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Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

© 2018 Santiago et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

After the publication of the article [1], it was brought to our attention that the GenBank Accession number for the EF1 is missing in Methods section. Please find the accession number in the erratum below.

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.

In the present study, we have analyzed transformed plants of the citrus rootstock Carrizo citrange constitutively expressing a Δ1-pyrroline-5-carboxylate synthetase mutant gene (p5cs), which encodes the rate-limiting enzyme of the proline biosynthetic pathway. Transgenic plants grew normally in vivo and accumulated high levels of proline in the leaves. In well-watered conditions, the photosynthetic rate of Carrizo citrange transgenic plants was not significantly different from the non-transformed control. The transgenic plants presented superior osmotic adjustment and significantly higher photosynthetic rate than the control plants after 15 days without watering. Under water deficit, the leaf water potential of non-transformed plants was close to zero after 17 days, and the photosynthetic rate was almost null 2 days later. On the other hand, in transgenic plants similar measurements were only observed after 24 days of water stress. The use of citrus rootstocks with increased proline accumulation appears to be a promising approach to maintain the productivity of plants under drought stress condition.

Transgenic plants of the important citrus rootstock Swingle citrumelo (Citrus paradisi Macf .×Poncirus trifoliata L. Raf.) were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Thin sections were co-cultured with Agrobacterium strain EHA105 carrying pBI121, a plasmid that contains the nptII (pnos) gene and the uidA (p35S) gene for scorable marker β-glucuronidase (GUS). The use of thin sections and the application of increasing selection pressure (25 and 50 mg l−1 kanamycin) reduced the occurrence of escapes. With the procedure described here, a transformation efficiency of 8.6% was achieved, with less than 10% of escapes. Stable integration of the transgene sequence was confirmed by histochemical assay of β-glucuronidase, PCR and Southern hybridization.

An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 μM 6-benzylaminopurine and 0.7 μM gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature.

A system for in vitro plant regeneration from thin transversal epicotyl sections of sweet orange (Citrus sinenis L Osbeck cvs Pera, Valencia and Folha Murcha) was developed. Thin section explants (1-2 mm) from seedlings produced adventitious shoot buds on MS medium supplemented with BA (0.5-5 μM). Optimal shoot induction was achieved on 2-5 μM BA. Explants with buds were transferred to elongation media in order to improve the recovery of normal shoots. Higher number of elongated shoots was obtained on medium with 0.5 μM BA. Elongated shoots were rooted on half strength MS medium without growth regulators and transferred to soil for acclimatization. The advantages of this system for transformation are discussed.

An in vitro system for studying the resistance response of cotton (Gossypium hirsutum L.) to Xanthomonas campestris pv. malvacearum was investigated. Cell suspension cultures, established from hypocotyl-derived callus of cotton cultivar 101-102B, were treated with bacterial extracellular polysaccharides (EPS) extracted from the incompatible race 18 of X. campestris pv. malvacearum. EPS at 600 mug/mL caused pronounced darkening of the suspension cultures, as indicative of cell death, 48 hours after incubation. Protein electrophoresis analysis of the time course of EPS-treated cells showed differential accumulation of several protein bands after 12-24 hours. The time course of protein accumulation and cell death was consistent with an elicitor-mediated hypersensitive response. Desenvolveu-se um sistema in vitro para estudar a resistência do algodoeiro (Gossypium hirsutum L.) à Xanthomonas campestris pv. malvacearum. Foram utilizados calos originados a partir de hipocótilos da cultivar de algodoeiro 101-102B para estabelecer culturas de células em suspensão, as quais foram tratadas com polissacarídeos extracelulares bacterianos (EPS) extraídos da raça incompatível 18 de X. campestris pv. malvacearum. O tratamento com EPS, na concentração de 600 mig/mL, causou acentuado escurecimento das culturas em suspensão, indicativo de morte celular, 48 horas após a incubação. A análise temporal do perfil eletroforético de proteínas extraídas das células tratadas com EPS mostrou um acúmulo diferencial de diversas proteínas após 12-24 horas. O acúmulo de proteínas e a morte celular ao longo do período estudado foram consistentes com um padrão de resposta de hipersensibilidade causada por elicitores.

Com o objetivo de conhecer os requerimentos nutricionais de suspensões celulares de algodoeiro foi feita uma análise da utilização de nutrientes no intervalo de 30 dias, normalmente utilizado entre subculturas. As culturas de células em suspensão de algodoeiro, genótipo 101-102B, foram estabelecidas e subcultivadas mensalmente em meio líquido. Foram analisadas o crescimento das culturas, as variações de pH do meio de cultura e a absorção de nutrientes. A matéria fresca das culturas de células foi superior a 6 vezes o valor inicial após 30 dias de cultivo. Houve aumento do pH no início da cultura, estabilizando-se em 6,2 próximo à fase estacionária. Durante o crescimento das suspensões, as células consumiram pelo menos 80% da concentração inicial da maioria dos nutrientes quantificados. A velocidade de absorção, entretanto, variou entre os elementos. Enquanto PO4(3-) foi praticamente esgotado do meio após 10 dias de cultivo, os íons K+, Ca2+, SO4(2-) e Mg2+ foram encontrados em concentrações próximas a 20% do valor inicial após 30 dias. As suspensões de células de algodoeiro consumiram 89% da concentração inicial de glicose. The nutrient uptake of cotton suspension cultures was studied during a 30 day-culture period. Cotton cell suspension cultures of the 101-102B genotype were established and monthly subcultured in liquid medium. Culture growth rate, nutrient uptake and pH variations in the medium were measured. A 6-fold increase in fresh weight of the cotton cell culture was observed on day 30. The majority of the nutrients were taken up at a high rate, and their consumption was at least 80% of the original concentration. Nevertheless, the uptake rate varied among the elements. While PO4(3-) was practically depleted from the medium on day 10, the ions such as K+, Ca2+, SO4(2-) and Mg2+ had a 20% residual at the end of the period. The pH of the culture medium increased initially and stabilized at pH 6.2 at the beginning of the stationary phase. In addition, the cotton cell suspension culture took up 89% of glucose added to the medium.

Estudos fenologicos de tres acessos morfologicamente distintos de pinhao-manso (Jatropha curcas L.) foram conduzidos no bioma Cerrado brasileiro. A taxa de crescimento da planta, florescimento, formacao e desenvolvimento dos frutos foram medidos, a fim de se estabelecer praticas de manejo eficientes para o aumento de produtividade. Os acessos estudados foram: CNPAE-102 (toxico, susceptivel a oidio), CNPAE-169 (nao-toxico, susceptivel a oidio) e CNPAE-259 (toxico, resistente a oidio). Os materiais considerados nao-toxicos nao possuem esteres de forbol detectaveis nos graos. No periodo de um ano (agosto de 2009 a julho de 2010) registraram-se, quinzenalmente, as variaveis agronomicas (altura de plantas, numero de inflorescencias, numero de frutos verdes e maduros por planta) de cada acesso, correlacionando-as com as variaveis climaticas, por meio da analise de correlacao de Pearson. O acesso CNPAE-102 apresentou a maior taxa de crescimento (0,76 cm dia-1), incremento 1,10 m na altura e altura final de 2,60 m; o CNPAE-169 apresentou menor incremento (0,63 cm dia-1), atingindo altura final de 1,85 m. Os acessos CNPAE-259 e CNPAE-102 foram os mais precoces, e o CNPAE-169 o mais tardio. A temperatura minima foi a variavel climatica que determinou o maior numero de inflorescencias.

Transgenic plants of Coffea canephora P. resistant to the herbicide ammonium glufosinate were regenerated from leaf explants after co-culture with Agrobacterium tumefaciens strain EHA105 harboring pCambia3301, a plasmid that contains the bar and the uidA genes both under control of 35S promoter. Direct somatic embryogenesis was induced on basal medium contained ¼ strength macro salts and half strength micro salts of MS medium, organic constituents of B5 medium and 30 g.L-1 sucrose supplemented with 5µM N6 - (2-isopentenyl)-adenine (2-iP). Ten µM ammonium glufosinate was used for putative transgenic somatic embryos selection. Presence and integration of the bar gene were confirmed by PCR and Southern blot analysis. Selected transgenic coffee plants sprayed with up to 1600 mg.L-1 of FinaleTM, a herbicide containing glufosinate as the active ingredient, retained their pigmentation and continued to grow normally during ex vitro acclimation.Plantas transgênicas de Coffea canephora P resistentes ao herbicida glufosinato de amônio foram regeneradas a partir de explantes foliares co-cultivados com Agrobacterium tumefaciens EHA105 contendo o plasmídio pCambia3301 que contém os genes bar e uidA ambos sob controle do promotor 35S. Embriogênese somática direta foi induzida no meio contendo ¼ da concentração de macro, metade da concentração de micronutrientes do meio MS, constituintes orgânicos do meio B5 e 30 g.L-1 de sacarose suplementado com 5µM N6 - (2-isopentenil)-adenina (2-iP) e 10 µM de glufosinato de amônio para seleção de embriões transgênicos putativos. A presença e a integração do gene bar foram confirmados pelas análises de PCR e Southern blot. As plantas transgênicas selecionadas de café, pulverizadas com 1600 mg.L-1 do herbicida FinaleTM que contém glufosinato como ingrediente ativo, mantiveram a coloração e continuaram crescendo normalmente na aclimatação ex vitro.

The objective of this study was to examine the effect of two culture systems, liquid medium associated to floating membranes and solid medium, both supplemented with different concentrations of 2,4-D, in the induction of somatic embryogenesis of cassava (Manihot esculenta Crantz). Only 28% of the young leaf lobes (with 9 µM 2,4-D) were induced to form organized embryogenic structures (OES) with membrane rafts, compared to 50% of the explants presenting this type of tissue in solid medium with 36 µM of 2,4-D. Despite the lower response observed in liquid medium with membrane, the amount of OES/explant in all 2,4-D concentrations was higher than solid medium. Based on the results and considering the high cost of the membrane rafts, this system was not distinctly superior than solid medium for inducing somatic embryogenesis in cassava.O objetivo deste estudo foi comparar a indução de embriogênese somática em mandioca (Manihot esculenta Crantz) utilizando o sistema de cultivo em meio líquido associado com membranas flutuantes com meio sólido, ambos suplementados com diferentes doses de 2,4-D. Utilizando membranas flutuantes, o melhor resultado foi obtido na concentração de 9 µM 2,4-D, onde apenas 28% dos explantes foliares apresentaram estruturas embriogênicas organizadas (OES). Por outro lado, em explantes cultivados em meio sólido suplementado com 36 µM de 2,4-D a frequência de OES foi de 50%. Embora a frequência de indução embriogênica tenha sido inferior em meio líquido associado com membranas flutuantes, a quantidade de OES por explante foi igual ou superior ao do meio sólido em todas as concentrações de 2,4-D testadas. Baseado nestes resultados, e considerando o elevado custo das membranas, este sistema de cultura não apresentou vantagens significativas para indução de embriogênese somática em mandioca em relação ao meio sólido.