Efficient plant regeneration via organogenesis in “Egusi” melon (Colocynthis citrullus L.)

Plant regeneration protocol of “Egusi” melon (Colocynthis citrullus L.) was established using three local (“Ejagham”, “Sewere” and “Barablackedge”) and one improved (NHC1-130) cultivars. Cotyledonary explants of different lengths (1/2, 1/4 and 1/6) excised from 4- or 8-day-old seedlings germinated in vitro were cultured on MS medium supplemented with different concentrations of 6-benzylaminopurine (BA). The best results were obtained when cotyledons from 4-day-old seedlings were cut into 2 (1/2) halves. Plant regeneration was optimal on medium containing 5 mg/l BA, yielding 86.3%, 77.0% and 76.3% shoot induction frequencies amongst the three local cultivars of “Ejagham”, “Sewere” and “Barablackedge”, respectively. In NHC1-130, the highest shoot induction frequency (85%) was obtained on medium containing 2 mg/l BA. Adventitious shoots were elongated on medium containing 0.1 mg/l BA and successfully rooted on hormone-free MS medium. Flow cytometric analysis revealed 70% of the plants to be diploid.     

In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

Efficient in vitro multiplication in Ornithogalum ulophyllum Hand.-Mazz. from twin scale explants

Ornithogalum ulophyllum Hand.-Mazz. with beautiful white flowers is an important medicinal and ornamental plant of the Middle Eastern countries and need exploitation for commercial propagation. The study reports in vitro mass proliferation of bulblets achieved from twin scales and “in vitro regenerated bulblet” explants on MS medium supplemented with various concentrations of BAP–NAA. The best regeneration on twin scales and “in vitro regenerated bulblets” was obtained on MS medium containing 2 mg l⁻¹ BAP–0.5 mg l⁻¹ NAA and 2 mg l⁻¹BAP–1 mg l⁻¹ NAA, respectively. However, bulb scales seemed to be more potent for bulblet regeneration. A large number of the developing bulblets rooted on the regeneration medium. Remaining non-rooting bulblets were rooted on MS medium containing 1 mg l⁻¹ NAA. All plants were acclimatized in the environmental chamber for 4 weeks and were transferred to the greenhouse for flowering. Regenerated bulblets developed into morphologically normal plants.     

Plant regeneration from stem segment-derived friable callus of “Fonio” (Digitaria exilis (L.) Stapf.)

“Fonio” (Digitaria exilis (L.) Stapf.) is a member of the grass family with excellent culinary and nutritional properties. In spite of its economic values, hardly has any improvement work been done. To enhance genetic improvement of this grain, plant regeneration protocol was developed using 8 cultivars. Stem segments of 5 mm long excised from 1 month-old seedlings germinated in vitro were cultured on 6 types of media for friable callus induction. Best result was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 g l⁻¹ casamino acid, where 91.3, 88.9 and 87.8% of the explants formed friable calli in cultivars ‘Kurelep’, ‘Churiwe’ and ‘Agyong’, respectively. Shoots appeared when friable calli were transferred to two regeneration media, i.e., MSBZ (MS medium + 0.022 mg l⁻¹ 2,4-D, 0 .22 mg l⁻¹ 6-benzylaminopurine (BA), 0.22 mg l⁻¹ zeatin) and MSBG (MS medium + 0.5 mg l⁻¹ BA, 0.1 mg l⁻¹ gibberellic acid). The highest frequency of plant regeneration was attained on MSBG, with 91.7% of the friable calli forming shoots in cultivar “Churiwe”. Regenerated plants were rooted on hormone free MS medium. Flow cytometric analysis revealed 100% of the regenerants to be diploid. The protocol developed here can be used in the transformation of “Fonio” to increase the yield potential of this crop by incorporating characteristics such as disease resistance and stress resistance.     

Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange

Shoot tips obtained from in vitro grown plantlets of guava (Psidium guajava L.) were encapsulated in calcium alginate beads for short-term storage and germplasm exchange. A gelling matrix of 3% sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Maximum percent response for conversion of encapsulated shoot tips into plantlets was obtained on growth regulator free full strength liquid MS medium. The regrowth ability of encapsulated shoot tips was affected by medium strength and sucrose concentrations in the medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 30 days with a survival frequency of 25%. After 60 days of storage under minimal growth conditions (sucrose lacking medium), about 75% encapsulated shoot tips were converted into plantlets when subcultured on 3% sucrose containing medium. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16 h in liquid medium with 0.3 M sucrose, then for 5 h in liquid medium with 0.7 M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20 min in a loading solution containing 1.9 M glycerol + 0.5 M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90 min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20 min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.     

Improved shoot regeneration due to prolonged seed storage

Regeneration in vitro from cotyledon explants of commercial squash (Cucurbita pepo L.) cultivars is generally efficient on Murashige and Skoog [Murashige, M., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15, 473–497] medium augmented with 4.4 μM benzyladenine. However, cotyledon explants from certain seed batches of cultivars Bareqet and Ma’yan could regenerate only buds, leaf primordia or very small shoots with storage for up to 2 years at 4 °C. Seed storage of cultivars Bareqet and Ma’yan at 4 °C for 6–8 years resulted in significant increases in shoot regeneration, shoot growth and explant growth, returning to the normal range of values for C. pepo. For example, for cv. Bareqet shoots regenerated per explant increased from 0.4 after storage for 2 years to 1.21 after storage for 8 years; shoot length increased from a mean of less than 2 mm after storage for 2 years to 22 mm after storage for 8 years. Additionally, the final explant fresh weight of cv. B increased from 181 mg after storage for 2 years, to 1389 mg after storage for 8 years. Similar responses were observed for seedling-derived explants of cv. Ma’yan following storage’ for 2–7 years. However, total regeneration (number of explants regenerating buds, leaf primordia or shoots) was not affected by prolonged storage for either cultivar. This is the first report of stimulation of in vitro shoot regeneration of a seedling-derived organ caused by prolonged seed storage. Moreover, the great improvement in regeneration due to long-term seed storage provides a new mechanism for the understanding of non-repeatability of plant tissue culture results.     

Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.     

Plant regeneration of an endangered medicinal plant Hydrastis canadensis L.

Goldenseal (Hydrastis canadensis L.) is an endangered medicinal plant used to treat sore eyes and mouths, cold and flu and also as a dye. The objective of this study was to develop an efficient in vitro propagation protocol for goldenseal. Significantly more shoots (26 shoots per leaf explants) were induced on a medium containing 2.5 μM thidiazuron (TDZ) and 5.0 μM 1-naphthaleneacetic acid (NAA) than any other treatment. Sub-culturing regenerated shoots on a medium with 5.0 μM 6-benzylaminopurine (BA) induced the maximum rate of shoot multiplication. Growth of the regenerated shoots in a temporary immersion bioreactor resulted in significant increases in biomass, shoot height and shoot multiplication. The regenerated shoots from the temporary immersion bioreactor formed roots when transferred onto a medium with 1.0–2.0 μM indole-3-butyric acid (IBA). Regenerated whole plantlets were acclimatized and maintained in standard greenhouse conditions for further growth. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of this rare, medicinally important species.     

Physiological and biochemical responses for two cultivars of Pisum sativum (“Merveille de Kelvedon” and “Lincoln”) to iron deficiency conditions

The aim of this work was to compare the tolerance and the physiological responses to Fe deficiency of two Pisum sativum cultivars (“Merveille de kelvedon” and “Lincoln”) commonly cultivated in Tunisia. We studied the effects of Fe deficiency on: (i) chlorophyll content, relative growth rate (RGR) and Fe status, (ii) rhizosphere acidification, (iii) changes under Fe-deficient conditions in the activities of two root enzymes, the first related to the proton extrusion (H⁺-ATPase) and the second to iron reduction mechanism (Fe(III)-chelate-reductase: FCR). Three treatments were used: C, control, complete nutrient solution (CNS) containing 30 μM Fe; DD, direct deficiency, CNS without iron; ID, indirect deficiency, CNS containing 30 μM Fe + lime. Fe deficiency led to a significant decrease of chlorophyll content in both cultivars. The below reduction was observed in Fe-deficient plants of Merveille de Kelvedon. In addition, relative growth of shoots and whole plant was not affected by Fe deficiency. H⁺-ATPase and FCR activities were more stimulated in Merveille de Kelvedon than in Lincoln, under DD and ID Fe deficiency. The capacity of this cultivar to maintain plant growth and to preserve adequate chlorophyll synthesis under iron-limiting conditions is related to its better Fe-use efficiency, in addition to its high acidification and root reducing capacities. This allows us to suggest that Merveille de Kelvedon is more effective in overcoming Fe deficiency than Lincoln.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus (Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.     

In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.     

Cryopreservation of Malus cultivars: Comparison of two droplet protocols

Two droplet procedures, droplet-vitrification (PVS2) and droplet (DMSO) were applied for cryopreservation of in vitro cultured apple (Malus domestica Borkh., cvs. Florina, Idared, Colmar and Rebra) plants. The highest post-thaw regrowth rates (70% for cv. Florina, 66% for cv. Idared, 63% for cv. Colmar and 60% for cv. Rebra) were achieved after using the droplet-vitrification (PVS2) protocol. The excised shoot tips (2–3 mm in length) were precultured in 0.5 M sucrose enriched media for 24 h. Subsequently they were transferred in PVS2 vitrification solution for 30 or 40 min (depending on cultivar) at 24 °C and then immersed in liquid nitrogen. Rewarming was performed in liquid MS medium at 24 °C. Plants regenerated from cryopreserved shoot tips did not display any sign of morphological alteration or abnormalities in growth in comparison with control plants.     

Changes in morphological phenotypes and essential oil components in lavandin (Lavandula × intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes

Hairy roots were induced from leaf-derived calli of lavandin (Lavandula × intermedia Emeric ex Loisel.) by infection with wild-type strains of Agrobacterium rhizogenes, A-5 (MAFF 02-10265) and A-13 (MAFF 02-10266). A-5-inoculated calli formed hairy roots more efficiently than A-13 ones. The transgenic shoots could be obtained from hairy root segments mediated by each Agrobacterium strain. However, different plant growth regulators were required for efficient adventitious shoot formation in each strain. In A-5, the most efficient adventitious shoot formation rate of 23.8% was observed in a medium with 4.4 × 10⁻⁶ M of 6-benzylaminopurine. On the other hand, a significantly higher rate of 13.2% was detected in a medium with 4.0 × 10⁻⁷ M of N-(2-chloro-4-pyridyl)-N′-phenylurea in A-13. Most of the regenerated plants showed dwarfism with closed internodes and extensive lateral branching, which were typical characteristics of ‘hairy root syndrome’. On the other hand, only nine of the 45 regenerated plants formed flower buds in early June, a delay of about one month compared with nontransgenic regenerated plants. The floral stalks and spikes of these plants were very short, resulting in a compacted form. Many regenerants showed a significantly lower productivity of essential oil than nontransgenic regenerants. Moreover, the relative percentage of the linalyl-cation-derived compounds, linalool and linalyl acetate, decreased in most of the regenerated plants. Compact plants with the ability of flower bud formation are assumed to be valuable not only for lavandin breeding, but also for clarifying the interaction between rol genes expression and essential oil production.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.