Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium

doi:10.13560/j.cnki.biotech.bull.1985.2024-0229

Abstract

Abstract:

【Objective】Lily mottle virus（LMoV）is one of the major viruses that jeopardize the important food and medicinal lily Lilium lancifolium Thunb, causing billions RMB yuan losses to the lily cultivation industry every year. We clarified the distribution of LMoV in the apical meristematic tissues of L. lancifolium by in situ hybridization to provide support for the cultivation of lily detoxification.【Method】Cultivated L. lancifolium planted in Qingzhen city, Guizhou province, was used as the test material. We identified the virus species carried by the test material by RT-PCR and LMoV isolates from different places were downloaded from the NCBI website to construct an evolutionary tree using the material of L. lancifolium. RNA flourescent probes were designed and prepared based on the LMoV genome sequence, and in situ hybridization was carried out in the stem tip and root tip regions to observe the distribution of LMoV.【Result】RT-PCR showed that the L. lancifolium material was infested with LMoV, and the construction of evolutionary tree revealed that the isolates in this experiment were most closely related to the LMoV isolates from Jilin and Liaolin, and in situ hybridization showed that the LMoV hybridization signals were mainly distributed in the tissues 0.15-0.2 mm below the shoot apical meristem, and there were weak viral hybridization signals in the base of the primary leaf adjacent to the first leaf primordium. The fluorescence signals of the virus were strong in the apices of mature leaves close to the primary leaves, and the size of the avirulent zone in the center of the LMoV-infected L. lancifolium meristem was (0.4-0.6) mm×(0.15-0.2) mm in longitudinal section. There was no distribution of hybridization signals in the 0.25 mm×0.2 mm meristem of the root tip. The signals were the strongest in the cortex and weaker in the root crown and root elongation zone.【Conclusion】No viral signals were found in the apical meristem and the closest apical leaf primordium[（0.4-0.6）mm×0.2 mm in size]. These regions can be used as a starting material for stem tip detoxification. The outer root crown of the apical meristem had virus signals and was not suitable for use as an explant in the culture of L. lancifolium detoxification.

Key words: Lilium lancifolium, lily mottle virus, virus distribution, in situ hybridization, shoot tip, root tip, RT-PCR

XU Xue-fei, YANG Pan-pan, ZHANG Wen-liang, BIAN Guang-ya, XU Lei-feng, LIU Hui-chao, MING Jun. Distribution of Lily Mottle Virus in the Shoot Tips and Root Tips of Lilium lancifolium[J]. Biotechnology Bulletin, 2024, 40(8): 212-220.

Figures/Tables 6

Fig. 1 In situ hybridization material of L. lancifolium A: Flower; B: bulbs after harvesting; C: bulbs after embedding. Bar = 1 cm

Table 1 Primer sequences for virus detection

引物名称Primer name	引物序列Primer sequence（5'-3'）	产物大小Product size/bp	参考文献Reference
CMV- Forward primer	ATGGACAAATCTGAATCAACCAG	657	[15]
CMV- Reverse primers	TCAGACTGGGAGCACTCCAG	657
LSV- Forward primer	GAAGAAGCACGCTGGACTG	171
LSV- Reverse primers	CGCCTGATGATCCCCTC	171
LMoV- Forward primer	GAAGAAGCACGCTGGACTG	428
LMoV - Reverse primers	CGCCTGATGATCCCCTC	428
Lily Actin- Forward primer	GCATCACACCTTCTACAACG	257	[31]
Lily Actin - Reverse primers	GAAGAGCATAACCCTCATAGA	257	[31]

Fig. 2 Electrophoresis diagram of three viruses via RT-PCR 1-4 are 4 lily samples; P: positive control; N: negative control

Fig. 3 Phylogenetic tree of LMoV-amplified fragments and other isolates

Fig. 4 In situ hybridization of LMoV in the shoot tips of L. lancifolium A: Negative control（LMoV-uninfected stem tips of L. lancifolium）; B: LMoV signal in the stem tip; C: virus distribution in the apical meristem and its surrounding tissues（enlarged white dashed box in B）; D: non-virus signal in the meristem and its closest apical leaf primordium（enlarged white dashed box in C）; E: image of the apical meristem dividing into new leaf primordia, where the base of the young leaves developed from the original first near-apical leaf primordium began to be infected（enlarged blue dashed box in B）; SAM: apical meristematic tissue; Le: leaf blade; Lp: leaf primordium; Bpl: the base of the primary leaf; Bml: the base of the mature leaf; Tml: the tip of a mature leaf; Pi: pith cell. B scale bar = 500 μm; C and E scale bar = 200 μm; A and D scale bar = 100 μm. Blue is nuclear staining and red is LMoV positive signal

Fig. 5 In situ hybridization of LMoV in the root tip of L. lancifolium A: Negative control（LMoV-uninfected stem tips of L. lancifolium）; B: LMoV distribution in the longitudinal profile of the apical center; C: distribution of LMoV in root tip cortical cells and vascular bundles; D: distribution of LMoV in root apical meristem（Am）and a small amount invades the root cap（RC）; VB: vascular bundles; Pi: pith cells; Co: cortex cells; Ez: elongation zone. The blue color is the nuclei staining, and the red color is the LMoV positive signal. A, B bars = 200 μm, C, D bars = 100 μm

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