Regeneration of Pinus halepensis (Mill.) through Organogenesis from Apical Shoot Buds

Abstract

Organogenesis and somatic embryogenesis have been widely applied as the two main regeneration pathways in plant tissue cultures. However, recalcitrance is still the main restriction in the clonal propagation of many woody species, especially in conifers. They undergo a “phase change” that leads to significant loss of vegetative propagation capacity, reducing the aptitude of tissues and organs to be regenerated in vitro beyond this point. In line with this, the in vitro regeneration of mature conifer trees has been a long-cherished goal in many laboratories worldwide. Based on previous works in Pinus species regeneration from adult trees, we now present data about the culture of apical shoot buds in an attempt to induce organogenesis and somatic embryogenesis to clone mature trees of Aleppo pine (Pinus halepensis). Reinvigorated axillary shoots were submitted to conditions usually applied to induce somatic embryogenesis through the manipulation of culture media, including the use of auxins such as 2,4-Dichlorophenoxyacetic acid and 1-Naphthaleneacetic acid, cytokinins (6-benzyladenine and kinetin), and phytosulfokine (50, 100, and 200 nM). Although somatic embryos could not be obtained, an embryogenic-like tissue was produced, followed by the emergence of actively proliferating non-embryogenic calli. Variations in the consistence, texture, and color of non-embryogenic calli were observed; especially those arising in the media containing phytosulfokine. Reinvigorated shoots, induced by 22 or 44 µM 6-benzyladenine, were obtained through organogenesis and acclimatized, and phenotypically normal plants were obtained.

Testing Explant Sources, Culture Media, and Light Conditions for the Improvement of Organogenesis in Pinus ponderosa (P. Lawson and C. Lawson)

“…In our experiments, BA at the concentrations tested (4.4, 22, and 44 µM) showed efficient organogenic response for NS, and, although not significant, a slightly higher response in whole zygotic embryos cultured in HLP supplemented with 22 µM BA was observed. A similar tendency was observed in several micropropagation protocols in Pinus where the culture medium was supplemented with BA (from 1 to 50 µM) to obtain bud induction [ 19 , 27 , 39 , 40 , 41 , 42 ].…”

“…The establishment of an aseptic in vitro culture is one of the most important factors of plant tissue culture and the effective elimination of contamination contributes to the better development of the explants [ 26 , 27 ]. When introducing P. ponderosa material in vitro, contamination rates ranging from 1% to 16% were obtained using hydrogen peroxide (protocol B).…”

“…Our results suggest that the protocol used in this work was adequate for this species and can be used routinely, being a valid procedure for the propagation of other Pinus species as well. The most important initial step in the micropropagation process is an adequate sterilization protocol that minimizes tissue damage to achieve the greatest number of aseptic explants ready to be used in the next phases of micropropagation [ 27 , 29 ].…”

Sensitivity in Callus Tissue of Plectranthus amboinicus (L.) through Mutation Induction with Colchicine

“…In vitro regeneration can be accomplished through two pathways: direct or indirect organogenesis and somatic embryogenesis (Yang et al, 2021). Organogenesis needs two different hormonal signals to instigate a shoot organ and a root organ (Pereira et al, 2021). However, this research's microscopic observations show that regeneration is more appropriate through an indirect organogenesis approach.…”

“…The characteristics of non-embryogenic cells can be seen with symmetrical cell division. Nonembryogenic cell aggregates grow into nodular structures and eventually form plantlets with roots and shoots (Pereira et al, 2021).…”