Optimization of In Vitro Regeneration, Calcium Bio-fortification and Mycorrhizal Biotization of Selected Seed Potato for Enhanced Performance and Bacterial Wilt Management

Abstract

Potato is an important tuber crop that plays a key role in achieving food security and alleviating poverty in sub-Saharan Africa. The Irish potato is a significant source of dietary energy, and is rich in essential vitamins and nutrients. In Kenya, the potato is the second most important food crop after maize, holding greater economic importance for smallholder cash crop farmers. Despite the increased demand for potato, production is hampered by constraints such as inadequate supply of clean and high quality seed, low soil fertility, pests and diseases such as Bacterial Wilt. The availability of clean and viable seed is the most constraining challenge necessitating the development of new production strategies that foster the production of high quality seed. Agronomic fortification with calcium during tissue culture and mycorrhizal inoculation are ways of stimulating rapid regeneration, while fostering plantlet growth under greenhouse conditions. Two laboratory experiments and a greenhouse experiment were set up to investigate the effect of calcium (Ca) fortification and AMF mycorrhizal inoculation on production of three potato varieties (Shangi, Unica and Dutch Robjyn). The first experiment involved sub-culturing single node cuttings of three varieties onto modified Murashige and Skoog (MS) media with five levels of Ca, from the macronutrient CaCl2; 8.8g/L (conventional MS medium), 10.4g/L, 12g/L, 13.6g/L and 15.2g/L Ca. In the second laboratory experiment, micro-tubers were initiated on MS media modified with the five Ca levels, 60g/L sucrose and 6mg/L 6-Benzylaminopurine. The experiments were set up in a completely randomized design and replicated five times. A pot experiment was established using non-Ca bio-fortified and Ca-bio-fortified plantlets (plantlets with highest mineral Ca content in root-zone section) from the first experiment. Calcium bio-fortified and non-Ca fortified plantlets were inoculated with a commercially available mycorrhizal inoculant (Rhizatech™) in a completely randomized design with six replications. The plantlets were then infected with Ralstonia solanacearum after one month of establishment. Performance determination was based on In vitro regeneration capacity, micro-tuber number, days to micro-tuber formation, micro-tuber fresh weight, root-zone mineral contents, plantlet height and leaf number, percent mycorrhizal colonization and Bacterial Wilt disease severity. Analyses of variance proved a significant and highly significant effect (P<0.01) on tested factors and their interactions on shoot and root development of the three varieties. MS medium containing 10.4g-13.6g/L Ca levels significantly (P<0.05) increased shoot and root number in the three varieties. Conventional MS medium resulted in a loss of apical dominance in Unica shoots. Each variety across all Ca treatments produced at most one micro-tuber. MS media containing 10.4g-13.6g/L Ca significantly (P=<0.0001) reduced the number of days it took to micro-tuber formation for Unica, Shangi, and Dutch Robjyn compared to the conventional MS medium containing 8.8g/L CaCl2. MS media containing 10.4g/L Ca completely inhibited micro-tuber formation in Unica, instead promoting the growth of etiolated shoots. MS media containing 13.6g/L Ca resulted in plantlets with the highest Ca content in the mid-stem and root-zone sections of Shangi, Dutch Robjyn and Unica. Analyses of variance proved a significant and highly significant effect (P<0.01) on tested factors and their interactions on the plantlet height and leaf number of the three varieties. Agronomic calcium bio-fortification combined with mycorrhizal inoculation resulted in 9% and 11% increase in Shangi and Dutch Robjyn plantlet height while simultaneously resulting in 40% reduction in Unica plantlet height compared to control plantlets. Mycorrhizal inoculation resulted in 21% and 25% decrease in Shangi and Unica plantlet height respectively. There were significant differences (P<0.05) in Shangi and Dutch Robjyn leaf numbers between treatments. Mycorrhizal inoculation using 2g/pot Rhizatech™ resulted in higher plantlet leaf numbers in the three varieties compared to the non-biotized treatments. Agronomic Ca bio-fortification combined with mycorrhizal inoculation resulted in an overall 35% increase in total chlorophyll content in Dutch Robjyn. Mycorrhizal inoculation using 2g/pot Rhizatech™ resulted in 12%, 16% 22% increase in total chlorophyll content in Shangi, Dutch Robjyn and Unica respectively, compared to non-biotized plantlets. All biotized plantlets showed 100% mycorrhizal colonization and there were strongly significant differences (P<0.01) in the number of mycorrhizal propagules found in 1cm root fragments of the three varieties. Agronomic Ca fortification combined with mycorrhizal inoculation decreased the number of mycorrhizal propagules in Shangi and Unica by 9% and 90% respectively and increased the number of fungal propagules by 21% in Dutch Robjyn. All plantlets across all treatments for the three varieties exhibited bacterial wilt disease symptoms. There were significant differences (P≤0.05) in bacterial wilt severity scores between treatments within the three varieties. Agronomic In vitro Ca bio-fortification combined with AMF biotization resulted in a 18.4% and 16.5% reduction in disease expression in Shangi and Dutch Robjyn while increasing severity in Unica by 43% compared to their control treatments. The obtained results confirm the benefits of In vitro agronomic Calcium fortification using 10.4-13.6g/L CaCl2, instead of conventional 8.8g/L CaCl2 on promoting shoot and root development, micro-tuber regeneration in the three varieties. Agronomic Ca bio-fortification combined with mycorrhizal inoculation positively influenced Shangi and Dutch Robjyn TC plantlet development and reduced bacterial wilt disease severity. The study findings point to the benefit of agronomic calcium bio-fortification during tissue culture, and when combined with mycorrhizal inoculation, improves seed potato growth, yield and disease suppression. Interactions between In vitro calcium bio-fortification combined with biotization should be investigated in the long term.