In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA), or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.     

In vitro cultivation of donor quince shoots affects subsequent morphogenesis in leaf explants

The effect of in vitro cultivation of donor shoots on subsequent morphogenesis in leaf explants of quince (Cydonia oblonga Mill.) clone BA29 was investigated. Proliferating donor shoots were cultured in ventilated or closed vessels under different photosynthetic photon flux densities (PPFD; 200 and 100 μmol m⁻² s⁻¹) with 0, 15, 30 g dm⁻³ sucrose. Shoots grown in ventilated vessels, especially with sucrose at 15 or 30 g dm⁻³, were better developed with fully expanded leaves compared to those in standard closed vessels. Leaves collected from pre-treated donor shoots were used to assess regeneration capacity. Somatic embryo production was highest in leaves harvested from shoots cultured in closed vessels with 30 g dm⁻³ sucrose and in ventilated vessels with 15 and 30 g dm⁻³ sucrose and under high PPFD which was, in comparison with the control treatment (closed vessel, 30 g dm⁻³ sucrose and low PPFD), about 2 to 2.5 times higher. A similar response was observed for root regeneration.     

In vitro plant regeneration from leaf explants of Ophiorrhiza japonica

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18–27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.     

Cotton somatic embryo morphology affects its conversion to plant

Somatic embryos differentiated from hypocotyl explant in cotton (Gossypium hirsutum L.) exhibited very divergent morphologies. Six different types of somatic embryos based on cotyledon development were observed. The growth hormones (2,4-dichlorophenoxyacetic acid and kinetin) used in induction and maintenance media did not affect embryo rooting and germination. The 95 % conversion of normal embryos (with two cotyledons) was achieved, while an overall conversion was only 38 %. Horn shaped embryos failed to exhibit shoot growth. Poorly developed apical meristems were responsible for lower conversion percentages in some of embryo classes. However, regenerated plants phenotypically resembled to seed grown control plants regardless of somatic embryo morphology.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis

Callus cultures initiated from shoot base explants of Curcuma aromatica Salisb. were maintained on Murashige and Skoog (MS) media supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid alone or with 0.5 mg dm⁻³ kinetin. Plantlets were regenerated from 60 and 180-d-old callus on MS media supplemented with 3 mg dm⁻³ benzyladenine and 0.5 mg dm⁻³ α-naphthalene acetic acid. Approximately 8–10 plantlets were produced after 30–40 d of culture per 50 mg of callus inoculated. Out of 113 regenerants analyzed 85 plants were exclusively diploid and 28 were predominantly diploid revealing presence of polyploid nuclei. Frequency of polyploid cells were more in regenerants obtained from 180-d-old callus then from 6-d-old callus which might be attributed to the ageing of callus.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

High frequency plant regeneration from the cotyledonary node of common bean

An efficient regeneration system for Phaseolus vulgaris was developed from mature seeds germinated on Murashige and Skoog (MS) medium supplemented with thidiazuron or N⁶-benzylaminopurine (BA) for 6 d. Using cotyledonary nodes, multiple buds were induced on the MS medium supplemented with 5.0 mg dm⁻³ BA with the induction frequency 71.9 % after 4-week culture. The buds were then transferred onto shoot formation medium containing 1.0 mg dm⁻³ BA, 0.1 mg dm⁻³ gibberellic acid and 2.0 mg dm⁻³ silver nitrate. The addition of AgNO₃ enhanced the frequency of the shoot formation from 61.3 to 87.6 %. Root induction medium was half-strength MS medium with 0.75 mg dm⁻³ indolebutyric acid and 0.02 mg dm⁻³ BA. The average root frequency was 84.3 %. The regenerated plantlets with healthy roots grew successfully when transferred to soil. Using this system we obtained over 10 regenerated plantlets from one explant.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides Pall. was here developed. The protoplasts were isolated directly from the leaves of the hairy root-induced plants. The highest yield of protoplasts was obtained from fully expanded leaves of young plants. Their viability was up to 72 ± 2.3 %. The highest division frequency (32.4 ± 0.13 %) and sustained divisions were obtained in Durand, Potrykus and Donn (DPD) medium supplemented with 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid, 0.2 mg dm⁻³ 6-benzylaminopurine, 0.3 M mannitol, 2 % sucrose and 500 mg dm⁻³ casein hydrolysate at the plating density of 3.0 × 10⁵ cm⁻³. The frequency of shoot differentiation from protocalli reached to 91.75 ± 3.1 %. Opine synthesis and polymerase chain reaction analysis confirmed that T-DNA still existed in the protoplast regenerated plants.     

Development and evaluation of microsatellite markers in Phoenix dactylifera L. and their transferability to other Phoenix species

Forty one simple sequence repeats were isolated from two microsatellite enriched libraries of date palm (Phoenix dactylifera L.). After screening, 17 selected microsatellite loci were characterized and evaluated on a set of 31 cultivars and clones from Algerian and Californian germplasm. All primer pairs produced an amplification product of the expected size and detected high polymorphism among the analysed samples. These nuclear simple sequence repeat (SSR) markers are expected to be a very effective tool for evaluating genetic diversity in date palm germplasm. Acrosstaxa amplification showed the usefulness of most SSR markers in 14 other species across the genus Phoenix.     

Determination of oxalate in plant tissues with oxalate oxidase prepared from wheat

A method for determination of oxalate with oxalate oxidase (OxO, EC 1.2.3.4) prepared from wheat bran, is based on specific oxidation of oxalate to produce H₂O₂. The H₂O₂ formed was colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H₂O₂. The new method was tested on rice, buckwheat, soybean and oxalis leaves, showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform. Good agreement could be obtained between this method and the HPLC.     

Nitrate reduction in ramets of a clonal plant Eichhornia crassipes responding to nitrate availability during clonal growth stage

Effect of nitrate availability on nitrate reduction was examined in inter-connected ramets of invasive clonal plant Eichhornia crassipes grown with two nitrate supply regimes during different clonal growth stage. Increase of nitrate availability accelerated nitrate reductase activity (NRA) in parent and offspring ramets of E. crassipes, and there was greatly different pattern in inter-connected ramets during clonal growth stage. Leaf NRA was lower in offspring than that in parent ramets in phase 1, while significantly higher leaf NRA in offspring ramets was detected during phase 2. The results indicated NRA in inter-connected ramets of E. crassipes was highly dependent on nitrate availability and growth stage.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Increased cold tolerance in Arabidopsis thaliana transformed with Choristoneura fumiferana glutathione S-transferase gene

A glutathione S-transferase (GST) gene cloned from the lepidopteran spruce budworm, Choristoneura fumiferana Clem. was transformed into the model plant Arabidopsis thaliana. The CfGST-transgenic and wild-type A. thaliana were subjected to 4 and 10 °C for 48 h and their cold resistance was studied. The GST activity of the transgenic plants was 46.6 and 35.7 % higher than that of the wild-type plants after 48 h under 4 and 10 °C, respectively. Relative membrane permeability and malondialdehyde content in the transgenic plants were lower while contents of the chlorophyll and proline were higher than those in the wild-type plants under 4 and 10 °C. The survival rate of the transgenic plants was 43.7 % for 24 h under 0 °C, while survival rate of wild-type plants was 28.3 %. The results indicated that the insect GST could enhance cold resistance in the transgenic A. thaliana.     

Cadmium-induced oxidative damage and antioxidant responses in Brassica juncea plants

Indian mustard (Brassica juncea L. cv. Vitasso) plants exposed to 10, 30, 50 and 100 μM of Cd for 5 d in hydroponic culture were analysed with reference to the distribution of Cd²⁺, the accumulation of biomass and antioxidants and antioxidative enzymes in leaves. Cd induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. Cd induced a decrease in catalase (CAT) and guiacol peroxidase (GPX) activities but an increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities. Enhancement in dehydroascorbate reductase (DHAR) activity was also at 10 μM Cd. Glutathione reductase (GR) activity showed pronounced stimulation after all treatments, but glutathione S-transferase (GST) and glutathione peroxidase (GPOX) activities decreased. The effectiveness of ascorbate-glutathione cycle (AGC) was determined by the ratio of ascorbate to H₂O₂. This ratio decreased in the Cd-treated leaves which indicated that the cycle was disordered.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.