Mammillaria in vitro. Regeneration, CAM and spine structure

A stem explant retains the ability to regrow

The 2018 study by Lázaro-Castellanos and coauthors compared Mammillaria hernandezii, M. dixanthocentron and M. supertexta (M. lanata in the publication). Plants of the World Online currently accepts the first two names and includes M. lanata among the synonyms of M. supertexta. The third group retained its own material identity and separate results in the experiment. [Lázaro-Castellanos et al. 2018, pp. 519–521; POWO, 11 October 2026][2-WWW][2-WWW][2-WWW]

The material was started from UNAM Botanical Garden seeds. Seedlings about 10 mm tall were obtained after twelve months in M. hernandezii, eight in M. dixanthocentron and six in the lanata group. They were maintained on MS with 30 g/l sucrose and 0,8% agar at 25 ± 1°C, under a sixteen-hour day and 55 µmol photons/m²/s. To accumulate material for comparison, preliminary multiplication with meta-topolin was performed before the main experiment. [Lázaro-Castellanos et al. 2018, pp. 519–521]

Shoots were divided into apical, basal and lateral parts. New shoots formed on apical and lateral explants, primarily from activated axillary meristems, retained zones capable of producing regrowth. Basal parts did not show this response. The methods provided four jars each for apical and basal explants and eight for lateral explants per treatment, with two explants in each jar. Explant position determined access to structures capable of regrowth and the subsequent response to the medium. [Lázaro-Castellanos et al. 2018, pp. 520–524, Table 2–4]

Multiplication efficiency and the presence of callus

The authors compared benzyladenine, kinetin, meta-topolin and thidiazuron. For lateral explants of M. hernandezii, 2,2 µM meta-topolin gave a mean of 7,4 ± 1,1 shoots per explant, with callus on 12,5 ± 6,5% of explants. In M. dixanthocentron, 1,1 µM meta-topolin gave 16,7 ± 0,4 shoots and callus on 80,0 ± 3,1% of explants. At 1,1 µM, the lanata material formed 17,9 ± 1,0 shoots, with callus on 75,0 ± 4,8% of explants. These are means and standard errors of separate experimental groups. [Lázaro-Castellanos et al. 2018, pp. 522–525, Table 2–4]

Callus is proliferating tissue with altered organization. In these experiments, it appeared alongside shoots arising from retained meristems. The high efficiency of meta-topolin in two groups coexisted with frequent callus formation. Axillary regrowth was therefore accompanied by callus proliferation. With thidiazuron, callus frequency and symptoms of hyperhydricity were greater. [Lázaro-Castellanos et al. 2018, pp. 522–527]

Lateral explants of M. dixanthocentron also produced a mean of 14,2 ± 2,1 shoots without added regulators, compared with 16,7 ± 0,4 at the selected meta-topolin concentration. In this group, retained regrowth capacity was therefore already high in the control. The control mean was 2,5 ± 0,9 for M. hernandezii and 5,6 ± 0,9 shoots for lanata material. Regulator choice had different significance in the three groups. [Lázaro-Castellanos et al. 2018, pp. 522–525, Table 2–4]

The publication described assessment time inconsistently. The methods list four weeks with cytokinin, four weeks on MS with activated charcoal and another eight weeks on fresh MS, totalling sixteen. Captions of tables 2–4 state twelve weeks. Table means refer to the specified treatments, with assessment time described inconsistently. [Lázaro-Castellanos et al. 2018, pp. 520–524, Methods and captions of Tables 2–4]

Rooted shoots and acclimatized plants

Separated shoots were rooted on MS without growth regulators. Rooting of 98,4% was reported after eight weeks in M. hernandezii, 94% after twelve weeks in M. dixanthocentron and 94,6% after ten weeks in the lanata group. These are rooting-stage results, separate from shoot number and later survival outside the vessel. [Lázaro-Castellanos et al. 2018, pp. 523–525]

Rooted plants about 10 mm tall were transferred to garden soil and pumice at 1:1. Greenhouse temperature was about 30°C. The first month under cover maintained 80–90% relative humidity, followed by a gradual transition to 50–60%. The authors reported survival exceeding 95% for the groups studied. After twelve months in the greenhouse, 80% of the described M. hernandezii flowered. Flowering in the other two groups was described after twenty-four months. Age counted from greenhouse transfer followed earlier seedling development and shoot culture. [Lázaro-Castellanos et al. 2018, pp. 520–521, 523–525]

SSR markers examine selected genomic sites

Similarity between starting material and daughter shoots was studied over three successive multiplication cycles in two years. Ten starting plants of each group and shoot samples from successive cycles were selected for analysis. Of nine primer pairs, repeatable results were obtained for seven loci in M. hernandezii and eight in the other two groups. SSR markers analyse length differences in short DNA repeats at selected genomic sites. [Lázaro-Castellanos et al. 2018, pp. 521, 525–526]

Comparisons indicated high similarity of daughter shoots to starting material while also revealing differences in some markers. Mean PIC, the polymorphism information content of a marker, was 0,04, 0,02 and 0,04 in successive cycles in M. hernandezii. In M. dixanthocentron, it was 0,10, 0,15 and 0,13, and in the lanata group 0,15, 0,12 and 0,08. PIC is a dimensionless measure of allele diversity in the marker set examined. [Lázaro-Castellanos et al. 2018, pp. 525–528, Table 6]

The SSR panel determines similarity of multiplied material at the seven or eight DNA loci examined. Shoot organization and metabolism were assessed by other methods in separate experiments on Mammillaria vetula material. [Lázaro-Castellanos et al. 2018, Lázaro-Castellanos et al., pp. 525–528; Balen et al., pp. 1884–1891; POWO, 11 October 2026][Balen et al. 2012, Lázaro-Castellanos et al., pp. 525–528; Balen et al., pp. 1884–1891; POWO, 11 October 2026][2-WWW]

The CAM rhythm in shoots and altered tissues

Balen and coauthors used Mammillaria vetula (M. gracilis in the publication). They compared five material types, comprising potted plants, normal in vitro shoots, spontaneous callus growing without added hormones, hyperhydric shoots from this callus and a tumour tissue line induced by Agrobacterium tumefaciens. The last group had a defined infectious origin and did not form normal organs. Hyperhydricity meant excessive hydration and altered shoot structure. The line's origin was identified at the level of the historical species name M. gracilis. [Balen et al. 2012, pp. 1884–1886; POWO, 11 October 2026][2-WWW]

Cultures grew on MS with 3% sucrose and 0,9% agar, without growth regulators, at 24°C around the clock. The day lasted sixteen hours at 90 µmol photons/m²/s. Potted plants remained under the same light and temperature conditions. Cultures were sampled two weeks after subculture, and one-year-old shoots were sampled from pots. Each measurement included six plants or flasks, and experiments were repeated during at least two material harvests. [Balen et al. 2012, pp. 1884–1886]

CAM links nocturnal carbon fixation with organic acid accumulation and their subsequent daytime use. In this study, normal in vitro shoots retained higher acidity at the end of the night than after eight hours of daytime. The difference was smaller than in potted plants because acidity remained high during the day. Callus, hyperhydric shoots and tumour tissue showed no significant daily difference in acidity. [Balen et al. 2012, pp. 1883–1884, 1887–1889, Fig. 2]

Activity of PEPC, phosphoenolpyruvate carboxylase involved in the first stage of CAM carbon fixation, was detected in all material types. The lowest activity was measured at the end of the night and the highest two hours after darkness began. Callus, hyperhydric shoots and tumour tissue then had greater enzyme activity than normal in vitro shoots, despite no significant nocturnal rise in acidity. The study therefore compared enzyme activity with the outcome of its action as a daily change in acidity. CAM assessment relied on these biochemical and fluorescence measurements within the metabolism of the tissues studied. [Balen et al. 2012, pp. 1887–1891, Fig. 2–5]

Fluorescence and microscopic spine structure

Maximum photochemical efficiency of photosystem II was measured by chlorophyll fluorescence as the Fv/Fm ratio. Means were 0,786 in potted plants, 0,730 in normal in vitro shoots, 0,640 in callus, 0,686 in hyperhydric shoots and 0,466 in tumour tissue. This index describes the maximum photochemical efficiency of photosystem II. Fluorescence, acidity and enzyme activity determine different features of photosynthetic apparatus function. [Balen et al. 2012, pp. 1885–1887, Table 2]

In a separate 2010 study, Peharec and coauthors compared spines of this historically identified M. gracilis material. Greenhouse plants originated from rooted and acclimatized normal in vitro shoots and then grew in a greenhouse for five years. They were compared with normal and hyperhydric shoots in culture. Scanning electron microscopy covered seventeen spines of each type. Sample size was stated for spines. The number of plants supplying material remained unspecified in the paper. [Peharec et al. 2010, pp. 79–80]

Mean spine length with standard error was 6,18 ± 0,17 mm in greenhouse plants, 2,48 ± 0,02 mm in normal in vitro shoots and 1,31 ± 0,02 mm in hyperhydric shoots. The surface of greenhouse spines had small epidermal protrusions. In both culture types, cells of this layer formed longer hair-like projections. The difference therefore also occurred on shoots considered normal. [Peharec et al. 2010, pp. 80–84, Fig. 3–5]

Hyperhydric shoots were translucent and swollen, with poorly defined tubercles and disordered spine arrangement. The comparison documented structure under different conditions and at different material ages. These factors changed together, and measurements covered shoot and spine morphology. Together with fluorescence and acidity, microscopy extends the description of characters of material obtained in culture. [Peharec et al. 2010, Peharec et al., pp. 80–85; Balen et al., pp. 1887–1891; Lázaro-Castellanos et al., pp. 525–528][Balen et al. 2012, Peharec et al., pp. 80–85; Balen et al., pp. 1887–1891; Lázaro-Castellanos et al., pp. 525–528][Lázaro-Castellanos et al. 2018, Peharec et al., pp. 80–85; Balen et al., pp. 1887–1891; Lázaro-Castellanos et al., pp. 525–528]