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Cryotherapy for Pathogen Free Planting Material

in Ornamental Crops

Abhay Kumar Gaurav10459

Ph. D. 1st Year

Seminar: FLA 691

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Pathogen-free stocks of planting materials: Pivotal for productivity and value of ornamental plants

Vegetatively propagated plants: Prone to accumulate pathogens, which are transmitted to new crops in infected propagules

Pathogen-free planting materials is required for Commercial cultivation

Germplasm collections in gene banks Breeding new cultivars for future needs

Traditional techniques: Shoot tip or meristem culture, , thermotherapy followed by shoot tip culture in vitro have been developed

They are widely used to produce pathogen-free plants although the results are not always satisfactory and reproducible

INTRODUCTION

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Shoot tips are structures that consists of the apical or lateral shoot meristem (0.1–1.5 mm in size) with three to four leaf primordia

The youngest meristematic cells are usually free of viruses/

phytoplasma/ bacteria. Excision and regeneration might result in pathogen-free plants

Regeneration ability: Proportional to size of the shoot tip, but pathogen eradication is more efficient using small shoot tips (0.2–0.4 mm)

Pathogen eradication using meristem culture is challenged by: Difficulty of excising very small meristems

Ensuring their survival and Regeneration of the tiny meristems

Problems of Meristem culture

Cryotherapy of shoot tips, i.e. brief treatment of shoot tips in liquid nitrogen: Based on cryopreservation techniques

It eliminates plant pathogens like viruses/ phytoplasmas/ bacteria by briefly treating shoot tips in liquid nitrogen using cryopreservation protocols

In cryotherapy, infected cells are eliminated by the lethal effects of the ultra-low temperature and/or subsequent warming

Healthy plants are regenerated from the surviving pathogen-free meristematic tissue

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Cryotherapy

• Brison et al. (1997) reported for the first time successful elimination of Plum pox virus (PPV) from an infected interspecific Prunus rootstock by cryotherapy of shoot tips

Advantages

It facilitates treatment of large numbers of samples because pathogen eradication is independent of the size of shoot tips used

[The frequency of virus elimination by shoot tip culture decreased from 31 to 12% when the size of shoot tips increased from 0.3 to 1.5 mm, while the size of shoot tips did not affect percentage (45-60%) of virus elimination by cryotherapy of shoot tips. Brison et al. (1997) ]

Easier excision of larger shoot tips is possible and the higher proportion of pathogen-free regenerants is obtained

Fewer regenerated shoots need to be pathogen-indexed to identify those free of the pathogen

Time period required to implement the whole cryotherapy procedure was shortest compared to thermotherapy and thermotherapy followed by shoot tip culture

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Chrysanthemum Stunt Viroid (CSVd) and Chrysanthemum Chlorotic Mottle Viroid (CChMVd Chrysanthemum Su Min Jeon et al., 2014

How does Cryotherapy

works

???

Elimination of infected cells by cryotherapy of shoot tipsIn

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Figure 1. (a) Overview of the main steps of the procedure

Cryotherapy depends on the availability of tissue culture and cryopreservation protocols for the plant of interest.

(Wang and Valkonen, 2008)

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The crucial step for achieving good survival in cryotherapy is the dehydration of cells

Vitrification is the preferred method for dehydration

LS is the liquid culture medium in which sucrose (0.4 mol/L) and the glycerol (2.0 mol/L) w

Most common vitrification solution: PVS2PVS2 contains 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethylsulphoxide (DMSO) and 0.4M sucrose in MS medium Fig. The protocol of vitirication method

( Sakai et al., 1990).

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In infected plants, the meristematic dome is either free or contains a very low concentration of viruses. Virus titer increases with increasing distance from the meristematic dome

When shoot tips freezes in liquid nitrogen, cells that are located in the lower part of the Apical Dome (AD) (infected by the virus) are killed

Cells that are located in the top part of the AD and in the first leaf primordia and are likely to be free from virus infection survive

Thus, plants regenerated after cryotherapy could be virus-free

Virus localization in the shoot tips demonstrated that viral signals were never detected in the apical dome and the first three leaf primordia, but only in more differentiated cells

(Wang and Valkonen, 2008a)

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Schematic illustration of the anatomical differences b/w the cells of the meristematic zone in apical dome and more differentiated cells in the shoot apex.

Abbreviations: AD, apical dome; HC, healthy cells; KC, killed cells; LP , leaf primordium: PIC, pathogen infected cells; SC, surviving cells.

(Wang and Valkonen, 2008)

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The sizes of cells and vacuoles increase, the nucleo–cytoplasmic ratio decreases and the propensity for injuries at ultra-low temperatures grows with increasing distance from the AD

For successful cryotherapy, lethal intracellular injury of the pathogen-infected cells should be promoted to remove them

Phytoplasma and Huanglongbing bacterium colonize only in the phloem tissues of the lower part of shoot tips

Thus do not invade the apical dome and the first two leaf primordia

Therefore, plants regenerated from cryotherapy could be free of phytoplasma and Huanglongbing bacterium. (Wang and Valkonen, 2008b)

CASE STUDIES

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Case Study-1

Objective:

Comparison b/w meristem culture and cryotherapy to eradicate the Pelargonium flower break virus and Pelargonium line pattern virus

Evaluation of Cryotherapy efficiency using 5 different Pelargonium cultivars, with the help of DAS-ELISA and immunolocalisation methods

Introduction

Pelargonium: Popular bedding and pot plant Pelargonium flower break virus (PFBV) and Pelargonium line

pattern virus (PLPV): Biggest threat in Pelargonium production

Transmission: Mechanical inoculation and Vegetative propagation

Symptoms of PFBV infection: Colour breaking on flowers and chlorotic

spotting of leaves etc.

PLPV infection: Yellow spots and line patterns on the leaves

But Pelargonium infected plants can stay symptomless, inducing an important risk of underestimating the spread of the diseases

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Plant Materials Five cultivars:

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Species Cultuvars

P. x hortorum Berg Palais

P. x hortorum Stellar Artic

P. x hortorum Cahors

P. x hortorum Elsie

P. x peltatum Balcon Rouge

‘Berg Palais’: DAS-ELISA negative Used for checking virus Immunolicalisation Not tested cryotherapy efficiency

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CryotherapySurface-disinfection of shoot with 70° ethanol Shoot apices (meristematic dome with the two most undifferentiated primordia) dissected in sterile conditions 20-minute immersion in LS Transferred for 15 min to PVS2 Placing on an aluminium foil and plunged into liquid nitrogen Thawing in the recovery solution (15 min at room temperature) RegenerationMeristem CultureIn parallel, apices were directly regenerated through tissue culture.

(Gallard et al., 2008)

DAS-ELISA Testing: Greenhouse material (considered as the control), Plants regenerated from apex culture and Plants regenerated from cryopreserved apicesImmunolocalisations: Greenhouse plants (control) apices, Apices just rewarmed after cryopreservation and Apices from plants regenerated after

cryopreservation with DAS-ELISA negative plant

Results and DiscussionIn

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The four cultivars being DAS-ELISA positive for at least one virus were tested for cryotherapy

After cryotherapy no shoot regenerated from ‘Elsie’

Virus elimination by meristem culture or cryopreservation evaluated for ‘Cahors’, ‘Stellar Artic’ and ‘Balcon Rouge’ by ELISA tests on 3-month old vitro plants

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PFBV-ELISA negative plants not obtained after apex culture

onlyLow or zero virus eradication rate by meristem culture: Use of meristems with their first two primordia.

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Three months after cryopreservation, immunolocalisation on the regenerated ELISA-negative plants were realised. Signals were detected in all the shoots indicating that PFPV and PLPV were not completely eliminated

Inferences

PFBV and PLPV are even present inside meristematic cells

Cryotherapy results in a higher probability to eradicate PFBV and PLPV compared to single apex culture

Cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally

In future research, thermotherapy and cryotherapy can be combine to investigate their impact on virus replication

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Case Study-2

Objective: To test how efficiently could SPFMV and SPCSV be eliminated

from sweet potato shoot tips by cryotherapy as compared to shoot tip culture

Whether synergism in plants co-infected with these viruses could affect the results

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Sweet-potato (Ipomoea batatas L.) important staple crop

Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) are imp virus hinders the production

Synergistic interactions of these two viruses cause the development of the severe sweet-potato virus disease (SPVD)

Symptoms: Leaf strapping and chlorosis symptoms, are stunted and may fail to produce any storage roots

Introduction

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Planting Material Healthy in vitro plantlets of the line 199004.2 from the CIP,

Lima

After establishment, the plants were side graft-inoculated with scions of sweet-potato cv. Tanzania infected with SPFMV, SPCSV or with both viruses to induce SPVD

Propagation: Vegetative method Constant supply of virus-free planting materials is pivotal The virus eradication efficiency depends: On size of shoot

tips excised Small meristems: Dificult to excise and lower regeneration

frequency Synergistic interactions of viruses influences elimination of

virus by shoot tip culture

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In vitro plantlets from single-node cuttings (1 cm) from the healthy stock plants, infected with SPFMV, infected with SPCSV or co-infected with both viruses

Shoot tips of 0.5mm with 2 leaf primordia, 1.0mm with 3LP and 1.5mm with 4LP were excised from 3-week-old in vitro cultures for shoot tip culture

Shoot tips (1-mm) 3LP excised from 3-week-old in vitro stock cultures were encapsulated and loaded in loading solution

Then beads were exposed to plant vitrification solution 2

Cryotubes having beads immersed in liquid nitrogen for 1 h and then thawed in water bath (40 ◦C for 3min), followed by washing with deloading solution

Method

Fig. 1. Production of virus-freesweetpotato plants using cryotherapy. (A)Sweetpotato line 199004.2 co-infected with SPFMV and SPCSV (B) Symptoms of chlorosis and malformation co-infected with SPFMV and SPCSV. (C) Cholorotic spots and diffuse chlorosis (D) Healthy leaf (E) Typical shoot tip used for cryotherapy. (F) A cryo-treated surviving shoot tip (G) Regrowth of a cryo-treated shoot tip(H) In vitro grafting of the top from a regenerated shoot (I) Establishment of regenerated plant after cryotherapy (J) An established healthy plant obtained from SPVD-affected plant using cryotherapy

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Virus detection by reverse transcription polymerase chain reaction (RT-PCR)

DAS-ELISA and TAS-ELISA used for detection of SPFMV and SPCSV from plants grown in the greenhouse

Results

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Fig. 4. Longitudinal sections of shoot tips of sweetpotato line 199004.2. (A) A non-treated shoot tip(B) A cryo-treated shoot tip after 1 day of post-culture Surviving cells were recognized by densely stained nucleoli (arrows) in nuclei located in well preserved cytoplasm. AD, apical dome; LP1, the first (youngest) leaf primordium; LP2, the second youngest leaf primordium.

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Fig. A cross-section of the shoot tip (A) Apical dome (AD) of the meristem and the six youngest leaf primordia (numbers 1–6)

(B) The cells of AD are thin-walled and isodiametric in shape i.e., not yet differentiated. Nucleoli in nuclei are densely stained

(C) In the LP1, cells are irregular in size/shape than AD & thicker cell wall (arrow), i.e., advanced differentiation

(D to G) LP2 to LP5, Development of anatomically more complex vascular bundles

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Shoot tip culture with shoot tips of 1mm or less in size was very efficient for the production of virus-free plants

But, through cryotherapy larger shoots tips can be excised easily & rapidly with high efficiency of virus elimination

Cryotherapy of shoot tips could be used for virus both elimination and preparation of virus free material for long-term preservation

Inferences

Conclusion:

Cryotherapy could be adopted in pathogen-eradication schemes with high efficiency for species and genotypes that are going to be cryopreserved

Cryotherapy-based procedures do not require any special equipment

It marginally add to the time and cost of the traditional procedures of shoot tip culture for pathogen eradication

The use of vitrification protocols and optimized regeneration of shoots will minimize the risk of somaclonal variation

However, studies on pathogen elimination by cryotherapy of shoot tips are still quite limitedIn

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Future thrust:

Very less work has been done on ornamental crops, so focus should be given

Cryotherapy depends on the availability of tissue culture and cryopreservation protocols for the plant of interest. So protocols, need to developed for different species/genotypes

Combination of thermotherapy and cryotherapy of shoot tips can enhance efficiency of virus elimination

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