A L I C E E S C A L A N T E D E C R U Z A N D N G S H E E P I N G

D E P A R T M E N T O F B I O S C I E N C E S

F A C U L T Y O F A P P L I E D S C I E N C E S

LOW COST OPTIONS FOR PLANT TISSUE

CULTURE TECHNOLOGY –

PRECONDITIONING OF DONOR PLANTS

March 22-23, 2018Nilai Springs Resort Hotel

Nilai, Malaysia

OUTLINE

• Introduction

• Objectives

• Methodology

• Results and Discussion

• Conclusion

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PLANT TISSUE CULTURE

• Applied on many commercially important crops such as banana, ginger, papaya and Jatropha.

• New avenues for agro-entrepreneurs as it can mass produce high quality, disease free plantlets in a short

time

• High cost due to chemicals, equipment, facilities and economic loss due to contamination

• Low cost options include simple, cheap and easily adopted approaches to reduce contamination

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COMMON STRATEGIES TO

PREVENT CONTAMINATION

• Surface sterilisation of explants using mercuricchloride• Seeds• Nodal segments • Leaves

• Preconditioning of donor plants• Treatment with contact and systemic chemicals

• Pruning

Stage 0

Preparation of donor plant

Stage I

Introduction and establishment

Stage II

Propagation of plants

Stage III

Rooting and preparation for ex vitro conditions

Stage IV

Ex vitro

acclimatisation

Stages in plant tissue

culture (García-Gonzales et

al, 2010)

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OBJECTIVE

• To investigate the effect of pruning and/or chemical treatment protocols on the occurrence of microbial contaminations in shoot tip and nodal cultures of Jatropha curcas.

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METHODOLOGY -

MICROPROPAGATION

OF J. CURCAS

J. curcas shoot

5- year old J. curcas

plants (donor plant)

grown in greenhouse

Shoot tip and nodal

explants

Explants air-dried in

laminar flowJ. curcas shoot-tip and nodal cultures

Sterilisation of

explants

Preconditioning

of donor plants

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Treatment Treatment Protocol

ControlDonor plants were not pruned.

Untreated shoots were collected and shoot culture was initiated.

Method I

(Chemical application only)

Donor plants were not pruned. Shoots sprayed with chemicals. Three days after chemical application, treated shoots were collected and shoot culture was initiated.

Method II

(Pruning only)

Donor plants were pruned. Three weeks after pruning, new shoots were collected and shoot culture was initiated.

Method III

(Pruning & chemical application)

Donor plants were pruned. Three weeks after pruning, new shoots were sprayed with chemicals. Three days after chemical application, shoots were collected and shoot culture was initiated.

Chemicals: fungicide (Benocide 50WP, 0.5g/L) and insecticide (CH Malaxion 84.0,

2.2ml/L).

Methodology – preconditioning of donor plant

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METHODOLOGY

Sterilisation of explants

• Washed with commercial detergent and 2-3 drops of Tween 20 for 5 minutes

• Rinsed under running tap water for 5 minutes • Further decontaminated in the laminar flow chamber in

• (i) 5% (v/v) commercial bleach + Tween 20 (3 hours)• (ii) 10% (v/v) commercial bleach + Tween 20 (25 min) and • (iii) 70% (v/v) Ethanol (2-3 min)

• Rinsed three times with sterile distilled waterCulture media and culture conditions• Full strength Murashige and Skoog‘s medium with 2% (w/v) sucrose and 0.8% (w/v) agar

• Incubated at 25 ± 1° C under 13/11 (light/dark cycle) and irradiance provided by cool-white fluorescent tubes

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METHODOLOGY

Assessment of cultures • The experiment was repeated three times. • A total of 155-156 nodal and shoot-tip explants were cultured per treatment

• Examined for microbial contamination every two weeks• Contaminated:

• Bacterial contamination - slimy appearance in colors such as white, cream, pink or yellow

• Fungal contamination - white or black ‘fuzzy’ appearance

• Clean: • Free of any fungal or bacterial contamination

Statistical analysis• Analysis of variance (ANOVA) using SPSS software version 20 (SPSS Inc., Chicago) and means were compared using the most significant difference test, Tukey’s test in which p<0.05.

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RESULTS

Bacterial contamination Fungal contamination Bacterial + Fungal

Contamination

• A total of 622 J. curcas cultures consisting of the four types of treatment protocols were produced.

• Contamination occurred mostly in cultures that utilized explants from Control and chemically treated donor plants (Method I).

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EFFECTS OF PRECONDITIONING TREATMENTS ON J.

CURCAS SHOOT-TIP AND NODAL EXPLANTS DERIVED

FROM GREENHOUSE-GROWN PLANTS, 4 WEEKS AFTER

CULTURE INITIATION

Treatment method

Total

number of explants

Percentage of

‘clean’ cultures ±SE

Percentage of

contaminated cultures ± SE

Method I - No Treatment

156 4.49 ± 0.02 a 95.51 ± 0.02 c

Method II -

Chemical application only

155 1.94 ± 0.01 a 98.06 ± 0.01 c

Method III -Pruning only

156 57.69 ± 0.04 b 42.31 ± 0.04 b

Method IV -

Pruning &

Chemical application

155 84.0.03 c 25.16 ± 0.03 a

Values are presented as means ± SE. Different letters within a column indicate significant differences atP<0.05 according to analysis of variance (ANOVA)

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RESULTS AND DISCUSSION

• Results indicate the existence of contaminants that are hard to eliminate in J. curcas.

• High percentage of explants (58%) derived from these newly developed shoots remained free from visible contaminants even after 4 weeks in culture

• Highest percentage of ‘clean’ explants (75%) was obtained from donor plants that have been pruned and applied with an insecticide and a fungicide (Method IV)

• Pruning with or without the application of fungicides and insecticides would effectively reduce the rate of contamination in shoot-tip and nodal cultures of J. curcas

• Both methods might have significantly reduced the microbial loads on the donor plants

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CONCLUSION

• Preconditioning the donor plants by pruning and/or application of insecticide and fungicide, were effective in obtaining a high percentage of ‘clean’ explants for in vitrocultures J. curcas.

• Adoption of these protocols in the establishment of in vitrocultures for J. curcas eliminates the use of antibiotics and hazardous chemicals like mercuric chloride

• Provides a low cost means of ensuring a higher percentage of ‘clean’ in-vitro cultures

• The elimination of fungal and bacterial contaminants from the explants of J. curcas would facilitate more productive and economical culture, mass propagation and genetic improvement of this useful plant

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ACKNOWLEDGEMENT

WE WOULD LIKE TO THANK:

• NILAI UNIVERSITY FOR EXTENDING THE SUPPORT TO CARRY OUT

THIS STUDY.

• GNANATHESIGAN MAYURATHAN, SHUBA SALVAM AND

DEEPAPHRIYA NAGARAJU FOR THEIR TECHNICAL ASSISTANCE.

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Thank You