Multiple morphogenic culture systems cause loss of resistance to cassava mosaic disease

Abstract

Background: Morphogenic culture systems are central to crop improvement programs that utilize transgenic and genome editing technologies. We previously reported that CMD2-type cassava (Manihot esculenta) cultivars lose resistance to cassava mosaic disease (CMD) when passed through somatic embryogenesis. As a result, these plants cannot be developed as products for deployment where CMD is endemic such as sub-Saharan Africa or the Indian sub-continent.

Result: In order to increase understanding of this phenomenon, 21 African cassava cultivars were screened for resistance to CMD after regeneration through somatic embryogenesis. Fifteen cultivars were shown to retain resistance to CMD through somatic embryogenesis, confirming that the existing transformation and gene editing systems can be employed in these genetic backgrounds without compromising resistance to geminivirus infection. CMD2-type cultivars were also subjected to plant regeneration via caulogenesis and meristem tip culture, resulting in 25-36% and 5-10% of regenerated plant lines losing resistance to CMD respectively.

Conclusions: This study provides clear evidence that multiple morphogenic systems can result in loss of resistance to CMD, and that somatic embryogenesis per se is not the underlying cause of this phenomenon. The information described here is critical for interpreting genomic, transcriptomic and epigenomic datasets aimed at understanding CMD resistance mechanisms in cassava.

Keywords: Cassava; Cassava mosaic disease; Meristem tip culture; Organogenesis; Somatic embryogenesis.

Fig. 1

Response of wild-type (left) and organized embryogenic structures (right) derived plants to inoculation with MeSPY1-VIGS to determine resistance to cassava mosaic disease. Silencing of MeSPY using MeSPY1-VIGS leads to shoot-tip necrosis and death of CMD susceptible cassava plants within 2–4 weeks after inoculation. a TME B7. b TMS 98/0002. c NASE 14. d Mbundamali

Fig. 2

Agrobacterium-mediated genetic transformation of TMS 98/0505 and response of transgenic plants to inoculation with the infectious geminivirus clone EACMV-K201. a transient GFP expression after 4 days co-culture with A. tumefaciens. b GFP-expressing callus line. c GFP-expressing somatic embryos on regeneration media. d Transgenic rooted plant. e Response of transgenic and micropropagated wild-type plants to EACMV-K201 at 33 days post inoculation

Fig. 3

Stable GFP-expressing transgenic events recovered from friable embryogenic callus (FEC) of different cassava cultivars. a Average number of GFP positive callus lines obtained after 5 weeks of co-culture. b Average number of GFP positive rooted events obtained after 4–5 months of co-culture. Values are Average ± SE, Number of experiments done = 2 and Replications = 3 per experiment

Fig. 4

Response of non-transgenic and transgenic cassava plants to inoculation with the infectious geminivirus clone EACMV-K201. Non-transgenic and transgenic plants of TMS 91/02324 and CMD3-type cultivar TMS 98/0505, respectively, were generated from FEC. a Percentage of cassava mosaic disease (CMD) symptomatic plants of FEC-derived and micropropagated TMS 91/02324. b Average CMD symptom severity scores (scale 0–5) on FEC-derived and micropropagated TMS 91/02324. c Percentage of CMD symptomatic plants of transgenic GFP expressing TMS 98/0505 and wild-type TMS 98/0505. d Average CMD symptom severity scores (scale 0–5) on GFP-expressing TMS 98/0505 and wild-type TMS 98/0505. Plant stems were cut back 48 days after biolistic inoculation and CMD assessed on new leaf growth. Breaks in the x axis indicate a lapse in shoot regrowth after stem cut-back

Fig. 5

Response of organogenesis-derived plants to inoculation with an infectious geminivirus clone EACMV-K201. a Percentage of CMD symptomatic plants of organogenesis-derived (ORG-TME 7) and wild-type CMD2-type cultivar TME 7. b Average CMD symptom severity scores (scale 0–5) on organogenesis-derived and wild-type TME 7. c Percentage of CMD symptomatic plants of organogenesis-derived (ORG-TME 204) and wild-type CMD2-type cultivar TME 204. d Average CMD symptom severity scores (scale 0–5) on organogenesis-derived and wild-type plants of TME 204. Plant stems were cut back at 48 days after biolistic inoculation and CMD was assessed on new leaf growth. Breaks in the x axis indicate a lapse in shoot regrowth after cut-back. n = 16 for ORG-TME 7 (resistant), n = 6 for ORG-TME 7 (susceptible), n = 7 for ORG-TME 204 (resistant), n = 4 for ORG-TME 204 (susceptible)

Fig. 6

Response of meristem tip culture-derived plants of TME 7, TME 14, TME 7, TMS 98/0505 to inoculation with MeSPY1-VIGS. Silencing of MeSPY using MeSPY1-VIGS leads to shoot-tip necrosis and death of the of CMD susceptible cassava plants within 2–4 weeks. a CMD resistant micropropagated TME 7 plant. b CMD susceptible meristem tip-derived TME 7 plant. c CMD resistant meristem tip-derived TME 7 plant. d CMD resistant wild-type TME 14 plant. e CMD susceptible meristem tip-derived TME 14 plant. f CMD resistant meristem tip-derived TME 14 plant

Fig. 7

Response of meristem tip-derived plants of cassava to inoculation with infectious geminivirus clone ACMV-CM. a Percentage of CMD symptomatic plants of meristem tip-derived CMD2-type cultivar TME 7 plants. b Average CMD symptom severity scores (scale 0–5) on meristem tip-derived TME 7 plants. c Percentage of CMD symptomatic plants of meristem tip-derived CMD2-type cultivar TME 14 plants. d Average CMD symptom severity scores (scale 0–5) on meristem tip-derived TME 14 plants. The FEC-TME 204 (FEC-derived) plants were used as CMD susceptible control and wild-type plants of TME 14 (WT-TME 14) and TME 7 (WT-TME 7) were used as CMD resistant controls. The MTC-TME7 and MTC-TME 14 are meristem tip-derived plants