Adventitious Shoot Production Research Articles

In Vitro Regeneration from Leaf Explants of Helianthus verticillatus, a Critically Endangered Sunflower.

Helianthus verticillatus (Asteraceae), a whorled sunflower, is a perennial species restricted to a few locations in the southeastern United States and is now considered endangered. Therefore, restoring and protecting H. verticillatus as a species is a priority. This study introduces a highly efficient in vitro adventitious plant regeneration system from leaf explants, utilizing five diverse specimens of H. verticillatus, each representing distinct genotypes with phenotypic variations in leaf and stem morphology. Key factors influencing in vitro morphogenesis, including genetic constitution, explant source, and plant growth regulators (PGRs), were identified. The study revealed a remarkably strong genotype-dependent impact on the regeneration efficiency of the investigated H. verticillatus genotypes, ranging from a lack of regeneration to highly effective regeneration. The selection of two genotypes with varying regeneration abilities provides valuable models for genetic analyses, offering insights into factors influencing the regeneration potential of this endangered species. Optimum adventitious shoot regeneration results were achieved using Murashige and Skoog basal media (MS) supplemented with 8.8 µM N6-benzyladenine (BA) and 1.08 µM α-naphthalene acetic acid (NAA). This combination yielded the highest adventitious shoot production. Subsequent successful rooting on ½ MS medium without PGRs further solidified the efficiency of the developed protocol. Regenerated plantlets, demonstrating robust shoots and roots, were successfully acclimatized to greenhouse conditions with a 95% survival rate. The protocol developed in this study is the first such report for this endangered species and is expected to contribute to future genetic manipulation and modification studies.

An Agrobacterium-mediated transformation via organogenesis regeneration of a facultative CAM plant, the common ice plant Mesembryanthemum crystallinum L

ABSTRACT The common ice plant, Mesembryanthemum crystallinum L. provides a useful model for the study of environmentally induced photosynthetic conversion and abiotic stresses tolerance. However, a procedure for the production of transgenic ice plant, which is essential for functional genomics, has not been fully established. Here we tested the factors on the transformation of cotyledonary nodes excised from the ice plant seedlings such as thidiazuron (TDZ), NaCl and phytosulfokine (PSK), a peptidyl plant growth factor using Agrobacterium tumefaciens strains EHA101 and EHA105 harboring binary vector plasmids pBI7EGFP and pCAMBIA1302, respectively. The established procedure is as follows: the explants (cotyledonary nodes) were co-cultivated with Agrobacterium for 3 days, and the explants were cultured in the medium with 0.5 mg l−1 kinetin and 100 mg l−1 carbenicillin for 72 h, and they were cultured in the medium with 0.5 mg l−1 kinetin and 100 nM PSKfor 4 weeks. Thidiazuron and NaCl enhanced the production of multiple adventitious shoot formation during regeneration but reduced the transformation efficiency due to the vitrification of adventitious shoots. PSK was effective in the production of healthy adventitious shoots. The transformation frequency at the stage of whole plants was 0.6% and 4.6% per inoculated cotyledonary nodes using the Agrobacterium strain EHA101 (pBI7EGFP) and EHA105 (pCAMBIA1302), respectively.

Chemical Constituents and Antimicrobial Activity of Essential Oils from Micropropagation and Field-Grown Plants of Wedelia biflora [L.

An efficient regeneration protocol through axillary and inter nodal explants was developed from Wedelia biflora. Explants were placed on MS medium supplemented with different treatments of cytokinins, 6-benzyladenine (BA) and kinetin (KIN) for adventitious shoot production. The highest multiple shoots (95 %; 26.4 shoots/ explant) and elongation (2.75 cm shoot length), was obtained in 1.0 BA mg L-1 in axillary node explants. The shoot was transferred to half-strength MS medium supplemented with 2.0 mg L-1 IBA showed the best root formation (85 %; 22.2 roots/shoot; 2.65 cm root length; propagate leaf d.w 1.42 g L-1 and root d.w 1.10 g L-1) and plantlets were successfully acclimatized in the green house. The compositions of essential oils were studied in in vitro and in vivo leaf explants and to determine their absolute and relative concentrations using GC-MS and GC-FID, respectively. In vitro and in vivo variations in composition among the essential oils were detected. Sesquiterpene and monoterpene are major components in the oil of in vitro and in vivo leaves of W. biflora. Essential oil quantification showed α-pinene (44.03 %); verbenone (15.37 %); δ-cadinene (5.48 %) of in vivo leaves; whereas α-pinene (5.02 %); verbenone (1.40 %) and δ-cadinene (3.13 %) of in vitro leave respectively. The in vitro and in vivo essential oils were analyzed by bacterial and fungal organism through disc diffusion and minimum inhibitor concentration (MIC) methods. The results were compared with reference drugs ciprofloxacin and nystatin respectively. In vitro developed plants exhibited higher sesquiterpene contents and antimicrobial activity than field grown plant.

Adventitious Shoot Production using Petiole Explants through Direct Regeneration in Sugar Beet

Adventitious Shoot Production using Petiole Explants through Direct Regeneration in Sugar Beet

High responsiveness in de novo shoot organogenesis induction of Passiflora cristalina (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L−1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L−1 BA; while for root segments, the highest mean values were observed with 1.0 mg L−1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.

The Effect of Heavy Metals on In Vitro Adventitious Shoot Production and Bacoside A Content in Bacopa Monnieri (L)

In the present study the effect of heavy metals (MnSO 4, ZnSO 4 and CuSO 4 ) on adventitious shoot regeneration, biomass and bacoside A accumulation in Bacopa monnieri was carried out. The leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg l -1 kinetin (Kin) with varying concentration of heavy metals (Control: Mn-0.10 mM, Zn-0.03 mM and Cu- 0.0001mM; Mn: 0.20, 0.40, 0.80 and 1.60 mM; Zn: 0.06, 0.12, 0.24 and 0.48 mM; Cu: 0.02, 0.05, 0.10 and 0.20 mM). Optimum number of adventitious shoots (123.50 shoot/explants), fresh weight (3.826 g) and dry weight (0.226 g) of Bacopa monnieri were obtained in the medium with 0.12 mM Zn concentration. The highest production

Physiological and biochemical effects of a tetrahydropyranyl-substituted meta-topolin in micropropagated Merwilla plumbea

In comparison to the most active cytokinins (CKs) previously reported for the micropropagation of Merwilla plumbea, we examined the effect of meta-topolin tetrahydropyran-2-yl (mTTHP—a novel aromatic CK derivative) on in vitro adventitious shoot production, rooting and photosynthetic pigment content of regenerated plants. Its carry-over effect on ex vitro growth, photosynthetic performance and antioxidant enzyme system of this bulbous medicinal plant was also investigated. The treatments with mTTHP and meta-topolin riboside (mTR) gave the highest number of adventitious shoots when compared to thidiazuron (TDZ) application and the control. The highest rooting frequency was observed in mTTHP treatments. Unlike in mTTHP treatments, an increase in mTR or TDZ concentration beyond 0.5 µM resulted in a significant decrease in the concentrations of all the photosynthetic pigments quantified. After 6 months of ex vitro growth, regenerated plants from 0.5 µM mTTHP treatment had the highest significant total leaf area, total leaf fresh weight and bulb size compared to all mTR and TDZ-treated plants. Plants regenerated from mTTHP or mTR treatments demonstrated a high capacity for energy dissipation in comparison to TDZ-regenerated plants with low photochemical quenching, PSII quantum efficiency and non-photochemical quenching. Despite a significant increase in the antioxidant enzyme activities, malondialdehyde concentration was significantly high in the leaves of TDZ-regenerated plants compared to other CK treatments. This finding indicated a high production of reactive oxygen species beyond the scavenging efficiency of the antioxidant enzymes leading to oxidative stress and subsequent low biomass accumulation in TDZ-derived plants.

Effect of cytokinins and amino acids on multiplication of Pelargonium cultivars

The effect of two cytokinins (BAP, m-Topolin) tested singly or in combination with a mixture of amino acids (glycine, glutamine, cysteine) on multiplication rate and quality of <em>Pelargonium</em> x <em>hederaefolium</em> and <em>Pelargonium</em> x <em>hortorum</em> shoots were investigated. For both <em>Pelargonium</em> cultivars, the application of cytokinins greatly increased the production of axillary and adventitious shoots compared with the shoots produced on the medium without growth regulators. In the case of <em>P</em>. x <em>hederaefolium</em> no significant differences it were found in the number of shoot formed for BAP and m-Topolin treatments. For <em>P</em>. x <em>hortorum</em> the most effective multiplication of shoots was obtained on the medium supplemented with m-Topolin (1.0 mg l<sup>-1</sup>). Moreover, in the presence of m-Topolin an improvement of shoot quality of both genotypes has been observed (well developed shoots with soft green leaves). It was particularly apparent for <em>P</em>. x <em>hortorum</em> shoots. The addition of amino acids to a cytokinin medium increased slightly the number of formed shoots of <em>P</em>. x <em>hederaefolium</em> and influenced the shoot quality of both <em>Pelargonium cultivars</em> (well developed leaf blades, uniform growth of shoots in clumps, slower process of shoot senescence).

Micropropagation of self-heading Philodendron via direct shoot regeneration

The present study describes a direct shoot regeneration-based micropropagation procedure for the self-heading cultivars of Philodendron. Three types of explant (i.e. leaf lamina, petiole, stem nodal segment) were screened for their shoot induction potential following a three months treatment with 0.5mgl−1 of either 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or both. Results indicated that the leaf laminas were poor candidates for shoot induction whereas the petioles showed potential for adventitious shoot production at frequencies of 2.8–11.1% in two of the cultivars tested. Stem nodal segments were the most responsive among the three as shoots formed directly following the TDZ treatment at frequencies of 16.7–41.7% depending on the cultivar. When comparing the effectiveness of different cytokinins to induce shoot proliferation on stem nodal segments, it was found that the 0.5 and 1mgl−1 of kinetin (Kn) and 6-benzyladenine (BA) treatments resulted in higher shoot formation percentages compared to the 0.5 and 1mgl−1 of TDZ treatments in two of the three cultivars. Furthermore, more shoots were produced on BA than on Kn-supplemented media in all the three cultivars. Shoots derived from the 0.5mgl−1 of BA treatment can be induced to root following one month incubation with 0.1–1mgl−1 of indole-3-butyric-acid (IBA). The rooted shoots showed 100% survival after acclimatization in the greenhouse. The procedure reported in the present study can assist in the large-scale multiplication of elite self-heading cultivars of Philodendron in the future.

GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta

A Geographical Information Systems (GIS)-facilitated approach for the in vitro propagation and ex situ conservation of the conservation priority species Achillea occulta is presented. To realize the species’ ecological requirements, the coordinates of the original habitat were linked with thematic layers derived from digital databases in a GIS environment. From wild plants, shoot tips were established in vitro in a basal medium with 4 μM 6-benzyladenine (BA) and 0.5 μM indole-3-butyric acid (IBA). A modified basal MS medium (modMS, double amount of Fe) proved to be the most effective for in vitro adventitious shoot production. The effect of BA in combination with α-naphthaleneacetic acid (NAA) or IBA on shoot proliferation was also tested. The highest number of new microshoots/explant (3.5), with 0.93 cm shoot height was obtained when the modMS was supplemented with 5 μM BA and 2.5 μM IBA. To evaluate the root induction ability, microshoots produced were transferred to modMS media supplemented with 0–20 μM IBA and 0–20 μM NAA. Rooting proved to be very difficult and only by adding 20 μM IBA, a 12.5% rooting percentage was achieved. Acclimatization succeeded only during early spring. Young plants transplanted at the Balkan Botanic Garden of Kroussia produced flowers and seeds in the first year. This GIS-approach provided useful guidelines for A. occulta’s (a) effective propagation (selection of greenhouse temperatures, temperatures during in vitro culture, suitable period for cuttings and acclimatization of plantlets), and (b) ex situ cultivation (selection of watering regime, temperatures, locations and exposures for growing sites).

Mixture screening and mixture-amount designs to determine plant growth regulator effects on shoot regeneration from grapefruit (Citrus paradisi macf.) epicotyls

The objective of this study was to improve shoot regeneration from grapefruit. Because many commercially grown citrus types are apomictic, important in vitro applications such as Agrobacterium-mediated transformation commonly use epicotyl explants from in vitro seedlings; thus, adequate adventitious shoot production is an important prerequisite for efficient use of these applications. Eight plant growth regulators were studied—six cytokinins (6-benzylaminopurine, kinetin, zeatin trans-isomer, 6-[γ,γ-dimethylallylamino] purine, zeatin riboside trans-isomer and meta-topolin) and two auxins (α-naphthalene acetic acid and indole-3-acetic acid). An iterative design strategy was followed that included mixture and mixture-amount experimental designs suitable for resolving proportional and concentration effects; in vitro effects of cytokinins and auxins are affected by both proportion and concentration. One-centimeter-long explants were excised from the epicotyl of etiolated, in vitro-grown seedlings. Explants were placed onto experimental formulations and cultured in growth cabinets at 27°C over 6 wk, which included 2 wk in the dark followed by 4 wk in the light. The results indicated that (1) 6-benzylaminopurine or zeatin riboside were the most effective cytokinins for inducing shoot regeneration in citrus; (2) zeatin riboside singly or in combination with indole-3-acetic acid resulted in the highest quality, the greatest number of explants with buds/shoots, and the greatest shoot number; and (3) 6-benzylaminopurine and indole-3-acetic acid improved shoot regeneration vs. 6-benzylaminopurine at a considerably lesser cost than zeatin riboside and indole-3-acetic acid.

The role of meta-topolins in alleviating micropropagation problems

Low shoot multiplication, morphological abnormalities, poor rooting frequency and high cost of production are among the factors challenging the micropropagation of ornamental perennials and garden plants. Most of these problems can be alleviated by using the appropriate type and concentration of plant growth regulator(s) (especially cytokinins) in developing efficient micropropagation protocols. In this study, we investigated the effects of five different aromatic cytokinins (BA, Kin, mT, mTR and MemTR) on adventitious shoot production from shoot-tip explants of B. greenii, a critically endangered plant with horticultural potential. Of all the cytokinin concentrations evaluated, the highest adventitious shoot production (5.88 ± 0.73 shoots/explant) was observed in cultures containing 7 μM MemTR. Low adventitious shoot production, which was not significantly different from that of the control, was observed at all the concentrations of kinetin (Kin), suggesting that it is a weak cytokinin for shoot production in this species. All the treatments with BA alone showed higher adventitious shoot production when compared to the BA treatments supplemented with NAA concentrations. At equimolar concentrations, however, all the BA concentrations had a higher abnormality index than the other cytokinins. It is noteworthy that the abnormality index in all the topolin treatments was much lower than that recorded at the lowest BA concentration. Almost all the abnormality indices recorded with mTR and MemTR concentrations were lower than that of the control. Given that the explants used were from BA-containing cultures, it is likely that the abnormalities recorded using mTR and MemTR were carry-over effects of BA. Culturing under 16 h light/8 h dark conditions resulted in a higher production of adventitious shoots with lengths greater than 10 mm compared to culturing under continuous light. This measure could help reduce the cost of production. Regenerated shoots were successfully rooted and acclimatized with a 65% survival frequency and no observable morphological variation. The developed micropropagation protocol has the potential for producing more than 60,000 transplantable shoots per year from a single shoot-tip explant of this critically endangered species.

In vitro regeneration from cotyledon explants in figleaf gourd (Cucurbita ficifolia Bouché), a rootstock for Cucurbitaceae

An efficient plant regeneration system has been developed for figleaf gourd (Cucurbita ficifolia Bouche), which is exclusively used as a rootstock for cucumber. The protocol is based on results obtained from a series of culture experiments involving different parts of the cotyledons and various media. The culture of cotyledon explants was critical for the enhancement of shoot regeneration frequency. The lower parts of the cotyledon excised at the plumule base were found to display a markedly enhanced production of adventitious shoots compared to other cotyledon regions. Culture in silver nitrate-supplemented Murashige and Skoog (MS) medium was not beneficial for shoot regeneration and suppressed root regeneration. Efficient shoot regeneration was obtained on MS medium containing 1.0 mg l−1 zeatin and 0.1 mg l−1 indole-3-acetic acid. Regenerated shoots successfully elongated and rooted in medium containing 0.1 mg l−1 1-naphthaleneacetic acid after 10–15 days of subculturing. The plantlets were satisfactorily acclimatized in a greenhouse and grew into normal plants without any morphological alterations.

Effects of temperature, photoperiod and culture vessel size on adventitious shoot production of in vitro propagated Huernia hystrix

High production costs due to low growth rate in vitro and high labour costs are among factors limiting commercial application of micropropagation techniques. The low growth rate could be due to unfavourable or sub-optimal environmental and chemical conditions of the cultures. The effects of temperature, photoperiod and culture vessel size were investigated on adventitious shoot production of Huernia hystrix. There were significant increases in shoot proliferation with increased temperature in cultures maintained under a 16 h photoperiod. Slow growth observed at low temperatures (15 and 20°C) offers a potential strategy for cost-effective in vitro storage of H. hystrix germplasm. The maximum adventitious shoots produced per explant and percentage of explants producing shoots (4.2 ± 0.74 and 94% respectively) were observed in cultures maintained at 35°C, the optimum temperature for photosynthesis in plants possessing crassulacean acid metabolism (CAM). The nocturnal accumulation of organic acids in cultures incubated under a 16 h photoperiod further suggest the presence of CAM in this species. On the other hand, cultures kept under continuous light appear to shift to a C-3 photosynthetic pathway. There was a significant decrease in fresh weight of adventitious shoots regenerated per explant as temperature increased. The use of larger culture vessels further increased the shoot proliferation to 5.6 shoots per explant with a potential production of 3,429 shoots per m2 in the growth room compared to 2,750 shoots per m2 using culture tubes.

Multigene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia

AbstractDuring a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3, tuf, putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI‐A and 16SrI‐B ribosomal subgroups and to rpI‐A and rpI‐B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non‐symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non‐epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes.

Effect of explants source on shoot proliferation and adventitious regeneration in 10 Buddleia cultivars

Viable shoot cultures and weaned plants were obtained from cultured apical meristems with 10 Buddleia cultivars giving viabilities of 32–72%. The number of shoots produced, the micropropagation rate and the root number produced in vitro was higher in meristem derived shoots compared to those derived from shoot-tips. The subsequent growth rate of meristem derived plants, in the greenhouse, was also greater. The number of roots produced by conventional cuttings collected from meristem derived plants was significantly higher than in cuttings which were collected from plants derived from shoot-tips or from the original stock plants. Endogenous bacteria were not detected in either shoot cultures derived from meristems or in 10-week-old weaned plants derived from meristems whereas those derived from shoot-tips showed the presence of endogenous bacteria when sterilized explants were cultured on nutrient agar or on tryptic soy broth. Factors affecting adventitious bud and shoot production in leaf and internode explants was determined for ‘Lochinch’, ‘Border Beauty’, ‘Ile de France’ and ‘Pink Delight’ using meristem derived shoot cultures. Adventitious shoots appeared after 4 weeks of culture, in both types of explant when cultured on MS supplemented with 0.5–5.0 μM TDZ. The highest percentage regeneration was achieved from bisected internode explants cultured on 0.5 μM TDZ, with 93–100% regeneration among the cultivars whereas BA was less effective. The best response was obtained using 5.0 μM TDZ which gave over 10–11 shoots per explant in all bisected explants for all cultivars.

Somatic embryogenesis and plant regeneration in purple food yam (Dioscorea alata L.)

A study was conducted to establish a reliable procedure for somatic embryogenesis and plant regeneration from callus cultures of purple food yam (Dioscorea alata L.). The procedure involved three steps; (1) culture of nodal stem segments from greenhousegrown plants to generate in vitro plantlets; (2) induction of callus from the leaf, petiole and nodal stem tissues; and (3) initiation of somatic embryo from callus. Results showed that the agar-solidified Murashige and Skoog (MS) medium containing 30 gl-1 sugar, 0.1 gl-1 α-cysteine , 10 mgl-1 calcium pantothenic acid, 2.0 mgl-1 asparagine, 2.0 mgl-1 arginine, 80.0 mgl-1 adenine sulfate (AdSO4) and 0.1 mgl-1 naphthalene acetic acid (NAA) effectively broke dormancy of lateral buds of nodal stem cultures from both ‘VU-2’ and ‘Kinampay‘ varieties. Production of multiple adventitious shoots occurred after transfer of in vitro nodal pieces to the same medium added with 1.0 mgl-1 benzylamino purine (BAP) or, MSA medium. Callus was effectively induced from the vegetative tissues in MS medium added with 1.0 mgl-1 2,4-Dichlorophenoxy acetic acid (2,4-D) or, with picloram. Among the three types of explants, the nodal stem was the most suitable which produced purplish nodular embryogenic callus. A higher percentage of nodal stem-derived calli produced globular embryos in MS medium containing 1.0 mgl-1 2,4-D and 0.5 mgl-1 BAP, or in 1.0 mgl-1 picloram and 0.5 mgl-1 BAP than, in the plant growth regulator-free medium (control). The maturation of embryos was facilitated by one-month culture in MS medium containing 0.1 mgl-1 ABA and 100 mgl-1 glutamine. This step improved the germination of somatic embryos in one-half strength PGR-free MS medium containing 100 mgl-1 glutamine (regeneration medium). All somatic embryoderived plantlets were morphologically normal and established well in soil.

Micropropagation and salt tolerance of in vitro grown Crithmum maritimum L.

Crithmum maritimum (Apiaceae), a perennial halophyte native in Greece, could be used as an alternative culture at problematic soils. It presents significant economical potentials as its essential oils are in high demand from the medicinal and cosmetic industry. The response of the species on in vitro conditions was studied. MS proved to be the most effective of the basal media tested for in vitro adventitious shoot production, resulting in significantly increased number of new microshoots/explant and higher shoots. 6-Benzyladenine (BA) at 2.5 μM increased shoot proliferation. The combination of α-Naphthaleneacetic acid (NAA) (1–2.5 μM) with BA (2.5 μM) had a positive influence at simultaneous proliferation and rooting resulting in high rooting percentage (82.5–95%) and increased number of roots. Rooting percentage reached 100% and number of roots increased significantly when 0.5 μM and 1 μM IBA was combined with ½MS and full strength MS. The in vitro response to salinity stress (0–300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher concentrations of NaCl but shoot height was enhanced. Acclimatization procedure was successful.

Effect of Sodium Chloride on Establishment of Callus and Organogenesis in Brassica napus L.

In order to produce salt tolerant canola (Brassica napus L.) plants hypocotyl segments of its that were excised from 7-days-old-seedlings cultured in MS medium with various concentrations of PGRs (NAA, IBA, 2, 4-D, KN and BA) and sodium chloride. The best media for induction and growth of callus (6 mg L(-1) 2, 4-D and 2 mg L(-1) BA), production of adventitious shoots (1 mg L(-1) IBA and 1 mg L(-1) BA) and roots (1 mg L(-1) NAA and 0.5 mg L(-1) KN) were determined. Then explants cultured in these media plus 0-273.53 mM L(-1) sodium chloride. Explants in these media established and in some of concentrations of sodium chloride (68.37-102.56 mM L(-1)) produced calli and adventitious shoots and roots better than the same media but free from sodium chloride. This finding suggests that somaclonal variation can yield stable-tolerant canola plants.

In vitro culture of rose species (Rosa spp.) via axillary bud growth

The utilization of nodal stem cuttings containing dormant axillary buds as explants for plant production of two rose species; Rosa chinensis cv.‘Old Blush’ and R. centifolia cv. ‘Petite de Hollande’ was demonstrated in this study. This propagation technique required the breaking of dormant axillary buds by aseptically culturing them in agarsolidified Woody Plant Medium (WPM) added with 0.5, 1.0, and 2.0 mg l-1 of 6benzylaminopurine (BAP) or, a combination of 2.0 mg l-1 BAP and 0.01 mg l-1 naphthalene acetic acid (NAA). Production of multiple adventitious shoots from one nodal stem explant was obtained after three months of culture in medium supplemented with 1.0 or 2.0 mg l-1 BAP. Four types of plant morphology; single shoot (type 1), multiple shoots with normal leaves (type 2), cluster of tiny shoots with curly leaves (type 3), and single shoot with callus at the base (type 4) were observed from the axillary bud-derived plantlets. The rooting of plantlets was induced in WPM containing 0.25 to 1.0 mg l-1 of indole-butyric acid (IBA) or, 2.0 mg l-1 of indole-3-acetic acid (IAA).