Annals of Botany 90: 11-19, 2002
© 2002 Annals of Botany Company
Photoautotrophic Culture of Coffea arabusta Somatic Embryos: Photosynthetic Ability and Growth of Different Stage Embryos
,1,1,11 Department of Bioproduction Science, Chiba University, Matsudo, Chiba 271-8510, Japan
* For correspondence. Fax 00 1 519 7670755, e-mail: szobayed{at}uoguelph.ca Present address: Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Received: 17 December 2001; Returned for revision: 1 February 2002; Accepted: 19 March 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 µmol m–2 s–1) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90–130 µg g–1 fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300–500 µg g–1 fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar-free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50 %, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100–150 µmol m–2 s–1) and increased CO2 concentration (1100 µmol mol–1) were found to be necessary for the development of plantlets from cotyledonary stage embryos.
Key words: CO2 enrichment, chlorophyll florescence, stomata, cotyledonary embryos.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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From an agricultural and forestry point of view, the most pressing reason for the development of somatic embryogenesis technology is its potential for genetic transformation and subsequent multiplication of transgenic plants (Jain et al., 1995). In the last decade, various techniques have been developed to improve the quality and productivity of somatic embryos. In addition, important advances have been made in recent years in the application of large-scale bioreactors that can produce somatic embryos in liquid media.
Photoautotrophic micropropagation of chlorophyllous plants can not only increase the growth of in vitro plantlets, but also minimize the risk of loss due to microbial contamination, reduce production costs, improve the physiological characteristics of the plantlets and enable better acclimatization ex vitro (Zobayed et al., 2000, 2001). Considerable effort has been devoted to full automation of somatic embryo development and micropropagation (Cervelli and Senaratna, 1995). The ability of somatic embryos to grow photoautotrophically will enable automation, which will lead to a reduction of production costs. Moreover, photoautotrophic growth can improve the quality of somatic embryos and possibly shorten and simplify the germination and plantlet development procedure.
Coffee plays a major role in the economy of many African, American and Asian countries. Only two species of coffee are commercially important, Coffea arabica and Coffea canephora. The low caffeine content and fine aroma of Coffea arabica was combined with the pathogen resistance of Coffea canephora (Berthouly and Etienne, 1999) in a new species named Coffea arabusta (Capot, 1972). C. arabusta has been propagated clonally to obtain genetically uniform transplants using micro cuttings (Dublin, 1980); however, the in vitro growth of micro cuttings is slow (Dublin et al., 1991). On the other hand, somatic embryogenesis is considered to be an effective method for mass clonal multiplication. The rapid progress of somatic embryogenesis research and its prospects for application to the subject of plant micropropagation prompted us to investigate the possibility of growing C. arabusta somatic embryos under photoautotrophic conditions. In this paper we describe the photosynthetic ability of different stage somatic embryos. Attempts were also made to culture these embryos under photoautotrophic conditions and to optimize different environmental conditions to maximize growth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Establishment of the culture
Nodal cuttings of coffee plantlets (Coffea arabusta) were cultured in a Magenta vessel containing hormone-free MS medium (Murashige and Skoog, 1962) supplemented with 20 g l–1 sucrose. After 4 weeks of culture, regenerated leaves were collected, cut into pieces (10 x 5 mm) and placed in a Magenta vessel containing MS medium (half strength MS salts and vitamins) supplemented with 200 mg l–1 KNO3, 70 mg l–1 KCl, 30 mg l–1 adenine, 10 mg l–1 BAP (benzylaminopurine)and 0·1 mg l–1 IBA (indolebutyric acid) (Nguen et al., unpubl. res.). Agar (8 g l–1; Kanto Chemical Co., Tokyo, Japan) was used as gelling agent and 30 g l–1 sucrose was added in the medium. Cultures were placed at 23 °C air temperature and under cool-white fluorescent lamps (National Co., Tokyo, Japan) providing a 16 h photoperiod and an 8 h dark period in a growth chamber. The ambient CO2 concentration was 400 µmol mol–1 and the photosynthetic photon flux (PPF) was 30 µmol m–2 s–1, measured on the empty culture shelf. Somatic embryos were developed within 9–12 weeks of culture (Fig. 1A and B). Globular (Fig. 1C), heart-shaped (Fig. 1D), torpedo (Fig. 1E and F), precotyledonary (Fig. 1F), cotyledonary (Fig. 1G) and germinated somatic embryos were selected and used as experimental materials.
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Measuring CO2 concentration, rate of CO2 uptake and production, chlorophyll content, chlorophyll florescence, and studying stomata
CO2 concentration in the culture vessel was measured using an infrared gas analyser (IRGA; S. W. and W. S. Burrage, Hustingleigh, Ashford, Kent, UK) and inserting a specially made CO2 sensor in the culture vessel headspace. The sensor consists of a cylindrical glass ring (length 10 mm, diameter 20 mm). The two open ends of the ring were covered by polycarbonate Nuclepore membrane (pore diameter 0·05 µm). Two separate flexible PVC tubes (length 50 mm, ID 3 mm) were connected to the sidewall of the ring; one end of each of these tubes opened in the inner airspace of the sensor and the other end was connected to either the inlet or the outlet pipe of the IRGA. The system thus acts as a closed circuit unit. The CO2 sensor (glass ring) was inserted in the vessel headspace through the sidewall of the vessel. The air inside the sensor represents the culture vessel air (equilibrium time is 30–60 s).
To measure rates of CO2 uptake and production by embryos (cm3 s–1 per embryo), vessels (volume 40 ml) were originally charged with sterile air (CO2 concentration 360 µmol mol–1) and were sealed tightly. Changes in CO2 concentration over time were recorded, and for maximum CO2 exchange rate the initial slope of the CO2 vs. time decay curve was used and was calculated using the following equation:
Maximum CO2 uptake/production rate =
Ca x V/(T xVm x E)
where Ca is the change in CO2 concentration (ml ml–1) over time interval T (s) over which CO2 concentration changes were recorded, V is the volume of the culture vessel, Vm is the molar volume of CO2 at the growth room temperature and E is the number of embryos per vessel.
Samples for chlorophyll measurement were collected from embryos at each stage of development and in every case an appropriate mass of embryos was soaked in 80 % ice-cold acetone for 3 d, centrifuged at 300 r.p.m. for 10 min and measured for light absorption between 400 and 700 nm in a spectrophotometer (Hitachi, Tokyo, Japan). From the absorption curves, the proportion of chlorophyll contents was evaluated according to the formula of Lichtenthaler and Wellburn (1983).
A fibre-optic based chlorophyll fluorimeter (Hansatech, King’s Lynn, Norfolk, UK) was used to analyse the photochemical activity of the somatic embryos. In dark-adapted samples (2 h), the maximal quantum yield of photochemistry through PSII (
pmax) was calculated from the ratio (Fm – Fo)/Fm (Kitajima and Butler, 1975). The actual quantum yield (p) of PSII photochemistry in light-adapted leaves was calculated from the steady-state level of chlorophyll fluorescence (Fs) and maximal fluorescence level: p = (Fm – Fs)/Fm (Havaux et al., 1991).
For studying stomata (on day 40), cotyledons from different embryo stages were collected and epidermal peels were taken from the abaxial (lower) surface. Stomata were observed and photographed using a pre-calibrated digital microscope (Keyence Corporation, Osaka, Japan). In the case of torpedo stage embryos, epidermal peels were taken from the polar region of the embryo. Scanning electron microscopy was also used to study stomata (for methodology see Zobayed et al., 2001b). Stomatal density (number per mm2) and stomatal length and width (with guard cells) were measured directly under the microscope for each treatment. The stomatal length refers to the distance between the ends of the guard cells, and the width is the distance transversely across them.
Statistical significance was determined by one-way or two-way ANOVA and least significant difference test (LSD). Experiments were repeated twice.
Photosynthetic ability of different stage coffee somatic embryos
Somatic embryos were established as described above. After 14 weeks of culture, a few vessels were transferred to a growth chamber under a high PPF of 100 µmol m–2 s–1 (pretreatment) and cultured for another 14 d with the aim of allowing the developing embryos to turn green (Fig. 1A and H). Embryos grown continuously under a PPF of 30 µmol m–2 s–1 for 16 weeks were used as control.
A microscope was used to classify/select somatic embryo stages, i.e. torpedo stage, precotyledonary stage, cotyledonary stage and germinated somatic embryos (Fig. 1). Selected embryos were transferred to glass vessels (volume 40 cm3; five embryos per vessel), each containing 10 cm3 MS medium (vitamins and sucrose were eliminated). Vessels were sealed with silicone rubber bungs. The CO2 uptake/production rate of each of the embryos was recorded over time. Chlorophyll fluorescence, chlorophyll content and stomatal studies were also conducted on each set/stage of embryos (ten replicates for each of the measurements).
Growth of different somatic embryo stages under photoautotrophic conditions
Pretreated (14 d with high PPF of 100 µmol m–2 s–1) torpedo, precotyledonary, coyledonary and germinated somatic embryos were transferred to plastic Petri dishes (volume 30 ml) containing MS medium (10 ml per Petri dish); agar (8 g l–1) was used as a gelling agent. For the photoautotrophic treatment, sucrose and vitamins were eliminated from the formulation and the Petri dishes were placed in a CO2-enriched growth chamber (1000 µmol mol–1) under 100 µmol m–2 s–1 PPF. To introduce natural ventilation, one Millipore filter membrane (pore diameter 0·5 µm; Nihon Millipore Ltd, Yonezawa, Japan) was used to cover a hole (10 mm diameter) in the lid of each of these Petri dishes (number of air exchanges 5·0 h–1). For the photomixotrophic treatment, 20 g l–1 sucrose was added in the medium and the Petri dishes were placed in a growth chamber with a CO2 concentration of 400 µmol mol–1 and a PPF of 50 µmol m–2 s–1. Each Petri dish contained five embryos of similar size and stage, and five Petri dishes were prepared per treatment. For both the treatments, ambient temperature and relative humidity were 25 °C and 80–85 %, respectively, during the photoperiod. The experiment was conducted for 60 d and growth (fresh and dry mass) of the somatic embryos was recorded.
Optimization of growth under photoautotrophic conditions
Effects of different supporting media on the photoautotrophic growth of cotyledonary stage somatic embryos.
For the photoautotrophic culture of cotyledonary stage somatic embryos, three different types of supporting media were investigated: (1) agar (8 g l–1; Kanto Chemical Co.); (2) vermiculite; and (3) Florialite [a mixture of vermiculite and cellulose fibre (described by Afreen et al., 2000)]. Cotyledonary stage somatic embryos were selected [previously grown in photomixotrophic conditions under a low PPF (30 µmol m–2 s–1) followed by 14 d pretreatment at high PPF (100 µmol m–2 s–1)] and two embryos were transferred to each Magenta vessel (volume 375 ml) containing 50 ml MS medium without any sucrose or vitamins to ensure photoautotrophic condition. Two Millipore filter membranes (Nihon Millipore Ltd) with a pore diameter of 0·5 µm were used to cover the holes (10 mm diameter) in the lid of each of these vessels; five vessels were prepared per treatment. The vessels were placed in a CO2-enriched (1000 µmol mol–1) growth chamber under a PPF of 100 µmol m–2 s–1. Growth of the embryos (fresh and dry mass) was recorded after 60 d of culture.
Effects of different PPF and CO2 concentrations on the photoautotrophic growth of cotyledonary stage somatic embryos.
Cotyledonary stage somatic embryos were cultured on MS medium [previously grown in photomixotrophic conditions under a low PPF (30 µmol m–2 s–1) followed by 14 d pretreatment at high PPF (100 µmol m–2 s–1)], and growth was studied under the following PPF and CO2 concentrations: (1) 50 µmol m–2 s–1 PPF and ambient CO2 concentration of 400 µmol mol–1; (2) 100 µmol m–2 s–1 PPF and 400 µmol mol–1 CO2; (3) 150 µmol m–2 s–1 PPF and 400 µmol mol–1 CO2; (4) 50 µmol m–2 s–1 PPF and 1100 µmol mol–1 CO2; (5) 100 µmol m–2 s–1 PPF and 1100 µmol mol–1 CO2; and (6) 150 µmol m–2 s–1 PPF and 1100 µmol mol–1 CO2.
For this experiment, somatic embryos were cultured in Magenta vessels and Florialite (9 g dry mass per vessel) was used as supporting material. Two Millipore filter membranes (Nihon Millipore Ltd) with a pore diameter of 0·5 µm were used to cover the holes (10 mm diameter) in the lid of each of these vessels. Two embryos were transferred per vessel and ten vessels were prepared per treatment. Culture conditions were the same as in the previous experiment. Growth (dry mass) of each of the embryos was recorded after 60 d of culture.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Photosynthetic ability of different stage coffee somatic embryos
CO2 evaluation in the culture headspace.
In those vessels containing torpedo and precotyledonary stage somatic embryos, an increase in CO2 concentration (compared with ambient) was noticed both in the photo- and dark period (Table 1). CO2 production rates for torpedo stage embryos with and without 14 d pretreatment were 2 x 10–6 and 2·2 x 10–6 cm3 s–1 per embryo, respectively, during the photoperiod (Table 1); values were similar during the dark period. CO2 production rates for precotyledonary stage embryos with and without pretreatment were 1·8 x 10–6 and 2·1 x 10–6 cm3 s–1 per embryo, respectively, in the photoperiod. In the dark, the CO2 production rate increased compared with that in the photoperiod (1·5- and 1·2-times with and without pretreatment, respectively). Results indicate that these embryos may have scavenged CO2 during the photoperiod, although the value may be too small to enable these embryos to grow sugar-independently (autotrophically).
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In the case of cotyledonary and germinated embryos, the CO2 concentration in the culture headspace was normally lower than ambient during the photoperiod (except cotyledonary embryos without pretreatment) (Table 1). The CO2 uptake rate in the cotyledonary stage embryo was approx. 1·3 x 10–6 cm3 s–1 per embryo with pretreatment. The values were even higher in germinated embryos: 2·9 x 10–6 and 2·1 x 10–6 cm3 s–1 per embryo with and without pretreatment, respectively. Therefore, these embryos showed carbon assimilation. In the dark period, values of CO2 production in both cotyledonary and germinated embryos were higher than those in other stages, probably because of the greater fresh mass of the embryos.
The above results clearly indicate that cotyledonary (with pretreatment) and germinated embryos (with or without pretreatment) are able to take up CO2. Pretreatment with high PPF increased the CO2 uptake rate in both cases. In the case of torpedo and precotyledonary stage embryos, respiratory activity masked any photosynthetic assimilation, probably because there was little chlorophyllous tissue but still a significant amount of non-chlorophyllous tissue with no stomatal development (see later). From the results it can be concluded that pretreatment of somatic embryos with high PPF increased the photosynthetic ability in almost all stages.
Chlorophyll florescence.
The potential activity of PSII, as estimated in the dark, was low (pmax = 0·45; Table 2) for the pretreated torpedo stage embryos. With high PPF pretreatment, the value of pmax increased in the precotyledonary stage (pmax = 0·69) followed by the cotyledonary stage (pmax = 0·84), and was highest in germinated embryos (pmax = 0·88) (Table 2). The actual photochemical efficiency of PSII is considered as a good estimate of the quantum yield of photosynthetic electron transport (Genty et al., 1989, 1992). As expected from their low PSII activity, torpedo stage embryos (with or without pretreatment) exhibited low electron-transport activity (p = 0·1). Again, an increase in the quantum yield for electron transport was observed in the cotyledonary and germinated embryos, with p reaching 0·17 and 0·29, respectively, in embryos without pretreatment, and 0·2 and 0·31 with pretreatment.
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pmax) of PSII photochemistry (in dark-adapted samples), actual quantum yield (The results clearly indicate the occurrence of photochemical activity in the cotyledonary (with high PPF pretreatment) and germinated embryos (with or without pretreatment).
Stomatal study.
Stomata generally did not form in torpedo and precotyledonary stage embryos despite 14 d of high PPF pretreatment. The absence of stomata in these stages was reflected in the limitation of photosynthesis in these embryos. However, in a very small number of cases, early stages of stomatal development were noted in the precotyledonary stage embryos. Stomata were present in cotyledonary and germinated stage embryos regardless of whether or not they were pretreated at high PPF (Fig. 2A–B). Generally, pretreated embryos had higher stomatal densities than embryos that were not pretreated. In cotyledonary stage embryos, stomatal density was 149 and 127 mm–2 cotyledon area with and without pretreatment, respectively (Table 2). The average length of each stoma was 29·5 and 24·5 µm in embryos that were and were not pretreated, respectively (Table 2). Well-developed stomata were observed in germinated embryos that were (Fig. 2C and D) or were not pretreated. The length and width of stomata in germinated embryos were greater than those of stomata from cotyledonary stage embryos (Table 2). The density was also higher: 199 (with pretreatment) and 170 mm–2 cotyledon area (without pretreatment).
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Development of stomata is essential for the physiological processes of plants; stomata act as portals for entry of CO2 into the leaf for photosynthesis (Willmer, 1983). As mentioned earlier, stomata were absent in the present experiment in torpedo and precotyledonary stage embryos despite pretreatment with high PPF. On the other hand, cotyledonary and germinated embryos usually developed stomata and thus may be able to photosynthesize.
Chlorophyll contents.
Low chlorophyll contents were recorded in the torpedo and precotyledonary stage embryos irrespective of high PPF pretreatment (Table 2). In the cotyledonary stage embryos, chlorophyll contents were higher than those in the torpedo or precotyledonary stage embryos. Chlorophyll a and b contents were 271 and 99 µg g–1 fresh mass, respectively, in the cotyledonary stage embryos with pretreatment; these values are, respectively, 1·3 and 1·1 times those in embryos without pretreatment. Chlorophyll a and b contents were highest in germinated embryos (385 and 129 µg g–1 fresh mass, respectively, in pretreated embryos, and 314 and 118 µg g–1 fresh mass, without pretreatment), but were still much lower than those noted in in vitro leaves.
During photosynthesis, green plants use light energy to produce chemical energy; chlorophyll is essential for this process. Our results demonstrate that cotyledonary (with pretreatment) and germinated C. arabusta embryos (with or without pretreatment) have the highest chlorophyll contents among the different embryo stages and treatments tested. This suggests that they are able to photosynthesize.
Growth of different stage somatic embryos under photoautotrophic conditions
In general, growth (fresh and dry mass) was greater in the photomixotrophic (PM) treatment than in the photoautotrophic (PA) treatment (Table 3). In case of torpedo and precotyledonary stage embryos, fresh mass was 1·9 and 1·7 times greater under PM compared with PA conditions, respectively, and dry mass was 2·3 and 1·8 times greater than under PA conditions. Similarly, the fresh masses of cotyledonary and germinated embryos were greater (40 and 42 mg per embryo, respectively) in PM treatment than those (25 and 32 mg per embryo, respectively) of embryos grown in PA conditions. Dry mass followed a similar trend.
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After 60 d of culture in the PA treatment, torpedo and precotyledonary stage embryos lost 20–25 % of their initial dry mass (Fig. 3). This loss could be due to continuous respiration and the low photosynthetic ability of the plant material (embryos were probably completely dependent on their own reserve food material). In contrast, in the PM treatment, the dry mass of each of the torpedo stage embryos increased by 200 % of their initial dry mass (Fig. 3). Under PA conditions, the dry mass of cotyledonary and germinated embryos increased by 10 and 50 %, respectively, of their initial dry mass (Fig. 3). These results are in agreement with previous findings that cotyledonary and germinated embryos show stomatal development, scavenge CO2 and have high chlorophyll contents.
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In the case of cotyledonary and germinated embryos, the first true leaves to unfold were observed after 4–6 weeks of culture in both PA and PM treatments. Precotyledonary stage embryos grown under PM or PA conditions did not produce any true leaves; however, well-developed unfolded cotyledons were observed in embryos after 6–7 weeks of PM treatment. When grown photoautotrophically, some embryos (54 %) also produced unfolded cotyledons by the end of the experiment, but these were smaller in size than those produced in the PM treatment. Interestingly, under PM treatment, some torpedo and precotyledonary stage embryos produced new embryos from the base. In general, normal formation of leaves was observed in embryos grown photoautotrophically; however, under PM treatment, hyperhydricity of leaves was quite obvious (Fig. 1I and J).
Under PM treatment, roots did not develop in torpedo or precotyledonary stage embryos, whereas 33 and 58 % of cotyledonary and germinated embryos, respectively, produced roots. When embryos were grown under PA-treatment, root formation was not observed in torpedo and precotyledonary stage somatic embryos. Some cotyledonary (15 %) and germinated embryos (25 %) produced roots.
Taking into account the growth results, it becomes increasingly apparent that coffee somatic embryos can be grown under photoautotrophic conditions. Results also indicate that cotyledonary and germinated embryos show growth increments (compared with initial growth) when grown under photoautotrophic conditions. Values of both potential activity of PSII and the actual photochemical efficiency of PSII of different stage embryos measured after 60 d culture were not significantly different among the photoautotrophic and photomixotrophic treatments (Table 3). In general, among the different stages values were greater in cotyledonary and germinated embryos in both treatments. Again, chlorophyll a and b contents of different stage embryos grown photoautotrophically were nearly the same as those of their photomixotrophic counterparts (Table 3).
Optimization of growth under photoautotrophic conditions
Effects of different supporting media on the photoautotrophic growth of cotyledonary stage somatic embryos.
Among the treatments, the greatest total fresh and dry masses were recorded in the Florialite treatment, with values being 1·3 times those of the agar treatment. With the exception of rooting percentage, there were no significant differences among growth parameters between the Florialite and vermiculite treatments (Table 4). Total root lengths were 7 and 8·2 mm per plantlet in Florialite and vermiculite supporting materials, respectively; these values are less than four times greater than those in the agar treatment. In agar-grown plantlets, percentage rooting was low; only 26 % of embryos produced roots and those roots produced were very short. Conversely, in Florialite and vermiculite, 69 and 56 % embryos, respectively, produced roots (Table 4). It should be noted that rooting of photoautotrophically grown cotyledonary stage embryos has recently been improved further by temporarily immersing the root zone and by using forced ventilation (to reduce relative humidity of the headspace and to increase CO2 concentration) (data not shown).
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To summarize, the use of Florialite and/or vermiculite can improve root and shoot growth during the development of plantlets from cotyledonary stage coffee somatic embryos under photoautotrophic conditions. The results are in agreement with those of earlier investigations in which root development and plant growth were improved by using fibrous supports, such as cellulose plugs and vermiculite in Eucalyptus (Kirdmanee et al., 1995), and a mixture of vermiculite and paper pulp in sweet potato (Afreen et al., 2000).
Effects of different PPF and CO2 concentrations on the photoautotrophic growth of cotyledonary stage somatic embryos.
In general, the dry mass of cotyledonary stage embryos was enhanced when CO2 was enriched. Under enriched CO2 (approx. 1100 µmol mol–1) and high PPF (100 µmol m–2 s–1), the dry mass was almost double the initial dry mass (Fig. 4). Increasing PPF further (150 µmol m–2 s–1) did not lead to any change in the dry mass of embryos. In contrast, in low PPF (50 µmol m–2 s–1) treatments there was hardly any increase in the dry mass (compared with initial dry mass), and the average dry mass was 3·8 mg per embryo compared with 4·4 and 4·5 mg per embryo in the 100 and 150 µmol m–2 s–1 PPF treatments, respectively. Therefore, results suggest that high PPF (100–150 µmol m–2 s–1) and an increased CO2 concentration (1100 µmol mol–1) are necessary for the development of plantlets from cotyledonary stage somatic embryos under photoautotrophic conditions.
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Our results represent the first step towards a clearer understanding of the photoautotrophic culture of somatic embryos. The conclusions drawn from these findings are: (1) cotyledonary and germinated embryos show photosynthetic ability, although high PPF (100 µmol m–2 s–1) pretreatment speeds up this process; (2) except in a very small number of cases, stomata do not develop fully in precotyledonary stage embryos and are absent in the torpedo stage; and (3) torpedo and precotyledonary stage embryos have very low chlorophyll contents (approx. 90–130 µg g–1 fresh mass), whereas cotyledonary and germinated embryos have higher contents (approx. 300–500 µg g–1 fresh mass). Due to the absence of stomata and very low chlorophyll contents, torpedo and precotyledonary stage embryos have very low photosynthetic capacity. Thus, we suggest that the earliest stage of somatic embryo that can be cultured photoautotrophically to develop plantlets is the cotyledonary stage.
Further experiments are necessary to extend our basic understanding of the role of photoautotrophy in the development of somatic embryos in order to develop plantlets using cotyledonary stage embryos in a limited time and space. However, the data presented here will be useful for the application of somatic embryogenesis of Coffea arabusta and will also provide valuable information for the development of this technology for the micropropagation of other commercially important species.
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ACKNOWLEDGEMENTS |
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We are grateful to C. Kubota (Chiba University) for critically commenting on the manuscript, Q. Nguyen (Institute of Tropical Biology, Vietnam) for supplying plant materials and M. Abe for culturing the somatic embryos. We are also grateful for financial support from the Japanese Society for the Promotion of Science (JSPS) Research-for-the-Future Program.
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LITERATURE CITED |
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Afreen F, Zobayed SMA, Kubota C, Kozai T, Hasegawa O. 2000. A combination of vermiculite and paper pulp supporting material for the photoautotrophic micropropagation of sweet potato. Plant Science 157: 225–231.
Berthouly M, Etienne H. 1999. Somatic embryogenesis of coffee. In: Jain SM, Gupta PK, Newton RJ, eds. Somatic embryogenesis in woody plants. Dordrecht: Kluwer Academic Publishers, 259–287.
Capot J. 1972. L’amelioration du cafeier en Cote d’Ivoire – Les hybrides ‘Arabusta’. Cafe Cacao The 16: 3–17.
Cervelli R, Senaratna T. 1995. Economic aspects of somatic embryogenesis. In: Aitken-Christie J, Kozai T, Smith ML, eds. Automation and environmental control in plant tissue culture. Dordrecht: Kluwer Academic Publishers, 29–64.
Dublin P. 1980. Multiplication vegetative in vitro de l’Arabusta. Cafe Cacao The 24: 281–290.
Dublin P, Enjalric F, Lardet L, Carron MP, Trolinder N, Pannetier C. 1991. Estate crops. In: Debergh PC, Zimmerman RH, eds. Micropropagation – technology and application. Dordrecht: Kluwer Academic Publishers, 337–361.
Genty B, Briantais JM, Baker NR. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochemica et Biophysica Acta 990: 87–92.
Genty B, Goulas B, Dimon B, Peltier G, Moya I. 1992. Modulation of efficiency of primary conversion in leaves, mechanisms involved at PSII. In: Murata N, ed. Research in photosynthesis IV. Dordrecht: Kluwer Academic Publishers, 603–610.
Havaux M, Strasser, RJ, Greppin H. 1991. A theoretical and experimental analysis of the qp and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events. Photosynthetic Research 27: 41–55.[CrossRef]
Jain SM, Gupta PK, Newton RJ. 1995. Somatic embryogenesis vol. 1 – history, molecular and biochemical aspects. Dordrecht: Kluwer Academic Publishers, 1–446.
Kirdmanee C, Kitaya Y, Kozai T. 1995. Effects of CO2 enrichment and supporting material in vitro on photoautotrophic growth of Eucalyptus plantlets in vitro and ex vitro: anatomical comparisons. Acta Horticulturae 393:111–115.
Kitajima M, Butler WL. 1975. Quenching of chlorophyll fluorescence and primary photochemistry by dibromothymoquinone. Biochemica et Biophysica Acta 376: 105–111[Medline]
Kozai T. 1991. Autotrophic micropropagation. In: Bajaj YPS. Biotechnology in agriculture and forestry, vol. 17. High-tech and micropropagation I. Berlin, Heidelberg: Springer-Verlag, 313–343.
Kozai T, Zobayed SMA. 2000. Acclimatization. In: Spier R, ed. Encyclopaedia of cell technology, section A. New York, NY: John Wiley & Sons, 1–12.
Lichtenthaler HK, Wellburn AR. 1983. Determinations of total carotenoids and chlorophylls a & b of leaf extracts in different solvents. Biochemical Society Transactions 11: 591–592.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15: 473–497.[CrossRef]
Willmer CM. 1983. Stomatal responses to environmental factors. In: Willmar CM. Stomata. New York: Longman Inc., 64–90.
Zobayed SMA, Afreen F, Kozai T. 2000. Quality biomass production via photoautotrophic micropropagation. Acta Horticulturae 530: 377–386.
Zobayed SMA, Afreen F, Kozai T. 2001a. Physiology of Eucalyptus plantlets grown photoautotrophically in a scaled-up vessel. In vitro cellular and developmental biology. Plant 37: 807–813.
Zobayed SMA, Armstrong J, Armstrong W. 2001b. Leaf anatomy of in vitro tobacco and cauliflower plantlets as affected by different types of ventilation. Plant Science 161: 537–548.[CrossRef]
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