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In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO 3 :NH~-balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stern explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.

Agave (Asparagaceae) includes cultivated and wild varieties of henequen used for hard fibre production. As part of a breeding programme to improve Agave production, species with different ploidy levels were genetically characterized: two diploids [A. tequiliana Weber and the hybrid H11648 ((A. amaniensis Trel. & Nowell ¥ A. angustifolia Haw.) ¥ A. amaniensis)], a triploid (A fourcroydes Lem. var. kitam ki), a tetraploid (A. angustifolia var. letona), three pentaploids (A. fourcroydes var. sac ki, A. fourcroydes var. yaax ki, and A. sisalana Perrine), and two hexaploids (A. angustifolia var. chelem ki from two locations). Chromosome spreading was used to determine the chromosome number, flow cytometry was employed to measure the genome size, and fluorescent in situ hybridization was performed using 45S and 5S ribosomal DNA (rDNA) and the telomeric sequences (TTAGGG)n and (TTTAGGG)n as genetic markers. There were proportional increases with ploidy level of the following: (1) chromosome number (from diploid 2n = 2x = 60 to hexaploid 2n = 6x = 180), including the number of large and small chromosomes in the bimodal karyotype of Agave; (2) genome size, with a mean monoploid genome size (1Cx) of 7.5 pg (range, 7.36-7.61 pg); and (3) the number and distribution of 45S and 5S rDNA loci, with one locus of each per basic, monoploid genome. Thus there was complete additivity in genome structure with increasing ploidy, as reported in some angiosperm polyploids. However, as other analyses of polyploids have revealed a decrease in 1Cx values with increased ploidy, possible explanations for the observed genomic stability were considered. With the (TTAGGG)n probe, the signal was localized at the telomeres, consistent with published data showing that many species in the order Asparagales have this type of telomere sequence. It is speculated that sporadic telomeric signals using the (TTTAGGG)n probe are probably derived from either errors in telomerase activity or relic ancestral-type telomeric sequences.

Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Cedrela odorata L. is a valuable tropical tree widely appreciated for its wood. This species confronts serious problems due to both overexploitation of its natural populations and its susceptibility to the Meliaceae borer Hypsipyla grandella, which destroys the apical meristems and produces structural deformations. The rapid introduction of new varieties through clonal forestry has been demonstrated to be the most effective way to improve the production of perennial plantation species. In this work, we report both a protocol for the rejuvenation of elite mature trees of C. odorata and the optimization of an in vitro culture system to scale up micropropagation. Several media formulations and the use of temporary immersion culture in bioreactors were evaluated. The addition of 20% coconut water to TY17 medium increased the number of adventitious shoots from hypocotyl segments to an average number of 4.68 shoots per explant. To replace coconut water and to define the culture medium, several cytokinins were tested at various concentrations; however, none of them produced the effect of coconut water. Rejuvenation of elite mature individuals was investigated by ex vitro grafting of mature tree twigs onto 3-mo-old juvenile trees.Although the grafting had a positive effect on the micropropagation of mature material, the multiplication rate of 1.5 new shoots per explant did not compare to the organogenic capacity of younger materials. Shoot and root elongation as well as acclimatization to ex vitro conditions were carried out in a temporary immersion culture of juvenile material using BioMINT® bioreactors. A 3.5-fold increase in shoot elongation and a 4-fold increase in root elongation were achieved compared to material cultured on semisolid media. Furthermore, this culture system allowed for 98% effectiveness in the soil adaptation of the in vitrogrown plants. The scaled-up multiplication capacity over a period of 6 mo calculated for the system is above 16,000 plants per mother plant with young materials but is only 125 with mature materials.

The pathways of micro- and megagametophyte development in Agave fourcroydes (henequén) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequén is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.

A new type of bioreactor system for plant micropropagation is described that incorporates a number of features specifically designed to simplify its operation and reduce production costs. The BioMINT unit is a mid-sized (1.2 L) reactor that operates on the principle of temporary immersion. It is built of polypropylene and is translucent, autoclavable, and reusable. It consists of two vessels, one for the plant tissues and the other one for the liquid culture media coupled together through a perforated adaptor piece that permits the flow of the liquid media from one vessel to the other. This flux is driven by gravity through a see-saw movement provided by equipment (SyB) consisting of electric motor powered platforms that change position. The structural simplicity and the modular and independent nature of the bioreactors simplify their operation and reduce the amount of hand labor required for transfers, thereby reducing the cost of the whole micropropagation process.

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