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In vitro plant regeneration of Mammillaria elongata normal and cristate forms
Abstract
In the normal form of Mammillaria elongata, shoots were regenerated in vitro, through callus, from tubercle explants excised from the upper part of the branch and cultured on Murashige and Skoog medium
(MS) with 1.07 μM α-napthaleneacetic acid (NAA) and 22.20 μM 6-benzylaminopurine (BA). A high percentage of tubercles explants
of the M. elongata cristate form, excised from the tip of the branch and cultured on MS with 0.54 μM NAA and 0.44 μM BA or 1.07 μM NAA, responded
by initially forming an inflated cristate shoot, which gave cristate and normal shoots, without callus intervention, when
transferred on basal MS. Callus formed on cristate tubercles explants gave both cristate and normal shoots when transferred
onto basal MS. Normal and cristate shoots were rooted in vitro on MS with 9.84 μM or 0.98 μM indole-3-butyric acid, respectively, and established ex vitro. In both normal and cristate form, the differential response appeared to be associated with the site of the explant excision.
The formation of shoots was influenced by the season of culture; i.e., explants excised in October had a higher shoot formation
rate than those excised February.
... Numerous micropropagation techniques have been applied such as regeneration through somatic embryogenesis, regeneration through direct or indirect organogenesis, and in vitro grafting (Figure 2). Those methods have been used for several species of cactus such as Mammillaria, Cereus, Hylocereus, Echinocereus, Astrophytum, Stenocactus, Schlumbergera, Coryphantha, and Copiapoa (Wakhlu and Bhau, 2000;Papafotiou et al., 2001;Mohamed-Yasseen, 2002;Al-Ramamneh et al., 2006;Lema-Rumińska and Kulus, 2012;Lema-Rumińska et al., 2013;Bhau and Wakhlu, 2015;Chornobrov and Bilous, 2021;Ivannikov et al., 2021). The regeneration through direct or indirect organogenesis appeared to be the most important and reliable method of in vitro propagation with about 80% of the conducted work on cacti (Figure 3). ...
... Indirect organogenesis has been successfully implemented for several cacti species such as Astrophytum asterias, Ariocarpus Kotschoubenyanus, Blossfeldia liliputiana, Strombocactus disciformis, Coryphantha elephantidens, Mammillaria elongata, Mammillaria gracilis, Mammillaria mathildae, Mammillaria pectinifera, Escobaria minima, and Pelecyphora aselliformis (Wakhlu and Bhau, 2000;Papafotiou et al., 2001;Giusti et al., 2002;Poljuha et al., 2003;del Socorro Santos-Díaz et al., 2006;García-Rubio and Malda-Barrera, 2010;Ivannikov et al., 2021; Table 1). Callus-mediated regeneration commonly occurs when the culture medium is supplemented with high amounts of auxin or cytokinin during the initial step of callus formation (Pérez-Molphe- Balch et al., 2015). ...
... Direct organogenesis is defined as the direct process of the production of new buds from the tissue without intervening callus stage. During the last decades, micropropagation through direct organogenesis has been widely implemented to ensure the micropropagation of many endangered species, including those belonging to the Cactaceae family (Papafotiou et al., 2001;Moebius-Goldammer et al., 2003;Dávila-Figueroa et al., 2005;de Medeiros et al., 2006;Ramirez-Malagon et al., 2007; Table 2). The success of this method of propagation relies mostly on the balance between cytokinin and auxin amounts. ...
Cacti are one of the most significant and diversified groups of angiosperms, distributed and cultivated globally, mostly in semi-arid, arid, and the Mediterranean climate regions. Conventionally, they are propagated by seeds or through vegetative propagation via rooted offshoots or grafting. However, these multiplication procedures remain insufficient for mass propagation. In vitro culture techniques are utilized to mass propagate endangered and commercial cacti species. These include somatic embryogenesis and plant regeneration through indirect or direct organogenesis. The latter is a promising tool for commercial clonal propagation of high-value species and has been successfully implemented for several species, such as Mammillaria, Hylocereus, Cereus, Echinocereus, and Ariocarpus. However, its success depends on explant type, basal nutrient formulation of culture medium, and types and concentrations of plant growth regulators. This study aimed to assess the potential of in vitro propagation methods applied to cacti species and discuss the different factors affecting the success of these methods. This study has also highlighted the insufficient work on Opuntia species for mass propagation through axillary buds' proliferation. The development of an efficient micropropagation protocol is thus needed to meet the supply of increasing demand of Opuntia species for human consumption as fruit, animal feed, and ecological restoration in semi-arid and arid zones.
... ex Vaupel) Moran y Hylocereus undatus (Haw.) Britton et Rose para favorecer el metabolismo celular de la semilla, activar el crecimiento del embrión y el proceso enzimático de los tegumentos, para que la cubierta de la semilla se rompa y emerja una nueva vitro-plántula (Papafotiou et al., 2001;Pelah et al., 2002) ...
... ex Vaupel) Moran and Hylocereus undatus (Haw.) Britton et Rose to stimulate seed cell metabolism, to activate the growth of the embryo and the enzymatic process of teguments, so that the seed cover brakes and emerges a new vitro-plant (Papafotiou et al., 2001;Pelah et al., 2002) (Figure 2). Figura 2. Establecimiento de semillas y aspecto de las plántulas de Turbinicarpus knuthianus después de 9 semanas de incubación. ...
... En el caso de la cactácea estudiada la máxima tasa de multiplicación en relación de 1:10:10:10 se obtiene in vitro en un medio de cultivo MIB con 4.4 mM de BA+ 0.413 μM (relación 1:10:10:10). Esta cifra es semejante a la que se ha registrado en especies de los géneros: Coryphantha, Echinocereus y Mammillaria (Clayton et al., 1990) y en Astrophytum myriostigma Lem. ( Villavicencio et al., 2006;2009)., pero que supera a la consignada en otras especies de cactáceas mexicanas como Mammillaria voburnensis Scheer y Mammillaria elongata DC. (Ordóñez, 2003;Papafotiou et al., 2001). ...
A four stage protocol was developed for the micropropagation of Turbinicarpus knuthianus , an ornamental cactus in risk status, in order to produce plants with standard commercial-sized pots in sufficient amount and good phytosanitary quality. The method here described is efficient compared to the traditional method of propagation; it is a new production technology that can be applied to the ornamental product system under the laboratory-greenhouse scheme. The seeds of this species are quiescent and can be established in vitro on MS medium at 50% supplemented with 8.65 2M of GA³, with 75% of germination. The induction of shoots and seedlings was obtained by setting segments of explants on MS medium with different treatments. It was determined that the type and concentration of phytohormone affect the multiplication rate and produce as much as 10 shoots/explant; kinetin (KIN) in interaction with AIB 10:1 in low concentrations favors this effect. During the acclimatization stage, it was observed that the application of 1.5 x10(6) CFU ml-1 of Azospirillum brasilense promoted a positive reaction of the rhizogenic process, as much as 6 roots 2.5 cm long per plant were formed. With this technology, species of ecological importance in risk status can be restored to the Chihuahuan Desert and the biological processes for the production of ornamental plants can be optimized.
... In the last decades, tissue culture has been implemented to propagate many threatened and endangered cacti, e.g., Obregonia denegrii (Malda et al., 1999); Coryphantha elephantidens (Wakhlu and Bahu, 2000); Mammillaria elongata (Papafotiou et al., 2001); Pelecyphora spp. (Pérez-Molphe-Balch and Dávila-Figueroa, 2002); Ariocarpus kotschoubeyanus (Moebius-Goldammer et al., 2003); Turbinicarpus spp. ...
... Micropropagation protocols commonly use an exogenous growth regulator to induce morphogenetic responses, particularly for the Mammillaria family (Papafotiou et al., 2001;Poljuha et al., 2003;Ramirez-Malagon et al., 2007). Growth regulator morphogenetic response commonly varies, so auxin-cytokinin ratio must be empirically determined for each species. ...
... Growth regulator-free MS medium was enough for in vitro shoot production of M. mathildae, contrary to several results reported for other Mammillaria species (Papafotiou et al., 2001;Poljuha et al., 2003;Ramirez-Malagon et al., 2007), where growth regulator complementation, especially cytokinins, is crucial for shoot generation. ...
A rapid shoot multiplication protocol was established for the endangered cactus Mammillaria mathildae to reintroduce it to its natural habitat. In vitro-germinated seedlings were used as the source of explants. Three explant sources (apical, lateral, and basal excised from in vitro-germinated seedlings) were tested. Shoot multiplication was induced in Murashige and Skoog (MS) medium supplemented with different 6-benzylaminopurine/indole-3-acetic acid (BA/IAA) combinations (0, 22.19, 44.39 and 0, 1.43,2.85, 5.71, respectively). Explants developed abundant callus in the presence of any BA/IAA concentration, whereas hormone-free media produced 0.59 ± 0.11 new shoots (with a 41% callus development) from basal explants. Apical and lateral explants produced 1.14 ± 0.07 and 4.09 ± 0.13 new shoots, respectively, without callus generation. Plantlets originating from lateral explants developed a vigorous rooting system after 2 months growing on MS medium supplemented with 30 g L-1 sucrose. Under greenhouse conditions, 98% of micropropagated M. mathildae survived. Plantlets were reintroduced in an experimental plot near to Juriquilla's wild population of M. mathildae; over 52% of the outplanted M. mathildae lot declined after 5 months. Water availability wasassociated with the decline of outplanted populations during the first month (43%).
... The amount and ratio of PGRs applied, as well as the genotype (or even site of explant excision), may determine the organogenesis pathway. For example, in the normal form of Mammillaria elongata (Papafotiou et al. 2001), shoots were regenerated indirectly, from tubercle explants inoculated on the MS medium with 1.07 µM NAA and 22.20 µM BA, whereas explants of M. elongata forma cristata, cultured on the MS medium with 0.54 µM NAA and 0.44 µM BA, resulted in shoots without callus formation (higher cytokinin concentration induced only the development of green calli). On the other hand, with Parodia magnifica, similar indirect regeneration efficiency (5-6 shoots per callus) was observed regardless of the medium applied (MS + 22.2 µM BA or MS + 4.6 µM Kin + 2.9 µM IAA; pH 5.7), although there was delayed shoot formation with this medium (Medeiros et al. 2006). ...
... adventitious shoots per explant for Mammillaria perbella and M. orcutii (MS with 5.7 µM IAA and 46.5 µM Kin) (Ramirez-Malagon et al. 2007), up to 269.78 shoots with Turbinicarpus laui (MS with 8.8 µM BA) (Rosas et al. 2001), which can also be affected by the season of culture. For Mammillaria elongata, shoot formation rate was higher for explants excised in October than in February (Papafotiou et al. 2001). With Cereus peruvianus the average increase in callus weight was greater in spring than in summer. ...
... Due to disruptions in photosystem II in plants grown in tissue culture under reduced (in comparison to natural) light conditions (Balen et al. 2012), such plants cannot be transferred directly into outdoor conditions. First microcuttings must be acclimatized for about 4 weeks to in vivo conditions in a light sand substrate (peat:perlite 1:2, sand:soil 1:1, or peat:sand 1:1 by volume) in the growth chamber (Papafotiou et al. 2001;Sayed et al. 2004;Resende et al. 2010) to harden them to higher temperatures and light intensity and lower air moisture in comparison with the culture vessel environment. Afterwards, plantlets can be transferred to in vivo conditions (Quiala et al. 2009). ...
In the past, members of the Cactaceae family were mostly propagated by seeds or vegetatively by cuttings and grafting. Seeds, however, do not guarantee genetic stability and, in the case of some cacti, seeds are difficult to obtain, their germination rates are low, or they need to be stored for a long time in specific light and temperature conditions. Traditional vegetative reproduction in vivo, on the other hand, may be less efficient insofar as a limited number of plants can be obtained. Major issues associated with traditional propagation methods may be solved by the use of plant tissue culture. Today micropropagation techniques are applied in order to produce large numbers of new high-quality plants in a relatively short time and space. This is especially important for endangered and desirable species such as cacti. This paper discusses the achievements, current state and future prospects of cactus micropropagation methods. We also provide an overview of cactus multiplication by areole activation, direct and indirect organogenesis (caulogenesis and rhizogenesis) and somatic embryogenesis, as well as consider acclimatization. Micropropagation of cacti is still sufficiently idiosyncratic that different protocols must be used for different species, even closely related ones.
... Reduction of the nitrogen nutrient concentration in the Murashige and Skoog (1962) medium to one-fourth affected cristate form stability. The in vitro behavior of cristated Euphorbia pugniformis resembles that of cristated Mammilaria elongata (Papafotiou et al. 2001) as far as explant type and plant growth regulators effect on cristate shoot regeneration are concerned. However, M. elongata cristated shoots were quite stable at normal MS nitrogen concentration (Papafotiou et al. 2001) as opposed to E. pugniformis. ...
... The in vitro behavior of cristated Euphorbia pugniformis resembles that of cristated Mammilaria elongata (Papafotiou et al. 2001) as far as explant type and plant growth regulators effect on cristate shoot regeneration are concerned. However, M. elongata cristated shoots were quite stable at normal MS nitrogen concentration (Papafotiou et al. 2001) as opposed to E. pugniformis. ...
... For example lower BA concentration (0.44 lM) was needed for shoot formation in cristate form of M. elongata whereas higher BA concentrations induced normal shoots The same study showed that Murashige and Skoog (1962) medium supplemented with 1.07 lM NAA or 0.54 lM NAA and 0.44 lM BA induced 100% of inflated cristate shoots in shoot-tip explants. The medium supplemented with 0.89 lM BA also promoted 100% cristate formation, but induced 50% hyperhydricity (Papafotiou et al. 2001). ...
Fasciation (or cristation) is a variation in the morphology of plants, characterized by the development of various widened and flattened organs. According to origin, fasciations are classified as physiological or genetic but comparatively little is known on their epigenetic or genetic nature at the molecular level. Physiological fasciations are caused by natural environmental factors or artificial treatments including exogenously applied growth regulators. CLAVATA genes (CLV1, CLV2, and CLV3) have been shown to be the main genetic factors associated with fasciation. Despite the great variety of fasciation-induction factors, fasciations have similar features of development during the first few weeks, i.e., increased mitotic activity and size of the apical meristem and an altered arrangement of cells in the meristematic zones, often leading to an increased number of organs and changes in the plastochron. The enhanced activity of apical meristem and cambium results in a significantly increased circumference of the stem and enlarged proportions of pith and cortical parenchyma, associated with a delayed differentiation of the vascular tissues. An elliptical or irregular shape of the cross section of a fasciated organ corresponds to a similar shape of the vascular cylinder. Later stages of the ontogenic development of fasciations are species-specific, may depend on the origin of fasciation, and in some cases may lead to deviations from the normal structure of the epidermis, shape of leaves, as well as altered development of axillary buds. Studying the causes and patterns of development of fasciations could provide a better understanding of the growth processes in the vegetative apex. Further anatomical and physiological research should focus on the structure and activity of meristems of fasciated shoots, as well as on their transcriptome analysis, in order to better understand the pattern of fasciation development.
... This study was focused on in vitro micropropagation of Mammillaria species, albeit combining it with in vivo conservation of regenerated plants. We assume, as many micropropagators of these species have claimed (Vyskot and Jára 1984; Clayton et al. 1990; Rubluo 1997; PerezMolphe et al. 1998; Papafotiou et al. 2001; Giusti et al. 2002), that conservation of threatened species should be supported by tissue culture biotechnology. The in vivo conservation of Cactaceae requires an adequate space and extensive labor force. ...
... According to Vyskot and Jára (1984), Rubluo et al. (1993), Rubluo (1997), and Pérez Molphe et al. (1998, the in vitro micropropagation of different Mammillaria species take place on a medium with cytokinin concentrations from 4.4 to 46.4 μM. Nevertheless, there are also reports on in vitro propagation of Mammillaria species either in the absence of PGR or in the presence of auxin alone (Papafotiou et al. 2001). For example, Papafotiou et al. (2001) reported the propagation of M. elongata in the absence of PGR or in the presence of 0.54 μM NAA. ...
... Nevertheless, there are also reports on in vitro propagation of Mammillaria species either in the absence of PGR or in the presence of auxin alone (Papafotiou et al. 2001). For example, Papafotiou et al. (2001) reported the propagation of M. elongata in the absence of PGR or in the presence of 0.54 μM NAA. Rubluo et al. (2002) reported the propagation of M. sanangelensis on culture medium with auxin alone, achieving the maximum proliferation rate at 34.2 μM IAA. ...
Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human
activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained
using kinetin at two levels: 27.9 and 46.5μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number.
For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5μM IAA/kinetin, yielding 4.8
and 4.7 shoots per explant, respectively, in 60d. Rooting of regenerated shoots was achieved by leaving the explants in their
shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved
by planting them in peat moss substrate after a desiccation treatment at room temperature for 3d.
... This production is significant for the regeneration of the species and lets a fast increment of the number of plants, compared to the traditional propagation method. This multiplication rate is over that acknowledged for other species such as Mammillaria voburnensis Scheer and Mammillaria elongata DC. (Papafotiou et al., 2001;Pelah et al., 2002;Ordoñez, 2003); also, it is higher than that recorded for the Coryphantha and Echinocereus genus (Clayton et al., 1990) as well as in Astrophytum myriostigma Lem. (Villavicencio, 2009), in which a rate of 1:10:10:10 has been registered. ...
... Esta producción es significativa para la regeneración de la especie y permite incrementar rápidamente el número de plantas, en comparación con el método tradicional de propagación. Dicha tasa de multiplicación supera a las reconocidas para otros taxa, como Mammillaria voburnensis Scheer y Mammillaria elongata DC. (Papafotiou et al., 2001;Pelah et al., 2002;Ordoñez, 2003); además, es mayor a la registrada en los géneros Coryphantha, Echinocereus (Clayton et al., 1990) y en Astrophytum myriostigma Lem. (Villavicencio, 2009), en las cuales se consigna un valor de 1:10:10:10. ...
Se desarrolló un protocolo para la micropropagación de Epithelantha micromeris, cactácea en estatus de riesgo, que comprende cuatro etapas: establecimiento, multiplicación, enraizamiento y aclimatación, a fin de obtener plantas con tamaño uniforme en cantidad suficiente y con buena calidad fitosanitaria. El método de propagación presentado es eficiente respecto al tradicional y constituye una nueva tecnología de producción cuya aplicación es factible en el sistema-producto ornamental, bajo el esquema de laboratorio-invernadero. Las semillas de E. micromeris son quiescentes y pueden establecerse in vitro en el medio MS a 50%, en el cual registra un PG máximo de 60%, que supera a su homólogo adicionado con fitohormonas (AG3); esto significa que el taxón no requiere promotores para germinar. La inducción de brotes se logró mediante segmentos de epicotilo, como explantes, en medio de cultivo MS con diferentes tratamientos. Se determinó que el tipo y la concentración de fitohormona influyen en la tasa de multiplicación, pues se formaron hasta 15 brotes por explante; la cinetina (KIN) en interacción 10:1 con AIB en baja concentración es la promotora de ese efecto. Durante el enraizamiento in vitro se observó que la aplicación de 1.5 x 106 UFC mL-1 de Azospirillum brasilense tiene un efecto positivo en el proceso rizogénico: se originaron hasta nueve raíces por planta, con 2.3 cm de longitud. A partir de esta metodología es posible regenerar especies en estatus de riesgo de importancia ecológica para el Desierto Chihuahuense y optimizar los procesos biológicos para la producción de plantas de ornato.
... La propagación in vitro de cactáceas mexicanas ya se ha realizado en diversos géneros como: Mammillaria san-angelensis (Martínez-Vázquez y Rubluo, 1989), Aztekium ritteri (Rodríguez-Garay y Rubluo, 1992), Lophophora williamsii (Ortiz-Montiel y Alcántara-García, 1997), Cephalocereus senilis (Flores-León y Ortiz-Montiel, 2000;Choreño-Tapia, 2002), Turbinicarpus laui (Mata et al., 2001), M. elongata (Papafotiou et al., 2001), Pelecyphora aselliformis y P. strobiliformis (Pérez-Molphe-Balch y , P. aselliformis (Santos-Díaz et al., 2003). El presente trabajo describe por primera vez, el sistema de propagación in vitro de M. schiedeana schiedeana permitiendo su reproducción clonal en un corto tiempo y su establecimiento exitoso en condiciones ex vitro como una alternativa para su conservación. ...
... L -1 dependiendo de la especie, por otra parte, el enraizamiento de M. elongata se logró en medio MS + AIB 2 o 0.2 mg . L -1 (Papafotiou et al., 2001). ...
Se reporta por primera vez la micropropagación de la subespecie Mammillaria schiedeana schiedeana, cactácea mexicana considerada como amenazada de extinción por la Norma Oficial Mexicana. Se obtuvo la formación de brotes por activación areolar utilizando explantes longitudinales procedentes de brotes regenerados de un ciclo previo de cultivo y a partir de plántulas germinadas in vitro los cuales se sembraron en medio basal Murashige y Skoog (MS) suplementado con 6-bencilaminopurina (BA) y ácido α-naftalenacético (ANA) a diferentes concentraciones. La producción de brotes en el medio de proliferación se evaluó después de dos meses. La mejor concentración para la formación de brotes fue de 2.2/0.53 μM (0.5/0.1 mgl–1) a partir de plántulas germinadas in vitro y de 4.4/0.53 μM (1/0.1 mg.l–1) para los brotes previamente regenerados. Después de dos a tres meses, los brotes se individualizaron y subcultivaron a medio MS al 50% de sus componentes adicionado con carbón activado 1 g.L–1 para inducir el enraizamiento. La sobrevivencia de las plántulas en condiciones ex vitro fue del 83% después de cuatro meses de su transferencia a suelo.
... 7 A ocorrência do processo oxidativo pode ser proveniente da liberação de exsudatos do próprio explante, o que se relaciona com a liberação de compostos fenólicos pelo explante após a realização de "ferimentos" para sua inoculação (Santos, et al. 2001). Segundo Souza et al. (2011) (Wakhlu & Bhau, 2000;Papafotiou et al., 2001;Giusti et al., 2002;Poljuha et al., 2003;del Socorro Santos-Díaz et al., 2006;Garcia-Rubio & Malda-Barrera, 2010;Ivannikov et al., 2021). ...
O objetivo deste trabalho foi avaliar a desinfestação, propagação in vitro e subcultivo de ora-pro-nóbis. No primeiro experimento, realizou-se a desinfestação dos explantes com diferentes concentrações de hipoclorito de sódio (2%, 4%, 6%). No segundo, foi realizada a propagação de 600 segmentos nodais, sob diferentes concentrações de meio MS (MS, MS/2, MS/4) e combinações de fitorreguladores: 1) ANA (0,5μM) + BAP (0,5μM); 2) ANA (0,5μM) + BAP (1,0μM); 3) ANA (0,5μM) + BAP (1,5μM); 4) ANA (0,5μM) + BAP (2,0μM); 5) ANA (0,5μM), com e sem adição de carvão ativado. No terceiro, foi realizado o subcultivo em meio MS a partir dos calos desenvolvidos no segundo experimento, submetidos a três tratamentos: T1 - ANA (2μM) + BAP (4μM); T2 - ANA (0,1μM) + BAP (1μM) e T3 - ANA (0,5μM). A concentração de 4% de NaClO foi mais eficiente, apresentando 5% de contaminação. As maiores taxas de multiplicação de calos foram observadas em meio MS/2 sem adição de carvão, suplementado com 0,5μM de ANA e diferentes concentrações de BAP (1,0μM, 1,5μM e 2,0μM) com médias de 0,85 e 0,80 calos por tratamento. Em 47,16% dos explantes ocorreram contaminações fúngicas e bacterianas e 23,5% oxidação. Há a necessidade de desenvolver protocolos para o estabelecimento da ora-pro-nóbis in vitro como, por exemplo, alternativas para controle da oxidação, contaminação e concentrações adequadas dos reguladores de crescimento.
... (Papafotiou et al., 2001).Nilsson et al., (1996) установяват, че фасциирани тъкани са наблюдавани при Populus tremula x Populus tremuloides на среда, съдържаща повишено количество на свободни цитокинини. Kitin et al. (2005) и Papafotiou et al. (2001)съобщават за появата на фасциирани леторасли при Prunus avium и Mammillaria elongata, когато хранителната среда е снабдена с БАП. ...
Summary
Common ash (Fraxinus excelsior L.) is important forest and ornamental tree species.
The aim was to find suitable methods for autovegetative and heterovegetative propagation of common ash and some its cultivars.
1. Autovegetative propagation
Semi-hardwood cuttings from 2-years-old seedlings and 45-years-old individuals were used. Common ash cuttings were taken on 10th, 20th and 30th of July and rooted in greenhouse. Using of auxins was essential for achieving strong root system and good rate of rooting in cutting from seedlings. The auxins indole-3-butyric acid (IBA) (0.6 0.8 and 1.0%) and -naphtalene acetic acid (NAA) (0.2, 0.4 and 0.6 %) were applied like powder to the basal part of the cuttings, before inserting in the rooting substratum (peat and perlite 2:1 v/v). The best rooting percent (90.00 ± 5.00) was obtained with cuttings taken on 20th of July and treated with 0.6% NAA.
2. Heterovegetative propagation
Grafting was carried out using 2 and 4-years-old rootstocks of Fraxinus oxycarpa Willd. and 2-years-old rootstocks of Fraxinus excelsior. Scions were collected from Fraxinus excelsior ‘Globosa’, ‘Pendula’ and ‘Diversifolia’. Spring and autumn chip budding ware investigated, summer chip and T-budding on Narrow-leaved ash rootstocks and summer chip and T-budding of ‘Pendula’ and ‘Globosa’ on common ash rootstocks. Summer chip budding of ‘Pendula’ and ‘Globosa’ cultivars gave high viabilities (‘Pendula’ - 93.33 ± 3.33 %; ‘Globosa’ - 93.33 ± 3.33 % on common ash rootstocks and ‘Pendula’ - 76.67 ± 4.41 %; ‘Globosa’ - 80.00 ± 8.66 % on Narrow-leaved ash rootstocks) with sufficient number of produced shoots in good condition (3.00 ± 0.43 to 7.25 ± 1.10 for ‘Pendula’ and 1.93 ± 0.33 to 2.66 ± 0.42 for ‘Globosa’). In ‘Diversifolia’ it is better to graft in spring (42.67 ± 3.33 %).
3. In vitro propagation
Isolated embryos, taken from dry seeds of common ash were placed in vitro and developed into viable seedlings. Generally, the formation of normally developed seedlings was significantly better on half strength MS medium (65.00 ± 2.89 %) than the other media used (full and half strength variants of MS, DKW, WPM and Knop media). For this reason this medium could be recommended as the most suitable for the germination of common ash embryos. Embryo cultures can provide material for micropropagation in whole year.
Apical and nodal segments and bud explants were taken from mature trees, grafted plants and stump shoots from Fraxinus excelsior. Only 4 bud explants from mature tree of Fraxinus excelsior ‘Pendula’ were established in vitro. After 2 months endogenous contamination appears in cultures and they died.
Micropagation is possible by using explants taken from juvenile material (epycotyl and hypocotil segments harvested from in vitro obtained 45-days old seedlings and apical and nodal segments from 2-yars-old seedlings). For inducing of shoot formation, MS and WPM supplement with 6-benzylaminopurine (BAP)(3.0 or 4.0 mg l-1), thidiazuron (TDZ)(0.1, 0.5 or 1.0 mg l-1) and zeatin (2.0 or 5.0 mg l-1) were studied. Shoot proliferation occurred on all media. Increasing of TDZ concentration promoted multiple shoot formation but shoots were short and often vitrified. Addition of indole-3-butyric acid (IBA) (0.01 mg l-1) promoted shoot elongation. Induction of high multiplication rates by increased concentrations of BAP (4.0 mg l-1) in the media resulted not only in the appearance of vitrification but, in some cases, in formation of fasciated shoots together with normal shoots. The structure of normal and fasciated stems, that have developed in vitro, was studied in semi-thin sections of paraffin-embedded samples by conventional and polarized light microscopy. The vascular cylinder in fasciated stems was not circular but elliptical in cross sections and the volume of fasciated stems, appeared considerably larger that that of normal stems.
Adventitious root formation was studied on half-strength WPM, supplemented with IBA (0.5 mg l–1), -naphtalene acetic acid (NAA) (0.5 mg l–1) or in combination (0.5 mg l-1 IBA and 0.5 mg l-1 NAA). After 7, 14 or 21 days on the above inductive media the cultures were transferred on half strength, hormone-free WPM (expressive medium). The results were compared with half strength WPM without or with the above mentioned auxin concentrations on which the shoots were cultivated without transfer. The best percentage of rooting (96.67 ± 3.33 %) was achieved on inductive medium (for 14 days) with 0.5 mg l-1 IBA and 0.5 mg l-1 NAA. In vitro obtained rootstocks were successfully transferred to greenhouse for acclimatization.
... Fasciation has been found in over 100 species of vascular plants (Iliev, Kitin 2011). Exogenously applied cytokinins can induce fasciation in some plant species (Varga et al. 1988;Iliev 1996;Papafotiou et al. 2001). However, shoot fasciation has not yet been reported in V. rotundifolia. ...
An efficient plant proliferation and regeneration system via fasciated stems was established for the first time in Vitex rotundifolia L. Aseptic nodal segments were used as explants and cultured on Murashige and Skoog medium supplemented with different plant growth regulators alone and in different combinations to investigate the induction, proliferation, axillary shoot initiation and elongation from fasciated stems and subsequent regeneration. The obtained results show that the growth of fasciated stems can be regulated by cytokinins. A higher concentration of cytokinins induced both the formation of axillary shoots from normal stems and the development of fasciated stems. Anatomical analyses revealed that fasciated stems (5 to 25 mm wide) were much wider than normal in planta shoot stems (2 to 3 mm wide). The pith cells of fasciated stems developed laterally to 0.05-0.08 mm in width, while normal stems were usually 0.02 to 0.04 mm in width. Flow cytometry indicated no obvious changes in chromosomes among the four types of shoots. This is the first report on the development of axillary shoots from fasciated stems in V. rotundifolia tissue culture. Our protocol serves as a new form of clonal plant regeneration.
... Al analizar la supervivencia en condiciones de aclimatización, se puede apreciar y corroborar lo planteado por otros autores como Gárces (2003) y Papafotiu et al. (2001), quienes plantean que las cactáceas son plantas que se adaptan fácilmente a diversos cambios. En la figura N.º 7 se pueden apreciar los resultados elevados obtenidos. ...
Escobaria cubensis, comúnmente denominado "cactus enano de Holguín" es un cactus endémico de la provincia de Holguín, que se encuentra amenazado por la actividad antrópica. Este es el objeto de la presente investigación y se hace con el fin de establecer la metodología para la propagación in vitro de Escobaria cubensis (Britton Rose) Hunts, a través de la inducción de brotes vía organogénesis a partir de plántulas germinadas in vitro y de areolas, provenientes de mamilas. Se emplearon semillas colectadas en la localidad de Purnio, en el municipio de Holguín. Se logró una desinfección de las semillas botánicas con hipoclorito de sodio (2%) en inmersión doble durante cinco minutos, y de las areolas durante cinco minutos en bicloruro de mercurio (0.2%). Así como su posterior siembra en medio de cultivo MS suplemento con sacarosa (30 g.L-1). Se obtuvo un 95.64% de germinación en medio de cultivo MS (25%), se logró además un 66.67% de supervivencia en las mamilas. En la multiplicación se evaluó diferentes concentraciones de Benzilaminopurina y se determinó que el mejor resultado, con 5.1 brotes por explante, fue el medio que contenía 13.3 uM Benzilaminopurina + 5.4 uM ácido naftalenacético. A partir del cuarto subcultivo, el número de brotes por explante disminuye. En la fase de enrizamiento se empleó el medio basal MS con 8.1 uM ácido naftalenacético + 2.5 uM ácido indolbutírico, obteniendo como promedio 2.5 raíces por planta de cultivo; en la aclimatización se empleó el sustrato Zeolita + Lecho de Bambú + Suelo y se obtuvo un 93.5% de supervivencia.
... ), benzyladenine(Papafotiou et al., 2001;Kitin et al., 2005;Chiruvella et al., 2014) but not with thidiazuron (TDZ)(Mitras et al., 2009) and can be overcome by a high concentration of the TDZ or Zeatin(Fukai et al., 2000). ...
Plant tissue culture techniques have become an integral part of progress in plant science research due to the opportunity offered for close study of detailed plant development with applications in food production through crop improvement, secondary metabolites production and conservation of species. Because the techniques involve growing plants under controlled conditions different from their natural outdoor environment, the plants need adjustments in physiology, anatomy and metabolism for successful in vitro propagation. Therefore, the protocol has to be optimized for a given species or genotype due to the variability in physiological and growth requirement. Developing the protocol is hampered by several physiological and developmental aberrations in the anatomy and physiology of the plantlets, attributed to in vitro culture conditions of high humidity, low light levels and hetero-or mixotrophic conditions. Some of the culture-induced anomalies become genetic, and the phenotype is inherited by clonal progenies while others are temporary and can be corrected at a later stage of protocol development through changes in anatomy, physiology and metabolism. The success of protocols relies on the transfer of plantlets to field conditions which has been achieved with many species through stages of acclima-tization, while with others it remains a challenging task. This review discusses various adjustments in nutrition, physiology and anatomy of micro-propagated plants and field grown ones, as well as anomalies induced by the in vitro culture conditions. K Ke ey yw wo or rd ds s: : Plant physiology, plant regeneration, in vitro culture, somaclonal variation, hyperhydric-ity, fasciation, acclimatization ACTA BIOLOGICA CRACOVIENSIA Series Botanica 57/2: 9–28, 2015
... The appearance of fasciation in in vitro-propagated silver birch may have been caused by p-fluorophenylalanine (FPA), because no fasciation was observed in the absence of FPA (Srivastava & Glock, 1987). Exogenously applied cytokinins induced fasciation in Kalanchoe blossfeldiana (Varga et al., 1988), Betula pendula (Iliev & Tomita, 2003) Prunus avium (Kitin et al., 2005) and Mammillaria elongata (Papafotiou et al., 2001). ...
Epicotyl segments from in vitro 45-day-old seedlings were used as explants. For inducing axillary shoot formation, WPM supplemented with IBA (0.01 mg l(-1)), BAP (3.0 or 4.0 mg l(-1)), or TDZ (0.1 or 0.5 mg l(-1)) were applied. Multiplication rate was low after 8 weeks of cultivation. During this period, TDZ promoted a significantly higher rate of multiplication than BAP. After 16 weeks, the multiplication increased in all variants studied (mean number of shoots ranged from 4.80 +/- 1.59 to 11.33 +/- 2.33), but there were no significant differences between the cytokinins and their concentrations. Increasing of BAP concentration to 4.0 mg l(-1) promoted multiple shoot formation, but shoots were short and often vitrified, and in some cases, formation of both fasciated and normal shoots was observed. Conventional and polarized light microscopy revealed anatomical differences between fasciated and normal shoots. For example, the vascular cylinder in fasciated stems was not circular but elliptical in cross section. Compared to normal stems, the cortex in fasciated stems appeared less differentiated on the 16th week of cultivation, and the stem thickness was considerably larger. Adventitious root formation was studied on half-strength WPM, supplemented with either IBA (0.5 mg l(-1)), NAA (0.5 mg l(-1)), or a combination of IBA and NAA (0.5 mg l(-1) and 0.5 mg l(-1), respectively). After 7, 14, and 21 days on these inductive media, parts of the cultures were transferred on half-strength, hormone-free WPM (expressive medium). The highest percentage of rooting (96.67 +/- 3.33%) and the most developed root system were achieved on an inductive medium with the combination of IBA and NAA for 14 days, and then transfer to a expressive medium.
... The callus tissue retained its regenerative capability over a lengthy period (18 subcultures), and the regenerated plants were morphologically similar to the explant donor plants. Papafotiou et al. (2001) reported indirect shoot regeneration from calli formed in tubercle explants of Mammillaria elongata DC. cultured on MS medium with 1.1 μM NAA and 22.2 μM BA. Giusti et al. (2002) investigated the responses of explants from Escobaria minima (Baird) D.R. Hunt, Mammillaria pectinifera F.A.C. Weber and Pelecyphora aselliformis Ehrenb. ...
Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.
... Mammillaria elongata produce brotes en medio MS adicionado con 1.07 μM ANA y 11.09 o 22.19 μM BA. Sin embargo, al tratar de inducirlos a partir de brotes en forma de cresta, se obtuvieron brotes hiperhidratados (Papafotiou et al., 2001). ...
... orta que la concentración de sacarosa influyó en el potencial de formación del callo, así cuando se obtuvo callo este mismo fue subcultivado en medio con sacarosa al 7%. En el 2002, el mismo autor, produce callo de la misma especie (C. elephantidens (Lem.)) a través del tallo como explante y utilizando medio MS complementada con (2,4-D) y kinetina.Papafotiou et al., 2003 generan plantas del género Mammillaria, específicamente M. elongata, utilizando el medio MS y los reguladores ANA y BAP, así mismo generan formación de raíz a través del 3-Ácido Indol Butírico.El cultivo in vitro de tejido meristemático de Opuntia ficus-indica L., cultivar Villa Nueva ha demostrado ser un explante adecuado para la infec ...
... During the multiplication stage, the treatments with higher concentrations of BA (19.9 and 26.6 mM) induced both the formation of normal and fasciated shoots with more robust constitution. The fasciation or cristation refers to a phenomenon that produces morphological variation of plant organs (White 1948, Papafotiou et al. 2001, Iliev and Kitin 2011). The variation is the result of changes in the physiology of the plants produced by the environmental conditions or the effect of plant growth regulators, in particular cytokinins (BA), or changes in genetic factors such as the activation and expression of the CLAVATA family genes (Iliev and Kitin 2011). ...
Ortegocactus macdougallii is highly appreciated ornamental and endemic Mexican cactus recently classified as
a threatened plant species by the Government of Mexico. In order to rescue this plant from the risk of extinction
and because conventional systems produce low propagation rates, we established a reliable and efficient
method of micropropagation based in tubercle culture. During the establishment stage, varies concentracions
of the combination of NAA (1.3, 4.4, and 13.3 μM) and BA (1.3, 4.4, and 13.3 μM) induced callus and shoot
formation when explants (tubercles and axillary meristems or areoles dissected from adult plants) were incubated
on Murashige and Skoog medium. In adition to this, the NAA free variants of the medium stimulated the
rooting during the establishment stage, in particular the treatments including low and medium concentrations
of BA (1.3 and 4.4 μM). During shoot proliferation, treatments including NAA (4.4 mM) and BA at 13.3, 19.9
or 26.6 mM produced between 5 and 6 shoots per explant; however, shoots obtained on medium with NAA
4.4 and BA 13.3 mM were statistically higher (5 mm). Rooting was produced on quarter- or half-strength MS
medium but half-strength MS medium supplemented with IBA (1.6 or 4.9 mM) resulted in higher percent of
rooted shoots (75%) and mean root number (8).
... A.F. Omar et al. 636 to the meristem and its immediate vicinity in fasciated stems has been suggested (Boke & Ross 1978). Previous reports demonstrated that exogenously applied cytokinins induced fasciation in Kalanchoe blossfeldiana (Varga et al. 1988), B. pendula (Iliev 1996), Mammillaria elongata (Papafotiou et al. 2001), and P. avium (Kitin et al. 2005). Also, it has been reported that fasciation in cotton is conditioned by GA 3 and genotype (Nadjimov et al. 1999). ...
Fasciation, a frequent phenomenon in Cactaceae, has been attributed to various causes. The present study reports on phytoplasma-induced fasciation in Euphorbia coerulescens (Euphorbiaceae), Orbea gigantea (Asclepiadaceae), Opuntia cylindrica (Cactaceae), and Senecio stapeliiformis (Asteraceae). DNA was extracted from symptomless and fasciated tissues and amplified by nested PCR using universal primers P1/P7 followed by R16F2n/R16R2 produced amplicons of 1.2 Kb. The nucleotide sequence analyses of the amplicons indicated that fasciated plants were infected by phytoplasma. Phylogenetic analysis placed the cacti fasciation phytoplasmas in 16SrII group. The hormonal content of symptomless and fasciated tissues including indole-3-acetic acid (IAA), kinetin (Kin), N-6-benzyladenine (BA), abscisic acid (ABA), and gibberellic acid (GA3) was quantified by high performance liquid chromatography (HPLC). The results indicated that fasciation in O. gigantea was correlated with the accumulation of Kin and IAA increasing five and two times, respectively, as compared to symptomless tissue. However, there was no consistent pattern of hormones in other fasciated species (E. coerulescens, O. cylindrica, and S. stapeliiformis), suggesting that different plant species might have different mechanism to develop fasciation associated with phytoplasma infection.
... Explants from adult plants showed a decrease of shoot regeneration capacity earlier in season, probably because adult plants cease growth earlier in summer compared to seedlings (Heide 1974). Seasonal influence of explant collection on establishment has been reported for other species as well (Papafotiou et al. 2001;Romano et al. 2002). ...
A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures
initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody
Plant Medium (WPM) salts, with 100mgl−1 myoinositol, 1mgl−1 thiamine, 0.5mgl−1 pyridoxine, 0.5mgl−1 nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for
their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44μΜ BA. IBA at 9.84μΜ in
the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting
results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material
of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized exvitro in soil from the natural
environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in
a compost–perlite (2:1 v/v) medium (up to 36%).
... Predieri et al. [18] observed the obvious effect of the season on the regeneration potential of the calli derived from in vitro cultured ovaries of some day-neutral strawberry varieties. The influence of season on the shoot regeneration ability of explants was reported in many other species, such as Mamillaria elongata [16], Gloxinia sp. [5], Dendrobium sp. ...
In order to develop a protocol for high efficiency in vitro propagation of two intergeneric Fragaria x Potentilla varieties, ‘Serenata’ and ‘Pink Panda’ respectively, the influence of season on the rate of multiplication was investigated in shoot cultures on Murashige and Skoog (MS) and Lee and Fossard (LF) media, supplemented with different combinations of growth regulators. In vitro performance of explants indicated a positive correlation between shoot proliferation and season in both genotypes of ornamental strawberry. The mean number of shoots formed per explant was higher when ‘Serenata’ and ‘Pink Panda’ varieties were subcultured on MS or LF media, in the active growing season, irrespective of the culture medium composition. In both ornamental strawberry varieties, the mean number of shoots formed per explant was slightly higher when subcultured on MS medium, in the spring and summer season, as compared to LF medium, which was proven to be the most effective in the cold season.
... Furthermore, analogously to sur1 and yucca mutants (Boerjan et al., 1995; Zhao et al., 2001), explants of the stf seedlings could be cultured on medium lacking phytohormones, whereas wild-type explants died under these conditions. In this respect, it is noteworthy that in-vitro-cultured explants of fasciated mutants of A. thaliana (Mordhorst et al., 1998) and Mammillaria elongata (Papafotiou et al., 2001) showed a different behaviour than the wild types, suggesting that in these genotypes a genetic mechanism might operate through a hormonal imbalance (Gorter, 1965; Boke and Ross, 1978). Hypocotyl disintegration concomitant with adventitious root development further supported that stf overproduced IAA. ...
Plant lateral organs such as leaves arise from a group of initial cells within the flanks of the shoot apical meristem (SAM). Alterations in the initiation of lateral organs are often associated with changes in the dimension and arrangement of the SAM as well as with abnormal hormonal homeostasis. A mutation named stem fasciated (stf) that affects various aspects of plant development, including SAM shape and auxin level, was characterized in sunflower (Helianthus annuus).
F1, F2 and F3 generations were obtained through reciprocal crosses between stf and normal plants. For the genetic analysis, a chi2 test was used. Phenotypic observations were made in field-grown and potted plants. A histological analysis of SAM, hypocotyl, epicotyl, stem and root apical meristem was also conducted. To evaluate the level of endogenous indole-3-acetic acid (IAA), a capillary gas chromatography-mass spectrometry-selected ion monitoring analysis was performed.
stf is controlled by a single nuclear recessive gene. stf plants are characterized by a dramatically increased number of leaves and vascular bundles in the stem, as well as by a shortened plastochron and an altered phyllotaxis pattern. By histological analysis, it was demonstrated that the stf phenotype is related to an enlarged vegetative SAM. Microscopy analysis of the mutant's apex also revealed an abnormal enlargement of nuclei in both central and peripheral zones and a disorganized distribution of cells in the L2 layer of the central zone. The stf mutant showed a high endogenous free IAA level, whereas auxin perception appeared normal.
The observed phenotype and the high level of auxin detected in stf plants suggest that the STF gene is necessary for the proper initiation of primordia and for the establishment of a phyllotactic pattern through control of both SAM arrangement and hormonal homeostasis.
A mass micropropagation method was developed for Mammillaria vetula ssp. gracilis cv. Arizonica Snowcap, a cactus species with high ornamental value. A culture media based in MS medium (Murashige and Skoog, 1962) supplemented with 0.1 mg l − 1 NAA + 0.3 mg l − 1 KIN + 5.0 mg l − 1 GA 3 + 30 g l − 1 sucrose (MSM-1), was developed specifically for the establishment and multiplication of Mammillaria vetula cv. Snowcap offshoots, let us to produce a proliferation rate of 34.9 ± 5.9 new offshoots per explant. The non-rooted offshoots were rooted in MSM-2, consisting in MS salts supplemented with 0.1 mg l − 1 NAA + 0.3 mg l − 1 KIN plus 5 mg l − 1 ancymidol and a lower dose (10 g l − 1 ) of sucrose, with a rooting rate of 100%. The rate of acclimatization of the rooted offshoots was 100%. The full process of micropropagation from aseptic establishment in vitro to the end of plant acclimation takes approximately between 5 and 6 months and the plant production can be maintained continuously during all year.
This study intended to develop an efficient in vitro micropropagation technique for the critically endangered ornamental cactus Mammillaria herrerae Werdermaan and to assess the phytochemical profile, antioxidant, and enzyme inhibitory activities of callus and shoot cultures. The greatest shoot induction (99.2 ± 0.8%), with more multiple shoots (15.4 ± 2.1), and maximum shoot length (0.83 ± 0.12) were achieved on nutrient medium Murashige and Skoog (MS) with N⁶-furfuryladenine (6-KN, 2.5 µM) and 3-indolebutyric acid (IBA, 1.0 µM). The maximum frequency of callus induction (100%) and callus growth (5.37 ± 0.22 FW g/100 mL) were obtained on a nutrient medium MS with 1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea (TDZ, 10.0 µM) and 2-(1-naphthyl) acetic acid (NAA, 5.0 µM). The highest number of roots (8.1 ± 1.6) and maximum mean length of root (4.2 ± 0.5) and shoot (1.4 ± 0.2 cm) were observed on a rooting medium containing IBA (0.5 µM). The in vitro-developed M. herrerae plantlets were acclimatized in the greenhouse with 92% survival. Shoots containing a higher number of phytochemicals than callus cultures. Thirty-five compounds were found in shoot extract and twenty-six compounds in callus extract. Also, the total flavonoid content of shoot (0.96 mg RE/g extract) was higher than callus (0.41 mg RE/g extract). The shoot extracts displayed the best metal chelating and acetylcholinesterase inhibitory activities. The protocol developed for M. herrerae could be utilized for bioactive compounds production, ex situ conservation, and commercial clonal propagation of this rare ornamental cactus.
Astrophytum asterias es una cactácea endémica de Nuevo León y Tamaulipas en México, y del sur de Texas en Estados Unidos, está considerada como vulnerable por la IUCN, se encuentra en el Apéndice I de la CITES y está listada como en peligro de extinción por la NOM-059-SEMARNAT-2010, por la destrucción de su hábitat, la colecta ilegal y limitantes biológicas como un lento crecimiento, una lenta maduración sexual, una obligada xenogamia, y una baja calidad en la polinización. Por lo cual son necesarias medidas que amortigüen el descenso de sus poblaciones naturales; una alternativa que ha resultado efectiva en distintas especies amenazadas, como las cactáceas, es el Cultivo de Tejidos Vegetales. En el presente estudio fue posible establecer las condiciones experimentales que permitieron la desinfección de semillas, la germinación in vitro y la posterior regeneración in vitro de A. asterias. Explantes (apicales, hipocótilos y raíces) obtenidos de 33 plántulas germinadas in vitro fueron sembradas en frascos con medio MS (Murashige y Skoog, 1962) adicionado con BAP/ANA 2/0.5 mg/l; KIN/ANA 2/0.5 mg/l y sin reguladores de crecimiento vegetal (CONTROL); con 11 réplicas por tratamiento. Las respuestas a los 12 meses de iniciada la inducción fueron: a) Formación de callo de color verdoso a marrón principalmente friable, pero en ocasiones compacto de color blanco o hialino, b) Respuesta organogénica principalmente de tipo indirecta a través de callo friable en el caso de los explantes ápice e hipocótilos, pero en ocasiones directa en el caso de raíces en el tratamiento control. La presencia de RCV no fue necesaria para el surgimiento de brotes, sin embargo al estar presentes aceleraban la aparición y el crecimiento de estos. La menor cantidad de brotes en explantes de tipo apical (22 brotes) y la aparición abundante de callo previa a la organogénesis, sugiere que A.asterias es una especie con una alta concentración endógena de auxinas. En el tratamiento CONTROL se obtuvo el mayor número de brotes con un total de 82, seguido del tratamiento BAP/ANA con 74 brotes. El explante con una mayor cantidad de brotes totales a través de los tres tratamientos fue el explante hipocótilo con 101 brotes. El explante que regeneró una mayor cantidad de brotes por tratamiento fue el explante hipocótilo del tratamiento control con 60 brotes seguido del explante hipocótilo del tratamiento BAP/ANA con 41 brotes. Los brotes más consolidados se individualizaron y se subcultivaron a frascos con medio MS a los que se les cambiaron las tapas plásticas por películas plásticas con un filtro de 0.02 μm, con el objetivo de reducir la hiperhidratación. Los resultados obtenidos en el presente estudio aportan al conocimiento y permiten observar el establecimiento de un método efectivo, pero mejorable, para la regeneración y conservación de A. asterias.
As a result of the work specific components needed to design a domestic ELISA test system for detecting Potato virus M were produced. The system is an integral part of the effective control of seed potato material at all stages of cultivation.
Our research goal has been to find the optimal nutrient media for initiation of the primary callus in the species of the Cactaceae family. Common methods of plant biotechnology were used. Primary explants of the cacti were cultivated on Murashige and Skoog medium (MS medium). The content of macro- and microelements has been diluted twice (½ MS) and the vitamins (B1 and B6 – 0.5 mg/l, PP – 1 mg/l) were added, as well as 100 mg/l meso-isonitol and 20 g/l of sucrose. It was determined that callus formation formed efficiently when cultivated on half MS media with 20 g/l sucrose, 3 mg/l 6- benzylaminopurine, 0,2 mg/l indole-3-acetic acid,0,1 mg/l α-napthaleneacetic acid and 5 mg/l ascorbic acid. It was discovered, that for initiation of tissue differentiation and cacti callus formation, high concentrations of cytokinine-active growth regulators are required.
Trabajos del Área Académica de biología de la UAEH enfocados a la Biología de la Conservación
Girdling was applied to 5-year-old potted beech individuals of early, intermediate and late phenological forms to block assimilate export from leaves. Phloem severance caused accumulation of soluble carbohydrates and starch in leaves and increased the C/N ratio. While the hexose content increased continuously until the end of the experiment, the sucrose and starch contents peaked earlier, depending on the plant's phenological features. Different rates of chlorophyll degradation and H2O2 and TBARS (thiobarbituric acid-reactive substances) production in different phenological forms implied that phloem girdling was the source of oxidative stress and, depending on the phenological form, accelerated leaf senescence to different degrees. The variable rate of the increase in soluble carbohydrate and starch content, characteristic of the different phenological forms, had different modifying effects on the antioxidant activity in leaves. Compared with the early phenological form, the late form was characterized by a smaller increase in H2O2 and TBARS content and delayed and slowed chlorophyll and carotenoid degradation. In conjunction with the larger increase in the activity of antioxidant enzymes (catalase, ascorbate peroxidase and superoxide dismutase) induced by carbohydrate accumulation and slower carotenoid degradation, these changes led to the late form having greater resistance to oxidative stress and slower senescence. © 2017 Polish Academy of Sciences and Jagiellonian University, Cracow.
A protocol which comprises four stages was made for the micropropagation of Epithelantha micromeris, a cactus in risk status, in order to obtain a good number of plants in a healthy phytosanitary condition, with uniform size to attain a successful acclimatization. The proposed propagation method is efficient compared to the traditional one. It is a new production technology which is feasible to apply in the ornamental product-system, under the scheme of laboratory-greenhouse. The seeds of this species are quiescent and can be established in vitro on a 50% MS medium in which it can get a maximum of 60% PG, which surpasses its counterpart supplemented with phytohormones (GA3); this means that the species does not need promoters for germination. The induction of shoots was obtained from epicotyl segments as explants in MS medium with different treatments. It was determined that the type and concentration of phytohormone has an influence upon the multiplication rate, generating up to 15 shoots per explant; kinetin (KIN) in interaction with AIB 10:1 in low concentration is the promoter of this effect. During the in vitro rooting it was observed that the application of 1.5 x 106 CFU ml-1 of Azospirillum brasilense has a positive effect on the rhizogenic process, generating up to 9 roots 2.3 cm long by plant. This methodology regenerates species in risk status of ecological importance for the Chihuahuan Desert and improves biological processes for the production of ornamental plants.
Euphorbia pugniformis, is a thornless small succulent that in its normal form has two to three rows of lateral shoots around the deepen tip of a main shoot. The laterals are cylindrical and bear tubercles with one leaflet each. The species has two cristate forms; one with a cristate central shoot and normal lateral shoots developed on it, and another with cristate lateral shoots only, without main shoot. As the cristate forms do not flower, the method for their propagation employed is by cuttings. In the present work a protocol for micropropagation has been developed. 80% of tubercle explants, from the tip of cristate lateral shoots, gave cristate shoots of both forms, when cultured on agar-solidified MS medium with 20 g l-1sucrose and 0.1 mg l -1 NAA and BA, while 20% gave normal shoots. 55% of in vitro regenerated cristate shoots rooted after three months culture on the initiation medium; 67% rooted when transferred to MS with 0.5 mg l-1 IBA. 95-100% of rooted shoots were acclimatized ex vitro in intermitted mist. During in vitro culture or after ex vitro establishment quite a lot of the cristate shoots reverted into the normal form. The degree of stability of cristate shoots in vitro was tested under low and high nitrogen concentrations. 75% reduction of the MS nitrogen salts concentration increased the percentage of cristate shoots regeneration to 90% and eliminated their reversion into normal form. 25 and 50% increase of nitrogen salts concentration reduced the percentage of explant cristate shoot formation to 60 and 20%, respectively.
Cactaceae is an American plant family found from Canada down to Argentina. Cacti have evolved anatomical and physiological adaptations, which allow them to grow and thrive under desert conditions. Therefore, cacti are a main part of the arid and semiarid landscape. The greatest cacti diversity, for genus and species (63 and 669, respectively), is located in Mexico, where approximately 78% of cacti is endemic. Cacti have been used since pre-hispanic times for food, medicines, fodder, and raw material. Furthermore, cacti are considered as one of the most important ornamental plants nowadays, given that they have beautiful flowers and low water requirements. Unfortunately, the meaningless exploitation, poachers and habitat destruction (for agriculture, grazing, housing development, etc.) have posed cacti on an unstable situation, near extinction. Pressure increases as time goes by, since more human developments are found everyday. Currently, more than 250 Mexican cacti are considered as threatened species. Cacti, usually, have a long life cycle and low growth rates, which are prone conditions for vulnerability. One of the main hindrances for cacti conservation is that they have a low multiplication rate, mostly in those species that are not asexually propagated. Cacti sexual multiplication has a low efficiency, and sometimes cacti seeds are very scarce. The precedent cacti multiplication dilemma has caused that enough cacti plants for reforestation are not easily available. In this context, in vitro plant tissue culture techniques are a feasible alternative to effortlessly propagate numerous cacti. These techniques use plant fragments, under lab and axenic conditions, to massively propagate cacti plants. The in vitro-obtained cacti can be hardened under greenhouse conditions, where they grow as wild type cacti. Plant tissue culture methods allow the fast asexual multiplication of cacti in a short time and in a reduced space, even starting from a scarce supply of plant material. Several successful evidences on cacti propagation using plant tissue culture protocols can be found in the literature; consequently these techniques may be valuable tools to overcome the cacti extinction.
Quercus euboica Pap., is a rare, endangered oak species, endemic in the Greek island Euboia. The species can be used as an ornamental plant in urban and suburban landscape. Its drought tolerance and capability to sprout again after fire and grazing makes it a desired species for reforestation in Mediterranean climates. A micropropagation method for the species was developed. Woody Plant Medium salts (WPM), with 100 mg/L myoinositol, 1 mg/L thiamine, 0.5 mg/L pyridoxine, 0.5 mg/L nicotinic acid and 3% sucrose was used as basal medium. The highest shoot multiplication rate was taken from one year old seedlings on basal medium supplemented with 1 mg/L BA. Seasonable influence of explants collection on establishment was observed; explants collected in beginning of May gave the highest multiplication rate compared to explants collected in July and September. IBA at 2 mg/L applied in the culture medium during the first week of culture followed by culture in hormone-free medium gave the best rooting results. Basal immersion of microshoots in IBA dense solutions, for various periods of time, followed by culture in vitro on WPM or ex vitro in soil, did not improve rooting in comparison to IBA added to the medium. Plantlets acclimatized at a higher percentage and had more vigorous growth in soil from the natural environment of the species than in a compost-perlite (2:1 v/v) medium.
Biotechnological tools have been successfully used with several plant species for improving existing cultivars and creating new ones. However, the application of such tools has been limited with ornamental plant species such as cactus. Cacti are known to possess a very slow growth rate and many of them are recalcitrant compared to other cultivated plant species thus creating problems in culturing or improving them. Some early work showed promise in establishing in vitro cultures with limited multiplication rates, but research findings recently indicate that Schlumbergera (Christmas cactus) and Rhipsalidopsis (Easter cactus) can be multiplied more rapidly via both axil-lary and adventitious shoots, and also via callus and somatic embryogenesis. These studies have discovered key factors that could enhance an understanding of morphogenesis in cacti. Studies were made on endogenous phytohormones and enzymes to elucidate the differences between the recalcitrant cultures and those with increased regenerative capacity of these plants. Recent studies have also shown that Agro-bacterium-mediated genetic transformation is possible and a transformation efficiency of about 23% in Rhipsalidopsis is reported. This transformation efficiency was based on transgenic shoots expressing the activity of the reporter β-glucuronidase gene (GUS) indicated by blue staining of the regenerated shoots. Transgenic shoots regenerated in these studies through callus phase. Transformation was confirmed in the selected transgenic callus lines by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for nptII gene). The stability of transgenic calli was confirmed through their continuous sub-culturing on media containing the selectable marker kanamycin followed by testing for kanamycin resistance, GUS analysis and Southern hybridization. The regeneration system adopted for transformation was reliable as shown by the stable regeneration of calli and shoots growing on media both on selection and control media. However, due to the slow growth rate of these plants, no attempts were made to verify transformation in the greenhouse-grown plants.
Propagación in vitro de un especie endémica cubana
Cereus peruvianus (Cactaceae) is an important medical plant. The study was carried out on callus induction of Cereus jamacaru f.monstrosus and Cereus hildmannianus fma monstrosa (Cereus peruvianus). Еxplants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (NAA), 2, 4-dichlorophenoxyacetic acid (2,4-D) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 1, 2, 3, 4 mg/l . The frequency of Callus induction of C. peruvianus was highest in medium containing 4 mg/l 2,4-D or 4 mg/l NAA. Areol was not effective for callus induction possitivly and regeneration.
Fasciated and normal stem segments of Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea were collected from the same plant and compared for their anatomy, water relations and genetic variations. Anatomical differences in terms of thickness of cuticle, vascular bundle, xylem and phloem were analyzed in both normal and fasciated stems. The mucilage cells were higher in the fasciated form of Opuntia microdasys than that in the normal form. Water status in terms of total water content (TWC), water deficit and relative water content (RWC) was influenced by fasciation. Genetic variations were tested in normal and fasciated stems using randomly amplified polymorphic DNA (RAPD) fingerprints and SDS-PAGE of soluble protein extracts. SDS-PAGE protein and RAPD analysis confirmed that normal and fasciated tissues were genetically different. Polymerase chain reaction (PCR) yielded different polymorphic banding patterns that were unique to each primer and distinguishable over all samples. The PCR results of normal and fasciated samples were significantly different in cases of primers P1, P2 and P3. These results indicate that occurrence of fasciation in Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea is an epigenetic mutation of tissues.
A combination of Murashige and Skoog’s medium and N
6−benzyladenine (BA) at various concentrations (0, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg l−1) was supplied to shoot tips from root cuttings of a 50-year-old wild-cherry tree (Prunus avium). The concentration of BA in the growing medium was a determining factor with respect to the number of proliferated shoots per explant in vitro.Normal and fasciated shoots were generated when BA was present at 0.5, 0.75, 1.0 and 1.25 mg l−1 in the medium and the mean numbers of normal shoots per explant were 3.63, 5.37, 8.93 and 7.30 respectively, and those of the fasciated shoots per explant were 0.03, 0.1, 0.47 and 0.4 respectively. Anatomical analysis by confocal microscopy of sections of paraffin-embedded specimens revealed that the cell structure and organization of the cortex and vascular cylinder in the fasciated shoots was similar to that in normal shoots. However, the cross-sectional area of stem of the fasciations was apparently greater than that of the normal shoots. In particular, the volume of vascular tissues, of pith and of some individual parenchyma cells in the cortex and pith was apparently greater in fasciated shoots than in normal shoots. Increases in cytokinesis and morphogenetic activity, such as the development of callus-like regions and the formation of adventitious shoots, were observed in the cortex and pith throughout the fasciations. The fasciated shoots had numerous buds and initiating new shoots at their apices while normal shoots had a single dominant axial bud.
Damos noticia de la existencia de un nuevo cultivar, encontrado en la localidad de Buñol (Valencia) a partir de ejemplares naturales, Sedum sediforme "Monstruosa", y citamos dos variedades hortícolas de especies de la familia Cactaceae, tribu Opuntioideae, escapadas de cultivo en la provincia de Valencia: Austrocylindropuntia subulata (Müehlenpfordt) Backeberg ¿Cresta¿ y Opuntia cylindrica (A. L. Jussieu ex Lamarck) DC. ¿Monstruosa¿ We cite one new cultivar, found in Buñol (Valencia, E. Spain), Sedum sediforme "Monstruosa", and two known cultivars naturalized, that belong to the Cactaceae family, tribe Opun-tioideae: Austrocylindropuntia subulata (Müehlenpfordt) Backeberg ¿Cresta¿ and Opuntia cylindrica (A. L. Jussieu ex Lamarck) DC. ¿Monstruosa¿.
In vitro propagation of Mammillaria elongata DC plants was successful using tubercle explants grown on a medium based on Murashige and Skoog’s high salts supplemented with various auxins and cytokinins. Optimum callus proliferation occured in response to 2,4-dichlor-ophenoxyacetic acid (2,4-D) (2-10 mg/liter) with either kinetin or 6-(dimethylallylamino)-purine (2iP) (1-2 mg/liter). Root initiation was optimized with either napthaleneacetic acid (NAA) or indolebutyric acid (IBA) (60 mg/liter). Shoot initiation was optimized by addition of 2iP (10 mg/liter) and indoleacetic acid (IAA) (1 mg/liter). The auxin:cytokinin balance required for shoot initiation appears to be unique for each species of Mammillaria studied. Shoots developed in vitro of M. elongata were successfully transferred to greenhouse conditions, where they rooted and continued to grow.
We have altered the growth and development of a deciduous forest tree by transforming hybrid aspen (Populus tremula x Populus tremuloides) with the Agrobacterium rhizogenes rolC gene expressed under the strong cauliflower mosaic virus 35S promoter. We demonstrate that the genetically manipulated perennial plants, after a period of dormancy, maintain the induced phenotypical changes during the second growing period. Furthermore, mass-spectrometrical quantifications of the free and conjugated forms of indole-3-acetic acid and cytokinins and several gibberellins on one transgenic line correlate the induced developmental alterations such as stem fasciation to changes in plant hormone metabolism. We also show that the presence of the RolC protein increases the levels of the free cytokinins, but not by a process involving hydrolysis of the inactive cytokinin conjugates.
The disease caused by Corynebacterium fascians can be imitated by treatment with kinetin. Cultures of the bacteria were grown on a purine-free medium of known composition. They require thiamine. Two assays for cytokinin were developed; one depends on the release of lateral pea buds from apical dominance and is highly specific but of only moderate sensitivity. The other depends on the retention of chlorophyll in senescing oat leaves and is very sensitive (detecting 2 × 10–4μg of kinetin equivalents), though it is somewhat less specific and, therefore, requires care in usage. Both give results in 3–4 days and are thus far more rapid than tissue culture methods. With these assays C. fascians has been shown to produce a chloroform-soluble cytokinin active in both tests. The substance is stable to heat in acid or basic media, is soluble in non-polar solvents, and behaves as a base. It is precipitated from water solution by Ag ions and may, therefore, be a purine derivative. Pea tissue infected with C. fascians, but not uninfected tissue, yields a compound of similar solubilities and biological activity. Reasons are given for believing that the synthesis of cytokinins may be important, not only for C. fascians, but also for many other plant parasites.
A main problem in the vegetative propagation of ornamental plants in vitro is the epigenetic instability of cells removed from their organized environment. With calluses of leaf explants of Kalanchoe blossfeldiana Poelln., cv. Yucatan, the role of plant growth regulators (PGRs) in the occurrence of fasciation was studied.
In various combinations of auxins and cytokinins, the auxin 2,4-D (2,4-dichlorophenoxyacetic acid) gave only deformed, inseparable shoot primordia. The most rapid callus induction with regeneration of well-developed sprouts was obtained with the natural IAA (indoleacetic acid) and Z (zeatin).
As a first symptom of fasciation, aberration in decussate phyllotaxis can be observed. At increasing concentrations of IAA + Z, this symptom gradually decreased but fasciation proper increased. The optimum concentration was at 1 μM for both PGRs. Reduction of exposure to the PGRs from six to three weeks reduced the epigenetic instability.
In vitro studies on Mammillaria elongata var. tenuis
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