In Vitro Shoot Culture of Sesuvium portulacastrum: An Important Plant for Phytoremediation

Sesuvium portulacastrum L., a member of the family Aizoaceae, is an important coastal halophyte. Due to its adaptability to salinity and heavy metals, S. portulacastrum has now been widely used for the phytoremediation of saline soils and wastewater and the protection of the coast from erosion. The increasing use of this plant requires a large number of propagules. Stem cutting propagation and seed germination cannot meet this demand, and such propagations can initiate and spread diseases. A recent occurrence of Bipolaris sesuvii J.Z. Zhang and Gibbago trianthemae E.G. Simmons in S. portulacastrum resulted in the substantial loss of the plants during the remediation of aquaculture wastewater. Thus, there is an urgent need for establishing efficient methods of propagating disease-free starting materials. In the present study, we evaluated different growth regulators in the induction of axillary shoots from nodal explants cultured on Murashige and Skoog medium and identified that zeatin (ZT) and α-naphthaleneacetic acid (NAA) was an appropriate combination for inducing high numbers of axillary shoots. The nodal explants were then cultured on MS medium supplemented with different concentrations of ZT and NAA, and the combination of ZT at 1.0 mg L−1 and NAA at 0.3 mg L−1 induced more than 12 axillary shoots per explant. The axillary shoots were excised to produce microcuttings or microshoots, which were rooted on half-strength MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The results showed that IBA at 0.6 mg L−1 induced 91.7% of the microcuttings to root with root numbers of over 36 per cutting. The rooted plantlets were healthy and true-to-type and grew vigorously in plug trays or plastic containers with a 100% survey rate in a greenhouse. Thus, this established protocol could be used for the rapid propagation of genetically identical and disease-free plants of S. portulacastrum for phytoremediation and the protection of shoreline soils from erosion.

Natural variation in the control of flowering and shoot architecture in diploid Fragaria species

In perennial fruit and berry crops of the Rosaceae family, flower initiation occurs in late summer or autumn after downregulation of a strong repressor TERMINAL FLOWER1 (TFL1) and flowering and fruiting takes place the following growing season. Rosaceous fruit trees typically form two types of axillary shoots, short flower-bearing shoots called spurs and long shoots that are respectively analogous to branch crowns and stolons in strawberry. However, regulation of flowering and shoot architecture differs between species and environmental and endogenous controlling mechanisms have just started to emerge. In woodland strawberry (Fragaria vesca L.), long days maintain vegetative meristems and promote stolon formation by activating TFL1 and GIBBERELLIN 20-OXIDASE4 (GA20ox4), respectively, while silencing of these factors by short days and cool temperatures induces flowering and branch crown formation. We characterized flowering responses of 14 accessions of seven diploid Fragaria species native to diverse habitats in the northern hemisphere, and selected two species with contrasting environmental responses, F. bucharica Losinsk. and F. nilgerrensis Schlecht. ex J. Gay for detailed studies together with F. vesca. Similar to F. vesca, F. bucharica was induced to flower in short days at 18°C and regardless of photoperiod at 11°C after silencing of TFL1. F. nilgerrensis maintained higher TFL1 expression level and likely required cooler temperatures or longer exposure to inductive treatments to flower. We also found that high expression of GA20ox4 was associated with stolon formation in all three species, and its downregulation by short days and cool temperature caused branch crown formation in F. vesca and F. nilgerrensis, although the latter did not flower. F. bucharica, in contrast, rarely formed branch crowns, regardless of flowering or GA20ox4 expression level. Our findings highlighted diploid Fragaria species as a rich source of genetic variation controlling flowering and plant architecture, with potential applications in breeding of Rosaceous crops.

Micropropagation of Raspberries (Rubus idaeus L.) in Liquid Media by Temporary Immersion Bioreactor in Comparison with Gelled Media

Temporary Immersion Bioreactor (TIB) is a suitable technique for large scale micropropagation of plant species. The aim of this work was to test the capacity of in vitro proliferation of the primocane-fruiting red raspberry cv Maravilla and floricane-fruiting red raspberry cv Willamette on gelled media compared to liquid media. The two varieties were cultured in vitro on two media, Murashige and Skoog 1962 (MS) and Driver and Kuniyuki walnut medium, 1984 (DKW), both supplemented with 0.5 mg/l 6-benzyladenine (BA). In the control cultures on gelled media the media were gelled with 5g/l Plant Agar, whereas for the cultures in liquid media Plantform bioreactors were used. After six weeks of in vitro culture we recorded the proliferation rates and lengths of the axillary shoots obtained in all the experimental treatments. The highest proliferation rate was 16 ± 2.03, obtained in cv. Willamette on gelled MS medium with 0.5 mg/l BA. The longest shoots (3.17 ± 0.32 cm) were obtained at cv. Maravilla on the DKW medium with 0.5 mg / l BA in the bioreactor. Our research highlighted that Rubus idaeus L. Maravilla and Willamette can be TIB propagated, although further research is needed to improve the efficiency of this method.

The Effect of Sucrose Supplementation on the Micropropagation of Salix viminalis L. Shoots in Semisolid Medium and Temporary Immersion Bioreactors

The effect of sucrose concentration on the micropropagation of axillary shoots of willow was investigated. The following factors were examined: the culture system (semisolid medium in glass jars versus liquid medium in temporary immersion bioreactors), the type of explant (apical and basal sections), the frequency of immersion, and CO2 enrichment. Shoots and leaf growth were significantly higher in RITA® bioreactors than in the jars for all the sucrose treatments. Apical or basal sections of willow cultured in bioreactors under high light intensity (150 µmol m−2 s−1) and ventilated six times a day with CO2-enriched air were successfully proliferated without sucrose, whereas shoots cultured in jars did not proliferate well if sucrose concentration was 0.5% or lower. More roots were formed when sucrose was added to the medium. Shoots cultured in bioreactors were successfully acclimatized irrespective of the sucrose treatment and the root biomass when transferred to ex vitro conditions. This is the first report of photoautotrophic willow micropropagation, our results confirm the importance of proper gaseous exchange to attain autotrophy during in vitro propagation.

Energy-Saving LED Light Affects the Efficiency of the Photosynthetic Apparatus and Carbohydrate Content in Gerbera jamesonii Bolus ex Hook. f. Axillary Shoots Multiplied In Vitro

An energy-saving light emitting diode (LED) system allows for adjustment of light quality, which affects plant development and metabolic processes in in vitro cultures. The study investigated the content of endogenous carbohydrates and the condition of the photosynthetic apparatus of Gerbera jamesonii Bolus ex Hook. f. Our aim was to analyze the effects of different LED light qualities—100% red light (R LED), 100% blue (B LED), a mixture of red and blue (7:3) (RB LED), and a fluorescent lamp as a control (Fl)—during the multiplication of axillary shoots. After 40 days, the culture measurements were performed using a non-invasive pulse amplitude modulation (PAM) fluorimeter. Sugar content was assessed with high performance liquid chromatography (HPLC). Two forms of free monosaccharides (glucose and fructose), two sugar alcohol derivatives (inositol and glycerol), and seven forms of free oligosaccharides were identified. Of those, glucose content was the highest. LEDs did not disturb the sugar metabolism in multiplied shoots. Their monosaccharides were three times more abundant than oligosaccharides; the same results were found in plants grown under control light. R light depleted the performance of the photosynthetic apparatus and caused its permanent damage. The RB LED spectrum ensured the most efficient non-photochemical quenching of the photosystem II (PS II) excitation state and high shoot quality.

Shoot Proliferation, Callus Induction And Plant Regeneration In Tripsacum Laxum Nash

The peduncles of Tripsacum laxum Nash were used as explants to induce axillary shoots. Multiple shoots were proliferated on Murashige and Skoog (MS) medium to establish, for the first time, efficient shoot proliferation and plant in vitro regeneration systems. Optimal shoot proliferation medium was MS with 3.0 mg/L 6-benzyladenine (BA) and 0.2 mg/L α-naphthaleneacetic acid (NAA), resulting in a shoot proliferation coefficient of 11.0 within 45 d. Optimal rooting medium was MS with 0.1 mg/L NAA and/or 0.1 mg/L indole-3-butyric acid (IBA), inducing 100% root formation from shoots within 30 d. When young roots, leaf sheaths and shoot bases were used as explants, MS medium with 1.0 mg/L thidiazuron (TDZ) and 0.2 mg/L BA induced most shoots, with the least callus. Shoot bases induced beige-white callus and shoots directly on MS medium with 1.0 mg/L TDZ and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), while leaf sheaths induced beige-white callus and shoots directly on MS medium with 1.0 mg/L TDZ and 0.2 mg/L BA. Rooted plantlets showed 99.3% survival when transplanted into a substrate of vermiculite: peat soil (1:3, v/v).

Development of the first axillary in vitro shoot multiplication protocol for coconut palms

The coconut palm or “tree of life” is one of nature’s most useful plants and the demand for its fruit is increasing. However, coconut production is threatened by ageing plantations, pests and diseases. Currently, the palm is exclusively propagated via seeds, limiting the amount of planting material. A novel micropropagation method is presented, based on axillary shoot formation. Apical meristems of in vitro coconut seedlings are cultured onto Y3 medium containing 1 µM TDZ. This induces the apical meristem to proliferate through axillary shoots in ~ 27% of the initiated explants. These axillary shoots are seen as white clumps of proliferating tissue and can be multiplied at a large scale or regenerated into rooted in vitro plantlets. This innovative micropropagation method will enable the production of disease-free, high quality in vitro plantlets, which will solve the worldwide scarcity of coconut planting material.

A Novel and Efficient In Vitro Organogenesis Approach for Ajuga lupulina Maxim

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of axillary shoots from nodal segment explants (10.2 shoots/explant) on MS medium containing 3.0 mg L−1 of 6-benzyladenine (BA). When BA and TDZ individually or in combination with NAA were employed for adventitious shoot regeneration, shoots and embryo-like structures (ELSs) were noted in the callus from leaf explants. The maximum response of 26.4 shoots /explant (81.6%) and 12.0 ELSs/explant were ascertained on MS medium with 4.0 mg L−1 TDZ and 0.1 mg L−1 NAA. The leaf despite browning still demonstrated a high regeneration capacity. TDZ (2.0 mg L−1) and BA (2.0 mg L−1) along with NAA (0.01 mg L−1) were found to perform well for shoot regeneration via callus from shoot tip explants. The best for rooting was MS medium (half-strength) containing indole-3-butyric acid (IBA: 1.5 mg L−1) and (NAA: 0.5 mg L−1) with the maximum number of roots (25.8 per shoot) and the highest rooting frequency (81.71%). The survival of the plantlets in the greenhouse was 78.2% indicative of successful acclimatization. This work is the first report of a consistent, definitive, and unique protocol for A. lupulina regeneration, paving the way for the in vitro preservation of such significant genetic resources and also further allied systems based on such callus-based or embryo-based approaches.

Preliminary study on in vitro shoot culture of Hibiscus coddii subsp. barnardii, an indigenous South African flowering plant

In vivo and in vitro grown plants of Hibiscus coddii subsp. barnardii were used as explant source for establishment of in vitro cultures. Nodal shoot explants derived from in vivo grown plants, both naturally and under controlled environmental conditions, showed high sensitivity to the surface disinfection treatment and poor survival in in vitro culture. In vitro grown seedlings proved successful as aseptic source of apical and basal shoot explants to establish contamination-free in vitro cultures. Sprouting of axillary buds was observed on 90% of apical shoot explants after four weeks of culture on full strength, plant growth regulator (PGR)-free Murashige and Skoog (MS) medium. However, further proliferation of short shoots, limited to the bud sprout at the explant base, occurred on only 50% of these explants. In contrast, all basal shoot explants attained 3-5 single primary axillary shoots (30-40 mm in length) while a clump of short (5-10 mm) shoots also formed at the base in 60% of these explants. In both explant types, addition of 0.25-1 mg L-1 6-Benzylaminopurine (BAP) to the MS medium resulted in a low frequency (10%-60%) of explants with short shoots (5-10 mm) that showed no further elongation. Moreover, explants cultured in the presence of BAP showed a high frequency of callus formation (up to 90%) and low survival (20%-60%). A lower frequency of callus formation (30%-40%) and higher survival (90%-100%) of both explant types occurred on BAP-free medium. Further subculturing of primary and secondary axillary shoots onto fresh MS medium (with and without BAP) did not improve shoot multiplication. Regenerated plantlets from PGR-free MS medium were successfully acclimatized and hardened-off.

Development of Micropropagation in Bigleaf Maple (Acer macrophyllum)

Natural populations of bigleaf maple (Acer macrophyllum Pursh) trees contain, at low frequency, individuals with stems that have attractive and valuable wavy grain in the wood. To maintain the genotype of these individuals, vegetative propagation is desired. To enable propagation from the limited amount of plant tissue that is often available, an in vitro micropropagation procedure was developed. A mix of wild trees was used as source material to generate a procedure that is genotype unspecific. Among tested basal media, DKW medium resulted in the highest frequency of growing shoots. For multiplication of shoots, removal of the apex of shoot explants was instrumental, presumably because this treatment broke a strong apical dominance in this species. Of tested hormone and hormone combinations, 0.1 μM thidiazuron produced the best results with an average of 3.2 axillary shoots per explant with an average of 3.7 nodes per axillary shoot after 1 month. Although rooting did not require hormone treatment, a 68% frequency of rooting was obtained on ½ MS supplemented with 1 μM IBA, 27% higher than hormone-free media. Taken together, we have developed a procedure for propagation of bigleaf maple from a limited amount of tissues that can be used to multiply various genotypes of interest.