Somatic embryogenesis

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Switchgrass somatic embryos Conger type callus 3ms White Light.TIF
Switchgrass somatic embryos

Somatic embryogenesis is an artificial process in which a plant or embryo is derived from a single somatic cell. [1] Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue. No endosperm or seed coat is formed around a somatic embryo.

Contents

Cells derived from competent source tissue are cultured to form an undifferentiated mass of cells called a callus. Plant growth regulators in the tissue culture medium can be manipulated to induce callus formation and subsequently changed to induce embryos to form the callus. The ratio of different plant growth regulators required to induce callus or embryo formation varies with the type of plant. [2] Somatic embryos are mainly produced in vitro and for laboratory purposes, using either solid or liquid nutrient media which contain plant growth regulators (PGR’s). The main PGRs used are auxins but can contain cytokinin in a smaller amount. [3] Shoots and roots are monopolar while somatic embryos are bipolar, allowing them to form a whole plant without culturing on multiple media types. Somatic embryogenesis has served as a model to understand the physiological and biochemical events that occur during plant developmental processes as well as a component to biotechnological advancement. [4] The first documentation of somatic embryogenesis was by Steward et al. in 1958 and Reinert in 1959 with carrot cell suspension cultures. [5] [6]

Direct and indirect embryogenesis

Somatic embryogenesis has been described to occur in two ways: directly or indirectly. [7]

Direct embryogenesis

occurs when embryos are started directly from explant tissue creating an identical clone. In other words without callus formation of embryo from explant, that is called direct embryogenesis.

Indirect embryogenesis

occurs when explants produced undifferentiated, or partially differentiated, cells (often referred to as callus) which then is maintained or differentiated into plant tissues such as leaf, stem, or roots. 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP) and Gibberellic acid (GA) has been used for development of indirect somatic embryos in strawberry (Fragaria ananassa) [8]

Plant regeneration by somatic embryogenesis

Plant regeneration via somatic embryogenesis occurs in five steps: initiation of embryogenic cultures, proliferation of embryogenic cultures, prematuration of somatic embryos, maturation of somatic embryos and plant development on nonspecific media. Initiation and proliferation occur on a medium rich in auxin, which induces differentiation of localized meristematic cells. The auxin typically used is 2,4-D. Once transferred to a medium with low or no auxin, these cells can then develop into mature embryos. Germination of the somatic embryo can only occur when it is mature enough to have functional root and shoot apices [3]

Factors influencing

Factors and mechanisms controlling cell differentiation in somatic embryos are relatively ambiguous. Certain compounds excreted by plant tissue cultures and found in culture media have been shown necessary to coordinate cell division and morphological changes. [9] These compounds have been identified by Chung et al. [10] as various polysaccharides, amino acids, growth regulators, vitamins, low molecular weight compounds and polypeptides. Several signaling molecules known to influence or control the formation of somatic embryos have been found and include extracellular proteins, arabinogalactan proteins and lipochitooligosaccharides. Temperature and lighting can also affect the maturation of the somatic embryo.

Applications

Applications of this process include: clonal propagation of genetically uniform plant material; elimination of viruses; provision of source tissue for genetic transformation; generation of whole plants from single cells called protoplasts; development of synthetic seed technology. [1]

Uses of somatic embryogenesis

The development of somatic embryogenesis procedures has given rise to research on seed storage proteins (SSPs) of woody plants for tree species of commercial importance, i.e., mainly gymnosperms, including white spruce. In this area of study, SSPs are used as markers to determine the embryogenic potential and competency of the embryogenic system to produce a somatic embryo biochemically similar to its zygotic counterpart (Flinn et al. 1991, Beardmore et al. 1997). [13] [14]

Grossnickle et al. (1992) [15] compared interior spruce seedlings with emblings during nursery development and through a stock quality assessment program immediately before field outplanting. Seedling shoot height, root collar diameter, and dry weight increased at a greater rate in seedlings than in emblings during the first half of the first growing season, but thereafter shoot growth was similar among all plants. By the end of the growing season, seedlings were 70% taller than emblings, had greater root collar diameter, and greater shoot dry weight. Root dry weight increased more rapidly in seedlings than in emblings during the early growing season

During fall acclimation, the pattern of increasing dormancy release index and increasing tolerance to freezing was similar in both seedlings and emblings. Root growth capacity decreased then increased during fall acclimation, with the increase being greater in seedlings.

Assessment of stock quality just prior to planting showed that: emblings had greater water use efficiency with decreasing predawn shoot water potential compared with seedlings; seedlings and emblings had similar water movement capability at both high and low root temperatures; net photosynthesis and needle conductance at low root temperatures were greater in seedlings than in emblings; and seedlings had greater root growth than emblings at 22 °C root, but root growth among all plants was low at 7.5 °C root temperature.

Growth and survival of interior spruce 313B Styroblock seedlings and emblings after outplanting on a reforestation site were determined by Grossnickle and Major (1992). [16] For both seedlings and emblings, osmotic potential at saturation (ψsat) and turgor loss point (ψtip) increased from a low of -1.82 and -2.22 MPa, respectively, just prior to planting to a seasonal high of -1.09 and -1.21 MPa, respectively, during active shoot elongation. Thereafter, seedlings and emblings (ψsat) and (ψtip) declined to -2.00 and -2.45 MPa, respectively, at the end of the growing season, which coincided with the steady decline in site temperatures and a cessation of height growth. In general, seedlings and emblings had similar ψsat and ψtip values through the growing season, and also had similar shifts in seasonal patterns of maximum modulus of elasticity, sympalstic fraction, and relative water content at turgor loss point.

Grossnickle and Major (1992) [16] found that year-old and current-year needles of both seedlings and emblings had a similar decline in needle conductance with increasing vapour pressure deficit. Response surface models of current-year needles net photosynthesis (Pn) response to vapour pressure deficit (VPD) and photosynthetically active radiation (PAR) showed that emblings had 15% greater Pn at VPD of less than 3.0 kPa and PAR greater than 1000 μmol m−2s−1. Year-old and current-year needles of seedlings and emblings showed similar patterns of water use efficiency.

Rates of shoot growth in seedlings and emblings through the growing season were also similar to one another. Seedlings had larger shoot systems both at the time of planting and at the end of the growing season. Seedlings also had greater root development than emblings through the growing season, but root:shoot ratios for the 2 stock types were similar at the end of the growing season, when the survival rates for seedlings and emblings were 96% and 99%, respectively.

Tracking and fate maps

Understanding the formation of a somatic embryo through establishment of morphological and molecular markers is important for construction of a fate map. The fate map is the foundation in which to build further research and experimentation. Two methods exist to construct a fate map: synchronous cell-division and time-lapse tracking. The latter typically works more consistently because of cell-cycle-altering chemicals and centrifuging involved in synchronous cell-division. [17]

Angiosperms

Embryo development in angiosperms is divided into several steps. The zygote is divided asymmetrically forming a small apical cell and large basal cell. The organizational pattern is formed in the globular stage and the embryo then transitions to the cotyledonary stage. [18] Embryo development differs in monocots and dicots. Dicots pass through the globular, heart-shaped, and torpedo stages while monocots pass through globular, scutellar, and coleoptilar stages. [19]

Many culture systems induce and maintain somatic embryogenesis by continuous exposure to 2,4-dichlorophenoxyacetic acid. Abscisic acid has been reported to induce somatic embryogenesis in seedlings. After callus formation, culturing on a low auxin or hormone free media will promote somatic embryo growth and root formation. In monocots, embryogenic capability is usually restricted to tissues with embryogenic or meristematic origin. Somatic cells of monocots differentiate quickly and then lose mitotic and morphogenic capability. Differences of auxin sensitivity in embryogenic callus growth between different genotypes of the same species show how variable auxin responses can be. [20]

Carrot Daucus carota was the first and most understood species with regard to developmental pathways and molecular mechanisms. [17] Time-lapse tracking by Toonen et al. (1994) showed that morphology of competent cells can vary based on shape and cytoplasm density. Five types of cells were identified from embryonic suspension: spherical cytoplasm-rich, spherical vacuolated, oval vacuolated, elongated vacuolated, and irregular shaped cells. Each type of cell multiplied with certain geometric symmetry. They developed into symmetrical, asymmetrical, and aberrantly-shaped cell clusters that eventually formed embryos at different frequencies. [21] This indicates that organized growth polarity do not always exist in somatic embryogenesis. [17]

Gymnosperms

Embryo development in gymnosperms occurs in three phases. Proembryogeny includes all stages prior to suspensor elongation. Early embryogeny includes all stages after suspensor elongation but before root meristem development. Late embryogeny includes development of root and shoot meristems. [18] Time-lapse tracking in Norway Spruce Picea abies revealed that neither single cytoplasmic-rich cells nor vacuolated cells developed into embryos. Proembryogenic masses (PEMs), an intermediate between unorganized cells and an embryo composed of cytoplasmic-rich cells next to a vacuolated cell, are stimulated with auxin and cytokinin. Gradual removal of auxin and cytokinin and introduction of abscisic acid (ABA) will allow an embryo to form. [17] Using somatic embryogenesis has been considered for mass production of vegetatively propagated conifer clones and cryopreservation of germplasm. However, the use of this technology for reforestation and tree breeding of conifers is in its infancy. [22] [23]

See also

Related Research Articles

Developmental biology is the study of the process by which animals and plants grow and develop. Developmental biology also encompasses the biology of regeneration, asexual reproduction, metamorphosis, and the growth and differentiation of stem cells in the adult organism.

<span class="mw-page-title-main">Meristem</span> Type of plant tissue involved in cell proliferation

The meristem is a type of tissue found in plants. It consists of undifferentiated cells capable of cell division. Cells in the meristem can develop into all the other tissues and organs that occur in plants. These cells continue to divide until a time when they get differentiated and then lose the ability to divide.

<span class="mw-page-title-main">Plant hormone</span> Chemical compounds that regulate plant growth and development

Plant hormones are signal molecules, produced within plants, that occur in extremely low concentrations. Plant hormones control all aspects of plant growth and development, from embryogenesis, the regulation of organ size, pathogen defense, stress tolerance and through to reproductive development. Unlike in animals each plant cell is capable of producing hormones. Went and Thimann coined the term "phytohormone" and used it in the title of their 1937 book.

<span class="mw-page-title-main">Plant nursery</span> Facility where plants are propagated and grown to usable size

A nursery is a place where plants are propagated and grown to a desired size. In a word, a nursery is a centre of seedling production where seedlings are produced and taken care until transplantation in the main field. Mostly the plants concerned are for gardening, forestry, or conservation biology, rather than agriculture. They include retail nurseries, which sell to the general public; wholesale nurseries, which sell only to businesses such as other nurseries and commercial gardeners; and private nurseries, which supply the needs of institutions or private estates. Some will also work in plant breeding.

<span class="mw-page-title-main">Auxin</span> Plant hormone

Auxins are a class of plant hormones with some morphogen-like characteristics. Auxins play a cardinal role in coordination of many growth and behavioral processes in plant life cycles and are essential for plant body development. The Dutch biologist Frits Warmolt Went first described auxins and their role in plant growth in the 1920s. Kenneth V. Thimann became the first to isolate one of these phytohormones and to determine its chemical structure as indole-3-acetic acid (IAA). Went and Thimann co-authored a book on plant hormones, Phytohormones, in 1937.

<span class="mw-page-title-main">Cytokinin</span> Class of plant hormones promoting cell division

Cytokinins (CK) are a class of plant hormones that promote cell division, or cytokinesis, in plant roots and shoots. They are involved primarily in cell growth and differentiation, but also affect apical dominance, axillary bud growth, and leaf senescence.

<span class="mw-page-title-main">Coleoptile</span> Protective sheath in certain plants

Coleoptile is the pointed protective sheath covering the emerging shoot in monocotyledons such as grasses in which few leaf primordia and shoot apex of monocot embryo remain enclosed. The coleoptile protects the first leaf as well as the growing stem in seedlings and eventually, allows the first leaf to emerge. Coleoptiles have two vascular bundles, one on either side. Unlike the flag leaves rolled up within, the pre-emergent coleoptile does not accumulate significant protochlorophyll or carotenoids, and so it is generally very pale. Some preemergent coleoptiles do, however, accumulate purple anthocyanin pigments.

<span class="mw-page-title-main">Callus (cell biology)</span> Growing mass of unorganized plant parenchyma cells

Plant callus is a growing mass of unorganized plant parenchyma cells. In living plants, callus cells are those cells that cover a plant wound. In biological research and biotechnology callus formation is induced from plant tissue samples (explants) after surface sterilization and plating onto tissue culture medium in vitro. The culture medium is supplemented with plant growth regulators, such as auxin, cytokinin, and gibberellin, to initiate callus formation or somatic embryogenesis. Callus initiation has been described for all major groups of land plants.

Organogenesis is the phase of embryonic development that starts at the end of gastrulation and continues until birth. During organogenesis, the three germ layers formed from gastrulation form the internal organs of the organism.

Plant embryonic development, also plant embryogenesis is a process that occurs after the fertilization of an ovule to produce a fully developed plant embryo. This is a pertinent stage in the plant life cycle that is followed by dormancy and germination. The zygote produced after fertilization must undergo various cellular divisions and differentiations to become a mature embryo. An end stage embryo has five major components including the shoot apical meristem, hypocotyl, root meristem, root cap, and cotyledons. Unlike the embryonic development in animals, and specifically in humans, plant embryonic development results in an immature form of the plant, lacking most structures like leaves, stems, and reproductive structures. However, both plants and animals including humans, pass through a phylotypic stage that evolved independently and that causes a developmental constraint limiting morphological diversification.

Kinetin (/'kaɪnɪtɪn/) is a cytokinin-like synthetic plant hormone that promotes cell division in plants. Kinetin was originally isolated by Carlos O. Miller and Skoog et al. as a compound from autoclaved herring sperm DNA that had cell division-promoting activity. It was given the name kinetin because of its ability to induce cell division, provided that auxin was present in the medium. Kinetin is often used in plant tissue culture for inducing formation of callus and to regenerate shoot tissues from callus.

<span class="mw-page-title-main">Micropropagation</span> Practice in plant tissue culture

Micropropagation or tissue culture is the practice of rapidly multiplying plant stock material to produce many progeny plants, using modern plant tissue culture methods.

<span class="mw-page-title-main">Seedling</span> Young plant developing out from a seed

A seedling is a young sporophyte developing out of a plant embryo from a seed. Seedling development starts with germination of the seed. A typical young seedling consists of three main parts: the radicle, the hypocotyl, and the cotyledons. The two classes of flowering plants (angiosperms) are distinguished by their numbers of seed leaves: monocotyledons (monocots) have one blade-shaped cotyledon, whereas dicotyledons (dicots) possess two round cotyledons. Gymnosperms are more varied. For example, pine seedlings have up to eight cotyledons. The seedlings of some flowering plants have no cotyledons at all. These are said to be acotyledons.

<span class="mw-page-title-main">Indole-3-butyric acid</span> Chemical compound

Indole-3-butyric acid (1H-indole-3-butanoic acid, IBA) is a white to light-yellow crystalline solid, with the molecular formula C12H13NO2. It melts at 125 °C in atmospheric pressure and decomposes before boiling. IBA is a plant hormone in the auxin family and is an ingredient in many commercial horticultural plant rooting products.

<span class="mw-page-title-main">Plant morphology</span> Study of the structure of plants

Phytomorphology is the study of the physical form and external structure of plants. This is usually considered distinct from plant anatomy, which is the study of the internal structure of plants, especially at the microscopic level. Plant morphology is useful in the visual identification of plants. Recent studies in molecular biology started to investigate the molecular processes involved in determining the conservation and diversification of plant morphologies. In these studies transcriptome conservation patterns were found to mark crucial ontogenetic transitions during the plant life cycle which may result in evolutionary constraints limiting diversification.

Important structures in plant development are buds, shoots, roots, leaves, and flowers; plants produce these tissues and structures throughout their life from meristems located at the tips of organs, or between mature tissues. Thus, a living plant always has embryonic tissues. By contrast, an animal embryo will very early produce all of the body parts that it will ever have in its life. When the animal is born, it has all its body parts and from that point will only grow larger and more mature. However, both plants and animals pass through a phylotypic stage that evolved independently and that causes a developmental constraint limiting morphological diversification.

Embryomics is the identification, characterization and study of the diverse cell types which arise during embryogenesis, especially as this relates to the location and developmental history of cells in the embryo. Cell type may be determined according to several criteria: location in the developing embryo, gene expression as indicated by protein and nucleic acid markers and surface antigens, and also position on the embryogenic tree.

<span class="mw-page-title-main">Plant tissue culture</span> Growing plant cells under known conditions

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used, to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

Embryo rescue is one of the earliest and successful forms of in-vitro culture techniques that is used to assist in the development of plant embryos that might not survive to become viable plants. Embryo rescue plays an important role in modern plant breeding, allowing the development of many interspecific and intergeneric food and ornamental plant crop hybrids. This technique nurtures the immature or weak embryo, thus allowing it the chance to survive. Plant embryos are multicellular structures that have the potential to develop into a new plant. The most widely used embryo rescue procedure is referred to as embryo culture, and involves excising plant embryos and placing them onto media culture. Embryo rescue is most often used to create interspecific and intergeneric crosses that would normally produce seeds which are aborted. Interspecific incompatibility in plants can occur for many reasons, but most often embryo abortion occurs In plant breeding, wide hybridization crosses can result in small shrunken seeds which indicate that fertilization has occurred, however the seed fails to develop. Many times, remote hybridizations will fail to undergo normal sexual reproduction, thus embryo rescue can assist in circumventing this problem.

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