Plant Cell, Tissue and Organ Culture Hort 515 Plant Regeneration Definitions and Background Information Morphogenetic Processes That Lead to Plant Regeneration Factors Affecting Morphogenesis in Vitro Genetic Variation of Plants Regenerated in Vitro Regeneration of Cereals

1. Definitions and Background Information Plant Morphology - form and structure of plants Plant Morphogenesis - differentiation and formation of organized structures; specifically processes that lead to plant regeneration from somatic cells, i.e., formation of shoots and roots (organogenesis) or somatic/adventive/asexual embryos (embryogenesis) Somatic cells - cells of the plant body, not gametes Regenerated plants are clones, within the limits of somatic cell variation, are exact genetic copies of the parent Genetic variation of regenerated plants is dependent on genetic constitution of somatic cells in the explant, and degree to which this variation increases in culture prior to regeneration.

2. Morphogenetic Processes that Lead to Plant Regeneration I. Organogenesis – formation of organs, i.e. shoots and roots Enhancement of axillary bud proliferation/ development Adventitious shoot formation Adventitious root formation II. Somatic (asexual/adventive) embryogenesis

Organogenesis - shoot initiation and development with subsequent formation of adventitious roots Adventitious - initiation from cells that are not normally the progenitors, e.g. non-meristematic cells Enhancement of axillary bud proliferation and development - stimulation of the shoot apical meristem differentiation and development, proliferation of lateral buds to shoots, example Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from nonmeristematic cells (one or more than one) either: Direct - cells of explant dedifferentiate (meristemoids) and/or then differentiate into adventitious shoots Indirect - callus is proliferated from the primary explant, dedifferentiates into meristemoids and then differentiate into shoots

Shoot Tip Propagation of Asparagus by Enhancement of Axillary Bud Development Six (6) shoots each passage Six (6) passages/year = 46656 plants/yr

Organogenesis - shoot initiation and development with subsequent formation of adventitious roots Adventitious - initiation from cells that are not normally the progenitors, e.g. non-meristematic cells Enhancement of axillary bud proliferation and development - stimulation of the shoot apical meristem differentiation and development, proliferation of lateral buds to shoots, example Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from nonmeristematic cells (one or more than one) either: Direct - cells of explant dedifferentiate (meristemoids) and/or then differentiate into adventitious shoots, example Indirect - callus is proliferated from the primary explant, dedifferentiates into meristemoids and then differentiate into shoots

Direct Adventitious Shoot Bud Development in Culture from Douglas Fir Cotyledon Explants

Organogenesis - shoot initiation and development with subsequent formation of adventitious roots Adventitious - initiation from cells that are not normally the progenitors, e.g. non-meristematic cells Enhancement of axillary bud proliferation and development - stimulation of the shoot apical meristem differentiation and development, proliferation of lateral buds to shoots, example Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from nonmeristematic cells (one or more than one) either: Direct - cells of explant dedifferentiate (meristemoids) and/or then differentiate into adventitious shoots, example Indirect - callus is proliferated from the primary explant, dedifferentiates into meristemoids and then differentiate into shoots

Tobacco Meristemoid Prior to Morphogenesis

Meristmoids - can give rise to several shoots, so those arising from one meristemoid may be clones, also meristmoids may derive from more than one cell leading to chimerism, example

Shoot Meristems Differentiated from an Individual Meristemoid of Potato Tuber Explants

I. Organogenesis - shoot initiation and development with subsequent formation of adventitious roots; adventitious - initiation from cells that are not normally the progenitors, e.g. non-meristematic cells Enhancement of axillary bud proliferation and development - stimulation of the shoot apical meristem in vitro that includes proliferation of lateral buds Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from non-meristematic cells (one or more than one) either directly or indirectly Adventitious root formation - roots are initiated adventitiously at the base of the shoot apex and a vascular continuum is established to complete plant regeneration, example

Adventitious Root Initiation in an Asparagus Shoot Tip Adventitious Root Initiation in a Rose Shoot

Somatic embryogenesis - embryo initiation and development from somatic cells Directly from cells in the explant, examples Indirectly via a callus intermediary; dedifferentiation is typically minimal but a meristemoid-like tissue can be formed in the latter case Histogenesis of somatic embryogenesis is characterized by the formation of a bipolar structure, in contrast to adventitious organogenesis Single cell origin of somatic embryos makes chimerism infrequent; adventitious shoots can arise from more than one cell

Direct Embryogenesis from Cacao Cotyledon Epidermal Cell

Somatic Embryogenesis from Cacao Cotyledons

Somatic embryogenesis - embryo initiation and development from somatic cells Directly from cells in the explant Indirectly via a callus intermediary; dedifferentiation is typically minimal but a meristemoid-like tissue can be formed in the latter case, examples Histogenesis of somatic embryogenesis is characterized by the formation of a bipolar structure, in contrast to adventitious organogenesis Single cell origin of somatic embryos makes chimerism infrequent; adventitious shoots can arise from more than one cell

Indirect Somatic Embryogenesis from Cacao Cotyledons

Somatic embryogenesis - embryo initiation and development from somatic cells Directly from cells in the explant Indirectly via a callus intermediary; dedifferentiation is typically minimal but a meristemoid-like tissue can be formed in the latter case Histogenesis of somatic embryogenesis is characterized by the formation of a bipolar structure, in contrast to adventitious organogenesis, example Single cell origin of somatic embryos makes chimerism infrequent; adventitious shoots can arise from more than one cell

Somatic Embryos of Cacao Illustrating Bipolar Meristematic Structures

Somatic embryogenesis - embryo initiation and development from somatic cells Directly from cells in the explant Indirectly via a callus intermediary; dedifferentiation is typically minimal but a meristemoid-like tissue can be formed in the latter case Histogenesis of somatic embryogenesis is characterized by the formation of a bipolar structure, in contrast to adventitious organogenesis Single cell origin of somatic embryos makes chimerism infrequent; adventitious shoots can arise from more than one cell

3. Factors Affecting Morphogenesis in Vitro I. Explant and explant source II. Culture medium III. Culture environment Totipotency, competence, determination and development Cell and tissue type Explant source age Genotype Genes that regulate morphogenesis leading to plant regeneration

Totipotency, competence, determination and development – cellular differentiation and developmental states in morphogenesis Totipotency - genetic potential of cells for embryogenesis; now extended to include organogenesis, as proposed by Haberlandt Ground state – ” cell steady state”, cells may be competent (meristematic or meristematic-like) or may be incompetent Competence – competent cells are undifferentiated and retain the capacity for differentiation and morphogenesis

Totipotency, competence, determination and development – cellular differentiation and developmental stages of morphogenesis Dedifferentiation – cells re-acquire morphogenetic competence Determination - competent cells become committed to a genetic program leading to morphogenesis induced - in response to a stimulus or permissive - preset determination is allowed to proceed Development – developmental pathway is fixed, i.e. organogenesis or embryogenesis Example - diagramatic example of cellular stages

Topics in Biotechnology/Plant Biology Lozovaya et al. (2006) Biochemical features of maize tissues with different capacities to regeneration plants. Planta 224:1385-1399 Vidi et al. (2007) Pastoglobules: a new address for targeting recombinant proteins in the chloroplast. 7;4

Cell Developmental Phases Leading to Morphogenesis

Certain cell types retain competence – regeneration practice dictates isolation of these for induced or permissive determination Cells may be “induced” to re-acquire competence by differentiation but this has been demonstrated for only a few genotypes, e.g. tobacco, carrot

Cell and tissue type - Explants for plant regeneration are composed of undifferentiated (meristematic) or differentiated, and morphogenetically competent or incompetent cells Explants must contain competent cells or cells capable of regaining competence (dedifferentiation) Axillary bud proliferation - shoot tip or shoot meristem or nodal explants Regeneration is based on facilitating differentiation and development leading to formation and development of axillary buds into shoots Cells of these explants are competent and committed to shoot development

Cell and tissue type Axillary bud proliferation Adventitious shoot formation or somatic embryogenesis Less differentiated cells tend to be morphogenetically competent Morphogenetic competence is associated with meiosis and its natural expression is during zygotic embryogenesis/embryogeny Somatic cells (maternal) associated with meiotic events, e.g. mircro- and mega-sporogenesis, and micro- and mega-gametogenesis, are a source of morphogenetically competent cells Hence, inflorescence, anther, ovule and ovary tissues, and embryos are sources of competent cells Presumably, expression of competence is repressed during embryogeny and de-repressed during meiosis, example

Embryogenic Competence of cells in citrus nucellar tissue Citrus varieties are mono-embryonic (zygotic embryo only) or poly-embryonic (zygotic and somatic embryos) Somatic embryos are produced from cells of the nucellus, inner ovular (maternal cells) tissue Embryo production from cultured nucellar tissue: Variety Intact Micropylar half Chalazal half Monoembryonic mono poly none Polyembryonic poly poly none These results indicate that cells in the micro-pylar half of the nucellus are competent for somatic embryogenesis in both monoembryonic and polyembryonic citrus types

Chalazal half of the nucellus from monoembryonic varieties Inhibits embryogeny of carrot callus Chalazal half of the citrus nucellus produces water soluble inhibitors of somatic embryogenesis

Citrus/carrot example illustrates that: Competence of cells in tissues is genetically determined Maternal cells associated with meiosis may be competent for embryogenesis Repression of totipotency/competence occurs during embryogenesis and embryogeny and “inhibitors” may regulate chemical and/or genetic repression

3. Factors Affecting Morphogenesis in Vitro I. Explant and explant source II. Culture medium III. Culture environment A. Totipotency, competence and determination B. Cell and tissue type C. Explant source age D. Genotype E. Genes that regulate morphogenesis leading to plant regeneration

Explant source age – developmentally immature organs are most likely to contain morphogenetically competent cells, i.e. less differentiated cells are more competent Within an organ, loss of competence is correlated with maturation, i.e. extent of differentiation, examples

Developmental Stage and Adventitious Shoot Formation from Tomato Leaf Discs Fig. 2: Changes in the shoot-forming capacity of the genotype PU 76-02 associated with changes in the developmental state of the explant source. The bars around each mean represent standard error values. A. Leaves were sampled in succession from the shoot apex, beginning with the 1st leaf from the apex at least 5 cm in length and continuing through the 5th consecutive leaf. Each point is an average number for 20 explants. B. The 2nd and 3rd leaves numbered in relation to the apical leaf closest to 5 cm in length were sampled from plants 6, 8, and 11 weeks after germination. Each point is an average number for 40 explants. C. Discs were excised from 3 positions on the leaflet; proximal to the petiole, midleaflet, and distal to the petiole. Each point is an average number for 30 explants. Z. Pflanzenphysiol. Bd. 102. S. 221-232. 1981.

Developmental Stage Positions in a Tomato Leaf

Genotype - Genotypes can be classified based on whether or not cells can be induced in vitro to reacquire competence, e.g. carrot/tobacco/potato vs. cereals/legumes Competent cells must be included in the cultured explant of species for which re-acquisition of competence cannot be induced Morphogenesis in vitro is an inherited trait, example

Adventitious Shoot Forming Capacity of Tomato Genotypes ● ● ● ● ● ● ●

Adventitious Shoot Forming Capacity Is Inherited in Tomato

Adventitious Shoot Forming Capacity of Tomato Is Quantitatively Inherited Source Crosses GCA SCA Reciprocal D.F. 5 9 15 Mean Squares 125.19 10.41 5.30 F 34.56** 2.87** 1.46 n.s. **significant at the 1% level.

Genes that regulate plant regeneration – identification of genes that regulate plant development may result in new approaches for plant regeneration in vitro Adventitious Shoot Formation: KNAT1 family transcription factors, example Cytokinin biosynthetic and signaling genes, etc. Somatic Embryogenesis: SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) Transcription factors: WUSCHEL, BABYBOOM, LEAFY COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT REGERATION1

KNAT1 family transcription factors Homeotic selector genes – encode a conserved family of gene regulatory proteins (transcription factors) that activate developmental programs in higher eukaryotes Homeodomain proteins – transcription factors that contain conserved DNA-binding domains, which interact with cis elements in promoters of genes necessary for developmental programs, e.g. vegetative and floral meristem development

KNAT1 - homeotic gene involved in shoot meristem differentiation in Arabidopsis Homeotic genes were first discovered in Drosophila, are conserved in higher eukaryotes and orthologs exist in other plant species KNAT1 is expressed naturally only in the shoot meristem Constitutive (35S-driven) ectopic expression resulted in shoot meristem initiation in leaves Chuck et al. (1996) Plant Cell 8:1277-1289 (examples)

35S::KNAT1 Expression Mediates Adventitious Shoot Formation in Arabidopsis Transverse section of a wild-type rosette leaf, including the midvein. Bar = 100 m. (B) Transverse action of a 35S::KNAT1 rosette leaf, including the midvein and enlarged secondary veins. Bar = 100 m. Close-up of a transverse section from the wild type showing palisade and spongy parenchyma. Bar = 50 m. Close-up of a transverse section from a 35S::KNAT1 leaf showing an abnormal vein, tightly packed cells, and lack of palisade parenchyma. Bar = 50 m. Transverse section of a cauline leaf from a 35S::KNAT1 transformant with an initiating inflorescence meristem over a vein. Bar = 25 m. Transverse section of a 35S::KNAT1 rosette leaf with an initiating vegetative meristem over a vein. A procambial strand is differentiating between the leaf primordia and the existing vein. Bar = 25 m.

KNAT1 Expression Is Correlated with Development of an Adventitious Shoot Meristem (Fig. 6A and B)

Genes that regulate plant regeneration – identification of genes that regulate plant development may result in new approaches for plant regeneration in vitro Adventitious Shoot Formation: KNAT1 family transcription factors Cytokinin signaling and shoot effector genes, etc., illustration Somatic Embryogenesis: SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) Transcription factors: WUSCHEL, BABYBOOM, LEAFY COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT REGERATION1

Positive and Negative Regulators of Shoot Formation in Arabidopsis (Cytokinin Overproducing) Howell et al. (2003) Trends Plant Sci 8:453-459

Genes that regulate plant regeneration – identification of genes that regulate plant development may result in new approaches for plant regeneration in vitro Adventitious Shoot Formation: KNAT1 family transcription factors Cytokinin singaling and shoot effector genes, etc. Somatic Embryogenesis: SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) Transcription factors: WUSCHEL, BABYBOOM, LEAFY COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT REGERATION1

II. Culture medium Basal medium - essential micro- and macronutrients, carbon source (sucrose/glucose), thiamine-HCl, and i-inositol NH4+ may facilitate organogenesis, NO3- favors embryogenesis, and glutamine/asparagine/proline favor embryogenesis. Growth regulators - auxin and cytokinin are the critical components for regulation of morphogenesis in vitro Hormonal requirements differ depending on the morphogenetic process Organogenesis Enhancement of axillary bud proliferation - (Wickson and Thimann, 1958) - cytokinin antagonizes apical dominance caused by auxin, example

Adventitious shoot/root formation (Skoog and Miller, 1957) High cytokinin/low auxin = shoot formation/caulogenesis Low cytokinin/high auxin = root formation/rhizogenesis Meristemoids are competent by not committed to development, examples

Auxin and Cytokinin Regulation of Organogenesis, Tomato Leaf Discs

Auxin and Cytokinin Regulation of Organogenesis (Gloxinia Leaf Segments) BA 0.3 0.3 0.3 0.3 0.3 (mg/L) NAA 0 0.1 0.3 1.0 3.0

Somatic embryogenesis (Reinert, and Steward et al., 1958) Carrot callus, 2,4-D either favors proliferation of competent cells or induces acquisition of competence by cells, example

Embryogenesis/Embryogeny of Carrot Is Regulated by 2,4-D (mg/L)

Gibberellin - inhibits differentiation of “meristmoids” but may be required for shoot development, example

Stage II: Stage I

III. Culture Environment Light Intensity - darkness or low light (20 mol m-2 s-1 or 1000 lux) Photoperiod - 16 hours daily Quality - blue wavelength favors shoot organogenesis, red wavelength favors root organogenesis Temperature Absolute - 24-27°C is standard, however, cool weather species may respond better to lower temperatures, e.g. potato - 18°C for shoot induction and 27°C for development Diurnal fluctuation - not usually important Dormancy – treatment can be in culture, e.g. bulbs and vernalization of scales, example

Cold Treatment of In Vitro Cultured Lily Bulb Scale Explants Satisfies Dormancy Requirement

4. Genetic Variation in Regenerated Plant Populations Background - Phenotypic variation in regenerated plant populations can be epigenetic (non-genetic/unstable) or genetic (inherited through gametes) Genetic variation in regenerated plants can arise because cells in the explant are genetically variant or tissue culture has proliferated variant cells or induced variation, example

4. Genetic Variation in Regenerated Plant Populations Regulating genetic variation in regenerated plant populations Low variability - highly meristematic explants Selection and reculture of meristmatic propagules Rapid regeneration without sustained periods in culture Simple media with low concentrations of growth regulators High variability - explants containing differentiated cells Long passage intervals where cells go through division and expansion phases Sub-culturing non competent cells, high concentrations of growth regulators, example

Regulating Genetic Variability in Regenerated Plants w/selection

Chimerism - plants exhibiting sectorial or periclinal chimeras can result from adventitious shoot initiation since morphogenesis may be initiated from more than one cell, example

5. Regeneration of Cereals Background - morphogenesis is focused on producing transgenic plants Isolation, culture and maintenance of competent cells and regeneration of transgenic plants Embryogenesis is preferred because of single cell origin. Phase/stages of culture leading to plant regeneration Induction Maintenance Regeneration Rooting

5. Regeneration of Cereals Phases/stages of culture leading to plant regeneration Induction Maintenance Regeneration Rooting Induction - explants are isolated that contain high frequency of competent cells and there is proliferation of pre-embryonically competent cells (PEDC) Medium with high auxin and, in some instances, asparagine/ proline/glutamine, examples

Embryogenic Competence of Sorghum Immature Inflorescences Inflorescence Size (cm) Numbers of Explants Embryogenic Callus (% of Explants) < 0.4 330 95 0.5 138 93 0.6 115 91 0.7 181 77 1.1 560 62 1.4 260 49 Notes: data obtained from experiments conducted in April 1995, with sorghum genotype PHB82.

Embryogenic Competence of Sorghum Immature Embryos < 1.0 398 29 Embryo Size (mm) Numbers of Explants Embryogenic Callus (% of Explants) < 1.0 398 29 1.0 - 1.4 339 45 1.5 - 2.0 141 28 > 2.0 168 15 Notes: data obtained from experiments conducted in April 1996, with sorghum genotype PH391.

Maintenance - competent cells continue to proliferate and differentiation occurs Cells tends to become non competent with time Visual selection pressure is applied Medium favors embryogeny and shoot formation (lower auxin + cytokinin), example Regeneration - plant development, lower cytokinin + auxin Rooting - root development in somatic embryos, minimal or no cytokinin and w/o or w/auxin

Induction and Maintenance of Embryogenic Callus from Sorghum Immature Inflorescences or Embryos

Regeneration - plant development, lower cytokinin + auxin Rooting - root development in somatic embryos, minimal or no cytokinin and w/o or w/auxin, example

Regeneration of Sorghum via Somatic Embryogenesis

Cereal Culture Plant Regeneration Stages, Sorghum Example w/selection for morphogenic callus w/selection