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.     

Mass Propagation of Eucalyptus camaldulensis in a Scaled-up Vessel Under In Vitro Photoautotrophic Condition

A scaled-up culture vessel was designed for the large-scalephotoautotrophic micropropagation of chlorophyllous plants.The culture vessel (volume 20 l) contained a plug cell traywith 448 plantlets, and had a forced ventilation system to supplyCO₂-enriched air. A nutrient-reservoir was connected to theculture vessel from which nutrient solution was circulated tothe culture vessel every 24 h. Nodal leafy cuttings of Eucalyptuscamaldulensis L. were cultured photoautotrophically in thissystem without sugar in the nutrient medium, but with an enrichedCO₂concentration and a high photosynthetic photon flux. Thegrowth and the net photosynthetic rate of the in vitro grownplantlets and the survival percentage of the plantlets aftertransplanting to ex vitro conditions were compared with thoseof plantlets grown photoautotrophically under natural ventilationin conventional small culture vessels (Magenta-type vessels;volume 0.4 l). Fresh and dry masses and net photosynthetic ratewere significantly higher in plantlets grown in the scaled-upvessel compared to plantlets grown in the conventional smallvessels (control). The environmental conditions created in thisscaled-up vessel (with forced ventilation) also facilitatedacclimatizationin vitro . Importantly, after transplanting tothe ex vitro condition, plantlets grew well without any specializedexvitro acclimatization treatment. Copyright 2000 Annals of BotanyCompany CO₂enrichment, Eucalyptus camaldulensis L., ex vitro, forced ventilation, natural ventilation, photoautotrophic, scaled-up vessel, survival percentage     

Development of a forced ventilation micropropagation system for large-scale photoautotrophic culture and its utilization in sweet potato

Summary A forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO₂ inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato (Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development.     

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.     

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.     

Insertion Profiles in Stomatal Density and Sizes in Nicotiana Tabacum L. Plantlets

Tobacco (Nicotiana tabacum L. cv. Samsun) plantlets were cultured in vitro on Murashige-Skoog medium photoautotrophically (without sucrose) or photomixotrophically (with 3 % sucrose) under two irradiances [70 or 230 µmol m^–2 s^–1]. Significant differences in stomatal density and sizes in leaves of different insertion levels (3^rd, 5^th and 7^th leaves from bottom) in photomixotrophic plantlets but not in photoautotrophic ones were found after 35 d of culture. Stomatal density was higher in upper leaves and on abaxial leaf side. Higher irradiance enhanced stomatal density in photoautotrophic plantlets. Stomatal sizes decreased with leaf insertion level but no significant differences between leaf sides were found. Abaxial stomata were more circular than the adaxial ones. In photomixotrophic plantlets stomata tended to be more elongated in the 3^rd and the 5^th leaves, whereas stomatal elongation in photoautotrophic plantlets was similar in all leaves.     

Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO₂ concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO₂ concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.     

Effects of sucrose concentration,supporting material and number of air exchanges of the vessel on the growth of in vitro coffee plantlets

Growth of coffee (Coffea arabusta) plantlets cultured in vitroas affected by sugar, types of supporting material and number of air exchanges of the vessel was investigated. Single node cuttings of in vitro coffee plantlets were cultured on half strength MS medium with or without 20 g l⁻¹ sucrose. Two types of supporting material, agar and Florialite, and two levels of air exchange expressed by number of air exchanges per vessel, 0.2 and 2.3 h⁻¹, were studied. At the end of a 40-day culture period, fresh weight, shoot length, root length and leaf area of plantlets when cultured on Florialite soaked in sugar-free medium and under the higher number of air exchanges were greater than those in sugar containing medium. Callus was observed at the shoot base of plantlets grown on agar medium containing sucrose. Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Promoting root induction and growth of in vitro macadamia (<Emphasis Type="Italic">Macadamia tetraphylla</Emphasis> L. ‘Keaau’) plantlets using CO<Subscript>2</Subscript>-enriched photoautotrophic conditions

In this study, a rooting protocol was developed for macadamia plantlets with healthy roots and enhanced growth performance, along with enhanced photosynthetic capability. In vitro-grown shoots rooted in vented vessels containing vermiculite as the supporting material exhibited 100% frequency of root induction, whereas when shoots were grown in non-vented vessels containing a solidified Murashige and Skoog (MS) medium, the frequency of root induction was less than 30%. The formation of root with callus, hyperhydricity, and leaf necrosis was observed in this photomixotrophic closed system. The modification of the vented photoautotrophic system with different concentrations of CO₂ and sucrose were investigated using vermiculite as the supporter. The number of roots, root length, root surface area, fresh weight, and dry weight were significantly higher in plantlets grown in CO₂-enriched (1,000 μmol CO₂ mol⁻¹) photoautotrophic conditions. The water content in both root and shoot tissues of plantlets cultured under photoautotrophic conditions was maximized. In addition, shoot and leaf performances were enhanced in plantlets cultured under CO₂-enriched photoautotrophic conditions. The supplementation of sucrose (29–88 mM) to culture media in both ambient and elevated CO₂ conditions affected a reduction in the shoot and root performance of in vitro plantlets. Chlorophyll a, chlorophyll b, and total carotenoids in the leaf tissues of plantlets acclimatized in CO₂-enriched photoautotrophic conditions were enriched, leading to increasing photosynthetic abilities, including chlorophyll fluorescence and net photosynthetic rate. From this investigation, a root induction protocol was established and the production of healthy macadamia plantlets was successfully implemented using CO₂-enriched photoautotrophic conditions.     

Growth and Photosynthetic Characteristics of Solanum tuberosum Plantlets Cultivated in Vitro in Different Conditions of Aeration, Sucrose Supply, and CO(2) Enrichment

Growth characteristics, oxygen exchange, and carbohydrate and chlorophyll contents were determined 30 days after subculturing of single node-derived plantlets of Solanum tuberosum cv Haig cultivated in vitro. Cultivation conditions were: (a) photomixotrophy in closed vessel, (b) photomixotrophy in closed vessel on medium supplemented with silver thiosulfate, (c) photomixotrophy in aerated vessel, (d) photoautotrophy in air, (e) photoautotrophy in CO₂-enriched air. In photomixotrophic conditions, aeration of the vessel enhanced sucrose utilization and had a positive effect on plantlet growth. In photoautotrophic conditions, growth of the plantlets was slow in air and was strongly enhanced by CO₂ enrichment of the atmosphere. Starch to sucrose ratios were higher in plants grown photoautotrophically than in plants grown with sucrose in the medium. Oxygen exchange characteristics on a chlorophyll basis were similar between the plantlets when measured under moderate light, and resembled those of greenhouse plant leaves. In high light, however, plantlets grown photoautotrophically in a CO₂-enriched atmosphere had higher oxygen exchange rates. We concluded from these results that potato plantlets in vitro in conditions (c), (d), and (e) developed C3-plant photosynthetic characteristics, which were in photoautotrophically grown plantlets comparable to those of field-grown plants.     

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.     

Influence of in vitro growth conditions on photosynthetic competence and survival rate of Rehmannia glutinosa plantlets during acclimatization period

The photosynthetic responses of Rehmannia glutinosa grown under photoautotrophic or heterotrophic conditions in vitro were investigated after transfer to greenhouse conditions. In addition, the changes in carbohydrate content and survival rates of the plantlets were evaluated. During six days after transplantation, the photosynthetic rate declined and photoinhibitory impairments represented by decrease of Fv/Fm and chlorophyll content were observed regardless of environmental conditions in vitro. Excessive transpiration was observed in plantlets grown under heterotrophic conditions during that period. Fructose and glucose content of the plantlets grown under photoautotrophic conditions increased with time and reached almost the same level of field grown plants after day 15. Under heterotrophic conditions, in contrast, the content of these sugars decreased continuously during that period. It is suggested that high survival rate of plantlets grown under photoautotrophic conditions has to be attributed to improvement of photosynthetic competence by imposed high light intensity and CO₂ concentration in vitro. The results strongly suggest that the control of transpiration during early stage after transplantation plays a key role in the acclimatization process, and photoautotrophic conditions could be a solution to solve the problems associated with transplantation stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

High irradiance can minimize the negative effect of exogenous sucrose on the photosynthetic capacity of <Emphasis Type="Italic">in vitro</Emphasis> grown coconut plantlets

There is increasing evidence that the sucrose normally added to the culture medium affects negatively the photosynthetic capacity of plantlets. At the same time, however, sucrose cannot be eliminated from the medium, as it is required for normal in vitro growth. We argue that this is true only under the conventional light conditions of growth-rooms. In the present paper irradiance of growth-rooms was increased 10 times and although the sucrose-inhibitory effect was found at high sucrose concentrations, it was possible to grow coconut (Cocos nucifera L.) plantlets without sucrose. Those plantlets showed both high photosynthetic capacity and comparable in vitro growth to those grown with sucrose in the medium under conventional growth-room irradiance. Nevertheless, the best growth was achieved under mixotrophic conditions where at high irradiance and moderate sucrose concentrations plantlets accumulated 27 % more biomass than plantlets grown without sucrose under high irradiance and 43 and 73 % more biomass than their counterparts at low irradiance with or without sucrose, respectively.     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

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).     

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.     

Induction of carotenoid pigments in callus cultures of Calendula officinalis L. in response to nitrogen and sucrose levels

In vitro carotenoid pigment production in callus cultures of Calendula officinalis L. was investigated using two basal media, semi-solid versus liquid media and varied concentrations of sucrose, ammonium, and nitrate nitrogen. Of the two explants that were evaluated, floret explants were best for callus induction using Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid under complete darkness. Carotenoid pigment induction was significantly augmented when the sucrose concentration was increased. Low sucrose concentrations in the culture medium deferred the onset of pigment induction and reduced the overall levels of carotenoid pigments produced. The highest amount of carotenoid pigments was observed when the callus was grown on the MS medium without ammonium nitrogen. The quantity of carotenoids was slightly elevated in cultures grown on semi-solid medium than those grown in liquid medium. In vitro carotenoid production was optimized by modifying the concentration of ammonium nitrogen to nitrate nitrogen in the culture medium and enhancing the sucrose concentration.     

Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.