Efficient elimination of Yam mosaic virus (YMV) from white yam (Dioscorea rotundata Poir.) by cryotherapy of axillary buds

Abstract

Dioscorea rotundata is an economically important food crop in many tropical countries as many people in this region depend on it for food and livelihood. Viral diseases, especially Yam mosaic virus (YMV), constitute a major constraint in the cultivation of this crop as they perpetuate through generations in the vegetatively propagated planting materials. Getting resistant or at least virus-free planting materials for farmers thus becomes crucial. This study was aimed at eliminating YMV in Dioscorea rotundata by cryotherapy of axillary buds. Enlarged axillary buds of YMV-infected TDr 2269 were frozen in liquid nitrogen for 1 h, re-warmed at 40 °C and cultured to regenerate plantlets. Approximately 76.33% plantlet regeneration and 100% YMV eradication were obtained for cryo-treated buds, against 95% and 0% obtained respectively for non-treated buds. RT-PCR and RT-qPCR analyses did not reveal detectable quantity of YMV in treated plants but did in control plants. Plants from cryo-treated buds showed no mosaic symptoms and produced slightly more tubers, and heavier mini-tubers (20.48±3.11 g) under greenhouse conditions contrary to non-treated plants that showed severe mosaic symptoms with significantly smaller tubers (1.91±0.39 g) (P < 0.05). This is the first report showing the elimination of YMV from infected white yam stock plant by cryotherapy and would be useful for producing clean planting materials.

Keywords: Axillary buds; Cryotherapy; Dioscorea rotundata; Yam mosaic virus.

Fig. 1

ART-PCR analysis of YMV-CP transcript from YMV-infected plants by RT-PCR. M, DNA marker; 1–4, leaf samples from four different plants; Mx, PCR master mix.

Fig. 2

Process of cryo-treatment and plant regeneration.

Fig. 3

Detection of YMV-CP in regenerated plants. (A) RT-PCR of plants regenerated from cryo-treated buds using YMV-CP specific primers; (B) Yam ubiquitin; (C) RT-PCR of plants regenerated from non-cryotreated buds using YMV-CP specific primers; (D) Yam ubiquitin; M – DNA marker; C_s – YMV-infected stock plant; Mx – PCR master mix; 1–20 – plants regenerated from cryo-treated axillary buds; 1–10 – YMV-infected plants regenerated from non-cryotreated axillary buds.

Fig. 4

Performance of Cryo-treated plants in the greenhouse. (A) Yam plants from cryo-treated bud showing no mosaic symptoms under greenhouse conditions; (B) Yam plants from non-cryo-treated bud showing mosaic symptoms under greenhouse conditions; (C) Mini-tubers produced by plants regenerated from cryo-treated buds under greenhouse conditions; (D) Mini-tubers produced by plants regenerated from non-cryo-treated buds under greenhouse conditions.

Fig. 5

YMV-CP relative expression in cryo-treated and control plants. Plants regenerated from YMV-infected non-cryotreated buds (CTRL); Plants regenerated from cryo-treated buds (C1–C20).