Benjamín Rodríguez-Garay

Efficient in vitro propagation of Agave parrasana Berger

An efficient method for the in vitro propagation of Agave parrasana Berger, an important ornamental plant species native to the state of Coahuila, México, was developed. Proliferation of good quality shoots was achieved on agar-solidified basal MS medium supplemented with L2 vitamins and 13.3 μM benzyladenine. Rooting was successful in the basal medium with no growth regulators; however, a light intensity of 100 μmol m-2 s-1 was found to promote better rooting than 25 μmol m-2 s-1.

Effect of light quality and culture medium on somatic embryogenesis of Agave tequilana Weber var. Azul

Somatic embryogenesis in Agave tequilana Weber var. Azul was affected by the interaction between the light regimes applied during the induction phase and the expression phase. When embryogenic calli was exposed to white or red light during the expression phase, an average of two germinated embryos per explant was obtained regardless of the light treatment used for callus induction. Conversely, the highest number of germinated embryos, an average of 18 per explant, was obtained when applying either white or red light during the induction phase and then wide-spectrum light during the expression phase. Culture medium had also a great influence in this process, with embryo germination being reduced by up to 70%, depending on the light treatment, when using Schenk and Hildebrandt (SH) medium instead of Murashige and Skoog (MS) medium.

Increased photosyntethic efficiency generated by fungal symbiosis in Agave victoria-reginae

A unique non-pathogenic strain of Fusarium oxysporum was isolated from field-collected plants of Agave victoria-reginae Moore. It was then re-introduced into micropropagated plants of the same species under greenhouse conditions. The fungic inoculation induced a 225% increase in the length of roots, 50% in the number of root branches and 50% in the number of stomata on the adaxial surface of leaves. Also, an increment of 167% in nocturnal acidity and a 122% in malic acid was observed and nocturnal pH was significantly more acid in the inoculated plants. Total chlorophyll and sugar content increased 14 and 172%, respectively. These results indicate a higher photosynthetic efficiency of the plants inoculated with the fungus than those plants which were not inoculated; therefore the association of this unique F. oxysporum strain with A. victoria-reginae Moore was considered as a beneficial symbiosis.

Detection of bacterial infection of agave plants by laser-induced fluorescence

Greenhouse-grown plants of Agave tequilana Weber var. azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot. We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies. The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora. A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range. Fluorescence maxima were at 690 and 740 nm. The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection. Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm. After 30 days there was no fluorescence. The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants. The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments. These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria. The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

Plant Somatic Embryogenesis: Some Useful Considerations

Embryogenesis has evolved as a successful reproductive strategy in higher plants. The life cycle of angiosperm plants (flowering plants) is divided into two phases: the diploid sporophytic phase and the haploid gametophytic phase (Fan et al., 2008). The functions of the gametophyte are short lived and less complex than those of the sporophyte and are only devoted to produce haploid male and female gametes (Fan et al., 2008; Reiser & Fischer, 1993; Yadegari & Drews, 2004). Male gametes or microgametophytes (pollen grains) are developed inside the anthers and are formed from a pollen mother cell which undergoes a meiotic process that gives rise to a tetrad of haploid cells called microspores. During the maturation towards the pollen formation, the microspore suffers an asymmetric mitotic division giving rise to a two new cells: the vegetative and the generative cells. The generative cell undergoes a second mitotic division producing two sperms, while the vegetative cell remains undivided and bears the capacity of producing the polen tube which

Nuclear genome size and cytotype analysis in Agave parviflora Torr. subsp. flexiflora Gentry (Asparagales, Asparagaceae)

This study presents the cytogenetic characterization by karyotyping and a determination of the DNA content by flow cytometry of wild adult plants, plants grown from seeds of wild plants and bulbils of Agave parviflora subsp. flexiflora from Bacadehuachi to Nacori Town, Sonora, Mexico. The analyzed plants were diploids (2n = 2x = 60) and had three different structural cytotypes. The cytotype observed in wild adult plants was 44m + 4sm + 10st + 2t, the cytotype found in plants grown from seeds was 48m + 8st + 4t and the cytotype displayed by bulbils was 44m + 2sm + 14st. Agave parviflora subsp. flexiflora showed a bimodal karyotype of 10 large + 50 small chromosomes. All diploid plants had a secondary constriction in one pair of the large chromosomes. The arm ratio, the proportion of different types of large and small chromosomes, the mean of genome length and the asymmetry index of karyotypes clearly varied among all three diploid cytotypes. The pattern of variation among all the types of plants is probably due to rearrangements in the large and small chromosomes of the complement. Differences in the amount of nuclear 2C DNA = 8.20 pg in diploid wild adult plants and 8.21 pg in bulbils were significantly different when compared to 2C DNA = 8.07 pg of diploid plants grown from seeds. The different types of plants displayed 1.7% variation in the 2C DNA content and the mean 2C DNA content was 8.16 pg; 1Cx value = 4.08 pg. The results here reported consist of basic and useful information to set conservation strategies and breeding approaches for Agave parviflora subsp. flexiflora.

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae)

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer’s-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures

We demonstrate the effectiveness of laser-induced fluorescence (LIF) for monitoring the development and stress detection of in vitro tissue cultures in a nondestructive and noninvasive way. The changes in LIF spectra caused by the induction of organogenesis, the increase of the F690/F740 ratio as a result of the stress originated in the organogenic explants due to shoot emergence, and the relationship between fluorescence spectra and shoot development were detected by LIF through closed containers of Saintpaulia ionantha.

Embryogenesis in Polianthes tuberosa L var. Simple: from megasporogenesis to early embryo development

The genus Polianthes belongs to the subfamily Agavoideae of the Asparagaceae family formerly known as Agavaceae. The genus is endemic to México and comprises about 15 species, among them is Polianthes tuberosa L. The aim of this work was to study and characterize the embryo sac and early embryo development of this species in order to generate basic knowledge for its use in taxonomy, in vitro fertilization and production of haploid plants and to complement studies already performed in other genera and species belonging to the Agavoideae sub-family. It was found that the normal development of the P. tuberosa var. Simple embryo sac follows a monosporic pattern of the Polygonum type and starts its development from the chalazal megaspore. At maturity, the embryo sac is of a pyriform shape with a chalazal haustorial tube where the antipodals are located, just below the hypostase, which connects the embryo sac with the nucellar tissue of the ovule. The central cell nucleus shows a high polarity, being located at the chalazal extreme of the embryo sac. The position of cells inside the P. tuberosa embryo sac may be useful for in depth studies about the double fertilization. Furthermore, it was possible to make a chronological description of the events that happen from fertilization and early embryo development to the initial development of the endosperm which was classified as of the helobial type.

Micropropagation of Agave Species

The genus Agave originates from the American continent and grows in arid and semiarid places, being México the center of origin. Many species of the genus are a source of diverse products for human needs, such as food, medicines, fibers, and beverages, and a good source of biomass for the production of biofuels, among many others. These plants are gaining importance as climate change becomes more evident as heat is reaching temperatures above 40 °C worldwide and rains are scarce. Many species of the genus grow in places where other plant species do not survive under severe field conditions, due to their CAM pathway for fixing CO2 where gas exchange occurs at night when stomata are open, allowing them to avoid excess loss of water. Most of the important species and varieties are usually propagated by offshoots that develop from rhizomes around the mother plant and by bulbils that develop up in the inflorescence, which are produced by the plant mostly when there is a failure in the production of seeds.Areas for commercial plantations are growing worldwide and therefore in the need of big amounts of healthy and good quality plantlets. Although many Agave species produce seeds, it takes longer for the plants to reach appropriate maturity and size for diverse purposes. Micropropagation techniques for the genus Agave offer the opportunity to produce relatively high amounts of plants year around in relatively small spaces in a laboratory. Here, a protocol for micropropagation that has proven good for several Agave species (including species from both subgenera) is presented in detail with two different kinds of explants to initiate the process: rescued zygotic embryos and small offshoots that grow around a mother plant.