Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum

A reproducible system for somatic embryogenesis and plantlet formation of sandalwood has been developed. A high frequency (100%) of somatic embryos were induced directly from various explants in MS (Murashige and Skoog, 1962) medium with thidiazuron (1 or 2 μM) or indirectly in medium containing 2,4-D plus thidiazuron. Within 8 weeks, white globular somatic embryos or friable embryogenic tissue developed on cultured explants. In S. album the globular somatic embryos were transferred to MS medium supplemented with IAA (6 μM) and kinetin (1 μM) where they developed further, multiplied and maintained friable embryogenic tissue. After 15–30 d, mature somatic embryos (1–2 mm) with well-developed cotyledons were separated and subcultured on to medium containing GA3 (6 μM) for germination. Once germinated, elongated somatic embryos (10–20 mm long) grew further in MS supplemented with lower GA3 (3 μM). In S. spicatum, the addition of casein hydrolysate and coconut milk was necessary for plantlet development from somatic embryos. From histological studies, it appeared that primary somatic embryos arose from single cells or had a multicellular origin from the epidermis or cortical parenchyma. Secondary somatic embryos and friable embryogenic tissue differentiated from groups of proembryogenic cells from a superficial layer of the primary somatic embryos

Intra- and inter-specific pollination of Santalum spicatum and S. album

The flower morphology, receptivity and sexual compatibility between genotypes and species were determined in Western Australian sandalwood (Santalum spicatum) and Indian sandalwood (S. album). The results showed that the stigma of both species became receptive at anthesis and reached a peak at 3 or 4 days after anthesis. Pollen tubes took 2 days to grow to the ovary when pollinated at anthesis, and 1 day when pollinated 2 or 3 days after anthesis. The egg apparatus matured at least 2 days after pollination and varied between genotypes. Fertilisation occurred 2 or 3 days following cross pollination. Although 10-40% of ovules were fertilised following intra-specific crosses of both species, the average initial fruit set was much lower: 4% in S. spicatum and 19% in S. album. Most immature fruit (75-80%) abscised following intra-specific pollination. The number of pollen tubes that grew in styles after self- and inter-specific pollination was lower than that for intra-specific pollination. Following serf and inter-specific pollination, growth of pollen tubes was arrested in the style, ovary and around the embryo sac; a few penetrated the embryo sac. Initial fruit set was low and developing fruit abscised prematurely. The results indicated that pre- and post-fertilisation mechanisms control self-incompatibility and inter-specific incompatibility between the sandalwood species

Reproductive biology of three commercially valuable Santalum species: development of flowers and inflorescences, breeding systems, and interspecific crossability

Santalum (sandalwood) spp. are hemi-parasitic trees, the heartwood of which produces valuable aromatic oil. There appears to be a significant commercial opportunity for establishment of a planted sandalwood resource. However, lack of basic biological knowledge is one constraint on such development. The study reported here addresses one such constraint. Controlled pollination using 13 genotypes of Santalum lanceolatum was undertaken to elucidate (i) self-incompatibility (ii) intraspecific cross-compatibility in the species, and (iii) interspecific cross-compatibility with S. album and S. austrocaledonicum. S. lanceolatum may be considered to have a facultative allogamous (incomplete outbreeding) breeding system. This study found variation between genotypes in the level of putative self-incompatibility: some (20%) were found to set seed following self-pollination, while the remaining 80% had no seed development with such pollinations. However, a significantly greater proportion of genotypes developed seed following intraspecific cross-pollination (62%) compared with self-pollination (20%). While total geographic isolation and significant morphological divergence exists between S. lanceolatum with each of S. album and S. austrocaledonicum this study found no indication of reproductive barrier(s) between them, indicating potential for use of interspecific hybridization in genetic improvement, but also suggesting the potential of undesirable gene flow between native and introduced species

Ploidy stability in embryogenic cultures and regenerated plantlets of tamarillo

Ploidy levels of short-term (1 and 2 years) and long-term (7 and 10 years) embryogenic cultures as well as of regenerated plantlets of tamarillo were analyzed by flow cytometry and chromosome counts. Embryogenic cultures were induced from expanding leaves cultured in the presence of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) and monthly subcultured on the same media. Embryo development and plantlets were obtained following subculture of the embryogenic tissue in auxin free medium containing gibberellic acid (GA3). Seedlings and rooted shoots from axillary shoot proliferation were used as controls. The results showed that in long-term embryogenic cultures the ability to develop somatic embryos and plantlets was reduced. Embryogenic tissues maintained for 10 years were mostly aneuploids of the tetraploid (2n = 4x = 48) level whereas those kept in culture for 7 years or less were also mostly aneuploids but of the diploid (2n = 2x = 24) level. The results obtained by flow cytometry were, in general, consistent with those obtained by chromosome counts. The chromosome alteration observed in the embryogenic tissues was already present after 1 year of culture and increased with culture age, hence impairing the maintenance of these tissues for long periods without affecting chromosome stability of the regenerated plantlets. However, the occurrence of triploids and tetraploids as well as aneuploids can be useful for breeding purposes. A value around 23 pg/2C was found for the genome size of tamarillo largely exceeding the value previously published (15.50 pg/2C).This work was supported by the Portuguese Foundation for Science and Technology (FCT).publishe