53 research outputs found

    Optimization of a plant regeneration protocol for broccoli

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    The factors which influence the regeneration of broccoli (Brassica oleracea var. italica) were studied using an orthogonal design. The results showed that the major factor was the explant type, followed by naphthylacetic acid (NAA), benzylaminopurine (BAP), sucrose and AgNO3 in turn. The maximum regeneration was on Murashige and Skoog (MS) medium + NAA 0.107 μM + BAP 17.76 μM + 2% sucrose + 0.8% agar. Hypocotyl proved to be the optimum explant source and its regeneration frequency reached 100%.Key words: Naphthylacetic acid (NAA), benzylaminopurine (BAP), AgNO3, hypocotyl, broccoli (Brassica oleracea var. italica)

    Manipulation of post-harvest physiology in broccoli through an optimised AgrobacteriumAgrobacterium rhizogenesrhizogenes-mediated transformation protocol

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    The aim of this project was to down-regulate ACC oxidaseoxidase (ACO) 1 and 2 and ACC synthasesynthase (ACS) in broccoli (BrassicaBrassicaoleraceaoleracea var. italicaitalica) to lengthen post-harvest shelf-life. The ACO 1 and 2 and ACS cDNAs of broccoli were ligated into pSCV1.0 in sense and antisense orientations in relation to a CaMV 35S promoter and nos terminator. They were electroporated into the AgrobacteriumAgrobacteriumrhizogenesrhizogenes co-integrate strain LBA 9402 pRi1855::GFP, and used to co-transform GDDH33, a doubled haploid line derived from the calabrese cultivar Green Duke. 150 transgenic hairy roots were identified by GFP fluorescence, and 18 were regenerated into whole plants. Four of these lines showed severe rolrol phenotype, which did not appear to be related to TL_L-DNA insert copy number. The floral buds from T0_0 broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. T0 lines with ACC oxidaseoxidase 1 and 2 constructs produced significantly less ethylene than the control plants, and chlorophyll loss was significantly reduced. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by the equation y= 0.2386x-23.041. There were two copies of aco1 and aco2 in the BrassicaBrassica oleraceaoleracea genome of which one was mapped for each to linkage group 1 and 8, respectively

    In vitro shoot regeneration from seedling explants in Brassica vegetables: Red cabbage, broccoli, Savoy cabbage and cauliflower

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    Brassica oleracea varieties (red cabbage, broccoli, Savoy cabbage and cauliflower) were tested for their ability to regenerate shoots in vitro. Cotyledon, hypocotyl and root explants of 7 day-old seedlings were incubated on Murashige and Skoog's (MS) medium supplemented with 1 mg l-1 6-benzyladenine (BA) or 6-furfurylaminopurine (KIN) in combination with 0, 0.1, and 0.2 mg l-1 indole-3-butyric acid (IBA). Hypocotyls showed the best explants in almost all varieties tested with a minimum regeneration potential of 75% and producing 3.5-7.4 shoots per explant. The BA-supplemented media were optimal for both shoot regeneration and multiplication. Shoots rooted maximally (100%) on plant growth regulator-free MS medium containing 2% or 4% sucrose. Increased sucrose content improved plant acclimation in the greenhouse.Ispitivana je sposobnost četiri varijeteta Brassica oleracea (crveni kupus, brokoli, kelj i karfiol) da regenerišu pupoljke in vitro. Eksplantati kotiledona, hipokotila i korenova, uzetih sa 7 dana starih klijanaca, su gajeni na Murashige i Skoog (MS) hranljivoj podlozi sa dodatkom 1 mg l-1 6-benziladenina (BA) ili 6- furfurilaminopurina (KIN) u kombinaciji sa 0, 0.1, i 0.2 mg l-1 indol-3-butirične kiseline (IBA). Eksplantati hipokotila su se pokazali kao najbolji za regeneraciju kod skoro svih testiranih varijeteta sa minimalnim regenerativnim potencijalom od 75% i sa produkcijom 3.5- 7.4 pupoljaka po eksplantatu. Podloge koje su sadržale BA su bile optimalne, kako za regeneraiju pupoljaka, tako i za njihovu kasniju multiplikaciju. Maksimalan procenat oživljavanja izdanaka (100%) je postignut na MS medijumu bez dodatih regulatora rastenja, a koji je sadržao 2% ili 4% saharozu. Povećan sadržaj saharoze u medijumu za ožiljavanje uticao je na poboljšanu aklimatizaciju biljaka u stakleniku.Projekat ministarstva br. 143026 i TR-20072

    The Highly Embryogenic Brassica napus DH4079 Line Is Recalcitrant to Agrobacterium-Mediated Genetic Transformation

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    [EN] Brassica napus is a species of high agronomic interest, used as a model to study different processes, including microspore embryogenesis. The DH4079 and DH12075 lines show high and low em-bryogenic response, respectively, which makes them ideal to study the basic mechanisms control-ling embryogenesis induction. Therefore, the availability of protocols for genetic transformation of these two backgrounds would help to generate tools to better understand this process. There are some reports in the literature showing the stable transformation of DH12075. However, no equivalent studies in DH4079 have been reported to date. We explored the ability of DH4079 plants to be genetically transformed. As a reference to compare with, we used the same protocols to transform DH12075. We used three different protocols previously reported as successful for B. napus stable transformation with Agrobacterium tumefaciens and analyzed the response of plants. Whereas DH12075 plants responded to genetic transformation, DH4079 plants were completely recalcitrant, not producing any single regenerant out of the 1784 explants transformed and cul-tured. Additionally, an Agrobacterium rhizogenes transient transformation assay was performed on both lines, and only DH12075, but no DH4079 seedlings, responded to A. rhizogenes infection. Therefore, we propose that the DH4079 line is recalcitrant to Agrobacterium-mediated transfor-mation.This research was funded by MCIN/AEI/10.13039/501100011033, grant number PID2020-115763RB-I00 to JMSS and by Generalitat Valenciana, grant number CDEIGENT 2018/023 to RM. ACS is recipient of a PhD contract (FPU17/00715) from the Spanish Ministerio de Educacion.Calabuig-Serna, A.; Mir Moreno, R.; Porcel, R.; Seguí-Simarro, JM. (2023). The Highly Embryogenic Brassica napus DH4079 Line Is Recalcitrant to Agrobacterium-Mediated Genetic Transformation. Plants. 12(10). https://doi.org/10.3390/plants12102008121

    The in vitro manipulation of cauliflower (Brassica oleracea L. convar. botrytis (L.) Alef. var. botrytis L.) meristematic tissues for utilisation in genetic improvement programmes

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    Merged with duplicate record 10026.1/629 on 27.02.2017 by CS (TIS)Cauliflower curd meristem activity (organogenic, plastochronic, phyllotactic) was analysed biometrically and confirmed that the curd is the product of a constant process of meristem production and branch ramification with little if any dominance between branch apices. A growth model based on curd branching pattern was developed and its mathematical expression enabled the estimation of the number of meristems carried by a curd at maturity to be over ten million which was previously widely underestimated. Analysis of the response to the in vitro culture of this meristematic tissues revealed that meristems are not predetermined to produce flower and that their shoot regeneration capacity is under several levels of control, the most important being explant physical property (size) and the culture system (nutrient supply). Optimisation of these parameters enabled the development of a low cost, semi-automated protocol for mass production of cauliflower propagules at an unprecedented scale with over 10000 propagules produced per curd. Micropropagules a few millimetres in length were produced, encapsulated in calcium alginate hydrogel, stored at 4°C for several months and used as an 'artificial seed' system of cauliflower propagation. The response to the procedure of micropropagule production is genotype-dependent with summer heading varieties being less reactive than winter heading varieties, this phenomenon was also associated with plasmalemma instability at the cellular (protoplast) level. Furthermore, this material was successfully cryopreserved in liquid nitrogen using a dehydration I vitrification method. The micropropagation protocol is of great interest when used as a regeneration system for experiments involving genetic manipulation such as genetic transformation. A preliminary study of genetic transformation by microprojectile bombardment, using the gus reporter gene, allowed transient expression in curd meristematic tissue. The fundamental and industrial implications for cauliflower breeding of the different protocols developed in this thesis are discussed

    In vitro adventitious shoot regeneration and acclimatisation of Brassica oleracea subsp. italica cv. Green Marvel

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    Cotyledonary explants of Brassica oleracea subsp. italica (broccoli) cv. Green Marvel were cultured on Murashige and Skoog (MS) medium containing different combinations of the growth regulators 6- benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) for shoot regeneration. The optimal medium for inducing shoots contained 3 mgl-1 BAP and 1 mgl-1 NAA, which produced a shoot induction percentage of 53.33% and a mean number of 0.43 shoot per explant. The shoots were subsequently rooted in MS medium that contained 0.2 mgl-1 of indol-3-butyric acid (IBA). Different potting media were assessed during plantlet acclimatization. The highest percentage of plant survival (83.33%) was on the medium that contained sand and soil (1:1), while maximum root length (4.37 cm) and plant height (7.87 cm) were attained in potting medium that consisted peat moss, perlite and vermiculite (3:1:1).Key words: Brassica oleracea, broccoli, 6-benzylaminopurine, α-naphthalene acetic acid, indole-3- butyric acid

    In vitro shoot regeneration from seedling explants in Brassica vegetables: Red cabbage, broccoli, Savoy cabbage and cauliflower

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    Brassica oleracea varieties (red cabbage, broccoli, Savoy cabbage and cauliflower) were tested for their ability to regenerate shoots in vitro. Cotyledon, hypocotyl and root explants of 7 day-old seedlings were incubated on Murashige and Skoog's (MS) medium supplemented with 1 mg l-1 6-benzyladenine (BA) or 6-furfurylaminopurine (KIN) in combination with 0, 0.1, and 0.2 mg l-1 indole-3-butyric acid (IBA). Hypocotyls showed the best explants in almost all varieties tested with a minimum regeneration potential of 75% and producing 3.5-7.4 shoots per explant. The BA-supplemented media were optimal for both shoot regeneration and multiplication. Shoots rooted maximally (100%) on plant growth regulator-free MS medium containing 2% or 4% sucrose. Increased sucrose content improved plant acclimation in the greenhouse.Ispitivana je sposobnost četiri varijeteta Brassica oleracea (crveni kupus, brokoli, kelj i karfiol) da regenerišu pupoljke in vitro. Eksplantati kotiledona, hipokotila i korenova, uzetih sa 7 dana starih klijanaca, su gajeni na Murashige i Skoog (MS) hranljivoj podlozi sa dodatkom 1 mg l-1 6-benziladenina (BA) ili 6- furfurilaminopurina (KIN) u kombinaciji sa 0, 0.1, i 0.2 mg l-1 indol-3-butirične kiseline (IBA). Eksplantati hipokotila su se pokazali kao najbolji za regeneraciju kod skoro svih testiranih varijeteta sa minimalnim regenerativnim potencijalom od 75% i sa produkcijom 3.5- 7.4 pupoljaka po eksplantatu. Podloge koje su sadržale BA su bile optimalne, kako za regeneraiju pupoljaka, tako i za njihovu kasniju multiplikaciju. Maksimalan procenat oživljavanja izdanaka (100%) je postignut na MS medijumu bez dodatih regulatora rastenja, a koji je sadržao 2% ili 4% saharozu. Povećan sadržaj saharoze u medijumu za ožiljavanje uticao je na poboljšanu aklimatizaciju biljaka u stakleniku.Projekat ministarstva br. 143026 i TR-20072

    Prijenos gena za proizvodnju opina (Agrobacterium pRi TL-DNA rolB i TR-DNA) u stanice zrnatog šćira Amaranthus spinosus L.

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    In vitro rhizogenesis occurred with a characteristic pattern typical of transformed roots following explant (internode/leaf) inoculation of Amaranthus spinosus L. with four different wild type Agrobacterium rhizogenes strains. The extent of rhizogenesis varied considerably with the explant type and source, and with the Agrobacterium strains employed; internodal segments performed better than leaves. Of the strains employed for cocultivation, A. rhizogenes LBA 9402 carrying pRi 1855 was the most virulent and infectious, causing hairy root induction in the maximum number of explants regardless of their type. Individual root clones (rhizoclones) were maintained on Murashige and Skoog\u27s basal medium without growth regulators. The physical presence of the rolB gene in the TL-DNA segment of the Ri plasmid of the infecting Agrobacterium in leaf tissues of plants regenerated from selected rhizoclones was confirmed by a positive PCR amplification. The ability of the genetically transformed plants to harbour and express TR-DNA specific opine synthase genes (man2 and ags) was substantiated by PCR and opine assay respectively, demonstrating the production of characteristic opines. Such findings are implicated in the context of pharmaceutical exploitation of transformed root cultures of A. spinosus and also towards protecting this nutraceutically important crop, amaranth, against biotic stress challenges via transgenic manipulations.Inokulacijom eksplantata (internodija ili listova) zrnatog šćira Amaranthus spinosus L. sa četiri divlja soja bakterije Agrobacterium rhizogenes dobiven je karakterističan uzorak transformiranog korijenja nakon in vitro rizogeneze. Stupanj rizogeneze uvelike je ovisio o upotrijebljenom tipu i porijeklu eksplantata te soju bakterije, pri čemu su bolji rezultati dobiveni inokulacijom internodija, nego pomoću listova šćira. Soj A. rhizogenes LBA 9042, koji posjeduje plazmid pRi 1855, bio je najvirulentniji i najinfektivniji, pa je uzrokovao pojavu čupavog (adventivnog) korijenja u maksimalnom broju eksplantata, neovisno o njihovom tipu. Pojedinačni klonovi transgenog korijenja uzgajani su na hranjivoj podlozi Murashige i Skoog, bez dodatka regulatora rasta. Prisutnost gena rolB u segmentu TL-DNA plazmida Ri iz bakterije Agrobacterium u lisnom tkivu biljaka regeneriranih iz odabranih klonova potvrđena je pozitivnim rezultatom PCR umnožavanja DNA. PCR umnožavanjem i analizom opina utvrđeno je da genetički transformirane biljke mogu primiti i eksprimirati TRDNA-specifične gene (man2 i ags) za sintezu karakterističnih opina. Rezultati upućuju na to da se transformirano korijenje šćira može upotrijebiti u farmaceutici, te da se transgenim uzgojem ova važna žitarica može zaštititi od biotičkog stresa

    Development of plant regeneration and transformation techniques towards reducing glucosinalbin biosynthesis in field pepperweed (Lepidium campestre L.)

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    Field pepperweed (Lepidium campestre L.) is a cruciferous winter annual wild edible plant. It has potential medicinal properties as it contains a considerable level of glucoraphanin, which is the precursor for sulforaphane, a phase 2 protein inducer. Phase 2 proteins either directly or indirectly promote the scavenging of strong oxidants, and thus decrease the risk of many complex disorders such as atherosclerosis and Alzheimer’s disease. However, field pepperweed plants also contain glucosinalbin, an antinutritional compound. For field pepperweed to become a green vegetable crop or source of functional food, it is desirable to reduce or eliminate glucosinalbin. The biosynthesis of glucosinalbin may be down-regulated through biotechnology. To that end, in the present studies, experimental protocols for plant regeneration and Agrobacterium-mediated transformation have been developed for field pepperweed. Establishment of such methods represents a vital first step in the process of engineering field pepperweed for enhanced nutritional value. The effect of explant type and various combinations of growth regulators on regeneration were evaluated in three accessions of field pepperweed (Ames 13179, 13180 and 15718). Among the three genotypes, accession Ames 13179 had the highest regeneration frequency under several conditions. Regeneration from hypocotyl explants was more rapid and prolific than regeneration from either mature leaf or cotyledonary explants. Segments from the acropetal end of the hypocotyls were more regenerable than those from the basipetal end. Evaluation of different hormonal combinations and concentrations identified an optimal growth regulator combination (3 mg L-1 thidiazuron / 0.1 mg L-1 naphthalene acetic acid) for shoot induction. The plant regeneration system established was adopted for field pepperweed transformation using the acropetal segments of hypocotyls as explants. Two plant expression constructs were tested for down-regulating by RNA interference with the expression of a field pepperweed cytochrome P450 gene named LcCYP79B2. This gene may be involved in biosynthesis of glucosinalbin. Conditions for transformation such as pre-culture, co-cultivation time, and antibiotic concentration were evaluated. Transgenic plants were obtained and confirmed by histochemical staining of the reporter â-glucuranidase activity and PCR (polymerase chain reaction) analysis of the NPTII gene. The current study has established efficient plant regeneration and transformation protocols for field pepperweed. They should be useful for future molecular biology studies and biotechnological applications in this species
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