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setacea seeds without aril, were submitted to pre-germination treatments: T1(control); T2 (pre-soaking in Promalin® solution [gibberellin (GA4+7) + N-(phenylmethyl)-1H-purine 6-amine (6-benzyladenine) both at a concentration of 18.8 g.L-1] in bainMarie, 45 °C/20 min.; T3 (osmopriming with KNO3 at a concentration of 2.6g.L-1 H2O, 25 °C/1 day, with aeration); T4 (osmopriming with KNO3 at a concentration of 2.6g.L- 1 H2O, 25 °C/2 days, with aeration); T5 (T3 followed by T2) and T6 (T4 followed by T2). The initial moisture contents, ranging from 6.7% to 8% did not differ from each other (α 0.05). The highest fresh mass increment at 168h and the highest moisture content at the end of the germination test were achieved by T2, 40.9% and 44.3%, respectively. The lowest fresh mass increment at 168h was T1 (13.1%). Osmopriming with KNO3 and aeration contributed to better water absorption in T3 and T4 than in T1, but did not stimulate the germination performance of the seeds. The highest germination percentages and germination speed indexes were obtained from T2 seeds (92% and 8, 4595), T5 (96% and 11.7438) and T6 (94% and 11.6728) as well as the lowest mean germination times T2 (11.73 days), T5 (9.49 days) and T6 (8 .81 days), which did not differ statistically from each other (α 0.05). However, the germination process took less time for T2 seeds, 21 days. The combination of aryl removal and phytohormone solution in a bain-marie at 45 °C/20min. with or without osmopriming with KNO3 stimulated the germination process of P. setacea seeds.

Aims: The objective of this research was to establish a cryopreservation protocol for shoot tips (ST) of in vitro P. glomerata using the droplet-vitrification technique. Study Design: The experimental design was a factorial, with four factors, arranged in a completely randomized design. Three vitrification solutions (PVS2, PVS3, PVS4), three times (20, 40, 60 min) and two temperatures (25 ± 2 °C and 0 °C) of treatment with the solutions, followed by freezing (LN+) or not (LN-) with liquid nitrogen (LN) were tested. All tests were performed using six replicates and the results analysed using Two-way ANOVA and Tukey’s tests and expressed as the mean ± the standard error of the means (SEM) deviation. Place and Duration of Study: Laboratory of Plant Cryobiology, Embrapa Genetic Resources and Biotechnology, over a two-year period. Methodology: ST excised from in vitro plantlets were pre-cultured overnight (19h), treated with a loading solution (LS) and three different vitrification solutions (PVS2, PVS3, PVS4) prior to freezing in LN. Treatment with the vitrification solutions was carried out at 0 or 25°C, for 20, 40 or 60 min. For freezing, drops of the vitrification solutions containing a single ST were dispensed on aluminum foil strips and the strips were submerged in LN (-196°C). For thawing, foil strips were submerged into unloading solution (US) at 40 ± 2°C, for three min. Thawed ST were transferred to regeneration medium and cultured in vitro. Results: Highest regeneration percentages after cryopreservation were 82% for ST treated with PVS3, at 0°C, for 60 min; 32% for ST treated with PVS4 at 25°C for 60 min or 0°C for 40 min and 22% for those treated with PVS2 at 0°C for 60 min. Conclusion: Droplet-vitrification is a suitable technique to ensure survival of P. glomerata ST after cryopreservation. This procedure can be applied to establish germplasm collections of this medicinal species in gene banks.

Amaryllidaceae include plant species that present alkaloids with analgesic, anti-cancer, anti-bacterial, anti-viral, anti-fungal and anti-malarial activities. Due to this pharmacological value, several species of this family have been widely studied and among them is White lilly, Crinum americanum. The objective of this work was to induce callogenesis on leaf explants of C. americanum cultivated in vitro for future production of alkaloids. Leaf explants were grown on a culture medium (solid) Murashige and Skoog (1962) supplemented with different concentrations and combinations of plant growth regulators, auxin 2,4-dichlorophenoxyacetic acid and cytokinin 6-benzylaminopurine and their effect on callogenesis assessed for percentage oxidation and explants responsive to callus induction. Callus formation started 10 days after hormone inoculation, and within 30 days after inoculation the best callogenesis and callus biomass growth were observed in medium containing 2.5 mg L-1 of 2,4-dichlorophenoxyacetic acid and 10 mg L-1 of 6-benzylaminopurine. The lowest percentage of oxidation was observed on explants cultivated on medium containing 5 mg L-1 of 6-benzylaminopurine and 2.5 mg L-1 of 2,4-dichlorophenoxyacetic acid. The calli obtained were compact and embryogenic. This work contributes not only to future studies on in vitro callogenesis of this species, but also to a possible protocol for the production of alkaloids of interest from cell suspension cultures produced in vitro. This is the first report of callus formation in Crinum americanum explants.

Hippeastrum goianum (Ravenna) Meerow, Amaryllidaceae, is an endemic species from the Cerrado, Brazil; there are only few studies about its chemistry or biological activity. This study aimed to investigate the occurrence of lycorine in extracts from in vitro H. goianum plantlets, as well as evaluate a possible inhibition of acetylcholinesterase. The ethanol extract of plantlets produced by in vitro seed germination and micropropagation of bulblets was obtained from seedlings from in vitro germination, while the ethanol extract micropropagtion of bulblets was obtained from a subculture of those seedlings. The presence of lycorine was detected in only in the micropropagation of bulblets. The micropropagation of bulblets was more active than the plantlets produced by in vitro seed germination, with an IC50 of 114.8 ± 0.95 μg/ml and IC50 386.00 ± 0.97 μg/ml, respectively. These results showed that both in vitro germination and micropropagation of H. goianum can lead to the biosynthesis of lycorine. Moreover, the micropropagation led to improved anticholinesterase activity. Keywords: Alkaloids, Biosynthesis, LC–MS, HPLC, Lycorine

This study presents the status of ex situ and in situ conservation for the crop wild relatives of rice, potato, sweet potato, and finger millet in Brazil, and the subsequent germplasm collection expeditions. This research is part of a global initiative entitled ?Adapting Agriculture to Climate Change: Collecting, Protecting, and Preparing Crop Wild Relatives? supported by the Global Crop Diversity Trust. Species of the primary, secondary, and tertiary gene pools with occurrences reported in Brazil were included: Oryza alta Swallen, O. grandiglumis (Döll) Prod., O. latifolia Desv., O. glumaepatula Steud., Eleusine tristachya (Lam.) Lam., E. indica (L.) Gaertn., Solanum commersonii Dunal, S. chacoense Bitter, Ipomoea grandifolia (Dammer) O?Donell, I. ramosissima (Poir.) Choisy, I. tiliacea (Willd.) Choisy, I. triloba L., and I. cynanchifolia Meisn. The status of the ex situ and in situ conservation of each taxon was assessed using the gap analysis methodology, and the results were used to plan 16 germplasm collection expeditions. Seeds of the collected material were evaluated for viability, and the protocols for seed germination and cryopreservation were tested. The final conservation score, resulting from the gap analysis and including the average of the ex situ and in situ scores, resulted in a classification of medium priority of conservation for all the species, with the exception of I. grandifolia (high priority). The total accessions collected (174) almost doubled the total accessions of these crop wild relatives incorporated in Embrapa?s ex situ conservation system prior to 2015. In addition, accessions for practically absent species were collected for the ex situ conservation system, such as Ipomoea species, Eleusine indica, and Solanum chacoense. The methods used for dormancy breaking and low temperature conservation for the Oryza, Eleusine, and Ipomoea species were promising for the incorporation of accessions in the respective gene banks. The results show the importance of efforts to collect and conserve ex situ crop wild relatives in Brazil based on previous gap analysis. The complementarity with the in situ strategy also appears to be very promising in the country. Made available in DSpace on 2021-04-07T12:34:02Z (GMT). No. of bitstreams: 1 agronomy-11-00638-v2.pdf: 2212936 bytes, checksum: 8c5b2d70dc1c6550dd47601dff681696 (MD5) Previous issue date: 2021

Seeds of Pyrostegia venusta (Ker Gawl.) Miers) were desiccated at 25 °C on silica gel for 0 h (T0), 7 h (T1) and 24 h (T2), reaching moisture contents of 6.5%, 4.4% and 3.8%, respectively. Seeds were frozen rapidly in liquid nitrogen (+LN) at -263 °C.min.-1 and after 72 h, they were thawed slowly at room temperature (25 ± 2 °C) at a speed of 5 °C.min.-1. Seed germinability evaluation before (-LN) and after freezing (+LN) was conducted at 25 °C, on paper roll substrate, and germinated at 16 hrs light/8 hrs dark, normal seedling counts for 38 days. The germinative percentages were 88% (T0-LN), 98% (T0+LN), 61% (T1-LN), 95% (T1+LN), 78% (T2-LN) and 89% (T2+LN). Mean days for seedling formation were 23 (T0-LN), 22 (T1-LN and T2-LN) and 30 days for seeds exposed to LN. Fast freezing, slow thawing, and the three tested moisture contents, were suitable for cryopreservation of Pyrostegia venusta seeds. Resumo Sementes de Pyrostegia venusta (Ker Gawl.) Miers) foram dessecadas a 25 °C, sobre sílica gel por 0 h (T0), 7 h (T1) e 24 h (T2), atingindo teores de água de 6,5%, 4,4% e 3,8%, respectivamente. As sementes foram congeladas em nitrogênio líquido (+NL), a -263 ºC.min.-1 e após 72 h foi descongeladas à temperatura ambiente de 25 ± 2 ºC, a 5 °C.min.-1. A avaliação da germinabilidade das sementes antes (-NL) e após congelamento (+NL) foi conduzida a 25 °C, substrato rolo de papel, fotoperíodo 16 h luz/8 h escuro e contagens de plântulas normais por 38 dias. Os percentuais germinativos foram de 88% (T0-NL), 98% (T0+NL), 61% (T1-NL), 95% (T1+NL), 78% (T2- NL) e de 89% (T2+NL). Os tempos médios para a formação de plântulas foram de 23 (T0-NL), 22 (T1-NL e T2-NL) e de 30 dias para as sementes expostas ao NL. O congelamento rápido, o descongelamento lento e os três teores de água testados foram adequados para a criopreservação de sementes de Pyrostegia venusta.

Jatropha seeds are classified as orthodox. However, since it is an oil seed species, adequate storage conditions are required to ensure their longevity. The objective of this work was to evaluate the physiological quality of jatropha seeds stored in different environments and packaging, for periods of 3, 9 and 15 months. Three types of seed packaging bags (high density plastic bag, aluminized envelope and multiwall paper bag) were used, and the storage environments were cold and dry chamber (20 °C and 15% RH, constant), refrigerator (7 ± 3 °C, 48 ± 8% RH) and laboratory conditions (25 ± 3 °C, 51 ± 7% RH). The initial moisture content and seed germination were 7.1% and 89%, respectively. During storage, the physiological quality (germination and vigor) and moisture content of the seeds were evaluated. Seed water content ranged from 3.3 to 7.7%, depending on the permeability of the packaging and the storage environment. The highest longevity (15 months) without loss of viability was observed for jatropha seeds with initial moisture of 7.1%, packed in semipermeable plastic. Seed vigor was maintained, regardless of the environment and the type of packaging used, for up to nine months of storage. Resumo: As sementes de pinhão manso são classificadas como ortodoxas, mas por ser uma espécie oleaginosa é preciso que as condições de armazenamento sejam adequadas, para garantir a sua longevidade. O objetivo nesse trabalho foi avaliar a qualidade fisiológica das sementes de pinhão manso armazenadas em diferentes ambientes e embalagens, por períodos de três, nove e 15 meses. As embalagens utilizadas para o acondicionamento das sementes foram saco plástico de alta densidade, envelope aluminizado e saco de papel multifoliado e os ambientes para o armazenamento foram câmara fria e seca (20 °C e 15% de UR, constantes), geladeira (7 ± 3 °C; 48 ± 8% de UR) e laboratório (25 ± 3 °C; 51± 7% de UR). A umidade e a germinação iniciais das sementes eram de 7,1% e 89%, respectivamente. Durante o armazenamento foram realizadas avaliações da qualidade fisiológica (germinação e vigor) e da umidade das sementes. O conteúdo de água das sementes variou de 3,3 a 7,7%, dependendo da permeabilidade da embalagem e das condições ambientais de armazenamento. Em período de armazenamento de nove meses houve manutenção do vigor das sementes, independentemente do ambiente e do tipo de embalagem utilizada. Sementes de pinhão manso com umidade inicial de 7,1% podem ser armazenadas por 15 meses, sem perda de viabilidade, desde que acondicionadas em embalagem plástica semipermeável.

Queen palm (Syagrus romanzoffiana [Cham.] Glassman) is a palm species best known as an ornamental tree for urban landscaping, but recently, it has been evaluated as a potential crop for biofuel production. The objective of the present work was to establish a cryopreservation technique for queen palm to ensure long-term conservation of this species. The cryopreservation protocol consisted of direct immersion in liquid nitrogen (LN) of whole endocarps with water contents ranging from 5.5 to 10.9%, followed by slow thawing at room temperature (25 ± 2°C) excision and in vitro culture of zygotic embryos. Viability of zygotic embryos isolated from endocarps with different water contents was evaluated before (control) and after freezing in LN using in vitro culture on Woody Plant Medium (WPM) medium. Germination percentages of zygotic embryos isolated from endocarps stored in LN varied from 84 to 93%, whereas those isolated from controls ranged from 55 to 71%. Germination rates were significantly higher for zygotic embryos excised from cryopreserved endocarps. The water content of control or frozen endocarps did not have a significant effect on germination percentages of zygotic embryos. Zygotic embryos excised from endocarps following cryopreservation in liquid nitrogen developed into normal plantlets after in vitro culture. The technique tested is simple, efficient, and can be used in plant gene banks as a routine approach for long-term conservation of queen palm germplasm.

Jatropha curcas L. is a plant species with many potential applications, especially medicinal uses (hypoglycemic, anti-inflammatory, haemostatic, healing, anti-tumor). The objective of this study was to test germination in moist paper rolls for whole seeds and in vitro for excised embryonic axes, in an attempt to identify the best method to assess the quality of J. curcas seed germplasm, cryopreserved with different water contents. The experimental sample with a 6.2% moisture content (MC) was divided in subsamples which were hydrated and dehydrated for 0 (control), 4, 8, 11 and 24h. The initial germination percentages were 63% for whole seeds and 81% for excised embryonic axes. After exposure to liquid nitrogen (LN), germination percentages were 48% (whole seeds) and 57% (excised embryonic axes). There was no significant difference between germination percentages in embryonic excised from seeds subjected or not subjected to freezing, with different MC. In contrast, there was a reduction of the whole seed germination percentage when exposed to LN (contrast = 0.17, standard error = 0.04, t = 4.09, p = 0.001) and not for the hydration and dehydration treatments. The methodology based on in vitro cultures of the embryonic axis isolated from seeds stored in LN with distinct MC values was more efficient than the standard germination test to evaluate the viability of J. curcas seeds before and after LN storage. RESUMO Jatropha curcas L., é uma espécie com várias aplicações potenciais, principalmente para usos medicinais (hipoglicemina, anti-inflamatório, homeostático, cicatrizante, antitumoral). O objetivo deste estudo foi testar a germinação em rolo de papel para sementes inteiras e in vitro para eixos embrionários excisados visando identificar o melhor método para avaliar a qualidade de germoplasma semente de J. curcas, criopreservado com diferentes teores de água. A amostra experimental com 6,2% de teor de água foi dividida em subamostras que foram hidratadas e desidratadas por 0 (controle), 4, 8, 11 e 24 h. Os percentuais de germinação inicial foram de 63% para sementes inteiras e de 81% para eixos embrionários excisados. Após exposição ao nitrogênio líquido (NL) os percentuais de germinação foram de 48% (sementes inteiras) e 57% (eixos embrionários). Não houve diferença significativa entre os percentuais de germinação de eixos embrionários excisados de sementes com diferentes umidades e submetidas ou não ao congelamento. Em contraste, houve redução de percentuais de germinação das sementes inteiras expostas ao NL (contraste = 0.17, erro padrão = 0.04, t = 4.09, p = 0.001), mas não aos tratamentos de hidratação e desidratação. A metodologia baseada em cultura de eixos embrionários in vitro isolados de sementes armazenadas em NL, com distintos teores de água foi mais eficiente que a germinação padrão para avaliar a viabilidade de sementes J. curcas antes e após a armazenamento em NL.

Pfaffia glomerata (Amaranthaceae), “Brazilian Ginseng”, is a medicinal plant used in folk medicine. Roots are used as a tonic to restore and enhance wellbeing and for treatment of arthritis, gastritis and rheumatism. Conservation of P. glomerata germplasm is a priority and cryopreservation is the most promising technique for long-term storage of plant genetic resources. Hence, the objective of this work was to develop a cryopreservation protocol for shoot tips of P. glomerata using vitrification techniques. For cryopreservation, shoot tips (ST) from in vitro grown plants were pre-cultured for 19 hr on MS medium containing 0.3 M sucrose, treated with loading and vitrification solutions prior to rapid freezing by direct plunge in liquid nitrogen, rapid thawing on a water bath at 38±2 °C and treatment with a dilution solution. Three vitrification solutions (PVS2, PVS3 and PVS4), three exposure times (20 min., 40 min. and 60 min.) and two temperatures (25 °C and 0 °C) were tested. After cryopreservation, rewarmed shoot tips were inoculated on MS growth medium and the best regeneration percentages were 63%, 42% and 65% for shoot tips treated with PVS2, PVS3 and PVS4, respectively, for 60 min., at 25 °C. The results obtained show that vitrification with PVS2 and PVS4, at 25 °C, for 60 min were the best treatments for successful cryopreservation of shoot tips of in vitro grown plantlets of P. glomerata and that cryopreservation is suitable for ex situ conservation of the germplasm of this medicinal species.