No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A highly efficient in vitro propagation protocol for elephant tusk cactus: Coryphantha elephantidens (Lem.) Lem
Abstract and Figures
Background: Elephant tusk cactus Coryphantha elephantidens (Lem.) Lem. is an important attractive ornamental cactus. The plant produces offshoots from tubercles very rarely, and the seedlings exhibit slow growth and susceptibility to damping off. Slow growth and high demand in the cactus industry lead to finding an alternate fast propagation method.
Results: An innovative in vitro technique based on axillary bud proliferation has been developed for an ornamental cactus C. elephantidens (Lem.) Lem. Four different explant types formed multiple shoots on Murashige and Skoog (MS) medium. Of the two cytokinins, 6-Benzylaminopurine (BAP) and Kinetin (KN), BAP proved to be more effective for multiple shoot induction and shoot growth from different explant types. Longitudinally cut stem explants, when cultured on MS medium supplemented with 6.6 μM BAP give maximum axillary shoot proliferation (12.4 shoots). Type of explant significantly influenced the micropropagation rate. Type of carbon source used in the medium imparted a profound effect on shoot growth and dry weight. The maximum dry weight gain of the shoot was observed with 9% sucrose.
Conclusion: Development of an efficient micropropagation protocol which can be used to produce more than 10,000 rooted plantlets in 150 days from a single longitudinally divided shoot explant.
Figures - available via license: Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Content may be subject to copyright.
... 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). ...
... Several disinfecting agents were applied for cacti surface sterilization, such as sodium hypochlorite (present in commercial bleach and other reagents), which has been widely used; however, its efficiency remains low. Combining sodium hypochlorite with ethanol appeared to be more effective to neutralize the exogenous microbiota of several cacti species; thus, this procedure has been widely employed (Estrada-Luna et al., 2008;Bhau and Wakhlu, 2015). In Coryphantha elephantidens, three-step disinfection was used for surface sterilization of young shoots, that is, thorough washing with tap water and 1% of detergent, a 1 min soaking in ethanol (70:30 v/v), and immersion in sodium hydrochloride for 15 min (Bhau and Wakhlu, 2015). ...
... Combining sodium hypochlorite with ethanol appeared to be more effective to neutralize the exogenous microbiota of several cacti species; thus, this procedure has been widely employed (Estrada-Luna et al., 2008;Bhau and Wakhlu, 2015). In Coryphantha elephantidens, three-step disinfection was used for surface sterilization of young shoots, that is, thorough washing with tap water and 1% of detergent, a 1 min soaking in ethanol (70:30 v/v), and immersion in sodium hydrochloride for 15 min (Bhau and Wakhlu, 2015). The aseptic culture of Opuntia robusta cladodes was obtained by soaking in a biocide solution (comprising 3 mg/L benlate, 3 mg/L captan, 1 ml/L previcur, 0.5 g/L amoxicillin, and 0.4% of ketoconazole) before the application of hypochlorite solution. ...
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.
... This is in agreement with the earlier report by Clayton et al. (1990) in which the addition of concentration more than 1.0 mg/l IAA to the BAP supplemented medium inhibited the rate of multiple shoot formation. In contrast, Bhau and Wakhlu (2015) and Khalafalla et al. (2007) found high shoot number per explant on 1.5 and 5 mg/l BAP, respectively. Due to the occurrence of high BAP concentration in medium, which decreases the role of endogenous auxin in stimulating cell elongation in salmon and AL Dabagh, the intention for the small numbers of shoots at high BAP concentration in medium (2000). ...
... Shoots with a high concentration of BAP responded to a deterioration in the shoot multiplication rate and shoot length similarly observed that following to reduction in the occurrence of BAP, nearby shoots were developed as normal shoots. Our finding on the effect of BAP on the shoot proliferation was contrary to other researchers such as Bhau and Wakhlu (2015), Martinez-Vazquez and Rubluo (1989), and Akram et al. (2013) who obtained the best multiplication rate and shoot length on the high concentration of BAP that was between 1.5 and 5 mg/l BAP. All different types of explants had a drop in the number of shoots as a result of the medium's higher cytokinin concentrations (BAP > 1 mg/l). ...
... Nowadays, humans use 57% of cacti diversity for horticultural, nutritional, medicinal, fodder, and handicraft manufacture (Goettsch et al., 2015). Plant cell, tissue, and organ culture technology has demonstrated to be a powerful tool for the micropropagation of cacti species (Angulo-Bejarano and Paredes-L opez, 2011;Bhau and Wakhlu, 2015;Elias et al., 2015;Wakhlu and Bhau, 2000), and for the production/elicitation of active plant metabolites (Dias et al., 2016;Hussain et al., 2012;Wang et al., 2017). Research focused on the generation of callus and cell suspension cultures, for the production of high-value secondary metabolites from medicinal plants has been reported in the last few years (Efferth, 2018;Yue et al., 2016); However, despite its medicinal properties, few studies have focused on callus induction for the study and production of metabolites from traditionally used cacti species (de Oliveira and Machado, 2003;Jacomini et al., 2015;Pires da Silva Assis Machado et al., 2004). ...
... Research focused on the generation of callus and cell suspension cultures, for the production of high-value secondary metabolites from medicinal plants has been reported in the last few years (Efferth, 2018;Yue et al., 2016); However, despite its medicinal properties, few studies have focused on callus induction for the study and production of metabolites from traditionally used cacti species (de Oliveira and Machado, 2003;Jacomini et al., 2015;Pires da Silva Assis Machado et al., 2004). Studies devoted to callus induction on cacti species have been carried out only to achieve systems for plant regeneration, including Coryphantha species (Angulo-Bejarano and Paredes-L opez, 2011;Bhau, 1999;Bhau and Wakhlu, 2015;Elias et al., 2015;Smith et al., 1991;Wakhlu and Bhau, 2000). Secondary metabolites obtained from tissue cultures, can be generated on a continuous production system, and without environmental constraints, since physical conditions such as temperature, light regime, nutritional availability, pH and so on, can be controlled under in vitro conditions (Hussain et al., 2012;Kozai et al., 1997). ...
Plant cell, tissue, and organ culture have become a powerful technology for the production of biomass, and for the research and biosynthesis of a variety of secondary metabolites. In this work, we report an efficient method for friable callus induction applied to the medicinal cactus Coryphantha macromeris, its kinetic behavior, and its phytochemical profile, assessed at the maximum biomass production phase. Callus cultures were obtained from stem discs inoculated on Murashige and Skoog medium (MS) supplemented with 6-ben-zylaminopurine (BAP; 2.2 mM) and picloram (4.14 mM). The highest biomass production (20.65 g DW L À1) was achieved at nine weeks of culture. Then, the phytochemical profile was analyzed using Ultra-High-Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). Chro-matographic and mass spectrometric analyses indicated the presence of 61 metabolites, with 52 being identified. Among these compounds, 11 organic acids, 16 phenolic acids, 8 flavonoids, and 17 metabolites of different classes were identified. Our results could significantly contribute to the current knowledge of tissue culture of cacti species, as well as the potential applications of the in vitro callus culture of C. macromeris. Furthermore , we report the presence of some metabolites in cell culture of cacti species and their fragmentation pattern for the first time.
... A high survival rate, similar to that recorded in Melocactus glaucescens (Resende et al., 2010), was observed in our experiment. A similar pattern was also recorded during the acclimatization of cacti of the genera Escobaria, Mammillaria, and Pelecyphora, cultivated in vitro (Giusti et al., 2002), and in micropropagated Coryphantha elephantidens seedlings (Bhau and Wakhlu, 2015). ...
Melocactus sergipensis, Melocactus violaceus, and Melocactus zehntneri are under threat of extinction. Their wild populations are exploited by predatory extraction, because the potential as ornamental plants. Thus, alternatives for sustainable production and conservation are needed. The present study investigated the influence of artificial (growth room) and natural light (greenhouse) on the in vitro germination and growth of these three Melocactus species, and their effects during the acclimatization. Plants were established in vitro and cultivated for 56 days under two lighting conditions (absence/ presence of light) and two light intensity (artificial light: 40 µmol m⁻² s⁻¹/ natural light: 160 µmol m⁻² s⁻¹). The samples were acclimatized in a greenhouse. The data were analyzed using an Analysis of Variance and compared by Tukey's test (P<0.05). The seeds of all three species depend on light for their germination, with natural light being the most effective environment, resulting in higher germination and growth rates. The seedlings produced in vitro, under natural light, also presented higher survival and growth rates during acclimatization, in particular M. zehntneri. The natural light in the greenhouse is viable for the production of M. sergipensis, M. violaceus, and M. zehntneri seedlings, from in vitro germination to the acclimatization.
... Debido a esto los RCV más usados han sido las citocininas en concentraciones relativamente altas (BAP, KIN) en combinación con auxinas (ANA, AIA) o en ausencia de estas. Los resultados obtenidos han sido diversos desde la obtención de brotes mediante organogénesis directa e indirecta, la obtención de embriones somáticos, hasta la obtención de un protocolo de alta eficiencia que permitiría obtener 10,000 plantas en 150 días en Coryphantha elephantidens (Bhau y Wakhlu, 2015); lo que demuestra que es posible la propagación masiva de cactáceas mediante CTV. Diversos ejemplos se muestran en la tabla 1. ...
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.
Edithcolea grandis is a succulent with branched, glabrous stems and pale yellow and maroon-purple flowers distributed in Yemen and sections of Africa, that is edible but there is greater potential in the commercial cultivation and trade of the succulent as an ornamental. The purpose of this current study was to establish an efficient protocol for the micropropagation of E. grandis by using stem cuttings. Young shoots were initiated for 6–8 weeks and then cultivated on two different types of multiplication media for 8 weeks to promote shoot multiplication. Shoots were transferred to Murashige and Skoog media augmented with various auxins: hormone-free, 1-Naphthaleneacetic acid (NAA), Indole-3-acetic acid and Indole-3-butyric acid to investigate the effect of each on root formation. The effect of various concentrations of NAA (0.5, 1.0, 1.5 and 2.0 mg/L) on root formation was evaluated. Medium B showed a better response in shoot growth producing 2.94 ± 0.142 shoots per explant, shoot length of 4.87 ± 0.101 cm and 100% shooting response. NAA generated the greatest number of roots (4.52 ± 1.047) and the highest rooting percentage (68%). No significant differences (p ≤ 0.05) were detected in the number of roots per shoot in varying concentrations of NAA. However, 2 mg/L NAA generated the greatest number of roots (23.09 ± 4.559) with a rooting percentage of 87.0%, followed by 1 mg/L NAA which produced 20.52 ± 2.296 roots with a rooting percentage of 95.7%.
In micropropagation, potassium nitrate (KNO3), an ACS reagent grade chemical, used in the preparation of growing mediums is expensive and its procurement depends on bureaucratic procedures, as it is controlled by the Brazilian Army. This research to assessed the effect of replacing the ACS KNO3 for a commercially available fertilizer (KNO3- based) on the micropropagation of the prickly pear cactus (Opuntia stricta (Haw.) Haw. cv. Elephant Ear. Treatments used six different fertilizer concentrations (0, 0.5, 1, 1.5, 2 and 2.5 g L-1) and a control consisting of 1.9 g L-1 KNO3, as shown in the MS salts. The survival, size and number of sprouts and the value of fresh biomass were evaluated. After seedling acclimation, we assessed the survival, number of sprouts, length, and number of roots, racket formation, average fresh biomass mass, macronutrient absorption and morphological changes of the seedlings. Explants inoculated with fertilizers at concentrations of 0.0; 2.0 and 2.5 g L-¹ did not grow. The response of explants at concentrations of 0.5 and 1.5 g L-1 of the fertilizer were the same as those developed in a KNO3 medium, and at a concentration of 1.0 g L-1, in all variables, the means were higher than those of the control medium. Therefore, it showed the feasibility of using fertilizers in the in vitro cultivation of the prickly pear cactus, which may remove bureaucratic barriers and reduce product costs by 99.12%.
Hershkovitz, M. A. & Zimmer, E. A.: On the evolutionary origins of the cacti. – Taxon 46: 217‐232. 1997. – ISSN 0040‐0262.
Understanding evolutionary responses of plants to desert environments depends upon phylogenetic knowledge of desert plants. The diverse American desert family Cactaceae has been presumed, on the basis of distinctiveness, to be phylogenetically isolated and relatively ancient (> 65 million years old). Using maximum likelihood and parsimony analyses of the rapidly evolving internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA), we show that the cacti are phylogenetically nested among other aridity‐adapted lineages of the angiosperm family Portulacaceae. The ITS divergence between pereskioid cacti and the genus Talinum (Portulacaceae) is less than that between many Portulacaceae genera. Synthesis of the ITS data with morphological and chloroplast DNA evidence suggests an origin of cacti in mid‐Tertiary, C. 30 million years ago, and a later Tertiary diversification coincident with development of the American desert. This, in turn, implies that the diversification rate in cacti was much higher than in their nearest relatives. The present results illustrate the central role of phylogenetic reconstruction in ecological and evolutionary theory.
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.
Summary A cell suspension culture of poplar (Populus deltoides (Marsh.) Bartr. var.occidentalis Rydb.), accumulating the anthocyanin pigment, cyanidin 3-glucoside, in the lag phase of culture growth, was subjected to
osmotic stress with glucose and mannitol. Osmotic stress treatments resulted in growth suppression and higher anthocyanin
accumulation compared with unstressed cells. Both an increase in the proportion of pigmented cells and an increase in the
concentration of anthocyanin in the pigmented cells were responsible for high anthocyanin content of cultured cells subjected
to osmotic stress. The osmotic stress induced by glucose suppressed growth more than that by mannitol and produced higher
anthocyanin levels. Only small amounts of [U-14C]mannitol were taken up and metabolized by the cells. Stressed cells accumulated sugars and free amino acids to a different
extent resulting in altered cell sugar-to-amino acid ratios. The accumulation of osmotically active solutes and cell growth
suppression may both be responsible for the accumulation of anthocyanin in stressed cells.
Higher plants grown in vitro are very seldom fully autotrophic. Therefore, such cultures are usually supplied with exogenous sugars. However, at higher sugar concentration a decrease in dry matter accumulation is observed which can be explained by a decrease in osmotic potential of the medium.To test this explanation a series of experiments with mannitol, a sugar alcohol often used for simulation of osmotic stress, were performed with excised wheat embryos, rape seedlings and potato stem segments grown in vitro. As the presence of mannitol in the medium caused a significant decrease in dry matter accumulation, the content of mannitol in the shoot tissues was determined using HPLC analysis to estimate the uptake and transport of mannitol from roots to shoots. Mannitol contents up to 30% of dry weight in wheat and 20% in rape and potato shoots were found, indicating that mannitol is easily taken up by in vitro plants and transported to shoots. There were no large changes in the content of glucose, fructose and sucrose caused by the presence of mannitol in the tissues. These data show that mannitol can not be used as an inert osmoticum in in vitro studies.
Cladode explants of Opuntia amyclaea were cultivated in Murashige and Skoog medium with different supplements. Benzyladenine was necessary for shoot development from pre-existing buds. Axillary proliferation was also stimulated in subsequent subcultures in the presence of benzyladenine and when the apical meristem was not present in the explant. The number of shoots and the total dry weight were maximum with 5% of sucrose in the medium. It was found that satisfactory rooting occurred when 510-5 M indole butyric acid was added to the medium. Vascular contact between roots and shoots was clearly shown by histological observations. The micropropagation system developed here allows the production in 100 days of 25 000 rooted plantlets from a single cladode, by the stimulation of axillary proliferation in the absence of apical dominance.
Hylocereus undatus (Haw.) and H. polyrhizus (Weber) are new fruit crops of the Cactaceae. In Israel, flowers of the two species, which are self-incompatible, are hand cross-pollinated. In order to ensure a current supply of compatible pollen and guarantee good yields, we have developed a procedure for long-term storage of pollen. Pollen for storage was collected in the evening or in the morning. Its moisture content ranged between 45% to 50% in the evening and between 18% to 22% in the morning. Pollen was first dehydrated in a vacuum desiccator until the moisture content was reduced to 5% to 10% and then stored at various temperatures (+4, –18, –70, –196 °C) for 3 or 9 months, after which it was used for cross-pollination. Percent fruit set and fruit fresh weight (FW) were affected by the temperature but not the duration of pollen storage; storage at +4 °C reduced fruit set, fruit FW, and seed number more than did storage at subfreezing temperatures. The FW of fruits produced by frozen pollen was similar to that produced by fresh pollen in commercial orchards. The rate of seed germination was high (≈90%) regardless of the temperature during pollen storage.
The genus Mammillaria includes many valuable species some of which are near to extinction. The beautiful white-spined M. san-angelensis has been reported to be extinct. Using the related species M. haageana in preliminary trials, the in-vitro mass propagation from young seedlings of the near-extinct M. san-angelensis has been achieved. Morphogenetic responses in M. haageana and M. san-angelensis were similar. The results were influenced mainly by two factors: the presence of the cytokinin 6-benzyladenine (BA) and the origin of the explant. Callus formation rarely occurred; however, shoots and multiple shoots were obtained when tip (apical) explants were exposed to different concentrations of BA and naphthalene acetic acid (NAA). Only multiple shoots were apparent when lateral explants were exposed to similar experimental conditions. Mass regeneration was most successful when lateral explants were subjected to three treatments: BA alone at 0.1 mg l−1; BA 0.1 mg l−1 combined with NAA at 0.01 and BA 1.0 mg l−1; or in combination with NAA 0.01 mg l−1.
Hylocereus undatus (Haw.) and H. polyrhizus (Weber) are new fruit crops of the Cactaceae. In Israel, flowers of the two species, which are self-incompatible, are hand cross-pollinated. In order to ensure a current supply of compatible pollen and guarantee good yields, we have developed a procedure for long-term storage of pollen. Pollen for storage was collected in the evening or in the morning. Its moisture content ranged between 45% to 50% in the evening and between 18% to 22% in the morning. Pollen was first dehydrated in a vacuum desiccator until the moisture content was reduced to 5% to 10% and then stored at various temperatures (+4, -18, -70, -196 °C) for 3 or 9 months, after which it was used for cross-pollination. Percent fruit set and fruit fresh weight (FW) were affected by the temperature but not the duration of pollen storage; storage at +4 °C reduced fruit set, fruit FW, and seed number more than did storage at subfreezing temperatures. The FW of fruits produced by frozen pollen was similar to that produced by fresh pollen in commercial orchards. The rate of seed germination was high (≃90%) regardless of the temperature during pollen storage.
Under water stress conditions, induced by mannitol solutions (0 to 0.66 M) applied to the apical 12 mm of intact roots of Zea mays L. (cv. LG 11), a growth inhibition, a decrease in the osmotic potential of the cell sap and a significant accumulation of abscisic acid (ABA) were observed. When the roots were placed in a humid atmosphere after the stress, the growth rate increased again, even if elongation had been totally inhibited. Under a stress corresponding to an osmotic potential of -1.09 MPa in the solution, growth was totally inhibited, which means that the root cell turgor pressure was reduced to the yield threshold. These conditions led to the largest accumulation of ABA. The effect of water stress on the level of ABA was studied for three parts of the root. The greatest increase in ABA (about 10 fold) was obtained in the growth zone and this increase was apparently independent of the hydrolysis of the conjugated form. With a mannitol treatment of 1 h equivalent to a stress level of -1.39 MPa, a 4-fold increase in ABA efflux into the medium was obtained. These results suggest that there are interactions between water stress, root growth, osmotic potential and the ABA level. The growth under conditions of stress and the role of endogenous ABA in the control of plant metabolism, specially in the growth zone, are discussed.
Explants from young joints of mature plants of tuna (Opuntia ficus-indica Mill.) were cultured on Murashige and Skoog (MS) medium containing 8.8 M benzyladenine (BA) and 0.5 M naphthaleneacetic acid (NAA). Shoots produced were utilized as secondary explants. Each shoot was cut longitudinally from apex to base into two explants, and some of these explants were cut transversely into proximal and distal explants. The size and number of shoots produced was affected by size and position of the explant within its source. The shoots were rooted in vitro or ex vitro and plants were successfully established in soil from both rooting methods.
Yellow pitaya (Mediocactus coccineus) seeds were sown on Murashige and Skoog (1962) mineral salt medium. After germination, epicotyls were placed on media enriched with a combination of naphthaleneacetic acid (NAA) (0.05, 0.27 or 0.54 M) and benzyladenine (BA) (2.2 or 4.4 M). The apical tip was excised from half of the shoots and the other half were kept intact. Different values for proliferation rate, shoot length and thickness were observed on each medium. The cotyledons and roots were placed on MS medium supplemented with NAA (2.7 or 5.4 M) and embryogenic calluses were formed. Somatic embryos were induced on these media and then they normally developed on a growth regulator-free medium.
Two Vitis species were cultured in vitro under photoautrophic (sucrose-free culture medium) and photomixotrophic (sucrose 15 g l-1) conditions during the period following microcutting rooting (day 34 to day 120). Several parameters were measured at the end of the culture: growth, plant dry weight, carbohydrate uptake from the medium and rates of photosynthesis and dark respiration. The two species behaved very differently. Under photoautotrophic conditions, dark respiration, net photosynthesis and daily CO2 fixation were higher in Vitis vinifera than in Vitis rupestris. Culture under mixotrophic conditions caused increase in growth, respiration and photosynthesis in Vitis rupestris. In contrast, photosynthesis decreased in Vitis vinifera under the same conditions.
Friable callus cultures were initiated from cotyledons and hypocotyls of Opuntia ficus-indica. Explants from cotyledons produced significantly more callus than those from hypocotyls. Optimum callus growth was observed
on Murashige & Skoog medium supplemented with 0.9 μM 6-furfurylaminopurine, 2.3 μM 2,4-dichlorophenoxyacetic acid, 1.0 μM
4-amino 3,5,6-trichloropicolinic acid, 400 mg l-1 casein hydrolysate and 3% sucrose. The same medium without agar was used for establishing cell suspensions.
Regeneration of Coryphantha elephantidens (Lem.) Lem. from callus cultures was obtained from (5–20 mm long) root explants of in vitro rooted shoots cultivated on the Murashige–Skoog medium, supplemented with different concentrations of growth regulators. Callus proliferation and shoot formation was optimal on a medium supplemented with 9 μM 2,4-D and 4.6 μM Kn. Explant size and sucrose concentration in the medium influenced the regeneration potential of the callus cultures significantly. Callus obtained from 10 mm long explant when cultured on a medium supplemented with 9 μM 2,4-D, 4.6 μM Kn and 7% sucrose induced maximum shoot differentiation. Regenerants were rooted on growth-regulator-free Murashige–Skoog medium and then acclimatized in a greenhouse. Regenerated plants showed 100% survival and performed better than seedlings.
Prolonged cultivation and the presence of exogenous growth regulators are factors suspected to induce genetic instability in vitro. In our previous work, we have achieved regeneration of a severely endangered cactus Mammillaria san-angelensis Sánchez-Mejorada from a long-term culture in the presence of auxins. The aim of this work was to investigate the cytogenetic characteristics of in vitro derived regenerants, analyzing nuclear DNA content, ploidy level and the extent of endopolyploidy. Plantlets grown for up to 7 years in MS medium alone were used as a source of explants which were cultured on MS in the presence of NAA, IAA, IBA, 2,4-D or Picloram at 2, 4, and 6 mg l−1. In vitro plantlets regenerated without auxins were used as controls. Adult plants grown in a greenhouse and in vitro young plantlets were both found to be diploids (2n=22) with the same karyotype, and no differences in DNA content were found between these two groups, both having a 2C DNA content=3.20 pg. However, differences in the frequency of endopolyploid cells were found between young and adult plants. On the other hand the extent of endopolyploidy (the frequency of cells with 2C, 4C, 8C, and 16C DNA content) in differentiated tissues was basically the same in the control as in plants regenerated in the presence of auxins, and only marginal differences were detected in five cases, without any pattern. Meiosis in adult plants was a normal behavior with eleven bivalents (n=11). This study demonstrated karyological stability of tissue cultured M. san-angelensis despite its origin from long-term subculture and the presence of auxins.
The Illustrated Guide to Cacti
- R Slaba
R. Slaba, in: The Illustrated Guide to Cacti, Chancellor Press,
London, 1992, pp. 40–41.
- J E Lazarte
- M S Gaiser
- O R Brown
J.E. Lazarte, M.S. Gaiser, O.R. Brown, Hort. Sci. 17 (1982) 84.
- J M Ribaut
- P E Pilet
J.M. Ribaut, P.E. Pilet, Physiologia Plantarum 81 (1991) 156-162.
- M R King
- J Succul
- M F Fay
- J Gratton
- G Palomino
- J Dolezel
- R Cid
M.R. King, J. Cact Succul. 29 (1957) 102–104.
[11] J. Lema-Ruminska, D. Kulus, Haseltonia 19 (2014) 46–63.
[12] M.F. Fay, J. Gratton, Bradleya 10 (1992) 33–48.
[13] G. Palomino, J. Dolezel, R. Cid, I. Brunner, I. Mendez, A.
Rubluo, Plant Science 141 (1999) 191–200.
- T Murashige
- F Skoog
T. Murashige, F. Skoog, Physiologia Plantarum 15 (1962) 473-497.
- B S Bhau
B.S. Bhau, J. Ind. Soc. Cacti Succul. 1 (1999) 12-15.
- H A Escobar
- M Vitor
- A Villalobos
- A Villegas
H.A. Escobar, M. Vitor, A. Villalobos, A. Villegas, Plant Cell
Tissue Organ Cult. 7 (1986) 269-277.
- B S Bhau
B.S. Bhau, Sci. Hort. 81 (1999) 337-344.
- M R King
M.R. King, J. Cact Succul. 29 (1957) 102-104.
- M F Fay
- J Gratton
M.F. Fay, J. Gratton, Bradleya 10 (1992) 33-48.
- Fay