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Cloning technology for plant production

With the increasingly demanding market for quality, standardization and phytosanitary seedlings, it is extremely important to ensure the success of the entire production chain. It is in this sense that the in vitro propagation of plants offers the producer clonal seedlings of high standard and in sufficient quantity to meet the demand.

The production of seedlings in vitro requires a great knowledge in the physiology to define a better route of multiplication of the materials, with the aim of generating a genetic change to the source material and with high vigor.

The present invention relates to a method for the production of seedlings in vitro and to the production of seedlings in vitro.

The methods used for in vitro vegetative propagation, which have more time, are genetically uniform and healthy, even from infected mother plants.

Steps in the technique used to extract and cultivate the meristematic apex of a passion fruit plant (Passi fl ora edulis) (a). (b) and (c) show shoot development at different concentrations of a growth regulator. (d) Propagation of aerial part in culture medium. (e) Preparation of an individual for root formation. Source: Efficient shoot regeneration from direct apical meristem tissue to produce virus-free purple passion fruit plants. (Accessed on September 18, 2017).

Micropropagation or cloning can be performed by means of three techniques: a) meristem culture; b) somatic embryogenesis; c) organogenesis.

a) Culture of meristems

Meristema are groups of undifferentiated cells in plant tissues, which show high division activity and are responsible for the growth of the different organs of the plant.

Meristem cultivation is a technology used to produce virus-free plants because these parts of the plant are the only non-virus infectious. Such capacity is due to the speed with which the cells multiply and the absence of a vascular system through which the virus can be disseminated.

Because of these peculiarities, meristem cultivation has become important in obtaining tissues for genetic transformation by biological agents, such as Agrobacterium, and in the conservation and exchange of germplasm.

b) Somatic embryogenesis

It consists of the production of embryos from somatic tissues that regenerate an entire plant, usually with a genetic constitution identical to that of the parent plant. These plants transmit the inserted genetic material to the next generations.

Somatic embryogenesis is considered a prerequisite in the production of transgenic plants. It has been a preferential regenerative system for such an objective since it is efficient and there is little chance of genetic abnormalities in regenerated plants.

Tissue culture is a set of techniques with several advantages and applications. Different parts of a plant can be isolated (explanted) and placed in culture medium with nutrient balance that will guide cell differentiation. These cells form tissues and organs for the establishment of new plants.

Organogenesis: formation of pineapple plants from the base of a leaf (explant) of the mother plant cultivated in vitro. (Photo: Thais R. Everbom)

c) Organogenesis

It is the formation of gems (and organs) directly from tissues (leaf bases, petioles, segments of roots, etc.) or indirectly from callus. This technique involves two phases: dedifferentiation, shortly after tissue isolation, which promotes rapid cell division, forming an agglomerate of undifferentiated cells; and redifferentiation, when the organ's primordium originates meristems. The newly formed organs are called adventitious.

Culture of protoplasms

Protoplast is the cellular material resulting from the enzymatic removal of the cell wall. After fusion of protoplasts, it is possible to regenerate whole plants from the selected tissues.

Protoplasts constitute a useful system for the study of the expression of isolated genes and their regulation in plants. Fusion of protoplasts can be used to produce somatic hybrids between different species, solving problems of sexual incompatibilities.

Artistic representation of protoplast fusion.

Culture of disorganized material: calluses and suspensions

Calluses are a mass of disorganized tissue, an alternative to obtain and select plants resistant to adverse conditions and different types of materials to produce compounds of interest.

Suspensions are cell proliferations isolated or in small agglomerates, obtained from the agitation of calluses, dispersed in a liquid medium.

These culture systems can be used to study cellular or biochemical processes related to cell division, growth and differentiation.

Tobacco corns (Nicotiana tabacum) in culture medium. (Wikimedia Commons, user: Igge)

Bibliographic References

CARVALHO, Julita Maria Frota Chagas; VIDAL, Márcia Soares. Noções de Cultivo de Tecidos Vegetais. Campina Grande: Embrapa Algodão, 2003. 39 p.

GAMBORG, Oluf L.. Plant tissue culture. Biotechnology. Milestones. In Vitro Cellular & Developmental Biology – Plant, v. 2, n. 38, p.84-92, mar. 2002.

NEUMANN, Karl-hermann; KUMAR, Ashwani; IMANI, Jafargholi. Plant Cell and Tissue Culture – A Tool in Biotechnology: Basics and Application. Heidelberg: Springer, 2009. 331 p.

OLIVEIRA, M. M. Aplicações e Avanços na Área da Biotecnologia Vegetal. Biotecnologia Molecular: Avanços e Aplicações. Boletim de Biotecnologia n. 66, Lisboa, p. 22-27, ago. 2000.

SUSSEX, I. M.. The Scientific Roots of Modern Plant Biotechnology. The Plant Cell Online, [s.l.], v. 20, n. 5, p.1189-1198, 20 maio 2008. American Society of Plant Biologists (ASPB).