Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips

Abstract

Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.

Figure 1

Immunolocalization of Raspberry bushy dwarf virus (RBDV) in raspberry shoot tips (Rubus idaeus, line Z13) (A) before and (B) 28 days after thermotherapy using antibodies to the viral coat protein (CP) antigen. Purple staining indicates cells and tissues containing the viral antigen. Note that the shoot tip and cells in B are approximately twice as large as those in A due to the heat treatment. (C) A shoot tip of a virus‐free in vitro plantlet of raspberry genotype TTA‐508. AD, apical dome; LP1, the first (youngest) leaf primordium; LP2, the second leaf primordium. (D) Detection of the viral RNA in shoot tips grown in vitro before and after thermotherapy by Northern blot analysis using the viral CP gene as a probe. The positions of viral RNA2 (2.2 kb) and RNA3 (0.9 kb) which corresponds to the 3′‐proximal part of RNA2 are indicated. RNA in lane 2 was extracted from shoot tips at the onset of thermotherapy and the RNA in lanes 3 and 4 after 5 and 8 days of thermotherapy, respectively. RNA in lane 5 was extracted from leaves before thermotherapy and in lane 6 after 5 days of thermotherapy. Lane 1, healthy control. The 18S RNA is shown at the bottom to control equal loading of RNA.

Figure 2

Detection of Raspberry bushy dwarf virus (RBDV) by (A) reverse transcription polymerase chain reaction (RT‐PCR) and (B–D) reverse transcription loop‐mediated isothermal amplification (RT‐LAMP). Primers were designed according to the coat protein (CP) gene of RBDV. (A) RT‐PCR: lane P, positive control (cloned RBDV CP gene); lane N, negative control (virus‐free raspberry clone TTA‐508); lanes 1–5, RBDV‐infected plants of raspberry line Z13. (B) Amplification of RBDV cDNA detected by turbidity caused by the magnesium pyrophosphate precipitate. P and N as above; B, reaction without template; 1–3, RBDV‐infected plantlets of line Z13. (C) The products of RT‐LAMP obtained in B analysed by agarose gel electrophoresis. The samples are in the same order as in the test tubes in B above. (D) Indexing of raspberry plants for RBDV by RT‐LAMP: P and N as above; 1 and 2, RBDV‐free plantlets of line Z13 obtained by thermotherapy followed by cryotherapy.

Figure 3

Longitudinal sections of shoot tips of raspberry (line Z13) stained with safranin and observed by light microscopy. Surviving cells were recognized by densely stained nucleoli (N, a few examples indicated) in nuclei located in well‐preserved and stained cytoplasm. (A) Untreated shoot tip. (B) Shoot tip following 28 days of thermotherapy. (C) Untreated shoot tip after cryotherapy and 1 day of post‐culture. (D) Heat‐treated (28 days) shoot tip after cryotherapy and 1 day of post‐culture. Note that the shoot tip and cells in B and D are approximately twice as large as those in A and C due to the heat treatment. Views including the meristem, the first (youngest) (LP1) and the second leaf primordium (LP2) are shown. AD, apical dome of the meristem; N, nucleolus.

Figure 4

Observation by transmission electron microscopy of subcellular changes in the second layer of cells in the apical dome and the base of the first (youngest) leaf primordium (LP1) of raspberry (line Z13) following 28 days of thermotherapy. (A,B) Untreated cells in the apical dome of meristem and in LP1, respectively. (C,D) Enlarged cells in the apical dome of meristem and in LP1, respectively, following thermotherapy. Scale bars = 1 µm. The sizes of the cells shown are directly proportional to the true differences in their sizes before and after thermotherapy. The most pronounced subcellular alterations observed in the heat‐treated tissues included enlarged vacuoles and the proportion of the cell volume occupied by them, especially in LP1. (E,F) Close‐up of plasmodesmata detected in the cell walls in the apical dome. Scale bars = 200 nm. CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; N, nucleus; NE, nuclear envelope; Nu, nucleolus; P, plastid; PD, plasmodesm; S, starch grain; V, vacuole.

Figure 5

A flow chart of the production of RBDV‐free raspberry plants using thermotherapy followed by cryotherapy of shoot tips.