Regulation of the <Emphasis Type="Italic">htpX</Emphasis> Gene of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> and Its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Xylella fastidiosa was the first phytopathogen to be completely sequenced, and its genome revealed several interesting features to be used in functional studies. In the present work, the htpX gene, which encodes a protein involved in the heat shock response in other bacteria, was analyzed by RT-PCR by using cells derived from different cultural conditions. This gene was induced after a temperature upshift to 37°C after growth in minimal medium, XDM, but showed constitutive expression in rich medium or in XDM plus plant extracts. Sequences upstream to the htpX gene, containing a putative regulatory region, were also transferred to E. coli, and the thermoregulation was maintained in the new host, since it was constitutively transcribed at 37°C or 45°C in all culture media tested, but not at 28°C in minimal culture medium. The gene was also cloned into the expression vector pET32Xa/LIC, and the expression of the corresponding protein was confirmed by Western blotting.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Purification and characterization of an<Emphasis Type="Italic"> N</Emphasis>-acetylglucosaminidase produced by a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain which controls<Emphasis Type="Italic"> Crinipellis perniciosa</Emphasis>

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate -nitrophenyl-N-acetylglucosaminide (NGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. K _m and V _max values for NGlcNAc hydrolysis were 8.06 moles ml^–1 and 3.36 moles ml^–1 min^–1, respectively, at pH 6.0 and 37°C. The enzyme was very sensitive to Fe³⁺, Mn²⁺ and Co²⁺ ions, but less sensitive to Zn²⁺, Al³⁺, Cu²⁺ and Ca²⁺. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

Androgenesis in the vine cacti <Emphasis Type="Italic">Selenicereus</Emphasis> and <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

Anther culture techniques were applied to develop a methodology for producing haploid vine cactus plants. Anthers with most microspores in the middle uninucleate developmental stage from the tetraploid species Selenicereus megalanthus and the diploid species Hylocereus polyrhizus and H. undatus were cultured on basal MS medium supplemented with picloram and 6-benzyladenine (BA). H. polyrhizus and H. undatus anthers were also cultured on MS medium containing either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid (2,4-D). Pro-embryo development started after three days of culture. A direct androgenic embryo response was achieved in S. megalanthus with and without picloram/BA, while H. polyrhizus exhibited non-regenerative callus formation. Only a single direct androgenic embryo was obtained in H. polyrhizus (with 0.1 mg/l TDZ). H. undatus required a cold pre-treatment of 4°C for 24 h in 0.3 M D-mannitol to produce a response, but only calluses were obtained and they did not regenerate. S. megalanthus and H. polyrhizus embryos converted into plantlets after transfer to MS medium in the light. Rooted plants were acclimatized successfully, and most plants showed normal phenotypes. Flow cytometry and cytological studies revealed monoploid, haploid, dihaploid, and mixoploid plants. This study showed that androgenesis is strongly species- and culture-medium-dependent, thus revealing new perspectives in the genetics and breeding of vine cactus species. To the best of our knowledge, this is the first report on the production of monoploid, haploid and dihaploid plants in Cactaceae.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

Metalloproteases Secreted by <Emphasis Type="Italic">Actinobacillus suis</Emphasis>

Actinobacillus suis secretes metalloproteases into its medium. These secreted proteins, when concentrated by precipitation with 70% (NH₄)₂SO₄ or methanol, displayed proteolytic activity at >200 kDa molecular mass bands in 10% polyacrylamide gels copolymerized with bovine casein (1%). They showed activity in a broad pH range (from pH 5 to pH 10) and were inhibited by 20 mM EDTA or EGTA, but could be reactivated by calcium. They were found heat stable at 40°C, 50°C, 60°C, and 70°C, but their activity diminished at 80°C or higher. They degraded pig and bovine IgG and cross-reacted with a polyclonal serum against a high molecular mass secreted protease from A. pleuropneumoniae. Extracellular proteases could play a role in diseases caused by A. suis.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

Induced androgenesis of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> L.

The aim of the research was to make a preliminary determination of the effectiveness of the induction of haploids in Capsicum frutescens L. In order to induce androgenesis red and yellow fruit forms of species were used, each bred by the researchers on their own. The experiment was performed in October. Anther cultures were conducted according to a modified method developed by Dumas et al. (1981) for C. annuum L. The anthers were laid on CP medium containing 0.01 mg dm⁻³ 2.4-D and 0.01 mg dm⁻³ kinetin, with the addition of 0.5 g dm⁻³ of activated carbon and 5 mg×dm⁻³ of silver nitrate, solidified with 8 g dm⁻³ of agar. The cultures were incubated in the dark at 35 deg C for 8 days. Next they were transferred to 25 deg C under a 12-hour photoperiod. After 14 days of induction, anthers were transferred to R₁ medium supplemented with 0.1 mg dm⁻³ kinetin. Obtained embryos were subsequently transplanted onto V₃ hormone-free medium and well growing plants were planted in greenhouses. The efficiency of androgenesis for both C. frutescens L. forms was relatively low and it did not exceed 5%. The ploidy level of the resulting plants was determined by flow-cytometric analysis. The regenerants consisted of about equal numbers of haploids and diploids. Additionally, among plants regenerated from anthers of yellow fruit forms, two mixoploids were observed.     

Evaluation of methods for celery (<Emphasis Type="Italic">Apium Graveolens</Emphasis> L.) transformation using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the <Emphasis Type="Italic">bar</Emphasis> gene as selectable marker

Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery. Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for 2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l⁻¹). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l⁻¹; calluses formed only with 0, 0.35 and 0.5 mg·l⁻¹ GS. All growing calluses from 0 and 0.35 mg·l⁻¹ and a few from 0.5 mg·l⁻¹, were divided and placed back on C + GS 0.35–0.5 mg·l⁻¹ for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l⁻¹. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected, but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine 2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l⁻¹ glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.)

Transient expression studies using blueberry leaf explants and monitored by -glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)₃AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 M AS. Explants were then placed on modified WPM supplemented with 1.0 mg l^–1 thidiazuron, 0.5 mg l^–1 -naphthaleneacetic, 10 mg l^–1 kanamycin (Km), and 250 mg l^–1 cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 E m^–2 s^–1 at 25°C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Activity of antioxidant enzyme during <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in <Emphasis Type="Italic">Crocus sativus</Emphasis>

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm⁻³ 1-naphthaleneacetic acid (NAA) + 1.0 mg dm⁻³ thidiazuron and 1.0 mg dm⁻³ NAA + 2.0 mg dm⁻³ kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm⁻³ NAA + 1.0 mg dm⁻³ benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.     

Homeothermy in adult salmon sharks, <Emphasis Type="Italic">Lamna ditropis</Emphasis>

Salmon sharks, Lamna ditropis, belong to a small group of sharks that possess vascular counter-current heat exchangers (retia mirabilia) allowing retention of metabolically generated heat, resulting in elevated body temperatures. The capacity of free-swimming lamnid sharks to regulate rates of heat gain and loss has not been demonstrated. Using acoustic telemetry, we recorded swimming depth and stomach temperature from four free-swimming salmon sharks in Prince William Sound, Alaska. Temperature data were obtained over time periods ranging from 3.8 to 20.7 h. Temperature profiles of the water column were obtained concurrently for use as estimates of ambient temperature. Mean stomach temperature among four individuals tracked ranged from 25.0 to 25.7°C. These sharks defended specific elevated temperatures regardless of changes in ambient temperature, which ranged from about 5–16°C. The maximum observed elevation of stomach temperature over ambient was 21.2°C. Because stomach temperatures were so strictly maintained relative to changes in ambient temperature, a thermal rate coefficient, k, (°C min^–1 °C thermal gradient^–1) for cooling of 0.053 min^–1 was obtained via a `control' experiment with a dead salmon shark. We show that free-swimming adult salmon sharks maintain a specific stomach temperature independent of changes in ambient temperature through a combination of physical and physiological means, and essentially function as homeotherms. This unique ability is probably the underlying factor in the evolutionary niche expansion of salmon sharks into boreal waters and in their ability to actively pursue and capture highly active prey such as salmon.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.