Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition

Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m⁻² s⁻¹) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h⁻¹ for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity, emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included 40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation method in photoautotrophic micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Number of air exchanges,sucrose concentration,photosynthetic photon flux,and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro

Rehmannia glutinosa plantlets were cultured for 4 weeks under different culture conditions to determine the optimum environment for in vitro growth and ex vitro survival. Plantlet growth increased with an increasing number of air exchanges of the culture vessel, exhibiting greatest shoot weight, total fresh weight, leaf area, and chlorophyll content at 4.4 h⁻¹ of air exchanges. High sucrose concentration (30 g l⁻¹) increased root weight but reduced shoot growth. Net photosynthetic rates of the plantlets were greatest when sucrose was not added to the medium. On the other hand, ex vitro survival of the plantlets was not influenced by sucrose concentration. In the experiment on difference in photoperiod and dark period temperatures (DIF) and photosynthetic photon flux (PPF), plantlet growth increased as DIF and PPF levels increased. Particularly, increasing PPF level had a more distinctive effect on plantlet growth than increasing DIF level. The interaction of DIF × PPF was also significant, showing the greatest plantlet growth in positive DIF (+8 DIF) and a high PPF (210 μmol m⁻² s⁻¹). In conclusion, the results of this experiment suggest that increased number of air exchanges of the culture vessel, decreased sucrose concentration, and positive DIF in combination with high PPF level enhanced growth and acclimatization of Rehmannia glutinosa plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoautotrophic growth of sugarcane plantlets <Emphasis Type="Italic">in vitro</Emphasis> as affected by photosynthetic photon flux and vessel air exchanges

Summary Explants of sugarcane, a C₄ plant, were cultured in vitro for 18d on Floridalite (a solid cube consisting of vermiculite and cellulose fibers) used as supporting material with sugar-free Murashige and Skoog liquid medium with double-strength KH₂PO₄, MgSO₄, FeSO₄, and Na₂-EDTA in the vessel with enhanced natural ventilation. CO₂ concentration in the culture room was kept at 1500 μmol mol⁻¹ (four times the atmospheric CO₂ concentration) during the photoperiod. A factorial experiment was designed with two levels of photosynthetic photon flux (PPF) and three levels of N (number of air exchanges of the vessel). The results were compared with those in the control treatment (photomixotrophic culture using sugar-containing agar medium under low PPF and low N). PPF and N showed significant positive effects on the growth of sugarcane plantlets in vitro. In the photoautotrophic (using sugar-free medium) treatments with relatively high PPF (200–400 μmol m⁻² s⁻¹) and high N (2–10 h⁻¹), the growth of plantlets was four to seven times greater than that in the control. Also, the culture period for multiplication and rooting was shortened from 30 d in the control to 18 d or less in the photoautotrophic, high PPF, and high N treatments. Use of porous supporting material in photoautotrophic treatments promoted rooting and plantlet growth significantly.     

Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically

Summary A comparative study was conducted to optimize the vegetative growth of sweet potato (Ipomoea batatas L. (Lam), cv. Beniazuma) plantlets cultured in vitro in five different types of supporting materials: agar matrix (a seaweed derivative; Kanto Chemical Co. Inc., Tokyo), gellan gum (a Pseudomonas derivative; Kanto Chemical Co. Inc., Tokyo), vermiculite (a kind of hydrous silicates), a mixture of vermiculite and cellulose fiber (Florialite; Nisshinbo Industries, Inc., Tokyo) and cellulose plug (Sorbarod; Baumgartner Rapiers SA., Switzerland). Single nodal cuttings were cultured photoautotrophically (without any sugar in the medium and with enriched CO₂ and high photosynthetic photon flux) for 21 d on MS basal medium. Plantlets exhibited the greatest growth when Florialite was used as supporting material. The leaf and root fresh and dry mass were 2.4× and 2.9×, and 2.2× and 2.8× greater, respectively, than those of the plantlets grown in the agar matrix (control). Plantlets cultured in Sorbarod supporting material exhibited the second greatest fresh and dry mass of leaves and roots followed by vermiculite and gellan gum supporting material. The most interesting feature was the development of a large number of fine lateral roots from the main adventitious root in the Florialite treatment. Among the treatments, the highest net photosynthetic rate was evident in the Florialite grown plantlets. The percent porosity of the supporting materials was highest in Sorbarod followed by Florialite and vermiculite. Plantlets transplanted from the Florialite supporting material exhibited the highest acclimatization percentage followed by that of the Sorbarod treatment.     

In vitro and ex vitro growth of grapevine rootstock `5BB' as influenced by number of air exchanges and the presence or absence of sucrose in culture media

Single node cuttings of grapevine rootstock `5BB' were cultured for 5 weeks under different numbers of air exchange (0, 0.8, 1.5, 2.5 and 4.4 h ^–1) with or without sucrose in the medium. To determine the effect of number of air exchanges related to presence or absence of sucrose, in vitro growth, net photosynthetic rate, stomata characteristics and ex vitro growth were investigated. Increasing numbers of air exchanges accelerated plantlet growth regardless of the presence or absence of sucrose in the medium; under the same number of air exchanges, plantlet growth and net photosynthesis were greater in sucrose-containing medium compared with sucrose-free medium. However, a high number of air exchanges (4.4 h^–1) inhibited the growth and photosynthesis in sucrose-containing medium compared to sucrose-free medium. A higher number of stomata was observed in sucrose-containing medium, and larger size stomata was observed in sucrose-free medium. These results emphasize the importance of ventilation (increased number of air exchanges) during in vitro culture, for ex vitro plantlet survival was greatly affected by with or without air exchanges. Without air exchanges ex vitro plantlet survival was less than 70%, while air exchanges increased ex vitro survival by more than 90% with greater growth.     

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.     

Factors influencing the production of hardened glaucous carnation plantlets in vitro

Medium type, its water status and the relative humidity in the culture vessel modified carnation leaf development in vitro. Carnation shoot apices cultured on liquid or on 0.8% agar solidified media developed into plantlets having succulent and translucent leaves which are not transplantable to non-aseptic conditions. Increasing the agar and/or sucrose concentration in the medium as well as decreasing the relative humidity in the culture vessel by a desiccant promoted glaucous leaf production. Increased water status (H₂O and relative humidity) increased shoot proliferation and translucency of leaves. Decreased water status reduced shoot proliferation but induced the formation of glaucous leaves. The culture of apices for 5–6 days on liquid medium prior to their sub-culture to 1.5% agar medium improved shoot proliferation and normal leaf development. An agar slant prevented the submergence of apices in water accumulating on the medium and thus reduced leaf translucency. Survival was further increased by the transfer of plantlets in uncapped culture vessels to a desiccator for 1–2 weeks prior to transplanting to soil.     

Similarity of growth patterns between plantlets and seedlings of Brassica campestris L. under different in vitro environmental conditions

Explants and seeds of Brassica campestris L. were cultured on Murashige & Skoog (1962) medium with and without sucrose in a vessel with different numbers of air changes per hour under different PPF (photosynthetic photon flux) conditions. The growth and development of plantlets in the vessel were similar to those of seedlings when cultured under the same in vitro environmental conditions. The growth and development of seedlings when cultured under the same in vitro environmental conditions. The growth and development of plantlets/seedlings were greater for treatments with a higher number of air changes per hour and a higher PPF regardless of the sucrose concentration in the culture medium.The CO₂ concentration in the vessel with a lower number of air changes per hour decreased to approximately 100 ppm during the photoperiod on day 21 due to the photosynthetic activities of the plantlets/seedlings. The low CO₂ concentration, in turn, reduced the net photosynthetic rate of plantlets/seedlings in the vessel, and thus affected their growth and development.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration in the culture room - MS mineral composition of Murashige & Skoog (1962) medium - PPF photosynthetic photon flux     

Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Net photosynthetic rates (P _n) of easy (EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes were examined under the following culture conditions: (1) photoautotrophic [sugar-free medium, high photosynthetic photon flux (PPF), high vessel ventilation rates and CO₂ enrichment, (PA)]; (2) modified photomixotrophic [sugar-containing medium diluted with sugar-free medium over time, high PPF, and high vessel ventilation rates (PM)]; (3) modified photomixotrophic enriched [same as PM with CO₂ enrichment, (PME)]; or (4) conventional photomixotrophic [sugar-containing medium, low PPF, and low vessel ventilation rates (control)]. Regardless of genotype, plantlets cultured under PA conditions died within 2 wk, whereas under PM and PME conditions, plantlets increased their P _n. After 6 wk, P _n per gram dry weight was 1.7 times greater in EK 16-3 than EK 11-1 plantlets cultured under PME conditions. In vitro-produced leaves of EK 16-3 plantlets were elongated with expanded blades, whereas EK 11-1 produced short leaves without expanded blades, especially under control conditions. After in vitro culture, EK 16-3 PME plantlets exhibited the highest dry weights among treatments. EK 16-3 PME and EK 16-3 PM had similarly high survivability, shoot and root dry weights and leaf lengths ex vitro compared to EK 16-3 control and EK 11-1 PM and PME plantlets. Ex vitro growth, survivability and P _n per leaf area of either genotype were not affected by CO₂ enrichment under modified photomixotrophic conditions. These results suggest that growth and survivability of sea oats genotypes with different acclimatization capacities can be enhanced by optimizing culture conditions.     

Physiology of Eucalyptus plantlets grown photoautotrophically in a scaled-up vessel

Summary Nodal cuttings of Eucalyptus camaldulensis L. plantlets were cultured photoautotrophically (sugar-free nutrient medium and with enriched CO₂ and high photosynthetic photon flux) in a scaled-up vessel (volume 4.0 liters) under forced ventilation (SV-treatment). After 28 d of culture, physiological aspects of the plantlets were compared with plantlets grown photomixotrophically (20 g l⁻¹ sucrose in the medium) in a Magenta vessel (volume 0.4 liters) under natural ventilation (control). In the SV-treatment net photosynthetic rates were enhanced, normal stomatal closing and opening were observed, and the epicuticular leaf-wax content was significantly higher than the control. The anatomical study showed well-organized palisade and spongy mesophyll layers of SV leaves. The SV-treatment also allowed in vitro acclimatization, and after transplanting ex vitro, the transpiration rate and the percent water loss was lower than those of the control and thus the SV plantlets acclimatized easily ex vitro.     

The Effect of In Vitro Culture Conditions on the Pattern of Photoinhibition during Acclimation of Gardenia Plantlets to Ex Vitro Conditions

We tested the effect of growing conditions during micropropagation on the fast kinetics of chlorophyll (Chl) fluorescence of Gardenia jasminoides Ellis plantlets during a 4-week acclimation to ex vitro. We studied whether photoautotrophic growing in vitro produced plantlets with less photoinhibition impairment during acclimation. Of the growing conditions stimulating photoautotrophy in vitro, only loose tube caps had a positive effect, whereas low sucrose or sucrose-free content in the medium and high PPFD showed a negative effect. Thus, plantlets cultured with 3 % (m/v) of sucrose were subsequently less photoinhibited throughout acclimation than those cultured with low sucrose (0.5 %) or sucrose-free media. Moreover, at the end of acclimation the former plantlets showed F_v/F_m and F_v/F₀ ratios typical of unstressed ex vitro plants as well as a higher Chl content and ratio of Chls to carotenoids. Plantlets cultured at a photosynthetic photon fluence density (PPFD) of 50 µmol m^–2 s^–1 also showed a better performance at the end of acclimation than those cultured at a higher (110 µmol m^–2 s^–1) PPFD. Thus except in the case of loose-tube closure, gardenia plantlets cultured in vitro under conventional sucrose concentration and PPFD are the least photoinhibited during acclimation. Nevertheless, significant interactions between the in vitro growing factors were observed at the end of acclimation.     

Micropropagation of <Emphasis Type="Italic">eclipta alba</Emphasis> (L.) hassk—An important medicinal plant

Summary An efficient and reproducible protocol for mass propagation of Eclipta alba (L.) Hassk, an important medicinal plant, was standardized by culturing shoot tips and nodal segments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on medium containing BAP (0.5 mgl⁻¹) and gibberellic acid (0.5 mgl⁻¹). Rooting was best achieved on MS medium supplement with 1 mg⁻¹ indole-3-butyric acid. Rooted plantlets attained maturity and flowered normally in the field.     

Multiple shoot induction and leaf and flower bud abscission of Annonacultures as affected by types of ventilation

Nodal explants of Annona squamosa L. and Annona muricata L. were cultured in vitro under various types of ventilation: airtight vessel (sealed condition; number of air exchange 0.1 h^–1), natural ventilation (via a polypropylene membrane; number of air exchange 1.5 h^–1), and forced ventilation (5.0 cm³ min^–1 in a 60 cm³ vessel; number of air exchange 5.0 h^–1). In both species, numbers of leaves, leaf areas and numbers of nodes per shoot increased with improving standards of ventilation, while leaf abscissions were substantially reduced; all the leaves had abscised in the airtight vessels after 12–15 days, but none had done so with forced ventilation. Flower-bud abscission in A. muricatashowed a similar trend after 21 days. These effects were associated with reductions in the accumulation of ethylene within the culture vessels, produced by increasing the efficiency of ventilation; ethylene was not detected in those fitted with a forced ventilation system. CO₂ concentrations in culture headspaces and the net photosynthetic rates of the plantlets were also evaluated. CO₂ concentrations decreased well below the ambient in the natural and airtight vessels; however, under forced ventilation, CO₂ concentrations were significantly higher during the photoperiod, compared to those of the natural ventilation and airtight vessel treatments. In general, net photosynthetic rates per unit leaf area increased with increasing photosynthetic photon flux (PPF) and rates were highest in plantlets grown under forced ventilation, intermediate under natural ventilation and lowest in the airtight vessels.Eighteen different media were investigated for their effects on multiple shoot induction in both species. The best medium for multiple shoot induction and growth in A. squamosa was Murashige and Skoog medium (MS) + 6-benzylaminopurine (BA; 1.5 mg l^–1) + casein hydrolysate (1.0 g l^–1) and for A. muricata MS + BA (1.0 mg l^–1) + naphthaleneacetic acid (NAA; 0.1 mg l^–1).     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Effect of water stress mediated through agar on <Emphasis Type="Italic">in vitro</Emphasis> growth of potato

With the objective to develop a practical method of screening potato for drought tolerance, shoot and root growth in plantlets raised in vitro (from nodal cuttings drawn from in vivo as well as in vitro grown plantlets) were studied in three genotypes with known root mass production under field conditions. Different levels of water stress were induced using five concentrations of agar in MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium. Water potential of various media ranged from −0.70 MPa to −0.98 MPa. Water stress in culture adversely affected plantlet growth, and the responses varied with genotype and explant source. Genotype IWA-1 was less affected than Konafubuki and Norin-1. In the experiment with explants from in vivo grown plants, the time to rooting was considerably delayed in Konafubuki and Norin-1 by an increase in agar concentration, but no such effect was observed in IWA-1. In all media, the mean number of roots and root length was greater in IWA-1 than Konafubuki and Norin-1, and the latter two genotypes were at par. At 10 gl⁻¹ agar, IWA-1 had taller plantlets, heavier foliage dry weight, root volume, as well as root dry weight than Konafubuki and Norin-1, whereas the latter two genotypes were at par for all these characteristics. This pattern was similar to the reported pattern of these genotypes for root dry weight under field conditions. However, such similarity in the in vitro and field behavior of the tested genotypes was not observed when nodal cuttings drawn from in vitro plantlets were used as explants. It is concluded that in vitro screening of potato under specific and limited water stress conditions by raising plantlets from nodal cuttings drawn from in vivo grown plants may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

Micropropagation of adult Swainsona formosa (Leguminosae: Papilionoideae: Galegeae)

Summary Explants of axillary buds excised from mature adult stems of Swainsona formosa (G. Don) J. Thompson (syn. Clianthus formosus) were cultured on Murashige and Skoog medium supplemented with a range of auxins, cytokinins, and sucrose concentrations. Auxins did not increase shoot or bud numbers above controls, and 2,4-dichlorophenoxyacetic acid was the only auxin to significantly increase callus production. Benzyladenine or thidiazuron incorporated into the medium at 0.1 μM stimulated shoot and bud production, and shoot growth occurred following removal of cytokinins from the medium after 4 wk. Shoot number increased linearly with sucrose concentration up to 40 g l⁻¹, but shoot height and the number of cytokinin-induced buds were optimal at sucrose levels of 20–30 g l⁻¹. Roots were initiated in vitro following treatment of cuttings with 0.1% indole-3-butyric acid and 0.1% α-naphthaleneactic acid. Plantlets were successfully established in soil but were plagiotropic and exhibited distichous phyllotaxy.     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum

Crown and leaf slices of in vitro plantlets of a non-flowering Vetiveria zizanioides from Java were used to induce compact calli and to regenerate plantlets. The influence of different growth regulators (2,4-dichlorophenoxy acetic acid, 6-benzylaminopurine), sucrose concentrations (10–100 g l⁻¹), cultivation in light or dark, and cultivation time on callus induction medium (6 or 12 weeks), on the induction of compact callus and the subsequent regeneration of plantlets was studied. Up to 75% of crown slices cultured on modified Murashige and Skoog medium supplemented with 2.26 μM 2,4-dichlorophenoxy acetic acid, 2.22 μM 6-benzylaminopurine and 75 g l⁻¹ sucrose developed compact callus. For subsequent regeneration of plantlets, callus induction in the light for 6 weeks on the callus induction medium containing 10 g l⁻¹sucrose, and subsequent transfer to the regeneration medium, was the best procedure, regenerating plantlets on around 60% of the crown or leaf slices, with up to 100 plantlets per slice. We have compared the efficiency of the above mentioned procedure with several other methods to regenerate plantlets. Our findings indicate that the procedure developed in this study was best in regenerating plantlets for the used vetiver variant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthetic characteristics of Cymbidium plantlet in vitro

The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO₂ concentration inside and outside of the culture vessels. The CO₂ measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m^-2 s^-1, a chamber air temperature of 15, 25 and 35°C and a CO₂ concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO₂ concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m^-2s^-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO₂ concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C₃ plants grown outside under shade. The results indicate that CO₂ enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration outside the vessel (in the culture room) - PPF photosynthetic photon flux