انسجة

Plant Tissue Culture Dr. Zakaria Al Ajlouni

Plant Tissue Culture Second semester, 2011 Lecture 1: Introduction, History and Terms

Introduction • What is plant tissue culture? Tissue culture is the culture and maintenance of plant cells or organs in sterile, nutritionally and environmentally supportive conditions (in vitro). • In commercial settings, tissue culture is often referred to as micropropagation. Micropropagation refers to the production of whole plants from cell cultures derived from explants (the initial piece of tissue put into culture) of (usually) meristem cells.

• Why Tissue Culture? Tissue culture offers numerous significant benefits over traditional propagation methods: • Much faster rates of growth can be induced in vitro than by traditional means.

• Why Tissue Culture? • It may be possible to multiply in vitro plants that are very difficult to propagate by cuttings or other traditional methods. • Large numbers of genetically identical clones may be produced.

• Why Tissue Culture? • Seeds can be germinated with no risk of damping off/predation. • Under certain conditions, plant material can be stored in vitro for considerable periods of time with little or no maintenance. • Tissue culture techniques are used for virus eradication, genetic manipulation, somatic hybridization and other procedures that benefit propagation, plant improvement, and basic research.

Plant tissue culture chronicle • 1838: MJ Schleiden hypothesized cell theory, suggesting the totipotency of cells. Totipotency: the cell contain all the information necessary for the organism to grow and reproduce in its environment. OR Every cell has the genetic potential to reproduce the entire organism. • 1882: Sachs reported that plants synthesizing organ-forming substances that are polarly distributed. • 1902: Haberlandt made the first (but unsuccessful) attempt at tissue culture. • 1904: Hanning attempted embryo culture. • 1909: Kuster achieved fusion of protoplasts although the products did not survive. Protoplast: a plant cell without its outer retaining cell wall.

Plant tissue culture chronicle Haberlandt

• 1921: Molliard cultured fragments of plant embryos. • 1922: Knudson germinated seeds in vitro; Robbins cultured root tips. • 1924: Blumenthal and Meyer achieved callus formation on carrot root explants. • 1925: Laibach used embryo culture to rescue interspecific hybrids of Linum. • 1934: White maintained long-term cultures of tomato roots. • 1941: Van Overbeek used coconut milk to support growth and development of very young Datura embryos; Braun cultured crown gall tissues in vitro. • 1946: E Ball produced the first whole plants from shoot tip cultures of Lupinus and Tropaeolum. • 1951: Nitch cultured excised ovaries in vitro; Skoog demonstrated chemical control of growth and organ formation in vitro.

• 1952: Morel and Martin produced first virus-free plants produced from meristem culture and first successful micrografts. • 1954: Muir regenerated calli from single cells. • 1957: Discovery of the regulation of organ formation by changing the ratio of auxin: cytokinin (Skoog and Miller); Culture of excised anthers of Allium (Vasil). • 1958: Regeneration of somatic embryos in vitro from the nucellus of Citrus ovules (Maheshwari and Rangaswamy); Pro- embryo formation in callus and cell suspensions of carrot. • 1960: Enzymatic degradation of cell walls to obtain large numbers of protoplasts (Cocking); Filtration of cell suspensions and isolation of single cells by plating (Bergmann). • 1962: Development of Murashige and Skoog medium. • 1964: Production of haploid plants from pollen grains of Datura (Guha and Maheshwari).

• 1970: First achievement of protoplast fusion (Power et al.). • 1972: First report of interspecific hybridization through protoplast fusion in two species of Nicotiana (Carlson et al). • 1973: Gene from African clawed toad inserted into plasmid DNA of bacteria (Herbert Boyer & Stanley Cohen) first recombinant DNA organism - beginning of genetic engineering; Somatic hybridization of tomato and potato, resulting in pomato (Melchers et al.). • 1974: Biotransformation in plant tissue cultures (Reinhardt). • 1975: Positive in vitro selection of maize callus cultures resistant to T toxin (Gengenbach and Green). • 1976: Shoot initiation from cryopreserved shoot apic of carnation (Seibert). • 1977: Successful integration of Ti plasmid from A. tumefaciens in plants (Chilton et al). • 1982: Naked DNA transformation of protoplasts.

• 1983: Transgenic plants were first created in the early 1980s • 1985: Infection and transformation of leaf discs with Agrobacterium tumefaciens and regeneration of transformed plants. • 1986: TMV virus-resistant tobacco and tomato ( transgenic plants developed using cDNA of coat protein gene of TMV. • 1988: Automated mass propagation with organogenesis and embryogenesis( Levi R et al). • 2000: Arabidopsis draft sequence completed.

Plant tissue culture terms • Aseptic: Absence of fungi, bacteria, viruses or other microorganisms. Bacteria, fungi, and other organisms can easily outcompete an isolated fragment of tissue from the plant in the relatively nutrient-rich environment of a culture flask. Therefore we need to remove competitor organisms from the culture. How? The medium and culture flasks used must also be sterile. Chemical surface sterilization of the explant. Sterile culture and working environment.

Plant tissue culture terms • Callus: An unorganized, proliferative mass of cells. Also defined as the undifferentiated growth of plant cells in vitro, rather like the way cells grow in tumor tissue. Callus naturally forms on a wounded plant part.

Plant tissue culture terms • Clonal propagation: Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual explant.

• Plant tissue culture terms Cybrid: An organism or cell which has the nuclear characteristic and chromosome number of one parent and the organellar traits of the other parents.

Plant tissue culture terms • Defined medium: A nutritive solution for culturing cells in which each component is of known chemical structure.

Plant tissue culture terms • Differentiated: Existing in a state of cell development characterized by cellular enlargement and elongation, cytoplasmic reduction and vacuolation and the presence of a nucleus.

Plant tissue culture terms • Embryo culture: In vitro development or maintenance of isolated mature or immature embryos. • Embryogenesis: The process of embryo initiation and development.

Plant tissue culture terms • Explant: Plant tissue taken from its original site and transferred to an artificial medium for growth or maintenance. • Epigenetic variation: Phenotypic variability which has a nongenetic basis.

Plant tissue culture terms • Induction: Initiation of a plant structure, organ or process. • In vitro propagation: Propagation of plants in a controlled, artificial environment, using plastic or glass culture vessels, aseptic techniques and a defined growing medium.

Plant tissue culture terms • In vitro transformation: A heritable change, occurring in cells in culture, either intrinsically or from treatment with chemicals, irradiation etc. and leading to the acquisition of altered morphological, proliferative or other properties. • Juvenile: A phase in the sexual cycle of a plant which may be characterized by differences in appearance from the adult and in general a lack of response to flower- inducing stimuli.

Plant tissue culture terms • Meristem culture: In vitro culture of a generally shiny dome-like structure, measuring less than 0.1 mm in length when excised, most often excised from the shoot apex. • Morphogenesis: Process of growth and development of differentiated structures. • Mutant: A phenotypic variant with a genetic basis for the change from the wild type character.

Plant tissue culture terms • Organ culture: Maintenance or growth of organ primordia, whole organs or parts thereof, in vitro. • Organized: Arranged into definite structure. • Organogenesis: Formation of a structure which shows natural organ form or function, or both.

Plant tissue culture terms • Pathogen free: Free from specific organisms based on specific tests for designated organisms. • Protoplast: A plant cell without its outer retaining cell wall. • Protoplast fusion: Technique in which protoplasts are fused into a single cell.

Plant tissue culture terms • Re-culture: Process by which an explant is transferred without subdivision, into fresh culture medium. • Shoot apical meristem (SAM): Undifferentiated tissue, located within the shoot tip, generally appearing as a shiny dome-like structure. SAM is a “stem cell” reservoir, the plant’s fountain of youth. Stem Cells: Stem cells are relatively undifferentiated cells defined by their abilities for self-renewal and for generating differentiated cells.

Shoot apical meristem How the stem cells are maintained?

Plant tissue culture terms • Shoot apex culture: An explant consisting of the shoot apical meristem plus one to several primordial leaves. • Somatic cell hybridization: In vitro fusion of protoplasts derived from somatic cells of genetically different sources. • Subculture: Process by which the tissue or explant is first subdivided, then transferred into fresh medium

Plant tissue culture terms • Suspension culture: A culture in which single cells or cell aggregates increase in number while suspended in a liquid medium. • Totipotency: A cell characteristic in which the potential for forming all the cell types in the adult organism is retained. • Variant: A culture exhibiting a stable phenotypic change, whether genetic or epigenetic

Plant tissue culture terms • Vegetative propagation: Reproduction of plant using a nonsexual process involving the culture of plant parts such as stem and leaf cuttings, floral parts or embryos. • Undifferentiated: Existing in a state of cell development characterized by isodiametric cells shape, prominent cytoplasm, a large nucleus and exemplified by cells comprising an apical meristem or embryo .

Introduction to Plant Reproduction Dr. Zakaria Al Ajlouni

Define Propagation • Propagation – The reproduction of plants either sexually or asexually.

Define sexual reproduction and the terms associated with it. • Sexual Reproduction: – The union of the female and male sex cells to produce a seed (embryo). • Ovule: female sex cell. • Pollen: male sex cell. • Embryo (seed-germ): an immature plant. – Sexual reproduction involves the creation of a genetically new individual.

Objective 1.6 Define pollination, fertilization • andPollination germination. – The transfer of pollen from an anther to a stigma of a flower of the same species. • Fertilization – The union of the pollen and ovule cells. • Germination – The sprouting of a seed.

Objective 1.7 Define asexual reproduction • Asexual Reproduction: – The reproduction of a plant without the uniting of a pollen and ovule. – Asexual reproduction is often referred to as vegetative propagation since no seed is involved in the formation of the new plant. – It is known as a clone. – Leaves, stems or roots may be used to grow a new plant. – *Produces a genetically identical plant.

Objective 1.8 List the benefits of vegetative propagation. • True traits of the parents – Maintains genetic purity with – 100% replication of parent plant. • No seed – Some plants do not produce a seed or the seeds are too small to work with. • Accelerates the time it takes to get a new plant to the market – Traditionally, it would take up to 40 years to get a new plant to the general public; however, micropropagation can yield marketable levels of plants within 8 to 12 years.

List and explain the different types of vegetative propagation. • Layering – Involves getting roots to grow from the stem. I.e., magnolia tree • Cutting – Using a short section of plant stems for propagation. • Budding – Taking a bud from one plant and moving it to another. • Grafting – Placing a section of a stem of one plant onto another plant. • Tissue culture – Taking a group of cells or a single cell and growing it to a plant.

Layering •

Cutting •

Plant Propagation by Cuttings a. Kinds of cuttings 1) Stem cuttings 2) Leaf-bud cutting (single-eye) 3) Root cuttings 4) Leaf cuttings b. Totipotency-The ability of an individual cell to reconstitute the entire plant part and functions c. Dedifferentiation-The capability of previously developed, differentiated cell to return to a meristem

Plant Propagation by Cuttings 1. Stem cuttings 2. Leaf-bud cutting (single-eye) 3. Root cuttings 4. Leaf cuttings

1. Stem Cuttings • Softwood Cuttings • Semi-Hardwood Cutting • Hardwood Cuttings • Herbaceous Cuttings

Softwood Cuttings

Shoftwood Cuttings - Hydrangea Butterfly cutting (top) Double-eye single node cutting (middle) Single-eye single node cuttings (bottom)

Softwood Cuttings-Peach - Actively growing shoots are used - Softwood cuttings are taken during spring and summer

Influence of IBA on Semi-Hardwood Cuttings - Cordia 1-Control, 2-50% ethanol, 3-100 ppm, 4-1000 ppm, 5- 2000 ppm, 6-4000 ppm, 7-6000 ppm, 8-8000 ppm, 9- 10000 ppm IBA

Semi-Hardwood Cuttings - Jojoba Treating cuttings in IBA Solution (top) Sticking IBA-treated cuttings in root substrate (bottom)

Bottom Heat System for Cutting Benches

Temperature Differential Helpful for Root Formation Ambient Temp - Rooting requires 60 oF carbohydrates (energy), auxin (growth hormone), and rooting cofactors (enzymes) Root zone Temp - Temperature 70 oF differential (10 oF) between the ambient air and root zone is helpful for faster rooting

Good sanitation is important Cutting on the left was infected with Alternaria and did not root Cutting on the right was healthy and rooted well