Explant Density Research Articles

Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50

Conventional in vitro propagation using semisolid Murashige and Skoog (MS) culture systems is costly, labor-intensive, and requires substantial space for large-scale plant production. This study investigated the application of a temporary immersion bioreactor (TIB) system for the micropropagation of the banana cultivar Kluai Numwa Pakchong 50, as a promising platform for economical commercial production. The cultivation parameters affecting plantlet multiplication, including plant growth regulator (PGR) use, explant density, and immersion frequency, were examined. Additionally, the ex vitro acclimatization of well-developed in vitro plantlets was also evaluated. Using liquid MS medium supplemented with 7.5 mg/L 6-benzylaminopurine (BAP) in the TIB system yielded significantly better results than the conventional semisolid MS control system, producing more shoots (5.60 shoots/explant) and leaves (2.80 leaves/explant) with longer shoot length (2.19 cm). Optimal conditions in the TIB system included an inoculum density of five explants per culture vessel and an immersion frequency of once every 6 or 8 h for 2 min. For root induction, 0.5 mg/L indole-3-butyric acid (IBA) proved more effective than 1-naphthaleneacetic acid (NAA). After 30 days of ex vitro acclimatization, plantlets regenerated from the TIB system demonstrated high survival rates, vegetative growth performance, and root formation efficiency comparable to those from the semisolid culture system. These findings establish the TIB system as a promising platform for the mass propagation of the Kluai Numwa Pakchong 50 banana. The protocol developed in this study could potentially be adapted for large-scale production of other banana varieties.

Micropropagation of iraca palm (Carludovica palmata Ruiz y Pav) using a temporary immersion system

Carludovica palmata is a Neotropical plant with a promising potential for the agroindustry. It is an important source of fibers used to manufacture handmade goods. Colombia is one of the leading countries in the production of this species; however, it lacks efficient techniques for its propagation. We developed a protocol for massive micropropagation of C. palmata using a temporary immersion bioreactor (TIB) system. Immersion frequency, immersion time, culture medium volume, and explant density were evaluated using a split-split-plot design. The variables evaluated were the number and length of shoots and roots, the number of leaves, and the dry weight of shoots. The performance of three micropropagation systems, TIB, semi-solid medium, and liquid medium, was evaluated using a generalized randomized block design. Murashige and Skoog (MS) culture medium with 1.0 mg L−1 of 6-benzylaminopurine, 0.5 mg L−1 of 1-naphthaleneacetic acid, and 20 g L−1 of sucrose added to it was used. The results obtained show a frequency of 12 h with an immersion time of 1 min improved the length of shoots, roots, and number of leaves, and dry weight. A volume of 20 mL/explant was found to be an adequate condition to increase the number and length of shoots, the number of leaves, and the dry weight. According to the optimization module, the factor levels that will maximize the evaluated variables are a 1-minute immersion of the tissues in the culture medium with a frequency of 12 hours each time, with 175 mL of culture medium per bioreactor and density of 10 explants. In conclusion, TIB was shown to be efficient for massive micropropagation of C. palmata compared with conventional methods.

Improving bacoside yield of Bacopa monnieri (L.) Wettst. in temporary immersion system by increasing immersion time and lowering the intervals

Bacopa monnieri (L.) Wettst. or Brahmi has been used in Ayurvedic medicine and currently use as an alternative medicinal herb for several therapeutic healing. Therefore, in vitro mass propagation of B. monnieri for high quality plant production using temporary immersion system (TIS) was necessarily examined. Explants of B. monnieri as regenerated shoots from leaf segments were used to determine the effects of culture system, immersion time and inoculum explant density on growth and bacoside production of B. monnieri. Statistically significant differences in growth and biomass accumulation were observed in the TIS compared with continuous immersion and semi-solid systems. Production of bacoside A, bacopaside II, bacopasaponin C, and total bacoside in B. monnieri obtained from microcontainer cultivation was significantly higher than TIS, whereas total yield of bacosides obtained from different culture systems showed no significant differences. Three immersions per day for 10 min duration promoted growth and biomass accumulation of B. monnieri, while cultivation with 20 explants per container offered optimal inoculum density for large-scale production. Various concentrations and yields of bacoside were obtained from different immersion interval conditions. Reducing immersion duration improved bacoside production. Five explants per container provided the highest bacoside content but yield per container was the lowest.

Evaluation of Flavonoid Content from Shoot Culture of Gynura procumbens in Balloon-type Bubble Bioreactors

Gynura procumbens has been widely used to treat several health disorders, such as hyperglycemia, hypertension, diabetes, and allergies. This study aimed to determine the flavonoid content of G. procumbens cultured in a balloon-type bubble bioreactor using various sucrose concentrations (10, 30, and 50 g/L) and explant densities (5, 10, and 15 explants). The plant parts used were stem nodes of G. procumbens, grown in a balloon-type bubble bioreactor using Murashige and Skoog (MS) liquid media with IAA 2 mg/L and BAP 4 mg/L. The total flavonoid content was measured using a UV-Vis spectrophotometer (λ= 510 nm), and the flavonoid profile was tested using thin-layer chromatography (TLC). The administration of 10 g/L sucrose produced the highest total flavonoid production. In the treatment of explant density, the most increasing total flavonoid production was obtained in the treatment of 5 explants. The TLC results showed that each treatment forms spots like standard. This study proved that sucrose and explant densities culture on balloon-type bubble bioreactor method had optimized the production of flavonoids of plant shoots of G. procumbens culture.

Effect of Explants Length and Density on in Vitro Rooting of Pineapple Ananas Comosus L Merr

Pineapple explants of different length (5, 10, 15, and 20 mm long) were cultured at different densities (one, two, three, four, and five explants per culture) on full strength, agar solidified MS medium supplemented with sucrose at 30 g/l and IBA at 2.0 mg/l for 60 days. The rooting ability (rooting %, root number, and length) of the explants were not affected by the explants' length and explants' densities per culture. Irrespective of length and densities, 82 % of the explants rooted and produced 4.1 roots each 17.7 mm long. Moreover, overall explants lengths, plantlet height was not affected by explants density and were about 50.5 mm tall. On contrary, overall density, explant of different lengths developed into plantlets of significantly different height. Plantlets' height increased from 39.2 to 49.6; 55.9 and 57.4 mm as the explants length increased from 5 to 10; 15 and 20 mm respectively. Keywords: Pineapple; Ananas comosus; Explants length; Explants density

Simple Protocol for the Micropropagation of Teak (<i>Tectona grandis</i> Linn.) in Semi-Solid and Liquid Media in RITA<sup>® </sup> Bioreactors and <i>ex Vitro</i> Rooting

In Latin America the forestry of exotic species such as teak has been increasing in recent decades, due to their advantages in wood quality, rapid growth; and the relative ease of producing clones and their multiplication with respect to native species. Therefore, there is great interest in developing larger-scale propagation strategies that reduce costs and intensive manual labor. Culture in liquid media with temporary immersion and the semi-automation of the system has raised expectations for large-scale micropropagation. We report a protocol for teak, which reuses the primary explants in several culture cycles in semi-solid medium to produce nodal explants for the multiplication phase in temporary immersion bioreactors (RITA® ). The control of factors such as cytokinin concentration, explants density, immersion frequencies and culture duration was analyzed. The number of shoots increased with 0.5 mg·l-1 of BA (6-Benzyladenine), alone or in combination with 0.5 mg·l-1 of Kinetin, with 2 daily immersions of 1 minute each; however, these shoots showed a high degree of hyperhydricity. When 0.05 mg·l-1 of BA was used with 1 immersion of 1 minute every 2 days, the hyperhydricity decreased. Although the number of shoots was lower, they showed good length to be used during multiplication and rooting ex vitro. Our results suggest that teak micropropagation can be simplified in two phases in vitro, the establishment and multiplication; followed by rooting ex vitro and acclimatization. This would imply a reduction in production costs, since most of the multiplication would take place in RITA® containers.

Effect of Incubation Periods, Medium Volumes and Explants Density on in Vitro Shoot Formation and Growth and Cost of Multiplication of Moris Pineapple (Ananas comosus L.)

This study was conducted to investigate the effect of combinations of four incubation periods (30, 45, 60 and 75 days), three explants density (one, two and three) and four volumes of medium (3, 6, 9 and 12 ml) in the in vitro shoot formation per explant, total shoots and total cost per liter of medium and cost per shoot of Moris pineapple. The highest shoot per explant was obtained in combination of three explants in 12 ml of medium incubated for 60 days, three explants in 9 and in 12 ml of medium and one explant in 3 and in 9 ml of medium incubated for 75 days but at different total shoots (2750, 3667, 2750, 3667 and 1222 shoots), total cost ( USA $25.85, 36.09, 28.39, 95.32 and 36.06) per liter of medium. Combinations in which three explants were used per culture resulted in lowest cost per shoot (1.0 cent) while combination in which one explant was used per culture, the cost per shoot was two to three times higher (2.6 and 2.9 cents). Combination of three explants, 3 ml of medium and 75 days of incubation, on the other hand, resulted in formation of only 6 shoots per explant (almost half of the highest obtained rate) but in highest total shoot per liter of medium (6000 shoots ) at highest total cost (USA $95.32) and intermediate cost per shoot (1.6 cents). Combination of three explants, 6 ml of medium and 75 days resulted in formation of 10 shoots per explant, production of 5000 shoots per liter of medium at lowest cost per shoot (1.0 cent) and intermediate total cost per liter of medium (USA $ 50.68). Using of one explant in 12 ml for 30 days resulted in formation of 5 shoots per explant, highest cost per shoot (5.0 cents) and lowest total shoots (417 shoots) and lowest total cost (USA $ 20.73) per liter of medium.

Effect of explant length and density on in vitro shoot formation and growth of Pineapple (Ananas comosus L. Merr.) cv Moris

The effect of explants length (5, 10, 15, 20, 25 mm long shoots) and density (one, two, three, four, five shoots per culture) on the in vitro shoot formation of Moris pineapple were assessed using average shoot formation per explant and total shoots and the frequency of shoots of different shoot length (£ 5; 6-10; 11-15; 16-20; 21-25 and ³ 26 mm long) per one liter of medium. Of all combinations of explants length and density, using of 25 mm long shoots at a density of one per culture resulted in highest shoot formation (8.3 shoots), and at density of three and four resulted in longest shoots (21.7 mm). The highest total shoots per one liter of medium (2800 shoots) obtained when 15 mm long explants were used at a density of four explants per culture. Using of 10 mm long shoots at a density of two per culture resulted in lowest shoot formation (2.7 shoots) and shortest shoots (5 mm long) per explant and lowest total shoots (233 shoots) per liter of medium. Overall explants density, the percentage of shoots of different length £ 5; 6-10; 11-15; 16-20; 21-25 and ³ 26 mm-long per liter of medium were 22.4; 21.9; 22.3; 16.9; 6.6 and 9.3% respectively. To obtain highest shoot formation per explant or ighest total shoots per liter of medium, the according to the length of available shoots.

IN VITRO MICROTUBERIZATION OF POTATO: EFFECT OF EXPLANT DENSITY, SOURCE, AND GENOTYPE

This study was conducted to examine explant density and source on production of in vitro potato microtubers, as well as survey of different newly-introduced potato cultivars for their microtuberization capacity at the Plant Tissue Culture Facility of the Department of Horticulture, Suez Canal University between 2014 until 2016. Results indicated that as culture density from single node explants increased, microtuber number and yield/petri dish were also increased. However, at the lowest density (10 explants/petri dish), the % tuberization and the average microtuber weight increased significantly over the higher densities (20, 25 and 30 explants/petri dish). Potato microtuber production from plantlets grown in vitroon microtuberization induction medium (liquid over solid media) was also studied using 5, 10 and 15 plantlets per jar.Results showed that microtuber number and yield/jar increased gradually with increasing plantlet density. However, on per plantlet basis, microtuber number increased at the lowest density. Microtuber yield/jar increased significantly at the highest density (1099 mg/jar) compared to 563.6 mg/jar at the lowest density. Average microtuber weight was also affected by culture density and genotype. At the lowest density, microtubers were significantly heavier than at the high density, and the cv. 'Safrane' recorded the highest weight per microtuber. Regarding the effect of explant source on microtuber production, results indicated that the use of single node explants derived from the top of the plantlet produced more microtubers than those taken from the basal part, or 3 node segments. Explant source also affected microtuber yield/dish. Single nodes from the top produced the highest yield compared to 3-nodes segment from the top (205 vs. 104.8 mg). The highest mean microtuber fresh weight was obtained from the culture of 3-node layered segment from the plantlet base, while single nodes from the top recorded lower microtuber fresh weight. The highest microtuber fresh weight was recorded in cv 'Universa' (114.3 mg) using single nodes from the plantlet base. Differences among the tested potato cultivars in microtuber production were detected.

Optimization of explants density for tissue culture propagation of banana cv. ‘GRANDE NAINE’

Cost of production is always stressed as the main obstacle for tissue culture. Nutrient media is one of the most costly input which accounts for 30-35 per cent of total cost of tissue culture propagation. Production cost, therefore, can be reduced by efficient utilization of culture media. To find out optimum quantity of media required for shoot proliferation, 1-5 multiple bud explants were incubated in each culture bottle. Explants density of 4/culture bottles was found best as it recorded higher total shoot production/l (291.94), shoot length (3.22 cm), number of leaves/shoot (2.81) and lower cost per shoot (Rs. 1.175). In vitro rooting was carried out with densities of 6, 8, 10 and 12 microshoots/culture bottle. The maximum response with regard to rooting had not yet been reached as there were no significant differences among the treatments. Incubation of 4 multiple bud explants and 12 microshoots per culture bottles is optimum for scaling-up of tissue culture production of banana cv. 'GRANDE NAINE'.

Effect of Explants Length and Segmentation and Separated and Cluster of Shoots on the in vitro Shoot Formation and Growth of Pineapple (Ananas comosus L. Merr.)

Two experiments were conducted. In one, multiple shoots complex from Smooth cayenne pineapple stock cultures were cut into cluster of two, three, four shoots and were also separated into individual shoots of different length 5, 10, 15, 20 and 25 mm long and cultured on agar solidified MS medium enriched with benzylaminopurine (BAP) at 2.23 mg/l. In the other one, 25 mm long shoots were cut longitudinally into halves and quarters and cultured at density of one explant and 15 mm long shoots were cultured intact at density of one, two, three and four explants per culture. Longer (25 mm) intact and halved explants produced the highest (10 shoots) while 5 and 10 mm long explants resulted in lowest (6 shoots) and 15 and 20 mm long explants resulted in an intermediate (8 shoots) shoot formation. Shoot formation whether the explants were cultured as separated intact or as cluster of shoots decreased from 10 to 4 shoots per explant as the density increased from one to four. Shoot length was not affected by using of separated intact explants of different length. However, increasing the explants density increased the shoots length particularly when cluster rather than separated shoot were used as explants.

MİKROÇOĞALTIMDA ÇEVRESEL KONTROL FAKTÖRLERİ

Micropropagation is a plant tissue culture technique that can yield a large number of genetically identical plantlets. This technique has been commercially used in horticulture, field crops, forestry and landscape architecture. Controlling in vitro environment is important for producing high quality plantlets with efficient use of energy. In this article, we evaluate environmental control factors currently used in micropropagation such as aerial environmental factors including light, carbon dioxide concentration, temperature, relative air humidity and oxygen, the effects of culture medium physical and chemical environmental factors including sugar concentration, concentrations of ion components and pH of the culture medium together with the effects of biological factors such as explant size, explant density and pathogens on the growth and photosynthesis of plantlets in vitro are discussed.

Efficient microtuber production of potato in modified nutrient spray bioreactor system

A laboratory scale bioreactor system has been developed using nutrient spray technology for in vitro mass production of potato microtubers. Its effectiveness on the production of microtubers was investigated and compared with conventional liquid and semi-solid culture systems through bioreactor. Optimal culture conditions such as spray intervals, varying concentrations of 6-benzylaminopurine (BAP) and explants density were determined for the NSB. In order to determine optimal spray intervals, liquid medium was sprayed inside the NSB at different intervals (½, 1–4h) of which the 1h interval resulted in the highest number of shoots (3.47) and length (8.99cm). Number of microtubers produced (5.13) was highest with 1h intervals and fresh weight of microtubers (0.90g) was highest for ½h interval. Different concentrations of BAP (0.5, 1.0 and 1.5mg/L) were used to evaluate its effect on microtuberization. It was observed that number and diameter of microtubers were increased (5.31 and 0.96cm) when 0.5mg/L BAP was supplemented in MS medium. We found fresh weight of microtubers (0.97g) was increased when 1.0mg/L BAP were added to the medium. In order to determine suitable explants density, single nodes grouped into five categories e.g 30, 45, 60, 75, and 90 and placed in the NSB system. A density of 60 explants resulted in increases in shoot length (17.5cm) number of internodes (12.5) and with highest amount of chlorophyll (40.2mg/g) as well as with highest number and fresh weight of microtubers (4.43 and 0.89g, respectively). Out of the three culture systems, the NSB performed best where 1.5–2.0 fold increases in shoot growth and microtuberization without hyperhydration. The NSB also produced the highest number (4.67), fresh weight (0.86g) and diameter (0.78cm) of microtubers. From this study we may conclude that the NSB system has good potential for commercial mass production of potato micro-tuber.

Produção de mudas de carobinha (Jacaranda decurrens Cham.) em sistema de imersão temporária com biorreatores do tipo R.I.T.A.

O objetivo deste trabalho foi averiguar a melhor densidade de explantes e o melhor tipo de sistema de cultivo visando desenvolver um protocolo de micropropagação de baixo custo para a Carobinha. Foram realizados experimentos de multiplicação in vitro com quatro tipos de frascos: R.I.T.A. (50 explantes/frasco), erlenmayer, (50 explantes/frasco), potes tipo maionese (6 explantes/frasco) e cubetas (1 explante/frasco). O co-cultivo de explantes, tanto em meio sólido quanto em meio líquido (R.I.T.A.), promoveu maiores taxas de explantes com brotação e de sobrevivência. O sistema de imersão temporária proporcionou melhores índices de desenvolvimento, brotação, sobrevivência e altura dos explantes. Concluímos que biorreatores podem ser utilizados eficientemente para a micropropagação de carobinha.

Optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte Kappaphycus

To improve the production of Kappaphycus plantlets in tissue culture, optimum media concentrations of an Ascophyllum nodosum extract (Acadian Marine Plant Extract Powder, AMPEP), plant growth regulators (PGR), pH–temperature combinations, and explant density were determined. Kappaphycus alvarezii var. tambalang purple (PUR), kapilaran brown (KAP), vanguard brown (VAN), adik-adik (AA), tungawan green (TGR), and K. striatum var. sacol green (GS) were used as explants. Based on the shortest period for shoot emergence and the economical use of AMPEP, the optimum enriched media was 3.0 mg L−1 AMPEP and 0.1 mg L−1 AMPEP + PGR 1 mg L−1 each phenylacetic acid (PAA) and zeatin for PUR, 1.0 mg L−1 AMPEP + PGR for KAP and GS, 0.1 mg L−1 AMPEP + PGR for VAN, and 3.0 mg L−1 AMPEP and 0.001 mg L−1 AMPEP + PGR for AA and TGR. Results showed that the addition of PGR to low concentrations of AMPEP hastened shoot formation. pH–temperature combinations for the most rapid shoot formation were determined for the brown (KAP) and purple (PUR) color morphotypes of K. alvarezii var. tambalang and the green morphotype of K. striatum var. sacol (GS) cultured in 1.0 mg L−1 AMPEP + PGR. The brown morphotype produced the most number of shoots at pH 7.7 at 20°C after as little as 20 days. Purple K. alvarezii showed an increased shoot formation at pH 6.7 at 25°C and the green K. striatum morphotype at pH 8.7 at 25°C. The optimum number of explants added to the culture media was also determined for tungawan green (TGR), brown (KAP), and tambalang purple (PUR) varieties of K. alvarezii in 1.0 mg L−1 AMPEP + PGR. The number of explants and the volume of the culture media combination were also tested. The highest average number of shoots formed occurred in two explants:1 mL culture media (2:1) for KAP and PUR (35.00% and 16.67%, respectively) and 1 explant: 2 mL culture media for the TGR (100.00%) with a range of 0.5–3.0 mm shoot length after 40 days in culture. The earliest shoot formation was observed after 21 days for the brown and 9 days for both the green and purple color morphotypes of Kappaphycus, in all densities investigated. This indicated that within the range tested, the density of explants did not have a significant effect on the rate of shoot formation but did influence the average number generated from the culture. The rate of production of new and improved Kappaphycus explants for a commercial nursery stock was improved through the use of AMPEP with optimized culture media pH, temperature, and density conditions.

Effect of Explants Density on the in vitro Proliferation and Growth of Separated and Cluster Shoots of Smooth Cayenne Pineapple (Ananas comosus L. Merr.)

Multiplication rate was always used as the sole parameter for assessment of the optimal treatment for in vitro shoot formation. However, beside its effect on the shoot formation, different treatments had different impact on the production cost. Separated shoots and shoot cluster of different sizes (1, 2, 3 and 4 shoots per cluster) of Smooth cayenne pineapple were cultured at density of 1, 2, 3 and 4 separated shoots and 1 cluster on agar solidified full strength MS medium enriched with sucrose at 30 g L-1 and BAP at 2.25 mg L-1 for 2 months to investigate the effect of explants density and explant types on shoot formation per explant, total per liter and cost per shoot. Density of one separated shoot per culture resulted in the highest shoot formations per shoot (11 shoots) but lowest total shoot per liter (550 shoots) and highest cost per shoot (13 cents), while density of 4 shoots per culture resulted in lowest rate (4 shoots) but higher total per liter (1400 shoots) and lowest cost per shoot (10 cents). Cluster of two shoots had higher rate (7 shoots) and lower cost (16 cents) than cluster of 4 shoots (4 shoots and 20 cents) but both resulted in equal total shoot per liter (750 shoots). Separated shoot had higher shoot formation capacity, higher total per liter and lower cost per shoots than cluster of shoots and using of total per liter instead of rate per explant was the best parameter for selection of treatment of lower cost and higher shoots production. © 2009 Asian Network for Scientific Information.

Comparison of Agar and an Agitated, Thin-film, Liquid System for Micropropagation of Ornamental Elephant Ears

Micropropagation of black-stemmed elephant ear (C. esculenta (L.) Schott `Fontanesii')' and upright elephant ear (A. macrorrhizos G. Don) were compared in semi-solid agar media and agitated, liquid thin-film bioreactor vessels at four explant densities (33, 100, 165, and 330 explants/L of media) using two growth regulator combinations: 1) 1 μm benzylaminopurine (BA)—growth medium, and 2) 3 μm BA plus 3 μm ancymidol—multiplication medium. The thin-film liquid system outperformed agar culture for most measured responses. Some exceptions were relative dry weights at higher explant densities and multiplication rate of Colocasia. When the thin-film liquid system was compared to agar culture, Alocasia explants produced their greatest biomass and had the least residual sugar at the highest explant density. Alocasia explants multiplied most rapidly and had the greatest relative dry weight on liquid media at the low explant densities. Alocasia plants were larger in growth medium than multiplication medium and larger in liquid medium than agar medium. When compared to agar, Colocasia in the thin-film liquid system produced the greatest biomass at the highest explant density in growth medium, had the greatest relative dry weight at the lowest explant density, and used the most sugar at the highest explant density. Alocasia and Colocasia would likely produce greater fresh and dry weight at the highest explant density if additional sugar were supplied during thin-film culture. Greater growth in thin-film culture of Alocasia and Colocasia is due in part, to greater availability of sugar in liquid compared to agar medium.

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated, Thin-Film Liquid System for Micropropagation

Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.

SOMATIC EMBRYOGENESIS AND BUD FORMATION ON CULTURED FAGOPYRUM ESCULENTUM HYPOCOTYLS

An indirect somatic embryogenesis via the development of proembryogenic cell complexes (PECC) was observed in the in vitro cultured hypocotyl explants of 4–5-day-old buckwheat (Fagopyrum esculentum Moench.) seedlings. PECC development was shown to depend on culturing conditions, including 2,4-D concentration and the period of explant exposure to 2,4-D, sucrose concentration, and explant density. The culturing protocol was designed to ensure the development of buckwheat somatic embryos in two-month period. The cytogenetic analysis demonstrated that this protocol did not affect the chromosome numbers in the regenerated plants.

Environmental Effects on the Growth of Plantlets in Micropropagation.

Research on environmental effects on the growth of plantlets in micropropagation is reviewed to give an overview on the subject conducted in the past decade, mainly to environmental biologists and engineers. Effects of aerial environmental factors including light, carbon dioxide concentration, temperature, relative humidity, oxygen and ethylene on the growth and photosynthesis of plantlets in vitro are discussed. Then, effects of culture medium environmental factors including sugar concentration, concentrations of ion components, osmotic potential and pH of the culture medium on the growth of plantlets in vitro are discussed. Lastly, effects of biological factors such as explant size and explant density on the growth of plantlets in vitro are discussed. Effects of aerial, culture medium and biological factors on the photoautotrophic (totally photosynthesis dependent) growth of plantlets are emphasized.