The aim of this study was to investigate the protein alterations in the transgenic kiwifruit (Actinidia deliciosa) overexpressing a transcription factor gene, OSH1. Although transgenic plant with introduced OSH1 indicates suppression of gibberellin (GA)-20 oxidase activity, application of GA to transgenic kiwifruit could not completely recover plant morphology and protein profiles. Eleven proteins decreased in the transgenic kiwifruit detected by two-dimensional polyacrylamide gel electrophoresis showed homologies to kiwifruit hypothetical protein, osmotin I and photosynthesis related protein. These results suggest that introduction of an OSH1 into kiwifruit caused wide-range alterations at protein level and alterations of protein accumulation should be considered to evaluate the substantial equivalence of plants transformed by a transcription factor.

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.

Exposure of barley plants (Hordeum vulgare L.) to soil flooding for 2 to 24 h reduced the net photosynthetic rate and transpiration rate. Stomatal conductance also decreased in flooded plants. Stomatal closure started within 2 - 6 h and stomata remained closed up to 24 h of treatment.

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO₃ ^-) or both nitrogen forms, nitrate and ammonium (NH₄ ⁺/NO₃ ^-). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH₄ ⁺/NO₃ ^- in comparison with NO₃ ^- grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH₄ ⁺/NO₃ ^- treatment. The PEG treatment decreased enzyme activities in NH₄ ⁺/NO₃ ^- grown plants, but in NO₃ ^- grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO₃ ^- grown plants, and lower activities of both, acidic and basic isoforms in NH₄ ⁺/NO₃ ^-grown plants.

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

In the conversion of oleic acid to linoleic acid, δ¹²-fatty acid desaturase (δ¹²-FAD) is involved. Based on the conserved oligo amino acid residues of the FAD2 genes from other plants, a new full-length cDNA (DiFAD2) encoding a δ¹²-FAD was cloned from Davidia involucrata Baill. Sequence analysis indicated that the DiFAD2 gene had an open reading frame (ORF) of 1 149 bp, coding for 382 amino acids residues of 44.3 kDa, pI of the deduced protein was 8.8. The deduced amino acid sequence of the cloned DiFAD2 showed high identities to those genes of other plant δ¹²-FAD. RT-PCR showed that DiFAD2 was expressed in all tissues and expression was abundant in young stems. Expression of DiFAD2 is not enhanced by low temperature and the altered polyunsaturated fatty acid content in leaves treated with low temperature may be due to the post-transcriptional regulation of the DiFAD2 gene or the other FAD2 gene family regulation.

Secondary embryogenesis from rapeseed microspore-derived embryos (MDEs) was studied in three Brassica napus L. cultivars Global, PF₇₀₄ and Option. The best results in terms of secondary embryogenesis percentage obtained in cultures of Global and PF₇₀₄ MDEs (75.88 and 65.97 %, respectively) and PF₇₀₄ produced the highest number of secondary embryos per each primary embryo (14.91 ± 2.18). After optimization of physical parameters, rapeseed hypocotyls of MDEs were bombarded with microcarriers coated with a plasmid containing GUS reporter gene. The highest levels of transient GUS expression were obtained using bombardment with gold particles of 1.6 µm, at helium pressure of 9.3 MPa, a bombardment distance of 9 cm, chamber vacuum pressure of 7.1 × 10^-6 kPa and single bombardment in bombardment medium containing 0.4 M mannitol.

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.

The genetic variability of Leucojum valentinum Pau (Amaryllidaceae), a vulnerable endemic species restricted to a small area in the region of Valencia (Eastern Spain), has been studied using random amplified polymorphic DNA (RAPD) markers. A total of 197 individuals from eleven populations were studied using 13 RAPD primers. Our results show high variability for the species, low differentiation among populations and uncorrelated levels of genetic variability and population size. Four groups in which three populations (SAG, PUG and COL) are separated from all the others were found, but without connection to geographical location.

The toxic aromatic compound 3-hydroxy-4-pyridone (HP) is an intermediate in both synthesis and degradation of mimosine, which is produced by the tree legume Leucaena leucocephala. The L. leucocephala root-nodule symbiont Rhizobium TAL1145 contains a dioxygenase (pydA) and a hydrolase (pydB) gene that produce enzymes necessary for the degradation of HP. In order to coordinately express both genes in plant tissues under a single promoter, three different pydA-pydB fusion constructs (G0, G3, and G7) with varying glycine linkers between the two genes were developed. Prior to transferring the fusion constructs into L. leucocephala, which is highly recalcitrant to genetic transformation, we tested the expression and activity of the hybrid proteins in Nicotiana tabacum, a model plant system that can be easily transformed and analyzed. Seven independent transgenic tobacco lines were generated by Agrobacterium-mediated transformation, and stable integration and expression of pydA-pydB in these transgenic lines were confirmed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and Western analysis. Only one of the fusion constructs, G3, containing a 9-nucleotide linker between pydA and pydB, provided significant levels of resistance to 3 mM HP, indicating that the hybrid protein produced by this fusion construct could degrade HP.

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm^-3 zeatin, 0.1 mg dm^-3 indoleacetic acid and 10 g dm^-3 sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0-2.0 mg dm^-3 gibberellic acid and 4.0-8.0 mg dm^-3 AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.

Both morphological characteristics and amplified fragment length polymorphism (AFLP) markers were used to validate the genetic fidelity of 1 080 field-grown Echinacea purpurea plants regenerated from leaf explants of donor T5-9. Morphological diagnosis revealed that 1 067 out of 1 080 regenerants were normal, while 13 regenerants were aberrant. AFLP analysis was further performed to assess DNA variations among donor, 43 sampled normal regenerants and all 13 aberrant regenerants. Seven primer combinations generated 471 fragments among donor and normal regenerants, of which 9 fragments were polymorphic. The same primer pairs generated 484 fragments for aberrant regenerants, of which 417 fragments were polymorphic. UPGMA clustering indicated that 42 normal regenerants and donor fell into same cluster at similarity scale of > 0.99, while all 13 aberrant regenerants and one morphologically normal regenerant comprised the other clusters. AFLP analysis indicated that these 14 regenerants are off-types.

Effects of the infection with tobacco mosaic virus (TMV) and potato virus Y (PVY) on chloroplasts from susceptible tobacco plants were determined. Changes in ribonucleases (RNases), phosphomonoesterase (PME), phosphodiesterase (PDE), glucose-6-phosphate dehydrogenase (G6P DH), 6-phosphogluconate dehydrogenase (6PG DH), glucokinase (GK), and fructokinase (FK) activities in thylakoid/envelope and stroma fractions were studied. Slight increase in the activities of PME, PDE, G6P DH and 6PG DH of thylakoid/envelope fraction as well as of RNases, PME, PDE, G6P DH, 6PG DH, GK and FK of stroma fraction was found in chloroplasts isolated from leaf tissues infected with PVY. Infection with TMV produced higher increase in enzymes activities in chloroplasts; especially, PME, G6P DH and 6PG DH in fraction of thylakoid/envelope, and RNases, PME, PDE, G6P DH, 6PG DH, and GK in stroma fraction.

The photosynthetic performance and related leaf traits of Incarvillea delavayi Bur. et Franch were studied at different water regimes to assess its capacity for photosynthetic acclimation to water stress. The initial response of I. delavayi to water stress was the closure of stomata, which resulted in down-regulation of photosynthesis. The stomatal limitation (S_L) represented the main component to photosynthetic limitations but non-stomatal limitation (NS_L) increased quickly with the increasing water stress, and had similar magnitude to SL under severe water stress (soil moisture 25-30 % of field capacity). Chlorophyll (Chl) a fluorescence parameters characterizing photosystem (PS) 2 photochemical efficiency (Φ_PS2), electron transport rate (J) and photochemical quenching (qP) decreased with the increasing water stress, indicating impaired photosynthetic apparatus. However, the water-stressed plants had a increased mesophyll CO₂ diffusional conductance, Chl a/b ratio, leaf nitrogen partitioning in RuBPCO and bioenergetics in later grown parts, indicating that I. delavay had a substantial physiological plasticity and showed a good tolerance to water stress.

The effect of 5-azacytidine (5-azaC) on the alleviation of damaging effects of NaCl treatment was studied in two wheat (Triticum aestivum L.) cultivars differing in salt tolerance (salt-tolerant Dekang-961 and sensitive Lumai-15). The plants were pre-treated or not with 50 μM 5-azaC and then subjected to salt stress induced by 100 or 150 mM NaCl. Salinity caused reduction in biomass accumulation and increase in malondialdehyde content in root tissues in both cultivars, but less in pre-treated seedlings. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in the roots of both cultivars increased during salt stress, but the rate of increase was higher in Dekang-961. Plants treated with 5-azaC had higher root SOD, CAT and POD activities under salt stress than untreated plants. Content of 5-methylcytosine (5mC) decreased in both cultivars under salt stress, and the level of demethylation was higher in Dekang-961 than that in Lumai-15. Moreover, the degree of methylation was lower in both cultivars under salt stress after 5-azaC application compared to only salt-treated groups. These findings suggested that 5-azaC could protect plants from salt stress.

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.

Effects of zinc (12-180 μM) alone and in mixtures with 12 μM Cd on metal accumulation, dry masses of roots and shoots, root respiration rate, variable to maximum fluorescence ratio (F_V/F_M), and content of photosynthetic pigments were studied in hydroponically cultivated chamomile (Matricaria recutita) plants. The content of Zn in roots and shoots increased with the increasing external Zn concentration and its accumulation in the roots was higher than that in the shoots. While at lower Zn concentrations (12 and 60 μM) the presence of 12 μM Cd decreased Zn accumulation in the roots, treatment with 120 and 180 μM Zn together with 12 μM Cd caused enhancement of Zn content in the root. Presence of Zn (12-120 μM) decreased Cd accumulation in roots. On the other hand, Cd content in the shoots of plants treated with Zn + Cd exceeded that in the plants treated only with 12 μM Cd. Only higher Zn concentrations (120 and 180 μM) and Zn + Cd mixtures negatively influenced dry mass, chlorophyll (Chl) and carotenoid content, F_V/F_M and root respiration rate. Chl b was reduced to a higher extent than Chl a.

Tubulins are basic components of microtubules and are encoded by a multigene family in eukaryotes. The expression of OsTUB4, one of eight β-tubulin isotypes identified in the rice genome, was characterized. OsTUB4 was expressed in root primodia and the shoot apical meristem in basal parts of the leaf sheath in rice seedlings. OsTUB4 transcript abundance in leaf sheath increased by treatment with gibberellic acid (GA₃) in a dose- and time-dependent manner. OsTUB4 transcript levels in gibberellin (GA)-deficient mutants were less than those of wild type rice. An OsTUB4 promoter::GUS assay also confirmed the responsiveness of OsTUB4 to exogenous GA₃, suggesting that OsTUB4 expression was regulated by GA and may be involved in GA-regulated leaf sheath growth. In addition, OsTUB4 could interact with different specific proteins in vitro as assayed by a yeast two-hybrid system, indicating that OsTUB4 may have diverse functions through interaction with different proteins.

The objective of the study was to investigate the effect of different arsenic concentrations on some physiological parameters of bean (Phaseolus vulgaris L.) cultivars Plovdiv 10 and Prelom in the early growth phases. Seedlings, grown in sand with Hoagland-Arnon nutrient solution in a climatic box, were treated with 0, 2, 5 mg(As) dm^-3 as Na₃AsO₄ (pH 5.5). After 5 d of As treatment, the changes in leaf gas-exchange, water potential, chlorophyll and protein contents, peroxidase activity and lipid peroxidation in roots were recorded. Physiological analysis showed a minor negative effect of arsenic at concentration 2 mg(As) dm^-3, but at the higher dosage of 5 mg(As) dm^-3 growth, leaf gas-exchange, water potential, protein content and biomass accumulation were reduced in both cultivars. The peroxidase activity and lipid peroxidation increased considerably at 5 mg(As) dm^-3, which is a typical reaction of the plants to a presence of oxidative stress.

Plantain (Musa ABB CEMSA 3/4) plantlets were micropropagated in temporary immersion bioreactors (TIB) or in gelled medium (GM). After ex vitro transfer ROS accumulation was determined by infiltrating leaves with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB). Stomatal cells were more stained with NBT and DAB in GM plants than in TIB plants, but the difference disappeared at the end of acclimatization. At the end of the in vitro phase, GM plantlets showed higher activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR), while activities of catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GT) were higher in TIB grown plantlets. At the end of acclimatization GT, SOD, CAT and MDHAR stabilized at low values of activity in plantlets derived from both treatments. Concerning the correspondent genes, GM plantlets showed higher expression of all transcripts with the exception of CuZnSOD. The immunobloting of peroxiredoxins (PRXs) showed that chloroplast-located PRXs were expressed at higher levels in TIB plantlets, some showing polymerization. In conclusion, TIB grown plantlets had an improved anti-oxidative response when compared with GM.

A DNA-based, inter simple sequence repeat (ISSR) markers were used to monitor genetic stability in micropropagated plantlets of Nothapodytes foetida. A total of 146 clear and distinct bands were produced using 26 primers resulting in 3 212 fragments. Out of 146, 135 bands (92.4 %) were monomorphic and 11 bands (7.53 %) were polymorphic which ranged from 200 to 21 226 bp in size. The number of bands per each primer varied from 1 to 11 with an average of 5.6 bands per primer. The banding pattern for each primer was uniform and comparable to mother plant from which the cultures had been established. The dendrogram based on the unweighted pair-group method with arithmetic averaging (UPGMA) depicted about 97 % homology between the mother plant and micropropagated plants. An attempt was made to reintroduce the micropropagated plants in the natural habitat and over 500 plants were successfully established.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = -26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = -28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = -14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = -21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = -28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.

The aim of the study was to examine the effect of exogenous 24-epibrassinolide on its uptake and content of endogenous brassinosteroids in wheat seedlings. 24-Epibrassinolide was applied at two concentrations (0.1 and 2.0 μM) and in three different methods: by soaking seeds, by drenching and by spraying plants. Brassinosteroids were determined by high-performance liquid chromatography combined with electrospray mass spectrometry. Three important brassinosteroids, 24-epibrassinolide, brassinolide and castasterone, were detected in the wheat leaves, but their contents varied with leaf insertion and plant age. Increased 24-epibrassinolide content in the leaf tissue was found when this hormone was applied by soaking or drenching. Additionally the seed treatment influenced brassinosteroid balance in seedlings. The growth response of wheat seedlings treated with 24-epibrassinolide has been also investigated.

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm^-3 benzylaminopurine and 1.5 mg dm^-3 kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm^-3 indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.

AP2 (APETALA2)/EREBPs (ethylene responsive element binding proteins) are the primary members of a family of transcription factors and OsAP211 was isolated from Oryza sativa L using the yeast one-hybrid system. It can specifically bind to the promoter containing three tandem repeats of DRE core sequence: TACCGACAT and activate the transcription of the downstream lacZ gene in the yeast one-hybrid system. OsAP211 contained a single open reading frame of 225 amino acids and encoded a protein containing a conserved AP2/EREBP domain featuring the DREB family. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated OsAP211 was strongly induced by low temperatures but not by NaCl and drought. It accumulates primarily in shoot tips during the tillering stage and young spikes during the booting stage. OsAP211 might function as a DRE-binding transcription factor in stress signal transduction pathways in rice.

The impacts of solar UV (280-400 nm) radiation on photosynthetic activities and polypeptide composition of thylakoids of cluster bean (Cyamopsis tetragonoloba L, UV-B sensitive) and black gram (Vigna mungo L., UV-B resistant) plants were compared. The activity of photosystem 1 and especially photosystem 2 increased in cluster bean while decreased in black gram, when either UV-B or UV-B + UV-A radiation was removed as compared to control plants. Exclusion of UV-B radiation caused changes in the protein composition of the thylakoids particularly in the 33, 23 and 17 kDa proteins of photosystem 2.

Tetraploidy was induced in vitro in mat rush (Juncus effusus L.) cultivar Nonglin-4 by exposure to colchicine (0, 50, 100 and 500 mg dm^-3) for 6, 12 and 24 h. Flow cytometric analysis was used to confirm the ploidy level. Anatomical and ultrastructural analyses at cellular and subcellular levels in tetraploid and diploid control plants revealed differences between diploid and tetraploid plants. The leaf epidermis had larger stomata but lower stomatal density in tetraploid plants. In addition, mesophyll cells in tetraploid plants appeared more compact and showed less intercellular spaces along with increased size of vascular bundles. However, a significant reduction of chlorophyll content was observed in tetraploid plants that might be the result of structural modification in the lamellar membranes of chloroplasts.

Expansins, found in the cell wall, have the unique ability to induce immediate cell wall extension. In this study, a β-expansin gene (TaEXPB23) isolated from wheat (Triticum aestivum L.) coleoptiles was transformed to tobacco (Nicotiana tabacum) to investigate its role in plant growth and development. TaEXPB23 was preferentially expressed in wheat coleoptile and a close correlation between TaEXPB23 expression and coleoptile growth was observed. The over-expression of TaEXPB23 in tobacco also resulted in accelerating growth of leaves and internodes at earlier developmental stages, and it was involved in regulating plant development.

An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and Skoog salts with B5 vitamins) containing 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm^-3 glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS medium supplemented with 0.5 mg dm^-3 2,4-D and 50 mg dm^-3 proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium containing 0.05 mg dm^-3 2,4-D, 20 mg dm^-3 Pro, 5 μM abscisic acid, 1000 mg dm^-3 KNO₃, 50 mg dm^-3 polyethylene glycol (PEG 6000) and 30 g dm^-3 D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished in MMS medium containing 30 g dm^-3 maltose, 0.5 mg dm^-3 benzyladenine and 500 mg dm^-3 KNO₃. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5 mg dm^-3 indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile.