SYSTEM DEVELOPMENT FOR IN VITRO REGENERATION AND GENE DELIVERY INTO COMMON BEAN (PHASEOLUS VULGARIS) By Kingdom Moses Kwapata A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Breeding, Genetics and Biotechnology - Crop and Soil 2011 ABSTRACT SYSTEM DEVELOPMENT FOR IN VITRO REGENERATION AND GENE DELIVERY INTO COMMON BEAN (PHASEOLUS VULGARIS) By Kingdom Moses Kwapata Common bean is an important staple food source for many people worldwide. Given the social economic and nutritional importance of this crop, the research presented in this dissertation focused on development of a novel system for in vitro regeneration using apical shoot meristem primordia explant and a gene delivery system for common bean. The research investigated methods for reducing recalcitrance of common bean towards in vitro regeneration. The results showed that a ratio of 2.5 mg L 0.1 mg L -1 -1 benzyladenine (BA) to of naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA) promoted -1 robust multiple shoot regeneration. The addition of 30 mg L of silver nitrate reduced the inhibitory effect of phenolic compounds. Standardized conditions for gene delivery into apical shoot meristem primordia were TM developed using both Biolistic bombardment and Agrobacterium tumefaciens with two marker genes, bar and gus. Results showed that transformation efficiency of the bar transgene with particle bombardment method was 8.4% using 7584 kPa helium pressure with a concentration of 1.5 µg of plasmid DNA per bombardment and bombarding the explants twice at a 24 hour interval. Effect of co-cultivation period for different strains of A. tumefaciens (EHA105, LBA4404 and GV3301) and genotypes of common bean were assessed. Transient and stable expression of the gus gene showed ‘Sedona’ to be more amenable to Agrobacterium transformation than ‗Matterhorn‘. A co-cultivation period of 15 days with Agrobacterium strain GV3301was most effective in producing the highest transient expression of 81% and stable expression of 0.68%. The above results show that the Biolistic TM gun delivery system is more efficient than Agrobacterium system for generating stable transgenes into common bean. Testing was conducted for stable integration and expression of two major agronomically valuable transgenes. The first was the barley (Hordeum vulgare) late embryogenesis abundant protein (HVA1) gene, which confers drought tolerance. Significant resilience of transformed plants versus wild type towards drought stress was observed with a corresponding increase in root length for transgenic genotypes ‗Matterhorn‘ and ‗Sedona‘. The second gene tested was the wheat (Triticum aestivum) germin gene (gf2.8) that produces an oxalate oxidase that reduce pathogenicity of Sclerotinia sclerotiorum the causal agent of white mold of common bean. Transfer of this gene delayed the onset of lesions caused by S. sclerotiorum for a period of 72 hours in leaves of transgenic ‗Matterhorn‘. In conclusion, the goals and objectives of the research were achieved by demonstrating the applicability of novel protocols that were developed for in vitro regeneration followed by gene delivery of two marker genes and two agronomically important genes into common bean. The novelty of this research is the utilization of apical shoot primordia cells that are actively dividing. The delivery of transgenes into these cells followed by their selection and regeneration resulted into stable transgenic common bean plants. DEDICATION I dedicate this work to my parents, Moses and Dorothy Kwapata for their encouragement and tireless support throughout my studies. iv ACKNOWLEDGEMENTS I would like to thank the almighty God for making it possible for me to reach this level. I would also like to thank my parents Moses and Dorothy Kwapata for their tireless support and encouraging me to go to school to obtain an education. I also thank my siblings, Victor, Ellenor and Moses for lifting my spirits when studies got tough. Special thanks go to my co-advisors Drs. Mariam Sticklen and James D. Kelly. I thank Dr. Sticklen for her tireless effort to see to it that my research project move ahead even during difficult times when there seemed to be no end in sight and for providing financial assistance to cover my health insurance. I also want to sincerely thank Dr. Kelly for his guidance and financial support when times became tough and the future of my research was being threatened by financial constraints. I thank Drs. Russell Freed and Ryan Warner for accepting to be members of my PhD guidance committee who were able to provide valuable advice in a timely manner when needed. I thank members of my research lab namely Sang, Thang, Jason and Jeap for being supportive. Thanks also go to Robab, our senior lab technician who was helpful in providing directions on development of in vitro regeneration system and the use of lab equipment such as the gene gun. I thank Gloria Katsande for being a supporting friend who helped me with greenhouse work. I would like to thank various donors who funded my research and studies at various stages namely the Fulbright commission, Mericle fellowship and the CANR Dean‘s office through the help of the Associate Dean Dr. R. Brandenburg. Finally, I would also like to thank Barbara Sawyer-Koch for helping with the editing of the dissertation. v TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .................................................................................................................... xii KEY TO SYMBOLS AND ABBREVIATIONS ................................................................. xviii CHAPTER I ................................................................................................................................... 1 LITERATURE REVIEW OF IN VITRO REGENERATION AND GENETIC TRANSFORMATION OF COMMON BEAN (PHASEOLUS VULGARIS) ....................... 1 1.0 Introduction ............................................................................................................... 1 1.1 In vitro Regeneration of Common Bean ................................................................... 3 1.2 Common Bean Genetic Transformation ................................................................... 7 CHAPTER II................................................................................................................................ 16 DROUGHT, SALT STRESS AND WHITE MOLD PATHOGENESIS............................ 16 RELATED TO COMMON BEAN ........................................................................................... 16 2.0 Drought and salt stress ............................................................................................ 16 2.1 White Mold (Sclerotinia sclerotiorum) ................................................................... 21 2.2 Germin Gene (gf2.8) .............................................................................................. 23 CHAPTER III .............................................................................................................................. 29 GOALS, OBJECTIVES AND METHODOLOGY ................................................................ 29 3.0 Goals....................................................................................................................... 29 3.1 Specific Objectives ................................................................................................. 29 3.2 Materials and Methods ........................................................................................... 30 3.2.1 In vitro regeneration protocol for common bean .............................................. 30 3.2.2 Genetic transformation ................................................................................... 36 3.2.3 Confirmation of transgene integration and expression .................................... 42 3.2.4 Biological activity tests ................................................................................... 45 CHAPTER IV .............................................................................................................................. 48 RESULTS AND DISCUSSION : SYSTEMS DEVELOPMENT FOR IN VITRO REGENERATION OF COMMON BEAN ............................................................................. 48 4.0 In vitro regeneration ................................................................................................ 48 4.1 Organogenesis and embryogenesis ......................................................................... 50 4.2 Discussion ............................................................................................................... 67 CHAPTER V ............................................................................................................................... 70 vi RESULTS AND DISCUSSION: GENETIC TRANSFORMATION SYSTEM DEVELOPMENT IN COMMON BEAN................................................................................ 70 5.1. Optimizing conditions for the BiolisticTM bombardment method using stable integration of the bar gene ............................................................................................. 70 5.2. Optimization of conditions used in developing Agrobacterium-mediated transformation method for transient expression of gus gene ........................................ 73 5.3 Confirmation of stable gene integration using PCR and Southern blot. ................. 78 5.4 Confirmation of gene expression using Northern blot analysis .............................. 84 5.5 Liberty herbicide resistance test of T2 and T3 of transgenic plants........................ 87 5.6 Confirmation of gus transgene expression using the GUS assay in T3 ‗Matterhorn‘ genotype ........................................................................................................................ 89 CHAPTER VI .............................................................................................................................. 93 RESULTS AND DISCUSSION: DROUGHT AND SALT STRESS TOLERANCE OF TRANSGENIC COMMON BEAN .......................................................................................... 93 6.1. Confirmation of HVA1 transgene integration in plants using PCR and Southern blot analysis ................................................................................................................... 94 6.2. Confirmation of HVA1 transcription in plants using Reversed Transcription PCR, followed by Northern blotting ....................................................................................... 97 6.3. Drought tolerance tests of transgenic versus control non-transformed plants ..... 100 CHAPTER VII .......................................................................................................................... 108 RESULTS AND DISCUSSION: WHITE MOLD STRESS TESTS OF TRANSGENIC COMMON BEAN .................................................................................................................... 108 7.1. Confirmation of germin transgene integration using PCR and Southern blot analysis ........................................................................................................................ 108 7.2. Confirmation of germin gene expression using RT-PCR and Northern blot analysis ..................................................................................................................................... 111 CHAPTER VIII ......................................................................................................................... 120 CONCLUSIONS AND FUTURE PERSPECTIVES ........................................................... 120 Appendix 1 ................................................................................................................................. 128 Appendix 2 ................................................................................................................................. 129 Appendix 3 ................................................................................................................................. 130 Appendix 4 ................................................................................................................................. 131 Appendix 5 ................................................................................................................................. 132 Appendix 6 ................................................................................................................................. 133 Appendix 8 ................................................................................................................................. 135 Appendix 9 ................................................................................................................................. 136 Appendix 10 ............................................................................................................................... 139 Appendix 11 ................................................................................................................................. 142 Appendix 12 ................................................................................................................................. 147 vii Appendix 13 ................................................................................................................................. 148 Appendix 14 ................................................................................................................................. 149 Appendix 15 ................................................................................................................................. 150 Appendix 16 ................................................................................................................................. 151 REFERENCES ............................................................................................................................ 152 viii LIST OF TABLES Table 1: A summary of in vitro regeneration of common and tepary bean ...... 5 Table 2: Grain legume transformation systems using different explants, genes and gene delivery methods.............................................................................. 12 Table 3: Summary of transgenic research using the germin gene in different crops ................................................................................................................ 25 Table 4: Ten genotypes of P. vulgaris representing the ten different commercial classes grown in Northern America ............................................ 31 Table 5: Effect of genetic origin as represented by gene pool and race on the efficiency of apical shoot meristem multiplication of 10 contrasting genotypes of common bean (Phaseolus vulgaris) ........................................................... 51 Table 6: Genotypic specific growth regulator combination promoting highest number of multiple shoots .............................................................................. 52 Table 7: Effect of anti-oxidants on apical shoot meristem qaulity in common bean genotype, ‗Condor‘................................................................................. 57 Table 8: Different treatment combinations (gene gun pressure, DNA plasmid concentration and bombardment frequency) used for optimizing BiolisticTM bombardment conditions ................................................................................. 72 Table 9: Summary of measurement parameters of drought stress test ......... 103 Table 10: Anova for statistical model ........................................................... 128 Table 11:Anova for treatment and interaction of treatments for type i and type ii sum of squares ........................................................................................... 129 ix Table 12: Least squares mean for the genotypic effect................................. 130 Table 13: Separation of means for genotypic effect ..................................... 131 Table 14 : Least squares mean for cytokinin effect ...................................... 132 Table 15: Least squares mean seperation for cytokinin effect ...................... 133 Table 16: Least squares mean for auxin effect ............................................. 134 Table 17: Least squares mean separation for auxin effect ............................ 135 Table 18: Means for the interaction between cytokinin and auxin ............... 136 Table 19: Means of multiple shoots for the interaction between genotype and auxin.............................................................................................................. 139 Table 20: Means of multiple shoot for the interaction between genotype and cytokinin ....................................................................................................... 142 Table 21: Anova for rooting experiment ...................................................... 147 Table 22: Least squares means for number of roots for dipping effect in IBA ....................................................................................................................... 148 Table 23: Least squares means for the effect of different levels of hormone concentration in media on the number of roots ............................................ 149 x Table 24 : Least squares means for the effect of IBA dipping on the length of roots (cm) ...................................................................................................... 150 Table 25: least squares means for the effect of growth regulators, NAA, IAA and IBA (hormone) on root length (cm) ....................................................... 151 xi LIST OF FIGURES Figure 1: Drought and salt stress biosynthetic pathway. (modified from Wang et al. 2003) ............................................................................................ 20 Figure 2: Linear map of pACT1F cassette (not drawn to scale). Rice actin promoter (Act), gus gene (UidA), and nopaline synthase terminator (Tnos) .. 36 Figure 3. Linear map of pBY520 cassette (not drawn to scale). Rice actin promoter (Act1), Barley or Hordeum vulgare (HVA1) LEA 3 gene, Cauliflower Mosaic Virus 35S promoter, bar gene and nopaline synthase terminator (Tnos) ............................................................................................ 37 Figure 4. Circular map of the 6.9 Kb of pBKSbar/gf2.8, not drawn to scale. Amp= ampicilin resistant marker, bar=herbicide selectable marker, pUC ori= origin of replication of the pUC 18 plasmid vector. ....................................... 38 Figure 5. Linear map of pCAMBIA3301 T-DNA cassette. LB/RB – left/right T-DNA border sequences; P35S/T35S – CaMV 35S promoter/terminator; bar – coding region of the phosphinothricin resistance gene; Tnos – nopaline synthase terminator; gus-intron –gusA gene coding region with intron sequence .......................................................................................................... 39 Figure 6: Effect of cytokinin-auxin combinations on apical shoot meristem multiplication of 10 P. vulgaris genotypes. Note: BAP/TDZ 1,2,3&4=1, 2.5, 5,10 mg L-1; NAA/IAA 1, 2, 3= 0.05, 0.1, 1 mg L-1 ( for supplemental data see appendix 9 and 10) .................................................................................... 53 Figure 8: In vitro apical shoot meristem multiplication performance of 10 P. vulgaris genotypes .......................................................................................... 54 Figure 9: Effect of 4 antioxidant treatments on the quality of multiple shoots ......................................................................................................................... 56 xii Figure 10: Effect of growth regulator combinations on morphogenesis pathway of in vitro cultures of P. vulgaris ..................................................... 59 Figure 11: Differentiation of somatic embryos, multiple shoots and regenerated mature greenhouse grown rooted ‗Olathe‘ common bean plants.60 Figure 12: Effect of dipping shootlets in IBA and culturing of shoots in different auxins on the number and the length of regenerated roots five weeks after transfer of shoots into rooting media. ..................................................... 63 Figure 13. In vitro response of rooting ability using different levels of IBA dipping system ................................................................................................ 64 Figure 14. Effect of co-cultivation period (1, 5, 10 and 15 d) on the transformation frequency of two genotypes of common bean, ‗Matterhorn‘ and ‗Sedona‘, using three different strains of A. tumefaciens (EHA105, GV3301 and LBA4404).................................................................................. 75 Figure 15. Effect of using different strains of A. tumefaciens (EHA105, GV3301 and LBA4404) with two common bean genotypes, ‗Matterhorn‘ and ‗Sedona‘, on the relative stable transformation frequency of T1 (second generation) plants after 15 days of co-cultivation........................................... 77 Figure 16: PCR T1 plants of genotypes; ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp ..... 78 Figure 17: PCR T2 plants of genotypes: ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp. .... 79 Figure 18: PCR T3 plants of genotypes: ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp ..... 80 Figure 19: Southern blot showing integration of bar gene in genotype ‗Condor‘. The (+) represents the plasmid DNA; Wt: Wild type non transgenic leaf DNA; C1 to C3: DNA taken from leaves of three transgenic plants of the xiii same transgenic line; C4: DNA taken from leaves of a different independent transgenic line. ................................................................................................ 81 Figure 20: Southern blot showing integration of bar gene in genotype ‗Matterhorn‘ line 2 (M2), digested with BamH1, the other line (M1) shows no integration. The results indicate that there is a single gene integration: Wt= Wild type; (+) = plasimid................................................................................ 82 Figure 21. Southern blot showing integration of bar gene in genotypes, Mat = ‗Matterhorn‘, Sed = ‗Sedona‘and Mont = ‗Montcalm‘, Wt = wild type, (+) = plasmid. Digestion was done with Hind III. The results indicate that there are four copies of the gene in ‗Matterhorn‘, three copies in ‗Sedona‘and two copies in ‗Montcalm‘. ..................................................................................... 83 Figure 22. Northern blot expression of bar gene in T2 plants; genotype C1-8 = ‗Condor‘, Wt= Wild type. ............................................................................... 84 Figure 23. Northern blot expression of bar gene in T3 plants: genotype ‗Matterhorn‘ (M2), ‗Condor‘ lines C1, C4, and C8. ‗Matterhorn‘ seems to have a higher expression than the ‗Condor‘ lines (C1-C8)............................. 85 Figure 24. Northern blot expression of bar gene in T3 plants; genotypes ‗Sedona‘ (S1-S3) and ‗Montcalm‘ (Mo1-Mo3). ‗Sedona‘ plants S2 and S3 have a higher expression than either ‗Montcalm‘ plants Mo2 and Mo3. The expression of ‗Montcalm‘ is extremely low. .................................................. 86 Figure 25: Bar tested T2 plants at a concentration of 150 mg L-1. ‗Condor‘ (A), ‗Matterhorn‘ (B), ‗Montcalm‘ (C) and ‗Sedona‘(D). ‗Matterhorn‘ seems to be better expressed. Transgenic plants are not 100% resistant, some leaves are scorched, and exhibit stunted growth. However, their survival is better than the wild type (Wt) plants ......................................................................... 87 Figure 26: T3 plants showing partial resistance to Liberty herbicide the genotypes used are ‗Condor‘, ‗Matterhorn‘, ‗Montcalm‘ and ‗Sedona‘. The tray on the left represents wild type non-transformed plants while that on the right are transformed plants. Each plastic container contains the four genotypes mentioned above. The spray rate of the herbicide liberty was 200 mg L-1 .............................................................................................................. 88 xiv Figure 27: Gus expression in ‗Matterhorn‘. All genotypes transformed, namely, ‗Matterhorn‘, ‗Condor‘, ‗Sedona‘, ‗Olathe‘, and ‗Montcalm‘, showed gus positive for both plants transformed using bombardment and Agrobacterium. However ‗Matterhorn‘ had the best expression; panel (A) in seed and (B) embryo. ...................................................................................... 89 Figure 28. PCR results of T3 transgenic plants of ‗Montcalm‘, ‗Condor‘, ‗Sedona‘and ‗Matterhorn‘ confirms the stability of HVA1 transgene integration. The expected band size is 670 bp. ............................................... 94 Figure 29: Southern blot showing integration of HVA1 gene in genotype ‗Condor‘(C8), digested with BamH1, the other lines show no integration. The results indicate that there is a double gene integration. .................................. 95 Figure 30. Southern blot showing integration of HVA1 gene in genotypes Co = ‗Condor‘, Mo = ‗Montcalm‘, Se = ‗Sedona‘and Ma = ‗Matterhorn‘ digested with BamH1. The results indicate that there is a double gene integration in all genotypes except ‗Montcalm‘ which has a single copy number. The Wt= wild type shows no transgene integration. .............................................................. 96 Figure 31. RT-PCR of HVA1 expression for T2 transgenic plants of S = ‗Sedona‘, C = ‗Condor‘, Mo = ‗Montcalm‘ and Ma = ‗Matterhorn‘. Expected band size is 670 bp for HVA1. The expression levels are the same for all four plants. Below is the cDNA loading control showing the expression of ubiquitin with an expected band size of 450 bp. ............................................. 97 Figure 32. RT-PCR showing expression of HVA1 T3 transgenic plants with expected band size of 670 bp. ‗Condor‘ completely lost its expression that was previously detected in the T2 generation. The expression of ‗Montcalm‘ declined while that of ‗Sedona‘ and ‗Matterhorn‘ remained stable. Below is the cDNA loading control showing the expression levels of ubiquitin the expected band size is 450 bp. .......................................................................... 98 Figure 33. Northern blot expression of HVA1 gene from T3 transgenic plants subjected to drought stress. Mat = ‗Matterhorn‘ and Sed = ‗Sedona‘ showed some expression. The remaining lanes, Wt = Wild type, Mon = ‗Montcalm‘ and Con = ‗Condor‘ showed no expression at all. .......................................... 99 xv Figure 34. Panel (A): ‗Matterhorn‘ plants before drought induction; (B) Plants after 21 days continuous no irrigation; (C) ‗Matterhorn‘ drought recovered plants after 3 days of water re-application; 1= control non-transgenic plant that was watered throughout the experiment; 2= ‗Matterhorn‘ transgenic plant after 21 days of no-irrigation, 3= Wild type non-transgenic plant after 21 days of no-irrigation; (D) root development of plants after 21 days of drought stress. 1: Control non-transgenic plant roots, these were watered daily, 2: Transgenic plant roots after 21 days of no-irrigation and 3: Wild type nontransgenic plant roots after 21 days of no-irrigation. ................................... 104 Figure 35. Salt stress test at 5 levels of concentration (0, 50, 100, 150 and 200 mM) on ‗Matterhorn‘ plants 10 days after salt treatment. Note that the two plants on the left side of each flat are control non-transgenic and the two plants on the right side of each flat are Northern blot positive transgenic. .. 106 Figure 36: PCR results of T3 transgenic plants of Ola 1-2 = ‗Olathe‘, Con = ‗Condor‘, Sed =‗Sedona‘and Mat 1-2 = ‗Matterhorn‘. The expected band size is 640 bp. ....................................................................................................... 109 Figure 37: Southern blot analysis showing integration of transgene in the T2 plants. Positive lines are S1 = ‗Sedona‘ with 2 gene inserts, Ma1 = ‗Matterhorn‘ with four gene inserts and Ola1 = ‗Olathe‘ with two gene inserts. The C1 or 2 = ‗‗Condor‘,‘ did not show positive integration of transgene and Wt=Wild type is also negative. ..................................................................... 110 Figure 38:RT-PCR of the germin gene for T2 plants has an expected band size 640 bp. All four genotypes transformed, S= ‗Sedona‘, C= ‗Condor‘, M= ‗Matterhorn‘ and O= ‗Olathe‘ show expression which is less than the positive control. Wt=Wild type is negative. Below is the ubiquitin loading control which shows equal amount of cDNA loading, with an expected band size of 450 bp............................................................................................................ 112 Figure 39:Northern blot of the germin gene from infected tissue of T3 plants. Only M= ‗Matterhorn‘ showed positive results for the expected band size of 640 bp. The rest Wt=Wild type, Ola= ‗Olathe‘, Co= ‗Condor‘ and Sed= ‗Sedona‘ have negative results...................................................................... 113 Figure 40. S. sclerotiorum fungus 72 hours after being grown on potato dextrose agar. ................................................................................................ 114 xvi Figure 41. White mold pathological test. The upper two panels shows the trifoliate leaf detachment assay with plugged mycelia on leaf surface incubated in Petri dishes containing agar media. The two bottom panels show the straw test in the greenhouse, with the straws containing the fungus inserted into the shoot tip of common bean. These two tests did not work because the fungus was unable to infect neither the transgenic nor the wild type plants. In both cases, the humidity was not conducive for the growth of the pathogen. ....................................................................................................................... 115 Figure 42. Relative rate of infection and development spread of pathogen as measured by lesion size on leaf surface of T2 ‗Matterhorn‘, ‗Sedona‘, ‗Olathe‘, ‗Condor‘ and wild type plants. ...................................................... 117 Figure 43. Fungal biological assay: trifoliate leaves placed on moist paper towel in a tray with plug mycelia on top of the leaves. Transgenic ‗Matterhorn‘ (gf2.8), on the left, shows delayed infection after 72 hours of inoculation, compared to wild type on the right. .......................................... 118 xvii KEY TO SYMBOLS AND ABBREVIATIONS ABA: Abscisic Acid Act1: Actin Rice Promoter BAP: Benzylaminopurine CamV35S: Cauliflower Mosaic Virus (CaMV) 35S Promoter CWDE: Cell Wall degrading Enzymes DREB: Dehydration Responsive Binding Element gf2.8: wheat germin gene HR: Hypersensitive response HSP: Heat Shock Protein HVA1: Hordeum vulgare late embryogenesis abandant protein gene IAA: Indole-3-acetic acid IBA: Indole-3-butyric Acid LB: Luria Bentani media MS: Murashige and Skoog media NAA: Naphthalene Acetic Acid nptII: neomycin phosphotransferase pBY520: plasmid containing bar and HVA1 gene pCAMBIA3301: Binary vector containing gus gene PCR: Polymerase Chain Reaction PGIP: Polygalacturonase Inhibiting Protein xviii pinIIt: potato proteinase inhibitor terminator ROS: Reactive Oxygen Species RT-PCR: Reverse Transcription- PCR TDZ: Thidiazuron tnos: nopaline synthase terminator UidA: β-glucuronidase xix CHAPTER I LITERATURE REVIEW OF IN VITRO REGENERATION AND GENETIC TRANSFORMATION OF COMMON BEAN (PHASEOLUS VULGARIS) 1.0 Introduction Common bean (Phaseolus vulgaris) is one of several crop species belonging to the Fabaceae family, commonly known as grain legumes or pulses. In total, there are about 650 genera and 18,000 species in the legume family (Hymowitz 1990). Common bean is a very important source of vegetable protein, especially in those regions of the world in which animal and fish protein is scarce. Common bean satisfy 22 % of the total protein requirement worldwide (Delgado-Sanchez et al. 2006) and account for over 50 % of all legumes consumed globally (Blair et al. 2006, McClean et al. 2004). Like most grain legumes, common bean is rich in the essential amino acid lysine. They are deficient, however, in methionine and cysteine, which are the sulphur-containing amino acids. These essential amino acid deficiencies alter the dietary protein balance (Babaoglu et al. 2000, Popelka et al. 2004). However, cereals that have relatively higher concentrations of these amino acids (Hymowitz 1990) can supplement these nutritional requirements. The need for the continuous improvement of traits in crop species remains an ongoing effort for crop scientists and farmers. Different plant species have their own set of 1 phenotypes that need to be improved in order to both add nutritional values and enhance economic gains for humankind. Common bean also present an array of traits which need improvement. In general, common bean like most grain legumes are lower yielding in comparison to cereals. This low yield is caused by three main factors. The first factor is photorespiration which consumes 30% of photo assimilates in grain legume crops. The second is nitrogen fixation which diverts 10% of carbohydrates fixed to be used by Rhizobium bacteria. The third and last factor is the photosynthetic energy relationship which takes more energy to produce oil and protein products than starch (Hymowitz 1990). These factors, which contribute to low yield production of common bean and other grain legumes, are also implicated in making these crops more susceptible to drought stress when compared to cereals (Dita et al. 2006). Despite these limitations, common bean and other grain legumes have a number of advantages over cereals, including the high protein content in their seeds and their ability to fix nitrogen. Further research for common bean improvement should focus on enhancing the positive traits while simultaneously addressing and improving the aforementioned limitations. Conventional breeding has contributed singularly to the improvement of cultivated common bean. Whilst plant breeding has contributed to the much needed genetic variation necessary for trait improvement, certain genes that can add to the value of agronomic traits in common bean do not exist naturally in its gene pool. Due to this limitation of plant breeding, new trait improvement approaches such as interspecific 2 horizontal gene transfer via genetic engineering, need to be utilized in order to complement the limitations encountered by conventional breeding (Aragão et al. 1996, 1998, 2001). 1.1 In vitro Regeneration of Common Bean A reliable and efficient plant in vitro regeneration system is a prerequisite to the development of an efficient genetic transformation system. A general feature of common bean genotypes is their recalcitrance to regenerate in vitro. This is because they produce significant amounts of phenolic compounds in vitro which inhibit their regeneration. A successful in vitro regeneration depends on three major factors. 1) The type of media formulation is crucial to creating a balance between levels of cytokinin and auxin. In general, a higher concentration ratio of cytokinin to auxin is required to promote shoot development. In the case of common bean, the ratio of 2.5 mg L -1 benzyladenine (BA) to 0.1 mg L -1 of naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA) promotes robust in vitro regeneration (Kwapata et al. 2009). 2.) The explant type also plays a role in either assisting or hindering in vitro regeneration. Grain legume explants that have been tested for in vitro regeneration include embryonic axes (Shroeder et al. 1993, 1995); cotyledonary nodes, stem nodal segments (de Kathen and Jacobsen 1990, Nauerby et al. 1991, Davies et al. 1993); and apical meristem (Pickardt et al. 1991, Russell et al. 1993). In vitro 3 regeneration of common bean using callus is extremely difficult and minimal success has been reported (Zambre et al. 1998, Arellano et al. 2009). 3.) The age of the explant is also critical. Young explants from emerging buds or growth points are most favorable to in vitro regeneration because they are actively dividing totipotent cells (Veltcheva et al. 2005). A summary of research on in vitro regeneration of common bean and tepary bean (P. acutifolius), a closely related genotype, is shown in Table 1. 4 Table 1: A summary of in vitro regeneration of common and tepary bean Genotype Explant Shoot Regeneration Media Shoot buds and embryo ‗Flor de for multiple Junio‘, shoot regeneration ‗Flor de Mayo- Anita‘ P. vulgaris MS salt, 100mg/l myo-inosital, 1mg/l thiamin HCl, 3% sucrose, pH 5.8, 1NKOH, 6.8g/l agar Rooting Media References MS but Delgadowithout any Sanchez et growth al. 2006. regulators BAP 10mg/l, Adenine hemisulfate 20mg/l Salt, 20g/l MS with no Zambre et P. acutifolius Callus from MS ‗A. Gray‘, embryonic sucrose, 8g/l bacto growth al. 1998 axis and Agar, pH5.7 regulators P.vulgaris cotyledon 0.1mg/l Thidiazuron ‗XAN-159‘ (TDZ), 0.05mg/l IAA BM with 10% coconut H2O, 1mg/l BA Multiple MS, BA 1mg/l, plantlets were Ahmed et Shoots from 0.1mg/l NAA, germinated on al. 2002 ‗Fonix‘ and Intact full MS, ‗Maxidor‘ pH5.7 seedlings B5vit, no (IS) and hormones, Cotyledonary pH5.7 nodes (CN) P. vulgaris P. vulgaris ‗Olathe‘ Multiple Full MS media, plus MS with no Aragão et shoots from 44.3 uM BAP growth al. 1998 apical regulators meristems with primary leaves removed. 5 Table 1: Continue….. Apical Full MS media no MS without Ana et al. meristems growth regulators or growth 1996 with hormones. regulators ‗Costa Rica‘, embryonic ‗Carioca‘, axis and root ‗GL11‘ tips P.vulgaris ‗Jalo‘, Salt, P. polyanthus Callus from MS buds without sucrose, 8g/l ‗Greenman‘ scales , Agar, pH5.7 (Year bean) 20g/l BM with Zambre et bacto 1mg/l BAP, al. 2001 100ml/l coconut Leafs, stem 0.1mg/l Thidiazuron water, nodal (TDZ), 0.05mg/l IAA segments 6 1.2 Common Bean Genetic Transformation Common bean is not only known to be recalcitrant towards in vitro regeneration, but also genetic transformation (Estrada-Navarrete et al. 2007). Although inefficient, approximately 90% of stable transformation of common bean has been achieved through biolistic TM gene delivery system (Veltcheva et al. 2005). This is in contrast to other grain legumes such as soybeans, chickpeas, pigeon peas and peas that have demonstrated to be more amenable to Agrobacterium-mediated transformation system (Popelka et al., 2004). The drawback of Biolistic TM method, also known as gene gun bombardment, is that it often causes multiple gene insertions, which are sometimes fragmented and result into instability of transgenes and low gene expression. A review article (Veltcheva et al. 2005), cites early attempts by several researchers from 1989-1997 who have reported transient expression, (rather than stable expression) of transgenes when Agrobacterium mediated transformation system was used. They further state that many of the Agrobacterium genetic transformation techniques of common bean have not been reproducible in other labs. Therefore, many researchers have abandoned Agrobacterium TM as a vehicle for gene delivery and have instead opted to use the biolistic bombardment method. Early attempts to develop a gene delivery system for common bean involved the use of electrical-discharge to accelerate DNA plasmid into meristems (Rech et al. 1991, McCabe and Christou., 1993, Aragão et al. 1993). Only Russell et al. (1993) were able to show stable transformation of the bar herbicide resistance gene using electrical-discharge 7 technique. However the recovery of transgenic plants was very low (0.03 %) to an extent that the technique used was rendered impractical for future work on the genetic transformation of common bean. Other researchers have unsuccessfully attempted to use DNA uptake by protoplast, either via polyethylene glycol or electroporation (Veltcheva et al. 2005). The sole report on the successful use of Agrobacterium-mediated transformation comes from Liu et al. (2005). They describe a procedure of transforming kidney bean with a group 3 LEA (late embryogenesis abundant protein) gene from Brassica napus. Their technique bypassed the tissue culture stage, due to poor in vitro regeneration, and directly transformed the beans with Agrobacterium using sonication and a vacuum infiltration system. Although the transformation efficiency was low, the transgenic plants exhibited a high growth rate under salt and water stress. Since then there has been no other reports of this procedure ever being repeated or of any other successful transformation technique developed using Agrobacterium. TM Using Biolistic bombardment, Aragão et al. (2002) developed transgenic common bean carrying the bar gene which conferred resistance to the herbicide glufosinate ammonium at concentrations of 500 g ha -1 in greenhouse and 400 g ha -1 in the field. Common bean have also been engineered to express viral antisense RNA, which results in a delay and attenuation of symptoms of Bean Golden Mosaic Gemini Virus (BGMGV) Aragão et al. 1998). A different approach was performed by Bonfim et al. (2007) using 8 RNAi-hairpin construct to silence the AC1 region of the viral genome of BGMGV. However, their transformation efficiency was so low that, out of 2,706 plants, only 18 putative transgenic lines were obtained, representing 0.66% transformation efficiency. Of the 18 putative transgenic plants, only one plant exhibited resistance to the virus. Field trials of the progenies of the putative transgenic plants showed partial resistance to BGMGV in the field (Aragão and Faria, 2009). The nutritional improvement of common bean was enhanced by a plasmid construct containing the fusion of the neo and gus genes which had been co-bombarded with the Brazilian nut methionine-rich 2S albumin gene. When the methionine expressing embryonic axes were also co-transformed with anti-sense sequences of AC1, AC2, AC3 and BC1 genes from the BGMGV, the co-transformation efficiency of unlinked genes was 40-50% (Aragão et al. 1996) and the methionine expression increased to 14 and 23% in two different transgenic lines (Aragão et al. 1999). TM A combination of both the Agrobacterium and Biolistic bombardment methods were used by Brasileiro et al. (1996) to stabilize the transformation of P. vulgaris ‗Jalo‘ that was bombarded with tungsten microprojectiles and inoculated with A. tumefaciens wild type (Ach5). The results showed that tumors were produced in 50-70% of the transgenic plants. When the bombarded meristems were also inoculated with the disarmed A. tumefaciens (LBA4404/p35SGUSINT), 44% of plants showed gus expression. Vianna et al. (2003) developed a novel approach of transforming the transgene assembly as 9 fragment pieces of DNA, as opposed to the entire plasmid. The transformation efficiency of using either an entire plasmid or a fragment of DNA has shown to be in the range of 0.2 to 0.8%, depending on plant genotypes. Genetic engineering of common bean has remained a challenge due to the inefficient and recalcitrant nature of the species towards in vitro regeneration. Low regeneration efficiency and frequency of multiple shoots ranging from 4 to 8 per explant (Ahmed et al. 2002) are common. A recent report by Kwapata et al. (2009) shows that common bean, cultured in vitro, could produce as many as 20 multiple shoots per explant. Though this is a relatively higher number, it is still very low from the desired numbers regenerated in other crop species such as corn and other cereals (Oraby and Sticklen, 2005). A closely related species to common bean, the tepary bean (P. acutifolius ), has been shown to be more readily transformable with Agrobacterium inoculation of callus. This is due to high efficiency of in vitro regeneration in tepary bean. Tepary bean callus lines were co-cultivated with A. tumefaciens strain C58CIRif (pMP90) harboring a binary vector with neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA) marker genes (DeClerq et al. 2002). In this experiment, the GUS activity was detected transiently in 5 of 6 genotypes tested. In another experiment, transgenic callus lines of genotype P. acutifolius ‗N1576‘ was transformed with a marker gene and the genomic fragment encoding the arcelin-5a protein from P. vulgaris, which confers resistance to Zabrotes subfaciatus pest (Dillen et al. 1997). This research was followed by Goosens et al. 10 (1999), who reported acelin-5 expression levels of the transgenic plants to be 25% of total soluble proteins. Optimization of the Agrobacterium transformation method in tepary bean has been explored using different selection methods under different temperatures, photoperiods, light conditions, Agrobacterium growth phases, cocultivation periods and using various concentrations of acetosyringones (DeClercq et al. 2002, Zambre et al. 2003, 2005). While tepary bean has been demonstrated to be more amenable towards in vitro regeneration and genetic transformation, they are of less value economically when compared to common bean. As a result, several researchers have proposed using tepary bean as a bridge of introducing foreign genes into the economically more valuable common bean (Dillen et al. 1997, Veltcheva et al. 2005). They suggest that this can be achieved by grafting the scion of common bean onto the root stock of tepary bean. This approach partially solves the problem of in vitro rooting, which is problematic in many grain legumes and especially in common bean (Krishnamurthy et al. 2000, Sarmah et al. 2004, Tewari-Singh et al. 2004, Sanyal et al. 2005). A summary of genetic transformation systems for grain legumes including common bean is presented in Table 2. 11 Table 2: Grain legume transformation systems using different explants, genes and gene delivery methods Grain Legume Arachis hypogaea (Peanut) Explant Transgenes embryo axis gus embryo axis embryo axis embryo gus gus gus uidA, bar uidA, bar cry1Ac, hph uidA, bar callus Transformation method Agrobacterium bombardment TSWV oxalate oxidase Ara2 protein gene DREB1A Cajanus cajan (Pigeon pea) shoot apices, cotyledonary node, embryonic axis, embryonic axis & cotyledonary node leaf cotyledonary nodes decapitated embryo axis, axillary shoot cotyledonary node shoot apices cotyledonary node embryonal segment uidA, nptII, nptII nptII hemagglutinin protein gene uidA, nptII uidA GFP, uidA, nptII hpt, rice chitnase hpt, uidA HN gene decapitated embryonic axis axillary bud of germinating seed nodal segment of embryos, plumule, cotyledon, shoot nodes embryo axis epicotyl PPRV, nptII nptII , cryI E-C, uidA, cryIAb cryIAb uidA hpt, uidA gus, nptII 12 Agrobacterium bombardment Reference Epen and George 1994 Mckently et al. 1995 Cheng et al. 1996 Egnin et al. 1998 Brar et al. 1994 Christou 1997 Singst et al. 1997 Livingstone and Birch, 1998 Yang et al. 1998 Livingsone et al. 2005 Dodo et al. 2005, 2008 Bhatnagar-Mathur 2007 Geetha et al. 1999 Lawrence and Koundal 2001 Satyavathi et al. 2003 Dayal et al. 2003 Thu et al. 2003 Mohan and Krishnamurthy, 2003 Kumar et al. 2004 Singh et al. 2004 Prasad et al. 2004 Surekha et al. 2005 Verma and Chand, 2005 Sharma et al. 2006 Surekha et al. 2007 Thu et al. 2003 Table 2: Continue…. Cicer arietinum (Chickpea) leaf and stem embryo embryonic axis embryonic axis embryonic axis and cotyledonary node plumule half embryo with one cotyledon embryo embryo slices embryonic axis embryo embryo axis seeds shoots nptII nptII,uidA, cryIAc, nptII,uidA uidA Agrobacterium bar, α-amylase inhibitor aspartate kinase gene nptII,uidA cryI Ac gene gus, hpt uidA, nptII pmi α -amylase inhibitor1 Sarmah et al. 2004 bombardment epicotyl hpt and GFP bar immature embryos cotyledonary node immature cotyledon embryonic axis cotyledonary node cotyledonary node cotyledonary node somatic embryos apical shoot meristem Lathyrus sativus (Grass pea) zygotic embryos immature embryos somatic embryos embryos Glycine max (Soybean) nptII cry1Ac, nptII, uidA hph BPMV-pCP hpt ahas ( imazapyr) CP4 Roundup gus G-OXO hph oleosin RNAi gus epicotyl uidA, nptII 13 Srinivasan et al. 1988, 1991 Fontana et al. 1993 Kar et al. 1996 Krishnamurthy et al. 2000 Sanyal et al. 2003 Senthil et al. 2004 Tewari-Singh et al. 2004 Polowick et al. 2004 Sanyal et al. 2005 Ignacimuthu and Prakash, 2006 Pathak and Hamzahm, 2008 Akbulut et al. 2008 Patil et al. 2009 Shivani et al. 2007 bombardment Christou et al. 1989 Santarem and Finer, 1999 Reddy et al. 2001 Frutani and Hidaka, 2004 Parrott et al. 1989 Zhang et al.1999 Agrobacterium Yan et al. 2000 Aragão et al. 2000 Clemente et al. 2000 Donaldson et al.2001 Olhoft et al. 2003 Schmidt et al. 2008 Govindarajulu et al. 2008 Barik et al. 2005 Agrobacterium Table 2: Continue…. Lens culinaris (Lentil) nptII, uidA decapitated embryo cotyledonary node cotyledonary node Lupinus angustifolius L.(Lupin) Phaseolus acutifolius (Tepary bean) Phaseolus vulgaris (Common bean) cotyledonary node and decapitated embryo cotyledonary node Agrobacterium Sarkar et al. 2003 bombardment intact axillary bud embryonic axis slices shoot apices uidA uidA, nptII Als (Lou Gehrig‘s disease) uidA 2S albumin bar Mahmaudian et al. 2002 Hassan et al. 2007 Akcay et al. 2009 Gulati et al. 2002 callus callus callus arcelin-5a uidA, nptII uidA, nptII Agrobacterium Dillen et al. 1997 De Clerq et al. 2002 Zambre et al. 2005 seed lea protein gene Liu et al. 2005 embryo axis embryo axis meristems uidA, bar, uidA, nptII, methionine rich albumin gene Ach5 methionine rich albumin gene bar BGMV Agrobacterium vaccumsonication bombardment embryo axis embryo axis embryo axis Pisum sativum (Pea) uidA shoot cultures hptII, bar, immature embryo slices cotyledon epicotyls electroporation Agrobacterium Agrobiolistics bombardment Agrobacterium α-amylase inhibitor1 SAF8 immature embryo slices cotyledonary node embryo uidA, Fv antibody Cahin, bar embryonic segments axillary meristem intact axillary bud nptII, uidA bar, PGIP, VST1 uidA, bar PEMV , uidA 14 injection electroporation Chowrira et al. 1996 Molvig et al. 1997 Pigeaire et al. 1997 Russell et al. 1993 Aragao et al. 1996 Brasileiro et al. 1997 Aragão et al. 1999 Aragão et al. 2002 Bonfim et al. 2007 Puonti-Kaerlas et al. 1990 Schroeder et al. 1993 Schroeder et al. 1994,195 de Kathen and Jacobsen, 1995 Perrin et al. 2000 Grant et al. 1995, 2003 Svabova et al. 2005 Richter et al. 2007 Krejci et al. 2007 Chowrira et al. 1998 Chowrira et al. 1996 Table 2: Continue… Vicia faba (Faba or broad bean) stem stem segments Ti plasmid uidA, lys C, methionine rich sunflower 2S albumin gene SFA8 gene, bar, lysC hpt 2S albumin PAT Agrobacterium Agrobacterium Pickardt et al. 1991 Saalbach et al. 1994; Pickardt et al. 1998 Agrobacterium PEG + electroporation bombardment Eapen et al. 1987 Kohler et al. 1987a,b hypocotyl nptII nptII cryIAc, nptII, uidA Vigna angularis (Azuki bean) epicotyl epicotyl hpt bar, hpt, GFP Agrobacterium EL-Shemy et al. 2002 Khalafalla et al. 2005 Vigna mungo (Blackgram) cotyledonary nodes shoot apex cotyledonary nodes uidA, nptII uidA, nptII uidA, nptII Agrobacterium Saini et al. 2003 Saini and Jaiwal, 2005 Saini and Jaiwal, 2007 Vigna radiata (Mung bean) cotyledons hypocotyl uidA., nptII uidA, nptII Agrobacterium Pal et al. 1991 Jaiwal et al. 2001 cotyledonary node, primary leaves callus leaf, cotyledonary node cotyledonary node nptII, uidA bar, α-amylase inhibitor1 nptII, uidA Agrobacterium Ignacimuthu, 2000 cotyledonary node uidA, nptII Agrobacterium cotyledonary node cotyledonary node bar α-amylase inhibitor 1 uidA uidA, bar Chaudhury et al. 2006 Popelka et al. 2006 Solleti et al. 2008a, 2008b Chowrira et al. 1996 Ikea et al. 2003 Ivo et al. 2003 embryo axis Vicia narbonensis (Narbon bean) embryogenic callus embryogenic callus Vigna aconitifolia (Moth bean) protoplast protoplast Vigna sesquipedalis (Asparagus bean) Vigna unguiculata (Cowpea) intact axillary bud embryonic axis embryonic axis 15 Jelenic et al 2000 Bottinger et al. 2001 Hanafy et al. 2005 Kamble et al. 2003 Tazeen and Mirza, 2004 Sonia et al. 2007 electroporation bombardment CHAPTER II DROUGHT, SALT STRESS AND WHITE MOLD PATHOGENESIS RELATED TO COMMON BEAN 2.0 Drought and salt stress Abiotic stresses, including drought, salinity and high temperatures, pose a major obstacle for crop yield and production, with more than 90% of arable land experiencing one or more of these stresses (Dita et al. 2006). In an effort to overcome or reduce these stress factors, plants have evolved to adapt by synthesizing low molecular weight osmolytes. Drought and salt stresses share a similar pathway (Figure 1). When drought occurs or high salt content is present, ionic and osmotic homeostasis of cells becomes inbalanced. As a result, plants lose cellular turgidity, followed by the aggregation and misfolding of proteins (Zhu 2002). The key input signal for drought is believed to be the loss in turgor pressure due to water + loss of the cells. The input signal for salt stress is the high concentration of Na having similar effect on the cells. These input signals are recognized by a plant‘s primary sensors, such as receptor-like kinases (RLK) and ion channels. The immediate effect of + sensing input signals by the primary sensors, such as those related to high levels of Na , is the activation of the Salt Overly Sensitive (SOS) pathway in which high amounts of Ca 2+ is released into the cytosol. + Na influx into cells is via non-selective cation channels known as cyclic nucleotide-gated ion channels (CNGCs). Ca 16 2+ plays a role in + inhibiting CNGC from allowing excessive entry of Na into the cells. SOS3 codes for a 2+ myristoylated calcium binding protein, which senses the Ca and interacts with a serine/ threonine protein kinase, SOS2. The interaction between SOS3 and SOS2 regulates the transport activity and expression levels of SOS1, which is a salt tolerant effector gene that codes for a plasma membrane Na+/H+ antiporter (Zhu 2002). 2+ Once secondary messenger elements, such as Ca , ROS or ABA, have accumulated to a threshold level in hyperosmotic stressed cells, the protein phosphorylation cascade is triggered in association with the MAP Kinase cascade. In tobacco, a MAPK called salicylic acid induced protein kinase (SIPK) is activated by osmotic stress (Xiong et al. 2002). This group also explains that, due to the elicitation of Ca 2+ during osmotic stress, calcium dependent protein kinases (CDPK) link the calcium signal to downstream responses. Phospholipid signaling, induced by osmotic stress, is catalyzed by phospholipases that cleave phospholipid messengers and generate a number of lipid messengers. These messengers function in guard cells to release more Ca 2+ from internal stores and cause stomatal closure, while some other messengers function to activate protein kinase C. The signaling mediated by phospholipid messengers is believed to be a double-edged sword: when the lipid signaling molecules are at low levels, they trigger downstream adaptive responses by activating transcriptional factors (TF‘s). On the other hand, when these signals increase due to drought or salt stress, they damage the cellular integuments. 17 Dehydration responsive transcription factor (DREB) and C-repeat binding factor (CBF) bind to the dehydration response element (DRE) and C repeat terminal (CRT) cis acting elements. Family members of these two TF‘s include CBF1, CBF2 and CBF3 or DREB1B, DREB1C and DREB1A, which are activated upon being induced by stress (Wang et al. 2003) The DREB or CBF are coded by certain multi-gene families and mediate the transcription of four groups of dehydration response genes.  Detoxification Genes: Serve to reduce the concentration of reactive oxygen - species (ROS), such as O2, H2O2 and OH , playing the role of antioxidants and preventing damage to membranes and macromolecules.  Osmoprotection Genes: Maintain turgor pressure of cells by driving the water gradient upwards towards the inside of the cell. These osmolytes fall into three categories: o Amino acids, e.g., proline o Quartery amines, e.g., glycine, betaine, and dimethylsulfoniopropionate o Polyol sugars, e.g., mannitol and trehalose  + + Antiporter genes: Mediate the exchange between H and Na across the cellular membrane. The last group are  Heat shock proteins (HSP): Regulated at the transcriptional level, trans acting heat shock factors (HSF) bind to cis acting elements (HSE's). HSP‘s are chaperones classified into one of five categories: HSP100, HSP90, HSP70, HSP60 or sHSP. These function to protect the protein from denaturing, misfolding and aggregation during osmotic stress (Wang et al. 2003). Late embryogenesis abundant (LEA) proteins are a class of HSP that are extremely 18 hydrophylic and resilient towards heat, such that they don't coagulate at boiling temperatures. These proteins may play a role in water binding, ion sequestration and macromolecule and membrane stabilization. HVA1 is a gene from barley that encodes a type III LEA protein (Xu et al. 1996). 19 Figure 1: Drought and salt stress biosynthetic pathway. (modified from Wang et al. 2003) 20 2.1 White Mold (Sclerotinia sclerotiorum) Sclerotinia sclerotiorum is the most devastating necrotrophic soil borne pathogen in the temperate region. Species of this pathogen cause both southern stem rot and white mold which result in the rotting of both seedling and pod in most grain legumes, especially in common bean, soybean, sunflower, lentils and peanut (Donaldson et al. 2001, Livingstone et al. 2005). This pathogen accounts for annual agricultural losses in the United States alone of more than $200 million (Bolton et al. 2006). The characteristics of S. sclerotiorum include its hyaline, septate, branched and multinucleate hyphae. It produces white mycelium with no asexual conidial. For prolonged survival under unfavorable growth conditions, it relies on the production of sclerotia, which is a compact mass of mycelium. This pathogen infects host plants primarily by producing ascospores from its apothecia (Steadman et al. 1983). The disease symptoms characterized by this fungus are water soaked lesions on leaves which spread to the petiole and stem. These lesions develop into necrotic tissue with fluffy white mycelia, which is the most obvious sign of the fungus infection and successful colonization (Bolton et al. 2006). For successful colonization to occur, cool temperatures o of about 10 C, coupled with damp and moist conditions, are required. Due to their preference of moist conditions, the pathogen establishes more readily under irrigation or during rainfall season (Clarkson et al. 2004). 21 Pathogenic fungi of plants produce a wide array of cell wall degrading enzymes (CWDEs), which include, pectinases, β-1,3-glucanases, glycosidases, cellulases, xylanases and cutinases (Annis and Goodwin, 1997). Carbon and nitrogen sources, as well as ambient pH, are the precursors for the activation of these CWDEs at the transcriptional level. S. sclerotiorum secrete pectinases in particular polygalacturonases (PGs) that are induced by the presence of galacturonic acid, which is a pectin monomer. This then degrades pectin to allow the fungus to penetrate the cell wall and feed on its carbon source (Fraissinet-Tachet and Fevre, 1996, Riou et al. 1992). Plants have an innate defense mechanism at the molecular level that can deter invading pathogens. Cell-wall-associated glycoproteins, such as polygalacturose-inhibiting protein (PGIP), have been shown to be effective in slowing down pathogen growth (Zuppini et al. 2005). Also, the secretion of endoPG‘s by pathogens elicits the hypersensitive response (HR), e.g., a rapid plant cell death aimed at stopping further invasion of the pathogen. Although HR has been shown to be more effective against biotrophic pathogens (pathogens that grow on living tissue), it promotes the growth and development of necrotrophic pathogens (pathogens that live on dead plant tissue) such as S. sclerotiorum (Govrin and Levine, 2000, Thomma et al. 2001). As a consequence, this necrotrophic fungal pathogen is more difficult to control than the biotrophic pathogens. Oxalic acid (ethanedioic acid), has been implicated as the main pathogenicity factor of S. sclerotiorum. During the early stages of pathogenesis, oxalic acid accumulates in host plant infected tissue. As the oxalic acid concentration increases, it lowers the pH to 4 or 5 22 (Bolton et al. 2006). It is this low pH produced by oxalic acid that allows S. sclerotiorum to escape the inhibitory action of plant defense PGIP‘s. The low pH also weakens the plant‘s defense system (Favaron et al. 2004). In addition oxalic acid chelates calcium and pectic material, which in turn allows polygalacturonase to hydrolyze pectates and disrupt the integrity of host cell walls (Smith et al. 1986, Kurian and Stelzig 1995). As a consequence calcium dependent plant defense response, production of polyphenol oxidases and the oxidative burst are compromised due to the action of oxalic acid (Cessna et al. 2000, Bolton et al. 2006). Oxalic acid also induces the wilting of leaves by preventing guard cells from closing the stomata and inhibiting ABA induced stomatal closure. Strong evidence implicating oxalic acid as the main pathogenicity factor of S. sclerotiorum derives from the fact that it has been recovered from host infected tissue (Ferrar and Walker 1993). The most compelling evidence is the fact that mutant strains of S. sclerotiorum that are incapable of producing oxalic acid and yet posses all the CWDE arsenal are non-pathogenic (Godoy et al. 1990). In view of this, an opportunity exists to develop transgenic plants that can inhibit the effect of oxalic acid. 2.2 Germin Gene (gf2.8) The germin gene (gf2.8) is a pepsin-resistant, homohexameric glycoprotein that is water soluble with a monomer molecular mass of ~22 kDa and an oligomer molecular mass of ~130 kDa (Lane, 2002). Germin protein is believed to promote plant cell hydration. Germin protein has been shown to correlate with germinating and maturing of embryos in wheat (Lane et al. 1992, 1993). Germin is an oxalate oxidase (G-OXO) which degrades 23 oxalic acid into CO2 and H2O2 (Schmitt 1991). H2O2 promotes localized hypersensitive response (HR) cell death, but most importantly, H2O2 is toxic to the oxalate producing pathogens such as S. sclerotiorum. H2O2 also promotes lignification and cross linking of cell walls, which provides a barrier against invading fungal pathogens. Germin-OXO is able to free-up chelated Ca 2+ bounded by oxalic acid (Luttrell et al. 1993, Apostol et al. 1989). As a consequence, the germin gene helps to promote plant defense against fungal pathogens such as S. sclerotiorum. Isoforms of the germin gene are present in all cereals, including wheat (Lane 2002). This is why most cereals are not susceptible to oxalate producing fungi such as S. sclerotiorum. While breeding initiatives are progressing to develop resistance to S. sclerotiorum, transgenic approaches have been attempted in a number of crops. Table 3 shows a summary of transgenic work performed in various crops using the germin gene to develop resistance to S. sclerotiorum. 24 Table 3: Summary of transgenic research using the germin gene in different crops Crop Method of Transformation Helianthus annuus Agrobacterium: Plants were transformed with the gf2.8 cDNA regulated by supermass promoters (Sunflower) Method to Confirm Transgenes Northern Blot showed a high level of transcription Method Used to Test Transgene Activity References 1.Sclerotinia sclerotiorum infection assay; Description: 6 -wk old plants were infected with mycelia of the fungus. Hu et al. 2003 2. Analysis of SA accumulation; Description; Total SA was extracted from 0.6g leaves tissue and samples were analyzed using liquid chromatography and UV light adsorption was measured using a photodiode array detector (model: 996 waters). 3. Histochemistry and microscopy Assay; Description; 5-wk leaf tissue was boiled in ethanol lactophenol 2:1 and viewed under an epifluorescence microscope. 4. Detection of oxalate oxidase (OXO) activity and H202 accumulation; Description: OXO activity was detected by analyzing H202 accumulation in the leaves in the presence of Oxalic Acid (OA) 25 Table 3: Continue… Populus fastigiata (Poplar tree) Agrobacterium: Plants were transformed with gf2.8 regulated by CAMV 35S promoter PCR SDS-Page gel showing the expression of germin gene (130 kDa). Oxalate Oxidase (OXO) Assay; Haiying et Description: Histochemical assay al. 2001 was done to localize the OXO in tissue. This was done by incubating 2.5mM OA in succinate buffer (25mM succinate acid, 3.5mM EDTA pH4.0 plus 4chloro-1-naphthol 0.6g mg/ml. OXO activity was measured at an extinction coefficient of A350. 2. Oxalic Acid tolerance assay; Description; Samples were treated with 200mM OA for 2h. Infected areas were measured using NIH image 1.61 program. Control samples were treated with HCl pH 1.15 with 5 replicates. 2. Detection of OA produced by fungus; Description; OA calculations were done as described by Boehringer Mannheim. 3. In vitro pathogen resistance test; Description; leaf discs 15 mm diameter were inoculated in 20 ul 6 of 10 conidia/ml and the severity of necrosis was analyzed. 26 Table 3: Continue… Glycine max (Soybean) Agrobacterium: Plants were transformed with gf2.8 regulated by CAMV 35S promoter Southern Blot was used as well as western blot with a polyclonal germin antisera antibody. The level of activity was significant. Histological screen for OXO activity; Description; leaf tissue were placed in detection solution as described above and samples with dark blue or purple color were designated positive. Donaldson et al. 2001 2.Microscopy of tissue stained for OXO activity ; Description; Zeiss Axiphot microscope with Ektacheme 64T tungsten film set at 100 ASA. 3. Quantitative OXO assay on pellet and supernatant fraction. Description; Protein fractions were incubated in OXO developing solutions and absorbance of 555nm was used to determine the OXO activity. Arachis hypogaea (Peanut) Biolistic: Callus was bombarded with gf2.8 regulated by CAMV 35S promoter. Southern blot was used northern blots showed significant expression. 4.Innoculation of samples with pathogen; Description; plant leaves and stems were wounded and the pathogen was inoculated on the wound sites. Oxalate Oxidase (OXO) activity Assay; Description; method same as one described above. 2.H202 was measured using the Amplex Red Kit as per manufacturers instruction. 3. Oxalic Acid (OA) Bio-assay; Description: protocol same as described above used to assess level of tissue tolerance in the presence of OA. 4.Fungal Bio-assay; Description: same protocol as described above was used to assess level of tissue tolerance in the presence of fungal pathogen. 27 Livingstone et al. 2005 Table 3: Continue… Solanum lycopersicum (Tomato) The binary vector pGPTV driven by the CAMV35S promoter was used with the Agrobacterium strain LB4404 PCR, SDS- polyacrylamide gel. The expected band size was 124kD 1.Histochemical Assay; Description: leaf tissue was placed in detection solution as described above and samples with dark blue or purple color were designated positive. 2.Wilting assay of leaf discs; Description: leaves were subjected to various levels of AO, 5,10, 20 and 30mM for a period of 24hrs. 3. Inoculation of samples with pathogen; Description; plant leaves and stems were wounded and the pathogen was inoculated on the wound sites. Pathogen 6 concentration was 10 conidia/ml and the severity of necrosis was analyzed. 28 Walz et al. 2008 CHAPTER III GOALS, OBJECTIVES AND METHODOLOGY 3.0 Goals The overall goals of this research were to develop novel protocols for an in vitro regeneration and gene delivery system for common bean (P. vulgaris). 3.1 Specific Objectives  Objective I: Develop an efficient and reproducible in vitro regeneration protocol for common bean.  Objective II: Develop a gene delivery system for common bean using gus screenable marker gene, bar herbicide resistance selectable marker gene, HVA1 drought/salt tolerance gene and germin-OXO (gf2.8) gene which confers resistance to fungal disease caused by S. sclerotiorum.  Objective III: Evaluate putatively transgenic plants for transgene integration and expression  Objective IV: Evaluate transgenic plants for their biological activity and function. 29 3.2 Materials and Methods 3.2.1 In vitro regeneration protocol for common bean 3.2.1.1 Plant material Ten genotypes of common bean were used in this research. They were provided by Dr. James D. Kelly of Michigan State University. These ten genotypes were used in order to explore the different genetic diversities and the different potential for in vitro growth and regeneration. These genotypes represent the nine main commercial classes, the two main gene pools and the four main races of common bean that are grown in North America. Table 4 is a summary of the genotypes used. Complete details on each genotype can be found at http://www.css.msu.edu/bean/Variety.cfm 30 Table 4: Ten genotypes of P. vulgaris representing the ten different commercial classes grown in Northern America GENOTYPE Commercial Gene Pool Race Class ‗Beluga‘ White kidney Andean Nueva Granada ‗Condor‘ Black Middle Mesoamerica American ‗Jaguar‘ Black Middle Mesoamerica American ‗Matterhorn‘ Great northern Middle Durango American ‗Merlot‘ Small red Middle Jalisco American ‗Montcalm‘ Middle Granada Middle Pinto Nueva American ‗Olathe‘ Dark red kidney Durango American ‗Redhawk‘ Dark red kidney Andean Nueva Granada ‗Seahawk‘ Navy Middle Mesoamerica American ‗Sedona‘ Pink Middle American 31 Jalisco 3.2.1.2. Seed sterilization and explant preparation Seeds were rinsed twice with sterile distilled water; immersed in 75% ethanol for 3 min; rinsed thrice with sterile distilled water; and immersed for 20 min in a solution of 25% −1 commercial Clorox, 5 ml L −1 Tween20 and 10 ml L of 0.02% HgCl2. Following sterilization, the seeds were rinsed five times in sterile distilled water and soaked for 20 hours. After soaking, the seeds were dissected and the embryos were excised. The hypocotyl and cotyledons were removed, leaving the epicotyl with apical meristem primordia. The excised epicotyl with apical meristem primordia were incubated in vitro o 2 for 5 days at 25 C with 16 hours photoperiod and light intensity of 45-70 µmol/m /sec in the culture media described below. 3.2.1.3. In vitro multiple shoot regeneration media −1 Regeneration culture media contained 4.43 g L −1 sucrose, 100 mg L −1 casein hydrolysate and 2.5 g L −1 (BA) and 0.1 mg L MS (Murashige and Skoog 1962), 3% −1 gelrite, 2.5 mg L benzyladenine −1 indole-3-acetic acid (IAA). Silver nitrate 30 mg L was added as an anti-oxidant to get rid of the phenolic compounds. After three weeks of visible shoot primordia growth the explants were transferred to shoot development media containing the above ingredients, excluding silver nitrate and adjusting BA and IAA to 1 mg L −1 each. Explants were kept on this medium for seven weeks before being transferred to rooting media, which contained all ingredients of the shoot development media excluding 32 −1 BA and adjusting IAA to 0.1 mg L −1 supplemented with 4 mg L of glufosinate of ammonium for selection. Shootlets were kept on this media for five weeks until firm roots developed. Growth regulators were added after autoclaving the media for 25 min at 120°C and 690 kPa. The final media combinations were then poured into 100 × 25 mm Petri dishes and solidified under a laminar flow hood. In vitro cultures were incubated at −2 −1 25°C with 16 hours photoperiod and light intensity of 45–70 μmol m s . Beacuse phenolic compounds were being produced in in vitro cultures of common bean, an experiment was designed to negate the inhibitory effects of these compounds. The experiment was conducted only with the most-phenolic producing genotype, ‗Condor‘. The apical meristem shoot multiplication experiment was repeated in media containing −1 4.43 mg L of MS salts and vitamins, 2.5 mg L −1 BAP and 0.1 mg L −1 IAA and four −1 different antioxidants which included ascorbic acid (2 mg L ), silver nitrate (30 mg −1 −1 −1 L ), activated charcoal (15 mg L ) and glutathione (5mg L ) based on modification of published data (Abdelwahd et al. 2008). 3.2.1.4. Statistical and experimental design for shoot regeneration The statistical design was a three-way factorial in a completely randomized design (CRD). In this design, ten genotypes were evaluated using nine levels of cytokinin (BAP and TDZ) and seven levels of auxin (NAA and IAA). The 10x9x7 factorial experiment 33 with 630 treatments was replicated three times. Each experimental unit (Petri dish) consisted of five P. vulgaris embryonic axes apical meristem primordia explants. After regeneration, three out of five samples were randomly selected for analysis. A total of 1,890 experimental units with 5,670 data points were analyzed using PROC GLM (SAS version 9.1.3). An Analysis of Variance (ANOVA) was used to test the statistical significance at an alpha level of 0.001. 3.2.1.5. In vitro rooting of regenerated shootlets The cut end of the regenerated shoots (2 cm long) were dipped (treated) for 30 s in −1 different concentrations (0.0 1.0, 5.0, or 10 mg L ) of indole-3-butyric acid (IBA). The −1 treated shoots were then cultured in 4.43 mg L MS medium containing different −1 concentrations (0.0, 0.05, 0.1 or 1.0 mg L ) of naphthalene acidic acid (NAA), indole-3acetic acid (IAA) or IBA to examine rooting potential. 3.2.1.6. Statistical and experimental design for rooting The statistical design for the in vitro rooting was a two factorial experiment in a CRD with the first factor being IBA dipping solution at four levels, and the second factor being auxin concentrations at ten levels. The 4x10 factorial experiment with 40 treatments was replicated in space three times. From each experimental unit (Petri dish), five explants where cultured and three plantlets were randomly selected for analysis. A total of 360 34 experimental units were analyzed using PROC GLM (SAS version 9.1.3). Analysis of Variance (ANOVA) was used to test the statistical significance at an alpha level of 0.001. 3.2.1.7. Morphogenesis studies via scanning electron microscopy Samples of in vitro multiplied shoot apices, grown from apical shoot primordia explants, were fixed, dehydrated and dried as described by Klomparens et al. (1986). These samples were then coated with gold particles and microphotographed with a JEOL JSM 31 (Tokyo, Japan) scanning electron microscope. 35 3.2.2 Genetic transformation 3.2.2.1 Gene Constructs pACT1F: The construct depicted below (Figure 2) was used as a selectable marker for transformation of β-glucuronidase (gus) into common bean. Figure 2: Linear map of pACT1F cassette (not drawn to scale). Rice actin promoter (Act), gus gene (UidA), and nopaline synthase terminator (Tnos) 36 pBY520: The construct depicted below (Figure 3) was used for the transformation of common bean with the HVA1 gene conferring drought and salt stress tolerance. This construct also contains the bar gene as a selectable marker. Xho1 BamH1/ HinIII /EcoR 1 Act15’ HVA1 EcoR 1 EcoRV Psf1 Xba1 PinII-3’ 35S-5’ bar Nos-3’ 1 kb Probe 2 Kb pBY520 (8.1kb) Xu et al. 1996 Figure 3. Linear map of pBY520 cassette (not drawn to scale). Rice actin promoter (Act1), Barley or Hordeum vulgare (HVA1) LEA 3 gene, Cauliflower Mosaic Virus 35S promoter, bar gene and nopaline synthase terminator (Tnos) 37 pBKSbar/gf2.8: The construct depicted below (Figure 4) was used for the transformation of common bean with the germin gene (gf2.8) that produces oxalate oxidase. This construct also contains the bar gene as a selectable marker. EcoR1 gf-2.8 bar pBKS bar/ gf-2.8 6.9 kb SspI Amp HindIII pUC ori Figure 4. Circular map of the 6.9 Kb of pBKSbar/gf2.8, not drawn to scale. Amp= ampicilin resistant marker, bar=herbicide selectable marker, pUC ori= origin of replication of the pUC 18 plasmid vector. 38 pCAMBIA3301: The binary vector depicted below (Figure 5), not drawn to scale, was used for Agrobacterium transformation of common bean with the gus and bar gene as a selectable marker. Hind III Figure 5. Linear map of pCAMBIA3301 T-DNA cassette. LB/RB – left/right T-DNA border sequences; P35S/T35S – CaMV 35S promoter/terminator; bar – coding region of the phosphinothricin resistance gene; Tnos – nopaline synthase terminator; gus-intron – gusA gene coding region with intron sequence 39 3.2.2.2. Transformation of plasmid vectors into E.coli competent cells All plasmid vectors of interest were transformed into E. coli for multiplication. In order to transform E. coli, 50 µl of DH5α E. coli competent cells (Sigma) were used which were mixed with 2 µl of plasmid DNA. The mixture was placed on ice for 25 min and 0 then heat-shocked for 45 s in a water bath at a temperature of 42 C. Immediately thereafter, it was placed on ice for 2 minutes. Luria Bertani (LB) medium 950 µl was added to the tubes containing the transformed E.coli. This was then incubated for 2 0 hours at 37 C with 150 rpm shaking. After two hours the cultures were plated on solid LB media with the appropriate antibiotic, kinamycin or ampicilin at a concentration of -1 15 mg L for bacterial colony selection. Colonies that grew on the LB selection media were putative transformants and single colony PCR was performed to see the presence of the gene(s) of interest. Upon confirming gene presence, the single colony was then 0 placed into glass flask containing 50 ml of LB media which was then incubated at 37 C with 280 rpm shaking for 48 hours. Thereafter, the plasmids where purified using Qiagen plasmid purification kit (Cat.No.12123) as per manufacturers instruction. TM Purified plasmid was used for either Biolistic transformation. 40 bombardment or for Agrobacterium TM 3.2.2.3 Biolistic bombardment Apical shoot meristems of mature embryos were excised and then bombarded with the helium particle delivery System (gene gun), model PDS-1000 (DuPont, Wilmington, −1 DE). The plasmid DNA was coated onto 50 µg L of 10 µm tungsten particles with 2.5 M calcium chloride and 0.1 M spermidine suspended in a solution of 1:1 (v/v) of 75% ethanol and 50% glycerol. Three levels of pressure were applied (3447, 6895 and 7584 kPa) to assess the most effective pressure. The concentration of plasmid DNA per bombardment was varied at 1.5 µg and 3.0 µg in order to see which concentration was most favorable. Three levels of bombardment frequency (1, 2 and 3) were used and plant tissues were kept for 24 hours before re-bombarding tissue. The plasmid vector pACT1F (Figure 2), containing the gus marker gene, was used in a mixture of 1:1 (v/v) with the plasmid vector pBY520 (Figure 3) containing the bar herbicide resistant selection marker gene. The plasmid vector gf2.8 (Figure 4) for white mold resistant was transformed independently. 3.2.2.4 Agrobacterium transformation Three strains of A. tumefaciens ( EHA105, GV3301 and LBA4404) were used, transformed with the pCAMBIA 3301 binary vector (Figure 5) containing gus gene driven by the 35S promoter with or without bar gene. These were cultured in 50 µl LB media in the dark at 37°C in a rotator at 280 rpm for 48 hours; OD600=1. These strains were co-cultivated with the explants for 1, 5, 10 and 15 days. The regeneration media 41 described above was supplemented with 500 mg L -1 of Timentin to kill the A. tumefaciens after the appropriate co-cultivation period. 3.2.3 Confirmation of transgene integration and expression 3.2.3. 1. Polymerase Chain Reaction (PCR) analysis Polymerase chain reaction (PCR) analysis for the detection of HVA1, gf.2.8 and bar genes was conducted on T0-T3 plants. Genomic DNA was obtained from leaf disks with diameters the size of the lid of a 1.5 ml Eppendorf tube. Extraction of DNA was done using REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, Cat No. XNA-P), as per manufacturer‘s instruction. The primers used were: bar F, 5`-ATG AGC CCA GAA CGA CG-3` (forward primer); bar R, 5`-TCA CCT CCA ACC AGA ACC AG-3` (reverse primer); and HVA1 F, 5`-TGG CCT CCA ACC AGA ACC AG-3` (forward primer); HVA1 R, 5`-ACG ACT AAA GGA ACG GAA AT-3` (reverse primer); gf2.8 F, 5`-ATG GGG TAC TCC AAA ACC CTA G-3` ( forward primer); gf2.8 R, 5`-CTA GAA ATT AAA ACC CAG CG-3`(reverse primer). The thermocycler (PerkinElmer/ Applied Biosystem, Forster City, CA) was used for DNA amplification. Optimized PCR conditions were 94°C for 3 min for initial denaturation; 35 cycles of 50 s at 94°C; 50 s at 56°C, 1 min at 72°C and a final 10 min extension at 72°C. The PCR product was loaded onto a 1% (w/v) agros, gel stained with 2 µl ethidium bromide and visualized under UV light. 42 3.2.3. 2. Southern Blot Hybridization Analysis The Southern blot hybridization analysis was conducted to determine the stability of the transgenic event and determine the gene copy numbers of HVA1, gf2.8 and bar gene. The DIG High Prime DNA Labeling and Detection Starter Kit ( Roche Co., Cat. No. 1 585 614 ), was used as per manufacturer‘s instructions. Transgenic and non-transgenic genomic DNA was isolated using the methods described by Saghai-Maroof et al. (1984). Hind III or BamHI restriction enzymes were used to digest 20 µg of genomic DNA, which was electrophoresed at 70 v on 1% agarose gel and transferred to a Hybond-N+ membrane (Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV Crosslinker 1800, Stratagene, CA ) at an energy level of 2,000 J. The DIG labeled probes that were used for bar, HVA1 and gf2.8 were synthesized using the primers for the specific gene as described above. 3.2.3.3. Reverse Transcription-PCR (RT-PCR) PCR positive plants of gf2.8 and HVA1 were used in the RT-PCR analysis. Leaf tissue weighing 200 mg was ground using liquid nitrogen. Trizol Reagent 1 ml (Invitrogen, Carlsbad, CA) was applied to homogenize samples. In each tube 0.2 ml chloroform was added and vortexed for a few seconds. The tubes were placed into a centrifuge and spun o at 12,000 xg for 15 min at 4 C. In fresh tubes containing 0.5 ml of isopropanol, 0.2 ml of aqueous phase was transferred. Samples were incubated at room temperature for 5 min 0 and centrifuged at 12,000xg for 10 min at 4 C. The supernatant was discarded, leaving 43 the RNA pellet. The pellet was washed with 1 ml 75% ethanol and flicked to better wash o it and later spun in a centrifuge at 7,500xg for 2 min at a temperature of 4 C. The RNA pellets were immediately dissolved in RNase-free water and quantified using a spectrophotometer. The RNA obtained was used for cDNA synthesis using the Superscript™ First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) as per the manufacturer‘s instructions. The same primers and PCR conditions for gf2.8 and HVA1 as described above were used but the number of cycles were reduced from 35 to 25 in order to detect differences in banding intensity. 3.2.3.4. Northern Blot Northern blot analysis was conducted using the DIG labeled Northern Starter Kit (Roche Co., Cat.No. 12 039 672 910 ), as per manufacturer‘s instructions, in order to assay the gene expression of the transgenic plants harboring the bar, HVA1 and gf2.8 transgenes. Total RNA from the leaves of transgenic and non-transgenic plants was isolated using TRI reagent ( Sigma-Aldrich, St. Louis, MO), as per manufacturer‘s instructions. A total of 30 µg of RNA per sample was loaded onto a 1.2% (m/v) agarose-formaldehyde denaturing gel as described by Sambrook et al. (1989) and transferred to a Hybond-N+ membrane (Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV Crosslinker 1800, Stratagene, CA) at an energy level of 200 J. An RNA or DNA DIG labeled probe, containing the coding region of the gene of interest, was used for detection of transcripts. 44 3.2.4 Biological activity tests 3.2.4.1. Histochemical gus assay Gus activity was tested on transgenic and non transgenic seeds and embryos using histochemical staining with 5-bromo-4-chloro-3-indoyl-β-D-glucuronicacid salt (X-gluc). Plant samples were dipped into gus substrate buffer, according to Jefferson et al. (1987), and incubated at 37°C for 24 hours. The tissue samples were washed with 100 percent ethanol to remove all coloration. 3.2.4.2. Biological assay for bar The herbicide Liberty (Aventis, Strasboug, France), with the active ingredient ammonium glufosinate, was used in both multiple shoot and rooting media, and applied to determine which plants were transgenic as well as to score the segregation ratios of the transgenic progeny. Plants were sprayed at different stages of growth and development ranging from three-week-old young seedlings to two and three-month-old plants. Different foliar application rates of the herbicide were assessed ranging from 50, 100, 250, and 350 mg L -1 of the Liberty herbicide. 45 3.2.4.3. Drought tolerance test Seedlings were raised in the growth chamber for three weeks or until trifoliate leaves appeared. They were then transferred to the greenhouse into 15 cm diameter clay pots containing BACCTO High Porosity Professional Planting Mix (Michigan Peat Company, Houston, TX). The plants were watered daily for three weeks, after which moisture was withheld for 21 days. Observations were recorded on plant survival, degree of leaf wilting, root length, plant growth and height. After the 21 days, moisture was applied to the plants continuously for 14 days in order for them to recover from the drought. The percentage of plants recovered was recorded. 3.2.4.4. Salt tolerance stress test Seedlings were raised in the growth chamber for two weeks. They were then transferred to the greenhouse into 8 cm diameter plastic pots containing BACCTO High Porosity Professional Planting Mix (Michigan Peat Company, Houston, TX). The plants were watered daily with normal tap water for one week in the greenhouse after being transferred from the growth chamber. Thereafter, moisture was withheld for a week to induce drought symptoms, after which five levels (0, 50, 100, 150 and 200 mM) of NaCl concentration was applied to the plants for 10 days. Observations were made and recorded on plant survival, degree of leaf wilting, root length, plant growth and height. After 10 days, water application without NaCl was applied daily for one week in order to recover plants injured by salt stress. 46 3.2.4.5 Pathogen resistance test: Fungal bio-assay Two methods were used to conduct the fungal bioassay. The first was the straw test, in which an agar plug of S. sclerotiorum mycelia was inserted into a straw and placed over the cut stem of the transgenic beans growing in the greenhouse. The second was the detachment of trifoliate leaves that were inoculated with S. sclerotiorum mycelia by placing a 6mm diameter agar plug with inoculum on center of the detached leaf. The inoculation was conducted either in a Petri dish or in a glass tray covered with a plastic paper containing agar or wet paper towel placed at the bottom to keep the leaves and the fungus moist during the infection process. The source of the S. sclerotiorum was obtained from Dr. J. Kelly‘s lab at Michigan State University. The fungal pathogen was grown at room temperature in the dark for 72 hours in medium containing potato dextrose agar (Difco, VWR, Montreal, Quebec, Canada). The protocol used was modified from Livingstone et al. (2005). 47 CHAPTER IV RESULTS AND DISCUSSION : SYSTEMS DEVELOPMENT FOR IN VITRO REGENERATION OF COMMON BEAN 4.0 In vitro regeneration In vitro regeneration of P. vulgaris poses the greatest obstacle and challenge limiting potential for an efficient genetic transformation system of common bean. Attempts have been made towards developing various in vitro regeneration protocols for P. vulgaris. Intact seedling (IS) and cotyledonary node (CN) tissue, cultured on full MS medium with 1 mg L -1 BA and 0.1 mg L -1 NAA resulted into buds and shoots being produced more from IS than CN (Ahmed et al., 2002). Cotyledon explants for P. vulgaris ‗XAN– 159‘, regenerated successfully as opposed to embryonic axis explants which failed. In contrast both explants of P. acutifolius genotypes ‗NI574‘ regenerated successfully with embryonic axis giving the best results. P. vulgaris had difficulty acclimatizing in the greenhouse due to poor in vitro rooting ability. However, P. acutifolius established easily in the greenhouse. In vitro grafting to harden P. vulgaris was done as a means to overcome this problem (Zambre et al., 1998). Arellano et al (2009) developed an in vitro regeneration protocol for P. vulgaris ‗Negro Jamapa‘ black bean using indirect organogenesis with 50% regeneration frequency. Apical meristems and cotyledonary nodes explants were used for callus induction on medium containing 1.5 µM 2,4 Dichlorophenoxyacetic acid and shoot development on medium containing 22.2 µM 6- 48 benzylaminopurine. Delgado-Sanchez et al. (2006) used embryonic axes of P. vulgaris ‗Flor de Junio Marcela‘ (FJM) and ‗Flor de Mayo Anita‘ (FMA) to regenerate whole plantlets with 83% and 50% regeneration efficiency respectively when cultured on MS supplemented with 5 or 10 mg L -1 benzylaminopurine (BAP). An efficient and repeatable in vitro plant regeneration protocol is the most important requirement for successful genetic transformation. In addition, in vitro regeneration is also important for the recovery of certain valuable germplasm which naturally have low germination potential. Furthermore, certain in vitro regenerations such as shoot apical meristem culture is also important for mass propagation of virus free and true-to-type plants which can be distributed to farmers in regions of the world where there is high prevalence of seed borne viral diseases (Delgado-Sanchez et al., 2006). The objective of this study was to develop a highly efficient and reproducible in vitro shoot apical meristem multiplication and somatic embryogenesis protocols for different genotypes of P. vulgaris that are commonly grown in the U.S.A using different combinations of concentration of cytokinin, auxin and antioxidants for optimization of efficient apical shoot meristem multiplication. 49 4.1 Organogenesis and embryogenesis The statistical model for the experiment was significant with an R-square value of 98% and a coefficient of variation of 17.5 with a root mean square of 77% (appendix 1). Statistically significant differences were observed for in vitro regeneration performance of different P. vulgaris genotypes (appendix 2). The separation of means for the different genotypes (appendices 3 and 4) showed that there were no statistically significant differences for the different genotypes within the races except for genotypes belonging to the race Durango which showed a significant difference (Table 5). The effect of growth regulators is also significant. For example, there are statistically significant differences among different cytokinin and cytokinin concentration levels (appendix 6). There are also significant differences among different auxins and auxin levels (appendix 8). As a result of interaction between growth regulators and genotype, the number of multiple shoot regeneration varies from genotype to genotype depending on the growth regulator combination used. Overall the most efficient growth regulator -1 combination for shoot multiplication was a combination of BAP 2.5 mg L and IAA 0.1 mg L -1 which produced an average of 12 multiple shoots per explant in all genotypes tested (Figure 6). Table 6, shows the genotypic specific growth regulator combination that gave the highest number of multiple shoots. The result in figure 7 further show that ‗Olathe‘ produced the highest number of multiple shoots followed by ‗Sedona‘, ‗Merlot‘, ‗Matterhorn‘, ‗Seahawk‘, ‗Jaguar‘, ‗Redhawk‘, ‗Beluga‘, ‗Montcalm‘ and ‗Condor‘. 50 Table 5: Effect of genetic origin as represented by gene pool and race on the efficiency of apical shoot meristem multiplication of 10 contrasting genotypes of common bean (Phaseolus vulgaris) Genotype Gene Pool Race Mean Number of Multiple Shoots ‗Montcalm‘ Andean Nueva Granada 3A ‗Redhawk‘ Andean Nueva Granada 3A ‗Beluga‘ Andean Nueva Granada 3A ‗Condor‘ Middle Mesoamerica 4B Mesoamerica 4B Mesoamerica 4B Durango 5C Jalisco 6D Jalisco 6D Durango 7F American ‗Jaguar‘ Middle American ‗Seahawk‘ Middle American ‗Matterhorn‘ Middle American ‗Merlot‘ Middle American ‗Sedona‘ Middle American ‗Olathe‘ Middle American Means with same letter are not different LSD0.001 was used to separate the means 51 Table 6: Genotypic specific growth regulator combination promoting highest number of multiple shoots Genotypes Growth regulator combination ‗Beluga‘ BAP 5 mg L ‗Condor‘ TDZ 1 mg L ‗Jaguar‘ TDZ 2.5 mg L ‗Matterhorn‘ BAP 5 mg L ‗Merlot‘ TDZ 2.5 mg L ‗Montcalm‘ BAP 5 mg L ‗Olathe‘ BAP 2.5 mg L ‗Redhawk‘ TDZ 2.5 mg L ‗Seahawk‘ TDZ 1 mg L ‗Sedona‘ BAP 2.5 mg L 52 -1 and NAA 0.1 mg L -1 and IAA 0.1 mg L -1 -1 -1 -1 and IAA 0.05 mg L-1 and IAA 0.1 mg L -1 -1 -1 and NAA 0.1 mg L -1 and NAA 0.05 mg L -1 -1 -1 and IAA 0.1 mg L -1 -1 and NAA 0.1 mg L and IAA 0.1 mg L -1 -1 and IAA 0.1 mg L -1 -1 Plant growth Regulator (PGR) treatment TDZ4-IAA3 BA4-Auxin0 TDZ4-Auxin0 Cytokin0-IAA3 Cytokin0-IAA2 Cytokin0-IAA1 Cytokin0-NAA3 Cytokin0-NAA2 Cytokin0-NAA1 Cytokin0-Auxin0 BA3-Auxin0 TDZ4-NAA3 TDZ3-Auxin0 TDZ2-Auxin0 BA1-Auxin0 TDZ3-IAA3 BA4-IAA3 BA3-NAA3 BA2-Auxin0 TDZ1-Auxin0 BA4-NAA3 TDZ1-IAA3 TDZ2-IAA3 TDZ3-NAA3 BA1-IAA3 BA1-NAA3 TDZ2-NAA3 TDZ1-NAA3 TDZ4-IAA2 BA2-NAA3 BA4-NAA1 TDZ4-NAA1 TDZ4-IAA1 BA4-IAA1 BA3-IAA3 TDZ1-NAA1 BA1-NAA1 BA2-IAA3 BA4-NAA2 TDZ3-NAA1 TDZ4-NAA2 TDZ3-IAA1 BA4-IAA2 BA1-IAA1 BA2-NAA1 BA2-IAA1 BA3-NAA2 BA1-NAA2 TDZ2-IAA1 TDZ1-IAA1 TDZ2-IAA2 TDZ3-IAA2 TDZ1-NAA2 TDZ3-NAA2 TDZ2-NAA1 BAP2-NAA2 BAP3-IAA1 BA3-IAA2 BA1-IAA2 BA3-NAA1 TDZ2-NAA2 TDZ1-IAA2 BA2-IAA2 0 2 4 6 8 10 12 Mean Number of Shoots 14 16 18 Figure 6: Effect of cytokinin-auxin combinations on apical shoot meristem multiplication 1 of 10 P. vulgaris genotypes. Note: BAP/TDZ 1,2,3&4=1, 2.5, 5,10 mg L- ; NAA/IAA 1, 1 2, 3= 0.05, 0.1, 1 mg L- ( for supplemental data see appendix 9 and 10) 53 20 15 10 5 0 Ol at he Se do na M at te rh or n M er lo M t on tca lm Ja gu ar Be lug a Se ah aw k Co nd Re or dh aw k Number of Shoots 25 GENOTYPE Figure 7: In vitro apical shoot meristem multiplication performance of 10 P. vulgaris genotypes 54 Overcoming phenolics from cultures: Phenolic compounds exuding from the excised site of the embryonic axis gave a characteristic browning and black color, which hindered normal growth and development of multiple shoots in vitro. To overcome this problem the MS media with growth regulators was supplemented with various antioxidants as described in the materials and methods. The qualitative comparison of control treatment that had no antioxidants and treated tissue with antioxidants, showed that silver nitrate -1 -1 (30 mg L ) and activated charcoal (15 mg L ) produced better quality multiple shoots (Figure 8) with reduced degree of browning and weight of secondary callus tissue (Table 7). 55 A Figure 8: Effect of 4 antioxidant treatments on the quality of multiple shoots       A an extreme case of an untreated explant failing to regenerate due to phenolics B control treatment with no antioxidant C treatment with ascorbic acid D treatment with glutathione E treatment with activated charcoal F treatment with silver nitrate 56 Table 7: Effect of anti-oxidants on apical shoot meristem qaulity in common bean genotype, ‗Condor‘ Antioxidants Percent Weight (mg) Browning Secondary Callus at of the Multiple Shoot Multiple Shoots Base No antioxidant 67±7 6.5±2 Ascorbic Acid 54±5 4.5±1 Silver Nitrate 24±4 1.2±0.6 Activated Charcoal 22±5 0.9±0.4 Glutathione 48±3 2.3±0.8 57 of Morphogenesis studies via scanning electron microscopy. Direct adventitious shoot primordia were formed (Figure 10A) after 3 weeks of culturing the embryonic axes in culture media A (Figure 9). Direct somatic embryogenesis (Figure 10B) occurred after the same duration of culturing the embryonic axis in culture medium B (Figure 9). Shoots developed from clumps of adventitious shoot primordia after 4 weeks on culture medium D (Figure 10C and D ) and from direct somatic embryos after 7 weeks on culture medium D (Figure 10C ). Rooted P. vulgaris plantlets were obtained 5 weeks by culturing of 2-3 cm long shootlets on culture medium E (Figure 10E). The type and concentration of growth regulators were the key factors in determining the morphological pathway of in vitro regeneration of P. vulgaris. Cytokinin, in particular BAP resulted into organogenesis, whereas, less cytokinin in particular TDZ resulted into initiation of embryogenesis (Figure. 9). 58 Mature Seeds 2d A Embryonic Axis Explant 3 wk B 3 wk C Direct Adventitious Shoot Primordia Direct Somatic Embryogenesis Shoot Development 5 wk E Rooted Bean Plant Media A 4.4mg/l MS B 4.4mg/l MS + C D 4.4mg/l MS + 4.4mg/l MS+ E 4.4mg/l MS 2.5mg/lBAP+ 1mg/l TDZ+ 1mg/l BAP/TDZ 0.1mg/l NAA/IAA 0.1mg/l NAA/IAA 0.5mg/l NAA/IAA 1mg/l NAA/IAA Figure 9: Effect of growth regulator combinations on morphogenesis pathway of in vitro cultures of P. vulgaris 59 D Figure 10: Differentiation of somatic embryos, multiple shoots and regenerated mature greenhouse grown rooted ‗Olathe‘ common bean plants. A. Scanning electron micrograph of a section of a multiple shoot clump 3 wk after in vitro culture of an embryonic axis. AvS, adventitious shoot; LP, leaf primordia; ST, shoot tip; LH, leaf hair. x 20; bar= 200µm 60 B. Scanning electron micrograph of a mixture of somatic embryos and organogenesis resulting 6 wk after in vitro culture of excised apical meristem of an embryogenic axis explant. IDSE, indirect somatic embryo; DSE, direct somatic embryo; LP, leaf primordia and ST, shoot tip resulting from organogenesis. . x 40; bar= 200µm C. An advanced regenerated shoot clump and embryogenic tissues 6 wk after in vitro culture of an embryonic axis. x 8 D. An advanced apical multiple shoot clump regenerated through organogenesis 10 wk after in vitro culture of an embryonic axis. E. Effect of 30 s dipping of the cut end of a single in vitro regenerated shoot in 1.0 mg/l IBA followed by 5 wk of culture in 0.1 mg/l NAA. F. Greenhouse grown mature plants produced from rooted shoots. 61 Rooting. The higher level of auxin and lower levels of cytokinin had the greatest effect on root establishment. Auxin level of 0.1 mg L -1 gave the best results while lesser -1 amount (0.05 mg L ) led to poor or no root development (Figure 11). High amounts of -1 auxin 1 mg L -1 in the presence of low amount of cytokinin 1 mg L , gave many roots with little or no shoots while the same high amount of auxin in the presence of high -1 concentration of cytokinin (5-10 mg L ) gave no roots and a few short shoots with many large leaves. There were no statistically significant differences among different auxin types based on the number and length of roots produced. However, there were significant differences among different concentration levels used on the root length and number of roots produced. The effect of dipping shootlets in IBA was also significant (Figure 12). Overall the best treatment that produced strong multiple root establishment -1 was dipping in 1 mg L IBA solution and then culturing of the IBA treated shootlets in media containing 0.1 mg L -1 of NAA, IAA or IBA. This resulted in the number of roots ranging from 1 to 28 and the root length ranging from 4 to 48 cm (for statistical and supplemental data on rooting see appendices 12, 13, 14, 15 and 16). 62 50.0 40.0 30.0 20.0 Mean number of roots +/- SD/ Mean root length +/- SD 60.0 10.0 0.0 IBA Dip 10.0 + IBA 0.1 IBA Dip 5.0 + IBA 0.1 IBA Dip 1.0 + IBA 0.1 No IBA Dip + IBA 0.1 IBA Dip 10.0 + IBA 0.5 IBA Dip 5.0 + IBA 0.05 IBA Dip 1.0 + IBA 0.05 No IBA Dip + IBA 0.05 IBA Dip 10.0 + IBA 0.01 IBA Dip 5.0 + IBA 0.01 IBA Dip 1.0 + IBA 0.01 No IBA Dip + IBA 0.01 IBA Dip 10.0 + IAA 0.1 IBA Dip 5.0 + IAA 0.1 IBA Dip 1.0 + IAA 0.1 No IBA Dip + IAA 0.1 IBA Dip 10.0 + IAA 0.5 IBA Dip 5.0 + IAA 0.05 IBA Dip 1.0 + IAA 0.05 No IBA Dip + IAA 0.05 IBA Dip 10.0 + IAA 0.01 IBA Dip 5.0 + IAA 0.01 IBA Dip 1.0 + IAA 0.01 No IBA Dip + IAA 0.01 IBA Dip 10.0 + NAA 0.1 IBA Dip 5.0 + NAA 0.1 IBA Dip 1.0 + NAA 0.1 No IBA Dip + NAA 0.1 IBA Dip 10.0 + NAA 0.5 IBA Dip 5.0 + NAA 0.05 IBA Dip 1.0 + NAA 0.05 No IBA Dip + NAA 0.05 IBA Dip 10.0 + NAA 0.01 IBA Dip 5.0 + NAA 0.01 IBA Dip 1.0 + NAA 0.01 No IBA Dip + NAA 0.01 IBA Dip 10.0 + No Auxin IBA Dip 5.0 + No Auxin IBA Dip 1.0 + No Auxin Control Auxin Combinations 63 Root length (cm) Number of Roots Figure 11: Effect of dipping shootlets in IBA and culturing of shoots in different auxins on the number and the length of regenerated roots five weeks after transfer of shoots into rooting media. A B C E F G I M J D H K N O L P Figure 12. In vitro response of rooting ability using different levels of IBA dipping system 64 Figure legend for figure 12 Label Treatment A Plain MS media without any hormones B 1 mg L IBA dipping solution C 5 mg L IBA dipping solution D 10 mg L IBA dipping solution E 0.05 mg L of NAA, IAA or IBA F 0.1 mg L G 1 mg L of NAA, IAA or IBA H 1 mg L IBA dipping solution -1 -1 -1 -1 -1 -1 -1 -1 0.05 mg L I of NAA, IAA or IBA plus -1 of either NAA, IAA or IBA in 4.4 mg L -1 1 mg L IBA dipping solution -1 plus MS media -1 0.1 mg L of either NAA, IAA or IBA in 4.4 mg L MS media J -1 1 mg L IBA dipping solution -1 plus -1 1 mg L of either NAA, IAA or IBA in 4.4 mg L MS media K -1 5 mg L IBA dipping solution -1 plus -1 0.05 mg L of either NAA, IAA or IBA in 4.4 mg L MS media L -1 5 mg L IBA dipping solution -1 plus -1 0.1 mg L of either NAA, IAA or IBA in 4.4 mg L MS media M -1 5 mg L IBA dipping solution -1 plus 1 1 mg L of either NAA, IAA or IBA in 4.4 mg L- MS media N -1 10 mg L IBA dipping solution -1 plus -1 0.05 mg L of either NAA, IAA or IBA in 4.4 mg L MS media O -1 10 mg L IBA dipping solution -1 plus -1 0.1 mg L of either NAA, IAA or IBA in 4.4 mg L MS media P -1 10 mg L IBA dipping solution -1 plus -1 1 mg L of either NAA, IAA or IBA in 4.4 mg L MS media 65 Acclimation of rooted plantlets and transfer to greenhouse. Rooted plantlets were removed from Petri dishes, the agar media was removed from the roots by direct rinsing under running tap water, and the washed rooted plantlets were transferred into small pots containing BACTO potting soil. The pots were covered with plastic bags to eliminate evaporation resulting in high humidity around the potted plantlets to mimic the high humidity in Perti-dishes. Potted covered plantlets were maintained under fluorescence light for three weeks or until new leaves emerged on the plants. Holes of approximately 3 mm in diameter, were punched in the plastic bag covers every other day to gradually reduce humidity and to eventually acclimate plantlets to the low humidity in the greenhouse. Acclimated plants were transferred into larger pots and kept in the greenhouse where they were grown to maturity and seeds were produced (Figure 10F). 66 4.2 Discussion This experiment has demonstrated that it is possible to efficiently regenerate multiple shoot meristems from excised apical shoot meristem primordia of embryonic axes of P. vulgaris. This is achievable through optimization of appropriate combinations and concentrations of cytokinin and auxin. Genotype played a significant role in apical shoot multiplication of P. vulgaris. Similar genotypic effects were also demonstrated in cereals by Sticklen and Oraby (2005). The results clearly show that closely related genotypes (belonging to the same race) perform similar as opposed to distantly related genotypes. Observations showed that genotypes that were less recalcitrant towards in vitro regeneration were those that were able to heal faster from the wounding caused by excising the hypocotyl and the cotyledonary nodes and those that produced less secondary callus tissue at the excision site. Cytokinin more than auxin was the key in accelerating wound healing of explants in vitro and reducing the amount of callus produced on the wounded explant, since auxins are well known to induce callus tissue in vitro. Moderate levels of cytokinin ranging from 2.5 to 5 mg L -1 favored the acceleration of wound healing and reduction of callus tissue which resulted in an increase in the number of multiple shoots. On the other hand -1 high levels of cytokinin (10 mg L ) delayed wound healing and inhibited the production of multiple shoots. 67 It was observed that in vitro plantlet development from somatic embryos in P. vulgaris is more difficult with low regeneration efficiency and takes a longer time as compared to shoot development from adventitious shoots followed by rooting. However if one should succeed, somatic embryogenesis has an advantage over adventitious shoots in that potentially more plantlets per explant can be regenerated in vitro. In this study, rooting of in vitro grown shoots was a challenge mainly because the base of the shootlets developed phenolic compounds in vitro causing blackening and death of cells which prevented rooting. Similar problems were encountered by other researchers (Mohamed et al. 1991, Santalla et al. 1998, Zambre et al. 1998). In order to overcome this problem, the effect of phenolic compounds was reduced by supplementing the -1 growth media with 15 mg L -1 activated charcoal or 30 mg L silver nitrate and dipping the base of the explant tissue in IBA solution for 30 sec. The rooting media lacked cytokinin as cytokinin was observed to delay root establishment. As had been indicated by other researchers (Ozyigit et al., 2008), callus combined with phenolic compounds are naturally produced following wounding to aid in healing of plant tissue and to prevent entry of microbes. However in the rooting studies, the greatest limiting factor for in vitro regeneration of P. vulgaris was its propensity to produce high amounts of callus tissue that blocked root formation and phenolic compounds that caused death of tissues due to oxidation of the tissue (Arnaldos et al., 2001). These oxidized phenols prevent multiple shoot development, rooting or 68 regeneration of the explants (Ozyigit et al., 2008). In this study, the anti-oxidants, activated charcoal and silver nitrate were able to effectively reduce the oxidative effect of the phenolic compounds resulting into more and better quality multiple shoots with increased in vitro regeneration efficiency. In conclusion, it is possible to successfully regenerate in vitro apical meristem primordia into multiple shoots and/or somatic embryos of P. vulgaris. In vitro regeneration of P. vulgaris is genotypic sensitive and therefore the media formulation has to be made specific for a particular genotype in order to obtain the maximum in vitro grown multiple shoots and/or somatic embryos. The excretion of phenolic compounds from wounds associated with high production of callus tissue is the greatest obstacle to in vitro regeneration of P. vulgaris. Supplementation of anti-oxidants to the culture media significantly improves the quality and increases the regeneration efficiency as well as the numbers of multiple shoots and the rooting ability of P. vulgaris plantlets in vitro. 69 CHAPTER V RESULTS AND DISCUSSION: GENETIC TRANSFORMATION SYSTEM DEVELOPMENT IN COMMON BEAN In this chapter results of experiments that were conducted in order to develop a gene delivery system for common bean are presented. The figures below show the optimization of Biolistic TM and Agrobacterium tumefaciens transformation systems followed by confirmation of transgene integration and expression in different common bean genotypes. In order to demonstrate and provide proof of concept for the genetic transformation system that was developed, the bar and gus genes were used as selectable and screenable markers respectively. 5.1. Optimizing conditions for the Biolistic integration of the bar gene TM bombardment method using stable In the bombardment method, conditions were optimized in order to obtain the maximum efficiency of transformation. The optimized conditions involved varying the pressure of the gene gun at three levels (3447, 6895 and 7584 kPa); varying the plasmid DNA concentration at two levels (1.5 and 3.0 µg); and finally, the number of times that embryonic tissue was bombarded varied at three levels (1, 2 and 3 times), each 24 hours apart. 70 To optimize the transformation efficiency of common bean, using the particle bombardment method, results suggest bombarding the plant twice, using a pressure setting of 7584 kPa with a concentration of 1.5 µg of plasmid DNA per bombardment. Such conditions yielded a transformation efficiency of 8.4% (Table 8). 71 Table 8: Different treatment combinations (gene gun pressure, DNA plasmid TM concentration and bombardment frequency) used for optimizing Biolistic bombardment conditions Bombardment Concentration Bombardment Mean Transformation Pressure (kPa) of plasmid DNA (ug) Frequency Percent 3447 1.5 1 0.1±0.04 3447 1.5 2 0.2±0.10 3447 1.5 3 0.4±0.30 3447 3 1 0.1±0.04 3447 3 2 0.6±0.32 3447 3 3 0.7±0.32 6895 1.5 1 2.9±0.67 6895 1.5 2 3.9±1.4 6895 1.5 3 5.1±1.2 6895 3 1 5.6±1.0 6895 3 2 8.1±0.3 6895 3 3 7.4±1.0 6895 1.5 1 7.2±0.70 7584 1.5 2 8.4±0.74 7584 1.5 3 8.2±0.50 7584 3 1 7.5±0.69 7584 3 2 4.8±0.93 7584 3 3 3.3±0.92 72 The gene gun pressure was the greatest determining factor for successful integration of transgene. Low gene gun pressure yielded very low and poor transformation efficiencies while increased frequency of bombardment damaged the explants. The transformation efficiency that was obtained was higher than those that have been reported by other researchers who have bombarded explants only once or used different gene gun pressures (Somers et al., 2003, Popelka et al., 2004). 5.2. Optimization of conditions used in developing Agrobacterium-mediated transformation method for transient expression of gus gene A. tumefaciens was used to evaluate its potential as a vehicle for gene delivery. Many researchers have failed to use A. tumefaciens as a vector for delivery of foreign genes into common bean (Velchelva et al. 2005). The approach used in this research was to optimize conditions for A. tumefaciens-mediated transformation. Different co-cultivation periods were assessed as well as different strains of A. tumefaciens, which included EHA105, LBA4404 and GV3301. The relative transformation efficiencies were also compared among ‗Sedona‘ and ‗Matterhorn‘ genotypes The results indicated that, for both transient and stable expression of gus gene, ‗Sedona‘ is more amenable to Agrobacterium transformation than ‗Matterhorn‘. The results also show that, for both transient and stable expression of gus gene, the Agrobacterium strain GV3301 is the most effective when compared to EHA105 or LBA4404. The most favorable co-cultivation period for high transformation frequency is 15 days. It was noted 73 that there was a significant discrepancy between transformation efficiencies of tissues that were transiently being expressed as compared to those with stable transformation. With a co-cultivation period of 15 days, using GV3301, transient expression efficiencies of gus were 51% with ‗Matterhorn‘ and 81% with ‗Sedona‘. Using the same cocultivation period and with the strain EHA105, transient expression efficiencies for gus of 66% and 69% were achieved for ‗Matterhorn‘ and ‗Sedona‘ respectively. Under the same conditions using LBA4404, 18% and 50% transient expression efficiencies were achieved for ‗Matterhorn‘ and ‗Sedona‘, respectively (Figure 13). 74 90 80 70 60 Transformation 50 Frequency % 40 30 20 10 1d 5d 10d 15d 1d 5d 10d 15d 1d 5d 10d 15d 1d 5d 10d 15d 1d 5d 10d 15d 1d 5d 10d 15d 0 GV3301 EHA105 LBA4404 GV3301 Matterhorn EHA105 LBA4404 Sedona Figure 13. Effect of co-cultivation period (1, 5, 10 and 15 d) on the transformation frequency of two genotypes of common bean, ‗Matterhorn‘ and ‗Sedona‘, using three different strains of A. tumefaciens (EHA105, GV3301 and LBA4404). 75 Despite having these relatively high frequencies of transient expression of the gus gene using the Agrobacterium method of transformation, there was much lower stable transformation frequencies. For example, with a 15 day co-cultivation period and using Agrobacterium strains GV3301, EHA105 and LBA4404, the efficiencies of stable transformation were 0.38, 0.31 and 0.1 percent, in that order, for the genotype ‗Matterhorn‘. The frequencies for the genotype ‗Sedona‘ were 0.68, 0.52 and 0.36 percent for the strains GV3301, EHA105 and LBA4404, in that order (Figure 14). The reasons why transient expression showed higher frequencies are most likely because (1) not every transiently expressed gene is stably integrated into the transgenic plants, and (2) the GUS staining can diffuse into plant tissues even if the plasmid expression vector is not integrated on the chromosome. Fifteen days of co-cultivation was more effective than fewer days of co-cultivation. In order to improve upon the relative frequencies of stable transformation, future research has to explore increasing the co-cultivation period beyond 15 days and also the use of chemicals such as acetosyringones or tobacco extract to increase the virulence of the Agrobacterium. 76 Stable transformation of common bean with gus gene: 0.8 0.7 0.6 0.5 Transformation frequency % 0.4 0.3 0.2 0.1 0 GV3301 EHA105 LBA4404 GV3301 EHA105 LBA4404 Matterhorn Sedona Figure 14. Effect of using different strains of A. tumefaciens (EHA105, GV3301 and LBA4404) with two common bean genotypes, ‗Matterhorn‘ and ‗Sedona‘, on the relative stable transformation frequency of T1 (second generation) plants after 15 days of cocultivation. 77 5.3 Confirmation of stable gene integration using PCR and Southern blot. PCR of bar transgene integration of T1 plants Figure 15: PCR T1 plants of genotypes; ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp 78 Segregation of T2 plants confirming the stability of bar transgene Figure 16: PCR T2 plants of genotypes: ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp. 79 Segregating T3 plants confirming the stability of bar transgene Figure 17: PCR T3 plants of genotypes: ‗Condor‘(C), ‗Sedona‘(S), ‗Montcalm‘ (Mo) and ‗Matterhorn‘ (Mat). Expected band size is 450 bp 80 Southern blot analysis confirming a single gene copy integration of bar transgene in T2 ‗Condor‘ plants Figure 18: Southern blot showing integration of bar gene in genotype ‗Condor‘. The (+) represents the plasmid DNA; Wt: Wild type non transgenic leaf DNA; C1 to C3: DNA taken from leaves of three transgenic plants of the same transgenic line; C4: DNA taken from leaves of a different independent transgenic line. 81 Southern blot analysis confirming a single gene copy integration of bar transgene in T2 ‗Matterhorn‘ plant. Figure 19: Southern blot showing integration of bar gene in genotype ‗Matterhorn‘ line 2 (M2), digested with BamH1, the other line (M1) shows no integration. The results indicate that there is a single gene integration: Wt= Wild type; (+) = plasimid 82 Southern blot analysis confirming different copy numbers of integration of bar transgene in T2 of different common beans genotypes Figure 20. Southern blot showing integration of bar gene in genotypes, Mat = ‗Matterhorn‘, Sed = ‗Sedona‘and Mont = ‗Montcalm‘, Wt = wild type, (+) = plasmid. Digestion was done with Hind III. The results indicate that there are four copies of the gene in ‗Matterhorn‘, three copies in ‗Sedona‘and two copies in ‗Montcalm‘. 83 5.4 Confirmation of gene expression using Northern blot analysis Northern blot analysis of bar transgene expression in T2 of ‗Condor‘ genotype C8 C7 C6 C5 C4 C3 C2 C1 Wt Figure 21. Northern blot expression of bar gene in T2 plants; genotype C1-8 = ‗Condor‘, Wt= Wild type. 84 Northern blot analysis of bar transgene in T3 of ‗Condor‘ and ‗Matterhorn‘ genotypes Figure 22. Northern blot expression of bar gene in T3 plants: genotype ‗Matterhorn‘ (M2), ‗Condor‘ lines C1, C4, and C8. ‗Matterhorn‘ seems to have a higher expression than the ‗Condor‘ lines (C1-C8). 85 Northern blot analysis of T3 transgenic ‗Sedona‘and ‗Montcalm‘ genotypes Figure 23. Northern blot expression of bar gene in T3 plants; genotypes ‗Sedona‘ (S1-S3) and ‗Montcalm‘ (Mo1-Mo3). ‗Sedona‘ plants S2 and S3 have a higher expression than either ‗Montcalm‘ plants Mo2 and Mo3. The expression of ‗Montcalm‘ is extremely low. 86 5.5 Liberty herbicide resistance test of T2 and T3 of transgenic plants -1 Figure 24: Bar tested T2 plants at a concentration of 150 mg L . ‗Condor‘ (A), ‗Matterhorn‘ (B), ‗Montcalm‘ (C) and ‗Sedona‘(D). ‗Matterhorn‘ seems to be better expressed. Transgenic plants are not 100% resistant, some leaves are scorched, and exhibit stunted growth. However, their survival is better than the wild type (Wt) plants 87 Liberty herbicide resistance test of T3 of transgenic plants Figure 25: T3 plants showing partial resistance to Liberty herbicide the genotypes used are ‗Condor‘, ‗Matterhorn‘, ‗Montcalm‘ and ‗Sedona‘. The tray on the left represents wild type non-transformed plants while that on the right are transformed plants. Each plastic container contains the four genotypes mentioned above. The spray rate of the herbicide liberty was 200 mg L -1 88 5.6 Confirmation of gus transgene expression using the GUS assay in T3 ‘Matterhorn’ genotype B A Figure 26: Gus expression in ‗Matterhorn‘. All genotypes transformed, namely, ‗Matterhorn‘, ‗Condor‘, ‗Sedona‘, ‗Olathe‘, and ‗Montcalm‘, showed gus positive for both plants transformed using bombardment and Agrobacterium. However ‗Matterhorn‘ had the best expression; panel (A) in seed and (B) embryo. 89 The integration of bar gene was demonstrated across four generations (T0-T3). The expected band size of this gene product is 450 bp, which confirms that there is successful bar gene integration in the genotypes transformed, which were ‗Condor‘, ‗Sedona‘, ‗Montcalm‘ and ‗Matterhorn‘. However, the Chi-square test of T2 and T3 plants reveal that the segregation of the bar gene does not follow Mendelian inheritance. The most probable explanation for this observation is that the transgenic plants are still chimeric, meaning that not all cells in the plant contain the bar transgene. Also, as another possibility, some of the introduced foreign plasmids could be residing in the cytoplasm as opposed to being integrated on the chromosomes. This scenario would still enable the plasmids to be transmitted from one generation to the next vegetatively, like organelle genomes or like viruses and bacteria that have symbiotic relationships with plant cellular systems. In order to solve this problem more generations of selfing are required in order for all the cells to acquire the transgene. The Southern blot analysis of T2 plants bombarded with a construct containing the bar gene shows integration of four different transgenic ‗Condor‘ plants with a single gene copy insert (Figure 18). Two ‗Matterhorn‘ independent transgenic lines showed successful integration of the bar gene (Figures 19 and 20) with a single and four copy numbers of the bar transgene. ‗Sedona‘and ‗Montcalm‘ showed three and two copy number of bar transgene respectively (Figure 20). 90 Northern blot analysis of T2 and T3 plants confirms the transcription of bar transgene (Figures 21, 22 and 23). The expression levels are not very high except for ‗Matterhorn‘ and ‗Sedona‘which shows a higher expression level than the others. This might result from differences in the integration site of the bar gene in the plant genome rather than the genotypes used. Resistance to Liberty herbicide was tested on two months old T2 plants and showed that they were still chimeric because certain portions of the leaves got burnt by the herbicide -1 three days after being sprayed with 150 mg L of Liberty herbicide (Figure 24). Testing of T3 plants was conducted to see if their level of resistance towards the herbicide had -1 improved. The foliar application of the herbicide was increased to 250 mg L . The observation noted is that the transgenic plants were still chimeric because some of the leaves got scorched by the herbicide (Figure 25). Stable expression of gus transgene is shown in seeds and zygotic embryos of T3 plants (Figure 26). The expression levels of gus in seeds and embryos of transgenic plants obtained from bombardment or Agrobacterium-mediated method was the same. This led to the conclusion that expression levels are not a function of method of transformation but maybe other factors such as genotype or promoter construct. Even though we had good expression of GUS protein (Figure 26) there were still a few small spots on the seeds and embryos not showing the expected blue color. These results suggest that the 91 transgenic plant material were still chimeric. This observation also helps us to correlate the previous observation made on Liberty treated transgenic plants that showed that some spots on the leaves were scotched while others were not. This clearly indicates chimeric expression. 92 CHAPTER VI RESULTS AND DISCUSSION: DROUGHT AND SALT STRESS TOLERANCE OF TRANSGENIC COMMON BEAN With the transformation protocol that was developed and optimized, 2,000 embryonic axis tissues were bombarded with tungsten microprojectile particles containing the plasmid DNA with HVA1 gene. The PCR results showed that 161 plants were positive which is about 8% transformation efficiency for putative transgenic material. However even though the PCR showed 8% positive in the T0 only five plants showed positive integration of transgene in T2 generation using southern blot analysis. The number of integrated transgenes ranged from one to two (Figures 28 and 29). PCR positives for these plants was demonstrated in the T3 population (Figure 27). RT-PCR analysis for T2 transgenic plants of ‗Montcalm‘, ‗Condor‘, ‗Matterhorn‘ and ‗Sedona‘ were positive and all four genotypes showed similar expression levels ( Figure 30). However the relative expression levels changed in the T3 generation with ‗Montcalm‘s‘ expression declining significantly when compared to the T2 generation and ‗Condor‘ completely losing its expression (Figure 31). Northern blot analysis was done on T3 plants and only ‗Matterhorn‘ and ‗Sedona‘ showed some expression, whilst no expression was detected for ‗Montcalm‘ and ‗Condor‘ (Figure 32). 93 6.1. Confirmation of HVA1 transgene integration in plants using PCR and Southern blot analysis Figure 27. PCR results of T3 transgenic plants of ‗Montcalm‘, ‗Condor‘, ‗Sedona‘and ‗Matterhorn‘ confirms the stability of HVA1 transgene integration. The expected band size is 670 bp. 94 Southern Blot Analysis confirming the copy number of HVA1 gene that have been integrated into ‗Condor‘ plants. Figure 28: Southern blot showing integration of HVA1 gene in genotype ‗Condor‘(C8), digested with BamH1, the other lines show no integration. The results indicate that there is a double gene integration. 95 Figure 29. Southern blot showing integration of HVA1 gene in genotypes Co = ‗Condor‘, Mo = ‗Montcalm‘, Se = ‗Sedona‘and Ma = ‗Matterhorn‘ digested with BamH1. The results indicate that there is a double gene integration in all genotypes except ‗Montcalm‘ which has a single copy number. The Wt= wild type shows no transgene integration. 96 6.2. Confirmation of HVA1 transcription in plants using Reversed Transcription PCR, followed by Northern blotting Figure 30. RT-PCR of HVA1 expression for T2 transgenic plants of S = ‗Sedona‘, C = ‗Condor‘, Mo = ‗Montcalm‘ and Ma = ‗Matterhorn‘. Expected band size is 670 bp for HVA1. The expression levels are the same for all four plants. Below is the cDNA loading control showing the expression of ubiquitin with an expected band size of 450 bp. 97 RT-PCR Figure 31. RT-PCR showing expression of HVA1 T3 transgenic plants with expected band size of 670 bp. ‗Condor‘ completely lost its expression that was previously detected in the T2 generation. The expression of ‗Montcalm‘ declined while that of ‗Sedona‘ and ‗Matterhorn‘ remained stable. Below is the cDNA loading control showing the expression levels of ubiquitin the expected band size is 450 bp. 98 Northern Blot Figure 32. Northern blot expression of HVA1 gene from T3 transgenic plants subjected to drought stress. Mat = ‗Matterhorn‘ and Sed = ‗Sedona‘ showed some expression. The remaining lanes, Wt = Wild type, Mon = ‗Montcalm‘ and Con = ‗Condor‘ showed no expression at all. 99 6.3. Drought tolerance tests of transgenic versus control non-transformed plants. When water was withheld for 21 days continuously, all the ‗Condor‘ plants regardless whether they were transgenic or non-transgenic wild types died within 12 days of treatment. No differences could be distinguished between the transgenic and the wild types. Similar results were also obtained for ‗Montcalm‘ plants that died within 16 days of treatment with no clear distinction between transgenic plants and wild types. On the other hand ‗Sedona‘ and ‗Matterhorn‘ transgenic plants persisted for 21 days without water. They showed symptoms of drought stress but soon recovered after three days when moisture application resumed. The wild types showed more severe symptoms of drought stress with most of the leaves wilted and dehisced (Figure 33B). Out of 30 plants that were planted for each genotype in the experiment, 15 were wild types and the other 15 were transgenic. Survival of wild type ‗Sedona‘ plants was only 2 out of 15 and the transgenic plants were 5 out of 15. Survival of ‗Matterhorn‘ wild type plants was 3 and transgenic plants were 8 out of 15. The percent leaf abscission was used as an indirect measure of the degree of plant wilting. Wilting was defined as the difference of ratios between the number of leaves on plant before 21 days of moisture withdraw and the number of green leaves on plant remaining after 21 days of moisture withdraw. The percent leaf abscission for transgenic ‗Sedona‘ plants was 78% and wild type was 91% and for ‗Matterhorn‘ it was 72% and 88% for transgenic and wild type respectively. It appears that ‗Matterhorn‘ possesses a 100 genotypic advantage over ‗Sedona‘in terms of tolerating drought as indicated by the results of the performance of their wild types (Singh 2007). The mean height or growth rate of transgenic versus non-transgenic plants did not differ significantly. For example before the experiment was conducted plants of uniform height (20 cm) were selected. After the treatment period height measurement was taken again. The results showed that the mean height for ‗Sedona‘ transgenic plants was 23 cm and that for wild type plants was 22 cm. There was a net growth after 21 days of treatment of three and two centimeters for transgenic and wild type plants. The mean height for ‗Matterhorn‘ transgenic plants was 24 cm and for wild type plants it was 23 cm. The net growth after 21 days was four and three centimeters respectively for transgenic and wild type plants. In contrast, the control normal watered plants grew to a height of 33 cm and had a net growth of 13 cm. This is an average of three-fold increase in growth compared to the plants under drought stress. The rooting ability was also examined and showed that the root growth of transgenic plants was more robust than wild type plants under stress but less developed than wild type plants under normal moisture regime (Figure 33D). The average root length measured after 21 days of treatment for ‗Sedona‘ transgenic plants was 15 cm and for wild type plants was 11 cm. For ‗Matterhorn‘ the average root length measured after the same treatment application was 17 cm for transgenic plants and 13 cm for wild type plants. In contrast, for control plants under normal irrigation the average root length was 101 28 cm. From the results of this experiment it was shown that transgenic plants engineered with HVA1 utilize their energy in developing and growing their root system as opposed to the above ground stem and canopy which exhibited little growth under drought stress conditions and showed no significant phenotypic difference between transgenic plants and wild types. (Figure 33C and D). A summary of the results of the drought experiment is shown in Table 9. 102 Table 9: Summary of measurement parameters of drought stress test Number Of Plants Surviving Per 15 Plants Genotype Percentage Of Leaf Abscission Transgenic Wt Transgenic Wt Plant Height (cm) After 21 Days of Drought Root Length (cm) After 21 Days of Drought Transgenic Wt Transgenic Wt ‗Matterhorn‘ 8 3 72 88 24 23 17 13 ‗Sedona‘ 5 2 78 91 23 22 15 11 ‗Condor‘ 0 0 100 100 21 21 8 7 ‗Montcalm‘ 0 0 100 100 22 22 9 9 103 B A D C Figure 33. Panel (A): ‗Matterhorn‘ plants before drought induction; (B) Plants after 21 days continuous no irrigation; (C) ‗Matterhorn‘ drought recovered plants after 3 days of water re-application; 1= control non-transgenic plant that was watered throughout the experiment; 2= ‗Matterhorn‘ transgenic plant after 21 days of no-irrigation, 3= Wild type non-transgenic plant after 21 days of no-irrigation; (D) root development of plants after 21 days of drought stress. 1: Control non-transgenic plant roots, these were watered daily, 2: Transgenic plant roots after 21 days of no-irrigation and 3: Wild type non-transgenic plant roots after 21 days of no-irrigation. 104 6.4. Salt stress test of HVA1 of T3 plants The salt stress test that was conducted did not distinguish the wild type plants from the transgenic plants. Five different regimes 0, 50, 100, 150 and 200 mM of NaCl solution were used. Beyond 150 mM all plants died and never recovered including the transgenic plants. At 100 mM there was partial recovery of both wild type, transgenic plants while at 50 mM both the transgenic, and wild type survived but showed symptoms of salt stress that were characterized by wilting and stunted growth (Figure 34). Although salt and drought stress share a similar pathway, there was no observable significant phenotypic differences between transgenic and wild type plants under salt stress. Only one transgenic plant showed a significant resistant phenotype from the rest of the plants at 50 mM application of NaCl. With this low statistical power it is difficult to tell whether this is a genuine resistant transgenic plant or an escape plant, further tests are required to be conducted to verify this observation. 105 Figure 34. Salt stress test at 5 levels of concentration (0, 50, 100, 150 and 200 mM) on ‗Matterhorn‘ plants 10 days after salt treatment. Note that the two plants on the left side of each flat are control non-transgenic and the two plants on the right side of each flat are Northern blot positive transgenic. 106 When comparing the salt stress test with the drought simulation test, very clear phenotypic differences were seen between transgenic and wild type plants, in particular ‗Sedona‘ and ‗Matterhorn‘, under drought stress test. A possible reason as to why there was an observable clear phenotypic difference between transgenic and non-transgenic plants under drought stress as opposed to salt stress maybe due to the functional class of HVA1 gene. This gene belongs to the group 3 LEA proteins that are members of HSP chaperones that have been known to be more effective towards heat and drought stress + rather than salt stress. Salt stress needs the addition of detoxification and Na antiporter genes such as SOS1. Therefore in order to detect meaningful phenotypic differences in salt tolerance between transgenic and non-transgenic plants members of such gene families may need to be engineered into common bean together with HVA1. In general, the plants under drought stress remained stunted and weak as compared to the control non-transgenic plants with normal watering. However, the transgenic plants, in particular ‗Sedona‘ and ‗Matterhorn‘ performed better than the rest of the other genotypes. In this experiment, HVA1 has been shown to be more effective in common bean in alleviating drought stress symptoms and not so effective for conferring resistance towards salt stress. 107 CHAPTER VII RESULTS AND DISCUSSION: WHITE MOLD STRESS TESTS OF TRANSGENIC COMMON BEAN Application of the optimized transformation protocol was developed using 2,000 embryonic axis tissues that were bombarded with tungsten micro projectile particles coated with DNA plasmid containing the germin gene (gf2.8). The PCR showed that 138 plants (i.e. less than 7% of the bombarded plant material) contained the gf2.8 insert in the T0 generation. 7.1. Confirmation of germin transgene integration using PCR and Southern blot analysis Even though the PCR tests were positive for T1 and T2 plants, only three plants showed positive integration of transgene using Southern blot analysis. Therefore, this may mean that the other plants were chimerically transgenic or the transformed plasmid is resident in the cytoplasm and not on the chromosome in the nucleus. In Southern blot, the number of integrated transgenes ranged from 2 to 4 copies (Figure 36). PCR positives for these plants were demonstrated in the T3 population along with other plants that did not show Southern blot positive (Figure 35). 108 Figure 35: PCR results of T3 transgenic plants of Ola 1-2 = ‗Olathe‘, Con = ‗Condor‘, Sed =‗Sedona‘and Mat 1-2 = ‗Matterhorn‘. The expected band size is 640 bp. 109 Figure 36: Southern blot analysis showing integration of transgene in the T2 plants. Positive lines are S1 = ‗Sedona‘ with 2 gene inserts, Ma1 = ‗Matterhorn‘ with four gene inserts and Ola1 = ‗Olathe‘ with two gene inserts. The C1 or 2 = ‗‗Condor‘,‘ did not show positive integration of transgene and Wt=Wild type is also negative. 110 7.2. Confirmation of germin gene expression using RT-PCR and Northern blot analysis The RT-PCR analysis for PCR positive T2 plants of ‗Olathe‘, ‗Condor‘, ‗Matterhorn‘ and ‗Sedona‘ was positive. The expression levels of these genotypes appeared to be the same for T2 plants using RT-PCR (Figure 37). However, when northern blot analysis of gf2.8 plants was carried out, none of the plants showed northern expression. This indicated that the expression levels were just too low to be detected by northern blot analysis, which is less sensitive than RT-PCR. When the same plants were inoculated with the fungal pathogen, northern blot analysis was done again using RNA collected from infected tissue. The results showed that only ‗Matterhorn‘ had limited expression and no expression was detected at all in the other plants (Figure 38). 111 Germin Gene RT-PCR S C M O Wt + 640bp 640bp S C M O wt 450 bp 450bp UBIQUITIN Figure 37:RT-PCR of the germin gene for T2 plants has an expected band size 640 bp. All four genotypes transformed, S= ‗Sedona‘, C= ‗Condor‘, M= ‗Matterhorn‘ and O= ‗Olathe‘ show expression which is less than the positive control. Wt=Wild type is negative. Below is the ubiquitin loading control which shows equal amount of cDNA loading, with an expected band size of 450 bp. 112 Northern Blot Figure 38:Northern blot of the germin gene from infected tissue of T3 plants. Only M= ‗Matterhorn‘ showed positive results for the expected band size of 640 bp. The rest Wt=Wild type, Ola= ‗Olathe‘, Co= ‗Condor‘ and Sed= ‗Sedona‘ have negative results. 113 7.3. Fungal biological assay of RT-PCR and Northern blot positive transgenic plants Prior to inoculation, the fungal pathogen was grown on PDA at ambient room temperature. Only the tip of the growing fungus was used for inoculation and the fluffy white mycelia shown (Figure 39) was not utilized because its mycelia had stopped actively growing. Figure 39. S. sclerotiorum fungus 72 hours after being grown on potato dextrose agar. The fungal biological assay was carried out on all RT-PCR positive transgenic plants that were compared to the control non-transgenic plants. Three systems were experimented on for directly testing the biological activity of germin transgene product (oxalate oxidase). The first experiment was the straw test (lower panel of figure 40), in which the growing mycelia were wedged into a straw, and placed onto an excised shoot tip of the common bean plant growing in the greenhouse. This test did not work because the greenhouse was too hot for the survival of the pathogen. A different approach was used in which trifoliate leaves were placed in a Petri dish containing plain agar and a plug of mycelia was placed on top of the leaves (upper part of Figure 40). This system failed because the agar was unable to provide adequate moisture for the growth of the pathogen. 114 Figure 40. White mold pathological test. The upper two panels shows the trifoliate leaf detachment assay with plugged mycelia on leaf surface incubated in Petri dishes containing agar media. The two bottom panels show the straw test in the greenhouse, with the straws containing the fungus inserted into the shoot tip of common bean. These two tests did not work because the fungus was unable to infect neither the transgenic nor the wild type plants. In both cases, the humidity was not conducive for the growth of the pathogen. 115 The third technique involved placing wet paper towels into a sterile tray covered with a transparent plastic wrap. This technique worked well because it provided adequate moisture for the fungal pathogen to grow and infect host tissue. The level of resistance of different independent transgenic lines that were inoculated with the fungal pathogen was compared to the non-transgenic wild type plants that were used as control. The RT-PCR positive transformed plant leaves showed little resistance against the pathogen when compared to the wild-type non-transgenic leaves which showed no resistance to the fungal pathogen. Among RT-PCR positive transformed plant leaves, the genotype ‗Matterhorn‘ performed the best. It displayed the longest delayed onset of lesions on the leaves. This was followed by ‗Sedona‘ then ‗Olathe‘ and finally ‗Condor‘ (Figure 41). Despite observing delayed establishment of the pathogen on ‗Matterhorn‘ transgenic plant leaves for a period of 72 hours (Figure 42), there were no observable differences between susceptibility of transgenic versus non-transgenic plants beyond the 72 hours time period. 116 35 30 25 Wt 20 Lesion Size (mm) Condor 15 Olathe Sedona 10 Matterhorn 5 0 1 2 3 4 5 Number of Days Figure 41. Relative rate of infection and development spread of pathogen as measured by lesion size on leaf surface of T2 ‗Matterhorn‘, ‗Sedona‘, ‗Olathe‘, ‗Condor‘ and wild type plants. 117 Figure 42. Fungal biological assay: trifoliate leaves placed on moist paper towel in a tray with plug mycelia on top of the leaves. Transgenic ‗Matterhorn‘ (gf2.8), on the left, shows delayed infection after 72 hours of inoculation, compared to wild type on the right. 118 This experiment has demonstrated that, even though the expression levels of germin gene in transgenic plants was significantly low as shown by northern blot analysis, the potential of using gf2.8 gene in common bean to confer resistance to white mold is feasible. Therefore, future research should use our transformation protocol and place more emphasis on exploiting the use of stronger cis and trans acting regulatory elements. These include different promoters or enhancer elements that have the capacity to increase expression levels. 119 CHAPTER VIII CONCLUSIONS AND FUTURE PERSPECTIVES The conclusions from these studies have confirmed that common bean still remains a crop species that is recalcitrant to in vitro regeneration and genetic engineering. Nevertheless, the research that has been presented here shows that great potential for efficient transformation of common bean can be further exploited and optimized by establishing suitable conditions for both in vitro regeneration and gene delivery into these plants. In this research effort emphasis was placed on resolving some of the obstacles associated with low transformation efficiencies, including the high rate of transgenic chimera as well as transgene loss in progressive generations. A significant breakthrough of this research dealt with one of the major causes leading to recalcitrance of in vitro regeneration which is, the prolific excretion of phenolic compounds. It was demonstrated that the application of anti-oxidants, silver nitrate in particular, is very effective against oxidation of phenolic compounds that turn into toxic oxides that kill tissue that is growing in vitro. Another cause of recalcitrance of common bean in in vitro regeneration is the lack of totipotency of cells. In work recently reported (Kwapata et al., 2009), it was shown that totipotency can be established by balancing accordingly the ratios of cytokinin and auxin. Using this approach, the number of multiple shoots per explant was increased to 20. This was significantly higher than 4 to 8 multiple shoots per explants that have previously been reported by a number of 120 researchers (Ahmed et al. 2002). However, the number of multiple shoots that were obtained is still lower than the number produced from cereal explants. In view of this work, more effort is needed to achieve higher numbers of multiple shoots per explant. Therefore, continued efforts to find more suitable media formulations and better methods for the removal of phenolic compounds from the culture media should be investigated. In vitro regeneration via direct organogenesis, using either cotyledonary nodes or embryogenic axis, are the current methods used by researchers. These methods have proven to be very inefficient (Popelka et al. 2004, Veltcheva et al. 2005). The system developed in this research of using multiple shoots from the apical meristem primordia offers a better alternative for an improved regeneration protocol that is amenable to a relatively efficient gene delivery system. An ideal alternative transformation protocol for common bean might involve the use of regeneratable embryogenic callus or cell suspension cultures. If successful, these explants can increase the efficiency of transformation and reduce the rate of chimeric transgenic plants based on the fact that somatic embryos originate from single cells while organogenesis originates from multiple cells. Unfortunately, no report exists of successfully using embryogenic callus or cell suspension cultures for genetic transformation of common beans. This is because callus explants produce phenolic compounds in vitro at a rate faster than the cells are able to multiply. These phenolic compounds suffocate the cells and hinder their growth. Future 121 research can explore the possibility of using the system that has been developed for eliminating phenolic compounds of multiple shoots grown in vitro and applying it to callus or cell suspension cultures. Another challenge associated with in vitro cultures of grain legumes, including common bean, is their prolonged regeneration period. This prolonged period invites opportunity for somaclonal variations that could adversely affect the phenotype and regeneration capacity. Developing protocols that expedite the regeneration processes may assist in alleviating these problems. Closely related to the problem of prolonged in vitro regeneration period of grain legumes including common bean, is the poor in vitro rooting, this presents a problem of establishing plantlets in soil and greenhouse. Due to the difficulty of regenerating roots of common bean in vitro, other researchers have opted to use grafting in order to solve this problem. However, this approach has been proven to be cumbersome and inefficient (Krishnamurthy et al. 2000, Sarmah et al. 2004, TewariSingh et al. 2004, Sanyal et al. 2005). In this research a better and more efficient method of rooting has been developed using an IBA dipping system. This method ensures healthy rooting, survival and establishment of in vitro grown plants in the greenhouse. The Agrobacterium-mediated gene delivery system that was developed is still in its early development phase and needs to be improved. The results have shown that common bean receptiveness towards genetic transformation with Agrobacterium is poor compared to most other docotyledoneous plants (e.g. potato, tobacco, etc). Despite this, there are some 122 encouraging signs. Even though the system exhibited low transformation efficiencies, some stable transformed plants were obtained. It was determined from the study that the most promising strains for Agrobacterium-mediated transformation are EHA105 and GV3301, both of which showed better results for gene delivery than LBA4404. In addition to the type of strains used in the studies, it was shown that the co-cultivation period also significantly affects the efficiency of Agrobacterium-mediated transformation systems. In this particular study, longer co-cultivation periods were shown to increase the chances of gene delivery. The results that were obtained can be used as a base for future work of improving Agrobacterium-mediated transformation systems. In order to improve on this work, virulence conditions for promoting transgene delivery should be investigated, such as the use of chemicals like acetosyringones or physical means like sonication. The results of the research have shown that Biolistic TM bombardment of multiple shoot offered a relatively better way of delivering transgene than Agrobacterium. The results obtained in this research were slightly better than those reported by other researchers who used either the bombardment method (Bonfim et al. 2007), electroporation or polyethylene glycol (PEG) treatment of protoplast (Babaoglu et al. 2000). However, the method that has been developed as a result of this research for multiple shoot bombardment still needs further improvement. This can be achieved by optimizing bombardment conditions such as the rapture disk pressure, concentration of plasmid DNA and the size and type of DNA micro-carrier. 123 In the research conducted, the potential of using the HVA1 transgene to alleviate symptoms of drought in common bean has been demonstrated. However, there was no distinct effectiveness of this gene for salt stress tolerance of transgenic plants when compared to wild type non-transgenic plants. The biochemical pathways for salt and drought stresses are similar, in the sense that common transcription factors and genes are switched on during the occurrence of these stresses. It has been shown, however, that salt related injuries to plants are more complex at a molecular level because many more genes are required to remove the salt toxicity, in addition to the restoration of homeostasis and turgor pressure of the cells (Kassem et al. 2004, Lee et al. 2004, Popelka et al. 2004, Sharma and Lavanya 2002). The four classes of osmotic stress related genes mentioned earlier, which are osmoprotectants, antiporters, aquaporins and chaperones, are required to work in concert in order to bring about an effective drought and salt stress tolerant response. Chaperones like HVA1 are more effective against heat and drought stress than they are to salt stress. This is why there were no observable phenotypic differences between transgenic and non-transgenic plants under salt stress. In order to fully optimize and develop high resistant genotypes of common bean towards drought and salt stresses, the other classes of osmotic stress related genes will need to be incorporated using a pyramiding or stacking scheme of transgenes. To further improve the resilience of the transgenic 124 common bean towards drought, future research needs to explore the genetic engineering of up regulation of drought-related transcription factors such as DREB1A. The research conducted has also demonstrated the potential of using the germin gene which expresses an oxalate oxidase. This gene product confers white mold resistance to common bean by inhibiting the pathogenic effect of S. sclerotiorum. The northern blot analysis conducted on the plants that were transformed with this gene showed very low expression levels. However, ‗Matterhorn‘ showed a relatively higher expression and the fungal bio-assay conducted on the transgenic plants showed that this genotype was able to delay the establishment of the fungus for a period of 72 hours when compared to the non-transformed wild type. However, beyond 72 hours, it was observed that the rate of fungal pathogen spread on the transgenic plants was as fast as the wild type. A possible explanation is that the necrotic tissue that developed on the plant leaf surface, as a result of plants HR cell death caused by the inoculated pathogen, conferred an advantage to this necrotrophic fungus to establish itself. In the previously chapters, it was discussed that HR cell death, despite being a natural defense mechanism for plants against invading pathogens, is only effective against biotrophic pathogens that need to keep the plant tissue alive for their survival. On the other hand, necrotrophs thrive on plants tissue that has died as a result of an HR plant cell death response. In conclusion, the goals and objectives set in this research have been met. Through this research the development of an efficient and reproducible in vitro regeneration protocol 125 for common bean has been achieved. Gene delivery systems for both Agrobacterium and TM Biolistic bombardment have been developed, although they still need further improvement. However dispite having achieved this the relative ease to which the two gene pools (Andean and Middle America) were able to regenerate in vitro and integrate the transgene significantly differed. Andean gene pool still remains more recalcitrant towards genetic engineering than Middle American gene pool. Better methods of manipulating the Andean gene pool to become more amenable towards genetic engineering need to be investigated in future research. The main problem encountered in the transformation systems developed is low expression of transgenes. The reasons why the transgenes had low expression could be due to poor promoter performance, positional effect variegation, methylation of transgenes or some other endogenous silencing activity within the bean genome, such as activation of transposable elements. Future research should replicate the transformation systems that have been developed from this research and investigate the causes for low gene expression. 126 APPENDICES 127 Appendix 1 Table 10: Anova for statistical model The SAS System The GLM Procedure Class Level Information Class Levels Values Genotype: 10 ‗Beluga‘, ‗Jaguar‘, ‗Merlot‘, ‗Montcalm‘, ‗Olathe‘, ―Red hawk‖, ‗Sedona‘, ‗Condor‘, ‗Matterhorn‘, ‗Seahawk‘ cytokinin 9 BAP1, BAP2, BAP3, BAP4, Cytokin0, TDZ1, TDZ2, TDZ3, TDZ4 Auxin 7 Auxin0, IAA1, IAA2, IAA3, NAA1, NAA2, NAA3 Number of Observations Read 1890 Number of Observations Used 1890 The SAS System 04:42 Tuesday, November 11, 2008 2 The GLM Procedure Dependent Variable: Shoots Source Sum of DF Squares Model 629 32766.02169 52.09224 Error 1260 743.33333 0.58995 Corrected Total 1889 33509.35503 Mean Square F Value R-Square Coeff Var Root MSE Shoots Mean 0.977817 17.51745 0.768080 4.384656 128 88.30 Pr > F <.0001 Appendix 2 Table 11:Anova for treatment and interaction of treatments for type i and type ii sum of squares Source DF Type I SS Mean Square F Value Pr > F Genotype 9 3307.873545 367.541505 623.01 Cytokinin 8 5168.397884 646.049735 1095.10 <.0001 Auxin 6 9937.121693 1656.186949 2807.35 <.0001 Genotype*cytokinin 72 2141.655026 29.745209 50.42 <.0001 Genotype*Auxin 54 1852.433862 34.304331 58.15 <.0001 Cytokinin*Auxin 48 4032.268783 84.005600 142.40 <.0001 Genoty*Cytokin*Auxin 432 Source DF Genotype 9 Cytokinin Auxin Genotype*Cytokinin 6326.270899 Type III SS 14.644146 <.0001 24.82 <.0001 Mean Square F Value 3307.873545 367.541505 623.01 <.0001 8 5168.397884 646.049735 1095.10 <.0001 6 9937.121693 1656.186949 2807.35 <.0001 50.42 <.0001 72 2141.655026 29.745209 Pr > F Genotype*Auxin 54 1852.433862 34.304331 58.15 <.0001 Cytokinin*Auxin 48 4032.268783 84.005600 142.40 <.0001 Genoty*Cytokin*Auxin 432 6326.270899 129 14.644146 24.82 <.0001 Appendix 3 Table 12: Least squares mean for the genotypic effect The GLM Procedure Genotype LSMeans Number ‗Beluga‘ 3.2 1 ‗Redhawk‘ 3.1 2 ‗Merlot‘ 5.7 3 ‗Montcalm‘ 3.0 4 ‗Olathe‘ 7.0 5 ‗Jaguar‘ 3.7 6 ‗Sedona‘ 6.0 7 ‗Condor‘ 3.6 8 ‗Matterhorn‘ 4.8 9 ‗Seahawk‘ 3.7 10 130 Appendix 4 Table 13: Separation of means for genotypic effect Least Squares Means for effect Genotype Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: Shoots i/j 1 1 2 3 4 5 6 7 8 9 10 0.2842 <.0001 0.0111 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 2 0.2842 <.0001 0.1409 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 3 <.0001 <.0001 <.0001 <.0001 <.0001 0.0011 <.0001 <.0001 <.0001 4 0.0111 0.1409 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 5 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 6 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.3153 <.0001 0.6393 7 <.0001 <.0001 0.0011 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 8 <.0001 <.0001 <.0001 <.0001 <.0001 0.3153 <.0001 <.0001 0.1409 9 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 10 <.0001 <.0001 <.0001 <.0001 <.0001 0.6393 <.0001 0.1409 <.0001 NOTE: To ensure overall protection level, only probabilities associated with pre-planned comparisons should be used. 131 Appendix 5 Table 14 : Least squares mean for cytokinin effect The GLM Procedure Shoots Cytokinin LSMean Number BAP1 4.8 1 BAP2 6.0 2 BAP3 6.3 3 BAP4 3.3 4 Cytokin0 1.0 5 TDZ1 5.3 6 TDZ2 5.7 7 TDZ3 4.5 8 TDZ4 2.6 9 132 Appendix 6 Table 15: Least squares mean seperation for cytokinin effect Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: Shoots i/j 1 1 2 4 5 6 7 8 9 <.0001 <.0001 <.0001 <.0001 <.0001 0.0012 <.0001 0.0019 <.0001 3 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 2 <.0001 3 <.0001 0.0019 4 <.0001 <.0001 <.0001 5 <.0001 <.0001 <.0001 <.0001 6 <.0001 <.0001 <.0001 <.0001 <.0001 7 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 8 0.0012 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 9 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 NOTE: To ensure overall protection level, only probabilities associated with pre-planned comparisons should be used. 133 Appendix 7 Table 16: Least squares mean for auxin effect The GLM Procedure Auxin Auxin0 Shoots LSMean 1.3 Number 1 IAA1 5.6 2 IAA2 7.4 3 IAA3 2.1 4 NAA1 5.5 5 NAA2 6.7 6 NAA3 2.1 7 134 Appendix 8 Table 17: Least squares mean separation for auxin effect Pr > |t| for H0: LSMean(i)=LSMean(j) Dependent Variable: Shoots i/j 1 3 4 <.0001 1 2 5 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.1045 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 1.0000 <.0001 <.0001 2 <.0001 3 <.0001 <.0001 4 <.0001 <.0001 <.0001 5 <.0001 0.1045 <.0001 <.0001 6 <.0001 <.0001 <.0001 <.0001 <.0001 7 <.0001 <.0001 <.0001 1.0000 <.0001 6 7 <.0001 <.0001 NOTE: To ensure overall protection level, only probabilities associated with pre-planned comparisons should be used. 135 Appendix 9 Table 18: Means for the interaction between cytokinin and auxin Cytokinin Auxin LSMean Number BAP1 Auxin0 1.6 1 BAP1 IAA1 5.8 2 BAP1 IAA2 9.6 3 BAP1 IAA3 2.5 4 BAP1 NAA1 4.9 5 BAP1 NAA2 6.6 6 BAP1 NAA3 2.5 7 BAP2 Auxin0 1.7 8 BAP2 IAA1 6.3 9 BAP2 IAA2 11.6 10 BAP2 IAA3 4.4 11 BAP2 NAA1 6.1 12 BAP2 NAA2 9.1 13 BAP2 NAA3 2.9 14 BAP3 Auxin0 1.2 15 BAP3 IAA1 9.3 16 BAP3 IAA2 9.4 17 BAP3 IAA3 4.2 18 BAP3 NAA1 11.7 19 BAP3 NAA2 6.5 20 BAP3 NAA3 1.7 21 BAP4 Auxin0 0.6 22 136 Table 18: Continued… BAP4 IAA1 4.2 23 BAP4 IAA2 5.8 24 BAP4 IAA3 1.7 25 BAP4 NAA1 3.5 26 BAP4 NAA2 5.4 27 BAP4 NAA3 1.7 28 Cytokin0 Auxin0 1.0 29 Cytokin0 IAA1 1.0 30 Cytokin0 IAA2 1.0 31 Cytokin0 IAA3 0.97 32 Cytokin0 NAA1 1.0 33 Cytokin0 NAA2 1.0 34 Cytokin0 NAA3 1.0 35 TDZ1 Auxin0 1.8 36 TDZ1 IAA1 6.8 37 TDZ1 IAA2 11.5 38 TDZ1 IAA3 1.8 39 TDZ1 NAA1 4.7 40 TDZ1 NAA2 7.4 41 TDZ1 NAA3 2.9 42 TDZ2 Auxin0 1.4 43 TDZ2 IAA1 7.0 44 TDZ2 IAA2 7.1 45 TDZ2 IAA3 1.9 46 TDZ2 NAA1 8.4 47 137 Table 18: Continued… Cytokinin Auxin LSMEAN Number TDZ2 NAA2 11.1 48 TDZ2 NAA3 2.9 49 TDZ3 Auxin0 1.3 50 TDZ3 IAA1 5.8 51 TDZ3 IAA2 7.3 52 TDZ3 IAA3 1.6 53 TDZ3 NAA1 5.6 54 TDZ3 NAA2 7.8 55 TDZ3 NAA3 2.4 56 TDZ4 Auxin0 0.8 57 TDZ4 IAA1 4.0 58 TDZ4 IAA2 3.0 59 TDZ4 IAA3 0.1 60 TDZ4 NAA1 3.6 61 TDZ4 NAA2 5.7 62 TDZ4 NAA3 1.2 63 138 Appendix 10 Table 19: Means of multiple shoots for the interaction between genotype and auxin Genotype Auxin LSMean Number ‗Beluga‘ Auxin0 1.2 1 ‗Beluga‘ IAA1 3.9 2 ‗Beluga‘ IAA2 5.2 3 ‗Beluga‘ IAA3 1.3 4 ‗Beluga‘ NAA1 3.7 5 ‗Beluga‘ NAA2 5.6 6 ‗Beluga‘ NAA3 1.7 7 ‗Jaguar‘ Auxin0 0.9 8 ‗Jaguar‘ IAA1 3.9 9 ‗Jaguar‘ IAA2 4.3 10 ‗Jaguar‘ IAA3 1.5 11 ‗Jaguar‘ NAA1 3.6 12 ‗Jaguar‘ NAA2 5.6 13 ‗Jaguar‘ NAA3 2.1 14 ‗Merlot‘ Auxin0 1.9 15 ‗Merlot‘ IAA1 6.5 16 ‗Merlot‘ IAA2 9.2 17 ‗Merlot‘ IAA3 2.3 18 ‗Merlot‘ NAA1 7.1 19 ‗Merlot‘ NAA2 9.9 20 ‗Merlot‘ NAA3 3.1 21 ‗Montcalm‘ Auxin0 1.0 139 22 Table 19: Continued… ‗Montcalm‘ IAA1 4.2 23 ‗Montcalm‘ IAA2 4.0 24 ‗Montcalm‘ IAA3 0.8 25 ‗Montcalm‘ NAA1 4.6 26 ‗Montcalm‘ NAA2 5.7 27 ‗Montcalm‘ NAA3 0.8 28 ‗Olathe‘ Auxin0 1.7 29 ‗Olathe‘ IAA1 9.2 30 ‗Olathe‘ IAA2 12.0 31 ‗Olathe‘ IAA3 4.4 32 ‗Olathe‘ NAA1 10.0 33 ‗Olathe‘ NAA2 8.2 34 ‗Olathe‘ NAA3 3.3 35 ‗Redhawk‘ Auxin0 0.7 36 ‗Redhawk‘ IAA1 6.6 37 ‗Redhawk‘ IAA2 5.4 38 ‗Redhawk‘ IAA3 0.8 39 ‗Redhawk‘ NAA1 5.2 40 ‗Redhawk‘ NAA2 6.3 41 ‗Redhawk‘ NAA3 1.0 42 ‗Sedona‘ Auxin0 1.8 43 ‗Sedona‘ IAA1 6.6 44 ‗Sedona‘ IAA2 11.5 45 ‗Sedona‘ IAA3 2.7 46 ‗Sedona‘ NAA1 7.6 4 140 Table 19: Continued… Genotype Auxin LSMean Number ‗Sedona‘ NAA2 9.2 48 ‗Sedona‘ NAA3 2.3 49 ‗Condor‘ Auxin0 1.2 50 ‗Condor‘ IAA1 3.8 51 ‗Condor‘ IAA2 5.9 52 ‗Condor‘ IAA3 1.7 53 ‗Condor‘ NAA1 4.2 54 ‗Condor‘ NAA2 5.7 55 ‗Condor‘ NAA3 2.7 56 ‗Matterhorn‘ Auxin0 1.7 57 ‗Matterhorn‘ IAA1 6.0 58 ‗Matterhorn‘ IAA2 9.0 59 ‗Matterhorn‘ IAA3 2.7 60 ‗Matterhorn‘ NAA1 5.2 61 ‗Matterhorn‘ NAA2 6.1 62 ‗Matterhorn‘ NAA3 2.7 63 ‗Seahawk‘ Auxin0 0.6 64 ‗Seahawk‘ IAA1 5.3 65 ‗Seahawk‘ IAA2 7.0 66 ‗Seahawk‘ IAA3 3.1 67 ‗Seahawk‘ NAA1 3.4 68 ‗Seahawk‘ NAA2 5.1 69 ‗Seahawk‘ NAA3 1.7 141 70 Appendix 11 Table 20: Means of multiple shoot for the interaction between genotype and cytokinin Genotype Cytokinin Lsmeans Number ‗Beluga‘ BAP1 4.3 1 ‗Beluga‘ BAP2 4.7 2 ‗Beluga‘ BAP3 5.7 3 ‗Beluga‘ BAP4 0.9 4 ‗Beluga‘ Cytokin0 1.0 5 ‗Beluga‘ TDZ1 3.7 6 ‗Beluga‘ TDZ2 4.5 7 ‗Beluga‘ TDZ3 2.7 8 ‗Beluga‘ TDZ4 1.3 9 ‗Redhawk‘ BAP1 4.0 10 ‗Redhawk‘ BAP2 4.8 11 ‗Redhawk‘ BAP3 3.0 12 ‗Redhawk‘ BAP4 1.0 13 ‗Redhawk‘ Cytokin0 1.0 14 ‗Redhawk‘ TDZ1 3.4 15 ‗Redhawk‘ TDZ2 5.4 16 ‗Redhawk‘ TDZ3 3.6 17 ‗Redhawk‘ TDZ4 1.9 18 ‗Merlot‘ BAP1 142 5.4 19 Table 20: Continued… ‗Merlot‘ BAP2 7.9 20 ‗Merlot‘ BAP3 7.9 21 ‗Merlot‘ BAP4 3.3 22 ‗Merlot‘ Cytokin0 1.0 23 ‗Merlot‘ TDZ1 7.5 24 ‗Merlot‘ TDZ2 8.2 25 ‗Merlot‘ TDZ3 6.6 26 ‗Merlot‘ TDZ4 3.6 27 ‗Montcalm‘ BAP1 1.7 28 ‗Montcalm‘ BAP2 4.3 29 ‗Montcalm‘ BAP3 7.3 30 ‗Montcalm‘ BAP4 2.8 31 ‗Montcalm‘ Cytokin0 1.0 32 ‗Montcalm‘ TDZ1 3.5 33 ‗Montcalm‘ TDZ2 1.9 34 ‗Montcalm‘ TDZ3 2.5 35 ‗Montcalm‘ TDZ4 2.1 36 ‗Olathe‘ BAP1 8.0 37 ‗Olathe‘ BAP2 10.0 38 ‗Olathe‘ BAP3 10.0 39 ‗Olathe‘ BAP4 8.8 40 ‗Olathe‘ Cytokin0 1.0 41 143 Table 20: Continued… ‗Olathe‘ TDZ1 6.6 42 ‗Olathe‘ TDZ2 7.6 43 ‗Olathe‘ TDZ3 7.7 44 ‗Olathe‘ TDZ4 3.2 45 ‘Jaguar’ BAP1 ‘Jaguar‘ BAP2 2.7 46 4.2 47 ‗Jaguar‘ BAP3 7.6 48 ‗Jaguar‘ BAP4 3.0 49 ‗Jaguar‘ Cytokin0 1.0 50 ‗Jaguar‘ TDZ1 5.2 51 4.3 52 ―‗Jaguar‘ TDZ2 ‗Jaguar‘ TDZ3 2.7 53 ‗Jaguar‘ TDZ4 2.6 54 ‗Sedona‘ BAP1 6.2 55 ‗Sedona‘ BAP2 9.0 56 ‗Sedona‘ BAP3 7.1 57 ‗Sedona‘ BAP4 5.0 58 ‗Sedona‘ Cytokin0 1.0 59 ‗Sedona‘ TDZ1 7.6 60 ‗Sedona‘ TDZ2 7.5 61 ‗Sedona‘ TDZ3 7.6 62 ‗Sedona‘ TDZ4 2.7 63 144 Table 20: Continued… ‗Condor‘ BAP1 4.4 64 ‗Condor‘ BAP2 4.1 65 ‗Condor‘ BAP3 3.1 66 ‗Condor‘ BAP4 3.1 67 ‗Condor‘ Cytokin0 1.0 68 ‗Condor‘ TDZ1 4.2 69 ‗Condor‘ TDZ2 ‗Condor‘ ‗Condor‘ 5.0 TDZ3 70 4.3 3.4 TDZ4 71 72 ‗Matterhorn‘ BAP1 6.6 73 ‗Matterhorn‘ BAP2 6.0 74 ‗Matterhorn‘ BAP3 6.3 75 ‗Matterhorn‘ BAP4 3.1 76 ‗Matterhorn‘ Cytokin0 1.0 77 ‗Matterhorn‘ TDZ1 5.9 78 ‗Matterhorn‘ TDZ2 7.1 79 ‗Matterhorn‘ TDZ3 4.3 80 ‗Matterhorn‘ TDZ4 2.9 81 ‗Seahawk‘ BAP1 4.6 82 ‗Seahawk‘ BAP2 5.3 83 ‗Seahawk‘ BAP3 4.5 84 ‗Seahawk‘ BAP4 1.9 85 145 Table 20: Continued… ‗Seahawk‘ Cytokin0 1.0 86 ‗Seahawk‘ TDZ1 5.1 87 ‗Seahawk‘ TDZ2 5.3 88 ‗Seahawk‘ TDZ3 3.4 89 ‗Seahawk‘ TDZ4 2.6 146 90 Appendix 12 Table 21: Anova for rooting experiment Dip*Hormone Effect Sliced By Hormone For Roots Source of Var. Hormone Sum of DF Squares Mean Square F Value Pr > F Hormone0 3 6.000000 2.000000 1.79 0.1555 IAA1 3 0.666667 0.222222 0.20 0.8968 IAA2 3 147.000000 49.000000 43.88 IAA3 3 758.250000 252.750000 226.34 IBA1 3 1.583333 IBA2 3 172.666667 57.555556 51.54 IBA3 3 775.333333 258.444444 231.44 <.0001 0.90 0.4473 0.527778 1.000000 0.47 <.0001 <.0001 0.7022 <.0001 NAA1 3 3.000000 NAA2 3 206.250000 68.750000 61.57 <.0001 NAA3 3 753.583333 251.194444 224.95 <.0001 147 Appendix 13 Table 22: Least squares means for number of roots for dipping effect in IBA IBA Dip LSMEAN Number 10mg/l 9.0 1 1mg/l 5.0 2 5mg/l 3.7 3 Dip0 7.2 4 148 Appendix 14 Table 23: Least squares means for the effect of different levels of hormone concentration in media on the number of roots Hormone LSMean Hormone0 1.7 1 IAA1 2.3 2 IAA2 7.8 3 IAA3 9.4 4 IBA1 2.3 5 IBA2 8.3 6 IBA3 9.7 7 NAA1 2.2 8 NAA2 8.3 9 NAA3 9.3 10 149 Number Appendix 15 Table 24 : Least squares means for the effect of IBA dipping on the length of roots (cm) Dip LSMeans 10mg/l 18.32 1 1mg/l 26.35 2 5mg/l 30.85 3 Dip0 17.15 4 150 Number Appendix 16 Table 25: least squares means for the effect of growth regulators, NAA, IAA and IBA (hormone) on root length (cm) hormone LSMEAN Number Hormone0 9.93 1 IAA1 19.95 2 IAA2 26.66 3 IAA3 27.08 4 IBA1 20.09 5 IBA2 26.80 6 IBA3 27.32 7 NAA1 20.08 8 NAA2 26.63 9 NAA3 27.13 10 151 REFERENCES 152 REFERENCES Abdelwahd R, Hakam N, Labhilili M, Udupa SM (2008) Use of an absorbent and antioxidants to reduce the effect of leached phenolics in in vitro plantlet regeneration of faba bean. African Journal of Biotechnology 7 (8): 997-1002 Acharjee S, Sen D P, Bordoloi S, Kumar S, Sarmah PA, (2003) Agrobacteriummediated genetic transformation of black gram for resistance against pod borers. Bioprospecting of commercially important plants. Proceedings of the national symposium on "Biochemical approaches for utilization and exploitation of commercially important plants", Jorhat, India Ahmed E, Bisztray GYD, Velich I (2002) Plant regeneration from seedling explants of common bean (Phaseolus vulgaris L.). Acta Biologica Szegediensis 46:(3–4):27–28 Akcay UC, Mahmoudian M, Kamci H, Yucel M, Oktem HA (2009) Agrobacterium tumefaciens -mediated genetic transformation of a recalcitrant grain legume, lentil ( Lens culinaris Medik) Plant Cell Rep. 28(3):407-417 Annis SL, Goodwin PH (1997) Recent advances in the molecular genetics of plant cell wall-degrading enzymes in plant pathogenic fungi. Eur. J. Plant Pathol. 103:1–14 Arnaldos TL, Munoz R, Ferrer MA, Calderon AA, (2001) Changes in phenol content during strawberry (Fragaria x ananasa, cv. Chandler) callus culture. Physiol. Plant. 113: 315-322 Apostol I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: role in defense and signal transduction. Plant Physiol 90: 109-116 Aragão FGL, Grossi de Sa MF, Davey MR, Brasiliero ACM, Faria JC, Rech EL (1993) Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device. Plant Cell Rep. 12:483–49 Aragão FJL, Barros LMG, Brasileiro ACM (1996) Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor Appl Genet 93: (1-2) 142-150 Aragão FJL, Ribeiro SG, Barros LMG (1998) Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus. Mol Breeding 4: (6) 491-499 153 Aragão FJL, Barros LMG, de Sousa MV (1999) Expression of a methionine-rich storage albumin from the Brazil nut (Bertholletia excelsa HBK, Lecythidaceae) in transgenic bean plants (Phaseolus vulgaris L., Fabaceae). Genet Mol Biol 22: (3) 445-449 Aragão FJL, Vianna GR, Albino MMC, Rech EL (2002) Transgenic dry bean tolerant to the herbicide glufosinate ammonium. Crop Science 42: (4) 1298-1302 Aragão FJL, Faria JC (2009) First transgenic geminivirus-resistant plant in the field. Nature biotechnology, 7(12):1086-8; Author reply 1088-9 Arellano J, Fuentes SI, Castillo-Espan P, Herna´ndez G (2009) Regeneration of different cultivars of common bean (Phaseolus vulgaris L.) via indirect organogenesis. Plant Cell Tiss Organ Cult, 96:11–1 Babaoglu M, Davey MR, Power JB (2000) Genetic engineering of grain legumes: Key transformation events. AgBiotechNet Vol 2, ABN050. http://www.agbiotechnet.com/reviews/june00/html/Babaoglu.htm Barik DP, Mohapatra U, Chand PK (2005) Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration. Plant Cell Rep. 24: 523-531 Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep. 16: 513–519 Bhalla-Sarin N, Bhomkar P, Debroy S, Sharma N, Saxena M, Upadhyaya CP, Muthusamy A, Pooggin M, Hohn T (2004) Transformation of Vigna mungo (blackgram) for abiotic stress tolerance using marker free approach. http://www.cropscience.org.au/icsc2004/poster/3/8/454_sarin.htm Bhatnagar-Mathur P, Devi J, Lavanya M, Vadez V, Serraj R, Yamaguchi-Shinozaki K, Sharma KK (2007) Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions. Plant Cell Rep. 26: 2071-2082 Blair MW, Caldas GV, Avila P, Lascano C (2006) Tannin content of commercial classes of common bean. Bean Improvement Cooperative Annual Report. 49:151-152 Bolton M D, Bart P, Thomma H J, Berlin D N (2006) Sclerotinia sclerotiorum (Lib.) de Bary: Biology and molecular traits of a cosmopolitan pathogens. Mol. Plant Pathology 7: 1-16 Bonfim K, Faria JC, Nogueira EOPL, Mendes EA, Aragão FJL (2007) RNAi-Mediated Resistance to Bean golden mosaic virus in Genetically Engineered Common Bean (Phaseolus vulgaris) MPMI 20:(6) 717–726 154 Bottinger P, Steinmetz A, Schieder O, Pickardt T (2001) Agrobacterium mediated transformation of Vicia faba. Mol Breed 8: 243–254 Brar G S, Barry C A, Carole V L, Johnson W (1994) Recovery of transgenic peanut (Arachis hypogaea L.) plants from elite cultivars utilizing ACCL technology. The Plant Journal 5: 745-753 Brasileiro ACM, Aragao FJL, Rossi S, Dusi DMA, Barros LMG, Rech EL (1996) Susceptibility of common and tepary beans to Agrobacterium spp. strains and improvement of Agrobacterium mediated transformation using microprojectile bombardment. J Am Soc Hort Sci. 121:810–815 Brinkmann S, Pickardt T, Mahmoodzadeh S, Schieder O, Meixner M (1997) Long time progeny analysis of transgenic Vicia narbonensis lines regarding the coexpression of two foreign genes located on a single t-DNA. Acta Hort. (ISHS) 447:301-312). T Cessna AS, Sears VE, Dickman MB, Low PS (2000) ―Oxalic Acid, a Pathogenicity Factor for Sclerotinia sclerotiorum, Suppresses the Oxidative Burst of the Host Plant.‖ The Plant Cell 12: 2191-2199 chnique Chen R, Tsuda S, Matsui K, Fukuchi-Mizutani M, Ochiai M, Shimizu S, Sakuradani E, Aoki T, Imaizumi R, Ayabe S, Tanaka Y (2005) Production of γ-linolenic acid in Lotus japonicus and Vigna angularis by expression of the Δ6-fatty-acid desaturase gene isolated from Mortierella alpine. Plant Sci. 169:599–605 Cheng M, Jarret RL, Li Z, Xing A, Demski JD (1996) Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens. Plant Cell Rep. 15: 653–657 Chaudhury D, Madanpotra S, Jaiwal R, Saini R, Kumar PA, Jaiwal PK (2006) Agrobacterium tumefaciens mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp) cultivar and transmission of transgenes into progeny. Plant Sci. 172:692–700 Chen SY (2004) Feedback-insensitive anthranilate synthase gene as a novel selectable marker for soybean transformation. Sheng Wu Gong Cheng Bao 20:646–651. Cheng M, Jarret RL, Li Z, Xing A, Demski JD, (1996) Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens. Plant Cell Rep. 15: 653–657 Chowrira GM, Akellan V, Fuerst E, Lurquin PF, (1996) Transgenic grain legumes obtained by in planta electroporation mediated gene transfer. Mol Biotechnol 5:85–96 155 Chowrira GM, Cavileer TD, Gupta SK, Lurquin PF Berger PH (1998) Coat protein mediated resistance to pea enation mosaic virus in transgenic Pisum sativum L. Transgenic Res. 7: 265–271 Christou P (1997) Biotechnology applied to grain legumes. Field Crops Res. 53: 83–97 Clarkson JP, Phelps K, Whipps JM, Young CS, Smith JA, Watling M (2004) Forecasting Sclerotinia disease on lettuce: toward developing a prediction model for carpogenic germination of sclerotia. Phytopathology 94: 268–79 Davies DR, Hamilton J, Mullineaux P (1993) Transformation of peas. Plant Cell Rep. 12: 180-183 Dayal S, Lavanya M, Devi P, Sharma KK (2003) An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.] using leaf explants. Plant Cell Rep. 21:1072–1079 De Clerq J, Zambre M, Van Montagu M, Dillen W, Angenon G (2002) An optimised Agrobacterium-mediated transformation procedure for Phaseolsus acutifolius A Gray (tepary bean). Plant Cell Rep. 21(4):333-340 De Kathen A, Jacobsen HJ (1990) Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors. Plant Cell Rep. 9: 276-279 De Kathen A, Jacobsen HJ (1995) Cell competence for Agrobacterium-mediated DNA transfer into Pisum sativum L. Transgenic Res. 184–191 Delgado-Sanchez P, Saucedo-Ruiz M, Guzman-Maldonado SH, Villordo-Pineda E, Gonzalez-Chavira M, Faire-VelAzquez S, Acosta-Gallegos JA, Mora-Aviles A (2006) An organogenic plant regeneration system for common bean (Phaseolus vulgaris L.). Plant Sci. 170: 822-827 Dillen W, DeClercq J, Goossens A, Van Montagu M, Angenon G (1997) Agrobacteriummediated transformation of Phaseolus acutifolius A Gray. Theor Appl Genet. 94(2): 151158 Dita MA, Rispail N, Prats E, Rubiales D, Singh KB (2006) Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147: 1–24 Dodo H, Konan K,Viquez OM (2005) A genetic engineering strategy to eliminate peanut allergy. Curr. Allergy Asthma Rep. 5:67–73 Dodo W, Konan KN, Chen FC, Egnin M, Viquez OM (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 Biotechnology Journal 6:135–145 156 Donaldson PA, Anderson T, Lane BG, Davidson AL, Simmonds DH (2001) Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotinia sclerotiorum. Physiol Mol Plant Pathol 59: 297–307 Eapen S, Kohler F, Gerdemann M, Schieder O (1987) Cultivar dependence of transformation rates in mothbean after co-cultivation of protoplasts with Agrobacterium tumefaciens. Theor Appl Genet. 75:207–210 Eapen S, George L (1994) Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.). Plant Cell Rep. 13: 582-586 Egnin M, Mora A, Prakash CS (1998) Factor enhancing Agrobacterium tumefaciensmediated gene transfer in peanut (Arachis hypogaea L). In vitro Cel Dev. Biol. Plant 34: 310–318 EL-Shemy H, Khalafalla M, Wakasa K, Ishimoto M (2002) Reproducible transformation in two grain legumes-soybean and azuki bean-using different systems. Cell Mol Biol Lett 7:709–719 Estrada-Navarrete G, Alvarado-Affantranger X, Olivares JE, Guillén G, Díaz-Camino C, Campos F, Quinto C, Gresshoff PM, Fast FS (2007) Efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes. Nature Protocols 2:18191824 Favaron F, Sella L, D‘Ovidio R (2004) Relationships among endopolygalacturonase, oxalate, pH, and plant polygalacturonaseinhibiting protein (PGIP) in the interaction between Sclerotinia sclerotiorum and soybean. Mol. Plant–Microbe Interact. 17:1402– 1409 Ferrar PH, Walker JRL (1993) O-Diphenol oxidase inhibition: an additional role of oxalic acid in the phytopathogenic arsenal of Sclerotinia sclerotiorum and Sclerotium rolfsii. Physiol Mol Plant Pathol. 43: 415–442 Fillipone E (1990) Genetic transformation of pea and cowpea by cocultivation of tissues with Agrobacterium tumefaciens carrying binary vectors. Cowpea Genetic Resources: Contributions in cowpea exploration, evaluation and research from Italy and IITA. IITA, Ibadan. 175-181 Fontana GS, Santini L, Caretto S, Frugis G, Mariotti D (1993) Genetic transformation in the grain legume Cicer arietinum L. (chickpea). Plant Cell Rep. 12: 194–198 Fraissinet-Tachet L, Fèvre M (1996) Regulation by galacturonic acid of pectinolytic enzyme production by Sclerotinia sclerotiorum. Curr. Microbiol. 33:49–53 157 Garcia JA, Hillie J, Goldbach R, (1986a) Transformation of cowpea (Vigna unguiculata) cells with an antibiotic resistance gene using Ti-plasmid derived vectors. Plant Sci. 44: 37-46 Garcia JA, Hillie J, Goldbach R (1986b) Transformation of cowpea (Vigna unguiculata) cells with a full length DNA copy of cowpea mosaic virus mRNA. Plant Sci. 44: 89-98 Geetha N, Venkatachalam P, Laksmisita G (1999) Agrobacterium mediated genetic transformation of pigeonpea (Cajanus cajan L Millsp.) and development of transgenic plants via direct organogenesis. Plant Biotechnol 16:213–218 Godoy G, Steadman JR, Dickman MB, Dam R (1990) Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiological and Molecular Plant Pathology 37: 179–191 Govrin E, Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Current Biology 10:751–757 Grant JE, Cooper PA, McAra AE, Frew TJ (1995) Transformation of pea (Pisum sativum) using immature cotyledons. Plant Cell Rep. 15:254–258 Grant JE, Thomson L, Pither-Joyce MD, Dale TM, Cooper PA (2003) Influence of Agrobacterium tumefaciens strain on the production of transgenic peas (Pisum sativum L). Plant Cell Rep. 2:1207–1210 Gulati A, Schryer P, McHughen A (2002) Production of fertile transgenic lentil (Lens culinaris Medik.) plants using particle bombardment. In vitro Cell Dev. Biol. Plant. 38:316–324 Hanafy M, Pickardt T, Kiesecker H, Jacobsen HJ (2005) Agrobacterium-mediated transformation of faba bean (Vicia faba L) using embryo axes. Euphytica 142:227–326 Hymowitz T (1990) ―Grain legumes‖, Advances in New Crops. Timber Press 54-57 PortlandIkea J, Ingelbrecht I, Uwaifo A Thottapilly G (2003) Stable transformation in cowpea (Vigna unguiculata Walp) using particle gun method. Afr J Biotechnol 2:211– 222 Ignacimuthu S (2000) Agrobacterium mediated transformation of Vigna sequipedalis Koern (asparagus bean). Indian J Exp Biol 38:493–498 Ignacimuthu S, Prakash S (2006) Agrobacterium mediated transformation of chickpea with α-amylase inhibitor gene for insect resistance. J Biosci 31:339–345 Islam R, Malik T, Husnain T, Riazuddin S (1994) Strain and cultivar specificity in the Agrobacterium-chickpea interaction. Plant Cell Rep. 13:561–563 158 Ivo NL, Nascimento CP, Vieira LS, Campos FAP, Aragao FJL (2008) Biolistic-mediated genetic transformation of cowpea (Vigna unguiculata) and stable Mendelian inheritance of transgenes. Plant Cell Rep. 27:1475–1483 Jacobsen H.J (1992) Biotechnology applied to grain legumes - current state and st prospects: Proceedings of 1 European Conference on Grain Legumes, Angers, France. 99-103 Jaiwal PK, Kumari R, Ignacimuthu S, Potrykus I, Sautter C (2001) Agrobacterium tumefaciens-mediated genetic transformation on mungbean (Vigna radiata L.Wilczek) a recalcitrant grain legume. Plant Sci. 161:239–247 Jaiwal PK, Singh RP (2003) Applied Genetics of Leguminosae Biotechnology. Kluwer Academic Publishers, Dordrecht. Jelenic S, Mit PT, Papes D, Jelaska S (2000). Agrobacterium-mediated transformation of broad bean (Vicia faba L.) Food Tech. 38:167-172 Kamble S, Misra HS, Mahajan SK, Eapen S (2003) A protocol for efficient biolistic transformation of mothbean (Vigna aconitifolia L. Marechal). Plant Mol Biol Report 21 :457 Kar S, Johnson TM, Nayak P, Sen SK (1996) Efficient transgenic plant regeneration through Agroabcterium-mediated transformation of chickpea (Cicer arietinum L.). Plant Cell Rep. 16: 32-37 Karchi H, Shaul O, Galili G (1993) Seed specific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco. Plant J. 3:721–727 Kim JW, Minamikawa T (1996) Transformation and regeneration of French bean plant by the particle bombardment process. Plant Sci. 117:131-138 Kiran K, Sharma M, Lavanya V, Anjaiah V ( 2006) Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L. millsp.) expressing the synthetic Bt Cry1Ab gene. In vitro Cell Dev. Biol. Plant 42:165–173 Khalafalla MM, El-shemy HA, Mizanur RS, Teraishi M, Ishimoto M (2005) Recovery of herbicide-resistant Azuki bean (Vigna angularis (wild) ohwi and ohashi). African. J Biotechnol. 4: 61–67 Klomparens KS, Flegler S, Hooper GR (1986) Procedures for transmission and scanning electron microscopy for biological and medical sciences – A laboratory manual, Ladd Research Industries, Burlington, Vt, USA 159 Kohler F, Golz C, Eapen S, Kohn H, Schieder O (1987) Stable transformation of mothbean (Vigna aconitifolia) via direct gene transfer. Plant Cell Rep. 6:313–317 Kohler F, Golz C, Eapen S, Kohn H, Schieder O (1987) Influence of plant cultivar and plasmid DNA on transformation rates in tobacco and mothbean. Plant Sci. Lett. 53:87–91 Kohler F, Cardon G, Pohlmann M, Gill R, Schieder O (1989) Enhancement of transformation rates in higher plants by low-dose irradiation : are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Mol. Biol 12:189-199 Kortt AA, Caldwell JB, Lilley GG, Higgins TJV, (1991) Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L). Eur. J. Biochem. 195: 329–334 Krejci P, Matuskova P, Hanacek P, Reinohl V, Prochazka S (2007) The transformation of pea (Pisum sativum L): Applicable methods of Agrobacterium tumefaciens mediated gene transfer. Acta Physiol. Plant 29:157–163 Krishnamurthy KV, Suhasini K, Sagare AP, Meixner M, de Kathen A, Pickardt T, Schieder O (2000) Agrobacterium-mediated transformation of chickpea (Cicer arietinum L.) embryo axes, Plant Cell Rep. 19:235-240 Kumar SM, Kumar BK, Sharma KK, Devi P (2004) Genetic transformation of pigeonpea with rice chitinase gene. Plant Breed. 123(5):485-489 Kwapata K, Sabzikar R, Sticklen MB, Kelly JD (2009) In vitro regeneration and morphogenesis studies in common bean. Plant Cell Tiss Organ Cult. 100:97–105 Lane BG, Cuming AC, Fregeau J, Carpita C, Hurkman WJ, Bernier F, Dratewka-Kos E, Kennedy TD (1992) Germin isoforms are discrete temporal markers of wheat development: Pseudogermin is a uniquely thermostable water-soluble protein in ungerminated embryos and like germin in germinated embryos it is incorporated into cell walls. Eur. J. Biochem. 209:961–969 Lane BG, Dunwell JM, Ray JA, Schmitt MR, Cuming AC (1993) Germin, a protein marker of early plant development, is an oxlate oxidase. J. Biol. Chem. 268:12239–12242 Lane BG (2002) Oxalate, germins, and higher-plant pathogens. International Union of Biochemistry and Molecular Biology Life 53: 67–75 Lawrence PK, Koundal KR, (2001) Agrobacterium tumefaciens-mediated transformation of pigeonpea (Cajanus cajan L. Millsp.) and molecular analysis of regenerated plants. Curr. Sci. 80 :1428–1432 160 Liu ZC, Park BJ, Kanno A, Kameya T (2005) The novel use of a combination of sonication and vaccum infiltration in Agrobacterium mediated transformation of kidney bean (Phaseolus vulgaris) with lea gene. Mol Breed. 16:189–197 Livingstone DM, Birch RG (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 DM, Hampton JL, Phipps PM, Grabau EA (2005) Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene. Plant Physiol. 137:1354–1362 Mahmoudian M, Yücel M, Öktem HA (2002) Transformation of lentil (Lens culinaris M.) cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens, Plant Mol. Biol. Rep. 20:251–257 Malik KA, Saxena PK (1992) Regeneration in Phaseolus vulgaris L.: High-frequency 6 induction of direct shoot formation in intact seedlings by N -benzylaminopurine and thidiazuron. Planta 186:384-3893; McCabe DE, Christou P (1993) Direct DNA transfer using electric discharge particle acceleration (Accell®) technology. Plant Cell Tissue Organ Culture 33:227–236H McKently AH, Moore GA, Doostdar H, Niedz RP (1995) Arobacterium mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants. Plant Cell Rep. 14:699–703 Metry EA, Ismail RM, Hussien GM, Nasr El-Din TM, El-Itriby HA (2007) Regeneration and microprojectile -mediated transformation in Vicia faba L. Arab J. Biotech. 10 (1):23-36 Mohamed MF, Read PE, Coyne DP (1991) Plant regeneration in vitro from the embryonic axes of common and tepary beans. Annual Report, Bean Improvement Cooperation 34:150-151 Mohan ML, Krishnamurthy KV (2003) Plant Regeneration from Decapitated Mature Embryo Axis and Agrobacterium Mediated Genetic Transformation of Pigeonpea. Biologia Plantarum 46 (4): 519-527 Molvig L, Tabe LM, Eggum BO, Moore AE, Craig S, Spencer D (1997) Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower albumin gene. Proc. Nat. Acad. Sci. USA 94:8393–8398 161 Muehlbauer FJ, Cho S, Sarker A, McPhee KE, Coyne CJ, Rebecca R, Fordey PN (2006) Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress. Euphytica 147:(1-2):149-165 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15: 473-497 R Muruganantham M, Amutha S, Selvaraj N, Vengadesan G, Ganapathi A (2007) Efficient Agrobacterium -mediated transformation of Vigna mungo using immature cotyledonarynode explants and phosphinothricin as the selection agent. In vitro Cell & Dev. Biol Plant 43:550-557 Nadolska-Orczyk A, Orczyk W, (2000) Study of the factors influencing Agrobacteriummediated transformation of pea (Pisum sativum L.). Mol. Breed. 6:185-194 Nauerby B, Madsen M, Christiansen J, Wyndaele R (1991) A rapid and efficient regeneration system for pea (Pisum sativum), suitable for transformation. Plant Cell Rep. 9:676-679 Obembe OO (2009) Exciting times for Cowpea genetic transformation research. Australian Journal of Basic and Applied Sciences 3(2):1083-1086 Ozyigit I I (2008) Phenolic changes during in vitro organogenesis of cotton (Gossypium hirsutum L.) shoot tips. African J. of Biotech. 7(8):1145–1150 Pal M, Ghosh U, Chandra M, Pal A, Biswas BB (1991) Transformation and regeneration of mungbean (Vigna radiata). Indian J. Biochem. Biophys. 28:(5–6):449–455 Perkins EJ, Stiff CM, Lurquin PF (1987) Use of alcaligenes eutrophus as a source of genes for 2,4,-D resistance in plants. Weed Sci. 35:12-18 Perrin Y, Vaquero C, Gerrard I, Sack M, Drossard J, Stöger E, (2000) Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy. Mol Breed. 6:345–352 Pickardt T, Meixner M, Schade V, Scheider O (1991) Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer. Plant Cell Rep. 9:535-538. Pigeaire A, Abernethy D, Smith PM, Simpson K, Fletcher N, Lu C.Y (1997) Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium mediated gene transfer to shoot apices. Mol. Breed. 3:341–349 Pniewski Tomasz, Józef Kapusta (2005) Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains. J. Appl. Genet. 46(2):139-147 162 Polowick PL, Baliski DS, Mahon JD (2004) Agrobacterium tumefaciens— mediated transformation of chickpea (Cicer arietinum L.): gene integration, expression and inheritance. Plant Cell Rep. 23:485–491 Popelka JC, Terryn N, Higgins TJV (2004) Gene technology for grain legumes: Can it contribute to the food challenge in developing countries? Plant Sci. 167:195–206 Popelka J.C, Gollasch S, Moore A, Molvig L, Higgins T.J.V (2006) Genetic transformation of cowpea (Vigna unguiculata L.) and stable transmission of the transgene to progeny. Plant Cell Rep. 25:304–312 Popelka JC, Higgins TJV (2007) Biotechnology in Agriculture and Forestry. vol. 59 Transgenic crop IV (ed. By E.C. Pau and M.R. Davey) Pounti-Kaerlas J, Eriksson T, Engstrom P (1990) Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens-mediated gene transfer. Theor. Appl.Genet. 80: 246-252 Prasad V, Satyavathi VV, Sanjaya Valli KM, Khandelwal A, Shaila MS, Sita GL (2004) Expression of biologically active hemagglutinin-neuraminadase protein of peste des petits ruminants virus in transgenic pigeonpea(Cajanus cajan (L) Millsp). Plant Sci. 166:199–205 Ramsay G, Kumar A (1990) Transformation of Vicia faba cotyledon and stem tissues by Agrobacterium rhizogenes: infectivity and cytological studies. J. Exp. Bot. 41: 841–847 Ravindra MB, Nataraja K (2007) Agrobacterium tumefaciens-Mediated genetic transformation of Vigna aconitifolia and stable transmission of the genes to somatic seedlings. Int. J. Agric. Res. 2:450-458 Rech EL, Vainstein MH, Davey MR (1991) An electrical particle acceleration gun for gene transfer into cells. Technique 3:143- 149. Richter A, Jacobson HJ, de Kathen A, de Lorenzo G, Briviba K, Hain R (2007) Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera). Plant Cell Rep. 25:1166–1173 Riou C, Freyssinet G, Fèvre M (1992) Purification and characterization of extracellular pectinolytic enzymes produced by Sclerotinia sclerotiorum. Appl. Environ. Microbiol. 58: 578–583 Rolletschek H, Borisjuk L, Radchuk R, Miranda M, Heim U, Weber H (2004) Seedspecific expression of a bacterial phosphoenolpyruvate carboxylase in Vicia narbonensis increases protein content and improves carbon economy. Plant Biotechnol. J. 2:211–220 163 Rolletschek H, Hosein F, Miranda M, Heim U, Götz KP, Schlereth A, Borisjuk L, Saalbach I, Wobus U, Weber H (2005) Ectopic expression of an amino acid transporter (VfAAP1) in seeds of Vicia narbonensis and Pisum sativum increases storage proteins. Plant Physiol. 137:1236–1249 Russell DR, Wallece KM, Bathe JH, Martinell BJ, McCabe DE (1993) Stable transformation of Phaseolus vulgaris L. via electric-discharge mediated particle acceleration. Plant Cell Rep. 12: 165-169 Saalbach I, Pickardt T, Machemeh LF, Saalbach G, Schieder O, Muntz K (1994) A chimeric gene encoding the methionine rich 2S albumin of Brazilnut (Berthlletia excelsa) is stably expressed and inherited in transgenic grain legumes. Mol. Gen. Genet. 242:226– 236 Saini R, Jaiwal PK (2005) Transformation of a recalcitrant grain legume, Vigna mungo L. Hepper using Agrobacterium tumefaciens—mediated gene transfer to shoot apical meristem cultures. Plant Cell Rep. 24:164–171 Saini R, Jaiwal PK (2007) Agrobacterium tumefaciens—mediated transformation of blackgram: An assessment of factors influencing the efficiency of uidA gene transfer. Biol. Plant 51:69–74 Saini R, Jaiwal S, Jaiwal PK (2003) Stable genetic transformation of Vigna mungo L. Hepper via Agrobacterium tumefaciens. Plant Cell Rep. 21:851–859 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual. 2nd ed. N.Y., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, pp. 1659 . ISBN 0-87969-309-6 Santalla M, Power JB, Davey MR (1998) Efficient in vitro shoot regeneration responses of Phaseolus vulgaris and P. coccineus. Euphytica 102:195-202 Sanyal I, Singh AK, Amla DV (2003) Agrobacterium tumefaciens mediated transformation of chickpea (Cicer arietinum L.) using mature embryogenic axis and cotyledonary nodes. Indian J. Biotechnol. 2:524–532 Sanyal I, Singh AK, Kaushik M, Amla DV (2005) Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) with Bacillus thuringiensis cryIAc gene for resistance against pod borer insect Helicoverpa armigera. Plant Sci. 168:1135–1146 Sarkar RH, Biswas A, Mustafa BM, Mahbub S, Haque MI (2003) Agrobacterium mediated transformation of Lentil (Lens culinaris Medik). Plant Tissue Cult. 13(1):1–12 Sarker A, Erskine W (2006) Recent progress in the ancient lentil. J.Agric. Sci. 144:19-29 164 Sarmah BK, Moore A, Tate W, Morvig L, Morton RL, Rees RP (2004) Transgenic chickpea seeds expressing high levels of a bean α-amylase inhibitor. Mol. Breed. 14:73– 82 Satyavathi V, Prasad V, Khandelwal A, Shaila MS, LakshmiSita G (2003) Expression of haemaglutinin protein of Rinderpest virus in transgenic pigeon pea (Cajanus cajan L. Millsp) plants. Plant Cell Rep. 21: 652-658 Saxena K. B. (2008) Genetic improvement of Pigeon pea – A Review. Tropical Plant Biology 1:159-178 Schroeder HE, Schotz AH, Wardley-Richardson T, Spencer D, Higgins TJV (1993) Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol. 101: 751-757 Schroeder HE, Gollasch S, Moore A, Tabe LM, Craig S, Hardie DC, Chrispeels MJ, Spencer D, Higgins TJV (1995) Bean alpha-amylase confers resistance to the pea weevil (Bruchus pisorum) in transgenic peas (Pisum sativum L.). Plant Physiol. 107:1233-1239 Senthil G, Williamson B, Dinkin RD, Ramsay G (2004) An efficient transformation system for chickpea. Plant Cell Rep. 23(5):297–303 Sharma KK, Lavanya V (2002) Recent developments in transgenics for abiotic stress in legumes of the semi-arid tropics. JIRCAS Working Report 61–73 Sharma KK, Lavanya M, Anjaiah V (2006) Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L Millsp) expressing synthetic Bt cryIAb gene. In vitro Cell Dev. Biol. Plant 42:165–173 Shaul O, Galili G, (1992) Threonine overproduction in transgenic tobacco plants expressing a mutant desensitized aspartate kinase from Escherichia coli. Plant Physiol. 100:1157–1163 Shivani I, Misra HS, Eapen S (2007) Genetic transformation of chickpea (Cicer arietinum L) with insecticidal crystal protein gene using particle gun bombardment. Plant Cell Rep. 26:755–763 Singh SP (2001) Broadening the genetic base of common bean cultivars: A Review. Crop Sci. 41:1659-1675 Singh S P (2007) Drought Resistance in the Race Durango Dry Bean Landraces and Cultivars. Agron. J. 99:1219–1225 Singh ND, Sahoo L, Saini R, Sarin NB, Jaiwal PK (2004) In vitro regeneration and recovey of primary transformants from shoot apices of pigeonpea using Agrobacterium tumefaciens. Physiol. Mol. Biol. Plants 10: 65–74 165 Singsit C, Adang MJ, Lynch RE, Anderson WF, Wang A, Cardineau G, Ozias-Akins P (1997) Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer. Transgenic Res. 6:169–176 Solleti SK, Bakshi S, Purkayastha J, Panda SK, Sahoo L (2008a) Transgenic cowpea (Vigna unguiculata) seeds expressing a bean a-amylase inhibitor-1 confer resistance to storage pests, bruchid beetles. Plant Cell Rep. 27:1841–1850 Solleti SK, Bakshi S, Sahoo L, (2008b) Additional virulence genes in conjunction with efficient selection scheme, and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation. Journal of Biotechnology 135: 97-104 Somers DA, Samac DA, Olhoft PM (2003) Recent advances in legume transformation. Plant Physiol. 131:892–899 Sonia, Saini R, Singh RP, Jaiwal PK (2007) Agrobacterium mediated transfer of Phaseolus vulgaris α-amylase inhibitor-1 gene into mungbean (Vigna radiata (L.) Wilczek) using bar as selectable marker. Plant Cell Rep. 26:187–198 Srinivasan M, Gupta N, Chopra VL (1988) Agrobacterium-mediated transformation of chickpea. Int Chickpea News 19:2–3 Srinivasan M, Mohapatra T, Sharma RP (1991) Agrobacterium mediated genetic transformation of chickpea Cicer arietinum. Indian J. Exp. Biol. 29:758–761 Steadman JR (1983) White mold:a serious yield-limiting disease of bean. Plant Dis. 67:346–350 Sticklen MB, Oraby HF (2005) Shoot apical meristem: a sustainable explant for genetic transformation of cereal crops. In vitro Cell Dev. Biol. Plant 41:187–200 Suraninpong P, Chanprame S, Cho Hyeon-Je, Widholm JM, Waranyuwat A (2004) Agrobacterium-mediated Transformation of Mungbean (Vigna radiata (L.) Wilczek). Walailak J. Sci. & Tech. 1(2):38-48 Surekha CH, Beena MR, Arundhati A, Singh PK, Tuli R, Dutta-Gupta A, Kirti PB (2005) Agrobacterium-mediated genetic transformation of pigeon pea (Cajanus cajan (L.) Millsp.) using embryonal segments and development of transgenic plant for resistance against Spodoptera. Plant Sci. 169:1074-1080 Surekha CH, Arundhanti A, Seshagiri Rao G (2007) Diffrential response of Cajanus cajan varieties to transformation with different strains of Agrobacterium. J. Biol. Sci. 7:176–181 166 Svabova L, Smykal P, Griga M, Ondrej V (2005) Agrobacterium-mediated transformation of Pisum sativum in vitro and in vivo. Biol. Plant 49:361–370 Tazeen S, Mirza B (2004) Factors affecting Agrobacterium tumefaciens mediated genetic transformation of Vigna radiata L. Wilczek. Pak. J. Bot. 36:887–896 Tewari-Singh N, Sen J, Kiesecker H, Reddy VS, Jacobsen HJ, Guha-Mukherjee S (2004) Use of a herbicide or lysine + threonine for non-antibiotic selection of transgenic chickpea. Plant Cell Rep 22:576–583 Thomma BPHJ, Penninck IAMA, Broekaert WF, Cammue BPA (2001) The complexity of disease signaling in Arabidopsis. Curr. Opin. Immunol. 13: 63–68 Timmerman-Vaughan GM, Pither-Joyce MD, Cooper PA, Russell AC, Goulden DS, Butler R, Grant JE (2001) Partial Resistance of Transgenic Peas to Alfalfa Mosaic Virus under Greenhouse and Field Conditions. Crop Sci. 41:846-853 Torisky RS, Kovacs L, Avdiushko S, Newman JD, Hunt AG, Collins GB (1997) Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens Strain Chry5. Plant Cell Rep. 17:102–108 Thu TT, Mai TTX, Dewaele E, Farsi S, Tadesse Y, Angenen G, Jacobs M (2003) In vitro regeneration and transformation of pigeon pea (Cajanus cajan L.) Millsp.). Mol. Breed. 11:159-168 Veltcheva M, Svetleva D, Petkova S, Perl A (2005) In vitro regeneration and genetic transformation of common bean (Phaseolus vulgaris L.):Problems and progress. Sci. Hortic. 107:2–10 Verma AK, Chand L (2005) Agrobacterium-mediated transformation of pigeon pea (Cajanus cajan L.) with uidA and cryIA(b) genes. Physiol. Mol. Biol. Plants 11:99–109 Vianna GR, Albino MMC, Dias BBA, SilvaL M, Rech E L, and Aragão FJL (2004) Fragment DNA as vector for genetic transformation of bean (Phaseolus vulgaris L.). Sci. Hortic. 99:371-378 Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity and extreme temperatures:towards genetic engineering for stress tolerance. Planta 218: 1–14 Weber H, Golombek S, Heim U, Rolletschek H, Gubatz S, Wobus U (2000) Antisenseinhibition of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation. Plant J. 24:33–43 167 Wisniewski JP, Brewin NJ (2000) Construction of transgenic pea lines with modified expression of diamine oxidase and modified nodulation responses with exogenous putrescine. MPMI 13(9):922–928 Xiong L, Shumaker K.S, Zhu J.K (2002) Cell signaling during cold, drought and salt stresses. Plant Cell 14:165-183 Yamada T, Teraishi M, Hattori K, Ishimoto M (2001) Transformation of azuki bean (Vigna angularis Willd. Ohwi and Ohashi) by Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult. 64:47–54 Yamada T, Moriyama R, Hattori K, Ishimoto M (2005) Isolation of two α-amylase inhibitor genes of tepary bean (Phaseolus acutifolius A. Gray) and their functional characterization in genetically engineered azuki bean. Plant Sci. 169:502–511 Yan Y, Huishen Y, Guocai L, Weijuan G, Hongmei J, Hongiu C, Mingchun J (2010) Expression of human cytomegalovirus pp150 gene in transgenic (Vicia faba L.) and immunogenicity of pp150 protein in mice. Biologicals 38: 265-272 Yang H, Singsit C, Wang A, Gonsalves D, Ozias-Akins P (1998) Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression. Plant Cell Rep. 17: 693–699 Zambre MA, De Clercq J, Vranova E, Van Montagu M, Angenon G, Dillen W (1998) Plant regeneration from embryo-derived callus in Phaseolus vulgaris L. (common bean) and P. acutifolius A. Gray (tepary bean). Plant Cell Rep. 17:626-630 Zambre MA, Geerts P, Maqeut A, Van Montagu M, Dillen W, Angenon G (2001) Regeneration of fertile plants from callus in Phaseolus polyanthus Greenman (Year Bean). Annals of Botany 88:371-377 Zambre M, Terryn N, De Clercq J, De Buck S, Dillen W, Van Montagu M, Van Der Straeten D, Angenon G (2002) Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells. Planta 216: 580-586 Zambre M, Goossens A, Cardona C, van Montagu M, Terryn N, Angenon G (2005) A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil. Theor Appl Genet 110:914–924 Zhu JK (2002) Salt and drought stress signal transduction in plants. Annual Rev. Plant Biol. 53:247-273 Zuppini A, Navazio L, Sella L, Castiglioni C, Favaron F, Mariani P (2005) An endopolygalacturonase from S. sclerotiorum induces calcium-mediated signaling and programmed cell death in soybean cells. Mol. Plant–Microbe Interact. 18: 849–855 168