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Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N&prime, phenylurea (CPPU). A medium containing 0.1 mgL&minus, 1 2,4-D and 0.25 mgL&minus, 1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL&minus, 1 2,4-D and 0.5 mgL&minus, 1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury&mdash, an emerging model in developmental biology.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

Background Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Results Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l−1 in darkness. TDZ at 3 mg l−1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95–100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. Conclusions In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore ‘Elsa’, was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures.

The development of protoplast derived from somatic embryos and some of their characteristics were compared with embryos from suspension and in vivo in the same B line. Embryos formed in a protoplast culture differed from others that their younger stages contained vacuolated cells, and older ones had altered morphological and histological structure. Somatic embryogenesis is more regular from suspension then from protoplasts. No distinct differences were observed in the rate of embryo development in vivo and in vitro, and in vitro embryos show a larger variation in size at the same stage. Embryos in vitro with fluorescence are generally larger than zygotic ones at each stage. The use of fluorescence is suggested for the selection of heterokariocytes after protoplast fusion.

Epidermal cell wall biogenesis with emphasis on cuticular layer formation during direct somatic embryogenesis in rice was examined by transmission electron microscopy. The first phenomenon noticed in the early globular stage of somatic embryos was a high activity of cell wall formation. In this stage, many endoplasmic reticula, Golgi bodies and their vesicles were present and epidermal cells were covered by fibrillar material. Cutin was initially deposited on the epidermal cell wall of late globular or early elongate stage embryos. In the late elongate stage, a lamellated cuticular layer formed, and in the late scutellar stage, a more homogenous continuous cuticular layer formed. The cuticular layer disappeared gradually from the scutellum cell wall as the somatic embryo germinated. The disappearance of the cuticular layer was similar to that of the zygotic embryo. There is no cuticular layer on the scutellar epithelial cell wall of the immature zygotic embryo 5 days after germination. Our observations lead to the conclusion that one of the structural and physiological markers of direct somatic embryogenesis is the transformation of fibrillar material into the cuticular layer.

Protocols for axillary shoot proliferation and somatic embryogenesis were developed for Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White. Shoot tips were cultured on a modified Murashige and Skoog (MS) media supplemented with benzyl aminopurine (BA) and gibberellic acid (GA 3 ) or BA, kinetin (Kin) and indole-3-acetic acid (IAA). The auxins indole -3-butyric acid (IBA) and IAA were used to induce rooting. Direct somatic embryogenesis was induced from leaf, internode’s stem and for the first time for chrysanthemums from petiole explants. Modified MS medium supplemented with 1 mg/L naphthalene acetic acid (NAA) or 2,4- dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L BA, 200 mg/L casein hydrosylate (CH) and 290 mg/L proline was used for induction. Proliferation rate of 3.2 new microshoots per one inoculated was achieved when BA (0.1 mg/L) was used in combination with GA 3 (0.5 mg/L). The number of roots per shoot was higher using IBA (0.5 mg/L), but IAA (2 mg/L) promoted longer roots. A high percentage of embryogenesis was induced by both combinations of plant growth regulators (PGRs). Leaf explants were most responsive, demonstrating the highest percentage of embryogenesis (97.9%), followed by petiole and internode’s stem explants (56.3 and 35.1%, respectively). The number of somatic embryos per embryogenic explant was also the highest on leaf explants; however, the best conversion rate (53.8%) of somatic embryos to plantlets was observed from petiole explants. For this reason, petiole explants are the most suitable type of explants for plant regeneration of chrysanthemum cv. Palisade White through somatic embryogenesis. Key words : Chrysanthemum, Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White, micropropagation, direct somatic embryogenesis, explant type.

Pepper (Capsicum L.) is a nutritionally and economically important crop that is cultivated throughout the world as a vegetable, condiment, and food additive. Genetic transformation using Agrobacterium tumefaciens (agrobacterium) is a powerful biotechnology tool that could be used in pepper to develop community-based functional genomics resources and to introduce important agronomic traits. However, pepper is considered to be highly recalcitrant for agrobacterium-mediated transformation, and current transformation protocols are either inefficient, cumbersome or highly genotype dependent. The main bottleneck in pepper transformation is the inability to generate cells that are competent for both regeneration and transformation. Here, we report that ectopic expression of the Brassica napus BABY BOOM AP2/ERF transcription factor overcomes this bottleneck and can be used to efficiently regenerate transgenic plants from otherwise recalcitrant sweet pepper (C. annuum) varieties. Transient activation of BABY BOOM in the progeny plants induced prolific cell regeneration and was used to produce a large number of somatic embryos that could be converted readily to seedlings. The data highlight the utility of combining biotechnology and classical plant tissue culture approaches to develop an efficient transformation and regeneration system for a highly recalcitrant vegetable crop. Electronic supplementary material The online version of this article (doi:10.1007/s00299-011-1018-x) contains supplementary material, which is available to authorized users.

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4 degrees C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9 degrees C and thereafter at 28 degrees C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 muM zeatin and 5 muM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.

With a four-step procedure, using the cassava clone M. Col 22, a cyclic system of somatic embryogenesis was developed. Primary embryos were induced in step 1 (2,4-D dependent) on young leaf lobe explants. Primary embryos were isolated in step 2 (BAP dependent) and were used as starting explant for secondary embryogenesis (steps 1 and 2 repeated). In this way, repetitive embryogenic cultures were established. It was shown that in all successive cycles embryo explants produced significantly more embryos than leaf lobe explants. According to their shape embryos were classified in two development stages; torpedo shaped embryos and germinated embryos. Germinated embryos produce more than three times as many embryos than torpedo shaped embryos. A third step in the embryogenic process was the development of shoots. The shoot conversion of embryos was increased if benzylamino purine (1 mg 1 -1 ) was added. In that case more than half of the isolated embryos developed into normal shoots. The shoot conversion rate of germinated embryos was higher than that of torpedo shaped embryos. Embryos were kept in culture for 9 months to give six cycles. The production of embryos from the sixth cycle was as efficient as that from the second cycle. The shoot conversion rate of embryos from the fourth cycle was comparable with that from the second cycle. Rooting of the embryo-derived shoots (step 4 of the procedure) occurred on a medium without BAP. More than 100 regenerants obtained from fourth cycle embryos were multiplied in vitro . They showed no phenotypical differences as compared to in vitro multiplied parental material.

In cassava a cyclic system of somatic embryogenesis was developed. Primary (torpedo shaped or germinated) embryos, originating from leaf lobes, could only be obtained after culture on solid medium. Cyclic embryos, originating from embryos, could be obtained in both liquid and on solid medium. The production of embryos in liquid medium was distinctly higher, faster and more synchronized than on solid medium. Lower densities and fragmentation of starting embryos improved the production significantly. The highest production found was 32.1 embryos per initial embryo. In all treatments the explants initiated multiple embryos. The production of single embryos was achieved by pressing starting embryos through a fine meshed sieve, indicating that embryos can be produced from a piece of tissue with a restricted number of cells. The shoot conversion rate of embryos from liquid medium was comparable with that of embryos from solid medium.