Mammillaria vetula ‘Snowcap’. Offsets and flowers in in vitro culture

Species and cultivar

The material described by Lopez-Granero and co-authors as Mammillaria vetula ssp. gracilis var. Arizonica cv. Snowcap belongs to a cultivar. POWO currently accepts the species Mammillaria vetula and treats the subspecies name gracilis as its synonym. The current form Mammillaria vetula ‘Snowcap’ combines the species with the cultivar name. The historical wording is retained in the publication title so that its material can be located. [2-WWW][2-WWW] [Lopez-Granero et al. 2023, pp. 759–760]

The plants studied had dense clusters of short white spines and numerous offsets giving the clump a rounded outline. The material came from a nursery in Almería, Spain. Whole young offsets 0,5–1 cm in diameter were used as primary explants. These were small plants already possessing their own apex and areoles. Their subsequent branching allowed the cultivar to be multiplied through lateral buds. [Lopez-Granero et al. 2023, pp. 760–761]

Medium, light and the importance of a specific stage

Vegetative culture was conducted on MS with vitamins, at 25 ± 1°C, with sixteen hours of light and eight hours of darkness. Lamps supplied 45 µmol photons/m²/s in the photosynthetically active radiation range of 400–700 nm. Each treatment used 35 explants, and experiments were repeated three times. Shoot maintenance, offset stimulation and root formation were studied separately. [Lopez-Granero et al. 2023, pp. 761–763]

Table 1 of the paper defines the shoot-maintenance medium as MS with NAA 0,5 mg/l, kinetin 0,3 mg/l and sucrose 20 g/l. GA₃ 5 mg/l was added for three days to stimulate offsets, while rooting used ancymidol 5 mg/l and a lower sucrose amount of 10 g/l. The abstract gives a different NAA concentration, sugar amount and medium numbering. The compilation below follows table 1 and the results description, in which the media are designated MSM-1, MSM-2 and MSM-3. [Lopez-Granero et al. 2023, pp. 759, 763–766, Table 1]

NAA is an auxin, kinetin a cytokinin, and GA₃ gibberellic acid. Their combination in this material had different effects from applying auxin or cytokinin alone. The authors rejected treatments that produced much callus and swollen tissue. Assessment of material for further multiplication therefore considered offset numbers, growth, roots and retention of appropriate shoot form. [Lopez-Granero et al. 2023, pp. 762–765, Table 2–4]

A short GA₃ pulse and many new offsets

Large explants 3–4 cm long and at least 1 cm wide were used for efficient offset stimulation. Shoots first had to grow to possess enough areoles capable of branching. After a three-day treatment with GA₃ 5 mg/l, all explants studied formed offsets. After eight weeks, the mean was 34,9 ± 5,9 offsets per explant, with a range of 28–46 given in the results. The abstract describes this result as being achieved in six to seven weeks. [Lopez-Granero et al. 2023, pp. 759, 761, 765, Table 5]

The higher tested GA₃ dose, 10 mg/l, also stimulated offsets on all explants, but their mean number was 13,3 ± 5,6. At 1 mg/l, 40 ± 2% of explants produced offsets, and without GA₃, 4 ± 2%. Assessment of effectiveness therefore includes offset number and the proportion of responding explants. The 5 mg/l treatment combined high offset numbers with a response in all explants studied. Developing offsets covered the surface of the starting plant. After their separation, it could produce further batches. [Lopez-Granero et al. 2023, pp. 761, 765, Table 5]

The authors describe the main multiplication pathway as direct activation of buds associated with areoles. The table for the best treatment also recorded callus in approximately 10% of explants. Its occasional co-occurrence was distinguished from regeneration of new plants through callus. The paper does not present molecular studies of all offspring obtained, so retention of the cultivar's appearance describes phenotypic monitoring and does not prove identity of all DNA sequences. [Lopez-Granero et al. 2023, pp. 761, 765, 767–769]

Rooting and leaving culture

The highest rooting percentage was obtained on MSM-3 with ancymidol. The 5 mg/l treatment gave 100% rooted shoots, while the control in table 7 gave 40 ± 6%. The effect was tested for shoots 1, 3 and 6 cm long. Ancymidol inhibits gibberellin biosynthesis. In the experiment, it limited elongation of the above-ground part and supported root-system formation. [Lopez-Granero et al. 2023, pp. 766, 768, Table 7]

Plants with developed roots were transferred to a disinfected mixture of sand, peat and perlite. Relative air humidity was gradually reduced from 80% to 60%, then 50%, with further transfers to larger containers. The results reported 100% survival after two months of acclimatisation. The methods, however, describe a four-month sequence and assessment after that period. This detail remains a discrepancy in assessment timing within the publication. The survival achieved applies to previously rooted plants. [Lopez-Granero et al. 2023, pp. 762, 766]

Flowers require different preparation

Large plants 4–6 cm long were selected for flowering experiments. Before regulators were applied, they underwent a period of limited medium nutrient availability, nighttime cold at 4–6°C and changes in day length. During the light phases, temperature was 25°C. A photon flux of 450 µmol/m²/s was reported for this stage, ten times higher than in vegetative culture. Preparation with cold, medium and photoperiod alone did not lead to flower initiation in the trials described. [Lopez-Granero et al. 2023, pp. 761–762, 766–767]

After preparation, paclobutrazol, another inhibitor of gibberellin biosynthesis, was applied. At 1 mg/l, flower initiation was found in 66 ± 6% of explants. In table 8, mean flower number per explant was 1,40 ± 0,16. Further increases in dose to 3 and 5 mg/l were associated with a decline in the initiation percentage to 21 ± 5% and 11 ± 4%. No flowers were recorded in the control or at 0,3 and 0,5 mg/l. [Lopez-Granero et al. 2023, p. 767, Table 8]

Table 8 describes a reading after six weeks, the methods mention eight weeks, and the abstract mentions one month of treatment and subsequent transfer. The durations of cold preparation and successive photoperiod phases also differ. The result identifies an effective treatment in the previously prepared cultivar. Fully reconstructing the experimental schedule requires these discrepancies to be resolved, so it was not presented as a uniform recipe of certain duration. [Lopez-Granero et al. 2023, pp. 759, 761–762, 766–767]

Developmental regulation and experimental scope

One regulator combination stimulated offsets, another supported rooting, and flowering required separate preparation and treatment. The authors link these results to the control of apical dominance and the balance of growth regulators. A detailed explanation of flowering through changes in endogenous hormones remains their hypothesis, because concentrations of these hormones were not measured in the trial presented. [Lopez-Granero et al. 2023, pp. 767–769]

The parameters concern tissue cultures of a specific cultivar. A sugar-containing medium, an aseptic vessel, a controlled photoperiod and explant size are part of the conditions producing the result. Developed offsets and the flowers obtained demonstrate the possibility of directing different developmental stages of this plant in the laboratory. Reproducing these processes also requires shoot production, rooting and flowering to be distinguished when analysing the data. [Lopez-Granero et al. 2023, pp. 761–769]