Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica

Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g−1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

Producing Black Pepper (Piper Nigrum L. cv. ‘Kuching’) Rootstock in a Deep-Water Culture Hydroponic System

Stem cutting is the common planting material for black pepper (Piper nigrum L.) farmers mainly because the method is cheap, easy to obtain, and produces satisfactory number of new plantlets, which are relatively genetically uniform to their parent. However, soil propagation of stem cuttings renders both the stem and developing roots susceptible to soil borne pathogens, ultimately compromising the quality of the plant. Good quality rootstock of the new plant promotes faster, safer, and better black pepper plant establishment. Hydroponic farming thus offers a good platform for producing quality rootstock of the new plants and has gained importance to many farmers due to its flexibility in manipulating plant growth conditions and timely pathogen management, thus safer, healthier, and faster growth. This study investigated the growing media suitable for rootstock growth of P. nigrum L. cv. ‘Kuching’ and compared the rooting ability between stem cuttings with adventitious roots at the time of planting and stem cuttings without any root at the time of planting. In a laboratory setting, a total of 210 stem cuttings were hydroponically planted in seven nutrient compositions, with each nutrient composition containing an equal number of stem cuttings with adventitious roots at the time of planting and stem cuttings without any root at the time of planting. Hoagland solution supplemented with 0.005 mM potassium silicate solution (T4) and Hoagland solution supplemented with 2 mM salicylic acid solution (T6) showed faster root initiation whereas T1 (Hoagland solution only) produced the highest increment in root length followed by T6. The least suitable nutrient composition was T5 [T4 + 6 mL of 1 M Ca (NO3)2.4H2O solution]. The total number of roots was highest in plants from stem cuttings which had some adventitious roots at the time of planting, whereas roots in plants from stem cuttings which did not have any root at the time of planting, increased in root length faster than plants withstem cuttings which had adventitious roots at the time of planting.

Induction and evaluation of secondary metabolite and antioxidant activity in adventitious root of Codonopsis javanica

In this study, the effects of auxin (IBA, NAA), explants, and culture conditions (light/dark) on adventitious root induction of Codonopsis javanica were investigated. The results showed that dark conditions were more suitable for adventitious root induction than light conditions. All three types of explants (internodes, leaves, and nodes) induced adventitious roots, and the appropriate concentration of auxin was 0.5 mg/l IBA. After 4 weeks of incubation under dark conditions, the rooting percentage and number of roots/explant of internode, leaf, and node segments on media supplemented with 0.5 mg/l IBA were 100% and 33.87 roots, 97.78% and 23.48 roots, 100% and 25.20 roots, respectively. These adventitious roots were analysed for the presence of alkaloids, carbohydrates, saponin, fixed oils and fats, phenol, flavonoids, gum, and mucilage. The total polysaccharide content, total phenolic content, and the antioxidant activity (IC50) of C. javanica adventitious root biomass were 16.98%, 1.876 (mg GAE/g DW), and 2.44 (mg/ml), respectively. These results indicate that the adventitious roots of C. javanica contain bioactive compounds, which can be used as a material source for multiplication in large-scale systems.

Aerenchyma Formation in Adventitious Roots of Tall Fescue and Cocksfoot under Waterlogged Conditions

The formation of aerenchyma in adventitious roots is one of the most crucial adaptive traits for waterlogging tolerance in plants. Pasture grasses, like other crops, can be affected by waterlogging, and there is scope to improve tolerance through breeding. In this study, two summer-active cocksfoot (Dactylis glomerata L.) cultivars, Lazuly and Porto, and two summer-active tall fescue (Lolium arundinaceum Schreb., syn. Festuca arundinacea Schreb.) cultivars, Hummer and Quantum II MaxP, were selected to investigate the effects of waterlogging on root growth and morphological change. Cultivars were subjected to four periods of waterlogging treatments (7, 14, 21 and 28 days), while comparable plants were kept under free drained control conditions. The experiment was arranged as a split–split plot design, with waterlogging treatments (waterlogged, control) considered as main plots, time periods (days of waterlogging) as subplots and cultivars as sub-subplots. Plants began to show signs of waterlogging stress 14–21 days after the onset of waterlogging treatments. There were no significant differences in shoot biomass between the waterlogged and control plants of any cultivar. However, waterlogging significantly reduced root dry matter in all cultivars, with greater reduction in cocksfoot (56%) than in tall fescue (38%). Waterlogging also led to increased adventitious root and aerenchyma formation in both species. Cocksfoot cultivars showed a greater increase in adventitious roots, while tall fescue cultivars had a greater proportion of aerenchyma. Both cultivars within each species showed similar responses to waterlogging treatments. However, an extended screening program is needed to identify whether there are varietal differences within species, which could be used to discover genes related to aerenchyma or adventitious root formation (waterlogging tolerance) for use in breeding programs.

Longitudinal Pattern of Aerenchyma Formation Using the Ti-Gompertz Model in Rice Adventitious Roots

The longitudinal pattern of root aerenchyma formation of its relationship with the function of adventitious roots in rice remains unclear. In this study, the percentage of the aerenchyma area to the cross-sectional area (i.e., aerenchyma percentage) was fit with four non-linear models, namely, W0-Gompertz, Ti-Gompertz, logistic, and von Bertalanffy. Goodness-of-fit criteria such as the R2, the Akaike information criterion (AIC), and the Bayesian information criterion (BIC) were used to select the model. The bias of the parameters was evaluated using the difference between the ordinary least squares-based parameter estimates and the mean of 1,000 bootstrap-based parameter estimates and the symmetry of the distributions of these parameters. The results showed that the Ti-Gompertz model, which had a high goodness-of-fit with an R2 close to 1, lower AIC and BIC values, parameter estimates close to being unbiased, and good linear approximation, provided the best fit for the longitude pattern of rice aerenchyma formation with different root lengths among the competing models. Using the second- and third-order derivatives according to the distance from the root apex, the critical points of Ti-Gompertz were calculated. The rapid stage for aerenchyma formation was from the maximum acceleration point (1.38–1.76 cm from the root apex) to the maximum deceleration point (3.13–4.19 cm from the root apex). In this stage, the aerenchyma percentage increased by 5.3–15.7% per cm, suggesting that the cortical cells tended to die rapidly for the aerenchyma formation rather than for the respiration cost during this stage. Meanwhile, the volume of the aerenchyma of the entire roots could be computed using the integral function of the Ti-Gompertz model. We proposed that the longitudinal pattern of root aerenchyma formation modeled by the Ti-Gompertz model helped to deeply understand the relationship between the anatomical traits and physiological function in rice adventitious roots.

Induction and Submerged Cultivation of Valeriana Jatamansi Adventitious Root Cultures for Production of Valerenic Acid and its Derivatives

In vitro adventitious roots were induced from Valeriana jatamansi to assess their quality as an alternative ingredient for extraction of secondary metabolites to meet the demand of phytopharmaceutical industries. A significantly (p ≤ 0.05) high root induction (90 %) was achieved on Schenk and Hildebrandt medium fortified with 9.84 µM indole-3-butyric acid. A maximum root biomass (144.09 ± 11.36 g/L) with high relative growth rate (2.01 ± 0.04) and growth index (13.41) was achieved in submerged cultivation. The total valerenic acid derivatives (1525.14 µg/g DW) and acetoxy valerenic acid (534.91µg/g DW) were significantly high in induced adventitious roots, with notable quantity of hydroxyl valerenic acid (919.57 µg/g DW) that otherwise not quantifiable in parent plant parts. In addition, 0.059% essential oil yield consisting of 24.00% patchouli alcohol was also obtained from induced adventitious roots. The phenolic acid derivatives were also significantly higher in adventitious roots (451.58 µg/g DW) as compared to rhizome (187.79 µg/g DW) and leaves (263.68 µg/g DW) of the parent plant. Notably, a substantial increase in phytochemicals was evident at subsequent culture stages with significantly reduced in vitro cultivation cycle (2 months) as compared to field grown plants (24 months). Conclusively, a comparable metabolic profile of in vitro induced V. jatamansi adventitious roots and considerably shorter growth cycle clearly determines its potential as a feasible source of phytoconstituents.