christou, P , eds ), Oxford Univ Press, Oxford., 71-99.
Christou, P. 1995. Strategies for variety independent genetic transformation of
important cereals, legumes and woody species utilizing particle
bombardment. Euphytica., 85: 13-27.
Chu, Y., P. Faustinelli, M. L. Ramos, M. Hajduch, S. Stevenson, J. J. Thelen, S. J.
Maleki, H. Cheng and P. Ozias-Akins. 2008. Reduction of IgE binding and
nonpromotion of Aspergillus flavus fungal growth by simultaneously
silencing Ara h 2 and Ara h 6 in peanut. J. Agri. Food Chem., 56 (112): 25-
33.
Chu. Y., X. Y. Deng, P. Faustinelli and P. Ozias-Akins. 2008. Bcl-xl transformed
peanut (Arachis hypogaea L.) exhibits paraquat tolerance. Plant Cell. Rep.,
27 (1) : 85-92.
Chumakov, M. I., N. A. Rozhok., V. A. Velikov., V. S. Tyrnov and I. V.
Volokhina. 2006. Agrobacterium-mediated in planta transformation of
maize via pistil filaments. Rus. J. Genet., 42: 893-897.
Clemente, T. E., T. G. I. Robertson, M. K. Beute and A. K. Weissinger. 1992.
Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic
148
embryos as recipient tissue for biolistic gene transfer. Transgenic Res., 1:
275-284.
Clemente, T.E., D. Robertson, T.G. Isleib, M.K. Beute and A.K. Weissinger. 1992.
Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic
embryos as recipient tissue for biolistic gene transfer. Transgenic Res. 1:
275-5284.
Clough, S. J. and Bent, A. F. 1998. Floral dip: a simplified method for
Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J.,
16: 735-743.
Cole, D. L. 1981. Diseases of groundnut (Arachis hypogaea L.) 1. Fungicide sprays
effects on Cercospora arachidicola and Phoma arachidicola leaf infection,
kernel yield and pod rots. Zimb., J. Agri. Res., 19: 101-110.
Culbreath, A. K., K. L. Stevenson and T. B. Brenneman. 2002. Management of late
leaf spot of peanut with benomyl and chlorothalonil: A study in preserving
fungicide utility. Plant Dis., 86: 349-355.
Dalton, S. J., A. J. E. Bettany, E. Timms and P. Morris. 1998. Transgenic plants of
Lolium multiflorum, Lolium perenne, Festuca arundiacea, and Agrostis
stolonifera by silicon carbide fibre-mediated transformation of cell
suspension cultures . Plant Sci., 132: 31 – 43.
149
Deane, J. 2004. Smoke point of olive oil. www.oliveoilsource.com.
Desfeux, C., S. J. Clough and A. F. Bent. 2000. Female reproductive tissues are the
primary target of Agrobacterium mediated transformation by the
Arabidopsis floral-dip method. Plant Physiol., 123: 895-904.
Dodo, H.W., K.N. Konan, F.C. Chen, M. Egnin and O.M. Viquez. 2008.
Alleviating peanut allergy using genetic engineering: the silencing of the
immunodominant allergen Ara h 2 leads to its significant reduction and a
decrease in peanut allergenicity. Plant Biotechnol J., 6:135–145.
Dunahay, T. G. 1993. Transformation of Chlamydomonas reinhardtii with silicon
carbide whiskers. Biotech., 15: 452-460.
Dunwell, J. M., B. Frame, M. MAIN, R. Schick and K. Wang. 2011. Genetic
Transformation Using Maize Immature Zygotic Embryos. In: Plant Embryo
Culture Methods and Protocols: Methods in Molecular Biology. Trevor A.
Thorpe and Edward C. Yeung (eds), Springer Science and Business Media,
LLC: 327-341.
Durham, R. E. and W .A. Parrott. 1992. Repetitive embryogenesis from peanut
cultures in liquid medium. Plant Cell Rep., 11:122-125.
150
Eapen, S. and G. Leela 1993. Plant regeneration from leaf discs of peanut and
pigeonpea: Influence ofbenzyladenine, indoleacetic acid and indoleacetic
acid-amino acid conjugates. Plant Cell Tiss. Org. Cult., 35: 223-227.
Eapen, S. and L. George. 1994. Agrobacterium tumefaciens mediated gene transfer
in peanut. Plant Cell Rep. 13: 582-586.
Egnin, M. A. Mora and C.S. Prakash. 1998. Factors enhancing Agrobacterium
tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.). In
vitro Cell Dev Biol Plant, 34(4):310-318.
Entoori, K., R. Sreevathsa, M. K. Arthikala, P. A. Kumar, A. R. V. Kumar, B.
Madhusudhan and U. Makarla. 2008. A chimeric cry1X gene imparts
resistance to Spodoptera litura and Helicoverpa armigera in the transgenic
peanut. Eurasia. J. BioSci., 2 (7): 53-65.
Feldmann, K. A. and M. D. Marks. 1986. Rapid and efficient regeneration of plants
from explants of Arabidopsis thaliana. J. Plant Sci., 47: 63-69.
Finer, J. J. and M. D. McMullen. 1991. Transformation of soybean via particle
bombardment of embryogenic suspension culture tissue. In vitro Cell.
Develop. Bio., 27: 175-182.
151
Folling, L. and A. Olesen. 2002. Transformation of wheat (Triticum aestivum L.)
microspore-derived callus and microspores by particle bombardment. Plant
Cell Rep., 20: 1098-1105.
Fontana, M.L., L.A. Mroginski and H.Y. Rey. 2009. Organogenesis and plant
regeneration of Arachis villosa Benth. (Leguminosae) through leaf culture.
Biocell, 33(3): 179-186.
Frame, B. R., P. R. Drayton, S. V. Bagnall, C. J. Lewnau, W. P. Bullock, H. M.
Wilson, J. M. Dunwell, A. Thompson and K. Wang. 1994. Production of
fertile transgenic maize plants by silicon carbide whisker-mediated
transformation. Plant J., 6: 941 – 948.
Gallo-Meagher, M. and J. E. Irvine. 1993. Effects of tissue type and promoter
strength on transient GUS expression in sugarcane following particle
bombardment. Plant Cell Rep., 12: 666-670.
Gamborg, O. L., R. A. Miller and K. Ojima. 1968. Nutrient requirements of
suspension cultures of soybean root cells. Exp. Cell. Res., 50: 151-158.
Garcia, G.M., H.T. Stalker and G. Kochert. 1995. Introgression analysis of an
interspecific hybrid population in peanuts (Arachis hypogaea L.) using
RFLP and RAPD markers. Genome, 38(1):166-176.
152
Geng, L., L. Niu, C. Shu, F. Song, D. Huang and J. Zhang. 2011. High-efficiency
regeneration of peanut (Arachis hypogaea L.) plants from leaf discs. Afri. J.
Biotech., 10 (5): 12680-12682.
Gibbons, R. W. 1966. Mycosphaerella leaf spots of groundnuts. FAO Plant
Protection Bulletin 14: 25-30.
Gill, B. S., J. S. Samra, H. S. Brar and S. P. Mehra. 1986. Studies on the critical
period of weed competition in groundnut. J .Res. Punj. Agric. Univ., India.
23: 394-397.
Gill, R. and P. Ozias-Akins. 1999. Thidiazuron-induced highly morphogenic callus
and high frequency regeneration of fertile peanut (Arachis hypogaea L.)
plants. In vitro Cellular & Developmental Biology – Plant, 35: 445-450.
Gill, R., P. K. Saxena. 1992. Direct somatic embryogenesis and regeneration of
plants from seedlings of peanut (Arachis hypogaea): promoter role of
thidiazuron. Can. J. Bot., 70:1186-1192.
Godoy, I. J., S. A. Moraes, A. R. Moraes, F. S. Kasai, A. L. Martin and J. C.
Pereira.2001. Yield potential of upright early maturing peanut lines with
and without control of foliar diseases. Bragantia, 60(2): 582-590.
153
Gorbet, D.W., D. A. Knauft and F. M. Shokes. 1990. Response of peanut
genotypes with differential levels of leaf spot resistance to fungicide
treatments. Crop Sci., 30: 529-533.
Govt. of Pak. 2008. http://www.pakistan.gov.pk/divisions/food-division/media/
statistical.Tables2008/Table-30.
Green, C. C. and J. C. Wynne. 1986. Field and greenhouse evaluation of the
components of partial resistance to early leaf spot in peanut. Euphytica., 35:
561-573.
Greenwood, N. N. and A. Earnshaw. 1984. Silicon carbide, SiC: chemistry of
elements. Oxford: Pergamon Press: 386.
Grichar, W. J., B. A. Besler and A. J. Jaks. 1998. Peanut (Arachis hypogaea L.)
response to leaf spot development under four disease management
programs. Peanut Sci., 25: 35-39.
H. Wang, M. K. Pandey, L. Qiao, H. Qin, A. K. Culbreath, G He, R. K. Varshney
and B. Guo. 2013. Genetic Mapping and QTL Analysis for Disease
Resistance Using F2 and F5 Generation-based Genetic Maps Derived from
Tifrunner × GT-C20 in Peanut (Arachis hypogaea L.). The Plant
Genome:doi: 10.3835/plantgenome2013.05.0018
154
Hagan, A. K. 1998. Foliar disease of peanuts. Alabama Cooperative Extension
System. http://www.aces.edu/pubs/docs/A/ANR-0369.pdf.
Hazra S., S.S. Sathaye and A.F. Mascarenha. 1989. Direct somatic embryogenesis
in peanut (Arachis hypogaea). Biotech., 7: 949–951.
He, X., S. C. Miyasaka, M. M. Fitch, P. H. Moore and Y. J. Zhu. 2008.
Agrobacterium tumefaciens-mediated transformation of taro (Colocasia
esculenta (L.) Schott) with a rice chitinase gene for improved tolerance to a
fungal pathogen Sclerotium rolfsii. Plant Cell Rep., 27: 903-909.
Hemmingway, J. S. 1957. The resistance of groundnuts to Cercospora leaf spots.
Empire J. Exp. Agri., 25: 60-68.
Herselman, L., R. Thwaites, F.M. Kimmins, B. Courtois, P.J.A Van Der Merwe
and S.E Seal. 2004.Identification and mapping of AFLP markers linked to
peanut (Arachis hypogaea L.) resistance to the aphid vector of groundnut
rosette disease. Theoret. Appl. Genet., 109(7):1426-1433.
Hooykas, P. J. J. and Schilperoort. 1992. Agrobacterium and plant genetic
engineering. Plant Mol. Biol., 14: 5-8.
155
Horne, C. W. 2005. Peanut disease control alternatives – a guide for producers. The
Texas Agricultural Extension Service. The Texas A and M University
System. Texas: 147-155
Horne, C. W., T. A. Lee and G. L. Philley. 1976. A system for improving control
of foliage disease on peanuts through weather monitoring. Texas
Agricultural Extension Service, College Station, Texas:37-41.
Horsch, R., J. Fry, N. Hoffman, M. Wallroth, D. Ekhholtz, S. Rogers and R. Fraley.
1984. A single and general method for transferring genes into plants. Sci.,
227: 1229-1231.
Huetteman, C.A. and J.E. Preece. 1993. Thidiazuron: a potent cytokinin for woody
plant tissue culture. Plant Cell. Tiss. Org. Cult., 33: 105-109.
Hui, Y.H. 1996. Peanut oil. Bailey’s Industrial Oil and Fat Product 2: 337-392.
Ijaz, M. 2011. Epidemiology and Management of Cercospora leaf spot of Peanut
(Arachis hypogaea L) in Punjab. Ph. D. Thesis, PMAS Arid Agri. Univ.,
Rawalpindi, Pakistan: 8-11
Ikea, J., I. Ingelbrecht, A. Uwaifo and G. Thotttappilly. 2003. Stable gene
transformation in cowpea (Vigna unguiculata L.) using particle gun method.
Afr. J. Biotech., 2: 211–218.
156
ISHS. Acta Horticulturae, http : //www. actahort. org/books/430/index. Htm.
Isleib, T. G., J. E. Bailey, P. W. Rice and R. W. Mozingo. 1999. Breeding for
Virginia type peanuts resistance to multiple diseases. American Peanut Res.
Soc., 31: 68.
Ismail, M. R. 1999. Genetic engineering studies in some plants. Ph. D. Thesis, Ain
Sham Univ., Egypt: 55-67.
Jacobi, J. C., P. A. Backman, D. P. Davis and P. M. Brannen. 1995. AU-Pnuts
Advisory I: Development of a rule-based system for scheduling peanut leaf
spot fungicide applications. Plant Dis., 79: 666-671.
Jahne, A., D. Becker and H. Lorz. 1995. Genetic engineering of cereal crop plants,
a review In: Cassells A.C. and P.W. Jones. The methodology of plant genetic manipulation: criteria for decision making. Dordrecht: Kluwer Academic Publ. 85pp.
Jauhar, P. P. 2001. Genetic engineering and accelerated plant improvement:
opportunities and challenges. Plant. Cell. Tiss. Org. Cult., 64:87–91.
Jenkins, W. A. 1938. Two fungi causing leaf spot of peanut. J. Agri. Res. 56: 317-
322.
157
John Wiley and Sons, Inc. Sanders, T.H. Caballero, B. Trugo, L. and L. Finglas.
2003. Groundnut oil.Encyclopedia of food science and nutrition, pp 2967-
2974.
Johnson, C.S. and M. K. Beute. 1986. The role of partial resistance in the
management of Cercospora leaf spot of peanut in North Carolina.
Phytopath., 76: 468-472.
Joshi M.V., N.A. Sahasrabudhe and S. Hazra. 2003. Responses of peanut somatic
embryos to Thidiazuron. Biol. Plant, 46:187–192.
Kaeppler, H. F. and D. A. Somers. 1994. DNA delivery to maize cell cultures using
silicon carbide fibers . In : M. Freeling & V. Walbot (Eds) . The Maize
Handbook. 610-613 Springer-Verlag, New York.
Kaeppler, H. F., D. A. Somers, H. W. Rines and A. F. Cockbum. 1992. Silicon
carbide fiber mediated stable transformation of plant cells. Theor. Appl.
Genet., 84: 560-566.
Kaeppler, H. F., W. Gu, D. A. Somers, H. W. Rines and A. F. Cockburn. 1990.
Silicon carbide fiber mediated DNA delivery into plant cells. Plant Cell
Rep., 9: 415-418.
158
Kanyand, M., A.P. Dessai and C.S. Prakash. 1994. Thidiazuron promotes high
frequency regeneration of peanut (Arachis hypogaea) plants in vitro. Plant
Cell Rep., 14: 1-5.
Katavic, V., G. W. Haughn, D. Reed, M. Martin and L. Kunst. 1994. In planta
transformation of Arabidopsis thaliana. Mol. Gen. Gene., 245: 363-370.
Khalafalla, M., H. A. El-Sheny, S. M. Rahman, M. Teraishi, H. Hasegawa, T.
Terakawa and M. Ishimoto. 2006. Efficient production of transgenic
soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic
(WSS) method. Afr. J. Biotech. 5 (18): 1594-1599.
Kishore, G. K., S. Pande and A. R. Podile. 2005. Biological Control of Late Leaf
Spot of Peanut (Arachis hypogaea) with Chitinolytic Bacteria. Phytopath.,
95 (11): 57-65.
Klein, T.M., T. Gradziel, M.E. Fromm and J.C. Sanford. 1988. Factors influencing
gene delivery into Zea mays cells by high velocity microprojectiles.
Biotech., 6: 559-563.
Knudsen, G. R., H. W. Spurr and C. J. Johnson. 1987. A computer simulation
model for Cercospora leaf spot of peanut. Phytopatho., 77: 1118-1121.
159
Kochert, G. 1996. Molecular markers and genome mapping. In: Darussamin A
(Ed) Current Status of Agricultural Biotechnology in Indonesia, AARD,
Jakarta, pp. 89–108.
Kokalis-Burelle, N., P. A. Beckman and R. Rodriguez-Kábana. 1992. Potential for
biological control of early leaf spot of peanut using Bacillus cereus and
chitin as foliar amendments. Biol. Ctrl., 2: 321-328.
Kumar, K. R. and P. B. Kirti. 2001. Differential gene expression in Arachis diogoi
upon interaction with peanut late leaf spot pathogen, Phaeoisariopsis
personata and characterization of a pathogen induced cyclophilin. Plant
Mol. Bio., 75: 497-513.
Kumar, S. M., B. K. Kumar, K. K. Sharma, and P. Devi. 2004. Genetic
transformation of pigeon pea with rice chitinase gene. Plant Breed., 123,
485-489.
Lacorte, C., F. J. L. Aragfio, E. R. Almeida, E. Mansur and E. L. Rech. 1997.
Transient expression of GUS and the 2S albumin gene from Brazil nut in
peanut (Arachis hypogaea L.) seed explants using particle bombardment.
Plant Cell Rep., 16: 619-623.
Langridge, P. 1992. Transformation of cereals Agrobacterium and the pollen
pathway: a critical assessment. Plant J., 2: 631-638.
160
Larkin, P. J. and W. R. Scowcroft. 1986. Somaclonal variation- a novel source of
variability from cell cultures to plant improvement. Theor. Appl. Genet. 60:
197-214.
Lei, Y., B. S. Liao, S. Y. Wang, Y.B. Zhang, D. Li and H. F. Jiang. 2006. A SCAR
marker for resistance to Aspergillus flavus in peanut (Arachis hypogaea L.).
Yi Chuan., 28: 1107-11.
Leitch, I. J. and M. D. Bennett. 1997. Polyploidy in angiosperms. Trends Plant Sci.,
2 : 470-476.
Lin, W., C. S. Anuratha, K. Datta, I. Potrykus, S. Muthukrishnan and S. K. Datta.
1995. Genetic engineering of rice for resistance to sheath blight. Biotech.,
13, 686-691.
Lioyd, A. M., A. R. Barnason, S. G. Rogers, M. C. Byrne, R. T. Fraley and R. B.
Horsch. 1986. Transformation of Arabidopsis thaliana with Agrobacterium
tumefaciens. Sci.. 234: 464-466.
Little, L. I., Z. V. Magbanua and W. A. Parrott. 2000. A protocol for repetitive
somatic embryogenesis from mature peanut epicotyls. Plant Cell Rep., 19:
351–357.
161
Liu, F., M. Q. Cao., L. Ya., Y. LI., C. Robaglia and C. Tourneur. 1998. In planta
transformation of pakchoi (Brassica campestris L. ssp. chinensis) by
infiltration of adult plants with Agrobacterium. Acta Horticulturae, 467:
187-192.
Livingstone, D. M. and R, G. Birch. 1999. Efficient transformation and
regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by
particle bombardment into embryogenic callus produced from mature seeds.
Mol. Breed., 5: 43–51.
Livingstone, D.M., J.L. Hampton, P.M. Phipps and E.A. Grabau. 2005. Enhancing
resistance to sclerotinia minor in peanut by expressing a barley oxalate
oxidase gene. Plant Physiol, 137:1354–1362.
Luo, M., P. Dang, M. G. Bausher, C. C. Holbrook, R. D. Lee, R. E. Lynch and B.
Z. Guo. 2005. Identification of Transcripts Involved in Resistance
Responses to Leaf Spot Disease Caused by Cercosporidium personatum in
Peanut (Arachis hypogaea). Phytopath., 95: 381-7.
Marion. J., L. Bach, Y. Bellec, C. Meyer, L. Gissot and J. D. Faure. 2008.
Systematic analysis of protein subcellular localization and interactionusing
high-throughput transient transformation of Arabidopsis seedlings. Plant J.
56 (1): 169-179.
162
Matsushita, M., M. Otani, M. Wakita, O. Tanaki and T. Shimida. 1999. Transgenic
plant regeneration through silicon carbide mediated transformation of rice
(Oryza sativa L.). Breed. Sci., 49: 21-26.
McKently, A. H. (1991). Direct somatic embryogenesis from axes of mature peanut
embryos. In vitro Cell Dev. Biol. 27: 197-200.
McKently, A. H., G. A. Moore, H. Doostdar and R. P. Niedz. 1995.
Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.)
embryo axes and the development of transgenic plants. Plant Cell Rep., 4:
699–703.
McKently. A. H., G. A. Moore and F. P. Gard. 1991. Regeneration of Peanut and
Perennial Peanut from Cultured Leaf Tissue. Crop Sci., 31(3): 833-837.
Mithila. J., S. J. Murch, S. Krishnaraj and P. K. Saxena. 2001. Recent advances in
Pelargonium in vitro regeneration systems. Plant Cell Tiss. Org. Cult.,
67:1–9.
Mohapatra, N. K. 1982. Post-infection changes in sugar content of groundnut
leaves infected with Cercospora personata. Geobios., 9(5-6): 246-248.
Monfort, W. S., A. K. Culbreath, K. L. Stevenson, T. B. Brenneman, D. W. Gorbet
and S. C. Phetek. 2004. Effects of reduced tillage, resistant cultivars and
163
reduced fungicide inputs on progress of early leaf spot of peanut (Arachis
hypogaea). Plant Dis., 88: 858-864.
Mroginski, E., H.Y. Rey, A.M. Gonzalez and L.A. Mroginski. 2004. Thidiazuron
promotes in vitro plant regeneration of Arachis correntina (Leguminosae)
via organogenesis. J. Plant Growth Reg. 23: 129-134.
Mroginski, L. A., K. K. Kartha. 1984. Tissue culture of legumes for crop
improvement. Pl. Breed. Rev., 2: 215–264.
Mroginski. L. A., K. K. Katha and J. P. Shyluk. 1981. Regeneration of peanut
(Arachis hypogaea) by in vitro culture of immature leaves. Can. J. Bot., 59:
826–830.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Plant Physiol., 15 (3): 473-497.
Murthy, B. N. S., S. J. Murch and P. K. Saxena. 1998. Thidiazuron: a potent
regulator of in vitro plant morphogenesis. In vitro Cell Dev. Biol. Plant., 34:
267–275.
Murthy, B. N. S., S. J. Murch, P. K. Saxena. 1995. Thidiazuron induced somatic
embryogenesis in intact seedlings of peanut (Arachis hypogaea):
164
endogenous growth regulator levels and significance of cotyledons. Plant
Physiol., 94: 268–276.
Nagatani, N., H. Honda, H. Shimada and T. Kobayashi. 1997. DNA delivery into
rice cells and transformation of cell suspension cultures. Biotech., 11: 471 –
473.
Nayche, L., Ivo, P. Cristina, Nascimento, S. L. Vieira, A. Francisco A. P. Campos,
F. J. L. Aragao. 2008. Biolistic-mediated genetic transformation of cowpea
(Vigna unguiculata) and stable Mendelian inheritance of transgenes Plant
Cell Rep., 27: 1475–1483.
Nigam, S. N. and S. L. Dwivedi. 2000. Enhanced resistance to leaf spot in
cultivated groundnut (Arachis hypogaea L.) on a partially-resistant peanut
(Arachis hypogaea L.) cultivar. ICRISAT-Patancheru, India. Soil and Crop
Sci. Soc. Fla. Proc., 50: 37-40.
Niu, C., Y. Akasaka-Kennedy, P. Faustinelli, M. Joshi, K. Rajasekaran, H. Yang,
Y. Chu, J. Cary and P. Ozias-Akins. 2009. Antifungal activity in transgenic
peanut (Arachis hypogaea L.) conferred by a nonheme chloroperoxidase
gene. Peanut Sci., 36:126–132.
165
Oakeley, E. J., A. Podesta and J. P. Jost. 1997. Developmental changes in DNA
methylation of two tobacco pollen nuclei during maturation. Proc. National
Acad. Sci., USA. 94: 11721-11725.
Olhoft, P.M., L.E. Flagel, C.M. Donovan and D.A. Somers. 2003. Efficient
soybean transformation using hygromycin B selection in the cotyledon-
node method. Planta, 216:723–735.
Omirrullah, S., A. Ismagulava, M. Karabaev, T. Meshi and M. Iwabuchi. 1996.
Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum
aestivum L.) mature embryos. Plant Cell Rep., 16: 133-136.
Opabode, J. T. 2006. Agrobacterium-mediated transformation of plants: emerging
factors that influence efficiency. Biotech. Bio. Rev., 1: 12-20.
Ozias-Akins, P. 1989. Plant regeneration from immature embryos of peanut. Plant
Cell Rep., 8:217-218.
Ozias-Akins, P. and R. Gill. 2001. Progress in the development of tissue culture
and transformation methods applicable to the production of transgenic
peanut. Peanut Sci., 28: 123–131.
166
Ozias-Akins, P., J. A. Schnall, W. F. Anderson, C. Singsit, T. E. Clemente, M. J.
Adang and A. K. Weissinger. 1993. Regeneration of transgenic peanut
plants from stably transformed embryogenic callus. Plant Sci., 93: 185-194.
Ozias-Akins, P., W.F. Anderson and C.C. Holbrook. 1992. Somatic embryogenesis
in Arachis hypogaea L.: genotype comparison. Plant Sci., 83: 103–111.
Ozudogru, E.A, E. Kaya and M. Lambardi . 2013. In vitro propagation of peanut
(Arachis hypogaea L.) by shoot tip culture. Methods Mol. Biol.,11(1):77-
87.
Pandey, M.K., E. Monyo, P. Ozias-Akins, X. Liang, P. Guimaraes, S.N. Nigam,
H.D.Upadhyaya, P. Janila, X. Zhang, B. Guo, D.R. Cook, D.J. Bertioli, R.
Michelmore and R.K. Varshney. 2012. Advances in Arachis genomics for
peanut improvement. Biotechnol. Adv.30 (3): 639–651.
Pattee, H. and H. T. Stalker. 1995. Advances in peanut science. American Peanut
Research and Education Society, Inc., Stilwater, OK 74078, USA: 163-175
Pattee, H. and Young, C. T. 1982. Peanut science and technology. Yoakum, Texas
77995, USA: 52-59.
Pauer, G. D., A. B. Van Jaarsveld, W. J. Jansen and S. W. Baard. 1983. Control of
groundnut leaf spot diseases with fungicides. Phytophylactica. 15: 1-6.
167
Pestana, M. C., C. Lacorte., V. G. de Freitas., D. E. de Oliveira and E. Mansur.
1999. In vitro regeneration of peanut (Arachis hypogaea L.) through
organogenesis.effect of culture temperature and silver nitrate. 35 (3): 214-
216.
Petolino, J. F., N. L. Hopkins and B. D. Kosegi. 2000. Whisker mediated
transformation of embryogenic callus of maize. Plant Cell Reports, 19, 781–
786.
Phillips, R. L. and S. M. Kaeppler and P. Olhoft. 1994. Genetic instability of plant
tissue culture: breakdown of normal controls. Proc. Nation. Acad. Sci.,
USA, 91: 5222-5226.
Phipps, P.M. 2000. Crop profile for peanuts in Virginia. State University of
Virginia,USA : 37-51.
Pittman, R. N., D. J. Banks., J. S. Kirby., E. D. Mitchell and P .E. Richardson.
1983. In vitro culture of immature peanut (Arachis spp.) leaves:
morphogenesis and plantlet regeneration. Peanut Sci., 10: 21– 25.
Ponsamuel, J., D. V. Huhman, B. G. Cassidy and D. Post-Beittenmiller. 1998. In
vitro regeneration via caulogenesis and brassin-induced shoot conversion of
dormant buds from plumular explants of peanut (Arachis hypogaea L.cv
`Okrun'). Plant Cell Rep., 17: 373–378.
168
Porter, D. M., D. H. Smith and R. Rodrígues-Kábana. 1990. Compendium of
peanut diseases. APS Press, 3340 Pilot Knob Road, St. Paul, Minnesota.
55121, USA.
Prasad, M. N. R. and M. V. C. Gowda. 2006. Mechanisms of resistance to tobacco
cutworm (Spodoptera litura F.) and their implications to screening for
resistance in groundnut. Euphytica., 149: 387–399.
Radhakrishnan, T., T. G. K. Murthy, K. Chandran and A. Bandyopadhyay. 2001.
Somatic embryogenesis in Arachis hypogaea: revisited. Aust. J. Bot., 49:
753–759.
Radhakrishnan, T., T. G. K. Murthy, K. Chandran and A. Bandyopadhyay. 2001.
Somatic embryogenesis in Arachis hypogaea: revisited. Aust. J. Bot., 49:
753–759.
Rao, P. V. S., D. McDonald and K. R. Reddy. 1993a. Perpetuation of peanut leaf
spots pathogens. Oléagineux, 48 (77): 2-82.
Rao, P. V. S., J. L. Renard, F. Waliyar, D. D. McDonald and R. Schilling. 1993b.
Variation in symptoms of Cercospora arachidicola isolates on some
groundnut genotypes. Oléagineux., 48 (5): 243-250.
169
Rasmussen, J. L., J. R. Kikkert, M. K. Roy and J. C. Sanford. 1994. Biolistic
transformation of tobacco and maize suspension cells using bacterial cells
as micro-projectiles. Plant Cells Rep., 13: 212-217.
Reddy L. R. and G .M. Reddy. 1993. Factors affecting direct somatic
embryogenesis and plant regeneration in groundnut, Arachis hypogaea L.
Ind .J. Exptl. Biol., 31: 57-60.
Reddy, L. J., S. N. Nigam, S. L. Dwivedi and R. W. Gibbons. 1987. Breeding
groundnut cultivars resistant to rust (Puccinia arachidis Speg.). Groundnut
rust disease proceedings of a discussion group meeting, 24-28 September
1984, ICRISAT., India., 17: 5-25.
Reddy, P. S. 1984. Groundnut in India: present status and strategy. Proc. Meeting
of Asian Reg. Res. on grain legumes ICRISAT: 32-35.
Rey, H. Y., A. M. Scocchi, A. M. Gonzalez and L. A. Mroginski. 2000. Plant
regeneration in Arachis pintoi (Leguminosae) through leaf culture. Plant
Cell Reports. 19: 856-862.
Robinson, P. J., S. Srivardhini and G. Sasikumar. 2011. Somatic embryogenesis
and plant regeneration from cotyledon tissue of Arachis hypogaea L. Res.
Plant Biol., 1(3): 21-27.
170
Rohini, V. K. and K. S. Rao. 2000. Transformation of peanut (Arachis hypogaea
L.): a non-tissue culture based approach for generating transgenic plants.
Plant Sci., 15: 41-49.
Rohini, V. K. and K. S. Rao. 2001. Transformation of peanut (Arachis hypogaea
L.) with tobacco chitinase gene: variable response of transformants to leaf
spot disease. Plant Sci., 160: 889-898.
Rohini, V. K. and R. K. Sankar. 2002. In planta strategy for gene transfer into
plants: Embryo transformation. Physiol. Mol. Biol. Plants., 8 (2): 161-169.
Russell, D. R., K. M. Wallace, J. H. Bathe, B. J. Martinell and D. E. McCabe.
1993. Stable transformation of Phaseolus vulgaris via electric- discharge
mediated particle acceleration. Plant Cell Rep., 12: 165- 169.
Russell, J.A., M.K. Roy and J.C. Sanford. 1992. Physical trauma and tungsten
toxicity reduce the efficiency of biolistic transformation. Plant Physiol., 98:
1050-1056.
Sanford, J. C. 1990. Biolistic plant transformation. Pzjro Plant 79: 206-209.
Sanford, J. C., F. D. Smith and J. A. Russel. 1993. Optimizing the biolistic process
for different biological applications. Methods Enzymol., 217: 483-510.
171
Sarkar, R.H. and A. Islam. 2000. Direct organogenesis from leaflet explants of
peanut (Arachis hypogaea L.). Bangladesh J. Bot., 29: 109-114.
Sato, S., C. Newell, K. Kolacz, L. Tredo, J. Finer and M. Hinchee, 1993.Stable
transformation via particle bombardment in two different soybean
regeneration systems. Plant Cell Rep. 12: 408-413.
Saxena, P. K., K. A. Malik and R. Gill. (1992). Induction by thidiazuron of
somatic embryogenesis in intact seedlings of peanut. Planta., 187:421-424.
Seitz, M. H., H. T. Stalker and C. C. Green. 1987. Genetic variation for
regenerative response in immature leaflet cultures of the cultivated peanut,
Arachis hypogaea. Plant Breed., 98:104– 110.
Sellars, R. M., G. M. Southward and G. C. Phillips. 1990. Adventitious somatic
embryogenesis from cultured immature zygotic embryos of peanut and
soybean. Crop Sci., 30:408-414.
Selvaraj, M.G., M. Narayana, A. M. Schubert, J. L. Ayers, M. R. Baring and M. D.
Burow. 2009. Identification of QTLs for pod and kernel traits in cultivated
peanut by bulked segregant analysis. Elect. J. Biotech. , 12(2)
http://www.ejbiotechnology.info/index.php/ejbiotechnology/
article/viewFile/v12n2-13/694
172
Sharma, K. K. and V. Anjajah. 2000. An efficient method for the production of
transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium
tumefaciens-mediated genetic transformation. Plant Sci., 159: 7–19.
Shokes, F. M. and D. W. Gorbet. 1990. Commercial fungicide use for leaf spot on
a partially-resistant peanut (Arachis hypogaea) cultivar. Soil. Crop Sci. Fla.
Proc., pp 50-61.
Siddharth, T. and T. Rakesh. 2012. Optimization of factors for efficient recovery
of transgenic peanut (Arachis hypogaea L.). Plant Cell. Tiss. Org. Cult.,109
(1): 111-121.
Singh, N.D., L. Sahoo, N.B. Sarin and P.K. Jaiwal. 2003. The effect of thidiazuron
on organogenesis and somatic embryogenesis in pigeonpea (Cajanus cajan
L. Millsp). Pl. Sci., 164: 341-347.
Singh. S., and S. Harza. 2009. Somatic embryogenesis from the axillary meristems
of peanut (Arachis hypogaea L.). Plant Biotechnol. Rep.,3(4): 333-340.
Singsit, C., M.J. Adang, R.E. Lynch, W.F. Anderson, A.M. Wang, G. Cardineau
and P. Ozias-Akins.1997. Expression of a Bacillus thuringiensis cry1A(c)
gene in transgenic peanut plants and its efficacy against lesser cornstalk
borer. Transgenic Res., 6:169–176.
173
Smith, D. H. and F. L. Crosby. 1973. Aerobiology of two peanut leaf spot fungi.
Phytopath., 63: 703-707.
Sobolev, V. S., B. Z. Guo, C. C. Holbrook and R. E. Lynch. 2007. Interrelationship
of phytoalexin production and disease resistance in selected peanut
genotypes. J. Agri. Food Chem., 55 (2): 195-200.
Srinivasan. T., K. Kumar and P. Kirti. 2010. Establishment of efficient and rapid
regeneration system for some diploid wild species of Arachis. Plant Cell
Tissue Org. Cult., 101(3): 303-309.
Subrahmanyam, P. and G. L. Hildebrand. 1997. Responses of peanut genotypes to
fungicidal control of early leaf spot in Malawi. Peanut Sci., 24: 73-77.
Subrahmanyam, P., D. McDonald, F. Waliyar, L. J. Reddy, S. N. Nigam, R. W.
Gibbons, R. V. Ramanatha, A. K. Singh, S. Pande, P. M. Reddy and P. V.
Subba Rao. 1995. Screening methods and sources of resistance to rust and
late leaf spot of groundnut. ICRISAT Info. Bulletin. 47: 1-20.
Subrahmanyam, P., J. H. Williams, D. McDonald and R. W. Gibbons. 1984. The
influence of foliar diseases and their control by selective fungicides on a
range of groundnut (Arachis hypogaea L.) genotypes. Ann. Appl. Bio., 104:
467-476.
174
Subrahmanyam, P., J. P. Bosc, H. Hassane, D. H. Smith, A. Mounkaila, B. J.
Ndunguru and P. H. Sankara. 1992. Groundnut diseases in Niger and
Burkina Faso. Oléagineux, 47 (3): 119-129.
Supartana, P., T. Shimizu, M. Nogawa, H. Shioiri, T. Nakijima, N. Haramoto, M.
Nozue and M. Kojima. 2006. Development of simple and efficient in Planta
transformation method for wheat (Triticum aestivum L.) using
Agrobacterium tumefaciens. J. Biosci. Bioeng., 100 (4): 162-170.
Swanevelder, C. J. 1998. Groundnuts – Always tops. ARC-Grain Crops Institute,
Potchefstroom. Kucharek, T. 2000. Peanut leaf spot and rust. Plant
Pathology Fact Sheet. Florida Cooperative Extension Service, Institute of
Food and Agricultural Sciences, University of Florida, USA: 40-52
Swathi, T. A., S. K. Jami, R. S. Dalta and P. B. Kirti. 2006. Genetic transformation
of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a
promoterless gus::nptII fusion gene based vector. J. Biosci., 31 (2): 235-
246.
Taylor, M. G., V. Vasil and I. K. Vasil. 1993. Enhanced GUS gene expression in
cereal/grass cell suspensions and immature embryos using maize ubiquitin-
based plasmid pAHC25. Plant Cell Rep., 12: 491-495.
175
Thu, T. T., T. T. X. Mai, E. Dewaele, S. Farsi, Y. Tadesse, G. Angenon and M.
Jacobs. 2003. In vitro regeneration and transformation of pigeonpea
(Cajanus cajan L.). Mol. Breed., 11: 159–168.
Tiwari, S. and R. Tuli. 2008. Factors promoting efficient in vitro regeneration
from de-embryonated cotyledon explants of Arachis hypogaea L. Plant Cell
Tiss. Org. Cult., 92: 15–24.
Tiwari, S. and R. Tuli. 2009. Multiple shoot regeneration in seed-derived immature
leaflet explants of peanut (Arachis hypogaea L.). Sci. Horti., 121: 223-227.
Touraev, A., E. Stoger, V. Voronin and E. Heberla-Borse. 1997. Plant male gene
line transformation. Plant J., 12: 949-956.
Tse, H. L. H. and G. Y. S. Chan. 2000. Pollen germination a challenging and
educational experiment. J. Bio. Edu., http://www.job.org/default.
ash?edname=457. htm&cont_id=13& n=7.
Tuggle, J. C., O. D. Smith and J. L. Starr. 1999. Variation in aggressiveness and
virulence among isolates of Cercospora arachidicola. Peanut Sci., 26: 94-
98.
176
Utomo. S. D., A. K. Weissinger and T. G. Isleib. 1996. High efficiency peanut
regeneration using a non-imbibed immature leaflet culture method. Peanut
Sci., 23: 71–75.
Valvekens, D., M. Van Montagu and M. Van Lijsebettens. 1988. Agrobacterium
tumefaciens mediated transformation of Arabidopsis thaliana root explants
by using kanamycin selection. Proc. Nat. Acad. Sci., USA 85: 5536- 5540.
Van Der L., L. M. Ven, J. M. Franken, V. Tuyl, A. J.V. Tunen and H. J. M. Dons.
2004, Transgenic lilies via pollen-mediated transformation. Acta Hort. 430:
529-530.
Van Wyk, P. S. and A. J. Cilliers. 2000. Grondboonsiektes en –plae/Groundnut
diseases and pests. ARC Grain Crops Institute, Potchefstroom, SA:110-123.
Vega, M., L. Bontoux and A. Liobell. 1999. Biotechnology for environmentally
safe agriculture. In vitro Cell Dev. Biol. Plant., 35: 432–435.
Venkatachalam, P., A. Subramaniampillai and N. Jayabalan. 1996. In vitro callus
culture and plant regeneration from different explants of groundnut (Arachis
hypogaea L.). Breed. Sci., 4: 315-320.
Venkatachalam, P., N. Geetha, K. Abha, M. S. Shaila and G. L. Sita. 2000.
Agrobacterium-mediated genetic transformation and regeneration of
177
transgenic plants from cotyledon explants of peanut via somatic
embryogenesis. Currn. Sci., 78: 1130-1136.
Venkataraman, S. and S. K. Kazi. 1979. A climatic disease calendar for ‘Tikka’ of
groundnut (Abstr.) J.Maharashtra Agri.Uni., 4: 91-94.
Verma, A., C. P. Malik, V. K. Gupta and Y. K. Sinsinwar. 2009. Response of
groundnut varieties to plant growth regulator (BAP) to induce direct
organogenesis. World. J. Agri. Sci., 5 (3): 313-317.
Victor, J. M. R., S. J. Murch, S. K. Raj and P. K. Saxena. 1999. Somatic
embryogenesis and organogenesis in peanut: the role of thidiazuron and N 6
-benzylaminopurine in the induction of plant morphogenesis. Plant Growth
Regul., 28: 9–15
Vidoz, M. L., P. Klusacek, H. Y. Rey and L. A. Mroginski. 2006. In vitro plant
regeneration of Arachis correntina (Leguminosae) through somatic
embryogenesis and organogenesis. Plant Cell Tiss. Org. Cult. 86:111–115.
Vijaya Laxmi, G. and C.C. Giri. 2003. Plant regeneration via organogenesis from
shoot base-derived callus of Arachis stenosperma and A. villosa. Current
Sci., 85: 1624-1629.
178
Waliyar, F., M. Adamou and A. Traoré. 2000. Rationale use of fungicide
applications to maximize peanut yield under foliar disease pressure in West
Africa. Plant Dis., 84: 1203-1211.
Wang A. M., H. L. Fan, C. Singsit and P. Ozias-Akins. 1998. Transformation of
peanut with a soybean vspB promoter-uidA chimeric gene I. Optimization
of a transformation system and analysis of GUS expression in primary
transgenic tissues and plants. Physiol. Plant, 102: 38–48.
Wang, K., P. Drayton, B. Frame, J. Dunwell and J. Thompson. 1995. Whisker-
mediated plant transformation: An alternative technology. In vitro Cell.
Dev. Bio., 31: 101-104.
Weeks, R., A. Hagan., W. Foshee., D. L. Hartzog and J. W. Everest. 2000. Peanut
pest management and scout manual. Alabama A & M and Auburn
Universities. Alabama Cooperative Extension System. www.aces.edu.
Wetzstesin, H. Y and C. M. Baker. 1993. The relationship between somatic embryo
morphology and conversion in peanut (Arachis hypogaea L.). Plant Sci., 92:
81-89.
Winans, S. C., R. A. Kerstetter and E. W. Nester. 1988. Transcriptional regulation
of the virA and virG genes of Agrobacterium tumefaciens J. Bacter.,
170: 4047–4054.
179
Woodroof, N. C. 1993. Two leafspots of the peanut (Arachis hypogaea L.).
Phytopath., 23: 627-640.
Yamamoto, T., H. Iketani, H. Ieki, Y. Nishizawa, K. Notsuka, T. Hibi, T.
Hayashi and N. Matsuta. 2000. Transgenic grapevine plants expressing a
rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep.
19, 639-646.
Ye, G. N., D. Stone., S. Z. Pang., W. Creely., K. Gonzalez and M. Hinchee. 1999.
Arabidopsis ovule is the target for Agrobacterium in planta vacuum
infiltration transformation. The Plant J., 19: 249-257.
Yong, L. L. Boshou, S.Y. Wang, L. Dong and H. Jiang. 2005. Identification of
AFLP markers for resistance to seed infection by Aspergillus flavus in
peanut (Arachis hypogaea L.). Acta Agronomica Sinica. 31:1349-1353.
Zhang, J., N. Y. Klueva, Z. Wang, R. Wu, T. H. D. Ho and H. T. Nguyen. 2000.
Genetic engineering for abiotic stress resistance in crop plants. In vitro Cell
Dev. Bio. Plant., 36: 108-114.
Zhang, S., M. S. Reddy, N. Kokalis-Burelle, L. W. Wells, S. P. Nightengale and J.
W. Kloepper. 2001. Lack of induced systemic resistance in peanut to late
leaf spot disease by plant growth-promoting Rhizobacteria and chemical
elicitors. Plant Dis., 85: 879-884.
180
Zhou, G. Y., J. Weng, Y. S. Zhen, J. G. Huang, S. Y. Qian and G. L. Liu. 1983.
Introduction of exogenous DNA into cotton embryos. In Methods in
Enzymology, Eds. Wu, R., Grossman, L. and Molddave, K., Recombination
DNA, Part C, Academic Press, New York, 101: 433-481.
Zuraida, A. R., K.1 Rahiniza, M. R. N. Hafiza, S. Roowi, Z. Zamri and S.
Subramaniam. 2010. Factors affecting delivery and transient expression of
gusA gene in Malaysian indica rice MR 219 callus via biolistic gun system.
Afr. J. Biotech. 9(5): 8810-8818.
PUBLICATION