Temporary Immersion System Research Articles

Overexpression of the chestnut CsTL1 gene coding for a thaumatin-like protein in somatic embryos of Quercus robur

Somatic embryos of Quercus robur derived from mature and juvenile trees were transformed with the CsTL1 gene coding for an antifungal protein. Clumps of somatic embryos were inoculated with Agrobacterium tumefaciens strain EHA105 harbouring a binary vector (pKWG2D-TAU) that included the green fluorescence protein (GFP) reporter gene. After 24 weeks of culture on selective medium, significantly higher transformation frequencies were obtained in a temporary immersion system (TIS) than in semi-solid medium (ranging from 1.4 to 9.6 % for the four genotypes). The first transformants appeared after 10 weeks and GFP activity was evaluated in all kan-resistant embryogenic lines. The transgenic nature of lines was confirmed by PCR and Southern blotting. CsTL1 transcripts were detected in all transgenic lines (TL) analyzed by quantitative real-time PCR, and levels of expression were significantly higher than in the wild type (wt), reaching up to 24 times higher in some genotypes. Sixty-two stable TL were obtained (38 derived from centenarian trees). Plant conversion rates of transformed somatic embryos were similar to those of the wt and were increased by use of the TIS. GFP fluorescence was detected in roots and shoots of transgenic plants, which confirmed stable transformation of regenerants.

Temporary immersion systems for Amaryllidaceae alkaloids biosynthesis by Pancratium maritimum L. shoot culture

The alkaloid patterns of sea daffodil (Pancratium maritimum L.) shoot culture, cultivated in a temporary immersion cultivation system were investigated. The shoots accumulated maximal amounts of biomass (0.8 g dry biomass/L and Growth Index = 1.6) at immersion frequency with 15 min flooding and 12 h stand-by periods. At this regime P. maritimum shoots achieved the highest degree of utilization of carbon source. Twenty-two alkaloids, belonging to narciclasine, galanthamine, haemanthamine, lycorine, montanine, tazettine, homolycorine and tyramine types were identified in intracellular and extracellular alkaloid extracts. The immersion frequency affected strongly the capacity of alkaloid biosynthesis in P. maritimum shoots and at the optimum conditions of cultivation, the total intracellular alkaloid content reached up to 3,469 μg/g dry biomass. The main biosynthesized alkaloids were haemanthamine (900.1 μg/g) and lycorine (799.9 μg/g). The obtained results proved that temporary immersion technology, as a cultivation approach, and P. maritimum shoots, as a biological system, are prospective for producing wide range bioactive alkaloids.

Global DNA methylation profiles of somatic embryos of peach palm (Bactris gasipaes Kunth) are influenced by cryoprotectants and droplet-vitrification cryopreservation

Regrowth capacity and genetic stability of plants recovered following cryopreservation are associated with changes in DNA epigenetics, particularly in DNA methylation levels. In this study, global DNA methylation profiles associated with frequency of regrowth of peach palm (Bactris gasipaes) somatic embryos following cryopreservation using droplet-vitrification were investigated. Somatic embryo clusters (SEC) subjected to plant vitrification solution 3 (PVS3) for different durations (0, 60, 120, 180, and 240 min) were evaluated for regrowth capacity. The highest frequency of regrowth (52.4 %) was obtained when SEC were incubated in PVS3 for 120 min prior to droplet-vitrification cryopreservation. Global DNA methylation profiles were influenced by both cryoprotectants and droplet-vitrification cryopreservation. Incubation of SEC in PVS3 for limited durations not only reduced frequency of regrowth, but also increased DNA methylations levels when compared with proliferating SEC grown in a temporary immersion system. Although SEC subjected to cryopreservation exhibited the highest DNA methylation variation, 120 min SEC incubation in a PVS3 solution resulted in the recovery of initial global methylation profiles after 24 weeks of regrowth.

Temporary immersion systems (RITA®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production

Somatic embryogenesis in cork oak (Quercus suber L.) is an efficient tool that allows the production of large number of embryos from selected quality and productive trees. Temporary immersion systems (TIS) are an alternative to semi-solid or liquid culture that combine the advantages of liquid culture and avoid the associated problems. Parameters that affect the TIS multiplication efficiency of Q. suber L. embryogenic cultures were evaluated. Immersion frequencies of 1 min every 6 or 4 h increased the fresh weight 3.7 or 7.5-fold compared with an immersion frequency of 1 min every 12 h or cultures on semi-solid medium, respectively. The cellular fate of embryogenic cultures was also affected by the immersion frequency, 1 min every 6 h was the best for mass propagation of proliferative developmental stages (embryogenic calli and embryo clusters) while 1 min every 4 h promoted the formation of single, fully developed cotyledonary embryos. An initial amount of 1.5 g fresh weight of proliferative tissues produced the best results in RITA® containers while 0.5 g of embryogenic callus was the best for semi-solid cultures.

High-efficiency Agrobacterium-mediated transformation in Quercus robur: selection by use of a temporary immersion system and assessment by quantitative PCR

An efficient protocol for genetic transformation of somatic embryos of Quercus robur by selection in a temporary immersion system is reported. The transformation frequency was 5 times higher than achieved by conventional culture on semi-solid medium, ranging between 6 and 26 % for the four genotypes evaluated. Clumps of globular or torpedo somatic embryos were precultured for 7–10 days, inoculated with Agrobacterium tumefaciens strain EHA105:p35SGUSINT and cocultivated for 4 days before being cultured for 4 weeks on semi-solid selection medium supplemented with 25 mg L−1 kanamycin. Explants were transferred to RITA® bioreactors and subjected to a two-step selection protocol involving immersion in liquid medium supplemented with 25 mg L−1 kanamycin, for 18 weeks, and then with 75 mg L−1 kanamycin. Putatively transformed explants appeared after serial transfer to selection medium over 12–16 weeks. The presence of neomycin phosphotransferase II and β-glucuronidase genes in the plant genome was confirmed by histochemical and molecular analysis, and the copy number was determined by Southern blotting and real-time quantitative polymerase chain reaction. Transformed somatic embryos were germinated and transferred to soil for acclimatization, approximately 8 months after inoculation of the original tissue with bacteria. As the limiting factor for recovery of plants from oak embryogenic lines is the low embryo conversion rate, axillary shoot lines were established from transformed germinated embryos. Transformed embryos and shoots were cultured in medium with or without kanamycin and the responses to several morphogenetic processes (recovery after cryopreservation, germination, shoot proliferation, and rooting) were evaluated.

Elicitation of galanthamine production by leucojum aestivum shoots grown in temporary immersion system

The influence of different elicitors (copper sulfate, silver nitrate, salicylic acid and methyl jasmonate), on both the growth and alkaloid production of Leucojum aestivum shoots grown in a temporary immersion system was studied. Seven Amaryllidaceae alkaloids and three protoalkaloids were quantitatively determined by GC-MS analysis in leaves and bulblets, separately. Methyl jasmonate was found to significantly improve the production of galanthamine (GAL) in both leaves and bulblets. The content of GAL released to the liquid nutrient medium was also measured. The release of GAL into the liquid medium took place mainly in the first 2 weeks determined by harvesting the liquid nutrient medium after 2 weeks and measuring the GAL content (1st subculturing step).

Nodule cluster cultures and temporary immersion bioreactors as a high performance micropropagation strategy in pineapple (Ananas comosus var. comosus)

Biotechnology tools based on tissue culture techniques allow mass clonal propagation of plants with desired genetic and sanitary features. In the present work a pineapple micropropagation protocol was established based on nodule cluster cultures (NC) associated with different culture systems. Leaf segments from in vitro-grown plantlets cultivated in culture medium supplemented with 2μM NAA and 8μM 2-iP or BAP resulted in the highest rates of NC induction. NC fresh weight increased ratio was more efficient in a culture medium supplemented with 2μM NAA and 2μM BAP in twin-flasks temporary immersion system (TIS-TF). This culture medium was also efficient for the microshoots development in RITA® temporary immersion system (TIS-RITA®). Microshoots elongation in permanent immersion system (PIS) was more efficient in a culture medium supplemented with 10μM GA3. Shoots longer than 2cm showed 92% survival rate when acclimatized. Free polyamine endogenous levels was measured and showed higher contents for the TIS-TF, followed by TIS-RITA®, and PIS, respectively. Putrescine levels also followed this pattern, being absent in PIS, which, on the other hand, showed higher levels of spermine and spermidine. The results indicate the establishment of efficient protocol for pineapple micropropagation, with a high multiplication and regenerative rates and great potential for agriculture application. The biochemical analysis suggests that besides culture medium composition, flask design and culture system also drastically affects the plant's metabolism toward multiplication and plant regeneration.

A comparative study between solid and liquid cultures relative to callus growth<br>and somatic embryo formation in date palm (Phoenix dactylifera L.) cv. Zaghlool

Tissue culture techniques enable mass propagation of elite cultivars of date palm (Phoenix dactylifera L.). The main limitations for date palm in vitro multiplication are the low rates achieved using solidified medium and the long period needed to produce acclimatized plantlets. This research focuses on the comparison between different culture types and plant growth regulator (PGR) combinations on callus growth and somatic embryo formation of cv. Zaghlool. For callus growth, 200 mg friable embryogenic callus dispensed in Rasotherm and Phytacon flasks containing 200 ml liquid (100 rotations per minute) and in temporary immersion system, RITA ® bioreactor (5 min immersion every 12 h), were compared with cultures grown on 200 ml solidified medium. For somatic embryo formation, 500 mg of friable embryogenic callus grown in Erlenmeyer flasks filled with 50 ml liquid or solid MS medium. The medium was supplemented with 0.1 mg l -1 Naphthaleneacetic acid (NAA), 1.5 g l -1 activated charcoal (AC) with or without 0.05 mg l -1 6-Benzyl amino purine (BAP) compared to PGR-free medium. Results proved that cell suspension cultures produced the highest callus fresh mass as compared to the other systems tested, and the callus fresh mass reached 4 g after 16 weeks. The temporary immersion system did not significantly enhance the fresh mass of callus compared to the solidified medium. For somatic embryo induction, the number of somatic embryos increased in cell suspensions 6-16 fold compared to the solidified medium. Using liquid MS medium enriched with 0.1 mg l -1 NAA and 1.5 g l -1 AC gave rise to the highest number of somatic embryos formed from 500 mg initial callus: 160 embryos. The number of somatic embryos was also affected by the callus source. The calli induced from leaflet segments excised from converted somatic embryos resulted in a lower somatic embryo number than those of shoot tip origin with about 60 somatic embryos per 500 mg callus. The formation of somatic embryos using liquid medium required only 6 weeks, thus considerably reducing the previously reported period of 18 weeks which is required for somatic embryo formation using solidified media.

ESTABLISHMENT OF A SHOOT PROLIFERATION PROTOCOL FOR BANANA (ABB GROUP) CV. ‘PISANG AWAK’ VIA TEMPORARY IMMERSION SYSTEM

□ Initial study of evaluating the factors affecting shoot proliferation of ‘Pisang Awak’ using temporary immersion system was carried out using four half shoots in each experimental unit as explants and repeated with four to seven experimental units for each factor. The inoculum size finally used for shoot proliferation was eight half-shoots for each experimental unit (250 mL culture flask) in MS liquid medium supplemented with 5.0 mg L−1 benzyladenine (BA) and 30 g L−1 sucrose, the shoot proliferation medium and an average of 4.9 ± 0.3 shoots was produced from each half-shoot explant after five weeks of culture. The final protocol for shoot proliferation using the temporary immersion system was established by inoculating eight half-shoot explants into the shoot proliferation medium adjusted to pH 5.7 with the inoculated shoots immersed once a day in the medium for 10 minutes. The cultures were incubated under continuous light for enhancement of shoot proliferation without vitrification.

The status of temporary immersion system (TIS) technology for plant micropropagation

The development of temporary immersion systems (TIS) for in vitro culture, in the late 1990s heralded new and semi-automated means of micropropagation of plants for agricultural and conservation purposes. By 2005, various systems had been developed and tested and their benefits were proven with a variety of commercially-important crops such as sugar-cane, coffee, banana, and yam. Progress in the field and the adoption of these systems in subsequent years has been reviewed. Improvements on previously-developed TIS protocols for economically-important species have been reported, and this continues to attest to their benefits when compared with protocols that employ semi-solid or liquid media. Though TIS protocols have recently been developed for other species, this list is limited. On the other hand, there appears to be an increasingly great interest in TIS technology for the production of secondary metabolites and for physiological studies.

Field performance and (epi)genetic profile of plantain (Musa AAB) clone ‘CEMSA ¾’ plants micropropagated by temporary immersion systems

Use of meta-topolin (mT) [6-(3-hydroxybenzylamino) purine] to scale up the plantain clone ‘CEMSA ¾’ in temporary immersion systems (TIS), when compared with conventional micropropagation protocols, has been shown to improve production and quality of microplants. To evaluate the impact of those innovations on plant field performance, in vitro plants of ‘CEMSA ¾’ were subjected to either an additional multiplication cycle using TIS or semi-solid medium (SS) in the presence of 4.44μM mT or N6-benzyladenine (BA). Growth and yield components in the microplants grown in field – plant crop – and in the developed plants of the following field generation – first ratoon – were evaluated. Also (epi)genomic traits were studied by flow cytometry, fluorimetry-based AFLP and HPLC analyses of global genomic DNA methylation. Neither in vitro microprogation systems – TIS and SS – nor cytokinins – mT and BA – had any factorial effect on yield productivity of the plant crop. In the first ratoon, significant differences in yield appeared, with TIS-BA plants being more productive than those generated from TIS-mT. For most of the measured variables, the plant crop had higher values than the first ratoon, but true-to-type and each off-type phenotype of the plant crop was always inherited by their corresponding offspring of the first ratoon. The proportion of true-to-type plants was higher for SS-BA and TIS-mT culture conditions. Irrespective of micropropagation protocol and phenotype, in the plant crop, average genomic DNA content was maintained and global genomic methylation levels were similar. Nevertheless, dwarf individuals showed more AFLP polymorphisms with respect to mother plants than the rest of the micropropagated plants, and a dwarfism-related AFLP marker is proposed. On the basis of the results obtained and previous reports, the innovative TIS-mT protocol was deemed to be the most suitable plantain multiplication technique among those tested.

Production of Galanthamine by Leucojum aestivum Shoots Grown in Different Bioreactor Systems

The production of galanthamine by shoots of Leucojum aestivum grown in different bioreactor systems (shaking and nonshaking batch culture, temporary immersion system, bubble bioreactor, continuous and discontinuous gassing bioreactor) under different culture conditions was studied. The influence of the nutrient medium, weight of inoculum, and size of bioreactor on both growth and galanthamine production was studied. The maximal yield of galanthamine (19.416 mg) was achieved by cultivating the L. aestivum shoots (10 g of fresh inoculum) in a temporary immersion system in a 1-L bioreactor vessel which was used as an airlift culture vessel, gassing 12 times per day (5 min).

Increased cardenolides production by elicitation of Digitalis lanata shoots cultured in temporary immersion systems

Digitalis lanata is an important source of cardenolides such as digoxin and lanatoside C, which have been widely applied in the treatment of cardiac insufficiencies. Elicitation is one of the most effective methods to enhance the biosynthesis of several secondary metabolites in medicinal plants. We studied the effect of elicitation with Chitoplant®, Silioplant® and methyl jasmonate on biomass and cardenolides accumulation in shoots of D. lanata cultivated in temporary immersion systems. Morphological response of the shoots was influenced by elicitors. A reduction in length and number of shoots was evident with all MJ concentrations. Regarding biomass production, Chitoplant® (0.1 g l−1) was found to impact significantly on fresh and dry weight of the shoots. HPLC analysis revealed a higher content of lanatoside C compared to digoxin in all treatments. The highest accumulation of lanatoside C was achieved with Chitoplant® (0.1 g l−1), which resulted in 316 μg g-DW−1 and with Silioplant® (0.01 g l−1; 310 μg g-DW−1), which accounted for a 2.2-fold increase in lanatoside C content compared to non-elicited shoot cultures. Additionally, elicitation of D. lanata shoots in temporary immersion systems resulted in an oxidative stress characterized by hydrogen peroxide and malondialdehyde accumulation. These observations point to a connection between hydrogen peroxide generation, lipid peroxidation and cardenolide accumulation. The optimization of elicitor treatment and culture conditions for cardenolide production as well as the advantages of TIS for this purpose are discussed.

Micropropagation and in vitro conservation of Jacaranda decurrens Cham.

Several pharmacological activities have been attributed to Jacaranda decurrens a medicinal plant species native to Brazilian Cerrado regions. Its root extracts have been used as anti-inflammatory, antiseptic and tonic agent and the infusion of its leaves has been efficacious in healing external ulcers. In this work, it was aimed at the development of protocols for J. decurrens micropropagation and in vitro conservation in germplasm bank. Asepsis of seeds and nodal segments was effective achieving 97 and 84% disinfestation, respectively and multiplication ratio ranged 6.98. Axillary buds used as explants, regardless of their position on the stem, produced vigorous in vitro plantlets. Multiplication rate of explants cultured in temporary immersion system RITA® was superior (9.61) compared to the proportion obtained in semi-solid culture medium. Optimized protocol for the in vitro conservation of J. decurrens in germplasm bank promoted 56.67% survival. Moreover, enhanced root formation (96.7%) was achieved. Key words: Jacaranda decurrens, Bignoniaceae, micropropagation, conservation.

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.

Improved growth and quality of Dioscorea fordii Prain et Burk and Dioscorea alata plantlets using a temporary immersion system

The effects of temporary immersion system (TIS) culture on the growth and quality of Dioscorea fordii Prain et Burk and Dioscorea alata plantlets were investigated. Results indicate that TIS promoted the growth and quality of D. fordii and D. alata plantlets. Proliferation rate, shoot length, fresh weight (FW) and dry weight (DW) of shoots, and total biomass production were significantly (P ≤0.05) higher in the TIS than in gelled and liquid medium, respectively. The TIS also promoted tuberization of D. fordii , and decreased vitrification of D. alata significantly. The healthy plantlets of D. fordii and D. alata obtained in the TIS would probably have positive effects on transplanting in large-scale commercial production.

Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.

Improving secondary embryogenesis in Quercus robur: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.

Diluição celular, características do meio de cultura e biorreatores de imersão temporária na diferenciação e regeneração de células em suspensão de bananeira

Altos custos de produção geralmente limitam o uso comercial da micropropagação. O uso de meios de cultura líquidos é considerado uma solução para a automação e redução de custos. Entretanto, dependendo da cultivar de bananeira, esse processo pode mostrar diferentes níveis de dificuldade, e adaptações nos protocolos são necessárias. Neste estudo, experimentos de diferenciação celular e regeneração de plantas foram desenvolvidos em células em suspensão de banana pela avaliação da densidade inicial de células, meios de cultura e sistemas de imersão temporária. Para tanto, uma sequência de três experimentos foi realizada: o primeiro avaliou os efeitos da densidade celular (0,5; 1 e 2 mL), meios de cultura (M1: 1/2MS, 0,1 g.L-1 de ácido ascórbico, 0,1 g.L-1 de L-prolina, 30 g.L-1 de sacarose e 10 µM de 2iP; M2: MS, 30 g.L-1 de sacarose, 2,2 µM de BAP e 11,4 µM de AIA) e períodos de diferenciação celular (40 e 130 dias); o segundo experimento analisou o efeito do tamanho dos propágulos diferenciados em meio líquido (aprox. 2,5; 5 e 10 mm em diâmetro) na formação de embriões somáticos ou na regeneração de plantas; finalmente, um terceiro experimento avaliou o efeito de sistemas de cultivo com papel-filtro cobrindo o meio de cultura semissólido e sistemas de imersão temporários na diferenciação dos propágulos e na regeneração de plantas. Não foram observadas diferenças significativas entre os meios de diferenciação, porém as melhores diluições de células para a diferenciação foram de 1 e 2 mL/30mL de meio de cultura, enquanto diluições de 0,5 mL/30 mL de meio aumentaram a oxidação celular. A extensão do período de diferenciação de 40 para 130 dias foi importante para produzir maior número de calos/propágulos uniformes e com pelo menos de 10 mm de diâmetro, que puderam ser usados em sistemas de imersão temporária (biorreatores) para a diferenciação de embriões somáticos e regeneração de plantas. Considerando todos os sistemas de regeneração, verificou-se que o uso de meios de regeneração de consistência semissólida com papel-filtro na superfície do meio é o mais responsivo para a diferenciação de embriões somáticos e regeneração de plantas.

Performance of yam microtubers from temporary immersion system in field conditions

The yam clones ´Pacala Duclos´ and ´Belep´ of Dioscorea alata were used to evaluate the performance of microtubers formed in temporary immersion systems (TIS) in field conditions. Previously sprouted microtubers with a fresh weight higher than 3.0 gFW were used while in vitro plants and tuber crowns from conventional propagation methods served as control. In both clones there were no significant differences in qualitative morphological characters between plants from microtubers and in vitro plants for all traits but both differed significantly from plants obtained from tuber crowns. The same trend was observed for number, length, diameter and fresh weight of tubers produced 36 weeks after field planting. The number of tubers formed per plant raised from microtubers doubled that raised from tuber crowns in both clones. Microtubers from temporary immersion systems can be grown on the field and used in original seed production programs. Key words: Microtuber, yam, field, temporary immersion system.