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Organogenic Nodule Formation in Hop
( Humulus lupulus var. Nugget)
Ana Margarida FORTES, Maria Salomé PAIS
Lab. of Plant Biotechnology, ICAT, FCUL, Campo Grande, 1749-016 Lisboa Portugal
Abstract. This work aimed to study some of the processes involved in organogenic
nodule formation in Humulus lupulus var. Nugget. Organogenesis and in vitro
somatic embryogenesis from differentiated plant cells are complex morphogenic
processes involving physiological, biochemical, molecular and elemental tissue and
cell changes. These morphogenic processes play pivotal roles in plant
biotechnology. Knowledge on the signals involved in their induction, formation and
development will enable in the future a controlled induction of morphogenesis.
In a first approach, it was studied the sequence of histological and histochemical events
occurring from internodes inoculation until the development of shoot buds [1]. Cell
division was observed in both cambial and cortical regions during the first week of culture
establishment. Divisions of cortical cells led to the formation of an incipient callus tissue.
Prenodular structures of cambial origin appeared surrounded by these calluses and gave rise
to nodules from which shoot buds were formed. Nodules kept separating into “daughter
nodules” from which arose an increasing number of shoot buds. Iodide staining showed a
strong starch accumulation in callus tissue and in prenodular structures. During shoot bud
primordia formation starch content decreased in nodules and was probably mobilized for
organ initiation and development. Control explants, which never gave rise to organogenic
nodules nor regenerated plantlets, accumulated starch at a much lower extent than explants
cultured on media with growth regulators. This suggested that a differential pool of sugars
could play an inductive role in organogenic nodule formation.
Previous studies carried out during induction of somatic embryogenesis in other
plant species suggested callose and cutin deposition as a way to isolate cells from the
surroundings, which might cause metabolic changes leading to embryo formation. With the
purpose of investigating if such a deposition occurred along morphogenesis induction in
hop, callose and cutin accumulation was followed by staining with Aniline Blue and Nile
Red and by immunolocalization using antibodies raised against callose [2]. A cutin layer
showing bright yellow autofluorescence appeared surrounding cells or groups of cells
committed to express morphogenic competence and enter mitosis. This cutin layer that
evolved to a randomly-organized network appeared underneath a callose layer and may
create a specific cellular environment with altered permeability and altered receptors
providing conditions for entering cell cycle. The fact that only an incipient callose
accumulation was observed in control explants suggested the involvement of callose in the
initiation of the morphogenic program leading to nodule formation. A scanning electron
microscopic study during the organogenic process showed that before shoot bud
regeneration, the cutin layer increased in thickness and acquires a smooth texture (Fig. 1).
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