Pelecyphora in vitro. From areole to micrografting

A small stem with many active areoles

An areole contains a meristem, tissue capable of forming new organs. In culture, a stem fragment with intact areoles can give rise to further plants. This property was used for the slow-growing Pelecyphora aselliformis and P. strobiliformis in an experiment published in 2002. The explant, a plant fragment placed in culture, was a seedling apex or a transverse segment without an apex. New shoots developed from existing areoles. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 73–76]

At the time, both plants belonged to the two-species genus Pelecyphora. The species with a cone-shaped body is also known as Encephalocarpus strobiliformis. Current POWO accepts a broad genus including species of the former Escobaria. The values described here remain results for two identified species and specific propagation material. [Sánchez et al. 2022, Sánchez et al., pp. 129–145. POWO, as of 11 October 2026][2-WWW][2-WWW]

Seedlings in a controlled environment

Pérez-Molphe-Balch and Dávila-Figueroa began with seeds obtained from two clones of each species. Fewer than one hundred seeds of P. aselliformis and fewer than one hundred of P. strobiliformis were available. Approximately half germinated under sterile conditions. MS medium, named after Murashige and Skoog, supplied mineral nutrients. For germination, it contained 30 g/L sucrose and 8 g/L agar, at pH 5,7. Sucrose provided a carbon source, and agar gave the medium a solid consistency. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 73–74]

The temperature was 25 ± 2°C. The photoperiod comprised 16 hours of light and eight hours of darkness, at a photosynthetic photon flux of 54 µmol m⁻² s⁻¹. The same basic chamber conditions were maintained in subsequent stages. Preliminary multiplication provided shoots 20–25 mm long, from which apical fragments and transverse discs 4 mm thick were obtained. The main multiplication comparison included two sucrose concentrations, 30 and 50 g/L, and different cytokinin concentrations. At this stage, the medium was solidified with 10 g/L agar. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, p. 74]

Cytokinin, apical dominance and successive cycles

Benzyladenine, BA, is a cytokinin used to stimulate shoot formation. In the 2002 study, fragments with an apex developed a large main shoot and smaller shoots at the base. Discs without an apex produced more uniform shoots. The authors linked this difference to apical dominance, the influence of an active apex on the growth of other meristems. Assessment of efficiency therefore depended on both the medium composition and the position of the fragment collected. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 75–76]

The first cycle lasted 60 days. The shoots were then transferred to fresh medium and multiplication was repeated. The compilation after 180 days expressed the number of shoots obtained per initial explant. This value describes the cumulative outcome of three cycles. Individual cytokinin treatments were compared using 20–30 explants of each type and two independent experiments for each species. Further cycles of three selected treatments used material derived from 15 initial explants per treatment. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 74–75]

The highest first-cycle result and the highest result after three cycles may come from different treatments. In P. strobiliformis, discs produced a higher initial shoot number, while apical fragments at a different sucrose concentration produced the highest number after 180 days. The abstract of this paper assigned the treatment giving 12,37 shoots to BA 8,8 µM. Table 2 places this result at BA 4,4 µM. The compilation above retains the values and conditions from the table, together with this discrepancy in the publication. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 73, 75, Table 2]

From a small shoot to a rooted plant

Shoots required elongation after intensive multiplication. After 60 days on MS with 3 g/L activated charcoal, the mean length of P. aselliformis was 12 ± 0,29 mm, and that of P. strobiliformis was 15 ± 0,37 mm. In the MS control, the values were 10 ± 0,24 and 11 ± 0,21 mm, respectively. The authors considered adsorption of residual regulators by charcoal as a possible explanation. They did not determine hormone composition in the shoots or hormone binding by charcoal in this experiment. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, p. 77, Table 3]

Rooting was conducted separately on MS supplemented with indoleacetic acid, IAA, or indolebutyric acid, IBA. Both are auxins used to induce roots. After six weeks, shoots whose roots had reached at least 15 mm were assessed. Across the treatments used, these accounted for 89% of P. aselliformis shoots and 87% of P. strobiliformis. After transfer to a 1:1 mixture of sand and soil, the plants remained under a humid cover for two to three weeks, then in a greenhouse. After 16 weeks, survival of the combined material of both species was 88%. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 74, 77–78]

Multiplication, rooting and survival after transfer outside culture are successive stages. Their percentages have different denominators. A high number of shoots obtained describes the first stage, while the subsequent outcome also depends on root development and the plant's adjustment to a new environment. In this study, hyperhydricity, excessive hydration and a glassy appearance of tissues, occurred in less than 5% of the shoots obtained. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 77–78]

Micrografting onto Opuntia ficus-indica

In 2016, Badalamenti and co-authors used a living rootstock as the growth environment for a small P. aselliformis scion. The seed material comprised 45 seeds of this species and 60 seeds of Opuntia ficus-indica. Under the experimental conditions, 73,3 and 75% germinated, respectively. Two types of scion were collected for micrografting, a 5-millimetre apex and a 3-millimetre subapical segment with at least one areole. The Opuntia fragments were 10 mm long and initially lacked roots. A rootstock diameter of 4–6 mm gave the best results in preliminary trials. [Badalamenti et al. 2016, pp. 2–3]

Cultures were maintained at 25 ± 1°C, with 16 hours of light per day and a photon flux of 50 µmol m⁻² s⁻¹. Rootstock production had previously included a medium with 2 µM BA. The micrografting stage itself was conducted on MS without added growth regulators. The method description therefore retains two separate stages with different medium compositions. [Badalamenti et al. 2016, pp. 2–3]

1. Scions of suitable size and fresh rootstock fragments were obtained from sterile seedlings and cultures. Areoles were retained on subapical segments.
2. A freshly cut scion was placed against a freshly cut rootstock under aseptic conditions. Both fragments were placed on medium for further growth and rootstock root formation.
3. Shoots growing from Opuntia areoles were removed periodically. Scion growth was assessed after three and six weeks, and the presence of living scions and new shoots after 60 days.
4. Developed micrografts were transferred to sterilised Jiffy peat pellets. Relative humidity was initially maintained at 95 ± 5%, until roots grew beyond the pellet.
5. The plants were then moved to a greenhouse with a temperature of 25 ± 2°C by day and 20 ± 2°C at night. Survival was checked two months after transfer outside culture.

This sequence describes the laboratory procedure of the published experiment. Subapical material yielded more fragments from a single seedling, because each retained segment with an areole could begin growth. [Badalamenti et al. 2016, pp. 2–3]

The apex and an areole-bearing segment give different results

The apex more often retained viability and began growing. The subapical segment had a lower success rate, but allowed a greater number of shoots to be obtained from areoles. Scion displacement was observed in some unsuccessful grafts. The way the seedling was used therefore determined the number of possible procedures, their success and subsequent multiplication at the same time. [Badalamenti et al. 2016, pp. 3–4, Table 1]

After six weeks, 90% of successful micrografts had roots. After transfer outside culture, 85 ± 0,8% of plants survived until assessment after two months. The table gives these two results jointly, without separate attribution to each scion type. The study therefore documents completion of a specific propagation and acclimatisation sequence, together with its observation period. [Badalamenti et al. 2016, pp. 3–4, Table 1]

Material for collections and conservation research

Stimulating existing areoles allows shoots to be multiplied, while sowing retains variation between new individuals. In the 2002 experiment, seed material came from only two clones of each species. The large number of shoots derived therefore does not define the size of the genetic pool represented. Preserving variation depends on the number and origin of starting plants, alongside the effectiveness of the technique itself. This conclusion follows from the scope of the material used in the study. [Pérez-Molphe-Balch and Dávila-Figueroa 2002, pp. 73–74]

The 2026 review concerning P. chihuahuensis discusses cryopreservation, storage at very low temperatures, and future applications of genomic tools. Its examples of culture and seed storage come mainly from other cacti. For P. chihuahuensis, the authors explicitly describe gene editing as a hypothetical direction. Validated procedures for the two classical Pelecyphora provide a concrete basis for planning trials for further species, with their responses determined separately. [Loweree-Rivera et al. 2026, pp. 10–14, 20]