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Plant biodiversity expresses one of the principal natural resources for all nations as it provides food, medicine, and shelter for all living besides its important role in balancing ecosystems and mitigating climate change. Jordan has paid great attention to its unique heritage of plant biodiversity since the foundation of the country, especially in terms of conservation. Notable conservation efforts have been made by Jordanian governments during the first hundred years of the country's foundation. These efforts were highly fruitful thanks to the unlimited governmental support to all projects related to the conservation of Jordan Flora. So, on the occasion of the first centenary of the country’s foundation, this review article was prepared to highlight some of the efforts made by Jordan during the past hundred years to conserve this national wealth of plant biodiversity.

Solanum villosum is a wild distinguished medicinal herb that has few studies describing its medicinal properties with no sustainable production and conservation studies. In this study, in vitro cultures of microshoots and callus of Solanum villosum were multiplied in vitro on Murashige and Skoog media supplemented with different growth regulators. The MS media that have 2,4 Dichlorophenoxy acetic acid (2,4-D) and Kinetin at the levels of (2.0, 1.2 mg/L; respectively) were used for the induction and multiplication of callus that yielded the highest fresh weight of (6.03 ± 0.15) g. Cryopreservation was applied successfully to seeds using the dehydration technique and for both microshoots and callus using v-cryoplate technique. The highest regrowth rate was obtained in callus (95 ± 0.13) after pre-culturing callus on 0.3 M sucrose for 5 days. The most inhibited bacteria strains by microshoots extracts were, Klebsiella pneumoniae and Staphylococcus epidermidis (30 ± 0.25 and 25 ± 0.17 mm; respectively) using the disk diffusion assay. In MIC method; The extract obtained from microshoots achieved best results of MIC values (9.77 and 39.06 (µg/ml) against (Micrococcus luteus and Klebsiella pneumonia; respectively). A sustainable protocol of callus culture and cryopreservation protocol for in vitro Solanum villosum has been set for the first time which makes this research novel. Furthermore, promising results of antimicrobial activity for both in vitro and wild Solanum villosum were obtained.

Paronychia argentea is a wild medicinal herbaceous herb that contains many secondary metabolites. It grows wild in Jordan under harsh high salinity soil conditions. This study was conducted to investigate the effect of salinity stress on growth, biochemical responses, and ion uptake of in vitro Paronychia argentea micro plants. Our results revealed a clear reduction in all growth parameters in response to an increase in salinity level used (0, 25, 50, 100, 200 mM). When the growth medium was supplemented with 200 mM NaCl; the fresh and dry weights were reduced by 40% compared with the control treatment. Moreover, micro plant uptake of phosphorus calcium and potassium ions decreased with increasing salinity levels. The Mg++ content was increased by 10% at the 25 mM level of NaCl compared with control, while it declined by 20% at 200 mM of NaCl. On the other hand, Na+ uptake increased with increasing salinity levels and reached a maximum value of (18328 ppm/D. W) at 200 mM NaCl. Moreover, proline content increased in response to salinity level to record maximum value (80.8 µmole/g) at 200 mM NaCl compared to (30.4 µmol/g) obtained in the control. Quercetins content in P. argentea micro plants was increased by 1.7-fold in concentration in response to salinity at 50 mM of NaCl, while an about 7-fold increase in the concentration of isorhamnetin content was obtained due to salinity at 100 mM.

Solanum nigrum L. is a medicinal plant of solanaceae family with distinguished therapeutic properties. Traditionally, S. nigrum. had been used as an anti-tumorgenic, antioxidant, hepatoprotective, diuretic, and antipyretic agent. The most important alkaloid member in this plant is solanine. Therefore, this study was conducted to utilize tissue culture techniques for the enhancement of solanine production in the in vitro grown cultures of this promising neglected plant. For callus growth and development experimental part, the highest callus growth parameters (callus diameter (21.4 mm) and callus fresh weight (2202.4 mg) were obtained in callus grown on Murashige & Skoog MS media supplemented with 2,4-Dichlorophenoxyacetic acid (2.0 mg·L-1) plus 1.5 mg·L-1 Thidiazuron. Similar trend was also obtained in cell suspension culture experiment, as maximum growth was recorded at similar hormone combination. Moreover, High-performance liquid chromatography analysis revealed that, solanine was affected by growth regulator type and concentration. The highest solanine levels were obtained when the explants were treated with 6-benzylaminopurine at level of 2.0 mg·L-1, as solanine content reached up to (2.61, 1.53 mg.g-1) for callus and cell suspension, respectively, while, microshoot contained the highest solanine (4.52 mg.g-1 DW) at 6-benzylaminopurine level of 1.6 mg.L-1. Additionally, carbon source had positively affected solanine level, where 0.2 M sucrose resulted in production of the highest amounts (3.13, 2.03 and 1.20 mg.g-1 DW) of solanine in microshoots, callus and cell suspension, respectively. Also, exposing microshoots and callus to light intensity of (100 µmol.m -2 s-1) yielded the highest solanine content (4.03 and 1.26 mg.g-1 DW, respectively),while the lowest solanine levels (1.50 and 0.48 mg.g-1 DW) were observed in plant material exposed to the lowest light intensity treatment (25 µmol.m -2 s-1) .

An efficient protocol is outlined for rapid and mass micropropagation of Ficus carica L. (fig). Shoot tips (5 mm) were obtained from mother plants stock grown on half strength Murashige and Skoog (½ MS) medium with the addition of 30 g/L sucrose. For shoot multiplication Benzyl amino purine (BAP) and kinetin produced differences number of new shoot per plant and shoot height. BAP at 0.4 mg/L in combination with 0.2 mg/L indole-3-butyric-acid (IBA) produce maximum in vitro propagation rate, with 4.2 shoots per ex-plant. Root initiation was experimented on MS medium containing different concentrations of mg/L, IBA, IAA (Indole-3-acetic-acid) (IAA) or Naphthalene acetic acid (NAA). Highest number of root (4.3) was resulted when 1.5 mg/L IBA was used. After acclimatization in a mixture of (1 soil: 1 perlite: 1 peat) survival rate of 80% was achieved. For in vitro conservation of F. carica was experimented as microshoots were stored for 40 weeks on MS medium containing different sucrose concentration. Medium supplemented with 3% sucrose gave the highest regrowth (89%) at 24 ± 2 °C. Culture grew slowly when transferred to new fresh medium after the storage periods.

In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm) and abscisic acid (0, 4, 8, 12 μm) in Murashige and Skoog (MS) media, while cryopreservation was conducted by using encapsulation dehydration, V-cryoplates and seeds. Significant differences were observed between tested lines grown on MS media supplemented with 200 mm sucrose where transgenic lines overexpressing SlAREB1 showed improved growth when compared with negative control. The addition of abscisic acid (ABA) to the preservation media affected negatively transgenic lines growth and development when compared with ABA-free media. In encapsulation dehydration, non-cryopreserved transgenic lines overexpressing SlAREB1 pretreated in 0.8 M sucrose for 1 day and subjected to different dehydration periods showed significantly higher survival percentages when compared with negative control. For V-cryoplates technique, cryopreserved transgenic lines overexpressing SlAREB1 treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly higher survival percentages when compared with the negative control. Seed cryopreservation was performed successfully with a clear reduction in germination percentage in transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines overexpressing SlAREB1 were found to improve tolerance against different abiotic stresses associated with different in vitro preservation protocols.

Solanum nigrum L. is a medicinal plant of solanaceae family with distinguished therapeutic properties. Traditionally, S. nigrum . had been used as an anti-tumorgenic, antioxidant, hepatoprotective, diuretic, and antipyretic agent. The most important alkaloid member in this plant is solanine. Therefore, this study was conducted to utilize tissue culture techniques for the enhancement of solanine production in the in vitro grown cultures of this promising neglected plant. For callus growth and development experimental part, the highest callus growth parameters (callus diameter (21.4 mm) and callus fresh weight (2202.4 mg) were obtained in callus grown on Murashige & Skoog MS media supplemented with 2,4-Dichlorophenoxyacetic acid (2.0 mg·L -1 ) plus 1.5 mg·L -1 Thidiazuron . Similar trend was also obtained in cell suspension culture experiment, as maximum growth was recorded at similar hormone combination. Moreover, High-performance liquid chromatography analysis revealed that, solanine was affected by growth regulator type and concentration. The highest solanine levels were obtained when the explants were treated with 6-benzylaminopurine at level of 2.0 mg·L -1 , as solanine content reached up to (2.61, 1.53 mg.g -1 ) for callus and cell suspension, respectively, while, microshoot contained the highest solanine (4.52 mg.g -1 DW) at 6-benzylaminopurine level of 1.6 mg.L -1 . Additionally, carbon source had positively affected solanine level, where 0.2 M sucrose resulted in production of the highest amounts (3.13, 2.03 and 1.20 mg.g -1 DW) of solanine in microshoots, callus and cell suspension, respectively. Also, exposing microshoots and callus to light intensity of (100 µmol.m -2 s -1 ) yielded the highest solanine content (4.03 and 1.26 mg.g -1 DW, respectively),while the lowest solanine levels (1.50 and 0.48 mg.g -1 DW) were observed in plant material exposed to the lowest light intensity treatment (25 µmol.m -2 s -1 ) .

Callus was induced from seeds of Colchicum hierosolymitanum Feib inoculated on the surface of MS media supplemented with 0.45 µM 2, 4-dichlorophenoxyacetic acid under dark conditions. Then callus was cultured on MS media supplemented with 4.52 µM 2, 4-dichlorophenoxyacetic for growth and maintenance. Friable callus from the fourth generation was transferred to liquid MS media supplemented with 0.54 µM 1-naphthaleneacetic acid to form cell suspension. Cells were successfully subcultured every 27 days on the same liquid media supplemented with 0.54 µM 1- naphthaleneacetic acid. Higher concentration (9 µM) of 6-benzyladenine with 0.45 µM 2, 4-dichlorophenoxyacetic acid resulted in higher cells fresh weight, while 1-naphthaleneacetic acid combinations with 6-benzyladenine had no effect on cell growth. On the other hand, the time for subculturing the cells into new fresh liquid media was determined to be after 27 days of incubation. (-) -Colchicine was identified in callus and cell suspension of C. hierosolymitanum by performing HPLC analysis against standard. Different ratios of NH +4 : NO -3 were used to study their effect on (-)-colchicine content, the highest colchicine content of 0.070 mg g -1 DW was obtained at 30 mM NH 4 + of total nitrogen. Colchicine alkaloid was highest, 0.090 mg g -1 DW, at 0.1 M of sucrose after 4 weeks incubation. (-) - Colchicine alkaloid was not detected in callus grown on sucrose free media. Maximum production of colchicine, 0.235 mg g -1 DW, was obtained in callus extracts of 60 days old callus grown under dark conditions. Cell suspension had 0.012 mg g-1 DW (-) -colchicine from suspended cells grown under dark. (-) -Colchicine content of callus incubated under dark (0.095 mg g -1 DW) was higher than light (0.070 mg g -1 DW) condition.

Somatic embryogenesis was obtained from callus of wild arum (Arum palaestinum). Callus was induced from sterilized corm bud sprouts cultured on basal medium containing 4.4 μM 6-benzyladenine and 5.4 μM 1-naphthaleneacetic acid. Callus was maintained under dark conditions using basal medium with 4.4 or 8.8 μM 6-benzyladenine and 5.4 or 10.8 μM 1-naphthaleneacetic acid. The highest callus weight and most desirable callus phenotype were achieved using basal medium containing 8.8 μM 6-benzyladenine and 5.4 μM 1-naphthaleneacetic acid. Friable calli were cultured in the dark on basal medium containing 4.5 μM 2, 4-dichlorophenoxyacetic acid, 0.46 μM 6-furfurylaminopurine, 5.4 μM 1-naphthaleneacetic acid, and 1.7 mM proline to induce embryogenesis before transfer to regeneration medium. Embryos that developed on regeneration medium were transferred to medium minus plant growth regulators for germination. Ninety percent of the germinating embryos developed into rooted plantlets. Rooted plants were grown in the greenhouse and acclimatized successfully with a 95 % survival rate. This is the first report of successful somatic embryogenesis and plant regeneration in A. palaestinum.

A protocol for in vitro propagation of the wild germander (Teucrium polium L.) was developed. In vitro plants were developed from ex vitro axillary buds. Then, shoot tips were excised and established on Murashige and Skoog medium. Proliferation of shoots was tested with different levels of 6-furfurylaminopurin, 6-benzyladenine, or thiadiazuron. The highest proliferation of T. polium was obtained when 6-benzyladenine and 6-furfurylaminopurin were used at 2.0 and 1.6 mg l−1, respectively. Thiadiazuron gave the lowest response for shoot proliferation. Rooting was experimented at different levels of Indol-3-butric acid, Indol-3-acetic acid, or 1-naphthaleneacetic acid. 1-Naphthaleneacetic was the only growth regulator which promoted root induction. Rooted plants were acclimatized successfully with 75% survival and grown in the greenhouse. In vitro- and in vivo-grown plants were analyzed for essential oil production. In vitro-grown T. polium on MS medium supplemented with 6-benzyladenine and 1-naphthaleneacetic gave higher oil yield than that grown on hormone-free Murashige and Skoog medium. In vivo (wild)-grown T. polium produced different oil yield when collected in different months (April and October). β-caryophyllene, used as a marker compound in the essential oil, was identified and quantified by gas chromatography (GC) analysis. Gas chromatography/mass (GC-MS) spectrometry analysis was also used to identify other components of in vitro cultures and to compare with in vivo-grown plants.

Physiological and biochemical responses of open-pollinated ‘Roma’ and dwarf F1 hybrid ‘Patio’ tomato (Lycopersicon esculentum Mill.) cultivars to in vitro induced salinity were examined in light of the possible contribution of ethylene to these symptoms. Salinity was induced by incorporating 0 (control), 50, 100, 150, or 200 mM NaCl into shoot culture media. Elevated salinity treatments significantly enhanced ethylene accumulation in the headspace and were accompanied by increased leaf epinasty in both cultivars. Growth, leaf cell sap osmolarity, leaf tissue viability and shoot soluble protein content were generally depressed with elevated salinity treatments, whereas electrolyte leakage, membrane injury, raffinose, and total sugars were concomitantly increased. Macronutrients N, P, K, Ca, Mg, and S decreased with elevated salinity in both cultivars and were accompanied by a significant increase in Na content and a sharp decrease in K/Na ratio. Tissue micronutrients, Fe, B, Zn, Mn, and Cu were generally decreased with elevated salinity especially at 100 mM or more. Incorporating ethylene inhibitors CoCl2 or NiCl2 at 5.0 or 10.0 mg/l into media supplemented with 100 mM NaCl significantly reduced ethylene accumulation in the headspace and prevented epinasty, but did not eliminate the negative impacts on growth and other physiological parameters caused by salinity treatment in either cultivar. Our results indicate that the increase in ethylene under salinity stress is not the primary factor contributing to salinity’s deleterious effect on tomato plant growth and physiology.

Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.

Branched broomrape (Orobanche ramosa) is a holo-parasitic flowering plant that attaches to the root of its host, green plant, by means of a specialized structure known as haustorium. Following successful contact and penetration on susceptible plant root, complex tissue of Orobanche cells is formed which is known as the tubercle. Newly formed tubercles contain high activity ofindole-3-acetic acid (IAA). Triiodobenzoic acid (TIBA), as an inhibitor of IAA polar transport, was utilized to investigate the supply and requirement of auxin to the developing O. ramosa on tomato plant. There was no significant reduction in the incidence of O. ramosa per pot of different TIBA treatments. However, infection severity in terms of the number of O. ramosa shoots that emerged per plant and number of attachments per plant root system were significantly reduced by 60 % and 45 % on TIBA treated plants, respectively. Histo-logical studies revealed conspicuous delay in the initiation of xylem vessel differentiation inside tubercles of the TIBA treated tomato plants. Also, differentiated vessels showed thinner secondary wall deposition, and improper alignment within bundles inside those tubercles. They were wider and shorter in diameter in comparison to those of untreated plants. These findings were attri-buted to the short supply of IAA required for normal development, and to the xylem vessel differentiation of O. ramosa tubercles on infected tomato. Hence, this parasitic flowering plant seems to depend upon its host in its requirements for IAA, in a source to sink relation-ship.

Seeds of broomrape (Orobanche cernua) were exposed to 0, 25, 50, 75, and 100 mM NaCl solutions during their preconditioning period (14 days of moisture) under laboratory conditions and induced to germinate by synthetic germination stimulant (GR24). There was significant reduction in seed germination with increased salt concentration as shown in 35.2, 32.5, 23.6, 14.3, and 9.2% germination, respectively. Exposure of Orobanche cernua seeds to 0.0, 1.0, 1.25, and 1.5 M levels of NaCl for 9 hours resulted in 29.4, 21.3, 20.5, and 17.4% germination, respectively. Water preconditioned seeds showed Heavier protein profile bands of 6.5-14.2 KDa than those of dry seeds. Seeds treated with 0.75 M NaCl showed profile similar with that of water preconditioned ones, plus an extra band at 29-36 KDa. The protein profiles of 1.0 and 1.5 M NaCl treated seeds showed weaker bands with the absence of 29-36 KDa band.

The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.

Silybum marianum L. is a wild medicinal herbal plant in Jordan. It is widely used in folk medicine due to its high content of silymarin. In vitro production of silymarin was experimented at different concentrations (0.0, 0.4, 1.0, 1.6 or 2.0 mg/l) of growth regulators (6-furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), or 6-(gamma,gamma- Dimethylallylamino) purine (2ip)) and different concentrations (15, 30,45,60 mg/l) of carbon sources (glucose, fructose, and sucrose). HPLC (High Performance Liquid Chromatography) analysis was used to identify silymarin components. In vitro grown S. marianum on MS (Murashige and Skoo 1962) medium supplemented with (1.6 mg/l) kinetin and (0.1 mg/l) 1-Naphthaleneacetc acid (NAA) gave the highest silymarin content of (0.84%) silybin and (0.49%) silydanin as compared with cultures grown on hormone-free MS media which contained (0.36%) silybin and (0.30%) silydanin. In vitro grown S. marianum on MS medium supplemented with (2.0 mg/l) BA and 0.1 mg/l NAA yielded (0.67%) silybin and (0.37%) silydanin, while MS media supplemented with (1.0 mg/l) of 2iP gave (0.72%) silybin and (0.24%) silydanin. Otherwise, the in vivo (wild) grown shoots of S. marianum gave (1.07%) for silybin and (0.46%) sildyanin. Among carbon source, glucose at (45 g/l) gave (1.63%) of silymarin content. Results indicated a significant use of in vitro grown cultures for silymarin production.

The influence of headspace ethylene on anthocyanin, anthocyanidin, and carotenoid accumulation was studied in suspension cultures of Vaccinium pahalae. Exogenous application of ethrel (an ethylene-releasing compound) significantly reduced growth and secondary metabolite production, whereas incorporation of 5.0 or 10.0 mg l-1 CoCl2 or NiCl2 effectively reduced ethylene accumulation and improved product accumulation, but AgNO3was toxic to cells. This study showed an overall negative impact of increased ethylene levels in the vessel headspace on phytochemical production in ohelo cell cultures.

Ohelo (V. pahalae Skottsb.) and bilberry (V. myrtillus L.) shoots were regenerated via direct organogenesis from whole leaves and leaf sections and also from hypocotyl explants of bilberry. Explants preincubated for 1 to 2 weeks in darkness yielded ≈75% regeneration frequencies and the highest number of regenerating shoots/explant on TDZ-supplemented media (0.9 to 2.7 μm). When 2iP or zeatin were substituted as the cytokinin source, frequencies of regeneration and shoot productivity were significantly lower. Explants held under constant illumination (no dark pretreatment) had significantly lower regeneration frequencies in all tested cytokinin-supplemented media. 2,4-D stimulated callus formation, but did not support regeneration from vegetative explants. Cells from callus and suspension cultures did not exhibit regeneration in any of the media that supported organogenesis from leaves. Regenerants were successfully micropropagated, although callus formation caused by zeatin and high 2iP levels interfered with shoot proliferation. Zeatin induced hyperhydricity in shoots from both species, but more severely in ohelo. Ex vitro rooting after treatment with 4.9 μm IBA or 5.4 μm NAA was 95% and 60% successful for bilberry and ohelo, respectively, and plants were readily acclimatized after an interval in a fog chamber. Bilberry microshoots also rooted in vitro in the absence of growth regulator treatment. Chemical names used: 1H-indole-3-butanoic acid (IBA); N-(3-methyl-2-butenyl)-1-H-purine-6-amine (2iP); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA); thidiazuron=1-phenyl-3-(1,2,3-thiadiazio-5-yl)urea (TDZ); 2,4-dichlorophenoxyacetic acid (2,4-D); 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

Successful in vitro multiplication of Capparis spinosa was achieved on Murashige and Skoog (MS) medium supplemented with benzyl amino purine (BAP) at 0.8 mg/L. The highest shoot length (35.6 mm) was obtained with the use of 0.4 mg/L BAP and 0.2 mg/L 1-naphthaleneanacetic acid (NAA). Kinetin at 2.0 mg/L produced a multiplication rate of 9.7 microshoots per explants with an average shoot length of 21.3 mm. In vitro rooting was successfully achieved on MS media supplemented with different concentration of indole-3-butyric acid (IBA), indole acetic acid (IAA) or NAA at various concentrations. However, rooting did not occur in the absence of IBA, IAA or NAA. A total of 85% survival was achieved when rooted explants acclimatized ex vitro using a mixture of 1 perlite: 1 peat. In another experiment, in vitro C. spinosa were successfully stored without serious losses by using MS medium supplemented with an appropriate concentration of osmoticum (sucrose, sorbitol, mannitol or glucose) at various concentration (0, 3, 6, 9 or 12%). Two types of plant material were used (in vitro plantlets and in vitro plantlets without tips). The results obtained show that the two type of plant material could be successfully maintained in vitro and optimum treatments were identified for each plant material. Further studies are still needed on medium term conservation to enhance the survival percentages of different plant material type. Key words: Clonal propagation, Capparis spinosa, medium term conservation, carbon source, roots formation.

Accumulation of essential oil in herbs depends on development stage. The aim of the present study was to investigate the effect of growth stages (pre-flowering and blooming stages) on the yield and composition of essential oil of thyme (Origanum syriacum L.). Three harvesting times (morning at 8 am, noon at 12 pm and evening at 6 pm) and two harvesting date (growth stage (pre-flowering stage) and blooming stage) were studied. Total yield of oil was higher in the plant at blooming stage in comparison with that at pre-flowering stage. According to the results of multivariate analysis, higher levels of volatile compounds were noticed during the blooming stage, except carvacrol, gamma-terpinene, alpha-terpinene and para-cymene that were found higher in pre-flowering stage. Unexpectedly, carvacrol as structural isomer of thymol depicts higher concentration at pre-flowering stage. Moreover, an accurate valid prediction equation of oil samples was built to predict the growth stage and quality control purposes of the oil. Key words: Carvacrol, essential oil, harvesting time, blooming stage, Origanum syriacum, thymol.

Successful clonal propagation of Stevia rebaudiana was achieved using microshoots as a primary step for in vitro conservation. Maximum proliferation was obtained on Murashige and Skoog (MS) medium supplemented with 1.5 mg L ‐1 benzyl amino purine and 0.2 mg L ‐1 indole-3-butyric-acid (IBA). Auxin increased rooting percentage of shoots at concentration of 0.4 mg L ‐1 IBA, indole-3-acetic-acid or naphthalene acetic acid and no rooting occurred without plant growth regulator. A survival of 90% was achieved when rooted explants were acclimatized in vivo in 1 soil: 1 perlite: 1 peat. In vitro S. rebaudiana shoots were successfully stored for up to 32 weeks on MS medium supplemented with an appropriate concentration of sucrose, sorbitol or mannitol, at 24 ± 2°C. After 32 weeks, 93.6% of the shoots were able to survive. Moreover, 89.3% of them were able to regrow when stored under light conditions. Cryopreservation by vitrif ication was successfully achieved (65.6% regrowth) when shoot tips were precultured on a medium supplemented with 0.4 M sorbitol for 2 d, followed by loading shoot tips with 80% concentrated plant vitrification solution 2 (PVS2) for 20 min; then dehydrated with 100% PVS2 for 60 min at 0°C prior to storage in liquid nitrogen. This procedure is easy to handle and produced a high levels of shoot formation. This protocol could be useful for longterm storage of S. rebaudiana germplasm.

Callus was initiated from leaves of Gladiolus cv. `Balady' on MS medium containing 1.0 mg/L NAA, 0.1 mg/L 2,4-D, and 0.5 mg/L kinetin. Organogenesis from callus was induced on medium containing 0.5, 1.0, 1.5, or 2.0 mg/L of either BA, kinetin, or TDZ. TDZ was more effective and resulted in a higher percentage regeneration and regenerant number. The microshoots produced were then propagated in vitro and cormel production was studied. Maximum shoot number (25.1) was obtained on medium containing 1.0 mg/L TDZ without auxin supplements in liquid shaking culture. In vitro cormel formation was significantly enhanced by B-9 and paclobutrazol. Increased sucrose concentration (4% to 5%) proved the most effective for cormel formation. Optimal dormancy break was obtained by storing cormels at 5°C for 1 month or by soaking them for 5 sec with 50 mg/L GA3. In-vitro rooting was achieved on solid medium containing NAA, IAA, or IBA, with higher root number recorded on NAA-treated cultures. Rooted microshoots were successfully acclimatized for ex vitro conditions and grown in the greenhouse. Plants produced from in-vitro propagation showed similar morphological characteristics of plants propagated by direct corm planting in the greenhouse.

Some factors that affect the in vitro conservation of wild pear (Pyrus syrica) microshoot cultures were studied. Sorbitol and mannitol at 0.2 to 4.0 M reduced growth significantly and extended the subculture intervals to 5 months when cultures where kept at 15°C. Increasing sucrose to 12% in the medium was not highly effective and the subculture intervals did not exceed 3.0 months. After 2 years of maintaining cultures on slow-growth medium, cultures grew slowly when transferred to fresh control medium. Shoots started to proliferate after three subcultures (6.0 weeks apart) on medium containing 1.0 mg/L BA and 0.1 mg/L NAA. New microshoots were rooted on medium containing 2.0 mg/L IBA and rooted microshoots gave 90% survival when acclimatized ex vitro under intermittent mist.

Osmotic adjustment in response to decreasing media water availability was observed for in vitro Chrysanthemum morifolium Ramat. cultivars Bright Golden Anne, Deep Luv, and Lucido. Water stress was induced by increasing sorbitol (0, 0.1, 0.2, 0.3, 0.4 M), mannitol (0, 0.1, 0.2, 0.3, 0.4 M), and sucrose (30, 45, 60, 75, 90 g·l-1) concentrations in modified MS media (2 mg·l-1 BA and 0.1 mg·l-1 NAA). Osmotic adjustment was evidenced by a significant reduction in measured cell sap osmotic potential (R2 = 0.78, 0.96, 0.91 for sucrose, sorbitol, and mannitol respectively) in all cultivars. Shoot length, weighted density (apparent mass), and proliferation were significantly reduced by sorbitol and mannitol treatments. Sucrose reduced shoot proliferation, increased length, and had an inconsistent effect on weighted density. Cultures grown on media without hormones showed tremendous increase in root number up to 60 g·l-1 sucrose. Sorbitol had a negligible effect on rooting at 0.1 M but no roots developed at higher sorbitol concentrations or in any mannitol treatments. Plants transferred to a non-water-stress media after they had experienced in vitro water stress exhibited no change in osmotic properties from the stress treatments.