Culture Morphology as a Biomarker for Somatic

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ACKNOWLEDGEMENTS, We would like to acknowledge and thank our advisors Professor Susan Roberts and Elizabeth. Cummings Bende for their continual support and guidance throughout this project Additionally. we would like to thank Michelle McKee for her advice and assistance in the lab. This research investigated possible predictive biomarkers for embryo yield of somatic. embryogenesis SE in Pinus taeda suspension culture Previous research in related systems. suggest that arabinogalactan protein AGP levels and or culture size morphology may serve as a. determinant for downstream embryo yield Size fractionation of culture was performed with a 710. m sieve and the embryo yield of each size fraction unfiltered control 710 m and 710 m. was determined 12 weeks after plating on developmental media Cell associated AGP. concentrations in each size fraction were also measured to determine if there is a difference in. between large and small aggregates Results demonstrated that the 710 m fraction developed. more embryos in two separate experiments additional size fractionation verification is. recommended to confirm this relationship Results also showed that larger aggregate have higher. concentrations of AGP when compared to smaller aggregate cultures opening up new avenues of. research in AGP analyses in the search for a predictive biomarker in early stage somatic. embryogenesis,TABLE OF CONTENTS,ACKNOWLEDGEMENTS ii. ABSTRACT iii,TABLE OF FIGURES v,INTRODUCTION AND BACKGROUND 1. Introduction to Somatic Embryogenesis 1,Initiation 1. Proliferation 1,Maturation 2,Optimizing Somatic Embryogenesis 4.
AGPs as biomarkers 5,Coulter counter 5,MATERIALS METHODS 7. Maintenance media 7,Development media 7,Culturing 7. Coulter Counter 8,Size fraction method development 8. Size fraction experiment methods 9,Dialysis buffer and protein extraction buffer 10. Cell associated protein extraction 10,Colorimetric Yariv assay 11.
RESULTS DISCUSSION 12,CONCLUSIONS 18,REFERENCES 19. TABLE OF FIGURES, Figure 1 60 Day time lapse tracking of development pathway of somatic embryogenesis in. Picea abies Norway spruce for cell line B41 Reproduced from Filonova et al 2000 2. Figure 2 Somatic embryo development in Picea abies Norway spruce Reproduced from. Filonova et al 2000 3, Figure 3 Embryo yield of loblolly pine suspension cultures vs volume of aggregates with. d 273 m mm3 Reproduced from Cummings Bende et al 2018 5. Figure 4 710 m sieve in collection dish 9,Figure 5 Size fraction experimental model 10. Figure 6 Experiment 1 Embryo yield per gram of growth n 5 14. Figure 7 Experiment 2 Embryo yield per gram of growth n 5 15. Figure 8 Concentrations of AGP normalized with fresh weight n 3 16. Figure 9 Media AGP vs Cell associated AGP n 3 17,INTRODUCTION BACKGROUND.
Somatic embryogenesis is the development of plant embryos in culture from somatic cells that are. not necessarily involved in plant reproduction These somatic embryos can then be germinated. into new plants with identical genotypes to the original somatic cells In recent years this process. has been researched as both a tool for understanding the mechanisms of embryo development and. a method for developing crops with ideal characteristics This area of research is of particular. interest to the forestry industry Crops that have genotypes that are cost effective and well suited. to agriculture are highly desirable to lumber cultivators Therefore a process such as somatic. embryogenesis that could produce a large scale source of genetically desirable plant embryos. ready to be germinated is of great interest to these cultivators While the use of somatic. embryogenesis is well established there remain limitations in its application the most notable. being unpredictable and variable embryo yields and a lack of understanding of the role of. morphological structures This section of the report discusses the key developments in somatic. embryogenesis and underscores areas requiring further attention. Introduction to Somatic Embryogenesis, Somatic embryogenesis SE was first observed in Daucus carota suspension cultures well over. fifty years ago Steward et al 1958 Since this time many researchers have independently studied. embryo development and the morphological characteristics in various species such as conifers. and other pines Filonova et al 2000 Steiner et al 2016 Generally there are three stages of. development from the explant to somatic embryos initiation proliferation and maturation von. Arnold 2002, Initiation Initiation of somatic embryo cultures begins with a sterilized cutting from the original. plant also known as an explant Cummings Bende et al 2018 For the cells to successfully. transition to embryos gene expression must be modified Research has demonstrated that this can. be induced by osmotic shock and addition of auxins such as 2 4 dichlorophenoxyacetic acid von. Arnold 2002 Furthermore successful initiation in embryogenic cultures requires careful. selection of the explant particularly for conifers It has been demonstrated that older explants and. vegetative tissues lead to lower competent embryos Roberts et al 1989. Proliferation Upon successful initiation a callus of dedifferentiated cells forms SE can be. induced at this phase known as direct SE or suspended in culture and induced later or indirect. SE von Arnold et al 2002 When suspended in culture cells proliferate Suspension in culture is. particularly advantageous for conifers and other industrially relevant crops as embryos can be. propagated at large scale The addition of abscisic acid ABA or other plant growth regulators. both promotes maturation and inhibits embryo formation Maturation is characterized by a bilateral. distribution of cell types into pre embryogenic heads and suspensor cells Filonova et al 2000. The suspensor cells are highly vacuolated and provide nutrients to the embryo proper It has been. suggested that programmed cell death of the suspensor cells leads to correct formation of the. embryo Smertenko and Bozhkov 2013, During the proliferation phase conifers and more generally gymnosperms progress through. three typical stages of cellular aggregates referred to as pre embryogenic masses PEMs I II and. III The simplest structure PEM I is characterized by a mass of pre embryogenic heads attached. to a single vacuolated suspensor cell The suspensor and head cell types continue to divide while. remaining aggregated giving rise to PEM II s Finally progression to PEM III morphologies is. distinguished by yet larger aggregates and budding embryo heads Filonova et al 2000 Figure. 1 In culture these individual morphologies can be distinguished and separated by their size In. Pinus taeda loblolly pine typical diameters for each stage are on the order of 300 m 300. 700 m and 700 m with some variance among genotypes Cummings Bende et al 2018. Figure 1 60 Day time lapse tracking of development pathway of somatic embryogenesis in Picea abies Norway. spruce for cell line B41 Reproduced from Filonova et al 2000 Aggregates smaller than 200 m were isolated. in agarose gel and supplied continuously with auxins The resulting development through each of the pre embryogenic. masses is indicated in the figure, Maturation PEMs can be transferred to a solid media where they are induced to form embryos. Traditionally this is accomplished by the addition of ABA to the media Pullman et al 2003 This. hormone has been demonstrated in Norway spruce to both promote SE while stunting growth of. less developed morphologies PEM I and PEM II Filonova et al 2000 Furthermore from time. lapse tracking studies Filonova et al 2000 hypothesized that PEM III s develop directly into. somatic embryos To reach full maturity the developing Norway spruce embryos are grown in the. dark for 8 12 weeks often showing development of cotyledons as early as 7 weeks Figure 2. This stage of development is comparable to natural zygotic embryo development for Picea abies. Filonova et al 2000, Figure 2 Somatic embryo development in Picea abies Norway spruce Reproduced from Filonova et al 2000.
The budding SE heads from PEM III start to develop finer structures such as the root apical meristem shoot apical. meristem and cotyledons, While somatic embryos undergo the maturation process they must go through a period of. desiccation This process occurs naturally within the plant reproductive system as a way of. preparing seeds for an inactive period von Arnold et al 2002 Furthermore this process induces. embryos to transition the metabolic pathways from maturation to germination Stasolla and Yeung. 2003 In vitro techniques have been developed that mimic the desiccation conditions within the. plant during zygotic embryogenesis Principally this is accomplished by the addition of an osmotic. agent such as salts and polyethylene glycols PEG However higher success has been achieved. with PEG as osmotic potential more closely resembles natural processes von Arnold et al 2002. The embryos can then be germinated and cultivated into full plants Each of these stages represent. a critical developmental change where conditions must be maintained for successful growth. Continued development and understanding of these stages will lead to higher and more predictable. embryo yields and quality,Optimizing Somatic Embryogenesis. In recent years an interest in developing consistent high value crops has grown into an important. research topic in plant biology This is particularly true for coniferous species and other woody. plants destined for wood products Traditional methods of breeding lead to variable crop yields. with undesirable characteristics such as poor wood quality and vulnerability to disease. Schmidtling 1983 Additionally conifers have long growth periods requiring plentiful resources. and space SE serves to alleviate these issues However SE is still limited for practical applications. by variable embryo yields and low embryo quality A body of research has manifested that. addresses these topics and offer potential solutions Continued research and improvements in these. topics serve to increase the effectiveness of SE for industrial applications. Embryo yield and embryo quality represent different points of optimization along the path to plant. regeneration However the former can be used more readily and efficiently as a prediction of. embryo success as results can be determined as early as the conclusion of the development period. around 10 12 weeks The latter requires experiments to last through the germination period which. can take anywhere from 6 8 months post the development period Furthermore it has been. suggested that success in the early developmental stages are indicative of SE success as a whole. Stasolla and Yeung 2003 Therefore the following discussion will focus on research. advancements that center on optimizing early culture conditions during the maintenance and. development periods that quantify success with embryo yield. A particularly advantageous area of research is determining early biomarkers that could indicate. later success Such indicators would allow for easy selection of valuable cell lines without. dedicating resources to undesirable outcomes Two factors that have been implicated in this. endeavor thus far are arabinogalactan proteins AGPs and PEM morphologies and sizes. PEMs as biomarkers Previous analysis of loblolly pine maintenance cultures has shown a. negative correlation between embryo yield and the total volume of aggregates with diameters less. than 273 m as shown in Figure 3 In other words a higher volume of small aggregates such as. PEM I s in culture appears to lead to a lower number of embryos during development This. correlation would agree with the working hypothesis that embryos develop directly from PEM. III s the largest and most developed aggregates in culture Filonova et al 2000. This apparent correlation could be tested by adjusting the morphologies of the cultures to. determine its effect on embryo yield One way of achieving a desired culture morphology is to. fractionate the culture by size by physically separating the different size aggregates using a filter. or sieve Size fractionation was previously performed for loblolly pine culture using nylon meshes. Cummings Bende et al 2018 and embryogenic hybrid tea rose cultures Kamo et al 2004. Figure 3 Embryo yield of loblolly pine suspension cultures vs volume of aggregates with d 273 m mm3. Reproduced from Cummings Bende et al 2018, AGPs as biomarkers AGPs are a broad class of proteins found in various species of plants. including cotton Gossypium hirsutum Poon et al 2012 carrot Daucus carota Kreuger and. Holst 1995 and loblolly pine Pinus taeda They are characterized by their high protein to. carbohydrate ratio with protein often making up less than 10 of the total weight Majewska. Sawka et al 2000 The carbohydrate side chains vary in composition and structure however the. major sugar residues are arabinose and galactose van Hengel et al 2001. AGPs have been demonstrated to play an important role in SE For instance SE was blocked in. D carota by precipitating out extracellular AGPs using glucosyl Yariv reagent showing AGPs. are critical in the maturation process von Arnold et al 2002 This importance was further. substantiated when added exogenous AGPs induced SE in Picea abies Egertsdotter et al 1995. While the role of AGPs in SE is not yet fully understoo. 4 Optimizing Somatic Embryogenesis In recent years an interest in developing consistent high value crops has grown into an important research topic in plant biology This is particularly true for coniferous species and other woody plants destined for wood products Traditional methods of breeding lead to variable crop yields

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