Effects of different spectral lights on <Emphasis Type="Italic">Oncidium</Emphasis> PLBs induction,proliferation, and plant regeneration

The effects of different spectral light distribution on in vitro induction and proliferation of Oncidium protocorm-like bodies (PLBs) and subsequent growth of plantlets were investigated. Shoot tips (5 mm in length) of proliferating shoots of Oncidium “Gower Ramsey” were vertically incubated on 1/2 Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 6-benzyladenine (BA), and grown under either monochromatic red light-emitting diodes (LEDs) (RR), blue LEDs (BB), yellow LEDs (YY) or green LEDs (GG). Cultures grown under fluorescent lamps (FL) were used as control. Selected FL-induced PLBs were cut into 3- to 4-mm sections and incubated on MS medium supplemented with 1.0 mg l⁻¹ BA and 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and grown under RR, BB, YY, GG, or FL. Moreover, FL-differented shoots (15 mm in length with two leaves) were incubated on 1/2 MS medium with 0.5 mg l⁻¹ NAA, and grown under either FL, RR, 10% blue + 90% red LEDs (1BR), 20% blue + 80% red LEDs (2BR), 30% blue + 70% red LEDs (3BR), BB, 80% red + 10% blue + 10% far-red LEDs (RBFr), or 80% red + 10% blue + 10% green LEDs (RBG). Overall, the red light spectrum enhanced induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths, while the blue spectrum promoted differentiation, protein accumulation, and enzyme activities in PLBs, as well as pigment content accumulation in PLBs and developing plantlets. The combination of red and blue LEDs resulted in higher energy efficiency as well as dry weight and enzyme activities in these plantlets.     

<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.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

<Emphasis Type="Italic">In vitro</Emphasis> fruiting and seed set of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. CV. Sweet banana

Summary Plantlets of Capsicum annuum L. ev. Sweet Banana regenerated via somatic embryogenesis from immature zygotic embryos were capable of producing flower, fruit, and seed when cultured in small tissue culture containers. In vitro floral buds were first formed on plantlets that grew on plantlet development medium [agar-gelled Murashige and Skoog (MS) basal medium containing 1 mgl⁻¹ (5.3 μM) α-naphthaleneacetic acid (NAA)] in a growth room at 22°C and continuous illumination. However, floral buds rarely developed further into mature flowers. This problem was overcome using the vented autoclavable plant tissue culture containers. In vitro fruit formation and ripening was observed when liquid half-strength MS basal medium supplemented with 5 μg ml⁻¹ silver thiosulfate, 1 mg l⁻¹ (5.3 μM) NAA, and 3% sucrose was added to the surface of the plantlet development medium. Hand-pollination improved fruit set. Further research in needed to determine why the pepper seeds formed in vitro failed to germinate.     

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

High-light-induced superoxide anion radical formation in cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach as detected by EPR spectroscopy

The generation of superoxide anion radical (O₂ ^·−) in the cytochrome b ₆ f complex (Cyt b ₆ f) of spinach under high-light illumination was studied using electron paramagnetic resonance spectroscopy. The generation of O₂ ^·− was lost in the absence of molecular oxygen. It was also suppressed in the presence of NaN₃ and could be scavenged by extraneous antioxidants such as ascorbate, β-carotene, and glutathione. The results also indicate that O₂ ^·−, which is produced under high-light illumination of the Cyt b ₆ f from spinach, might be generated from a reaction involing ¹O₂, and the Rieske Fe-S protein could serve as the electron donor in the O₂ ^·− production. The mechanism of photoprotection of the Cyt b ₆ f complex by antioxidants is discussed.     

<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.     

<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">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.     

Removal of <Emphasis Type="Italic">p</Emphasis>-chlorophenol by the marine microalga <Emphasis Type="Italic">Tetraselmis marina</Emphasis>

The ability of Tetraselmis marina, a green coastal microalga, to remove chlorophenols under photoautotrophic conditions was investigated. T.marina was able to grow in the presence of 20 mg L⁻¹ of the phenolic compounds tested. The EC₅₀ (growth rate) value of p-chlorophenol (p-CP) to T.marina was found to be 25.5 mg L⁻¹. The microalga was able to remove chlorophenols, showing higher efficiency for p-CP. The effect of photoregime and NaHCO₃ concentration on p-CP removal was investigated. Under continuous illumination with 1 g L⁻¹ NaHCO₃ initial concentration T.marina removed 65% of 20 mg L⁻¹ in a 10-day cultivation period.     

Camptothecin accumulation in various organ cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne grown in different culture systems

Levels of camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) were determined in different cultures of Camptotheca acuminata grown either in a Temporary Immersion System (TIS) or on solid medium. CPT was also detected in liquid culture medium. HPLC analysis showed significant differences in CPT contents in all tissues analysed and the highest CPT contents were found in shoots grown on solid medium and in TIS with a mean of 2.2 and 2.5 mg g⁻¹ DW, respectively. The highest content of CPT detected in seedlings was 1.96 mg g⁻¹ DW; while that of somatic embryos at cotyledonary stage and regenerated plants were 0.87 and 1.23 mg g⁻¹ DW, respectively. It was also shown that shoots cultured in TIS secreted substantial amount of CPT into the liquid medium. After 4 weeks in culture a mean of 6, 05 and 12, 6 μg g⁻¹ FW were determined at 4 and 8 immersion cycles daily (IC d⁻¹), respectively. This aspect opens new possibilities regarding the isolation of CTP using TIS culture systems.     

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.     

<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.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Improved growth and quality of <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Siraitia grosvenorii plantlets were investigated. The TIS promoted the growth and quality of S. grosvenorii plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤ 0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also decreased callus formation at the base of shoots. Callus diameter was significantly (P ≤ 0.05) lower in the TIS (3.30 mm) than in gelled medium (6.31 mm) and liquid medium (6.77 mm), respectively. FW (50.83 mg) and DW (7.08 mg) of callus in the TIS were also significantly (P ≤ 0.05) lower than those in gelled medium (80.00 and 10.56 mg, respectively) and liquid medium (218.75 and 23.75 mg, respectively). During rhizogenesis, minimal callus was evident at the base of shoots in the TIS, with a well-developed root system. However, the plantlets in gelled medium just produced thick, brown and easily broken roots with obvious callus and fewer secondary roots. The natural-like plantlets of S. grosvenorii obtained in the TIS would probably have positive effects on ex vitro rooting and transplanting in large-scale commercial production.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.