1. 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

2. 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.

3. 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

4. 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

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

6. 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

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

8. 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

9. Tobacco Meristemoid Prior to Morphogenesis

10. 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

11. Shoot Meristems Differentiated from an Individual Meristemoid
of Potato Tuber Explants

12. 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

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

14. 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

15. Direct Embryogenesis from Cacao Cotyledon Epidermal Cell

16. Somatic Embryogenesis from Cacao Cotyledons

17. 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

18. Indirect Somatic Embryogenesis from Cacao Cotyledons

19. 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

20. Somatic Embryos of Cacao Illustrating Bipolar Meristematic Structures

21. 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

23. 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

24. 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

25. 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

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

27. Cell Developmental Phases Leading to Morphogenesis

28. 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

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. Developmental Stage and Adventitious Shoot Formation from Tomato Leaf Discs
Fig. 2: Changes in the shoot-forming capacity of the genotype PU 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 S

37. Developmental Stage Positions in a Tomato Leaf

38. 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

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

40. Adventitious Shoot Forming Capacity Is Inherited in Tomato

41. 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.

42. 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

43. 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

44. 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: (examples)

45. 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.

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

47. 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

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

49. 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

50. 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

52. 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

53. Auxin and Cytokinin Regulation of Organogenesis,
Tomato Leaf Discs

54. Auxin and Cytokinin Regulation of Organogenesis (Gloxinia Leaf Segments)BA
(mg/L)
NAA

55. 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

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

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

58. Stage II:
Stage I

59. 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 °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

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

61. 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

63. 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

64. Regulating Genetic Variability in Regenerated Plants
w/selection

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

67. 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

68. 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

69. 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.

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

71. 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

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

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

74. Regeneration of Sorghum via Somatic Embryogenesis

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