Phenylmethylsulphonyl fluoride (PMSF), a well known inhibitor of both thiol- and serine-type proteases, in aqueous solutions either alone or with the plant growth regulators, methyl ester of jasmonic acid (MeJA) and N⁶-benzyl-aminopurine (BAP), significantly inhibited the growth of excised Cucurbita pepo L. (zucchini) cotyledons. SDS-PAGE analysis of the protein profiles showed that PMSF suppressed the gradual decline of the main 20 - 25 kDa polypeptide group and the low molecular mass polypeptides (below 15 kDa) while leupeptine was not able to affect the electrophoretic pattern of cotyledon proteins. On the other hand, in the presence of PMSF, the content of the polypeptides with higher molecular mass including the 97.4 kDa polypeptide and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (55 kDa) decreased. Besides, when applied together with MeJA, PMSF prevented the appearance of the jasmonate-induced polypeptides (JIPs; 69, 60 and 43 kDa) thus suggesting that JIPs are synthesized from aminoacids released during the breakdown of cotyledon storage proteins.

The effect of different radiation sources - blue (B), red (R), R plus B (RB), B plus far red (BFr), R plus far red (RFr) - was tested on the growth of hairy roots and betalain accumulation in Beta vulgaris L. (red beet). Light emitting diodes were used as radiation sources. The growth of hairy roots under different radiation treatments depended on radiation quality. Highest biomass accumulation was under the BFr treatment. BFr treatment efficiently induced betalain pigmentation in hairy roots. Total sugar and sucrose contents of hairy roots were also greater in this treatment. Thus, the betalain pigmentation in the cultured hairy roots can be influenced by radiation quality and BFr is most suitable for accumulation of betalains.

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 µM benzylamino-purine (BAP) and 0.3 µM indole-3-acetic acid (IAA) or with 0.5 µM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 µM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 µM BAP.

Callus induction was obtained on Murashige and Skogg agar medium with 45 μM 2,4-dichlorophenoxyacetic acid under dark at 25°C. Among the four explant types investigated, the best callus induction was obtained from two-week old fronds to which a surgical incision was applied in the basal (meristematic) region. This treatment resulted in 89.11% of fronds producing callus which continued to proliferate for another 24 months. To obtain plant regeneration pieces of calluses were transferred onto Murashige and Skoog agar medium containing 22 μM indole-3-acetic acid and 4.6 μM kinetin and maintained under 16-h photoperiod (irradiance of 30 μmol m^-2 s^-1) at 23°C. Green fronds formed on all callus pieces. The regenerated fronds were later transferred onto Wang medium where they formed roots. The regenerated Lemna minor L. plants obtained through indirect organogenesis did not differ morphologically from individuals forming the stock collection.

Salt tolerant callus and cell suspension cultures of Brassica oleracea L. var. botrytis were obtained by the selection of cells from cultures growing in medium supplemented with 85, 170, and 255 mM NaCl. Salt adapted calli and cell suspensions differed in their RNA and protein concentrations. These concentrations tend to diminish in calli and increase in cell suspensions, both at one or three weeks periods of growth in NaCl. Contents of sucrose and reducing sugars, however, accumulate similarly both in calli and cell suspensions after NaCl treatments. The activity of sucrose synthase was higher in salt adapted cells than in controls. Calli exposed to 255 mM NaCl for six months synthesized a 27 kDa polypeptide, while a 13 kDa polypeptide present in control conditions was absent under salinity. Several high molecular mass polypeptides (> 200 kDa) were visualized in control calli and at moderate salt concentrations, when conditions of the gel were modified.

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased.

Dunaliella salina V-63 was cultivated in different concentrations of NaCl (0.5, 1.0, 2.5, 3.0, or 4.0 M) and at two irradiances (170 or 220 μmol m^-2s^-1). Concentration-dependent suppression of growth was observed above 1 M NaCl, and elevated salinity induced formation of salt-containing vacuoles. However, the changes in the chloroplast ultrastructure following changes in salinity and irradiance (increase of invaginations and protuberances, numerous grana with low number of thylakoids, less number of starch grains, etc.) appeared to be of primary importance.

The effects of different paraquat, alachlor and metolachlor concentrations on superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX) activities and pigment contents in lettuce, bean and pea seeds and leaves were studied. Under paraquat (1.0 and 2.0 μM) treatment declined SOD and CAT activities were observed in seeds and undetectable ativity of GPX. Germination of all investigated seeds was completely inhibited. All used concentrations of alachlor and metolachlor inhibited antioxidant enzyme activities in seeds but did not prevent germination and growth. In leaves, lower concentrations of these herbicides increased activities of antioxidant enzymes but at the highest herbicide concentrations (200 μM) activities of investigated enzymes declined. The pigment contents the leaves decreased due to alachlor and metolachlor treatment in a concentration dependent manner.

Seasonal dynamic of total nonstructural saccharides (TNS) and individual saccharides (starch, sucrose, glucose, fructose, fructans) was followed in rhizomes and stem bases of Calamagrostis epigeios (L.) Roth at two types of meadows communities in the South Moravia (Czech Republic): cnidion and molinion alliances, which differ in their water regime. The TNS were formed mainly by fructans and starch, while glucose, sucrose and fructose were low. The amount of TNS in rhizomes and stem bases of plants from wet cnidion site was higher than in plants from drier molinion site. The seasonal trends of all saccharides were similar in the both sites. During growing season (June to October) the main storage sugar was fructan (18 - 21 % of dry biomass). At the beginning of September the content of fructan decreased to 10 - 12 % and simultaneously the content of sucrose increased from 1 to 3 %. This may increase frost resistance. The content of TNS in the stem bases was lower than in the rhizomes. During winter time the stem bases contained 2 to 2.5 % of sucrose. Plant height and aboveground biomass were also higher in molinion site.

Higher amylase activity in cotyledons of kinetin treated salt stressed (75 mM NaCl) chickpea (Cicer arietinum L. cv. PBG-1) seedlings, as compared to salt stressed seedlings was observed during a growth period of 7 d. The activities of acid and alkaline invertases were maximum in shoots and minimum in cotyledons under all conditions. The reduced shoot invertase activities under salt stress were enhanced by kinetin with a simultaneous increase in reducing sugar content. Kinetin increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in both the cotyledons and shoots of stressed seedlings. Kinetin appears to increase the turnover of sucrose in the shoots of stressed seedlings.

Isolated pistils of dimorphic buckwheat (Fagopyrum esculentum Moench.) flowers were treated with phosphatase inhibitors (ocadaic acid and cantharidin) and with calcium antagonists (verapamil, La³⁺, and A23187). They were subsequently cross- or self-pollinated, and the growth of pollen tubes was observed under the fluorescence microscope. All treatments suppressed inhibition of pollen tubes growth suggesting that protein phosphatases and calcium signaling may be involved in self-incompatibility signal transduction in buckwheat.

Influence of boron on somatic embryogenesis in papaya (Carica papaya L.) cv. Honey Dew was investigated. Immature zygotic embryos were grown in the induction medium containing Murashige and Skoog basal salts, with B₅ vitamins, picloram (1 mg dm^-3) or 2,4-dichlorophenoxy acetic acid (2 mg dm^-3) and different concentrations of boric acid (30 to 500 mg dm^-3). Maximum somatic embryo initiation was observed at 62 mg dm^-3 boric acid irrespective of the growth regulator used. The cotyledonary stage somatic embryos were germinated on MS basal medium devoid of growth regulators. The regenerated plantlets were hardened under greenhouse conditions and transferred to field.

Induction of both somatic embryogenesis and organogenesis in presence of thidiazuron is reported in peanut tissues. However the histological evidence of thidiazuron induced somatic embryogenesis was unclear. Thidiazuron triggered multiple shoot differentiation in the plumule of the embryos. Keeping in view the ability of thidiazuron to induce both organogenesis and embryogenesis in peanut tissues, experiments were conducted to define the pathway of morphogenesis in the plumule of rooted somatic embryos. On exposure to thidiazuron, projections appeared from the plumule. These projections closely resemble the somatic embryos. However histological examination revealed that these are caulogenic buds and not somatic embryos. In concurrence with the earlier reports on thidiazuron induced organogenesis it is concluded that this growth regulator induces organogenic response in peanut tissues.

Effects of four culture media on callus induction, regeneration and number of plants per unit culture were studied with mature seeds from five indica rice genotypes as explants. Based on the morphology, the calli were classified into four types as I to IV. Type I and type II are most suited to initiate suspension cultures or as target material for transformation. Number of plants regenerated per unit culture, formation of easily dissociating cell clusters and frequency of type I and type II calli were highest on NBKNB medium. Thus NBKNB medium is suitable for in vitro culture of even the hitherto recalcitrant indica genotypes.

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.

Transgenic rice plants harbouring Bacillus subtilis protoporphyrinogen oxidase (Protox) gene, which is targeted into plastid, were generated by Agrobacterium-mediated transformation using a rice (Oryza sativa L. cv. Dongjin) and their gene integration at T₁ generation by Southern and mRNA expression in T₂ generation by Northern blotting were analyzed. Their herbicide-resistant trait was further confirmed by in vitro leaf segment assay and in planta bioassays such as seed germination assay and measurement of growth inhibition. The herbicide oxyfluorfen resistance in transgenic rice plants was not very high. The results showed that the Protox from B. subtilis can not be applicable as a gene source to generate a high level oxyfluorfen tolerance in plants.

A liquid culture technique has been developed to study lipid metabolism in seeds of Brassica campestris L. grown in vitro from terminal inflorescences detached 4 to 46 days after anthesis. Seeds developed under these conditions exhibited pattern of growth, deposition of storage products and lipid composition similar to those from intact plant.

X-rays at doses between 2.5 and 20 Gy were applied to Allium cepa L. bulbs containing either dormant root primordia (before water imbibition) or actively proliferating meristems. Irradiation of the primordia that were enriched in G₀ cells neither delayed proliferation onset nor root sprouting. Under both protocols, irradiation decreased the final length of the roots to about 60 % (at 20 Gy) of that reached by the unirradiated controls. Irradiation of the proliferating meristems increased the mitotic index at some fixation times. This could not be due to a rise in the cell entry into mitosis, as the rate of root growth decreased simultaneously. The increased mitotic index should be the consequence of a delay in the relative time taken by mitosis in the whole cycle time. Lengthened mitosis probably allows the post-replicative repair of most DNA lesions, as the frequency of interphases with micronuclei was higher in the cells which were irradiated when still dormant than in those irradiated when cycling. Thus, the mitotic delays should be the consequence of a checkpoint pathway activated by the presence of DNA damage. This feedback mechanism seems only to develop after cell proliferation is restored.

In vitro regeneration of Melia azedarach L. was studied. Shoots were regenerated from calli initiated from leaflets of in vitro growing plants. The best medium for establishment of cultures was Murashige and Skoog (MS) medium with 4.44 μM benzylaminopurine (BAP) + 0.46 μM kinetin (KIN) + 16.29 μM adenine sulphate (ADE). Regenerated shoots were multiplied in MS + 0.44 μM BAP + 0.37 μM KIN + 3.26 μM ADE. Maximal rooting of 89 % was achieved by culture of regenerated shoots in MS + 12.26 μM indole-3-butyric acid for 3 d and subsequently in MS lacking growth regulators for 27 d. Rooted shoots were acclimatized and successfully transferred to soil.

The effects of cadmium and copper on in vitro growth of Bacopa monniera (L.) Wettst. was monitored. Cadmium (25 and 50 µM CdCl₂) inhibited plantlet growth and addition of 50 or 100 µm CuSO₄ partially alleviated this negative effect. Cadmium increased both protein and proline contents, but to a lesser extent with the additional supply of CuSO₄.

In vitro bulblet formation was studied using solid, liquid and bioreactor culture (immersion and periodic immersion in liquid media using ebb and flood) in order to develop a cost effective method for the mass propagation of Lilium oriental hybrid 'Casablanca'. Although the percent of bulblet formation was higher in solid culture, the increased growth rate and production of large number of bulblets in bioreactor makes it suitable for mass propagation. Four times per day and 15 min of medium supply was optimal for bulblet formation in ebb and flood bioreactor. Bulblet formation was also found to be effective in 16-h photoperiod. It was also observed that bulblet formation in the medium with 1.0 mg dm^-3 BA and 0.3 mg dm^-3 NAA was higher than in the medium without growth regulators, but formation of abnormal bulblets was higher in medium with BA and NAA.

Studies on stem (and leaf) structure and histology of a semi-natural grassland community, permanently growing in mini-FACE rings under elevated concentrations of atmospheric CO₂ (560 μmol mol^-1) are presented. Histochemical analysis of stem sections from legume plants grown under high CO₂ concentration revealed both a reduction of lignin deposition in spring vascular bundles of Trifolium repens L., and a decrease in size of the xylem vessels in Vicia hybrida L. and Vicia sativa L. Thus, the effects of elevated CO₂ on the stem histology of the species investigated are rather species-specific and/or organ-specific, and of major account especially in the early phases of vegetative growth, in particular as regards lignin deposition mechanisms. In leaves, neither differences as to lignification nor any other anatomical structure modification were found under CO₂ enrichment.

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 μM benzylaminopurine (BAP) and 2.46 μM indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 μM IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^-3 BA in combination with 0.1 mg dm^-3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^-3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.

The effects of gibberellic acid (GA3) and N1-(2-chloro-4-pyridyl)-N2 phenylurea (4-PU-30) on maize seedling growth, photosynthetic parameters, and leaf protein composition were investigated. The agents used alone or in combination increased leaf growth and photosynthetic rate of the seedlings. Chlorophyll and total nitrogen contents in leaves as well as the quantity of individual protein fractions increased simultaneously. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins (albumins and globulins) revealed quantitative differences between 4-PU-30-treated plants and the other experimental variants. They differed in polypeptide composition associated with changes in soluble proteins and amino acids. However, GA3 did not induce similar changes in polypeptide composition of soluble proteins.

Chlamydomonas reinhardtii was grown in medium with different carbon (acetate, CO₂, or both), and nitrogen (ammonium chloride, peptone, urea) sources and under light of different spectral composition. The light-dark cycles were found more suitable for mixotrophic growth than continuous irradiation. Both blue (BR) and red (RR) radiations decreased photosynthetic capacity of mixotrophic cells compared to "white light" (WL). Effect of RR was associated with photon distribution favouring photosystem 1 (PS1) suggesting increased cyclic phosphorylation. Mixotrophic growth in 10 mM NH₄Cl increased photosynthetic oxygen evolution compared to standard concentration of 5 mM NH₄Cl used for growing C. reinhardtii. Autotrophic growth stimulated the photosynthetic capacity compared to mixotrophic one. However, higher photosynthetic capacity was achieved for mixotrophic cells by growing them at high NH₄ ⁺/K⁺ ratio and high phosphate concentration.

Plasmid DNA (pChlCOD), containing the selectable hygromycin phosphotransferase hpt gene for hygromycin B resistance and the Arthrobacter globiformis codA gene for choline oxidase which catalyzes the direct conversion of choline to glycinebetaine, was delivered into rice plants using Agrobacterium-mediated gene transfer via scutellum-derived calli. Southern, Northern and Western blot analyses demonstrated that the foreign gene had been transferred, integrated into rice chromosomal DNA and expressed. Drought test indicated that glycinebetaine acts as an osmoprotectant and its production in transgenic rice plant helped the cells to maintain osmotic potential and increased root growth, and thus enhanced the ability of the plants to tolerate water deficit

An Agrobacterium rhizogenes-mediated transformation system for Aesculus hippocastanum L. has been developed. Wounded androgenic embryos of A. hippocastanum were inoculated with bacteria containing the pRiA4 plasmid, with the uid A sequence as a reporter gene. The hairy roots emerging from the wounded sites of androgenic embryos were isolated and maintained in Murashige and Skoog's (MS) liquid hormone-free medium. Five hairy root lines have been maintained in vitro for 4 years with unchanged growth rate and might be a suitable source for secondary metabolite production. The transformation events have been confirmed by a polymerase chain reaction specific to the rol A, B, C and D genes. The absence of residual contaminating bacteria has been shown by a polymerase chain reaction specific to the vir D1 sequence.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 μM benzylaminopurine (BAP) and 0.57 μM indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 μM BAP and 0.57 μM IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 μM indole-3-butyric acid and 2.88 μM gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.

Embryogenic cultures of chir pine (Pinus roxburghii Sarg.) were cryopreserved successfully in liquid nitrogen. It was found that using sorbitol and dimethyl sulfoxide (DMSO) as cryoprotectants was essential for the survival of the tissue. Among the different concentrations of the cryoprotectants used, the most effective treatment was observed to be 0.3 M sorbitol and 5 % DMSO. On staining the cryopreserved tissue with fluorescein diacetate, it was observed that only a few meristematic embryo heads survived and resumed growth after a very short initial lag phase. The recovered cultures showed normal regrowth on proliferation medium and, it was also observed that washing off the cryoprotectants was necessary for the cultures to survive. The results indicate that cryopreservation can be used for conserving the germplasm, and in maintaining the embryogenic capacity of the tissue.