Mammillaria mathildae. From in vitro culture to natural habitat

Material from many maternal plants

Mammillaria mathildae is a Mexican cactus associated with Guanajuato and Querétaro in the current POWO treatment. García-Rubio and Malda-Barrera studied material from Cañada Juriquilla near Querétaro. In their 2010 account, the site lay on steep slopes within well-preserved tropical forest that sheds its leaves in the dry season, at an elevation of approximately 1890 m above sea level. The regional nature of the experiment allows its results to be linked to a specific population. [2-WWW] [García-Rubio and Malda-Barrera 2010, pp. 934–935]

The starting material consisted of seeds from 62 maternal plants. A seed mixture was used to include many genetic sources from the local population in propagation. Sowing under aseptic conditions therefore preceded shoot multiplication. The resulting seedlings could represent different genotypes, and fragments of an individual seedling subsequently produced its clonal offspring. The number of plants obtained and the number of starting lines represented describe two different aspects of material for population restoration. [García-Rubio and Malda-Barrera 2010, p. 934]

Regeneration without added growth regulators

After surface disinfection, seeds germinated on a medium with half-strength Murashige and Skoog salts, called MS, and 15 g/l sucrose. After a month, seedlings were transferred to full-strength MS with 30 g/l sucrose. The medium supplied mineral compounds and an available carbon source, and agar formed a solid substrate. Culture was conducted at 26 ± 1°C, with a sixteen-hour photoperiod and a photon flux of 120–130 µmol/m²/s. [García-Rubio and Malda-Barrera 2010, p. 934]

Three-month-old seedlings, approximately 15 mm long, were divided into explants. An explant is a piece of plant placed in culture. A 5 mm long apex, a 7 mm long longitudinal lateral section without an apex, and a basal fragment were compared. BA, the cytokinin 6-benzylaminopurine, was tested at 0, 22,19 and 44,39 µM, and the auxin IAA at 0, 1,43, 2,85 and 5,71 µM. Each treatment comprised 11 apical and basal explants or 22 lateral explants. The experiment was performed twice. [García-Rubio and Malda-Barrera 2010, pp. 934–935]

The most favourable shoot regeneration was obtained on MS without added BA and IAA. After 60 days, lateral explants produced a mean of 4,09 ± 0,13 new shoots, apical explants 1,14 ± 0,07, and basal explants 0,59 ± 0,11. Values were reported as the mean and standard error. Lateral and apical sections regenerated without callus, while approximately 41% of basal sections formed this mass of proliferating cells. Added regulators increased callus and generally greatly reduced shoot formation. [García-Rubio and Malda-Barrera 2010, p. 935, Table 1]

Rooting as a separate stage

Shoots 10–15 mm long were placed on full-strength or half-strength MS with different doses of the auxin IBA. Rooting was considered positive when a shoot produced at least three roots 15 mm long. The comparison included 15 shoots per treatment and two experiments. Approximately 97% of shoots rooted on full-strength MS without IBA, and approximately 63% on half-strength MS. The root system developed within seven to eight weeks. [García-Rubio and Malda-Barrera 2010, pp. 935–936, Table 2]

The presence of IBA gave a poorer result in this trial. On full-strength MS with 4,9 µM IBA, approximately 10% of shoots rooted and most formed callus. At higher tested doses, callus was recorded without positive rooting. This relationship is specific to the material and regeneration stage studied. It helps explain why selecting conditions involves the explant source, medium salts and growth regulators together. [García-Rubio and Malda-Barrera 2010, p. 936, Table 2]

Moving from jar to greenhouse

Rooted plants were transferred to small pots with a mixture of peat substrate and perlite in the 1:1 ratio given in the paper. During the first week, a plastic-film cover limited drying, and watering was provided every third day. By the end of the second week, plants had been moved to a greenhouse. They were watered weekly for a further two months. During the eight weeks before planting in the habitat, 98% of plants in this batch survived. [García-Rubio and Malda-Barrera 2010, p. 935]

Acclimatisation changes the conditions of water and energy exchange around the plant. In a jar, tissues develop under high humidity and controlled light, with sugar supplied in the medium. Transfer to air brings roots into operation in a new substrate and requires maintenance of the water balance. In the paper discussed, greenhouse survival was the directly measured outcome of this transition. The authors did not separately state the size of the greenhouse batch. [García-Rubio and Malda-Barrera 2010, pp. 934–937]

The environment changes for one hundred plants

In October 2007, at the end of the rainy season, 100 marked plants were planted in a 75 × 10 m experimental area in Cañada Juriquilla. The timing was intended to limit the washing of young plants from the slope by heavy rain and the effects of herbivores. Plant condition was checked every other day during the first week, then weekly for a month and subsequently at monthly intervals. Survival data were compared with rainfall records from a meteorological station. [García-Rubio and Malda-Barrera 2010, p. 935]

In the first month, 43% of the planted plants were lost. After five months, mortality was approximately 52%, meaning that approximately 48 plants survived from the initial one hundred. A pronounced decline followed the cessation of rain. This observation links water availability with the difficulty of establishing young plants at the site. The study had one described cohort and a specified planting date. The recorded course was not converted into a fixed success percentage for all reintroductions of this species. [García-Rubio and Malda-Barrera 2010, pp. 935–937, Fig. 3]

What explains the field result

The authors consider the physiological state of tissues after in vitro culture and a possible contribution from microorganisms associated with roots. The experiment discussed did not compare plants inoculated with mycorrhizae with a control or measure their microbiological composition. The role of symbionts is an interpretative hypothesis. The direct data concern shoot production, rooting, acclimatisation and subsequent survival in relation to the course of rainfall. [García-Rubio and Malda-Barrera 2010, pp. 936–937]

Spontaneous formation of new shoots on roots was also noticed during rooting. The authors document corresponding regeneration in the field and indicate that a plant may rebuild its above-ground part after stem damage. This ability allows propagation to be discussed together with restoration of lost tissues. Survival, growth and reproduction then matter for population restoration. In the cohort presented, observation covered the initial establishment stage, up to five months. [García-Rubio and Malda-Barrera 2010, pp. 936–937, Fig. 2]