Plant Cell, Tissue and Organ Culture 37: 83-86, 1994. (~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.

Research Note

Somatic embryogenesis and plant regeneration of Nerium oleander

Isabel Santos , Isabel Guimar~es & Rober to Sa l ema Institute of Botany and Centre of Experimental Cytology, 4100-Porto, Portugal

University of Oporto, Rua Campo Alegre, 823,

Received 14 January 1993; accepted 17 January 1994

Key words: micropropagation, tissue culture

Abstract

Leaf explants of Nerium oleander L. produced masses of callus when both an auxin and a cytokinin were included in the medium. Leaves cultured on the B5 medium of Gamborg et al. supplemented with 2,4-dichlorophenoxyacetic acid (2,4-9; 9.05 ~tM) plus benzyladenine (BA; 4.4 laM) produced callus and profuse rhizogenesis was observed from callus developed from older leaves. On Murashige & Skoog medium (MS) with the same concentration of 2,4-D and BA, explants from young and mature leaves produced callus, but only that from young leaves was embryogenically competent. Globular somatic embryos were obtained when embryogenic cells were cultured on MS medium without growth regulators. Both normal and anomalous development of embryos occurred in either liquid or gelled medium. Plantlets were produced faster when mature embryos were cultured on either solid medium or placed on Sorbarod plugs soaked with this same medium but with 1% sucrose. Plantlets with three nodes were transferred to pots and acclimatized in a growth chamber and afterwards transferred to garden beds.

Nerium oleander L. (oleander) is a vegetatively prop- agated ornamental plant valued for evergreen foliage and showy terminal flower clusters that are available in different colours. This species also produces secondary metabolites (Paper & Franz 1989), some of which are of pharmacological interest.

In vitro culture of plants has gained in importance during recent years because, besides other applications, this technique can be used for the rapid multiplication of some plants (Tisserat 1987). As far as we are aware, there are no published reports about micropropagation of Nerium oleander and the aim of the present work was to determine the culture conditions for microprop- agation of this plant.

Seeds of Nerium oleander were disinfested first with 70% ethanol (2 min) followed by sodium hypochlorite (0.6%) prepared from commercial bleach plus 0.1% (w/v) Tween 20. Seeds were gently agitat- ed for 10 min after which they were washed five times with sterile bidistilled water and germinated under ster- ile conditions in half-strength Murashige & Skoog salt medium (1962; MS) with 1% sucrose. From 6-week-

old-seedlings young leaves (first and second leaf pair from the apex, 8 and 10 mm long, respectively) and developed leaves (seventh leaf pair from the apex, 20 mm long) were used as explant sources. For the cul- ture of these explants, two different basal media were used, MS and B5 (Gamborg et al. 1968). Both were supplemented with 2% sucrose and with growth reg- ulators as follows: MS-1 and B5-1 with 9.05 ~tM 2,4- D dichlorophenoxyacetic acid(2,4-D); MS-2 and B5- 2 with 9.05 I~M 2,4-D plus 4.4 ~tM 6-benzyladenine (BA). MS medium with no growth regulators (MS- 0), either gelled or liquid, was used for culture of embryogenic callus and globular embryos produced in MS-2 medium. The pH of all media were adjusted to 5.8 before autoclaving and 0.615% of agar (Sico- mol, Portugal) was used for gelling. The apical and basal region of the young leaves and also 1 mm of the margin of the developed leaves were discarded in the preparation of the explants; the resulting pieces were directly used as explants except on the latter material, which was further divided into two simi- lar portions. The explants were cultured in disposable

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petri dishes (90 mm) containing 25 ml of medium. For each experiment 10 petri dishes for each medi- um were used with 6--8 explants per dish. The petri dishes were sealed with parafilm and incubated in a growth room at 27 °C with a 12-h photoperiod (23 ~nol m -2 s - l , cool white fluorescent tubes). Por- tions of callus isolated from explants were subcultured every 3--4 weeks. The embryogenic callus and embryos produced in MS-2 were transferred to both solid or liquid MS-0 medium; the suspension was maintained in Erlenmeyer flasks (250 ml) containing 100 ml of medium, which were shaken on an orbital Certomat M (Braun) at 150 rpm. Well-developed embryos as well as very young plantlets were taken from MS-0 medium and transferred to Sorbarod plugs (Baumgartner Papi- er SA, Lausanne), soaked with the same medium but with only 1% sucrose, for further development. After the plantlets reached three nodes, they were transferred to pots with vermiculite and grown for 6 weeks in a growth chamber for acclimatization, after which they were transferred to garden beds. Experiments were repeated three times.

The response of the cultured leaf explants was dependent on both the composition of the culture medi- um and the age of the leaves. In fact, the callus tis- sue produced on the four different media used dis- played a very different morphology as well as dif- ferent organogenic and embryogenic capacities. Frag- ments of young and developed leaves produced cal- lus tissue on their periphery in all media used after 14 days of culture. At this time, the only difference registered among them concerned the colour of the tissue, which was slightly darker on either MS-1 or B5-1 media than on the others. From this initial period on, the behaviour of the explants cultured on different media was notably different. The tissue developed on B5-1 from both young and developed leaves darkened and after about 6 weeks degenerated, became necrotic and finally died. Callus formed on explants cultured on MS-1 also darkened; that on developed leaves died, whereas that on young leaves was rhizogenic produc- ing numerous roots, which also darkened. However, in media with both 2,4-D and BA, the callus developed on either MS-2 or B5-2 appeared healthy after 2 months of culture although morphologically different. Profuse rhizogenesis was observed in callus from developed leaves on B5-2, whereas young leaves produced callus that was compact and whitish or pale green. Explants from developed leaves cultured on MS-2 produced cal- lus that rarely formed roots and was somewhat less compact than the callus produced by young leaves on

B5-2. In contrast, explants of young leaves on MS- 2 produced callus that was embryogenic, white and nodular in appearance, but easily disaggregated into small clusters of cells (Fig. 1A). This callus produced numerous somatic embryos (Fig. 1B). Embryogenic capacity appeared in 72% of the callus produced by explants from the first leaf pair and in 57% of that derived from the second leaf pair. Embryogenic callus could be maintained as a proliferating mass of embryo- genic cells for at least 3 months when it was subcul- tured every 3 - 4 weeks. Compact callus produced by young leaves on B5-2 medium was not embryogenic. Our results showed that both the basic composition of the culture medium and the leaf developmental phase conditioned the type of the response as others have found (e.g. Vasil & Vasil 1986).

Contrary to what has been described for carrot explants, BA did not inhibit the production of embryo- genic cells; indeed, it seems that BA was even neces- sary for the induction of embryogenic cells in N. ole-

ander as observed for other species (Halperin 1986; Vieitez et al. 1990).

Embryogenic callus, when maintained for more than 1 month on the same medium (MS-2), produced nodules, most of which formed many globular embryos (Fig. 1B). When masses of embryogenic cells were either transferred into liquid or on solidified MS-0 medium, numerous somatic embryos also developed under both culture conditions (Fig. 1D ); globular embryos transferred to these conditions developed fur- ther (Fig. 1C). In liquid medium most embryos devel- oped the root pole earlier than the shoot, which inhib- ited shoot development. Only a few somatic embryos developed the radicle and the shoot and these struc- tures developed into plantlets faster when they were transferred to solid medium with sucrose reduced to 1% or placed on the Sorbarod plugs soaked with this same medium.

Curiously, the development of embryoids on MS- 0 solid medium (Fig. 1D) differed from that above described when using liquid medium because a large number of globular embryos showed earlier develop- ment of the shoot pole and they either evolved develop- ing a root pole or root growth was inhibited (Fig. 1D). Anomalous embryos with their hypocotyl region fused together were also observed (Fig. 1E) and these did not develop into normal plantlets; however, numerous embryos developed into normal plantlets (Fig. IF). Possibly in liquid medium the embryos developed a root pole earlier due to the agitation, since the con- centration of 02 can affect embryo development (Car-

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Fig. 1. Regeneration of plants from callus cultures derived from young leaves of N. oleander. (A) embryogenic callus produced by a leaf explant on MS-2 medium and subcultured on the same medium; note the nodular texture that easily disaggregates into small clusters of cells (Bar=-0.7mm). (B) differentiation of somatic embryos from embryogenic cells after 1 month of culture on MS-2 (Bar=lmm). (C) somatic embryos at an early cotyledonary stage developed on MS-0 medium (Bar=2.2mm). (D) general view of the embryogenic culture developed on solid MS-0 (Bar=-4mm); note the embryos at different stages of development, secondary embryogenesis (left inset - arrowhead; Bar=8.5mm) and embryos that developed the shoot pole first (right inset; Bar=6.5mm). (E) anomalous development of embryos due to the fusion of their hypocotyl regions (Bar--4mm). (F) young plantlet developed on solid MS-0 medium. (Bar=-4mm)

m a n 1990). Inhib i t ion of shoot development due to early root format ion has been found in other species ( De Touchet et al. 1991 and references therein). In both solid and l iquid m e d i u m secondary embryogene- sis was observed (Fig. ID) on the cotyledons of the pri-

mary somatic embryos, which compromised the con- version of the primary embryos into a whole plantlet. This phenomenon was probably due to the remain ing effect of 2,4-D, as described in cultures of oil pa lm and chestnut (De Touchet et al. 1991; Vieitez et al. 1990).

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When regenerated plantlets reached three nodes and had a vigorous branched root system, they were potted in vermiculite and grown in a growth chamber for 6 weeks for acclimatization; then they were transferred to garden beds. The regenerated plants appeared to be morphologically normal and 90% of them survived.

As far as we are aware, this is the first report of regenerating N. oleander by somatic embryogenesis. Previous work (Pal et al. 1990) with cultured leaves of N. oleander only described callus formation and rhizogenesis but no regeneration of plants. In spite of the high embryogenic capacity of the embryogenic callus produced by young leaves cultured on MS-2 medium, the number of normal plantlets obtained was low, very likely due to anomalous embryo development and secondary embryogenesis. The latter situation led to the appearance of multiple intermingled embryos in different phases of growth, which makes it very difficult to manually separate plantlets.

References

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De Touchet B, Duval Y & Pannetier C ( 1991) Plant regeneration from embryogenic suspension cultures of oil palm (Elais guineensis jacq.) Plant Cell Rep. 10:529-532

Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158

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