Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Carbon and water relations for developing fruits of Opuntia ficus-indica (L.) Miller, including effects of drought and gibberellic acid

Growth, gas exchange rates, and carbohydrate content were studied for developing fruits of the cultivated cactus Opuntia ficus-indica (L.) Miller, including effects of drought and exogenous gibberellic acid (GA3). Fruit development required 110 d from the time of bud differentiation to ripening at 80 d after anthesis, when the fruit mass averaged 67 g. Stomatal conductance and net CO2 uptake rates for fruits were higher during the night; they were maximal at 7 d before anthesis and decreased as development progressed. Fruits undergoing drought, imposed by detaching terminal stems bearing fruits, were 50% smaller than the control at 80 d after anthesis and did not ripen. Fruits injected with 2 ml of 500 ppm GA3 were 30% smaller than the control at 80 d after anthesis; they contained a large proportion of aborted seeds that produced a weak sink signal for dry mass accumulation. Gas exchange was higher at 21 d after anthesis for fruits treated with GA3. Total soluble sugars represented 40% of the fruit's dry mass until 45 d after anthesis, when the sugar content rapidly increased, reaching 90% at 73 d after anthesis. Such an increase was not observed for fruits treated with GA3, and the sugar content for fruits undergoing drought remained low throughout development. Starch content increased for developing fruits of O. ficus-indica until 14 d after anthesis and, except for the fruits undergoing drought, decreased thereafter. Fruit development for O. ficus-indica is apparently regulated by water availability as well as hormonal signals originating both within and outside the fruit.     

Cladode development for Opuntia ficus-indica (Cactaceae) under current and doubled CO2 concentrations

Morphological and anatomical changes for first-order daughter cladodes (flattened stem segments) of a prickly pear cactus, Opuntia ficus-indica, were monitored to determine the effects of a doubled atmospheric CO₂ concentration on their development and mature form. For daughter cladodes developing in controlled environment chambers for 60 d, maximal elongation rates were similar under a photosynthetic photon flux density (PPFD) of 6 mol m⁻² d⁻¹ and a CO₂ concentration of 370 μl liter⁻¹, an increased PPFD (10 mol m⁻² d⁻¹), and an increased PPFD and a doubled CO₂ concentration. These maximal rates, however, occurred at 20, 15, and 12 d, respectively. The maximal relative growth rate under the doubled CO₂ concentration was about twice that under the other conditions. For cladodes at 60 d as well as after 4 and 16 mo in open-top chambers, doubling the CO₂ concentration had no effect on final length or width. At 4 mo, cladodes under doubled C0₂ were 27% thicker, perhaps allowing the earlier production of second-order daughter cladodes. The chlorenchyma was then 31% thicker and composed of longer cells. At 16 mo, the difference in cladode thickness diminished, but the chlorenchyma remained thicker under doubled CO₂, which may contribute to greater net CO₂ uptake for O. ficus-indica under elevated CO₂ concentrations. Two other persistent differences were a 20% lower stomatal frequency and a 30% thicker cuticle with more epicuticular wax for cladodes under doubled CO₂, both of which may help reduce transpirational water loss.     

Vegetative Parthenocarpy in the Cactus Pear Opuntia ficus-indica (L.) Mill.

The dependence of fruit development on fertilization was studiedin two clones of Opuntia ficus-indica, Ofer and BSI. Fruitsof the clone Ofer bear fully developed seeds, whereas fruitsof the clone BSI contain only degenerated seeds and it was suspectedthat BSI is parthenocarpic. The two clones differed in theirpattern of fruit development. The increase in fruit fresh weightin Ofer was a result of both peel and pulp growth, whereas inBSI fruit growth was mainly due to pulp growth and the peelhad reached its final weight almost at anthesis. Pulp growthin BSI commenced earlier and was faster than in Ofer, but thefinal pulp weight in BSI was only 64% of that in Ofer. Seedgrowth in BSI was limited to the development of semilignifiedand lignified seed coats, whereas in Ofer 43·5% of theseeds were completely developed, the remainder being similarto those found in BSI. Fruits bearing well-developed seeds werealso found in a 'mutant' of BSI that, like Ofer, contained smallerovules at anthesis than flower of the regular BSI genotype.Germination in vivo and in vitro were similar in BSI and Ofer,but pollen tubes failed to reach the ovules in the regular BSIgenotype, while penetrating the ovules in Ofer and the small-ovuledgenotype of BSI. Good-quality fruits similar to Ofer fruitsin weight but with a higher peel to pulp ratio developed inBSI after flower-sterilization in the spring and in the autumn.In Ofer, sterilized flowers failed to develop fruits in springand partially set fruit in the autumn; the fruits consistedalmost exclusively of peel tissue. It was concluded that BSIis a vegetative parthenocarpic clone, i.e., that pollinationis not required for fruit set and development.Copyright 1993,1999 Academic Press Opuntia ficus-indica, cactus pear, fruit development, parthenocarpy, pollen-tube-growth, fluorescence microscopy     

The origins of an important cactus crop, Opuntia ficus-indica (Cactaceae): new molecular evidence

Opuntia ficus-indica is a long-domesticated cactus crop that is important in agricultural economies throughout arid and semiarid parts of the world. The biogeographic and evolutionary origins of this species have been obscured through ancient and widespread cultivation and naturalization. The origin of O. ficus-indica is investigated through the use of Bayesian phylogenetic analyses of nrITS DNA sequences. These analyses support the following hypotheses: that O. ficus-indica is a close relative of a group of arborescent, fleshy-fruited prickly pears from central and southern Mexico; that the center of domestication for this species is in central Mexico; and that the taxonomic concept of O. ficus-indica may include clones derived from multiple lineages and therefore be polyphyletic.     

Irrigation magnifies CAM-photosynthesis in Opuntia basilaris (Cactaceae)

Summary Measurements of acid metabolism and gas exchange were carried out four times during a year to assess the relative importance of temperature and the accompanying seasonal change to the carbon metabolism of Opuntia basilaris Engelm. & Bigel. plants growing in situ under irrigated and natural (control) conditions. Our experiments showed that this cactus did not change its pattern of carbon assimilation when continuously irrigated under field conditions. Non-irrigated cacti had maximum acid accumulation after periods of precipitation. Maximum acid accumulation in irrigated cacti occurred when there was a large difference between day/night temperatures (i.e., 16°C), and when nighttime temperatures were moderate (14C). Irrigated cacti had greater duration of stomatal opening and lower resistance to ¹⁴CO₂ uptake. When temperatures were low, daytime stomatal resistance to ¹⁴CO₂ uptake decreased (to 20–40 s cm^-1), but never to the level of the nocturnal resistances (5–10 s cm^-1). During periods of drought, nonirrigated cycti changed to a pattern in which organic acids fluctuated. Irrigated cacti continued to have ¹⁴CO₂ uptake when nighttime temperatures were as high as 33°C. ¹³C/¹²C isotope composition ratios, determined after two years of irrigation, were near -12 in irrigated and non-irrigated plants. Therefore, under conditions of continual irrigation, seasonal and temperature changes affected the degree of dark CO₂ fixation and acid metabolism, but these cacti did not change from CAM to CO₂ fixation in the light.Abbreviations C₃ reductive pentose phosphate cycle - C₄ dicarboxylic acid cacle - CAE carbon assimilation efficiency - CAM Crassulacean acid metabolism - THO tritiated water - T/P transpiration ratio - vpd vapor pressure deficit - water potential This study was supported in part by National Science Foundation grant OIP 74-15673. Under the auspice of this grant a cooperative research project was carried out between the Australian National University, Canberra, and the Philip L. Boyd Deep Canyon Desert Research Center, University of California, Riverside. The studies involved a comparison of the photosynthetic pathways employed by succulents during growth in their native environment (the Sonoran Desert of southeastern California) and in a favorable introduced environment (Queensland and New South Wales, Australia). Studies carried out in Australia are under the direction of Dr. C.B. Osmond     

Opuntia fragilis (Cactaceae) verified in Illinois flora

Opuntia fragilis has been reported only once from Illinois. Recent workers have not been able to find a substantiating specimen and have excluded the species from the flora. The missing specimen has now been found and is referable to this species.     

The prickly pear (Opuntia ficus-indica [Cactaceae]) in South Africa: Utilization of the naturalized weed,and of the cultivated plants

In spite of successful biological control efforts, vast areas of South Africa remain infested with the spiny prickly pear weed, Opuntia ficus-indica. These remnant populations, which vary from sparse to dense and which are more or less stable, are confined mainly to the eastern Cape. They provide a popular fruit and a limited source of income for certain sectors of the population in these areas. In terms of existing legislation, there are, however, restrictions on the large-scale utilization of these fruit. A novel method for mass-rearing the cochineal insect, Dactylopius coccus, for the commercial production of a red dye (carminic acid) has been developed. The cultivated spineless prickly pear has recently been enjoying renewed attention from researchers and growers alike. Whereas it has traditionally been cultivated mainly as a droughtresistant fodder crop, and is still popular as such, it is now increasingly recognised as a fruit, in its own right, with considerable promise as a commercial crop for local and export markets. There is also a need to encourage the use of very young cladodes (nopalitos) as a highly nutritious vegetable for human consumption in South Africa.     

Lactic Acid Fermentation of Cactus Cladodes (Opuntia ficus-indica L.) Generates Flavonoid Derivatives with Antioxidant and Anti-Inflammatory Properties

Cactus pear (Opuntia ficus-indica L.) is widely distributed in the arid and semi-arid regions throughout the world. In the last decades, the interest towards vegetative crop increased, and cladodes are exploited for nutraceutical and health-promoting properties. This study aimed at investigating the capacity of selected lactic acid bacteria to increase the antioxidant and anti-inflammatory properties of cactus cladodes pulp, with the perspective of producing a functional ingredient, dietary supplement or pharmaceutical preparation. Preliminarily, the antioxidant activity was determined through in vitro assays. Further, it was confirmed through ex vivo analysis on intestinal Caco-2/TC7 cells, and the profile of flavonoids was characterized. Cactus cladode pulp was fermented with lactic acid bacteria, which were previously selected from plant materials. Chemically acidified suspension, without bacterial inoculum and incubated under the same conditions, was used as the control. Lactobacillus plantarum CIL6, POM1 and 1MR20, Lactobacillus brevis POM2 and POM4, Lactobacillus rossiae 2LC8 and Pediococcus pentosaceus CILSWE5 were the best growing strains. Fermentation of cladode pulp with L. brevis POM2 and POM4 allowed the highest concentration of γ-amino butyric acid. Lactic acid fermentation had preservative effects (P<0.05) on the levels of vitamin C and carotenoids. Two flavonoid derivatives (kaemferol and isorhamnetin) were identified in the ethyl acetate extracts, which were considered to be the major compounds responsible for the increased radical scavenging activity. After inducing oxidative stress by IL-1β, the increased antioxidant activity (P<0.05) of fermented cladode pulp was confirmed using Caco-2/TC7 cells. Fermented cladode pulp had also immune-modulatory effects towards Caco-2 cells. Compared to the control, fermented cladode pulp exhibited a significantly (P<0.05) higher inhibition of IL-8, TNFα and prostaglandins PGE2 synthesis. The highest functional effect was found using ethyl acetate extracts. In conclusion, fermentation, especially with L. plantarum strains and L. brevis POM4, enhanced the antioxidant and immune-modulation features of cladode pulp.     

Rapid propagation of tuna (Opuntia ficus-indica) and plant establishment in soil

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.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) medium - NAA naphthaleneacetic acid     

Intriguing thigmonastic (sensitive) stamens in the Plains Prickly Pear Opuntia polyacantha (Cactaceae)

The movement of sensitive stamens in flowers of the Plains Prickly Pear (Opuntia polyacantha) is described in detail along with the external and internal filament anatomy. The goals of this investigation were: (1) to provide a synthesis of floral phenology and determine whether this rather unique stamen movement is nastic or a tropism and (2) to conduct macro- and micro-morphological analyses of filaments to determine if there are anatomical traits associated with this movement. To better understand the internal and external structure in sensitive filaments of O. polyacantha, we performed comparative anatomical analyses in two additional species from the Opuntioideae with stamens lacking such sensitivity. The consistent unidirectional movement of stamens, independent of the area stimulated, indicates a thigmonastic response. This movement serves multiple purposes, from enhancing pollen presentation to facilitating cross-pollination, protecting pollen and preventing insects from robbing pollen. Anatomically, the sensitive and non-sensitive filaments exhibit different tissue organization. Cuticle thickness, presence of capsular structures, two layers of curved cells, and more and larger intercellular spaces are characteristic of sensitive filaments. A thin unicellular epidermal layer is characteristic in sensitive filaments versus 2–3 epidermal layers in non-sensitive filaments. Another striking feature in sensitive filaments is the presence of papillae and capsular structures. We believe that these elements are related to water mobility with subsequent contraction during the thigmonastic response. Capsular structures might have a role in fluid mobility according to the stimulus of the filaments. We hypothesize that the thigmonastic response is controlled by cells with elastic properties, as evidenced by the plasmolyzed curved and contracted cells in the filaments and the fact that the movement is activated by changes in cell turgor followed by contraction as a result of plasmolysis.     

A Stereological Analysis of the Succulent Tissue 5Opuntia ficus-indica (L.) Mill.: I. DEVELOPMENT OF MUCILAGE CELLS

A correlated stereological and developmental analysis of thesucculent tissue of Opuntia ficus-indica was carried out. Thestereological parameters related to mucilage cells were describedas a function of their distance from the apical meristem. Polynomesof the second and third degree were found to describe the developmentalprocess. Three zones of development could be distinguished.The relation of the structure of the tissue to its physiologicalfunction and mucilage secretion is discussed.     

The acid-labile,peripheral chains of the mucilage of Opuntia ficus-indica

Partial hydrolysis of the mucilage of O. ficus-indica affords O-β-d-galactopyranosyl-(1→6)-d-galactose, the polymer-homologous trisaccharide, and fourteen oligosaccharides that contain arabinose and most of which have xylosyl end-groups. O-β-d-Xylopyranosyl-(1→5)-l-arabinofuranose and O-β-d-xylopyranosyl-(1→5)-O-α-l-arabinofuranosyl-(1→5)-l-arabinofuranose were the oligosaccharides isolated in greatest amount. The most-important structural features found in the peripheral chains in the mucilage are discussed.     

Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae)

Background and Aims

The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described.

Methods

The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD.

Key results

PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption.

Conclusions

Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.     

Evaluation of three varieties ofOpuntia ficus-indica (L.) Miller as hosts of the Cochineal insectDactylopius coccus costa (Homoptera: Dactylopiidae) in a semiarid area of northeastern Mexico

Three varieties of prickly pearOpuntia ficus- indica (Villanueva, Copena, and Jalpa varieties) were evaluated in their capacity to be host of the cochineal insect (Dactylopius coccus Costa) in two localities of northeastern Mexico. The experiment was carried out for two seasons, winter and summer, in 1998. Variables under evaluation were: fresh weight of grana, dry weight of grana, and number of insects. The Villanueva variety showed the highest production for the three variables and there were no differences between the other two varieties. Iturbide locality was superior to Linares for the three variables. Higher production (both dry and fresh weight of grana) was obtained during winter and there were no differences between seasons for number of insects. Figures obtained for the Villanueva variety are slightly higher than those reported in literature for the most productive cochineal areas in Mexico.     

Seed anatomy and water uptake in relation to seed dormancy in Opuntia tomentosa (Cactaceae, Opuntioideae)

BACKGROUND AND AIMS: There is considerable confusion in the literature concerning impermeability of seeds with 'hard' seed coats, because the ability to take up (imbibe) water has not been tested in most of them. Seeds of Opuntia tomentosa were reported recently to have a water-impermeable seed coat sensu lato (i.e. physical dormancy), in combination with physiological dormancy. However, physical dormancy is not known to occur in Cactaceae. Therefore, the aim of this study was to determine if seeds of O. tomentosa are water-permeable or water-impermeable, i.e. if they have physical dormancy. METHODS: The micromorphology of the seed coat and associated structures were characterized by SEM and light microscopy. Permeability of the seed-covering layers was assessed by an increase in mass of seeds on a wet substrate and by dye-tracking and uptake of tritiated water by intact versus scarified seeds. KEY RESULTS: A germination valve and a water channel are formed in the hilum-micropyle region during dehydration and ageing in seeds of O. tomentosa. The funicular envelope undoubtedly plays a role in germination of Opuntia seeds via restriction of water uptake and mechanical resistance to expansion of the embryo. However, seeds do not exhibit any of three features characteristic of those with physical dormancy. Thus, they do not have a water-impermeable layer(s) of palisade cells (macrosclereids) or a water gap sensu stricto and they imbibe water without the seed coat being disrupted. CONCLUSIONS: Although dormancy in seeds of this species can be broken by scarification, they have physiological dormancy only. Further, based on information in the literature, it is concluded that it is unlikely that any species of Opuntia has physical dormancy. This is the first integrative study of the anatomy, dynamics of water uptake and dormancy in seeds of Cactaceae subfamily Opuntioideae.     

A Stereological Analysis of the Succulent Tissue 5Opuntia ficus-indica (L.) Mill.: II. ULTRASTRUCTURAL DEVELOPMENT OF THE MUCILAGE CELLS

Five stages of the development of the cytoplasm of mucilagecells were followed by stereological analysis and analysed togive numerical (N_v), volumetric (V_v) and surface (S_v) densitiesof organelles and membranes. A quantitative evaluation of membrane areas, membrane sites,and mucilage secretion was obtained based on morphological andhistochemical data. The developmental process from early differentiationto complete degeneration of the cytoplasm is discussed in quantitativeterms.