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Characterization Of Radiotolerance In Potato And Development Of A Gamma Radiation Phytosensor., Robert Graham Sears Dec 2023

Characterization Of Radiotolerance In Potato And Development Of A Gamma Radiation Phytosensor., Robert Graham Sears

Doctoral Dissertations

As humans pursue space travel and nuclear energy, the risk of harm from ionizing radiation increases. On Earth or in space, plants are essential to our personal and environmental health. Plants serve as sentinels, bioremediators and food sources in areas of high ionizing radiation, therefore it is essential to understand how ionizing radiation affects plant biology. This work aimed to understand plant responses to ionizing radiation in the potato chassis and apply that knowledge to generate novel phenotypes for nuclear energy and space applications. The first gamma radiation phytosensor was developed for monitoring at standoff distances greater than three meters. …


Characterization Of Lignin Structural Variability And The Associated Application In Genome Wide Association Studies, Nathan D. Bryant Dec 2023

Characterization Of Lignin Structural Variability And The Associated Application In Genome Wide Association Studies, Nathan D. Bryant

Doctoral Dissertations

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall …


Regulation Of Protein Synthesis In Arabidopsis Thaliana Through A Bioinformatic And Mathematical Lens, Ricardo Andres Urquidi Camacho Dec 2023

Regulation Of Protein Synthesis In Arabidopsis Thaliana Through A Bioinformatic And Mathematical Lens, Ricardo Andres Urquidi Camacho

Doctoral Dissertations

Organisms exist under constantly varying environmental and internal conditions, which necessitate the differential regulation of gene expression. To synthesize proteins, the ribosome translates the information encoded in the nucleotide sequence of an mRNA into the final, functional amino acid sequence. Knockouts of ribosomal proteins lead to lethality. One such protein is the ribosomal protein 6 of the small subunit (eS6/RPS6). We confirmed that the knockout of either one of two eS6 paralogs in Arabidopsis leads to stunted growth and chlorosis. Here, these phenotypes have been further characterized in seedlings by precisely quantifying the ribosome loading of mRNAs as well as …


Functional Analysis Of Soybean Proteinase Inhibitor Genes And Cyst Nematode-Inducible Synthetic Promoters For Insects And Nematode-Resistance In Plants, Mst Shamira Sultana Dec 2022

Functional Analysis Of Soybean Proteinase Inhibitor Genes And Cyst Nematode-Inducible Synthetic Promoters For Insects And Nematode-Resistance In Plants, Mst Shamira Sultana

Doctoral Dissertations

Proteinase inhibitors (PIs) from legumes have the potential for use as protectants in response to pests and pathogens. Soybean (Glycine max) contains two trypsin inhibitors (TIs): Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. In addition to the three known TIs (KTI1, KTI2 and KTI3), novel inhibitors KTI5, KTI7, and BBI5 were identified in soybean. Their functional role was further examined by overexpression in soybean and Arabidopsis. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory …


Genome Evolution In The Salicaceae: Genetic Novelty, Horizontal Gene Transfer, And Comparative Genomics, Timothy Yates Aug 2022

Genome Evolution In The Salicaceae: Genetic Novelty, Horizontal Gene Transfer, And Comparative Genomics, Timothy Yates

Doctoral Dissertations

Genome evolution is a powerful force which shapes genomes over time through processes like mutation, horizontal transfer, and sexual reproduction. Although questions which aim to explore genome evolution are broad, they are all understood through the discovery and comparison of genetic variation. For example, genetic diversity may explain differences in phenotypes, etiology of disease, and is essential for phylogenomic analysis. Recently, the democratization of next generation and third generation DNA sequencing technologies have allowed for genomics to produce large amounts of sequence data. This has facilitated the capture of genetic variation at species and population scales.

Populus and Salix are …


Poa Annua: An Annual Species?, Devon E. Carroll May 2022

Poa Annua: An Annual Species?, Devon E. Carroll

Doctoral Dissertations

Poa annua L. is ranked the most troublesome turfgrass weed but can also be a highly desirable turfgrass species. As the Latin name annua implies, the species is thought to persist via an annual life cycle; yet there are many reports in literature of P. annua persisting perennially. Considering that P. annua senescence patterns do not align with other true annual species such as Triticum spp. and Zea mays L., we hypothesized that P. annua presents itself similarly to other perennial, cool-season, turfgrass species that are subject to a confluence of environmental factors in summer that can cause mortality. …


Nodulin 26 Like Intrinsic Proteins: Structurally Similar Membrane Channels With Diverse Functions In Plant Hypoxia Stress, Metalloid Nutrition & Toxicity, Zachary Beamer May 2022

Nodulin 26 Like Intrinsic Proteins: Structurally Similar Membrane Channels With Diverse Functions In Plant Hypoxia Stress, Metalloid Nutrition & Toxicity, Zachary Beamer

Doctoral Dissertations

Plant nodulin 26 intrinsic proteins are categorized into three groups (NIP I, II, and III) based on pore architecture. NIP II and III participate in metalloid nutrition, whilst the function of a third (NIP I) is less understood. Here we investigate the physiological function of one NIP I protein (Arabidopsis thaliana NIP2;1) as a lactic acid channel, and also explore the structural basis for metalloid and water permeability of NIP I and NIP II proteins in general. In addition, a strategy was developed for the purification and crystallization of soybean nodulin 26 as a step towards structure determination of a …


Plant Community Responses To Interactive Anthropogenic Disturbances Along A Natural-Wildland-Urban Gradient And Undergraduate Students’ Attitudes Toward Disturbances, Mali M. Hubert May 2022

Plant Community Responses To Interactive Anthropogenic Disturbances Along A Natural-Wildland-Urban Gradient And Undergraduate Students’ Attitudes Toward Disturbances, Mali M. Hubert

Doctoral Dissertations

Anthropogenic disturbances are defined as any change caused by human activity that alters biodiversity. Wildfire and urbanization disturbances are among the most influential on the landscape because of their individual and interactive properties. Areas deemed wildland-urban interfaces (WUI; area where environment intermingles with human-built structures) are increasing near protected lands because of human population growth and movement, which often facilitates fire ignitions by humans. Houses that are adjacent to or overlap with wildland vegetation can complicate protection of urban development and wildlands from fires. The expansion of the WUI due to population growth will exacerbate fire risk, which can ultimately …


Calmodulin Like 38 Is Required For Autophagy Of Hypoxia-Induced Cytoplasmic Rna Granules In Arabidopsis Thaliana, Sterling Field Dec 2021

Calmodulin Like 38 Is Required For Autophagy Of Hypoxia-Induced Cytoplasmic Rna Granules In Arabidopsis Thaliana, Sterling Field

Doctoral Dissertations

In response to the energy crisis resulting from submergence stress and hypoxia, the model plant Arabidopsis thaliana limits non-essential mRNA translation, and accumulates cytosolic stress granules. Stress granules are phase-separated mRNA-protein particles that partition transcripts for various fates: storage, degradation, or return to translation after stress alleviation. Another response by the plant cell to low oxygen stress is the induction of the turnover pathway autophagy. Stress granule regulation by autophagy occurs by a ‘granulophagy’ pathway in yeast and mammalian systems through which parts or whole stress granules are degraded. Whether this occurs in plants has not been investigated.

A connection …


Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro Dec 2021

Erecta Family Genes Regulate The Shoot Apical Meristem And Organ Formation, Daniel A. Degennaro

Doctoral Dissertations

Plants are sessile and must adjust their organ growth to their environments. A reservoir of stem cells in the shoot apical meristem (SAM) supplies cells for differentiation into organs. The SAM must balance organ production with stem cell maintenance. The ERECTA family (ERfs) encodes the leucine-rich repeat receptor-like kinases ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERL2. ERf signaling regulates organ initiation and stem cell maintenance. Results presented in this work include the following:

1) WUSCHEL (WUS) and CLAVATA3 (CLV3) make up a negative feedback loop to maintain SAM size. WUS and CLV3 expression localization is critical for …


Identification And Functional Characterization Of Plant Small Secreted Proteins During Arbuscular Mycorrhizal Symbiosis, Xiaoli Hu Aug 2021

Identification And Functional Characterization Of Plant Small Secreted Proteins During Arbuscular Mycorrhizal Symbiosis, Xiaoli Hu

Doctoral Dissertations

Plant small secreted proteins (SSPs) are sequences of 50 – 250 amino acids in size which are transported out of cells to fulfill multiple functions related to plant growth and development and response to various stresses. With the development of more accurate and affordable genome sequencing technology, an increasing number of SSPs have been predicted using diverse computational tools based on machine learning. Although experimentally validated plant SSPs are still limited, some studies have reported that plant SSPs can be induced and involved in mutualistic relationships between plants and microbes. In Chapter I, known SSPs and their functions in various …


Epigenetic Mechanisms Governing Plant Growth, Development, And Responses To Nematode Parasitism, Meredith M. Bennett May 2021

Epigenetic Mechanisms Governing Plant Growth, Development, And Responses To Nematode Parasitism, Meredith M. Bennett

Doctoral Dissertations

Epigenetic mechanisms, including histone and DNA methylation and microRNAs, play key roles in mediating transcriptional changes during plant development and stress responses. However, how these interconnected epigenetic components regulate gene expression in a spatiotemporal fashion remains partially known. Here, I generated 15 transgenic Arabidopsis GUS reporter lines for genes involved in DNA methylation and demethylation pathways. The spatiotemporal expression patterns of these genes were profiled in various plant organs during development, exogenous phytohormone response, and plant-parasitic nematode pathogenesis. The analyses revealed unique and overlapping expression patterns in roots, shoots, and reproductive organs, emphasizing the importance of a DNA methylation—demethylation equilibrium. …


Root Phosphomonoesterase As A Vital Component Of Increasing Phosphorus Availability In Tropical Forests, Kristine Grace Manno Cabugao Dec 2020

Root Phosphomonoesterase As A Vital Component Of Increasing Phosphorus Availability In Tropical Forests, Kristine Grace Manno Cabugao

Doctoral Dissertations

Tropical forests, relative to other terrestrial ecosystems, exchange the largest amount of carbon with the atmosphere and also constitute a significant carbon sink. However, nutrient limitation, particularly of phosphorus (P), could limit growth of tropical forests and their function with the global carbon cycle. Thus, understanding root mechanisms to acquire P is necessary to representing the P cycle and corresponding interactions with plant growth. A large portion of total soil P in tropical forests occurs in organic forms, only accessible through root and microbial production of phosphatase enzymes. These phosphatase enzymes mineralize organic P into orthophosphate, the form of P …


Non-Canonical Signaling From The Etr1 And Etr2 Ethylene Receptors In Arabidopsis Thaliana, Arkadipta Bakshi Dec 2017

Non-Canonical Signaling From The Etr1 And Etr2 Ethylene Receptors In Arabidopsis Thaliana, Arkadipta Bakshi

Doctoral Dissertations

The gaseous phytohormone ethylene regulates several physiological and developmental processes in higher plants. There are five ethylene receptor isoforms that mediate the responses to ethylene in the model plant Arabidopsis thaliana. Prior research has shown that these five ethylene receptor isoforms in Arabidopsis have both overlapping and non-overlapping roles in regulating diverse responses such as growth in air, growth recovery after removal of ethylene, and ethylene stimulated nutational bending. Functional divergence of ETR1 has been determined in controlling some of these traits and in some of these cases, ETR1 subfunctionalization requires the receiver domain. Using homology modeling and sequence …


Evaluation Of Off-Type Grasses In Interspecific Hybrid Bermudagrass [Cynodon Dactylon (L.) Pers. X C. Transvaalensis Burtt-Davy] Putting Greens, Eric Hall Reasor May 2017

Evaluation Of Off-Type Grasses In Interspecific Hybrid Bermudagrass [Cynodon Dactylon (L.) Pers. X C. Transvaalensis Burtt-Davy] Putting Greens, Eric Hall Reasor

Doctoral Dissertations

The economic impact of the golf industry in the United States (U.S.) in 2011 was estimated to be $176.8 billion. Interspecific hybrid bermudagrasses [Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy] are some of the most widely utilized grasses on golf courses throughout tropical, subtropical, and temperate climates. In 2007, bermudagrass was grown on 80% of putting green acreage in the southern U.S. ‘Tifgreen’ and ‘Tifdwarf’ were two of the first widely established cultivars on putting greens, but their genetic instability led to the occurrence of phenotypically different off-type (OT) grasses. Several OT grasses were selected and released as …


Computational Analyses Of Mrna Ribosome Loading In Arabidopsis Thaliana, Joseph Benjamin Ernest Aug 2016

Computational Analyses Of Mrna Ribosome Loading In Arabidopsis Thaliana, Joseph Benjamin Ernest

Doctoral Dissertations

Translation of mRNA into protein is a critical step in gene expression, but the principles guiding its regulation at the genome level are not completely understood. Translation can be quantified at a genome scale by measuring the ribosome loading of mRNA—the extent to which mRNA is associated with ribosomes. In this dissertation, I present investigations into how genome-wide ribosome loading is controlled in Arabidopsis thaliana. In chapter 1, I give an overview of regulation of ribosome loading and translation. In chapter 2, I present research demonstrating for the first time that genome-wide ribosome loading in plants is partially controlled by …


Modification Of Carbohydrate Active Enzymes In Switchgrass (Panicum Virgatum L.) To Improve Saccharification And Biomass Yields For Biofuels, Jonathan Duran Willis Aug 2016

Modification Of Carbohydrate Active Enzymes In Switchgrass (Panicum Virgatum L.) To Improve Saccharification And Biomass Yields For Biofuels, Jonathan Duran Willis

Doctoral Dissertations

The natural recalcitrance of plant cell walls is a major commercial hurdle for plant biomass to be converted into a viable energy source as alternative to fossil fuels. To circumvent this hurdle manipulation of carbohydrate enzymes active in the cellulose and hemicellulose portions of the plant cell wall can be utilized to improve feedstocks. Production of cellulolytic enzymes by plants have been evaluated for reducing the cost associated with lignocellulosic biofuels. Plants have successfully served as bioreactors producing bacterial and fungal glycosyl hydrolases, which have altered plant growth to improve saccharification. A bioprospecting opportunity lies with the utilization of insect …


Decoding The Cellular Zipcode: Functional Analysis Of Transit Peptide Motifs And Mechanistic Implications In Plastid Targeting And Import, Kristen N. Holbrook Aug 2016

Decoding The Cellular Zipcode: Functional Analysis Of Transit Peptide Motifs And Mechanistic Implications In Plastid Targeting And Import, Kristen N. Holbrook

Doctoral Dissertations

Eukaryotic organisms are defined by their compartmentalization and various organelles. The membranes that define these organelles require complex nanomachines (known as translocons) to selectively mediate the import of proteins from the cytosol where they are synthesized into the organelle. The plastid, (specifically the chloroplast) which is characteristic of plant cells, possibly represents the most complex system of protein sorting, requiring many different translocons located in the three membranes found in this organelle. Despite having a small genome, the vast majority of plastid-localized proteins are nuclear-encoded and must be post-translationally imported from the cytosol. These proteins are encoded as a larger …


Computational Identification Of Terpene Synthase Genes And Their Evolutionary Analysis, Qidong Jia May 2016

Computational Identification Of Terpene Synthase Genes And Their Evolutionary Analysis, Qidong Jia

Doctoral Dissertations

Terpenoids, the largest and most structurally and functionally diverse class of natural compounds on earth, are mostly synthesized by plants to be involved in various plant environment interactions. Some terpenoids are classified as primary metabolites essential for plant growth and development. Terpene synthases (TPSs), the key enzymes for terpenoid biosynthesis, are the major determinant of the tremendous diversity of terpenoid carbon skeletons. The TPS genes represent a mid-size family of about 30-100 functional genes in almost all major sequenced plant genomes. TPSs are also found in fungi and bacteria, but microbial TPS genes share low levels of sequence similarity and …


Calmodulin-Like Protein 38: A Component Of Ribonucleoprotein Particles During Hypoxic Stress Responses In Arabidopsis, Ansul Lokdarshi Aug 2015

Calmodulin-Like Protein 38: A Component Of Ribonucleoprotein Particles During Hypoxic Stress Responses In Arabidopsis, Ansul Lokdarshi

Doctoral Dissertations

Waterlogging stress leads to a crisis in energy metabolism and the accumulation of toxic metabolites due to the hypoxic and/or anoxic environment associated with this condition. To respond and adapt to this situation, higher plants employ an integrated genetic program that leads to the induction of anaerobic response polypeptide genes that encode metabolic and signaling proteins involved in altering metabolic flow and other adaptive responses. The study presented here shows that the Arabidopsis thaliana calmodulin-like protein CML38 is calcium sensor protein that serves as a member of the core anaerobic response gene family and is involved in modulating the survival …


Pore Selectivity And Gating Of Arabidopsis Nodulin 26 Intrinsic Proteins And Roles In Boric Acid Transport In Reproductive Growth, Tian Li Dec 2014

Pore Selectivity And Gating Of Arabidopsis Nodulin 26 Intrinsic Proteins And Roles In Boric Acid Transport In Reproductive Growth, Tian Li

Doctoral Dissertations

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional channels of uncharged metabolites and water. They share the same canonical hourglass fold as the aquaporin family. The aromatic arginine (ar/R) selectivity filter controls transport selectivity based on size, hydrophobicity, and hydrogen bonding with substrates. In Arabidopsis thaliana, NIP II subclass proteins contain a conserved ar/R “pore signature” that is composed of Alanine at the helix 2 position (H2), Valine/Isoleucine at the helix 5 position (H5), and an Alanine (LE1) and an invariant Arginine (LE2) at the two loop E positions. In this study, …


Toward Direct Biosynthesis Of Drop-In Ready Biofuels In Plants: Rapid Screening And Functional Genomic Characterization Of Plant-Derived Advanced Biofuels And Implications For Coproduction In Lignocellulosic Feedstocks, Blake Lee Joyce Aug 2013

Toward Direct Biosynthesis Of Drop-In Ready Biofuels In Plants: Rapid Screening And Functional Genomic Characterization Of Plant-Derived Advanced Biofuels And Implications For Coproduction In Lignocellulosic Feedstocks, Blake Lee Joyce

Doctoral Dissertations

Advanced biofuels that are “drop-in” ready, completely fungible with petroleum fuels, and require minimal infrastructure to process a finished fuel could provide transportation fuels in rural or developing areas. Five oils extracted from Pittosporum resiniferum, Copaifera reticulata, and surrogate oils for Cymbopogon flexuosus, C. martinii, and Dictamnus albus in B20 blends were sent for ASTM International biodiesel testing and run in homogenous charge combustion ignition engines to determine combustion properties and emissions. All oils tested lowered cloud point. Oils derived from Copaifera reticulata also lowered indicated specific fuel consumption and had emissions similar to the ultra-low sulfur diesel control. Characterization …


Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham May 2013

Development And Application Of Mass Spectrometry-Based Proteomics To Generate And Navigate The Proteomes Of The Genus Populus, Paul Edward Abraham

Doctoral Dissertations

Historically, there has been tremendous synergy between biology and analytical technology, such that one drives the development of the other. Over the past two decades, their interrelatedness has catalyzed entirely new experimental approaches and unlocked new types of biological questions, as exemplified by the advancements of the field of mass spectrometry (MS)-based proteomics. MS-based proteomics, which provides a more complete measurement of all the proteins in a cell, has revolutionized a variety of scientific fields, ranging from characterizing proteins expressed by a microorganism to tracking cancer-related biomarkers. Though MS technology has advanced significantly, the analysis of complicated proteomes, such as …


Functional Genomic Studies Of Soybean Defenses Against Pests And Soybean Meal Improvement, Jingyu (Lynn) Lin Dec 2011

Functional Genomic Studies Of Soybean Defenses Against Pests And Soybean Meal Improvement, Jingyu (Lynn) Lin

Doctoral Dissertations

Soybean [Glycine max (L.) Merr.] is an important crop worldwide. It has been widely consumed for protein, oil and other soy products. To develop soybean cultivars with greater resistance against pests and improved meal quality, it is important to elucidate the molecular bases of these traits. This dissertation aims to investigate the biochemical and biological functions of soybean genes from four gene families, which are hypothesized to be associated with soybean defense against pests and soybean meal quality. There are three specific objectives in this dissertation. The first one is to determine the function of components in the salicylic …


Developing Biocontainment Strategies To Suppress Transgene Escape Via Pollen Dispersal From Transgenic Plants, Hong Seok Moon Aug 2011

Developing Biocontainment Strategies To Suppress Transgene Escape Via Pollen Dispersal From Transgenic Plants, Hong Seok Moon

Doctoral Dissertations

Genetic engineering is important to enhance crop characteristics and certain traits. Genetically engineered crop cultivation brings environmental and ecological concerns with the potential of unwanted transgene escape and introgression. Transgene escape has been considered as a major environmental and regulatory concern. This concern could be alleviated by appropriate biocontainment strategies. Therefore, it is important to develop efficient and reliable biocontainment strategies.

Removing transgenes from pollen has been known to be the most environmentally friendly biocontainment strategy. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco …


Developmental Evolution Of The Progamic Phase In Nymphaeales, Mackenzie Lorraine Taylor May 2011

Developmental Evolution Of The Progamic Phase In Nymphaeales, Mackenzie Lorraine Taylor

Doctoral Dissertations

The period between pollination and fertilization, or the progamic phase, is a critical life history stage in seed plants and innovations in this life history stage are hypothesized to have played an important role in the diversification of flowering plants. Over the course of this dissertation research, I investigated programic phase development in Nymphaeales (water lilies), an ancient angiosperm lineage that diverged from the basalmost or next most basal node of the angiosperm phylogenetic tree and that is represented in the oldest angiosperm fossil record. I used field experiments and microscopy to document pollination biology, breeding system, and reproductive developmental …


Spatiotemporal Dynamics In A Lower Montane Tropical Rainforest, Robert Michael Lawton Aug 2010

Spatiotemporal Dynamics In A Lower Montane Tropical Rainforest, Robert Michael Lawton

Doctoral Dissertations

Disturbance in a forest’s canopy, whether caused by treefall, limbfall, landslide, or fire determines not only the distribution of well-lit patches at any given time, but also the ways in which the forest changes over time. In this dissertation, I use a 25 year record of treefall gap formation find a novel and highly patterned process of forest disturbance and regeneration, providing a local mechanism by examining the factors that influence the likelihood of treefall. I then develop a stochastic cellular automaton for disturbance and regeneration based on the analysis of this long term data set and illustrate the potential …


In Vitro Propagation, Regeneration, Attempted Tetraploid Induction, And Agrobacterium-Mediated Transformation Of Euphorbia Pulchurrima ‘Winter Rose’™, Kimberly Ann Pickens Dec 2004

In Vitro Propagation, Regeneration, Attempted Tetraploid Induction, And Agrobacterium-Mediated Transformation Of Euphorbia Pulchurrima ‘Winter Rose’™, Kimberly Ann Pickens

Doctoral Dissertations

Poinsettia, Euphorbia pulchurrima, is the number one potted flowering plant in the United States. ‘Winter Rose’™ is a very popular cultivar with more than one million plants sold each year. To further improve this cultivar, particularly for larger flower heads and free branching, this research aimed at establishing some in vitro systems for application of biotechnology to poinsettia genetic improvement.

A protocol was established for in vitro axillary bud proliferation using greenhouse grown terminal buds. Buds were placed on Murashige-Skoog (MS) basal medium supplemented with benzlyaminopurine (BA). Explants produced the greatest number of axillary buds on medium containing between 2.2-8.8 …