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1 IN VI R TO AND IN VIVO EVALUATION OF Arachis paraguariensis AND A. glabrata GERMPLASM By OLUBUNMI OLUFUNBI AINA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQU IREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011
2 2011 Olubunmi O. Aina
3 To my late father Isaac O. Ajani
4 ACKNOWLEDGMENTS It is a great privilege to be mentored and advised by such a knowledgeable and experienc ed professor like Dr Kenneth H. Quesenberry I sincerely thank him for the opportunity to join his team as well as for his patience and support. His desire to see his student succeed in every aspect of life is worthy of acknowledgment and emulation I w o uld like to express my gratitude to a ll the members of my supervisory committee Drs. Mike Kane, Barry Tillman Maria Gallo, and Yoana Newman for giving me so much support, reassurance and inspiration throughout this project. I am also thankful to Dr Fre dy Altpeter whose assistance always exce eded expectation The staff at the University of Florida College of Medicine Ele ctron Microscopy Core Facility is acknowledged for their assistance with the histological studies. I am thankful to my past and present lab members as well as Judy Dampier, Gearry Durden Justin McKinney and Jim Boyer for their help with the field evaluation aspect of this study. I am thankful to April Bensa and their entire family as well as the families of other soccer mom s who hav e invite d my son to their home for sleepovers when I need ed to work overnight on this dissertation. Kathy, Rebecca, Iris, Christiana, Christine, Lena, Dorothy, Cheryl, and their wonderful families are greatly acknowledged for all their help and support in various ways. I thank my mo ther a nd my brothers, Femi and Bode for supporting all my academic efforts especially for their financial inputs. Lastly, I am thankful to my precious little son, Peace for good behavior and cooperation throughout my studies.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Hypotheses ................................ ................................ ................................ ............. 17 Objectives ................................ ................................ ................................ ............... 17 Rationale ................................ ................................ ................................ ................. 18 2 LITERATURE REVIEW ................................ ................................ .......................... 19 The Genus Arachis ................................ ................................ ................................ 19 Origin and Distribution ................................ ................................ ...................... 19 Reproductive Biology ................................ ................................ ....................... 21 Wild Arachis in Forage Systems ................................ ................................ ............. 22 The Uniqueness of Rhizoma Perennial Pe anut ................................ ................ 22 Limitations to Genetic Improvement of Rhizoma Perennial Peanut .................. 23 Wild Arachis in Gene Introgression ................................ ................................ ......... 23 Potential Uses of Arachis paraguariensis ................................ ......................... 24 Hybridization Barriers ................................ ................................ ....................... 25 Introgress ion Pathways ................................ ................................ .................... 25 In Vitro Tissue Culture of Wild Arachis ................................ ................................ .... 27 Morphogenic Pathways ................................ ................................ .................... 27 Signal Transduction ................................ ................................ .......................... 30 The Challenges of In Vitro Tissue Culture of Wild Arachis ............................... 34 In Vitro Ploidy Manipulat ion ................................ ................................ .............. 36 Ploidy Detection ................................ ................................ ............................... 37 3 OPTIMIZING IN VITRO REGENERATION CONDITIONS FOR Arachis paraguariensis ................................ ................................ ................................ ........ 39 Materials and Methods ................................ ................................ ............................ 41 Explant Source and Sterilization ................................ ................................ ....... 41 Experimental Design and Data Collection ................................ ........................ 42 Statistical Analysis ................................ ................................ ............................ 43
6 Histological Analysis ................................ ................................ ......................... 44 Tissue Culture Regeneration ................................ ................................ ............ 44 Experiment I ................................ ................................ ............................... 45 Experiment II ................................ ................................ .............................. 45 In Vitro Rooting ................................ ................................ ................................ 45 In Vitro Flowering ................................ ................................ ............................. 46 In Vivo Evaluation ................................ ................................ ............................. 47 Results ................................ ................................ ................................ .................... 48 Experiment I The Role of 2,4 D in Induction of Embryogenesis ..................... 48 Experiment II The Role of TDZ in Induction of Emb ryogenesis and Organogenesis ................................ ................................ .............................. 49 Rooting and Post Acclimatization Survival of In Vitro derived Plantlets ........... 50 The Effect of Photope riod on In Vitro Flowering ................................ ............... 52 Performance of Plantlets in the Greenhouse and in the Field .......................... 53 Discussion ................................ ................................ ................................ .............. 54 4 IN VITRO INDUCTION OF TETRAPLOIDY IN Arachis paraguariensis .................. 69 Materials and Methods ................................ ................................ ............................ 70 Plant Materials ................................ ................................ ................................ .. 70 Tissue Culture Initiation and Establishment ................................ ...................... 71 Culture Condition ................................ ................................ .............................. 72 Ploidy Determination by Flow Cytometry ................................ .......................... 72 Morphological and Fertility Observations ................................ .......................... 73 Experimental D esign, Data Collection and Analysis ................................ ......... 73 Results ................................ ................................ ................................ .................... 74 Tetraploid Induction by Colchicine Application to Quartered Seeds and Shoot Ti ps ................................ ................................ ................................ ..... 74 Regeneration of Mixoploids from Colchicine Treated Callus ............................ 75 Morphology, Fertility and Survival of Induced Tetraploid and Mixoploid ........... 76 Discussion ................................ ................................ ................................ .............. 77 5 SEED PRODUCTION IN A. glabrata AND TISSUE CULTURE REGENERATION FROM THE SEED DERIVED EXPLANTS ................................ 84 Materials and Methods ................................ ................................ ............................ 86 Seed Production ................................ ................................ ............................... 86 Seed Quality Evaluatio n ................................ ................................ ................... 86 Tissue Culture Regeneration ................................ ................................ ............ 87 Statistical Analysis ................................ ................................ ............................ 89 Resu lts ................................ ................................ ................................ .................... 89 Seed Production ................................ ................................ ............................... 89 Seed Quality Evaluation ................................ ................................ ................... 90 Tissue Cult ure Regeneration ................................ ................................ ............ 91 Discussion ................................ ................................ ................................ .............. 92 Concluding Remarks ................................ ................................ ............................... 95
7 APPENDIX: PROCEDURE FOR PLASTIC (GLYCOL METHACRYLATE BASED) EMBEDDING ................................ ................................ ................................ .......... 99 LIST OF REFERENCES ................................ ................................ ............................. 102 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 115
8 LIST OF TABLES Table page 3 1 The identity and geographical origin of the six genotypes of A. paraguariensis used for the study. ................................ ................................ .............................. 66 3 2 The influence of 2,4 D and BAP concentrations and combinations on tissue culture regeneration from deembryonated cotyledon explant of A. paraguariensis. ................................ ................................ ................................ ... 66 3 3 The effects of TDZ and BAP concentrations and combinations on shoot regeneration across all the six genotypes. ................................ ......................... 67 3 4 The effects of TDZ and 2ip concentrations and combinations on tis sue culture regeneration across all the six genotypes. ................................ .............. 67 3 5 Root formation as affected by auxin type and concentration after 6 weeks of culture in PETG vessel. ................................ ................................ ...................... 68 4 1 The induction of polyploidy from different explants of A. paraguariensis. ........... 82 5 1 Percent germination and viability of rhizoma perennial peanut obtained fr om four different tests. ................................ ................................ .............................. 96 5 2 Effect of combination of different cytokinins on callus formation and shoot regeneration from quartered seed explant of rhizoma perennial peanut. ........... 96
9 LIST OF FIGURES Figure page 3 1 In vitro regeneration of A. paraguariensis via non zygotic embryogenesis and organogenesis. ................................ ................................ ................................ ... 59 3 2 In vitro rooting of A. paraguariensis as affected by auxin and culture vessel treatments. ................................ ................................ ................................ .......... 60 3 3 In vitro flowering, peg formation and ex vitro seed formation in A. paraguariensis ................................ ................................ ................................ ... 61 3 4 The influence of culture vessel type on the rooting efficiency across six genotypes of A. paraguariensis ................................ ................................ ......... 62 3 5 The effect of culture vessel type on the root characteristics of in vitro derived plantlets. ................................ ................................ ................................ ............. 62 3 6 Ex vitro survival of rooted shoots in response to various auxin treatments at 6 w k after culture initiation in PETG vessel.. ................................ ......................... 63 3 7 The rooting response and survival of six genotypes of A. paraguariensis across all the auxin t reatments inside a PETG vessel. ................................ ....... 63 3 8 The time to initiate flower buds in vitro across five genotypes of A. paraguariensis as affected by photoperiod ................................ ......................... 64 3 9 In vitro flowering r esponse of five genotypes of A. paraguariensis .................... 64 3 10 Leaf spot disease scores for two genotypes of A. paraguariensis and a cultivated peanu t susceptible variety Florunner ................................ .................. 65 3 11 Seed yield of in vitro derived plantlets and germinated seedlings of two genotypes of A. paraguariensis inside pot in the greenhouse and in the field.. .. 65 4 1 The effect of colchicine concentration and treatment duration on timing of in vitro shoot formation from quartered seed explants of A. paraguariensis .......... 79 4 2 Effect of colchic ine concentration and treatment duration on tetraploid induction and quartered seed explants viability in A. paraguariensis ................. 79 4 3 Representative flow cytometric analysis showing DNA histograms of A. paraguariensis ................................ ................................ ................................ .... 80 4 4 Imprints of A. paraguariensis leaf showing trichomes ................................ ......... 81 4 5 Abaxial surface of upper leaf from acclim atized diploid and tetraploid plants showing increased density of trichomes on the tetraploid. ................................ 81
10 4 6 Leaf characteristics of diploid and tetraploid A. paraguariensis ......................... 82 5 1 Vegetative and reproductive growth of 2 rhizoma perennial peanut cultivars from 2009 to 2010. ................................ ................................ ............................. 96 5 2 Seed weight and yield of RPP in fall 2009 and 2 010. ................................ ......... 96 5 3 Seed production and tissue culture regeneration from seed derived explant of RPP.. ................................ ................................ ................................ .............. 97
11 LIST OF ABBREVIATION S 2ip 6 ( Dimethylallylamino ) purine AFLP Amplified fragment l ength p olymorphism ANOVA A nalysis of variance ARF Auxin Response Factors Aux/IAA Auxin/ Indole 3 acetic acid BAP 6 benzylaminopurine cGMP Cyclic Guanosine Monophosphate CIM callus induction medium DNA Deoxyribonucleic acid EST E x pressed sequence tag IBA Indole 3 butyric acid ICRISAT International Crops Research Institute for the Semi Arid Tropics ITS I n ternal transcribed spacers MS Murashige and Skoog N 2 O Nitrous Oxide NAA N aphthalene acetic acid PETG Polyethylene Terephthalate Gl ycol rDNA Ribosomal deoxyribonucleic acid RFLP Restriction fragment length polymorphism SRAP Sequence related amplified polymorphism SSR Simple Sequence Repeat TDZ T hidiazuron TIR1 Transport Inhibitor Response Protein 1
12 Abstract of Dissertation Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IN VIRTO AND IN VIVO EVALUATION OF Arachis paraguariensis AND A. glabrata GERMPLASM By Olubunmi Olufun bi Aina M ay 2011 Chair: Kenneth H. Quesenberry Major: Agronomy Apart from p o ssessing important traits needed for improvement of the cultivated peanut ( A rachis hypogaea L.), wild Arachis species are grown for forage and ornamental purposes. In the first p hase of this research a series of in vitro experiments led to the development of high frequency regeneration procedure s for A rachis paraguariensis Chodat and Hassl and A rachis glabrata Benth. Both auxin type and concentration in the culture medium had s ignificant influence on root formation although application of auxin may not always be necessary for rooting, it is essential for early emergence of root primordia. Plantlets that were allowed to form roots on medium lacking auxin showed significantly enha nced plantlet vigor and root length, even though root emergence was delayed. For A. glabrata the frequency of regeneration was improved along with reduced pro duction of phenolic compounds when 6 benzylaminopurine (BAP) was substituted with 6 (Dimethylallylamino) puri ne (2ip) in the culture medium. The most suitable regeneration procedure was then combined with the antimitotic agent colchicine to induce chro mosome doubling of diploid A. paraguariensis The induced autotetraploids from A. paraguariensis had fewer stomata, but more hair like
13 trichomes per unit leaf area compared to their diploid precursor. The best results in which 39% and 43% of the explants p roduced tetraploid plants resulted from treatment with 0.5% colchicine for 4 h and 8 h, respectively. Treating explants with high concentrations of colchicine for 24 h proved to be very lethal. In the second phase, in vivo evaluations of A. paraguariensis and A. glabrata germplasm with specific emphasis on forage yield, disease resistance and seed production were conducted. In vivo evaluation of A. glabrata led to the discovery of the Additionally, the result from field evaluation of two genotypes of A. paraguariensis confirmed higher resistance to C. arachidicola leaf spot than cultivated peanut. The findings from this study should contribute towards genetic improvement of the wild Ar achis species as well as the enhancement of gene introgression into cultivated peanut.
14 CHAPTER 1 INTRODUCTION Genetic variation describes the variation of alleles which exist among the total genetic loci of a population. On the other hand, genetic var iability measures the tendency of individual genetic traits in a population to vary from one another (Sleper and Poehlman, 2006; Smith, 1977). Generally, most efforts of plant breeders are tailored toward the evaluation and preservation of existing variabi lity as well the creation of new genetic variation. The diversity existing within the genus Arachis is enormous as observed by the large differences in genome size, botanical characters, and important agronomic traits (Singh et al. 1998 a ). In addition, wi de intraspecific variation has been reported between and within several accessions (N bile et al. 2004; Herselman, 2003 ; Upadhyaya et al., 2011 ) Several breeding approaches including sexual hybridization, ploidy manipulation, somatic hybridization, genet ic transformation and natural/ induced mutation have been exploited as important means of producing new genetic variation within crop species (Sleper and Poehlman, 2006). While sexual hybridization is mostly applicable to the closely related species that e xhibit complete chromosome pairing, the technique of peg or embryo culture may be applied to eliminate partial sterility of F 2 hybrids that are derived from more distant species. Other techniques such as in vitro fertilization and somatic hybridization are mostly explored in marginally sexually compatible species with severe F 1 sterility. Furthermore, gene transfer via transgenic methods is currently the only feasible approach for species in the quaternary gene pool (Simpson, 2001 ; Upadhyaya et al., 2011 )
15 Currently both conventional and molecular plant breeding methods are increasingly relying on in vitro tissue culture as an important tool that provides strategic opportunities for overcoming hybridization barriers ( Loberant and Altman 2010 ). For instance results from in vitro selection for peanut cell lines with resistance to Cercosporidium personatum the causual agent of late leaf spot, proved that tissue culture technique s could be combined with field evaluations to develop disease resistant cultivars Development of pest and disease resistant cultivars based on this approach can lead to a reduction in cost, time, space, labor and other resources (Venkatachalam et al. 1998). Other studies have also demonstrated that embryo and peg tip culture may be v ery useful for rescuing embryos of hybrids derived from interspecific crosses of A. hypogaea and other wild species including A. villosa Benth. (Bajaj et al. 1982), A. duranensis Krapov. and W.C. Gregory, A. batizocoi Krapov. and W.C. Gregory, and A. vali da Krapov. and W.C. Gregory (Feng et al. 1996). Although, s omatic hybridization is yet to be successfully applied to the breeding of Arachis species, high frequency of plant regeneration from protoplasts of A. paraguariensis Chodat and Hassl w as reported by Li et al. (1993). Additionally, several methods utilizing in vitro tissue culture regeneration have been described for the production of transgenic peanut plants (Bhatnagar et al. 2010; Anuradha et al. 2006; Athmaram et al. 2006). Al though haploid pl ants have not been derived from any of the Arachis species, results from some studies (Bajaj et al. 1981; Croser et al. 2006) indicate that anther culture is a promising tool for the development of haploid plants in this genus. Likewise, in vitro inducti on of tetraploidy is another vital technique that could broaden the available gene
16 pool as well as eliminat e hybridization barriers that are due to differences in ploidy levels. This study presents results from detailed in vitro and in vivo evaluations of two important wild Arachis species ; A. glabrata and A. paraguariensis The rhizoma perennial peanut (RP P ) ( A. glabrata Benth.) is well adapted to the Florida climate and is being cultivated for its high quality forage as well as for ornamental purposes. Previous evaluations of A. glabrata have shown significant phenotypic variation for several agronomic traits including yield potential (Freire et al. 2000; Butler et al. 2006), rate of establishment (Kelly and Quesenberry, 1993; Williams et al. 1997), a nd winter hardiness (French and Prine, 2006). Since, most accessions of RP P rarely produce seeds (Prine et al. 1981), hybridization has not been effective for breeding this species. The agronomic potential of RPP and the lack of information on its seed pr oduction potential were the justification for a major part of this study. The second species, A. paraguariensis is a potential source of novel genes for the genetic improvement of cultivated peanut. The species is of significant importance because some of its accessions show high levels of resistance to early leaf spot Cercospora arachidicola (Subrahmanyam et al. 1985). Almost all the potential pathways for introgression of desired genes from wild Arachis into cultivated peanut involved at least one phas e of ploidy manipulation (Simpson, 2001) This is because while the cultivated peanut is tetraploid (2n = 4x = 40), most of its wild relatives are diploid (2n = 2x = 20). Traditional in vivo application of antimitotic agents such as colchicine to plant sho ots, meristems, seeds, or seedlings has long been a common method for generating
17 polyploids ( Nebel and Ruttle, 1938). S everal studies have shown that in vitro chromosome manipulation led to improved efficiency of polyploidy induction, and reduced the occur rence of chimeras compared with in vivo methods (Cohen and Yao, 1996; Adaniya and Shirai, 2001). Stalker and Wynne (1979) achieved in vivo chromosome doubling of some diploid Arachis while Singsit and Ozias Akins (1992) obtained chimeric plants in an atte mpt to induce polyploidy in vitro but there is still no clearly described in vitro chromosome doubling procedure for any of the diploid wild Arachis Certainly, with the availability of a reliable tissue culture regeneration system, coupled with the appro priate method of gene transfer, genes expressing desired traits can be successfully transferred from A. paraguariensis and other diploid wild species into cultivated peanut. Given th is information, hypotheses, objectives and rationale for this research are hereby presented. Hypotheses In vitro tissue culture conditions could be modified to achieve high frequency tissue culture regeneration of A. paraguariensis and A. glabrata The tissue culture regeneration system derived for A. paraguariensis at the init ial phase of this study can be successfully combined with antimitotic agent colchicine to derive tetraploid plants. Seed production in A. glabrata is limited by a high rate of embryo abortion in dense canopies and rhizomes due to competition for energy res erves between sexual (flower/seed) and asexual (rhizome) reproduction. There is considerable variation in germination and vigor of seeds of rhizoma peanut cultivars UF Tito and UF Peace Objectives To optimize in vitro tissue culture regeneration proce dure for high frequency regeneration of A. paraguariensis and A. glabrata through defined regeneration pathways.
18 To induce tetraploidy in A. paraguariensis through in vitro tissue culture methods. To assess the seed production potential of RPP cultivars UF Tito and UF Peace To determine the germination percent and vigor of seed produced by the two RPP cultivars. Rationale Very low frequency of tissue culture regeneration using leaf derived explant s was observed by McKently et al. (1991) and Vidoz et al. (2004). T herefore an efficient regeneration procedure for A glabrata is lacking. In addition, there is no tissue culture procedure for regeneration of A. paraguariensis through a defined regeneration pathway. If high frequency of regeneration is achieved through a tissue culture regeneration procedure for A. paraguariensis at the initial phase of this study, successful induction of tetraploidy by colchicine should be expressly achieved. If the high rate of embryo abortion in dense canopies with extensive rhizomes as a result of competition for resources w as the main obstacle to seed production in RP P cv. Florigraze (Niles 1989 ; Williams, 1993), then this should be applicable to cultivars UF Tito and UF Peace as well. The result of the study conducted by Venuto et al. (1997) proved that there w as considerable variation among five genotypes of RP P for seed germination and seedling vigor. It is therefore likely that such variation would also be observed between cultivars UF Tito and UF Peace
19 CHAPT ER 2 LITERATURE REVIEW The Genus Arachis The g enus Arachis L belonging to the Fabaceae family, Papilionoideae subfamily, Stylosanthinae subtribe, Aeschynomeneae tribe has enormous economic and nutritional importance as a source of oil seed, food and fod der. Various Arachis spp. spread over more than 2.6 million km 2 of the South American continent. Several of the species have also been introd uced to Africa and Asia. Most of the species are perennial or annual legumes that are either trifoliate or tetrafol iate. Arachis are peculiarly recognized for their production of under ground fruits and there is wide range of genetic diversity within and between the species (Krapovickas and Gregory, 2007). While cultivated peanut ( Arachis hypogaea L ) is a major oilsee d crop grown in warm and subtropical regions of the world, several of the wild Arachis are also important sources of novel genes and therefore possess the potential for use in genetic improvement of cultivated peanut. In addition, several wild species in t he sections Rhizomatosae, Arachis Erectoides, Procumbentes, Caulorrhizae and Triseminatae have been evaluated and found to produce forage that is persistent under grazing and comparable or even superior to other tropical forage legumes in terms of yield a nd quality ( Krapovickas and Gregory, 2007 ; Stalker and Simpson, 1995) Origin and Distribution Indigenous to several parts of South America including Argentina, Brazil, Bolivia, Uruguay and Paraguay, the goecarpic nature, as well as the numerous types of r oot systems possessed by Arachis are essential for persistence in these diverse environments. S everal species were initially described and conserved by the early
20 European plant explorers who later transported them to other continents (Kochert et al. 1991) I t was further germplasm collection by 20 th century plant scientists that played a significant role in identifying the origin and geographical distribution of the genus Over the years, the categorization of the genetic variability within genus Arachis has been facilitated by intensive cooperate efforts of several academic institutions, government bodies and international organizations. There are currently over 15,000 accession s from 93 countries that are being conserved at the RS Paroda G ene Bank in ICR ISAT, Patancheru, India. The USDA Southern Regional Plant Introduction Station, Griffin, Georgia holds approximately 8747 accessions. In addition to these, North Carolina State University, Raleigh, N.C. and the Texas A & M University, College Station, TX. maintain a few hundred wild Arachis accessions. The geographical distribution and various types of environments associated with various Arachis species were studied by Ferguson et al. ( 2005) through the use of Geographic Information Systems (GIS). Singh and Simpson (1994) used the gene pool concept in describing Arachis gemplasm. Members of the primary gene pool consist of cultivated peanut A. hypogaea and its wild relative A. monticola while the secondary pool represents the diploid species of the sec tion Arachis that are cross compatible with cultivated peanut. The species of other sections that have demonstrated low levels of compatibility with cultivated peanut were placed in the tertiary pool. Apart from these classifications, interspecific and in traspecific variations in the genus Arachis have been described on a molecular basis. S everal molecular markers including restriction fragment length polymorphism (RFLP) (Gimenes et al., 2002), amplified fragment length polymorphism (AFLP) (Milla et al., 2005), Simple sequence
21 repeat (SSR) (Koppolu et al., 2010; Barkley et al., 2007), sequence related amplified polymorphism (SRAP) (Ren et al., 2010), and internal transcribed spacer (ITS) ribosomal DNA sequencing (Wang et al., 2010; Bechara et al., 2010) ha ve been used to explain these variations. Additionally, high density oligonucleotide microarray utilizing 49,205 publicly available expressed sequence tags (ESTs) have been developed for cultivated peanut (Payton et al., 2009). Reproductive Biology All of the species of Arachis produce only subterranean fruit. The perfect papilionaceous flowers of Arachis are sessile and are usually borne on a long stalk like tubular hypanthium consisting of fused lower portions of the calyx, corolla and filaments. The ov ary which contains two to three ovules is usually located within the base of the hypanthium. Because the release of pollen from the anthers norm ally occurs very early in the morning before the flowers open, successful controlled cross pollination in breedi ng requires emasculation prior to pollination (Krapovickas and Gregory, 2007). Although self pollination is common to all the species, a low occurrence of cross pollination by insects and parthenogenesis is possible. After self pollination and fertilizati on, the pedicel curves downward and the cells underneath the ovary start to divide to produce a gynophore or peg that forces the ovary into the ground to form mature fruit. The influence of abiotic factors especially photoperiod and temperature on the repr oductive efficiency and allocation of plant resources has been reported specifically for cultivated peanut (Bell et al., 1991). These authors indicate that both temperature and photoperiod have independent effects on growth and development, and that temper ature affects the phenology while photoperiod affects the effectiveness of reproduction and the distribution of plant assimilates.
22 Wild Arachis in Forage Systems Apart from possessing important traits needed for impro vement of cultivated peanut, wild Arach is spp. are grown for forage and ornamental purposes. Although several wild Arachis produce green forage that is palatable to grazing animals much emphasis has been given to rhizoma perennial peanut ( A. glabrata Benth.) and A. pintoi Krapov. & W.C. Greg because they are highly persisten t under grazing and trampling. They compete well with several weed species and they are resistant to stress factors such as cool temperatures, flooding, diseases, and pest infestations (Freire et al., 2000 ; Prine et al., 1 981 ) T he Uniqueness of Rhizoma Perennial Peanut Certainly, both the rhizomatous A. glabrata and the stoloniferous A. pintoi are sources of high quality persistence forage and are suitable for intensive grazing Additionally, A. glabrata is well adapted to the Florida climate and provides forage with high nutritive value that is similar to a lfafa. Its dense rhizomatous masses are also beneficial for stabilizing soil s (Freire et al., 2000) The rhizoma perennial peanut was originally added to the USDA nation al germplasm system in 1936, but intensive research on its evaluation started in the early 1960s. Florigraze (PI 421707) which is one of the most widely grown cultivar s was released after repeated evaluation of an off type line (Prine et al., 1981). Ano ther important cultivar Arbrook (PI 262817) was later released (Prine et al. 1990) based on adaptation to Florida soil and weather condition s Later, Arblick and Ecoturf with low groundcover were released as germplasm by the University of Florida (Prine et al., 2010) for forage and ornamental purposes. Recently, the cultivars UF Tito (PI 262826) and UF Peace (PI 658214) both with improved yield, persistence, and tolerance to
23 virus diseases were released by the Florida Agricultural Experimental Station (Quesenberry et al. 2010 b ). Limitations to Genetic Improvement of Rhizoma Perennial Peanut Based on past observations ( V enuto et al. 1997; Niles, 1989) most accessions and released cultivars of the se ction Rhizomatosae produce few, if any seed. Hence, their propagation and germplasm exchange have always been carried out through vegetative means. Consequently crop improvement efforts in the past have been restricted to improving establishment, evaluati ng new germplasm collections (Freire et al., 2000), and increasing their performance under grazing (Garay et al., 2004; Rice et al., 1995) However, the approach to genetic improvement of this species might change if enough viable seeds can be induced or p roduced. Wild Arachis in Gene Introgression Gene introgression through i nterspecific hybridization has led to the development of a few important peanut cultivars Important genes have been transferred from A. cardenasii Krapov. & W. C. Greg A. diogoi Hoe hne and A. batizocoi Krapov. & W. C. Greg. into cultivated peanut leading to the development of cu ltivars COAN and NemaTAM that are resistant to Meloidogyne spp. of the root knot nematode ( Simpson and Starr, 2001; Simpson et al. 2003 ). Another culti var Tifguard with high resistance to both tomato spotted wilt topo virus and root knot nematode has been released by the USDA ( Holbrook et al., 2008 ). Furthermore, accessions of A. diogoi, A. stenosperma Krapov. & W. C. Greg A. duranensis Krapov. & W. C. Greg A. cardenasii and A. correntina (Burkart) Krapov. & W. C. Greg. have been found to be highly resistant to rust caused by Puccinia arachidis while A. paraguariensis Chodat & Hassl A. diogoi, A. stenosperma and A. cardenasii
24 are highly resistan t to early leaf spot caused by Cercospora arachidicola and late leaf spot caused by Cercosporidium personatum Potential Uses of Arachis paraguariensis Arachis paraguariensis (Section Erectoides ) is a long lived perennial wild species with a deep and tube rous taproot. Wide gene tic and physiological variations exist between and within accessions of this species Some accessions are highly resistant to early leaf spot caused by Cercospora arachidicola which is an economically significant and widespread dise ase of cultivated peanut (Subrahmanyam et al. 1985). Furthermore, there are accessions that have been found to display resistance to root knot nematode ( Meloidogyne javanica (Treub) Chitwood Race 3 ), an important nematode parasite of cultivated peanut (Sh arma et al. 2002 ) High resistance to attack by tobacco armyworm ( Spodoptera litura Lepidoptera: Noctuidae), an other important pest of peanut has also been reported for this species (Stevenson et al., 1993) In addition to these, A. paraguariensis has bee n described as being very persistent under grazing systems, tolerant to drought, and adaptable to wide soil conditions ( Krapovickas and Gregory, 2007 ). Various a ttempts were made to transfer genes coding for important traits from A. paraguariensis to the c ultivated peanut. But crosses made between A. paraguariensis and other wild Arachis of the section Erectoides, Rhizomatosae, Procumbentes, Caulorrhizae Heteranthae, and Trierectoides led to the production of sterile hybrids with low pollen germinability (R ao et al. 2003; Krapovickas and Gregory, 2007 ). In a previous report, ( Singh, 1998 b ) made crosses between several accessions of tetraploid A. hypogaea and diploid A. paraguariensis S uccessful pollination leading to fertilization was achieved but there was impaired development of the resulting
25 proembryos because they aborted due to hypertrophic growth of nucellar tissue into the embryo sac. Hence, identification of an efficient method for combining the genomes of A paraguariensis or other Arachis specie s still needs to be performed, as this could greatly enhance the breeding of Arachis species. Hybridization Barriers E xtensive use of wild Arachis for genetic improvement of cultivated peanut has been hampered by several barriers including linkage drag, pl oidy barriers and sexual incompatibility ( Subrahmanyam et al. 2001). Additionally, there is need for more efficient and reliable tools for validating hybrid identities and monitoring introgressed chromosomal segments. G ene introgression from wild relative s belonging to compatible and incompatible gene pool s can expand the genetic base of Arachis and also lead to the development of new and important cultivars. Introgression Pathways Several possible options for transferring genes from wild Arachis spp. int o cultivated peanut have been proposed (Simpson, 2001) However, the decision to follow a particular pathway is largely dependent on the breeding goals, availability of resources and other essential technologies. The potential pathways for introgression o f genes from wild Arachis into cultivated peanut are discussed below Hexaploid pathway Th e hexaploid pathway involves crossing diploid Arachis directly with cultivated peanut followed by chemically induced chromosome doubling of the triploid F 1 hybrid to derive a fertile hexaploid. The hexaploid is then backcrossed to cultivated peanut several times in order to eliminate enough chromosomes t o lead to restoration of the tetraploid condition (Simpson, 2001)
26 Diploid tetraploid pathway. Several disease and insect resistant breeding lines were developed through this pathway (ICRISAT, 1990). It involves crossing two or more diploid wild species followed by induction of tetraploidy in the resulting hybrid before crossing it to cultivated peanut. Backcrossing us ually follows to carry out selection for the desired trait. The pathway is most suitable when the progenitor wild species of cultivated peanut are being utilized, as in the case of the development of cultivar COAN (Simpson and Starr, 2001) This pathway can also be modified such that the chromosome number of two wild species is doubled before they are hybridized. If the derived tetraploid hybrid is fertile, it is then crossed to cultivated peanut. Although, several attempts to utilize this modified route have been reported ( Simpson, 2001 ) to date none have been successful due to high levels of sterility in the hybrids. Transformation p athway Agrobacterium mediated transformation, v irus or chemical mediated microinjection, el e ctroporation, particle bombar dment and other transformation methods can be used for directly inserting genes into different species ( Sleper and Poehlman 2006 ). For traits from species in sections of wild Arachis that are not closely related to cultivated peanut, the use of molecular insertion of genes through biotechnology techniques likely will be the only feasible method of gene introgression ( Chenault et al., 2008) Currently, e fficient method s for producing transgenic peanut plants through Agrobacterium mediated genetic transforma tion (Sharma and Anjaiah, 2000 ; Bhatnagar et al., 2010 ) and particle bombardment (Ozias Akins et al., 1993 ; Niu et al., 2009 ) have been established, but only limited genotypes of cultivated peanut are amenable to transformation.
27 In Vitro Tissue Culture of Wild Arachis Tissue culture r egeneration refers to the development of organized structures such as roots, shoots, flower buds and somatic embryos from cultured cells or tissues that are called explants. Usually, regeneration is carried out in vitro such t hat the environment and growth medium composition can be altered to achieve high frequency regeneration Through the application of tissue culture and biotechnology techniques, the genes controlling disease, insect, and nematode resistance found in wild A rachis spp. may be transferred into cultivated peanut. In vitro tissue cultures are also capable of inducing stable somaclonal variation (Larkin and Scowcroft, 1981) that can become a useful source of genetic variation. However, the success of such genetic improvement program s is highly dependent on the ability to successfully regenerate plants from tissue cultures. Limited success has been achieved in the regeneration of plants from callus originating from various explants including immature embryos, cotyl edons, epicotyls, hypocotyls, and leaves of several Arachis species (Gagliardi et al., 2000; Pacheco et al., 2007; Pacheco et al. 2009 ; Pache co et al. 2008) Plantlet regeneration has also been achieved from short term cultures by in vitro organogenesis of a few species of Arachis ( Gagliardi et al., 2002; Pacheco et al., 2009) However, some wild species including A. paraguariensis and A. glab rata still lack efficient tissue culture regeneration procedures that can be successfully employed for somatic hybridization, in vitro ploidy manipulation and genetic transformation. Morphogenic Pathways Correct identification of developmental pathways of regeneration is important to identify the most suitable system to achiev e specific research goals. The three major
28 developmental pathways for in vitro plant regeneration are propagation from pre existing meristems (shoot culture or nodal culture), shoot o rganogenesis, and non zygotic embryogenesis or somatic embryogenesis Propagation from pre existing meristems The propagation of shoots or nodal segments is a common method for mass production of plantlets through in vitro vegetative multiplication. The p rocess usually consist of four stages (Fossard, 1987) starting with the selection and inoculation of explant into nutrient medium, followed by multiplication and growth of culture with repeated subculture. Next is the stage involving rooting and acclimatiz ation of plantlets before the final transfer to soil. Even though, propagation from pre existing meristems has minute application to plant breeding, it is the major method of plant propagation at industrial level where the goal is typically to propagate an d maintain large population of homogenous plantlets (Dodds and Roberts, 1985). Organogenesis. Shoot organogenesis (adventitious shoot formation) involves propagation from explants without pre existing meristems through production and subsequent rooting of adventitious shoots (Schwarz and Beaty, 2000) It is a common method of micropropagation and shoots can be directly induced in vitro from different explants such as leaf, petiole, stem, and other organs obtained from intact plants. Such shoots can arise d irectly on explants or indirectly through an intermediary callus phase. The advantages of shoot organogenesis when compared to other in vitro regeneration pathways include : 1) efficiency and ease of propagation, 2) more synchronous shoot production than so matic embryogenesis, 3) importance of its often single cell or igin in
29 genetic transformation, and 4) the relatively higher ex vitro survival of derived plantlets ( Preece, 2000 ). In general, the cellular or tissue origin of shoots derived from organogenesis appear s controversial. There are reports that have used histological and morphological analysis to describe the formation of adventitious shoots of plants from different cellular origins The model proposed by Broertjes and Van Harten (1978) Broertjes a nd Keen (1980) and Verma and Mathur (2011) stating that directly regenerated adventitious shoot meristems are always formed from one or a few daughter cells originat ing from a single cell has not been widely accepted. T here was an opposite proposition tha t such regenerated plants are of multicellular and multihistogenic origin ( Norris e t al., 1983 ; Zhu et al., 2007 ) Since the exact origin of shoots derived from organogenesis has led to inconsistent observations, it is recommended th at definition of the o rigin of shoots emerging from explants should not be generalized. On the other hand, investigation s should be carried out with the aid of histological and molecular tools as required to determine the exact origin for each genotype or species. This approach will be worthwhile especially when there is need to determine the most suitable pathway for specific crop improvement methods such as genetic transformation and ploidy manipulation Non zygotic embryogenesis This is an important pathway for producing som aclonal variants, developing artificial seeds, and synthesizing metabolites. Non zygotic embryogenesis involves regeneration of embryos from cells, tissues or organs other than the zygote (Steward et al. 1958). Embryos can be initiated either directly
30 fro m explants without callus formation in a manner usually described as direct embryogenesis or indirectly when callus mass es form on explant s before embryos are developed. The developmental patterns of non zygotic embryos in vitro have been described based on studies in Arabidopsis (Zimmerman, 1993; Yasuda et al., 2000) F irst there is asymmetric division followed by formation of a globular structure which transforms into an oblong. Next is the formation of a heart shaped structure, then torpedo and cotyle donary shape structures, and finally the mature embryo. Non zygotic embryos originate from a single cell, and are bipolar with both shoot and root poles. They also lack vascular connection s between the callus mass or explant thus making their separation ea sy ( Stephen et al., 2000) Signal Transduction Definitely cellular dedifferentiation is characterized by remarkable changes in the pattern of gene expression ; t here are specific genes involved in dedifferentiation, acquisition of competence and induction of in vitro regeneration ( Henry et al., 1994 ) It is also possible that genes are regulated by epigenetic mechanisms, including certain chromatin effects S hoot regeneration efficiency in tissue culture has been described as a quantitative trait that ofte n varies between plant species and within a plant species among subspecies, varieties, cultivars, or ecotypes. Consequently, shoot regeneration often becomes difficult especially when a different regeneration procedure s ha ve to be developed for different g enotypes within the same species (DeCook et al. 2006 ). The quantitative trait loci (QTL) associated with variation in shoot regeneration efficiency in Arabidopsis w ere identified by Schiantarelli et al. (2001), while c andidate genes for regeneration in se veral species have also been defined through the use of QTL
31 mapping (Bolibok and Rakoczy Trojanowska 2006; Taguchi Shiobara et al., 2006) and microarray analyses (Bao, 2009). Totipotency, genotype, and explant source. The concept of totipotency is fundam ental in understanding the process of in vitro tissue culture regeneration. It is the potential of living cells to express the total genetic potential of the parent plant in regenerat ing to whole new individuals T otipotency is highly dependent on genotype explant type, and the presence of appropriate stimuli (Dodds and Roberts, 1985 ). There are easy to regenerate species as well as recalcitrant ones. Several legumes and woody species have been described as recalcitrant Generally, Arachis was described a s one of these recalcitrant leguminous species (Cheng et al., 1994; Mroginski et al., 2004) But advancements in tissue culture research in recent years have led to establishment of various regeneration procedures for cultivated peanut a nd many of the wilds Arachis spp. (Dunbar and Pittman, 1992; Faustinelli et al., 2009; Li et al., 1993; Pacheco et al., 2009) Often the effects of genotype on developmental pathway s growth rate s and callus characteristics are observed in vitro Similar ly, the amount and type of endogenous plant hormone possessed by different plant genotypes influence their tissue culture response. T he explant source and age has been found to be crucial for success ful tissue culture regeneration as well. As a rule, expla nts obtained from less differentiated, immature tissues are easier to regenerate while explants derived from highly differentiated tissues are less responsive in vitro In reality, the capacity of cells to become competent for regeneration may be completel y lost once they over mature. M oreover, the orientation and positioning of explants on the culture medium often
32 becomes important for absorption of nutrients and growth regulators (Papafotiou and Martini, 2009) Endogenous and exogenous growth regulators Endogenous growth regulators or phythohormones are signal ing molecules produced within the plant in extremely low concentrations. These molecules play very important roles in regulat ing cellular processes inclu ding the formation of somatic embryos, shoots, stems, leaves and flowers. Other processes such as seed growth, time of flowering, senescence the direction of ti ssues grow th plant longevity, and death are also heavily regulated b y these growth regulators (Gan et al., 2004). Growth regulators are usually produced by plants within the meristem atic zone where high concentration of actively dividing cells exist s before any specialized tissue is differentiated. They are then transported to other plant parts to induce specific changes or they are deposited in cells for future release and utilization. Exogenous growth regulators may be artificially synthesized and are used to regulate plant growth in vitro or in vivo The type and quantity of plant growth regulat ors ( PGRs ) in the tissue culture medium is critical for determin ation of the morphogenic pathway of cultured cells. The five main classes of PGRs that are commonly used in plant cell and tissue culture are auxins, cytokinins, gibberellins abscisic acid a nd ethylene. Certainly, auxins and cytokinins affect most key development al episodes in plants. They both have pronounced and synergistic effects on plant growth and development especially in apical dominance and the development of root s and shoot s ( Eklf et al. 199 7 ). Specifically, cytokinin signaling modulate s auxin signaling and transport for
33 determining root meristem size in Arabidopsis (Dello Ioio et al. 2007). The balance of auxin and cytokinin in the medium is therefore important for determination of the morphological fate of explants D ifferent organ types can also be achieved by regulating the auxin to cytokinin ratio O rdinarily, a relatively high auxin to cytokinin ratio promotes regeneration of root s while the reverse leads to the production o f shoot s Typically, explant cells will proliferate to form callus when they are exposed to a nutrient medium containing approximately the same level of auxin and cytokinin. Environmental stimuli Various environmental factors, such as temperature, light conditions, and humidity may function as signals thereby causing groups of cells to redefine their fate Hence, abiotic factors inside culture vessel s and in growth chamber s can influence cellular and physiological processes involved in tissue culture rege neration These factors interact wit h the cellular factors to produce either a positive or negative effect. In general, temperature has to be optimal and constant while light intensity should be adequate and of the right quality and duration. When humidit y is too high, plantlet growth can be retarded (Chen, 2004). The design of the culture vessel is very much associated with ventilation, humidity, and accumulation of gasses such as ethylene that could hinder shoot or root elongation and also induce leaf ab scission in plantlets (Kumar et al., 2009). A dditionally, the type of support matrix can determine how water relations, humidity, temperature and some other physical factors are manifested. C learly there is a complex interact ion within and between the phy sical, chemical and p hysicochemical environment of explants.
34 The Challenges of In Vitro Tissue Culture of Wild Arachis The problems facing tissue culture regeneration of Arachis species are specific for each genotype, explant type, and regeneration pathway and cannot be oversimplified. For instance, tissue culture of some wild Arachis spp. including A. glabrata has not been very successful due to lack of a suitable explant source. On the other hand, A. paraguariensis possesses very high morphogenic potentia l; successful regeneration from protoplast and callus cultures (Li et al., 1993; Still et al., 1987) s upport its potential suitability as an ideal model system for studying in vitro regeneration and morphogenesis of leguminous species. However, failure of regenerated shoots to produce root s either in vitro or ex vitro has constituted a major limitation to the production of a large number of surviving plantlets. For most breeding programs utilizing in vitro tissue culture technique s the final aim is succes sful establishment of derived plantlets i n the field or in the greenhouse. However, a substantial number of plantlets do not survive du e to stress encountered dur ing the acclimatization process. Lack of roots or poor formation prior to acclimatization can also result in a low survival rate. M ore importantly, there is need to identify specific challenges that are being faced by each of the Arachis spp. and devise appropriate methods for overcoming them. Root f ormation. Some plants easily form roots in vitro while others do not. Events involved in formation of a root during embryogenesis or organogenesis are controlled by interactions of m any e xogenous and endogenous factors such as sugars, calcium, auxin s cytokinin s polyamines, ethylene, nitric oxide, hydr ogen peroxide, carbon monoxide, c yclic g uanosine m onophosphate (c GMP ), and peroxidase. Specifically, a uxin signaling promote s the expression of cytokinin signaling inhibitors
35 while cytokinin signaling also promotes the expression of auxin signaling inhibi tors (Bishopp et al. 2011) Adventi tious roots are post embryonic; their formation involves a process of de differentiation, in which determined cells change their morphogenetic pathway and then act as precursor cells for the root primordia It has been c learly established that auxin response f actors ( ARF ) transcription factors and Aux/ IAA proteins play crucial roles in auxin signaling These two proteins facilitate the auxin gene expression response. The Aux/IAA proteins normally function as repressors b y binding ARF transcription factors to prevent the activation of promoters and other control sequences of genes that are turned on or off by auxin. The binding of auxin to TIR I receptor leads to the formation of a unique complex that binds proteins that a ttaches ubiquitin to Aux/IAA proteins for their targeted destruction. Hence gene transcription can commence (Teale et al., 2006 ; Kepinski and Leyser 2002). Even though it is more cost effective to root plantlets ex vitro than in vitro it is not always a feasible option for leguminous species that are difficult to root and acclimatize. Acclimatization and p lantlet survival. Certainly, an acclimatization process is required for most in vitro derived plantlets to ensure high survival rates. The ex vitro env ironment generally ha s significantly lower relative humidity high er light intensity and is non a septic Hence, plantlets undergo numerous stresses during transplanting. Besides, the negative carryover effects of synthetic growth regulators on acclimatizat ion can also exert a negative impact on plantlet survival rates ( Valero Aracama et al. 2010 ) Rooting is an important determinant of ex vitro survival but in vitro derived roots are prone to damage during acclimatization Moreover, shoots are usually expo sed to
36 12 24 hours of photoperiod p rior to acclimatization to improve their photosynthetic efficiency and also to prepare them for ex vitro acclimatization. In Vitro Ploidy Manipulation Polyploidy occurs when the number of chromosomes in a cell becomes m ultiplied. It is a key evolutionary mechanism that often leads to creation of new species from different progenitors. There are two common forms of polyploidy. Autopolyploidy is the formation of a polyploid with more than one diploid chromosome set from th e same species genome while allopolyploidy involves the combination of multiple genomes into one single nucleus. Several autopolyploids occur spontaneously or naturally through the fusion of 2n gamete s However, it is also possible to chemically induce pol yploidy by using antimitotic agents such as colchicine, oryzalin or trifluralin (Quesenberry et al. 2010 a ) Chemical induction of polyploidy has been successfully utilized to transfer useful traits between several s pecies (Stalker and Simpson, 1995) Doub ling of chromosome numbe r s in this way can also be used to facilitate the crossing of diploid, tetraploid and hexaploid species and to improve fertility of some hybrids (Adams and Wendel, 2005; Castillo et al., 2009). A utopolyploids often display polysomic inheritance because they have more than two copies of each chromosome with equal ch ance of recombining at meiosis. T herefore, crossovers can occur between more than two homologues, resulting in multivalent arrangements of homologous chromosomes at metapha se I and chromosome abnormal segregation at anaphase ( Burnham 1966) Consequently, anomalous chromosome segregation may lead to partial sterility B ut some autopolyploids are nearly completely fertile with mostly bivalent configurations observed at meiosi s I. The formation of bivalents in fertile autopolyploid s is a random event, and
37 appears to be under genetic regulation (Cifuentes et al. 20 10 ). Nevertheless the underlining genetic basis for the reduction in formation of multivalents still needs to be i nvestigated. Colchicine induced polyploidy can produce changes in plant morphology and is considered suitable for crop improvement. G ene duplication occur s as a result of polyploidy and such duplicated genes may be expressed equally or unequally. Likewise, s ilencing of one copy of a duplicated gene is a common response to polyploidy (Adams and Wendel, 2005) Ploidy Detection The u se of flow cytometry for ploidy analysis in plant breeding is widel y accepted. The technique can easily be used to distinguish parent plants, screen offspring after interploidy crosses, monitor ploidy levels during seed multiplication or evaluate occurrence of polyploidisation ( Leus et al. 2009). Flow cytometric analysis involves the estimation of DNA content by using the fluorescence and light scatter properties of distinct particles such as cells, nuclei, and chromosomes during their passage inside a narrow, liquid stream The nuclei to be analyzed are usually released into cell homogenates by chopping or by lysis of protoplasts. Flow cytometr y of nuclear DNA is appropriate for cell cycle analysis because the nuclear DNA content reflects the position of each cell within the cell cycle Hence it is possible to determine t he fraction of the cell popula tion in the G1, S and G2 phases of the cell cycle. Usually, computer software is used for such analysis to derive a DNA content distribution curve with corresponding peaks. For nuclear genome size determination the G1 peak co rresponding to the unknown sample nuclei is compared relative to nuclei isolated from another plant of known DNA content
38 Knowledge of leaf stomata size and frequency can be used to define incidence of tetraploidy Typically, the stomata in the tetraploid s are clearly larger and more dispersed than those of the same diploid species. Observations on the size and frequency of stomata on leaf surfaces have suggested that they can be successfully utilized as rapid indirect methods for preliminary i dentif ication of ploidy level in several species (Beck et al., 2003 ; Quesenberry et al., 2010 a ) This is most useful when screening a large number of plants for polyploidy Finally, traditional chromosome cytological examination is usually carried out for verification of induced polyploids. Ordinarily, chromosomes from preparations of r oot tip s or pollen mother cells are observed and counted under the microscope. For instance, a normal tetraploid plant should have two times the number of chromosome of the diploid. Coun ting of chromosomes is laborious and prone to errors especially in species having a large number of chromosomes of small size. Results from a study on c hromosome doubling of Trifolium s pecies with the use of N 2 O (Taylor et al., 1976) revealed that ploidy l evel classification based on pollen size was almost as effective as subsequent cytological verification via counting of chromosomes
39 CHAPTER 3 OPTIMIZING IN VITRO REGENERATION CONDITI ONS FOR Arachis paraguariensis Tissue culture and biotechnology tech niques are central to several innovative concepts for overcoming hybridization barriers between cultivated peanut ( Arachis hypogaea L.) and several of its wild relatives. Of significant importance is the diploid Arachis paraguariensis Chodat & Hassl, a wil d species with novel traits including disease, insect, and nematode resistance. Although tissue culture of cultivated peanut has witnessed a remarkable improvement in the past few decades, many of the wild Arachis species appear recalcitrant to similar met hods (Srinivasan et al., 2010). Limited success has been achieved in the regeneration of plants from mature leaflets (Dunbar and Pittman, 1992), protoplast s (Li et al., 1993), immature zygotic embryos (Sellars et al. 1990), stamens (Still et al., 1987), cotyledons, embryonic axes and embryonic leaflets of A. paraguariensis (Pacheco et al., 2009). Identification of the tissue culture regeneration pathway and the specific factors influencing morphogenetic potential of a given species is important for develo ping a highly efficient regeneration system. This is because during the process of cellular dedifferentiation, the developmental fate of explants can easily be altered by endogenous hormones as well as several environmental factors including stress, exogen ous growth regulators, light, temperature and humidity. The formation and activities of meristems are highly dependent on these factors. Consequently, the morphogenic pathway for the tissue culture explant is highly flexible and open to alternative routes. Generally, the two common pathways of plant tissue culture regeneration are organogenesis and non zygotic (somatic) embryogenesis. During organogenesis
40 adventitious organs or axillary buds form directly or indirectly on the explant. N on zygotic embryogen esis involves a notable developmental pathway with anatomical and physiological features of embryos that are highly comparable to zygotic embryos (Zimmerman 1993; Mandal and Gupta, 2002). Due to the single cell origin of non zygotic embryos, they are prefe rred in several regeneration systems for clonal propagation, ploidy manipulation, gene transfer and synthetic seed production. However, tissue regeneration via organogenesis has proved to be advantageous for studying regulatory mechanisms of plant developm ent (Hicks, 1994). The role of thidiazuron (TDZ) in bio regulation of in vitro morphogenesis is well recognized. For instance, the auxin 2,4 D has been used to induce embryogenesis in many cultivars of peanut, however, replacing 2,4 D with TDZ lead to the development of the most efficient genotype independent peanut non zygotic embryogenic system. (Saxena et al. 1992; Sharma and Anjaiah, 2000). In addition, Murthy et al. (1995) reported that culturing seedlings for just two days on TDZ supplemented medium was sufficient to induce embryogenesis. The only report currently existing on in vitro non zygotic embryogenesis of A. paraguariensis utilized immature zygotic embryo explants but the induction of embryogenesis did not commence until 120 days after culture initiation (Sellars et al., 1990) A major problem in previous studies utilizing organogenesis was difficulty in regenerat ing shoots to produce roots either in vitro or ex vitro Still et al. (1987) first reported that root formation of A. paraguariensis plantlets rarely occurred in agar or liquid cultures, therefore, grafting to stems of rooted seedlings was the method used to obtain plants from regenerated shoots. In another study, Li et al. (1993) observed shoots that failed to form roots or normal plan tlets after 90 days of
41 culture initiation. Consequently, porous root cubes saturated with nutrient medium and auxin w ere used before rooting could be achieved. Flowering in vitro is a phenomenon that has been reported in A. paraguariensis tissue cultures ( Still et al., 1987; Li et al., 1993) but the environmental and genetic factors involved are unknown Apart from being a significant event for potential gene transfer methods such as in vitro fertilization, flowering in vitro can be used as an experimental system for studying the molecular mechanisms of flowering (McDaniel et al., 1991 ; Kane et al., 2000 ) in Arachis Therefore this study was designed to : 1) optimize an in vitro regeneration procedure for A. paraguariensis through clearly defined morphogeni c pathways, 2) increase rooting efficiency of the regenerated shoots over that previously achieved, and 3) identify the influence of photoperiod on in vitro flowering. Materials and Methods Explant Source and Sterilization Seeds of A. paraguariensis were obtained from the USDA Plant Genetic Resources Conservation Unit, Griffin Georgia a component of the Germplasm Resources Information Network (GRIN) National Plant Germplasm System (NPGS). Identification and further information on the origin of the six ge notypes of A. paraguariensis used for this study are presented in Table 3 1. Shelling of seeds was done manually before they were surface sterilized by rinsing in 70% ethanol for 1 min followed by treatment with 0.1% (w/v) aqueous mercuric chloride for 10 min. Thorough washing with sterile distilled water was performed before seeds were soaked in sterile water for 4 h in a laminar airflow chamber. The seed coat was then carefully removed before each type of explant was derived. The quarter ed seed explants w ere obtained
42 after removal of the seed coat and each seed was aseptically dissect ed into four equal longitudinal pieces. T he deembryonated cotyledon explants were obtained after the removal of the entire embryonic axis from each seed followed by longitudin al dissection into four equal sections. Culture Media and Environment All media formulations consisted of MS basal salts ( Murashige and Skoog, 1962) supplied by Sigma Aldrich, St. Louis, MO. V itamins (Sigma # G1019 ) were as in Gamborg B5 medium (Gamborg et al. 1968 ) All media also contained 30g L 1 sucrose (Sigma # S5390 ) 0.8% (w/v) agar (Sigma # A7921 ), and were supplemented with different concentrations and combinations of auxin, cytokinins. The pH of each medium was adjusted to 5.8 using 0.1 N KOH prio r to autoclaving at 121 o C with 1.06 kg cm 2 pressure for 20 min while stock solutions of each plant growth regulator were first filter sterilized through a double 0.2 m filter before being added to the autoclaved media in sterile bottles. Routinely, 1 ml L 1 of Plant Preservative Mixture (PPM TM Plant Cell Technology, Washington DC, USA) was added to the culture medium. Liquid medium ( 25 ml ) w as dispensed into each 2.5 10 cm petri dish while each Phytatray TM culture vessel (Sigma # P5929) contained 50 ml of medium formulation. Initial culture incubation of explants was performed at 261C under continuous lighting provided by cool white fluorescent lamps at 60 molm 1 s 1 light intensity. A medium (MS0) that lacked growth regulator was used as the control treatment for the explant regeneration experiments. Experimental Design and Data Collection This study consisted of five separate experiments that were designed as follows :
43 Tissue culture regeneration (Experiment I) 6 (genotypes) x 2 (explants) x 3 (2,4 D concentrations) x 3 (BAP concentrations) factorial experiment in a completely randomized design with sub sampling with 2 replications Tissue culture regeneration (Experiment II). 6 (genotypes) x 3 (TDZ concentrations) x 4 (BAP concentrations) or 4 (2ip concentrations ) factorial experiment in a completely randomized design with sub sampling having 4 replications In vivo evaluation. Two genotypes evaluated in a randomized complete block design with 2 replications In vitro rooting experiment 6 (genotype s) x 3 (auxin types) x 3 (auxin concentrations) x 2 (culture vessel) factorial experiment in a randomized complete block design with 4 replications In vitro flowering experiment 6 (genotypes) x 3 (photoperiod) factorial experiment in a completely randomiz ed design with sub sampling having 2 replications Statistical Analysis SAS PROC MIXED (SAS Institute, 2010) was used for all data analysis according to a mixed effects model while mean separation was performed using the Tukey's Honestly Significant Differe nce Test (P = 0.05). In the tissue culture regeneration experiments, each petri dish contained four explants or shoots to constitute a subsample. Means for shoot characteristics for each medium genotype replicate were computed and analyzed. Data on the eff ect of medium on regeneration and shoot characteristics were analyzed according to a completely randomized design with sub sampling. Growth regulator concentration and explant type were treated as fixed effects while genotype, replicate and the interaction s of the growth regulators with growth regulator and genotype were treated as random effects. A separate analysis within each genotype was also implemented to confirm the results of the overall analysis. The data on the effect of culture vessel, auxin type and auxin concentration on rooting, survival, and plantlet characteristics were analyzed according to a randomized
44 complete block design with each replicate being a block. Culture vessel, auxin type and auxin concentration were treated as fixed effects w hereas genotype, replicate and the interactions were considered as random effects. In order to determine the effect of photoperiod on flowering, the data were subjected to analysis according to a completely randomized design with sub sampling. Genotype and photoperiod were treated as fixed effects while replicate and the interactions were analyzed as random effects. The test for significance of treatment effects for the greenhouse and field evaluation studies were carried out according to a mixed model whi le means were separated based on Tukey's Honestly Significant Difference Test (P = 0.05). Histological Analysis Histological studies were performed at the University of Florida College of Medicine Electron Microscopy Core Facility according to the method o f Yeung and Saxena, (2005). C allus masses at different developmental stages were fixed in a mixture of 1.6% paraformaldehyde and 2.5% glutaraldehyde in a 0.05M phosphate buffer at pH 6.8 for 24 h. T issues were then dehydrated using methyl cellosolve follo wed by two changes of absolute ethanol before infiltration and embedding in glycol methacrylate based plastic molding cups Serial sections were obtained with a glass knife using a Leica 2040 rotary microtome with a retractable return stroke. T hin sections were mounted on glass slides and stained with toluidine blue while photographs were taken with Nikon light microscope Tissue Culture Regeneration Each explant was carefully implanted on the appropriate medium inside petri dishes with their cut ends embe dded in the medium. Observations for greening,
45 swelling, and formation of callus, leaf, or globular structure s on the explants were recorded for each subsample while the frequency of shoot and root formation as well as plantlet survival and were observe d throughout the regeneration studies. Experiment I In the first experiment, deembryonated cotyledon and embryonic axis explants were established on MS medium containing 2, 4 D (0, 2.2 and 4.4 mg L 1 ) or BAP (0, 2.2 and 4.4 mg L 1 ) alone or combined in a fa ctorial manner inside 2.5 x 10 cm P etri dishes. After callus formation, the maturation, conversion of embryo and rooting took place o n MS medium without any growth regulator inside PhytatrayTM culture vessels. Experiment II The second experiment involved testing of quarter ed seed explant on MS medium containing TDZ (0, 2.2 and 4.4 mg L 1 ) alone or in combination with either BAP (1.1, 2.2 and 4.4 mg L 1 ) or 2ip (1.1, 2.2, 4.4 mg L 1 ) inside 2.5 x 10 cm petri dishes. Elongation of buds was performed inside Phy tatray TM culture vessels containing 50 ml of MS medium that was supplemented with 2 mg L 1 BAP and 1 mg L 1 However, the plantlets were rooted on MS medium devoid of growth regulator under the same conditions except that the photoperiod was reduced to 16 h. In Vitro Rooting Rooting was tested on semi solid MS medium supplemented with IAA IBA or NAA each at concentrations of 0.2, 0.6 and 1 mg L 1 These media were compared in 11.4 cm 8.6 cm 10.2 cm polyethylene terephthalate glycol (PETG) vessels and in 2.5 cm 15 cm glass tubes. Micro shoots (Figure 3 1a) of 1 2 cm height were transferred into each vessel type. Each PETG vessel contained four micro shoots on 50 ml of medium while there was only one micro shoot on 10 ml of medium per glass tube. The mic ro
46 shoots were derived according to the regeneration procedure developed from the previous regeneration experiment utilizing MS salts with vitamins, 3% (w/v) sucrose, 0.8% (w/v) agar, 2.2 m g L 1 TDZ and 4.4 mg L 1 BAP. The number of rooted shoots and the num ber of roots per plantlet were recorded after six weeks of culture initiation. The maximum root length was determined from the mean of the three longest roots per plantlet. Afterwards, plantlets were transferred into J iffy peat pellets (Jiffy, Lorain, OH ) and acclimatized for two weeks in a humidity chamber. The acclimatized plants were transplanted into pots containing Metro mix 300 (Sun Gro Horticulture, Canada Ltd.) and sand in a ratio of 1:1 (v/v). After four weeks, the number of surviving plants wer e counted. In Vitro Flowering R egenerated micro shoots derived from quarter ed seed explants maintained on semisolid MS medium supplemented with 3% (w/v) sucrose, 0.8% (w/v) agar, 2.2 m g L 1 TDZ and 4.4 mg L 1 BAP were used for this study. Micro shoots of 1 2 cm height ( four per culture vessel) were placed into Phytatray TM culture vessels containing 50 ml of MS medium with 3% sucrose (w/v) and 0.8% (w/v) agar but without growth regulator s C ultures were maintained in three separate growth chambers at 26 2 o C under 12, 16 or 24 h photoperiod s with illumination of 60 molm 1 s 1 provided by daylight type florescent lamps. After the appearance of the first flower bud, observations and data collection were performed weekly until the plantlets ceased to flower. Flo wering plantlets were acclimatized and transplanted into pots filled with Metro mix 300 and sand in a ratio of 1:1 (v/v) (1:1) in the greenhouse.
47 In Vivo Evaluation After two to three roots have been formed, the plantlets derived from the experiment utili zing TDZ (Experiment II) were transplanted into J iffy peat pellets and acclimatized for two weeks inside a humidity chamber before they were transferred to Metro mix 300 and sand in a ratio of 1:1 (v/v) in 16.5 (depth) 16 cm (diameter) pots under greenh ouse condition s One hundred an eight p lants that had already been acclimatized and established inside pots in the greenhouse were transplanted to the field in the summer of 2009. The field study was carried out at the Plant Science Research and Education Unit, Citra, Florida in a randomized complete block design with two replications. A cultivated peanut ( A. hypogaea ) variety Florunner and seedlings of A. paraguariensis that had never been subjected to any in vitro tissue culture procedure were plant ed as control treatment s Evaluation of the plants was carried out in 2009 and 2010. L eaf spot disease caused by C. arachidicola w as rated using the Florida peanut leaf spot scoring system (Chiteka et al., 1988) where 1 = no disease, 2 = very few leaf sp ots in canopy, 3 = few leaf spots in lower and upper leaf canopy, 4 = some leaf spotting in lower and upper canopy with light defoliation (<10%), 5 = leaf spots noticeable in upper canopy with some defoliation (<25%), 6 = leaf spots numerous with significa nt defoliation (<50%), 7 = leaf spots numerous with heavy defoliation (<75%), 8 = numerous leaf spots on few remaining leaves with severe defoliation (<90%), 9 = very few remaining leaves covered with leaf spots and severe defoliation (<95%), and 10 = plan ts defoliated or dead. Harvesting of seeds was performed manually by hand lifting of the pedicels. The pods were removed, washed and air dried in a cool, dry area. The seed yield of plants
48 grown inside pots in the greenhouse was then compared with the yie ld attained by plants that were established i n the field. Results Two experiments were designed to investigate the roles and interactions of different genotypes, explant sources, and growth regulators on tissue culture regeneration of A. paraguariensis. T he study was also aimed at identifying the different regeneration pathways involved during the in vitro regeneration process. Experiment I The Role of 2,4 D in Induction of Embryogenesis Indirect non zygotic embryogenesis was achieved after the inductio n of embryogenic callus from the deembryonated cotyledon explants. Greening and swelling of explants began after 24 h of explant incubation followed by callus formation after 2 wk. The calli displayed great variability in color, texture and proliferation r ate depending on the culture medium. Sub culturing of callus mass es was carried out every 2 wk, however, embryo formation did not commence until after one month of culture initiation. When the callus masses were observed under a stereomicroscope, embryogen ic masses with proliferating shoots were observed. Consequently, the calli were transferred onto MS medium lacking growth regulators for maturation. Minimal callus formation was observed on the embryonic axis explants after 2 w k of culture incubation on t he MS medium formulations containing 2, 4 D and BAP but no embryos or shoots were formed. The calli that formed turned black and died after 4 wk of culturing. Likewise, no callus or shoot formation was observed on any of the explants that were cultured on the MS medium (control). The explants however remained fresh until the entire medium dried up.
49 The effects of 2,4 D and BAP concentrations and combinations on tissue culture regeneration from deembryonated cotyledon explant s of A. paraguariensis are prese nted in Table 3 2 Induction of non zygotic embryo s was observed in all the concentrations of 2,4 D that were tested. However, the earliest embryo initiation occurred when deembryonated cotyledons were cultured onto MS medium containing 4.4 mg L 1 2,4 D or 2.2 mg L 1 BAP The highest number of plantlets was recovered from medium that was supplemented with 4.4 mg L 1 2,4 D and 4.4 mg L 1 BAP. All the plantlets produced lack ed vigor, exhibited poor root formation, and were short For example heights ranged from 1.7 2.8 cm (Table 3 2). Consequently, only a few of them survived initial acclimatization and s urvived post acclimatization stress in the greenhouse. Experiment II The Role of TDZ in Induction of Embryogenesis and Organogenesis Callus formation was obs erved after 24h of culturing the quarter ed seed explants onto all the media containing 4.4 mg L 1 of TDZ in combination with any of the levels of BAP tested (Figure 3 1b). Green globular structures were observed o n callus masses as early as one week after c ulture initiation (Figure 3 1c). These globular structures were distinct (Figure 3 1d) and after a week of further development, it was easy to remove them as individuals from the explant. H istological analysis revealed that the structures were bipolar soma tic embryos lacking a suspensor cell, but having a well defined protoderm (Figure 3 1e). E mbryos differentiated to form shoots after they were transferred to MS medium lacking growth regulator s Table 3 3 shows the effects of TDZ and BAP concentrations and combinations on shoot regeneration across all six genotypes. The results indicate that the application of 4.4 mg 1 of TDZ in combination
50 with the same or lower levels of BAP was necessary for formation of non zygotic embryos. However, combining 1.1 mg L 1 of BAP to the optimal concentration of TDZ resulted in the highest mean number (14.8 1.6 ) of plantlets per explant cultured and the tallest plantlets (7.3 0.7 cm) at acclimatization. Besides, the time (6 0.3 days) taken for buds to appear was relativ ely short. In general, the explants on media that were supplemented with TDZ and BAP were more prolific with regard to callus, globular structure and shoot formation than the explants cultured on media containing TDZ alone or in combination with 2ip ( Tabl e 3 3 and Table 3 4) Interestingly, the application of TDZ in combination with 2ip (Table 3 4) resulted in the overall highest number of plantlets recovered per explant of 20.2 1.8 but with higher mean number of days (13.9 1.0) to bud initiation than for the treatment utilizing BAP. The regeneration pathway for tissues originating from the experiment utilizing TDZ in combination with BAP differs depending on the concentration, but not the genotype. Regeneration of the explants cultured on medium conta ining 2.2 mg L 1 of TDZ in combination with any of the concentrations of BAP did not follow a specific pathway; non zygotic embryo as well as organogenic callus developed simultaneously on the same explant. Nonetheless, regeneration via shoot organogenesis was observed in the explants that were cultured on medium containing TDZ only. Contrariwise, the histological analysis of tissues from the experiment utilizing TDZ in combination with 2ip revealed that the regeneration pathway was purely non zygotic embryo genesis. Rooting and Post Acclimatization Survival of In Vitro derived Plantlets The results from the rooting experiment revealed that the culture vessel type has a significant (P = 0.05) influence on rooting efficiency across all the genotypes studied.
51 A cross all the auxin treatments, plantlets in wide PETG vessels showed a significantly higher rooting ( 68 %) and ex vitro survival (38 %) than those in glass tubes 17% and 6 % respectively (Figure 3 4). In vitro flowering was observed in several of the shoots even though they lacked roots (Figure 3 2c). The PETG vessel also gave plantlets with a higher number of roots per plantlet and were faster to initiate root ing (1 4.9 vs 27 .3 ) than the glass tubes. Additionally roots of plantlets grown in PETG vessel were significantly longer than those in glass tubes (3.7 vs 1.2 cm) across all the genotypes and auxin treatments (Figure 3 5). The influence of auxin type and concentrations on root formation inside PETG vessel is presented in Table 3 5. The treatment which g ave the most desirable rooting performance based on a combination of number of roots, maximum root length and number of days to root initiation was the 0.2 mgL 1 of IBA. T he control treatment of MS medium devoid of auxin gave the maximum root length (7.1 1.2 cm), and the highest number of days to rooting of 26.6 1.8. The ex vitro survival of plantlets in response to the various auxin treatments inside PETG vessel is shown in Figure 3 6. Highest post acclimatization survival was recorded for the non tre ated control and the IBA or IAA treatment of 0.2 mg L 1 The rooting response and survival of the six genotypes were evaluated across all the auxin treatments inside PETG vessel and the result is presented in Figure 3 7. Only PI 468365 appeared to be a diff icult to root genotype with 40% rooting and 17% survival rates. The addition of 1 mg L 1 of NAA to the medium led to early emergence of root and the highest number of root per plantlet, nonetheless, the roots produced by the plantlets
52 were thick and short ( Figure 3 2b) while there was also severe leaf abscission of the plantlets (Figure 3 2d). Consequently, these plantlets failed to survive during ex vitro acclimatization and further transplanting to soil. Conversely, plantlets that were allowed to form root s on MS medium without any growth regulator were vigorous with long flexible roots (Figure 3 2e). The Effect of Photoperiod on In Vitro Flowering High frequency of flowering was observed for all the genotypes that were studied in vitro (Figure 3 3a b). Eac h flower appeared normal and was borne on an erect pedicel (Figure 3 3c). A pollen germination study was therefore conducted to determine if the pollen was fertile. All the pollen samples tested germinated well in vitro A further confirmation of the plant in vitro peg formation (Figure 3 3d). The effect of photoperiod on in vitro flowering in the five genotypes is summarized in Table 3 6. The highest flowering percentage of 65% recorded across all the genotypes for pla ntlets exposed to 12 h photoperiod is indicative that flowering induction actually occurred. However, the overall mean flowering frequency across all the genotypes and photoperiod treatments was 48%. The effect of photoperiod treatments on the time taken t o initiate flower buds starting from after the explant incubation is displayed in Figure 3 8. The result suggested that the length of photoperiod had a significant influence (P=0.05) on the time taken for the plantlets to initiate flower buds in vitro Bes ide, flowering response differed among the five genotypes (Figure 3 9), but the plantlets exposed to the12 h photoperiod were the earliest to initiate flower buds. This result was consistent across all the genotypes that were studied.
53 Performance of Plant lets in the Greenhouse and i n t he Field The result from two years combined data for C. Arachidicola leaf spot disease incidence in field cultivated plantlets and seedlings of A. paraguariensis is presented in Figure 3 10. A cultivated peanut ( A. hypogaea L .) variety Florunner was planted along as a control treatment. Although there was no difference in disease incidence scores among the genotypes and between plantlets and seedlings, nevertheless, there was a wide variation in the incidence C. Arachidicola l eaf spot between A. paraguariensis and A. hypogaea Both the i n vitro regenerated plantlets and the in vivo derived seedlings produced seeds in the greenhouse and i n the field. Contrary to the situation in the greenhouse, seed harvesting was laborious i n t he field because the field cultivated plants ha d very long pedicels (Figure 3 3e). Seed formation was observed in the greenhouse cultivated plants as early as three mo after transplanting. However, the plantlets and seedlings in the field did not produce s eeds until the following Spring. Hence the first harvesting of seeds was carried out when the seedlings and the plantlets had been established in the greenhouse and in the field for 9 months. Only mature pods (Figure 3 3f) were harvested for a comparison o f the seed yield of in vitro derived plantlets and the germinated seedlings. From the results in Figure 3 11, Grif 15201 had higher (P< 0.001) seed yield than Grif 15208 in the greenhouse and in the field for both plantlets and seedlings T he mean yield ov er genotypes did not differ between the plantlets and seedlings in the greenhouse, but the seedlings i n the field produced more seeds than the in vitro derived plantlets.
54 Discussion This study was undertaken with the objective of developing an efficient and rapid protocol for the regeneration of A. paraguariensis through defined morphogenic pathways followed by a field evaluation of the in vitro derived plantlets. The study also examined the roles of auxin and culture vessel on rooting and post acclimati zation survival of plantlets. Finally, the effect of photoperiod on the induction of in vitro flowering was investigated. Though some tissue culture regeneration procedures have been developed for A. paraguariensis scanty information is available on the morphogenic pathways involved. The importance of making use of the appropriate tissue culture regeneration pathway for breeding techniques such as embryo rescue, ploidy manipulation and genetic transformation cannot be overemphasized. A number of explan ts including Stamens (Still et al., 1987), mature leaflets (Dunbar and Pittman, 1992), protoplast (Li et al. (1993) and embryonic axis (Sellars et al., 1990) have been used with limited success in tissue culture regeneration of Arachis Therefore prelimina ry studies were conducted to develop and identify the most suitable explant. The morphogenic response observed in the quarter ed seed explant was high and rapid. Further observations showed that the quarter ed seed can be advantageous in shoot regeneration o f these six genotypes of A. paraguariensis as compared to other explants reported earlier. However, the simultaneous occurrence of organogenesis and embryogenesis on the same callus mass that was observed might be due to the nature of the explant since it is made up of tissues from the embryonic axis as well as the cotyledon. The cytokinin like action of TDZ has proved to be more effective than BAP for inducing shoot regeneration in the cultivated peanut (Gill and Saxena 1992). Besides, Li
55 et al. (1994) re ported that it is possible to achieve organogenesis in peanut after a short period of culturing explants onto medium containing TDZ. The result from the present study suggests that continuous exposure of explants to TDZ is not required for shoot induction as bud initiation from the callus mass was observed after 5 days of explant incubation in medium containing TDZ. Furthermore, prolonged exposure of explants to TDZ has been found to inhibit shoot formation and rooting of plantlets in A. hypogaea (Akasaka e t al., 2000). Hence, explants should be removed from culture medium containing high concentration of TDZ immediately after bud formation in order to prevent the inhibitory action of TDZ on shoot elongation. The present work shows that culturing quarter ed s eed explant for one week onto MS medium that have been supplemented with 4.4 mg L 1 TDZ and 1.1 to 2.2 mg L 1 of BAP or 2ip is effective for high frequency regeneration via non zygotic embryogenesis. The use of 2ip in combination with TDZ led to the recover y of more plantlets; however, bud initiation was delayed. It has been shown in several studies involving different species that genotype plays a critical role during in vitro regeneration (Bailey et al. 1993; Srinivasan et al. 2010). Yet, it is desired that a regeneration protocol be applicable to a wide range of genotypes or even species. The protocols developed in this study were suitable across six different genotypes of A. paraguariensis Further research might be needed to determine the applicabilit y of the protocols to other genotypes or species. Two of the genotypes with varying morphology were selected for greenhouse and field studies. The seed production potential and resistance to C. Arachidicola leaf spot disease in in vitro derived plantlets and in vivo derived seedlings were evaluated. The
56 result revealed the wide variation in seed production potential of the two genotypes. Hereafter, future research is needed to identify the genotypes that are high yielding so that seed derived explants can be made available for tissue culture regeneration. Based on the results from the rooting experiment, the use of PETG vessel significantly enhanced the rooting of plantlets in the presence or absence of auxin treatment. Similar results have been reported by Lucchesini and Mensuali Sodi (2004); during rooting, the use of ventilated vessels in comparison with the closed ones enhanced development of roots, and doubled the dry weight of Phyllirea latifolia plantlets. Water relations in the tissue culture vessel is dependent on factors such as the culture medium and environmental factors including temperature, photosynthetic photon flux, and the exchange of gas in and out of the culture chamber. In addition, a high auxin to cytokinin ratio at the site of root init iation has been found to be capable of promoting early root induction (Gaspar et al., 1996). Specifically, the use of NAA at concentrations between 0.5 and 1.0 mg L has been effective for rooting plantlets of wild Arachis species (Gagliardi et al. 2000) However, the results from this study revealed that supplementing the rooting medium with 1 mg 1 of NAA led to severe leaf abscission as well as inhibition of root elongation. Besides, there was a great deal of callus proliferation at the base of the shoot s. Consequently, it is suggested that plantlets be allowed to form root on MS medium lacking any growth regulator when there is no need for rapid root emergence, otherwise, the plantlets should be rooted on a medium containing 0.2 mg L 1 of IBA. An in vitro flowering system is a convenient tool to study specific aspects of flowering, and floral organ development (Wang et al., 2002). Different cytokinins,
57 sucrose concentrations, photoperiod, and subculture time have been used to promote flowering in vitro in many species (Vu et al., 2006; Narasimhulu and Reddy, 1984). In vitro flowering has been reported in A. paraguariensis (Still et al., 1987; Li et al., 1993), however, the factors influencing the event are yet to be understood. In the present study, the hig h frequency of flowering recorded across all the genotypes for plantlets exposed to 12 h photoperiod could be attributed to short day length induced in vitro flowering, an event that has also been reported in Kinnow mandarin culture (Singh et al 2006) In summary, high frequency and rapid regeneration procedures have been established for tissue culture regeneration of A. paraguariensis via indirect shoot organogenesis and non zygotic embryogenesis. Supplementing the culture medium with 4.4 mgL 1 TDZ in c ombination with 2.2 mgL 1 2ip resulted in the overall highest number of plantlets recovered per explant but it took 2 wk for buds to form on explants I t is also possible to recover a moderately high number of plantlets per explant cultured on medium conta ining 4.4 mgL 1 TDZ in combination with 1.1 to 4.4 mgL 1 BAP within a week of culture initiation Root formation in the in vitro derived plantlets has been improved over what was achieved in previous studies. Wide PETG vessel is recommended at elongation a nd rooting stage of in vitro regeneration while supplementation of the medium with 0.2 mgL 1 IBA may be carried out to induce early emergence of roots. Finally, in vitro flowering in A. paraguariensis appeared to be influenced by of photoperiod ; 12 h photo period was most effective for induction of in vitro flowering.
58 Further observations in the greenhouse revealed that some morphological variation existed among the in vitro derived plantlets that originated from quarter ed seed explants through indirect orga nogenesis. These variations was most easily seen in the shape of leaves and the branching habit of the plants and are probably due to somaclonal variation (Larkin and Scowcroft, 1982) that could be genetic or epigenetic in origin. Since this is a common ph enomenon especially in plants regenerated via the callus phase, further assessment using molecular markers should reveal the origin and the genetic stability of these plantlets.
59 Figure 3 1. In vitro regeneration of A. paraguariensis via non zygotic emb ryogenesis and organogenesis. (A) Quarter ed seed explants culture on medium containing 4.4 mg L 1 TDZ and 2.2 m g L 1 2ip. (B) Callus formation on explants after 24hr of culture initiation. (C) Green globular structures on the callus mass after one wk of cult ure initiation, bar= 1mm. (D) Distinct g lobular structures on explants after 3 wk of culture bar=1mm. (E) Histological analysis of embryogenic tissue revealing bipolar somatic embryo with a well defined protoderm, and (F) Organogenic callus mass from explant culture on medium containing 4.4 mgL 1 TDZ bar=1mm. (G) Histological analysis of organogenic tissue showing the formation of a meristematic dome and va scular connection between explant and the
60 Figure 3 2. In vitro rooting of A. paraguariensis as affected by auxin and culture vessel treatments. (A) Micro shoots used for the rooting experiment were excised from quarter ed seed explant s that were cultured on MS medium containing 2.2 m g L 1 TDZ and 4.4 mg L 1 BAP, S=shoot, NM=nutrient medium, while C= callus ( B ) Roots from plantlets growing on medium containing 1 mgL 1 NAA were thicker and shorter than those cultured on medium dev oid of auxin. ( C ) A plantlet inside a glass tube on medium containing auxin flowered without root formation ( D ) Plantlets treated with 1 mg L 1 of NAA suffered severe leaf abscission as well as inhibition of root elongation ( E ) P lantlets cultured on MS me dium without growth regulator were vigorous with long flexible roots
61 Figure 3 3. In vitro flowering, peg formation and ex vitro seed formation in A. paraguariensis (A B) High frequency in vitro flowering observed in genotype Grif 15201 and PI 262 842 respectively (C) The flowers were normal with viable pollen. (D) Peg formation in vitro was observed in genotype Grif 15201. (E) In vitro regenerated plantlets produced seeds after transplanting to pots in the greenhouse. (F) Mature pods and seeds of A. paraguariensis were obtained 3 months after transplanting.
62 Figure 3 4. The influence of culture vessel type on the rooting efficiency across six genotypes of A. paraguariensis Mean percentage followed by the same letter(s) within each parameter (%) are not significantly different at P=0.05 Figure 3 5. The effect of culture vessel type on the root characteristics of in vitro derived plantlets. Means followed by different let ter(s) within each parameter are difference (HSD) test.
63 Figure 3 6. Ex vitro survival of rooted shoots in response to various auxin treatments at 6 wk after culture initia tion in PETG vessel. Means followed by the same letter(s) within each concentration of auxin are not significantly different at Figure 3 7. The rooting response and survival of six genotypes of A. paraguariensis across all the auxin treatments inside a PETG vessel. Bar = SE
64 Figure 3 8 The time to initiate flower buds in vitro across five genotypes of A. paraguariensis as affected by photoperiod. Means followed by the same honestly significant difference (HSD) test. Figure 3 9. In vitro flowering response of five genotypes of A. paraguariensis Means followed by the same letter(s) are not significantly different at P=0.05 when
65 Figure 3 10. Leaf spot disease scores for two genotypes of A. paraguariensis and a cultivated peanut susceptible variety Florunner. Means follo wed by the same honestly significant difference (HSD) test. Figure 3 11. Seed yield of in vitro derived plantlets and germinated seedlings of two genotypes of A. paraguariens is inside pot in the greenhouse and i n the field. Means followed by the same letter(s) within each section are not significantly (HSD) test.
66 Table 3 1. The identity and geograph ical origin of the six genotypes of A. paraguariensis used for the study. NPGS/GRIN Accession No Taxonomic authority Origin Grif 15201 A. paraguariensis Chodat & Hassl. Paraguay Grif 15208 A. paraguariensis Chodat & Hassl Paraguay PI 262842 A. p araguariensis subsp. paraguariensis Brazil PI 468155 A. paraguariensis subsp. paraguariensis Brazil PI 468362 A. paraguariensis subsp. paraguariensis Paraguay PI 468365 A. paraguariensis subsp. paraguariensis Paraguay Table 3 2. The influence of 2,4 D and BAP concentrations and combinations on tissue culture regeneration from deembryonated cotyledon explant of A. paraguariensis. Each value represents the mean 2 replicates. Means followed by the same letter(s) within each column test. 2,4D (mg L 1 ) BAP (mg L 1 ) No. of d ays to embryo initiation No. of p lantlets recovered/ explant H eight (cm) at acclimatizat ion 0 0 0 0 0 2.2 0 0 0 4.4 0 0 2.2 0 0 0 2.2 2.2 81a 1.3b 1.7a 2.2 4.4 62b 2.3b 2.5a 4.4 0 78a 3.7b 2.7a 4.4 2.2 3 6 c 3.8b 2.4a 4.4 4.4 4 1 c 8.1a 2.8a
67 Table 3 3. The effects of TDZ and BAP concentrations and combinations on shoot regeneration across al l the six genotypes. TDZ (mg L 1 ) BAP (mg L 1 ) No. of d ays to bud initiation No of plantlet s / explant Height (cm) at acclimatization Regeneration pathway 0 0 0 0 NR 0 2.2 0 0 NR 0 4.4 0 0 NR 2.2 0 5c 2.0c 2.5b SO 2.2 1.1 10 ab 4.6c 2.3b O+E 2.2 2.2 10 ab 4.1c 2.6b O+E 2.2 4.4 10 ab 3.5c 3.1b O+E 4.4 0 1 1 a 3.9c 2.8b SO 4.4 1.1 6bc 14.8a 7.3a E 4.4 2.2 6 bc 10.4b 6.2a E 4.4 4.4 6 bc 8.1b 6.3a E Means followed by the same letter(s) within each column are not significantly different at P=0.05 when SO = Shoot organogenesis; O+E= Organogenesis and Embryogenesis (simultaneously on the same callus mass); E= Embryogenesis (non zygotic); NR = No response Table 3 4. The effects of TDZ and 2ip concentrations and combinations on tissue culture regeneration across all the six genotypes TDZ (mg L 1 ) 2ip (mg L 1 ) No. of d ays to bud initiation No of p lantlet s / explant Height (cm) at acclimatization Regeneration pathway 0 0 1.1 2.2 0 0 0 0 NR NR 0 4.4 0 0 NR 2.2 1.1 1 1 b 15.7b 8.1ab E 2.2 2.2 13ab 7.3c 7.1ab E 2.2 4.4 15a 18.7ab 8.5ab E 4.4 1.1 1 4 a b 18.9ab 10.8a E 4.4 2.2 1 4 ab 20.2a 7.8ab E 4.4 4.4 12ab 19.7ab 5.6b E Means followed by the same letter(s) within each column are not significantly different at P=0.05 when E = Embryogenesis (non zygotic); NR = No response
68 Table 3 5. Root formation as affected by auxin type and concentration after 6 weeks of culture in PETG vessel. Auxin Conc. (mg L 1 ) No of roots/ plantlet Max. root length (cm) at acclimatization Days to ro ot initiation IAA 0.2 7.1ab 5.6abc 11.3c 0.6 1.7b 2.0cd 11.9bc 1 6.0b 3.2bcd 12.0bc IBA 0.2 5.4b 6.4ab 12.2bc 0.6 1.4b 1.8d 16.5b 1 2.8b 5.0abcd 14.6bc NAA 0.2 4.3b 2.5cd 11.5c 0.6 4.8b 2.5cd 16.2bc 1 13.6a 1.6d 11.3 c Means followed by the same letter(s) within each column are not significantly different at P=0.05 when Table 3 6. The effects of photoperiod on in vitro flowering in five genotypes of A. paraguariensis. Photoperiod (h) Genotype No. of plantlets evaluated No. of flowered plantlets Flowering frequency (% SE ) 12 Grif 15201 40 34 85 7 Grif 15208 40 25 63 5 PI 262842 40 37 93 6 PI 468362 40 20 50 6 PI 468365 40 14 35 4 Mean 40 26 65 5 16 Grif 15201 40 24 60 6 Grif 15208 38 25 66 4 PI 262842 40 30 75 5 PI 468362 40 16 40 5 PI 468365 40 8 20 1 Mean 40 21 52 4 24 Grif 15201 39 16 41 4 Grif 15208 40 10 25 5 PI 262842 40 21 53 2 PI 468362 40 2 5 2 PI 468365 38 4 11 3 Mean 39 11 27 3 Overall Mean 40 19 48 3
69 CHAPTER 4 IN VITRO INDUCTION OF TETRAPL OIDY IN Arachis paraguariensis At least one step involving ploidy manipulation is required along many of the potential pathway s for introgression of disease resistant genes from wild Arachis species into cultivated peanut ( Arachis h ypogaea L.). This is because while cultivated peanut is tetraploid ( 2n = 4x = 40 ) many of its wild relatives are diploid (2 n = 2x = 20 ) species (Simp son, 2001; Mallikarjuna et al., 2004). Results from previous studies (Stalker and Wynne, 1979; Simpson, 2001) indicate that creating autotetraploids or amphidiploids and subsequently crossing the tetraploid plants with A. hypogaea can be a more efficient p athway to germplasm introgression than producing triploid hybrids through direct crossing of A. hypogaea to the wild species. Arachis paraguariensis is a wild species from the section Erectoides. Some of its accessions are highly resistant to early leaf s pot, an economically important and prevalent fungal disease of cultivated peanut caused by Cercospora arachidicola (Subrahmanyam et al., 1985). Tolerance to root knot nematode, Meloidogyne javanica Race 3, which is an important nematode parasite of cultiva ted peanut (Sharma et al., 1999) as well as resistance to tobacco armyworm ( Spodoptera litura Lepidoptera: Noctuidae), another important peanut pest (Stevenson et al., 1993) have also been reported in several accessions of A. paraguariensis However, att empts (Singh, 1998 b ; Rao et al., 2003) to generate a fertile hybrid after crosses with cultivated peanut have not been successful due to hybridization barriers. Induced chromosome doubling has been useful for facilitating the crossing of diploid, tetraplo id and hexaploid species and also to improve the fertility of hybrids (Brubaker et al., 1999). The induction of tetraploidy through the use of colchicine is a
70 suitable method to be explored because it does not require special equipment and it is relatively safe (van Harten, 1998). Importantly, the method often leads to the disruption of cellular mitosis through inhibition of the spindle fibers that segregate replicated chromosomes into daughter cells. In many cases, the resulting polyploids show changes in plant morphology (Taylor and Quesenberry, 1996), and such plant materials can be used as genetic bridges for the transfer of desirable features into other species (Recupero et al., 2005). Traditional application of colchicine in vivo to plant shoots, meris tems, seeds, or seedlings has long been a well known method for generating polyploids (Nebel, 1938), nevertheless, research ha s shown that in vitro chromosome manipulation is a more efficient way of inducing polyploidy than the in vivo methods (Cohen and Yao, 1996; Adaniya and Shirai, 2001). Autotetraploids of several wild Arachis species have been produced ( Simpson, 2001 ; Singh, 1986) via in vivo colchicine treatment. However, there is no clearly described procedure for in vitro chromosome doubling of sev eral diploid Arachis species including A. paraguariensis Consequently, the first objective of this study was to develop an in vitro chromosome doubling procedure for A. paraguariensis while the second objective was to evaluate the fertility and morphology of the induced autotetraploids. Materials a nd Methods Plant Materials One year old mature greenhouse grown plants were the source of mature seed derived explants that were used in this study. Seeds harvested from the plants were washed, air dried and she lled manually before storage in an air tight container at 4C for 6 months.
71 Tissue Culture Initiation a nd Establishment Seeds were surface sterilized by rinsing in 70% ethanol for 1 min followed by treatment with 0.1% (w/v) aqueous mercuric chloride for 10 min. Thorough washing with sterile distilled water was performed before seeds were soaked in sterile water for 4 h in a laminar airflow chamber. The seed coat was then surgically removed and each cotyledon with the embryonic axis still attached was cut in to vertical halves to obtain the quarter ed seed explant. The callus explant was derived by culturing quarter ed seed explants onto semi solid callus induction medium (CIM) containing MS inorganic salts, B5 vitamins, 3% (w/v) sucrose, 0.8% (w/v) agar, 4.4 m g L 1 TDZ and 2.2 m g L 1 2ip. Green and compact callus masses each weighing 0.2 g were selected as explants after 3 wk of culture incubation. P lantlets for deriving shoot tips were obtained by allowing some of the callus masses to develop into shoots on MS me dium lacking growth regulators. Each shoot tip consisting of a terminal bud plus two expanded leaves was then excised. A fresh stock solution of colchicine (Sigma) was prepared by dissolving an appropriate amount of powdered colchicine in 2 ml of 95% eth anol. The different explants were then immersed in the aqueous solutions of colchicine at concentrations of 0.05%, 0.1%, 0.2% and 0.5% (w/v) for 4, 8 16, 20 and 24 h while controls were held in sterile, distilled water for similar durations. Shoot regene ration of the quarter ed seeds was allowed to take place on semi solid CIM inside Phytatray TM culture vessels for 2 wk. The shoots were then sub cultured on to MS basal medium without growth regulators. T reated callus and shoot tip explants were cultured sep arately onto MS basal medium without growth regulator s When two to three roots formed, each plantlet was
72 transplanted into a J iffy peat pellet and acclimatized in a humidity chamber for 2 wk. Hardened plants were later transferred into soilless mix in a greenhouse. Culture C ondition The pH of all media was adjusted to 5.8 prior to autoclaving while the plant growth regulator solutions were filter sterilized through a double 0.2 m filter before they were added to the autoclaved media in sterile bottles. Each Phytatray TM (Sigma) culture vessel contained four explants on 50 ml of medium formulation while culture incubation was performed at 261C under a 16 h photoperiod provided by cool white fluorescent lamps at 60 E/m 2 /s light intensity. Ploidy D etermin ation by F low C ytometry Flow cytometric analysis of tissue sample s from regenerated A. paraguariensis diploid plants w as carried out using a Partec PAII Ploidy Analyzer according to the method of Quesenberry et al. (2010 a ). Approximately 0.5 cm 2 of leaves root, and a small piece of soft meristematic tissue were excised. In order to discharge the intact nuclei, a standard edge razor blade was used to chop the tissue inside a plastic petri dish containing 500 The nuclei were then washed into the tilted side of the dish and siphoned up with a micro pipet before 1500 diamidino 2 pheny lindole) to the vial, samples were allowed to stain for 3 min before analysis. The untreated diploid control w as sampled and used as a known 2x reference standard while samples from in vitro regenerated cultivated peanut ( A. h ypogaea ) and rhizoma peanut ( A. glabrata ) were used as known 4x reference standards. The reference standards were run each time plants were processed for analysis while the gain of the ploidy analyzer was always
73 adjusted to produce G2 peaks at near 200 and 400 for the diploid and tet raploid standards respectively. At least 10,000 nuclei per sample were analyzed at least twice on different days. Morphological and Fertility Observations Stoma and trichome observations were made on plants that were identified as tetraploids based on the ir flow cytometry profiles while the samples used as control were obtained from plants that have not been treated with colchicine. The frequency and size of stoma and trichome from upper and basal leaves from greenhouse plants were determined by coating th e abaxial side of leaves with clear transparent nail polish. The polish was allowed to dry before being peeled off, and dry mounted on microscope slides. For each leaf position, the number of stomata and trichomes per 1 mm 2 of 20 plants were determined wit h the aid of an ocular micrometer of a compound light microscope. Additionally, the morphology of 20 putative tetraploid, and 20 untreated diploid plants were assessed. Leaf measurements of breadth/width ratios were based on the means of 2 upper and 2 basa l leaves from 6 months old greenhouse plants while the plant height was measured from the soil level to the base of the apical bud. Observations for flowering, peg and seed formation were also recorded. Experimental D esign, D ata Collection and A nalysis Th e experiment was laid out in a split plot with 4 (colchicine concentration) x 5 (treatment duration) factorial main plots and 3 explant types (quarter ed seed, callus and shoot tip) as the subplots. Observations for callus shoot and root formation as well a s plantlet survival were carried out throughout the in vitro regeneration studies. Ploidy level analysis was first performed at 2 months after culture initiation and repeated 2 months after ex vitro acclimatization. The DNA histograms and nuclear DNA conte nt
74 were estimated on the basis of a linear scale with the aid of FlowJo V.7.6.3 for Windows (Treestar, Ashland, OR, USA). Data on frequency of tetraploid induction were analyzed using GLIMMIX procedure in SAS statistical program (SAS Institute, 2010). A fa ctorial logistic regression model was used since the dependent variable (tetraploidy) is dichotomous. The explant type was considered as a fixed effect while colchicine concentration, treatment duration and their interaction were the random effects. Moreov er, the plant morphology data was analyzed separately using a mixed model approach. Results Tetraploid Induction by Colchicine Application to Quarter ed Seeds a nd Shoot Tips After the exposure of quarter ed seed explant s to the various concentrations and tr eatment durations of colchicine, the overall frequency of regeneration obtained was lower than that of the water treated controls, especially when higher levels of colchicine were applied for longer durations. The time required for shoot formation was also significantly increased (Table 4 1). All the quarter ed seed explants survived every level of colchicine concentrations for the 4 h and 8 h treatment durations. However the best results in which 39% and 43% of the explant produced tetraploid plants were 0. 5% colchicine for 4 h and 8 h respectively. The proportion of induced tetraploids and explant lethality followed a linear pattern with increasing colchicine concentrations at 4 h and 8 h treatment durations. Besides, application of the two highest concent rations of colchicine to explants for 24 h proved to be lethal to the quarter ed seeds (Figure 4 2). The shoot tip explants yielded a relatively low frequency of plantlet formation and tetraploid induction. T he three tetraploid plants that were derived out of the 25 plantlets
75 that regenerated (Table 4 1) did not survive ex vitro acclimatization due to poor root formation. The representative flow cytometric DNA histograms of DAPI stained nuclei preparations of the diploid and the induced tetraploid A. parag uariensis are shown in F igures 4 3 a and 4 3 b, respectively. The dominant peak indicates the ploidy level. Overall, quarter ed seed explants gave the highest frequency of plantlet regeneration and tetraploid induction, as well as the lowest mortality rate ( Table 4 1) Additionally, the flow cytometric analysis of induced tetraploids from quarter ed seeds revealed that the plants were true to type with absence of chimeras. Therefore, quarter ed seed proved to be the best explants for in vitro induction of tetra ploidy in A. paraguariensis Regeneration o f Mixoploids f rom Colchicine Treated Callus The capabilities of the different explants to induce tetraploidy in vitro is summarized in Table 4 1. The lowest frequency of plantlet regeneration and highest explant m ortality rate were observed from the colchicine treated callus explants. Only chimeras were produced despite exposure to the various colchicine treatments. Results from the flow cytometric analysis revealed two patterns of peaks for the chimeric plants. T he histograms for two of the plants revealed approximately equal amount of 4x and >5x nuclei (Figure 4 2c) while the other three plants displayed profiles having three peaks. The first of the three peaks corresponds to G1 nuclei of the 2x cells, and was fo llowed by a peak that represents both the G2/M nuclei of 2x cells and the G1 nuclei of the 4x cells while the third peak corresponds to the G2/M nuclei of 4x cells (Figure 4 2d).
76 Morphology Fertility and Survival o f Induced Tetraploid a nd Mixoploid The fl ow cytometry analysis of DAPI stained cell nuclei of the induced tetraploid agreed with the stomata and trichome density observations. In other words, increasing the ploidy level from 2x to 4x in A. paraguariensis resulted in fewer stomata but more trichom es per unit leaf area (Figure 4 4a d). Additionally, the upper leaves of both the diploid and tetraploid plants have a significantly higher stomata density than the basal leaves (Figure 4 5 ) while there was no significant difference in plant height and lea f breadth/width between the diploid and tetraploid plants. Flower size (Figure 4 7e) and density of trichomes (Figure 4 5) on upper leaves were the noticeable and consistent morphological differences observed between the two ploidy levels. All the diploid plants generated during this study were fertile and produced viable seeds inside pots after six months of transplanting to the greenhouse, but flowering was observed in only two of the tetraploid plants. Flowers on the tetraploids were larger than those of the diploids (Figure 4 7e) but their fertility could not be verified due to lack of sufficient pollen. During the summer months of 2010, all the tetraploid and mixoploid plants were adversely affected by severe heat stress. Temperature fluctuations a resu lt of faulty air conditional occurred in the greenhouse where the plants were kept. Brown spots first appeared on leaves of the affected plants followed by the rolling up of the leaf margins, and finally, total defoliation results (Figure 4 7a c). Tissue s amples from plants showing these symptoms were submitted to the University of Florida Plant Disease Clinic for analysis, but, no disease pathogens were isolated from the samples. Interestingly, all the diploid plants recovered from the heat stress (Figure 4 7d) while only nine of the tetraploids remained alive.
77 Discussion The flow cytometry results concurred with stoma and trichome observations, thus highlighting the practicality of flow cytometry for the analysis of DNA content as well as ploidy level in A rachis species. Although changes in ploidy level are often accompanied by morphological variations (Taylor and Quesenberry, 1996), the only noticeable and consistent morphological differences between the diploid and the induced tetraploid A. paraguariensis were flower size and the density of trichomes on the upper leaves. Hence, the use of morphological features alone may not be ideal for identification of putative tetraploids. Ploidy determination using stoma and trichome characteristics alone could also b e misleading because only ploidy level of the L1 meristem layer can be determined. Other methods such as root tip chromosome counts and chloroplast counts in guard cell have been portrayed as very consistent and reliable for ploidy determination (Singsit a nd Ozias Akins, 1992; Quesenberry et al., 2010 a ). Nevertheless, these methods require certain degree of technicality and often prove to be laborious. The inverse relationship between colchicine concentration and explant survival was anticipated as well as the delayed shoot formation in colchicine treated explants. Colchicine as an anti mitotic agent is capable of acting on dividing cells by breaking down the spindle fibers during C mitosis so that the chromatids lay within the same cell without subsequent cell plate formation. Usually, the tissue has to recover from the interference before the chromosome number is altered. In vitro colchicine treatment during callus phase of tissue culture regeneration has been effective for chromosome doubling in other spe cies (Quesenberry et al., 2010 a ; Wu and Mooney, 2002), however, the colchicine treated callus explant used in this study failed to yield any true tetraploid
78 plants. Additionally, the tissue culture regeneration follows a pathway of non zygotic embryogenesi s which due to its single cell origin should decrease the possibility of chimerism, yet all the 5 plants derived from the callus explants were chimeras. The failure to induce tetraploidy in the callus explants might be associated with the developmental sta ge of the callus explants at the time of treatment especially because nodular shoot buds had already been formed on the callus at the time of colchicine application. Callus at this stage was selected because it is more compact and can remain solid for seve ral hours in the aqueous solution of colchicine. Future studies should aim at determining the optimal stage of colchicine application during callus induction and proliferation so as to increase the frequency of polyploidy induction. The chimeric plants ob tained from colchicine treated callus were identified based on their flow cytometric profiles but the exact type of chimerism was not verified. The occurrence of chimerism is quite typical in somatic polyploidization. It is important to identify the type o f chimera produced from colchicine induced tetraploid since the apical meristem is made up of 3 distinct cell layers usually referred to as LI, LII and LIII. The most stable type of chimera is periclinal resulting from mutation in the entire portion of one or more meristem layers. (Zhu et al., 2010). In this study, an efficient procedure for production of tetraploid A. paraguariensis was demonstrated. The flow cytometry analysis of DNA allows rapid evaluation of hundreds of putative tetraploids. The tetrap loid plants grown in the greenhouse have larger flower, as well as higher stomata and trichome density than the diploid plants. Nonetheless, there is still the need to evaluate the stability and fertility of the induced tetraploids.
79 Figure 4 1. The eff ect of colchicine concentration and treatment duration on timing of in vitro shoot formation from quartered seed explants of A. paraguariensis Different letters on top of each bar indicate a significant difference according to Tukey's Honestly Significant Difference Test (P = 0.05). Error bars represent SE. NA = data not available due to death of explant materials. Figure 4 2. Effect of colchicine concentration and treatment duration on tetraploid induction and quarter ed seed explant s viability (Mean S E) in A. paraguariensis
80 Figure 4 3. Representative flow cytometric analysis showing DNA histograms of A. paraguariensis ; (A) diploid with normal 2n ploidy, (B) induced tetraploid with 4n ploidy, (C) mixoploid with 4n+>5n ploidy, and (D) mixoploid wit h 2n+4n ploidy. The G1 peak of the diploid was approximately on channel 100 while the tetraploid showed a G1 peak on channel 200. The G2 peak of the diploid was approximately on channel 200 while the tetraploid showed a G2 peak on channel 400.
81 Figure 4 4 Imprints of A. paraguariensis leaf showing trichomes from 2n = 2x = 20 (a) and from 2n = 4x = 40 (b), stomata from 2n = 2x = 20 (c) and from 2n=2x=40 (d). Bar=100 m (a b) and 50 m (c d). Figure 4 5. Abaxial surface of upper leaf from acclimatized dip loid and tetraploid plants showing increased density of trichomes on the tetraploid.
82 Figure 4 6. Leaf characteristics of diploid and tetraploid A. paraguariensis Different letters on top of each bar within each parameter indicate a significant differenc e according to Tukey's Honestly Significant Difference Test (P = 0.05). Error bars represent SE.
83 Figure 4 7 Ex vitro performance of diploid and induced tetraploid A. paraguariensis (a) tetraploid plants 2 weeks after acclimatization before the occu rrence of heat stress; (b) a tetraploid plant severely affected by heat stress; (c) defoliation of tetraploid plant 5 days after the heat stress; (d) diploid plants recovered and remain healthy after heat stress; (e) flowers from induced tetraploid (4x) an d diploid (2x) plants of A. paraguariensis Table 4 1. The induction of polyploidy from different explants of A. paraguariensis. Explant source No. of e xplants treated No. of p lantlet s produced No of t etraploid s (%) No. of m ixoploid ( % ) Quarter ed se ed 160 408 75 (18)a 0 (0) Shoot tip 160 25 3 (12)b 0 (0) Callus 160 19 0 (0)c 5 (26) Values in parentheses represent percentage of induced tetraploid or mixoploid. Values with identical a factorial logistic regression model.
84 CHAPTER 5 SEED PRODUCTION IN A. glabrata AND TISSUE CULTURE REGENERATION FROM THE SEED DERIVED EXPLANTS The rhizoma perennial peanut ( Arachis glabrata Benth.) (RPP) is an important forage and ornamental crop in t he southern US. Its introduction into the US dates back to the 1930s, but today over 150 accessions have been introduced mostly from Brazil, Paraguay and Argentina (USDA ARS National Genetic Resources Program, 2010). Although Florigraze and Arbrook are the two most successful cultivars, the recent release of cv. UF Tito and cv. UF Peace based on increased yield, persistence, and tolerance to virus diseases was part of the strategy to address potential genetic vulnerability in this species (Garay e t al., 2004 ; Quesenberry et al. 2010 b ). Numerous field observations and previous research efforts have shown that most accessions of RPP produce very few seeds (Venuto et al., 1997; Niles, 1989). The seeds produced often do not germinate well, and the re sultant seedlings often fail to survive due to lack of vigor (Venuto, 1997). Therefore, large scale field establishment is usually carried out by vegetative propagation using rhizomes. Apart from the fact that establishment from rhizomes is slow and prone to stand loss from drought their purchase, transportation and sprigging can be expensive when compared to other forage crops. Niles and Quesenberry (1992) reported that poor pollen germinability was not the reason for low seed production in RPP but the two main constraints were desiccated pollen and high rate of embryo abortion as a result of competition in dense canopies. Furthermore, Williams (1994) attributed the low sexual reproductive effort to the need for partitioning energy resources between rh izome and seed reproduction. Even though these hypotheses were put forward to explain the reproductive behavior of RPP, the factors that regulate every stage involved in viable seed production remain
85 largely unknown. This lack of significant seed productio n coupled with prevalent hybridization barriers, and very low frequency of tissue culture regeneration observed in vitro have limited genetic improvement of RPP to conventional breeding efforts through germplasm evaluation and capturing of occasional favor able mutations (Williams et al. 2004). Biotechnology approaches such as genetic transformation and somatic hybridization provide an alternative avenue for overcoming hybridization barriers through direct introduction of genes across taxa. However, an eff icient cell and tissue culture regeneration procedure is usually a major requirement for these procedures The few papers that have investigated tissue culture regeneration of RPP from leaf derived explants have reported very low frequency and a long durat ion of regeneration. For instance, McKently et al. (1991) and Vidoz et al. (2004) reported low conversion rates of 10% of meristems to shoots and 6% of embryo s to shoots respectively. Consequently, it is evident that an efficient tissue culture regenerati on system for RPP is still lacking. Seed derived explant s ha ve been a suitable starting material for high frequency in vitro regeneration of the cultivated peanut ( A. hypogaea L.) (Sharma and Anjaiah, 2000 ) and several other wild Arachis species (Sriniva san et al. 2010) that were previously considered recalcitrant. This appears to be because seeds possess the precursor cells for self renewing, totipotent cells that are needed to generate whole plants. Thus, the use of seed derived explant s can be an appr opriate strategy for enhancing tissue culture regeneration. The objectives of this study were: (1) to evaluate the seed production potential of two RPP cultivars and (2) to a ssess the quality of
86 seeds that were produced. A third objective was to develop a n improved tissue culture regeneration procedure for RPP using seed derived explants. Materials and Methods Seed P roduction An area of approximately 0.34 ha each at the UF Agronomy Department Forage Research Unit near Hague, Florida was planted to the R PP cv s UF Tito and UF Peace by vegetative propagation using rhizomes in February 2009. Rhizome planting material was distributed in shallow furrows approximately 1.8 m apart and covered with soil. Elimination of perennial grasses and broadleaved wee ds prior to planting was achieved chemically while other cultural practices for RPP according to IFAS recommendation ( Ferrell, J. and B. Sellers, 2009 ) were followed Monthly visual evaluations were made to assess plant survival, vegetative growth, and ons et of sexual reproduction Data on reproductive and vegetative characteristics were collected at 8 and 20 mo after planting which was approximately 2 mo nths after the onset of peg initiation each year Within each plot, an area of 1.0 m x 0.5 m was random ly selected and dug with a shovel to loosen the soil around the plants. The plant material was then hand lifted and inverted with the pods and rhizome pointed upwards. Pods were removed, washed and air dried in a cool, dry area. For each sampled area, the canopy/rhizome spread, plant height, number of opened flower s number of pegs, pedicel length and number of harvested seeds were recorded while t he weights of dry seeds were determined one month after harvest. Seed Quality Evaluation Four different but c omplementary germination tests were conducted in a completely randomized design consisting of 3 replicates of 10 seeds each. For each of
87 the experiment s cultivated peanut (AT VC 2) was used as a control treatment All seeds were stored for about 10 months at 5 C and were surface sterilized with 1% mercuric chloride to eliminate fungi contamination. For filter paper germination, the seeds were placed on moist filter paper inside a plastic bag and then incubated at 261 o C. T he in vitro study involved pretre at ment of the seeds with 2.2 mg L 1 TDZ for 24 h before placement in Phytatry TM culture vessels containing semisolid MS basal medium. Culture vessels were wrapped with parafilm and placed in a growth chamber at 27 o C and 12 h photoperiod. Germination in s and medium was tested in vivo by planting seeds directly into a tray containing sand in a laboratory growth room at 261 o C After 3 wk, germinated seeds were grouped as normal or abnormal while non germinated seeds were classified as fresh or dead. The ge rmination percentage was calculated based on normal seedlings according to the method of I STA (2010 ). Prior to the tetrazolium test, seeds were physiologically prepared by soaking in a beaker of water for 18 hours at 27 C. For each seed, the seed coat was removed and the cotyledons were separated with the embryonic axis still remaining attached to one of them. Each pair of cotyledon from the same seed were covered with 1% tetrazolium solution in a 6.0 x 1.5 cm petri dish and allowed to stain for 8 hours at 30 C. Seed by seed analyses were performed based on staining patterns. Tissue Culture R egeneration To test if the tissue culture conditions developed for A. paraguariensis using seed derived explants could be modified to achieve high frequency of in vitro regeneration of RPP. The two media formulations that were successful for shoot induction of A. paraguariensis in previous experiments were tested. Prior to culturing, seeds were
88 surface sterilized by rinsing in 70% ethanol for 1min followed by treatment wi th 0.1% (w/v) aqueous mercuric chloride for 10min. Thorough washing with sterile distilled water was performed before seeds were soaked in sterile water for 4 h. Quarter ed seed explants were derived after the removal of seed coat by aseptically dissecting each seed into four equal longitudinal pieces while the embryonic axis explants were surgically removed from a different set of seeds. Each explant was carefully implanted on semi solid MS medium containing 3% (w/v) sucrose ( Sigma # S5390 ) Gamborg B5 vita mins ( Sigma # G1019) 0.8% (w/v) agar (Sigma # A7921), 4.4 mg L 1 TDZ and 2.2 mg L 1 BAP or 2ip. Three MS media formulations containing one of either 4.4 mg L 1 TDZ or 2.2 mg L 1 BAP or 2.2 mg L 1 2ip and a fourth medium (MS0) that lacked growth regulator were used as control treatments. All media were solidified with 0.8% (w/v) agar and adjusted to pH 5.8 before autoclaving for 20 min at 121 C and 1.1 kg cm 2 Approximately 20 ml of each media formulation was poured into 100 25 mm petri dishes. Explants w ere implanted on the different media inside petri dishes with cut ends embedded in the medium. The incomplete 2 (cultivar) x 2 (explants) x 2 (media) factorial experiment in a completely randomized design with sub sampling was repeated twice. Each petri di sh contained four explants to constitute a subsample while 2 dishes were evaluated per explant per cultivar for each replication. Culture incubation was at 261C under continuous lighting provided by white cool fluorescent lamps with 60 Em 2 s 1 light int ensity. Daily observations for greening, swelling, and formation of callus or globular structures on explants were recorded for each subsample unit. When shoots appeared, they were transferred to into Phytatray TM (Sigma) culture vessels containing 50 ml o f
89 MS medium without growth regulator and incubation continued under same conditions except that the photoperiod was reduced to 12 h. After two weeks, elongated shoots were rooted on MS medium that was supplemented with 2 mg L 1 of NAA. When 2 to 3 roots hav e been formed, plantlets were transplanted into jiffy pellets and acclimatized for two weeks inside a humidity chamber. Shoots were evaluated based on their in vitro vigor, rooting ability and ex vitro survival. To enhance the frequency of plantlet regene ration and eliminate browning of explants due to oxidation of phenolic compounds, another experiment was carried out to determine if supplementation of the culture media with activated charcoal w ould improve the frequency of shoot formation. Hence, s upplem entation of the nutrient medium with different concentrations of activated charcoal (0.5 and 1.0 g L 1 ) was incorporated into the same procedure used in the first experiment. Statistical Analysis All data were analyzed according to MIXED procedure (SAS, 20 11) for ANOVA. 0.05) while the means obtained from the seed viability tests and in vitro tissue culture experiments were separated based on Turkey's honest significant diffe rence (HSD) at the 5% levels. Percent germination data were first subjected to arcsine transformation before analysis and then back transformed for presentation in tabular form. Results Seed Production It wa s evident from the observations on vegetative an d reproductive growth of the two RPP cultivars between 2009 and 2010 that an increase in vegetative growth appeared to lead to a decrease in se ed p roduction (Fig ure 5 1 ). Observations from seed
90 yield and seed weight of the two cultivars during 2009 and 201 0 harvest seasons are presented in Fig. 5 2. 57 Kgha 1 ) was higher 1 ). However, these yields declined approximately 65% (145kgha 1 ) for cv. UF Peace and 100% (nil) for cv. UF Tito from the preceding year. Interestingly, highest 100 seed weight (28.8 2.5) was recorded for cv. UF Tito during the first year while seed weight was not determined in the second year due to no seed being harvested in the sampled areas In addition, the more extensive rhizome mat present made harvesting of seeds more difficult and laborious during the second year than in the previous year when the seeds were still firmly attached to their pedicels and in open soil not yet filled with rhizom es (Fig. 5 b.). Seed Quality E valuation Differences were evident in the color, size and weight of seeds obtained from both cultivars as well as the pod characteristics (Fig. 5 a ). P od s of cv. UF Tito appeared more reticulated harder and darker than t hose of UF Peace Results from the four different viability tests are summarized in Table 1. Germination rates for all the viability tests ranged from 7 73% for cv. UF Peace 10 44% for cv. UF Tito and 53 98% for the cultivated peanut (control). From the ANOVA, considerable variation existed among cultivars for percentage seed germination in vitro (44 73%). S ignificant difference in germination rates between tests also existed for each of the cultivars. Obviously, germination was highest in vitro and lowest in sand for all the cultivars while the. standard germination test on filter paper gave values intermediate between the in vitro media germination and the in vivo sand germination However, a few of the seeds that germinated in vitro formed multiple shoots on the MS medium, but there was no significant callus formation. Rupture of RPP seed coats and emergence of radicle on
91 sand and on moist filter began after two weeks when the germinated peanut (control) plants were already at the two leaf stage. O n the other hand, germination of seeds on MS nutrient medium occurred as early as three days after planting irrespective of the cultivar. Tissue C ulture R egeneration While the embryonic axis failed to respond to any medium treatment in vitro callus formati on and plantlet regeneration from quarter ed seed explant occurred approximately 6 wk after culture initiation (Fig 5 c e). Production of phenolic compounds reduced the frequency of regeneration in medium containing a combination of TDZ and BAP but regenerat ion of shoots w as enhanced when BAP was replaced with 2ip (Fig ure 5 3f g). An experiment was therefore carried out to determine if supplementation of the culture media with activated charcoal will improve the frequency of shoot formation especially in medi um containing BAP. Even though browning of explants decreased when activated charcoal was added to the medium, shoot formation was not achieved. Dark green callus was formed which regenerated into individual leaves without buds. The effect of combination s of different cytokinins on callus formation and shoot regeneration from quarter ed seed explant of RPP is presented in Table 5 2 The ANOVA revealed highest number of shoots per explant (4.38 0.47) for cv. UF Peace cultured on MS medium containing TDZ and 2ip while cv. UF Tito gave lowest result (0.54 0.49) on medium containing TDZ and BAP. With 3.00 0.26 acclimatized plantlets per explant ccultured, the overall performance of cv. UF Peace undoubtedly surpassed that of cv. UF Tito The four ne gative control treatments failed to produce callus or shoots thus indicating that a combination of T DZ and BAP or 2ip is required for callus induction and shoot regeneration Acclimatization of rooted shoots was achieved
92 within two weeks in a humidity cham ber but some of the plantlets failed to survive due poor root development. Nevertheless acclimatized plantlet s (Fig. 5 h) thrived well after transplanting into pots containing sand in the greenhouse and flowering was obseved after 2 months. Discussion The objectives of this study were to evaluate the seed producing potential and seed quality of 2 RPP cultivars, and to determine optimal tissue culture regeneration conditions for RPP using seed derived explants. Increased vegetative growth resulted in the sup pression of sexual reproduction in the two cultivars evaluated. This is in agreement with the general hypothesis that plant resource allocation to a specific biological function is traded off against investment in other function(s). The p resence of this tr ade off between sexual (flower and seed) and asexual (rhizome) reproduction is a strong indication that that both functions are dependent on the same resource pool. Williams ( 1994 ) o bserved that RPP flower production increased when rhizome production was m oderately restricted but attempt s to increase seed production through monitoring of the defoliation frequency was unsuccessful. In addition, excessive defoliation was found to be detrimental to flower and seed production. In a recent finding, Narbona and Dirzo (2010) reported that defoliation affects male but not female reproductive performance of C. suberosus because monoecious plants can allocate resources separately to male and female functions more easily than hermaphrodites. While it is expected that manipulation of resource allocation through experimental defoliation should result in an increased seed production in a hermaphroditic plant such as RPP, the factors that regulate partitioning of these resources are still largely unknown. Additionally, th ere is need for proper understanding of how certain
93 c ompensatory mechanisms influence resource uptake in such a way that the reproductive trade offs become unnoticeable. The wide difference in seed production potential of the two cultivars could be conside red a genotype effect. Although results of seed yield were based on data obtained for two consecutive years, the results are still preliminary due to inability to evaluate the cultivars under a suitable experimental design. The field plots which were initi ally established as a source of foundation rhizome stock were planted in several rows without blocks. Even though proper randomization was performed during sub sampling of plots, it was impossible to adequately eliminate experimental errors through blockin g of confounding factors. Nevertheless, production of a substantial amount of seed in the two RPP cultivars was truly a novel and exciting discovery. P ursuing this further there is need to conduct a more extensive evaluation of RPP germplasm to identify and carry out selection for adapted cultivars with high seed yields. Observations from the germination tests defeated the common expectation that l arger seeds should yield higher germination percentages. While Venuto et al. (1997) detected considerable var iation in seed germination and seedling vigor among five genotypes of RPP results from the present study did not show differences in the frequency of seed germination among these two RPP cultivars except on MS medium under in vitro condition. However, the germination percent for both RPP cultivars were significantly lower in comparison to the cultivated peanut. The experiments involving seed germination in sand was terminated after 45 days while the filter paper germination test lasted for 30 days. Because most of the non germinated seeds remained fresh until the end of the study, t he low germination recorded for the RPP
94 could possibly be due to either seed dormancy or seed persistence in the soil, or a combination of both factors. Besides that, insufficien t levels of calcium in the soil has been found to be connected to seed abortion and poor seed germination in cultivated peanut ( A. h ypogaea ), but addition of s upplementary calcium in combination with irrigation was found to greatly improve the seed maturit y and germination of some peanut cultivars. (Tillman and Stalker, 2009) In this study, the amount of calcium in the soil was not determined and supplementary calcium was not applied. When seeds were germinated on MS nutrient medium, multiple shoot buds we re formed even though no growth regulator was present in the medium. This may be as a result of the 24 h pretreatment with TDZ. The roles of TDZ in breaking of dormancy in seeds and in promoting direct embryogenesis from explants have been reported in many species including A. hypogaea (Gill and Saxena, 1992). The frequency of shoot regeneration obtained when both TDZ and 2ip were included in the tissue culture media for regenerating explants exceeds previous reports (McKently et al. 1991; Vidoz et al. 2 004). Cotyledon seed sections were highly suitable as an explant source for high frequency tissue culture regeneration of RPP. Moreover, they were easy to disinfect, store and can be made available for deriving explants all year long. Inhibit ion of shoot regeneration when BAP was present in the medium led to brown discoloration of explants as a result of excessive production of polyphenol This has been reported in tissue cultures of Arachis species (Li et al. 1993; Medina Bolivar et al. 2007). Common me thods of alleviating this problem include increasing the number of subcultures and adding activated charcoal to the tissue culture medium. Activated charcoal can reduce or prevent discoloration by rendering polyphenol oxidase
95 and peroxidase inactive. On th e contrary, cytokinins such as TDZ, BAP and 2ip may have great adsorption affinity for activated charcoal thus reducing their effect on cultured explants. Conclu ding R emarks This preliminary study on seed production demonstrated that increased allocation o f resources towards vegetative growth in RPP led to obvious reduction in sexual reproduction. Therefore, it is possible to obtain a substantial amount of viable seeds during the first year of establishment when the below ground vegetative growth is still m inimal. Afterwards, the seed yield rapidly declined due to increased rhizome and canopy spread. The observation of considerable variation in seed production potential of cv. UF Tito and cv. UF Peace calls for intensive breeding and selection for culti vars that are good producers of high quality seeds. The seeds could be utilized for rapid stand establishment as well as an easy source of explants for high frequency tissue culture regeneration. However, due to the competition between rhizomes and seeds, any seed production for propagation might be restricted to an initial year after planting. The discovery of substantial amount of seed production in cultivars has the potential of chang ing the current approach to genetic improveme nt and breeding of RPP. For example, breeding program s for RPP may be able to utilize methods such as mass selection, backcross breeding and pedigree selection in the near future. I n final consideration, the propagation, breeding, conservation and germplas m dissemination of RPP should be greatly enhanced.
96 Figure 5 1. Vegetative and reproductive growth of 2 rhizoma perennial peanut cultivars from 2009 to 2010. P= cv. UF Peace while T= cv. UF Tito Different letters on top of each bar for the same p arameter indicate a significant difference according Duncan's Multiple Range Test (P = 0.05). Error bars =SE. Figure 5 2. Seed weight and yield of R PP in fall 2009 and 2010. Different letters on top of each bar for the same parameter indicate a significant difference according to Duncan's Multiple Range Test (P = 0.05). Error bars =SE.
97 Figure 5 3. Seed production and tissue culture regeneration from seed derived explant of RPP. (a) variation in pod and seed characteristics of cv. UF Peace and UF Tito ;(b) mature pods s till firmly attached to pedicels during 2009 harvest season; (c) quarter ed seed explants on MS medium containing 4.4 mg L 1 TDZ and 2.2 mg L 1 2ip; (d) embryogenic callus mass on a quarter ed seed explant after 2 wk in culture; (e) elongated shoot of cv. Peac e on MS medium without growth regulator at 4 w k after culture initiation; (f) brownish discoloration of explants due to production of polyphenols in MS medium containing BAP; (g) regeneration without much interference of polyphenols when BAP was substitute d with 2ip in the culture medium; (h) acclimatized plantlet of cv. UF Peace at 6 wk after culture initiation.
98 Table 5 1. Percent germination and viability of rhizoma perennial peanut obtained from four different tests. Germination/ viability (%) Species Cultivar In vitro Warm S and T etrazolium A. glabrata UF Peace 73aB 25bB 7cB 30bB UF Tito 44aC 18bB 10bB 26abB A. hypogaea AT VC 2 (control) 98aA 85aA 53bA 77aA Percentages in the same row for the same cultivar followed by the same lower case letter are not significantly different at P = 0.05 when subjected to Tu Percentages in the same column for the same parameter followed by the same upper case letter are not significantly different at P= 0.05 when subjected to HSD test. Table 5 2. Effect of combination of different cytokinins on callus formation and shoot regeneration from quarter ed seed explant of rhizoma perennial peanut. Cultivar Cytokinin Callus rating (1 5) Callus weight (g) Shoots/ explant Acclimatized plant/explant UF Peace TDZ + BAP 1.68 (0.12)b 4.27 (0.40)a 2.77 (0.47)b 0.4 2 (0.26)b TDZ + 2ip 2.68 (0.13)a 2.76 (0.39)ab 4.38 (0.47)a 3.00 (0.26)a MS0/TDZ/BAP / 2ip (Control) NA NA NA NA UF Tito TDZ + BAP 1.71 (0.12)b 3.27 (0.40)ab 0.54 (0.49)c 0.31 (0.27)b TDZ + 2ip 1.96 (0.13)b 3.79 (0.41)a 0.85 (0.49)c 0.88 (0.27)b MS0/TDZ/ BAP / 2ip (Control) NA NA NA NA Each value represents the mean (SE) of 2 replicates. Means followed by the same letter(s) are not NA = data not available due to lack of callus/shoot formation.
99 APPENDIX PROCEDURE FOR PLASTIC (GLYCOL METHACRYLATE BASED) EMBEDDING Paraformaldehyde stock solution A stock solution of paraformaldehyde (16 %) is prepared by adding the appropriate weight of paraformaldehyde into a beaker containing boiling distilled water in which a few drops of 1 N KOH have been added. The solution is stirred continuously with heat to dissolve the powder. After approximately 5 minutes, the solution should be clear with a few undissolved particles. The final volume of the solution is then adjusted. The solution should then be filtered and stored in a tightly capped glass bottle. Fixative To prepare 100 ml of fixative, 50 ml of 0.1 M phosphate buffer is mixed with 10 ml of each paraformaldehyde and glutaraldehyde stock solutions and 30 ml of distilled water. Using the above procedure, the final concentration of the fixative is a 1.6 % paraformaldeyde, 2.5% glutaraldehyde in 0. 05 M phosphate buffer. The preparation of the paraformaldehyde solution and fixative solution were carried out in the fume hood Explant/ t issue preparation The explants and organogenic/ embryogenic tissues are collected from culture vessels and the appr opriate part is carefully excised and trimmed to the desired orientation with a sharp double edge razor blade. Fixing and vacuuming The tissues are fixed at room temperature for 1 2 h prior to a vacuuming step. After vacuuming, the fixative is replaced a nd the vials are transferred to a refrigerator and left overnight at 4C. The total fixation time should be no more than 24 h. Dehydration After fixation, the specimen is dehydrated with methyl cellosolve followed by two changes of absolute ethanol. Dehy dration should take place at 4C to
100 minimize extraction of macromolecules from cells. After the completion of dehydration, if the specimens are not processed immediately, they can be stored in the freezer and used at a later date. Infiltration To prepare the infiltration solution, one packet of activator (benzoyl peroxide powder, moistened with 20% H2O, supplied in packets of 0.5 gm) should be dissolved in 50 ml of the basic resin. The infiltration solution should be kept at 4 C but bottle should be allo wed to warm up to room temperature prior to its use in order to prevent condensation of water vapor from the air. Infiltration should be carried out gradually with a mixture of absolute ethyl alcohol and infiltration solution in a ratio of 2:1, 1:1, 1:2 be fore transferring to the pure infiltration solution. The duration of infiltration depends on the size and the density of the specimens. Embedding Plastic molding cups are used for the embedding of specimens. For embedding the embryos/tisssues are poured together with the infiltration solution into one or more wells. Once the embryos are in place, the embedding solution is prepared. This is done by mixing 15 ml of infiltration solution with 1 ml of the hardener. This solution should be used immediately be cause polymerization begins as soon as it is prepared. The embryos are rinsed briefly with the embedding solution by adding a small quantity of the embedding solution into each well and then removed immediately. After rinsing, the embedding solution is add ed so that it fills the wells close to their rim. Working quickly, the orientation of the embryos within the wells should be checked using a stereomicroscope if necessary, prior to the addition of the round plastic specimen adapter. Once the embryos are pr operly arranged within the well, a plastic block holder is placed gently on top of each well to exclude air from the surface of the
1 01 mold as oxygen interferes with the polymerization process. T he entire tray is then left on the bench for at least two hours, by which time the embedding solution should be polymerized.
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115 BIOGRAPHICAL SKETCH Born to a nuclear family in Nigeria, West Africa, Olubunmi Aina (Bunmi) has two older brothers. Her f ather was a high school principal while her mother was more involved in mixed farming than her teaching job As a result, she developed an interest in caring for plants early in childhood After a BS in a griculture from L adoke University, Bunmi worked as a memb er of the National Staff at the International Institute of Tropical Agriculture (IITA) Ibadan, and later as a consultant to the International Livestock Research Institute (ILRI). Following this further, s he earned two m aster s degrees; o ne was in a gronomy from the University of Ibadan, and the other was in organic food chain management f rom the University of Hohenheim Germany A strong interest in creating new things was a major reason why she chose to pursue doctoral studies in plant breeding and genetics Her decision to start a doctoral program at The University of Florida in Spring of 2008 was due to an interest in research projects that faculty members in Agronomy Department were focusing on. Her current research on in vitro and in vivo evaluation of w ild peanut germplasm is aimed at contributi ng towards sustainability. To facilitate the exploration of her research questions she has utilized in vitro methods for doubling the chromosome number of a wild peanut species and determined how this approach co uld be used to remove the barriers existing in gene transfer between the wild species and the cultivated peanut Curious by nature, Bunmi enjoyed the fact that her research work allow ed her to discover, think and inquire. As a research assistant in the Agr onomy D epartment, she gained hands on experiences both in the lab and on the field. A unique accomplishment in this capacity was identifying the correct timing in abundant seed production in rhizoma peanut. This novel and exciting discovery has the potenti al of chang ing the
116 current approach to the genetic improvement and breeding of this forage crop Bunmi has earned a Doctor of Philosophy in a gronomy from The University of Florida in 201 1.