Snježana Mihaljević

Increase of root induction in Pinus nigra explants using agrobacteria

SummaryWounding of explanted Pinus nigra primary explants followed by infection with Agrobacterium rhizogenes wild strains 8196, 15834, or with the pRiA4abc transconjugant strain of A. tumefaciens (C58 chromosomal background) resulted in adventitious root induction. Roots were formed in 60–97% of explants (1–3 roots/explant) but without a hairy root phenotype. The presence of T-DNA of pRi8196 or pRiA4abc in regenerated roots was confirmed by the opine (mannopinic acid) content. Transformation response was influenced by the bacterial strain, age of explant and period of co-cultivation. Both the aggregate state (liquid) of medium and the season of the year (spring) had a positive effect on the root induction and their development. Histological analysis of the transformed roots showed that complete elements of primary and secondary root structures were present but roots were always triarch or tetrarch in the central cylinder as opposed to the primary roots of the untransformed seedling wich are diarch.

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): Control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.

Somatic and zygotic embryos share common developmental features at the onset of plant embryogenesis

In many vascular plants, zygotic reproduction regularly alternates with different types of asexual reproduction, so embryos can develop not only from fertilized egg cells, but also from induced somatic cells through the process of somatic embryogenesis. Although somatic and zygotic embryogenesis are not directly correlated, their common features are presented, demonstrating that the origin and development of the somatic embryo morphologically and physiologically resemble zygotic embryogenesis at certain points. To initiate embryogenesis, both competent egg and somatic cells require activation either by fertilization or specific environmental signals, respectively. During induction of somatic and zygotic embryogenesis, modulation of DNA methylation, activation of particular hormonal and stress-related mechanisms and changes in cell wall properties are triggered. Here, we give an overview and discuss the most recent research in the field of plant somatic and zygotic embryogenesis, with special attention given to the onset of embryogenesis and early embryo development as well as to embryogenesis-related interconnections between plant hormones, stress responses, DNA methylation and regulatory gene expression.

Root formation in micropropagated shoots of Sequoia sempervirens using Agrobacterium

Improved rooting was demonstrated in coast redwood (Sequoia sempervirens (D. Don.) Endl.) using an Agrobacterium-mediated gene transfer system. Microcloned shoot-tips of redwood (clone CA3) were inoculated with Agrobacterium rhizogenes wild strain 8196 or with the pRi A4 transconjugant strain of Agrobacterium tumefaciens (C58 chromosomal background) at the basal-cut end. Roots were formed in 58–69% of explants, but without a hairy-root phenotype. Rooting frequency and the number of adventitious roots on inoculated shoot-tips were slightly moderated by the bacterial strain, duration of inoculation and explant support system. Growth of excised roots was very slow on hormone-free medium and opine production could not be confirmed. Callus cultures derived from putative transformed roots were established on medium supplemented with 1.13 μM 2,4-D and 2.22 μM BA. PCR analysis of callus tissues DNA confirmed that integrative transformation had occurred in Sequoia adventitious roots generated after infection with Agrobacterium strains 8196 or A4. This is the first report of transgenic Sequoia tissue using A. rhizogenes-mediated gene transfer.

Omorika Spruce (Picea omorika)

This book contains 46 chapters, divided into 4 sections : A) 12 chapters on conifers, B) 14 chapters on fruits, C) 14 chapters on angiosperms, and D) 6 chapters on histology, bioencapsulation, protoplasts, cryopreservation, double staining and thin cell layer sectioning. The book provides stepwise protocols for somatic embryogenesis of a range of selected woody plants in order to assist researchers to initiate somatic embryogenic cultures without too much alteration in protocols. Each chapter provides information on initiation and maintenance of embryogenic cultures ; somatic embryo development, maturation, and germination ; acclimatization and field transfer of somatic seedlings. Some chapters include applications of somatic embryogenic cultures, e.g. protoplasts, encapsulation, cryopreservation, genetic transformation, genetic fidelity with molecular markers, and bioreactor.