In vitro regeneration of Ariocarpus kotschoubeyanus. From tubercle to rooted plant

Seedling tubercles as starting material

Moebius-Goldammer, Mata-Rosas and Chávez-Avila began with 250 seeds of Ariocarpus kotschoubeyanus from the UNAM Botanical Garden collection. After preparation for aseptic culture, about 70% germinated within 8–50 days. The first or second tubercle developed after 13–15 weeks. Tubercles 2–7 mm long were cut at their base from these seedlings and placed on a culture medium. The authors recorded continued seedling development after these fragments had been taken. [Moebius-Goldammer et al. 2003, pp. 388–390]

Cultures were maintained at 26 ± 2°C under sixteen hours of light with a photon flux density of 50 µmol m⁻² s⁻¹. The base was Murashige and Skoog medium with 30 g/l sucrose and pH 5,7. Combinations of growth regulators were compared, including BA (6-benzylaminopurine) with NAA (1-naphthaleneacetic acid) and kinetin with 2,4-D. Each treatment comprised three vessels, each containing two tubercle explants. These conditions apply to sterile culture of small plant fragments. [Moebius-Goldammer et al. 2003, p. 390, Table 1]

Two routes to shoot formation

In direct organogenesis, new shoots developed from the areoles of the excised tubercle. The first became visible after about five weeks on media containing BA, a cytokinin. Indirect organogenesis began with callus, tissue forming on the explant. After six weeks, more compact nodular areas appeared in it, followed by small rosettes of tubercles forming a new shoot bud. [Moebius-Goldammer et al. 2003, pp. 389–391, Fig. 1, Table 1]

The response was stronger with BA and NAA than with kinetin and 2,4-D. The highest mean for indirect organogenesis was 6,3 ± 5,6 shoots per explant with 13,3 µM BA and 5,4 µM NAA. Variation was reported as the standard error. A similar mean of 6,2 shoots was obtained with 4,4 µM BA alone. With six explants per treatment and wide variation, these results describe the response of the particular cultures and material used in the study. [Moebius-Goldammer et al. 2003, pp. 390–391, Table 1]

After isolated shoots were transferred to a medium without regulators, some retained the ability to form further shoots from their areoles. The authors saw this as a possible way to maintain propagation through direct organogenesis after initial induction. The callus route also produced hyperhydrated shoots, which sometimes reverted to callus. The appearance of the regenerated plant therefore mattered for subsequent culture management. [Moebius-Goldammer et al. 2003, pp. 391–392]

Rooting was a separate stage

Shoots 0,5–1,5 cm tall were separated and transferred to rooting media. Full-strength and half-strength MS salts, the addition of activated charcoal and vessel closures were compared. Some shoots were placed on the medium immediately after cutting, while others were left in an empty vessel in darkness for three days to allow the cut surface to heal. [Moebius-Goldammer et al. 2003, pp. 390–391]

Among material suitable for rooting, about 25% of shoots rooted after immediate transfer and about 30% after the healing period. Highly hydrated shoots did not form roots. Adding 1 g/l activated charcoal and using filter closures, which allowed better air exchange, favoured rooting and reduced hyperhydration. This stage markedly limited the number of plants that could leave culture. [Moebius-Goldammer et al. 2003, pp. 390–391]

Somatic embryos developed more slowly and less successfully

After about four months, the authors described somatic embryos on about 20% of explants from media containing 8,9–22,2 µM BA combined with 0,5–5,4 µM NAA. Small nodular structures appeared first, followed by globular forms and then a stage with two cotyledons. Such embryos arise from plant tissue cells outside the fertilization process. Images and descriptions of development formed the basis for their identification in this study. [Moebius-Goldammer et al. 2003, pp. 389, 391, Fig. 1]

Some forms began developing prematurely or had abnormal structures and therefore did not form plants. The best maturation and subsequent development were described on a medium with half-strength MS salts, activated charcoal and a filter closure. This route yielded few plants compared with organogenesis. Formation of an embryonic structure and obtaining a developing plant from it were thus separate outcomes. [Moebius-Goldammer et al. 2003, pp. 391–392]

Plants in soil and the significance of seed-derived material

Rooted regenerated plants were transferred to pots 8 cm in diameter and kept in a greenhouse at high relative humidity. The authors reported 95–100% survival in this group and showed a plant after one year of growth in soil. This is the survival rate of plants that had already passed through regeneration and rooting. The efficiency of the whole method also depended on the limited proportion of shoots that formed roots. [Moebius-Goldammer et al. 2003, pp. 389, 391, Fig. 1]

Starting cultures from seeds makes it possible to retain multiple material lines, while multiplying shoots from one explant replicates a particular line. The authors proposed regeneration as a way to supply plants to collections and as a possible tool for species conservation. Genetic fidelity of the regenerated plants and their lasting return to natural populations were not studied. The reported result covers plant regeneration in culture and subsequent growth under greenhouse care. [Moebius-Goldammer et al. 2003, pp. 391–392]