Direct Somatic Embryogenesis Research Articles

The relationship between developmental stages of zygotic embryos at explanting and embryogenic frequency on hickory (Carya cathayensis Sarg.)

In recent years, more attention has been paid than previous to clonally propagating hickory via methods of tissue culture. However, most of such methods have failed. Somatic embryogenesis is an alternative system that includes the simultaneous production of both an organized root and a shoot axis. Abscisic acid (ABA) plays a key role in zygotic embryo development at each stage, in both a stimulatory and an inhibitory manner. Thus the influences of the development stages of immature zygotic embryo on somatic embryogenesis was studied. ABA concentration of immature zygotic embryos was also detected. Immature embryos were microscopically visible at 8–9 weeks postpollination with pale yellow liquid endosperm. Cotyledons expanded rapidly at 10–12 weeks postpollination. The immature embryos were actively growing and cotyledons were fully elongated at weeks 14 weeks post-pollination. Cotyledons were filled with of kernel, and endosperm was utilized at 15–16 weeks post-pollination. The concentration of ABA ranged from 29.1 to 166.8ngg−1 FW within the development of zygotic embryos. The ABA content levels at 8–9 weeks and 16 weeks postpollination were significantly lower and higher, respectively, than ABA contents at all otherdevelopmental stages. For callus induction, the frequency gradually increased with the development of the zygotic embryos from 8 to 10 weeks post-pollination followed by a decrease from 10 weeks to 16 weeks post-pollination. The highest frequency (66.67%) of embryogenic callus formation was obtained at the stage of 10 weeks post-pollination, with low levels obtained before or after this period. For direct somatic embryogenesis, frequency increased with the development of zygotic embryos from 8 to 11 weeks post-pollination followed by a decrease from 11 to 16 weeks post-pollination. The highest frequency of somatic embryogenesis was 46.67%, obtained with explants collected 11 weeks postpollination.

Effect of Activated Charcoal, Carbon Sources and Gelling Agents on Direct Somatic Embryogenesis and Regeneration in Sugarcane via Leaf Roll Segments

Efficient plant regeneration is an essential pre-requisite for a complete genetic transformation protocol. Direct somatic embryogenic cultures were established in commercial sugarcane varieties viz. CoJ 83 and CoH 119. Problem of browning of media was observed mainly in cultures of CoJ 83 as compared to CoH119. To study the effect of adsorption of phenolics by activated charcoal, two treatments were assayed in cv. CoJ 83 i.e. on Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA; 5 mgl−1) and Kinetin (Kin; 0.5 mgl−1) (control) and MS + NAA (5 mgl−1) + Kin (0.5 mgl−1) + charcoal (0.2% w/v) medium (treatment). In addition to control of browning of medium by phenolics, per cent somatic embryogenesis (SE) increased from 80.21 to 84.88 and per cent regeneration was also enhanced from 75.15 to 81.22 in sugarcane variety CoJ 83, when the medium was supplemented with 2,000 mgl−1 of activated charcoal (AC). Further, the effect of addition of different carbon sources i.e. sucrose and maltose in four different concentrations 20, 25, 30 and 35 gl−1 was studied and maximum per cent SE (98.35% in CoJ 83 and 93.12% in CoH 119; respectively) and maximum per cent regeneration (94.17% in CoJ 83 and 89.67% in CoH 119; respectively) was observed on MS + NAA (5 mgl−1) and Kin (0.5 mgl−1) medium supplemented with 25 gl−1 maltose. Direct SE and regeneration was also affected considerably by varying the gelling agent (agar or gelrite) and its concentration. Best results were observed with increases in per cent SE and regeneration on medium solidified with agar (1.2%) compared to gelrite, when used as a gelling agent. Thus, this fairly simple activated charcoal (2% w/v), maltose (25 gl−1) and agar (1.2%) containing direct SE medium provided an alternative method for improving SE and hence, regeneration frequency of sugarcane.

Direct somatic embryogenesis from hypocotyl segments of Digitalis trojana Ivan and subsequent plant regeneration

This study describes, for the first time, an in vitro protocol for the direct development of somatic embryos and subsequent plant regeneration from hypocotyl segments excised from 21-days-old in vitro-germinated seedlings of Digitalis trojana Ivan (Helen of troy foxglove). Two sets of experiments were carried out, the first compared different concentrations of four cytokinins: N6-benzyladenine [BAP], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-(4-hydroxy-3-methylbut-2-enylamino) purine [zeatin] alone, while the second set tested TDZ or BAP combinations with IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA (α-naphthalene acetic acid). In the first set of experiments, TDZ was found the most effective at 1.0mg/l concentration, producing a mean of 10.7 somatic embryos per explant. In the second set, a combination of 1.0mg/l TDZ with 0.5mg/l IAA produced significantly more somatic embryos per explant (13.8 embryos) than with BAP (8.8 embryos). During subculture on growth regulator-free half-strength MS medium, somatic embryos gradually developed into plantlets. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for a large-scale production of cardenolides and genetic transformation of this valuable medicinal plant for its further improvement.

Direct somatic embryogenesis from leaf and petiole explants of Spathiphyllum ‘Supreme’ and analysis of regenerants using flow cytometry

This study established a method of regenerating Spathiphyllum ‘Supreme’ through direct somatic embryogenesis. Somatic embryos occurred in leaf and petiole explants cultured in the dark on a Murashige and Skoog basal medium supplemented with 2.27, 4.54, or 9.08 μM N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) in combination with 1.08 μM α-naphthalene acetic acid or 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Explants with somatic embryos were transferred to fresh medium containing the same concentrations of growth regulators under lighted conditions for embryo conversion. The highest frequencies of leaf explants with somatic embryos and embryo conversion were both 84.4 %, which were induced by 9.08 μM TDZ with 2.26 μM 2,4-D. The frequencies for somatic embryo induction and embryo conversion were both 100 % when petiole explants were induced by 4.54 μM TDZ with 2.26 μM 2,4-D. The number of plantlets produced per leaf explant and petiole explant were as high as 67.4 and 74.4, respectively. Plantlets after transplanting to a soilless substrate grew vigorously in a shaded greenhouse. Liners were stable without phenotypic variation. Flow cytometry analysis of randomly selected plants showed that they all had a single identical peak. The mean nuclear DNA index for ‘Supreme’ was 1.568, and the nuclear DNA content was 14.222 pg 2C−1. The estimated genome size for ‘Supreme’ was 6,954.5 Mbp 1C−1 with a CV at 4.008 %. The results suggest that the regenerated plants have a stable ploidy level and this established regeneration method can be used for highly effective propagation of uniform Spathiphyllum ‘Supreme’.

The micropropagation of chrysanthemums via axillary shoot proliferation and highly efficient plant regeneration by somatic embryogenesis

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.

Plant regeneration via direct somatic embryogenesis from leaf and petiole explants of Epipremnum aureum ‘Marble Queen’ and characterization of selected variants

Leaf and petiole explants of Epipremnum aureum ‘Marble Queen’ were cultured on Murashige and Skoog basal medium containing three concentrations of either N-(2-chloro-4-pyridl)-N’-phenylurea (CPPU) or N-phenyl-N’-1, 2, 3-thiadiazol-5-ylurea (TDZ) with 1.07 μM α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–6 weeks of culture. TDZ at 4.54 μM with 1.07 μM NAA induced 75% of leaf explants to produce somatic embryos and 62.5% of explants to produce germinated embryos. Both 8.07 μM CPPU and 9.08 μM TDZ, respectively, with 1.07 μM NAA induced 100% of petiole explants to produce somatic embryos and 78.1 and 91.7% of explants to produce germinated embryos. Plantlets with completely green, variegated, and whitish leaves were identified among regenerated individuals. Some variegation patterns differed greatly from the parent ‘Marble Queen’ within the variegated plants. Flow cytometry analysis of stock plants and selected variants showed that all plants had one identical peak. Analysis of simple sequence repeats amplified from 14 universal chloroplast primer pairs showed no variation between stock plants and selected variants. RT-PCR analysis of EaZIP, a marker gene involved in leaf variegation of E. aureum ‘Golden Pothos’, suggested little expression difference between green and whitish plants or between green and whitish sectors of a variegated leaf. However, SDS-PAGE analysis of proteins showed differences in band intensity and patterns. These results suggest that mechanisms underlying leaf variegation of ‘Marble Queen’ differ from the closely related ‘Golden Pothos’. The established regeneration system and identified variants could be important materials for further investigation of leaf variegation in E. aureum.

Primary and repetitive secondary somatic embryogenesis in Rosa hybrida ‘Samantha’

This study describes a plant regeneration system for Rosa hybrida ‘Samantha’, a mainstream commercial cultivar, via direct somatic embryogenesis. Somatic embryogenesis was induced from in vitro-derived leaf explants, achieving a frequency of 7.5% following 8 weeks of culture on a Murashige and Skoog (MS) medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g/L glucose. We evaluated the effects of various types and concentrations of carbohydrates and plant growth regulators (PGRs) on the proliferation and germination of secondary somatic embryos. MS medium containing 1.0 mg/L 2,4-D, 0.05 mg/L 6-benzyladenine (BA) and 60 g/L glucose was found to be the most effective in promoting the proliferation of somatic embryos. The highest germination rate (56.2%) of somatic embryos was observed on MS medium containing 1.0 mg/L Thidiazuron (TDZ) and 0.5 mg/L BA. Whole plantlets were obtained by culturing germinated shoots on rooting medium comprising 1/2-strength MS salts supplemented with 0.05 mg/L α-Naphthalene acetic acid (NAA). Plantlets grew vigorously with normal vegetative and flowering characteristics.

Direct somatic embryogenesis and plant regeneration from seed derived protocorms of Cymbidium bicolor Lindl.

A protocol for the induction of direct somatic embryogenesis from seed derived protocorms was developed in Cymbidium bicolor. Ninety-day-old protocorms cultured on half strength MS medium alone or supplemented with auxin and cytokinin induced direct somatic embryogenesis. The best response was observed in protocorms cultured half strength MS medium supplemented with BAP at 1.0mg/L and 2-4,D at 2.0mg/L. Both epidermal and sub epidermal cells were involved in the formation of embryos. The pro-embryos developed into globular stage and subsequently developed into protocoms. Complete plantlets were formed after 60 days culture. The plantlets were acclimatized in plastic pots containing sterilized vermiculite. Survival rate was 90% when maintained in culture room condition (25±2°C).

In vitro regeneration of Eucalyptus camaldulensis.

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2mgl(-1) benzyl aminopurine (BAP) and 0.1mgl(-1) naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5mgl(-1) BAP, while root induction and elongation was superior in 1/2 MS + 1mgl(-1) Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100% coco peat resulted in maximum survival (80%) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60-148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.

An improved protocol for plant regeneration from leaf- and hypocotyl-derived protoplasts of carrot

An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 106 and 106 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds.

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses—caulogenesis, direct somatic embryogenesis, and callusing—were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08 μM TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46 μM indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.

Experimental research on in vitro propagation through direct somatic embryogenesis of basil (Ocimum basilicum L.).

Induction, generation and development of embryogenic somatic phenomena are matters that can be modeled by monitoring the interplay between exogenous phytohormones and explants. As a result, this work presents experimental studies that illustrate the influence that exogenous cytokinins (BAP and TDZ), and auxins (NAA or IBA) have, added in the composition of the artificial nutritive medium, for modeling the in vitro propagation through direct somatic embryogenesis in two cultivars of basil (Ocimum basilicum L. var. Marseille and Ocimum basilicum L. var. Red Rubin), starting from different types of explants (leaf, cotyledon, epicotyl, hipocotyl and radicle). The best embryogenic response was recorded for experimental variant consisting in TDZ cytokinin and IBA auxin in quantities of 1 mg/l respectively 0,5 mg/l, and cotyledon type explant.

The effect of auxin 2,4-D and cytokinin 2-ip on direct somatic embryogenesis formation of Coffea arabica L. leaf explant

One of the propagation technique for coffee plant production is tissue culture. Tissue culture technique for Coffea arabica L. faces some problems, mainly in the planlet formation regenerated from explants. The objective of this experiment was to examine the effect 2,4-D and 2-ip combination on the formation of direct somatic embryogenesis of Coffea arabica L. in leaves explant. Auxin (2,4-D) and cytokinin (2-ip) concentrations of, respectively, 1; 5 µM and 5; 10; 15; 20 were used as treatments. This research was conducted using completely randomized design with 10 replications. Observation to induce somatic embryos was done by quantitatively on number of callus from explant and number of embryogenic callus. Beside that, observation by qualitative descriptive was also done on deve lopment of embryogenesis. The results showed that Arabica coffee leaves explant of AS 2K clones could be induced in all medium combination except 5µM 2,4-D and 20µM 2-ip combination. Arabica coffee leaves explant of S 795, Sigararutang and AS 1 varieties could be induced in all medium combination. The highest frequency of callus formation was found in AS 2K, Sigararutang and AS 1 varieties on medium containing 1µM 2,4-D in combination with 10µM 2-ip, whereas for the S 795 variety on medium containing 5µM 2,4-D in combination with 10µM 2-ip. The highest frequency of embriogenic callus in all Arabica coffee variety could be reached on medium containing 5µM 2,4-D in combination with 15µM 2-ip. Key words : Coffea arabica L., somatic embryogenesis, 2,4-D, 2-ip, tissue culture, leaves, callus embryogenic.

Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis

A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 mo from culture initiation. Biolistic gene transfer into cross-sections of immature leaf whorl explants followed by direct somatic embryogenesis resulted in the stable genetic transformation of the commercially important sugarcane cultivar CP 88-1762. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Southern blot analysis revealed simple transgene integration patterns ranging from one to five hybridization products. NPTII-ELISA confirmed that most of the transgenic plants expressed the transgene stably in vegetative progeny. Using a minimal, linear expression cassette (MC) without vector backbone sequences for the biolistic gene transfer and reducing the amount of MC to 10 ng per shot may have led to simple transgene integration and stable transgene expression. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events.

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.

Establishment of regeneration and transformation system of sugarcane cultivar GT54-9 (C9)

Plant regeneration protocols for sugarcane GT54-9(C9) cultivar were developed for direct organogenesis and indirect somatic embryogenesis, using young leaf segments as explants by studying the influence of different concentrations and types of cytokinin and auxin hormones. For the callus formation from young leaves, a medium containing 4mg/l 2,4-D was found very effective. For embryo formation, MS medium supplemented with 1mg/l Kin and 0.5 mg/l 2,4-D was used. While in the case of direct organogenesis protocol, the medium containing 1mg/l BAP and 2mg/l NAA was the best for direct shoot formation. Data showed that the best shoot regeneration and elongation medium for direct organogenesis and indirect somatic embryogenesis was obtained on medium with 2 mg/l Kin and 0.1 mg/l BAP. Root induction was best performed on 2mg/l NAA and complete plantlets were hardened in the greenhouse before transferring to the field for further evaluation. For transformation, young leaf segments of sugarcane from the cultivar GT54-9(C9) were inoculated and co-cultivated with Agrobacterium tumefaciens strain LB4404 harboring the binary vector pISV2678 with the bar and the gus-intron genes. The obtained putative transgenic plantlets were able to grow under bialaphose containing medium. Stable integration of the bar gene into the plant genomes was tested by PCR and Southern blot hybridization. Histochemical assay and leaf painting analysis were carried out to study the expression of the gus and bar genes in transgenic plants, respectively. The results indicated that the direct organogenesis produced a higher yield of regenerated plants (22% more) within shorter time (4 weeks less). Therefore, this method is recommended for sugarcane regeneration and for further use in genetic transformation via A. tumefaciens with desired genes.

Somatic Embryogenesis and Plant Regeneration from Hypocotyl and Leaf Explants of Brassica Oleracea var. Botrytis (Cauliflower)

We investigated direct and indirect formation of somatic embryogenesis in Brassica oleracea var. botrytis (cauliflower), a very important vegetable crop worldwide. Direct somatic embryogenesis, which is rather rare, was achieved in culture of 2-week-old hypocotyl explants of Brassica oleracea var. botrytis On MS medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5; 1.0; and 1.5 mg/l kinetin. Initial induction of embryogenic callus was achieved on MS supplemented with very low concentrations of 2,4-D (0.05 mg/l and 0.1 mg/l). Indirect somatic embryogenesis from leaf sections was obtained on MS supplemented with 0.05 or 0.1 mg/l 2,4-D. We examined various stages of somatic embryos (globular, heart, torpedo, cotyledonary). More embryos per explant were produced through the indirect pathway (23-25) than through the direct pathway (14-19). The number of embryos produced was nigh. There is a potential for recurrent, repeated or secondary somatic embryogenesis, possibly an unlimited source for mass propagation and ideal for synthetic seed production in this species. Plant regeneration was achieved on half-strength MS medium without any hormones.

Optimization of Two Different Morphogenesis Pathways in Three Iranian Cumin Landraces with the use of an Embryo

ABSTRACTThe best two morphogenesis pathways in Iranian Cumin landraces were optimized in this experiment. The first method is based on callus induction from zygotic embryo extracted from mature seeds and the second method includes direct somatic embryogenesis without callus phase and regeneration to stem. Both methods led finally to development of whole plantlets. Various concentrations of plant growth regulators (0.0, 0.1 mg/l BAP, 0.2, 0.4, 0.6 mg/l NAA and 0.2, 0.4 mg/l IAA) in twelve hormonal compositions on B5 medium were used. The experiment was conducted as factorial based on completely randomized design (CRD) with 8 replications (8 Petri dishes) and 5 explants per each Petri dish. Khorasan landrace showed the best response with both pathways exhibiting high callus production (27.5%) and direct shoot regeneration (67.7%).

Crambe tataria: actions for ex situ conservation

In vitro micropropagation by direct organogenesis and somatic embryogenesis via callus was developed for Crambe tataria (Brassicaceae). C. tataria is an endemic species of the Pontic-Pannonic region, but it is also present in Italy, where it is localized in Friuli on a characteristic grassland formation, called “magredi”. C. tataria is regarded as an endangered species. Leaf and root explants were subjected to plant regulator treatments, which invoked different morphogenic responses. Leaf explants produced more callus than root explants and a higher amount of callus was obtained with 1 mg l−1 2,4-D in combination with 2 mg l−1 Kin. Somatic embryogenesis was obtained in calli maintained in a delayed subculture regime on media containing BAP in combination with NAA. Root explants cultured with BAP combined with NAA developed adventitious rosette shoots. Shoots rooted on half-strength MS media, and the number of roots per plantlet and their length were heavily dependent on sucrose content. The in vitro regenerated plantlets were acclimatized ex vitro and a mean of 50% of the plantlets survived and showed a true-to-type growth habit. This study describes the development of two in vitro micropropagation protocols, via direct organogenesis and via embryogenesis from callus, that are the basis for the application of in vitro tools for the establishment of basal collections with representative genetic diversity and for the long-term storage of plant genetic material.

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.