Technique establishment for in vitro selection of drought tolerant sugercane genotypes.

Abstract

There is a need to have constant supply of sugarcane varieties adapted to different South African regions. However, the genetic improvement and selection of sugarcane cultivars with superior traits, e.g. drought tolerance, are difficult due to its complex polyploid and aneuploid genome. Biotechnology approaches are being investigated for the selection and/production of drought tolerant cultivars. Towards this end, the aim of this study was to establish: 1) the best source of meristematic explant for initiation and mass propagation of in vitro shoots; 2) in vitro conditions to screen and select for drought tolerance; and 3) physiological parameters as indicators of drought tolerance in vitro. Sugarcane stalks and shoots from single-budded setts of NCo376 were used. From the former, 1.3 cm-long meristems were isolated and used for shoot induction, shoot multiplication and rooting. The single-budded setts (approx. 50 mm) were first germinated in 20 ml sterile water or sterile moist paper, resulting in 100% and 60% sett contamination, respectively. With 1 mg lˉ¹ methylene blue (MB) there was 30% sett contamination, whilst 1 mg lˉ¹ MB in combination with 1 ml lˉ¹ Previcur® or 1 ml lˉ¹ BRAVO® resulted in 40% and 7% contamination, respectively. The uncontaminated germinated shoots (approx. 1 - 2 cm) were excised after 10 days in culture and used as the other source of meristems. Meristems from both sources were multiplied and rooted in vitro and their plantlet yield was 60 shoots/sugarcane stalk meristem and 10 shoots/meristem from in vitro-germinated sett. NCo376 and N41 varieties were used to determine the effect of mannitol (204, 326, 448 and 569 mM) on in vitro plantlet shoot and root re-growth. For both, increased mannitol in the media delayed shoot and root re-growth, with NCo376 being affected first. Stress was more significant on root than on shoot re-growth. For NCo376 plantlets, there were significant differences in root re-growth between 0 and 204 mM and the other tested treatments. For N41 plantlets, % root re-growth at day 10 on 569 mM mannitol was significantly higher than that at the other treatments. At 4 – 10 days, % shoot re-growth of NCo376 on 0 and 204 mM mannitol was greater than that at 326, 448 and 569 mM mannitol. Similar results were observed with N41 plantlets. The LD₅₀ and LD₉₀ for mannitol were 332 and 606 mM for NCo376, and 851 and 1493 mM for N41. There was no differences between the effects of polyethylene glycol-6000 (PEG-6000) and mannitol on root re-growth at the same osmotic potential. However, PEG-6000-containing cultures required to be aerated. As at 87 mM PEG-6000, NCo376 plantlets showed 50% root re-growth compared to 10% in non-aerated cultures, mannitol was used in subsequent investigations. Mannitol concentrations equivalent to LD₅₀ and LD₉₀ for NCo376 and N41 were used to screen N12, N36, N19 and N26 varieties. Based on the results obtained, the varieties were ranked on their tolerance to mannitol stress: N41 > N26 > N36 > N12 > N19 > NCo376. Leaf electrolyte leakage, leaf chlorophyll content measured with Soil Plant Analysis Development (SPAD) measurements, and histochemical detection of hydrogen peroxide (H₂O₂) (with nitroblue tetrazolium) and superoxide anion (O₂⁻˙) (with 3, 3’-diaminobenzidine) production were evaluated as indicators of stress using N41, N26, N19 and NCo376 on 332, 606 or 851 mM mannitol. N19 and NCo376 plantlets on 332 mM mannitol showed a higher % electrolyte leakage at day 5 (70%) than at day 10 (40 – 50%) of culture than N41 and N26 plantlets. A slight decrease in chlorophyll content was recorded at day 10 of culture in 332 and 851 mM mannitol, with no differences between NCo376 and N19, and N41 and N26. NCo376 and N19 accumulated more H₂O₂ than N41 and N26. O₂⁻˙ accumulation was also greater in NCo376 and N19 than in N41 and N26. All these parameters detected stress at lower levels of mannitol (332 and 606 mM), but not at 851 mM. It was concluded that mannitol stress in vitro (332 – 606 mM), in combination with the physiological assays allow for the discrimination of in vitro osmotic stress among sugarcane varieties. Further work is necessary before recommendations can be made regarding the use of the other stress biomarkers.