Invitro regeneration of plantlets of Canary Island pine (Pinuscanariensis)

Abstract

A protocol is described for plantlet formation in juvenile tissues of Pinuscanariensis Sweet ex K. Spreng. (Canary Island pine). Adventitious buds were induced on 3-day-old cotyledonary explants cultured on Bornman's MCM medium supplemented with cytokinin. The concentration of benzylaminopurine, the use of other cytokinins alone or in combination with benzylaminopurine, and the concentration of mineral salts strongly affected the bud forming capacity of the cotyledonary explants. Also, the age of the explants significantly influenced the frequency of adventitious bud formation. Bud development was enhanced by the elimination of phytohormones, a reduction of mineral salts and sucrose, and the inclusion of activated charcoal in the medium. The conditions used during the induction phase strongly affected the ability of the induced buds to develop into vigorous rootable shoots. Vitrification problems were eliminated by transferring the shoots to the elongation medium solidified with Gelrite, and shoot remultiplication was enhanced by removing the apical bud. Maximum rooting was obtained by pulsing shoots with a high concentration of indolebutyric acid and by using peat–vermiculite or peat–vermiculite–perlite as substrates. Roots developed within 6–8 weeks, and the regenerated plantlets were transferred to soil under nonsterile conditions, where further development occurred.

Effect of various bud induction treatments on elongation and rooting of adventitious shoots of Canary Island pine (Pinus canariensis)

“…Seeds were surface disinfested and seed coats removed as described by Martinez Pulido et al (1990). For embryo germination, about one-third to one-half of the megagametophyte (incision made parallel to the embryo axis) was removed and the remaining portion containing the embryo was plated on water agar plates with 10 g 1-1 sucrose and 8 g 1-1 Difco Bacto-agar® (Martinez Pulido et al 1992).…”

“…All cultures were incubated at 27 + 1 o C under a 16-h photoperiod (Sylvania Gro-Lux ® F40712 Gro-WS lights, 80-100 Ixmol m-2 s-1), but embryos were germinated under 40-50 ~tmol m -2 s -1 . For bud induction, cotyledons were cultured in petri plates (100 x 15 mm) containing MCM mineral salts (Bornman 1983) modified according to Martinez Pulido et al (1990). N 6-benzyladenine (BA), zeatin (filtersterilized), kinetin and 2-isopentenyl adenine (2iP) were added singly at 10 gM or in combination, with each cytokinin added at 10 or 5 ~tM.…”

“…After 4 weeks, explants were transferred to glass storage jars (100 × 80 mm) containing 100 ml of half-strength Schenk and Hildebrandt (SH;1972) medium supplemented with 20 g 1-1 sucrose, 0.5 g 1-l activated charcoal (Sigma, C4386) and 8 g 1 -I Difco Bacto-agar ®. Initially, separated shoots were also maintained on this medium; however, when shoots were about 10 nun in length they were transferred to the same medium gelled with 4 g 1-1 Gelrite ® (Scott Labs Inc., Carson, CA) to prevent hyperhydric transformation (Martinez Pulido et al 1990). All jars were sealed with Stretch'n Seal ® .…”

“…However, it appeared from these studies that the bud induction treatments had an influence on shoot quality as well as rooting (Martinez Pulido et al 1990). In this paper, we present the results of studies done on the effects of various bud induction treatments on subsequent plantlet regeneration.…”

“…The subtropical conifer Pinus canariensis Sweet ex K. Spreng, has been successfully micropropagated using cotyledonary explants cultured in vitro (Martinez Pulido et al 1990). However, it appeared from these studies that the bud induction treatments had an influence on shoot quality as well as rooting (Martinez Pulido et al 1990).…”