Obregonia in vitro. From areole to independent plant

From seed to starting material

Obregonia denegrii grows slowly from seed, and older accounts identified pressure from the removal of specimens for collections and the loss of habitat. Tissue culture propagation therefore offered a way to seek more efficient methods of producing new plants. An explant is a fragment used to initiate a culture. In this species, it could be part of a seedling, a tubercle with an areole or tissue capable of forming callus from which further structures developed. [Cardarelli et al. 2010] [Malda et al. 1999b]

Cardarelli, Borgognone and Colla began their study with seeds. At 25 ± 1°C, with 16 hours of light at approximately 40 µmol photons m⁻² s⁻¹, germination on sterile, moist filter paper reached 72,7 ± 1,4% after 28 days. The trial comprised five replicates of 25 seeds. In a separate series on solid MS medium, with three replicates of 25 seeds, prior soaking in water gave 22,3 ± 1,0%, and GA₃ treatment gave 85,2 ± 0,9% after seven days. Variation was reported as the standard deviation. The methods and table give different GA₃ concentrations, so the comparison documents the effect of the treatment without establishing an unambiguous dose. [Cardarelli et al. 2010, p. 30, Table 1 p. 32]

MS medium supplies mineral salts. In this series, it also contained 30 g/l sucrose as an additional carbon source and 9 g/l agar to form a gel. After four weeks, the seedlings were transferred to treatments with growth regulators. Auxins and cytokinins shape the developmental response of tissues. Depending on the combination used, a plant may increase in size or form shoots, roots or callus. The outcome of an individual stage describes only the response that was actually assessed. [Cardarelli et al. 2010, pp. 30–33]

Cutting the shoot and activating areoles

Five types of explant were prepared from four-month-old plants. Whole apical, basal and root fragments, longitudinally divided apices and individual tubercles were compared. The common CM medium contained MS, 4,4 µM BAP, 10,7 µM NAA, sucrose and agar. After 45 days, longitudinally divided apices formed approximately 3,3 shoots per explant, and tubercles approximately one. The other three types showed no proliferation at this stage. [Cardarelli et al. 2010, pp. 30–33]

New shoots developed from the areoles. Cutting the apex changed the plant's growth pattern and, according to the authors, reduced apical dominance, promoting the activation of lateral buds. Comparing whole and longitudinally divided fragments demonstrated the importance of material preparation. Hormone concentrations within the areoles were not investigated. The anatomical interpretation is based on the position of the new shoots and the response to cutting. [Cardarelli et al. 2010, pp. 32–33]

Medium additives and the quality of new shoots

The next series retained the two effective explant types and compared CM with three additives. Each experimental treatment used four replicates of 24 explants. The material was transferred to fresh medium in successive 45-day subcultures. CPPU, a synthetic cytokinin, increased shoot numbers to 5,0 ± 0,4 per explant. Putrescine, a polyamine, gave 2,4 ± 0,6 and was associated with the highest proportion of spontaneously rooted explants. Silver nitrate gave a shoot number similar to CM. [Cardarelli et al. 2010, pp. 31–34]

CM was already a medium containing BAP and NAA. All three substances were therefore assessed as additives to this combination. The symbol “—” preserves the indication that a given phenomenon was absent in the source table. The highest shoot number with CPPU was accompanied by the highest proportion of explants with callus. During subculture, the authors removed callus from the cut surfaces, directing subsequent culture towards the direct activation of areoles. Regeneration from pre-existing meristems and regeneration through callus are different developmental pathways. [Cardarelli et al. 2010, pp. 33–34]

In this series, hyperhydricity affected an average of approximately 10–23% of explants, depending on the medium, and differences between treatments were not statistically significant. Excessive tissue hydration therefore needs to be considered alongside the number of new shoots. In a separate seedling growth stage, media containing TDZ produced more callus and hyperhydricity than treatments without TDZ. A useful micropropagation outcome includes tissue quality and subsequent rooting. [Cardarelli et al. 2010, Tables 2–3, pp. 33–34]

Embryos arising from vegetative tissues

In the 1999 study by Malda, Suzán and Backhaus, apical portions of two-month-old O. denegrii seedlings produced lateral shoots, adventitious shoots and somatic embryos. A somatic embryo arises from vegetative tissue without fertilisation. On MS medium with 0,5 mg/l BA and 0,1 mg/l NAA, the table recorded 11,4 ± 6,2 shoots and 9,2 ± 5,3 embryos per explant after four months, as means ± standard error. Callus transferred to fresh medium without regulators retained its capacity for embryogenesis for further months. [Malda et al. 1999b, pp. 74–75, Table 1]

Micropropagation through buds replicates the starting genotypes. Regeneration through callus and embryogenesis may also involve somaclonal variation arising during culture. The authors considered these differences in conservation planning. Many plants from a single explant form a large clone. Representing population diversity requires a broader range of starting material, including many seeds or plants of known origin. The study did not include a genetic analysis confirming the stability of each line. [Malda et al. 1999b, p. 83]

Tissue acidity and CAM metabolism

The cultures in the 1999 study were maintained at 25–27°C, with 16 hours of light and a photon flux of approximately 120–130 µmol m⁻² s⁻¹. CAM metabolism involves the nocturnal accumulation of organic acids and their use during the light period. The authors took one-gram tissue samples at different times in the cycle, in three replicates, and titrated the extracts to pH 7. The graph for O. denegrii shows increasing acidity in darkness and decreasing acidity in light, with differences between somatic embryos, in vitro shoots and one-year-old seedlings grown outside culture. [Malda et al. 1999b, pp. 73–75, Figure 3a p. 79]

Titratable acidity measures the amount of acids in tissue. In this publication, it served as an indirect indicator of metabolic changes, without directly measuring CO₂ flux in Obregonia. A separate study by Malda, Backhaus and Martin concerned Pelecyphora minima (published as Coryphantha minima). The available abstract describes an analysis of gas exchange and CO₂ uptake in light and darkness during culture, followed by a return to the typical CAM rhythm after transfer outside the vessel. The gas-exchange result applies to that species and that separate experiment. [Malda et al. 1999b, Malda, Suzán and Backhaus pp. 74–75 and 81, Malda, Backhaus and Martin abstract] [Malda et al. 1999a, Malda, Suzán and Backhaus pp. 74–75 and 81, Malda, Backhaus and Martin abstract] [2-WWW]

Acclimatisation and independent growth

Malda and co-authors rooted new shoots both in culture and after transfer to a substrate. In O. denegrii, medium containing IBA gave 64% rooting, and spontaneous rooting in culture reached 28%. After this phase, the plants were transferred to a 1:1:1 mixture of peat, perlite and coarse sand. Air humidity was gradually reduced by removing covers. Subsequent development therefore involved a change in the entire environment of the roots and shoot. [Malda et al. 1999b, p. 74, Table 2 p. 76]

Four months after acclimatisation, survival was 98% for plants that had previously rooted spontaneously in vitro, 75% for those rooted outside culture and 93% for six-month-old plants obtained through somatic embryogenesis. The article does not give unambiguous sample sizes for these groups. The results describe the ability to continue living under the cultivation conditions used after culture. Reintroduction into natural habitat is a further stage requiring a separate assessment of survival, genetic origin and reproductive capacity. [Malda et al. 1999b, Table 3 p. 78, discussion pp. 83–84]