Plant Cell Reports

, Volume 36, Issue 11, pp 1717–1730 | Cite as

Use of Plant Preservative Mixture™ for establishing in vitro cultures from field plants: Experience with papaya reveals several PPM™ tolerant endophytic bacteria

  • Pious Thomas
  • Mukta Agrawal
  • C. B. Bharathkumar
Original Article


Key message

Prevalence of diverse PPM™-tolerant endophytic bacteria in papaya, the broad-spectrum microbicide specified for use in plant tissue cultures, capable of surviving covertly in MS-based medium, with implications in contamination management.


Plant Preservative Mixture™ was employed for establishing papaya (Carica papaya) tissue cultures from field explants. Comparing three recommended practices for controlling endogenous microbial contaminants, axillary shoot tips (1.0–1.5 cm) from cv. Arka Prabhath were treated with PPM™ 5% for 4 h (T1), 50% for 10 min (T2) or 100% for 10 min (T3) and cultured in MS-based papaya establishment medium (PEM). By 4–6 weeks, all treatments proved non-rewarding with cultures succumbing either to microbial contamination (80% in T1) or phytotoxicity effect/contamination (90% in T2 and 95% in T3). Another trial adopting a multi-step surface sterilization treatment (carbendazim–cetrimide–HgCl2) followed by culturing in 0.05% PPM-supplemented PEM showed 35% obvious bacterial contamination compared with 40% in control. Single colonies from pooled bacterial growths were tested on 0.1% PPM-incorporated nutrient agar (NA) registering 60% isolates as PPM sensitive. Twenty PPM-surviving isolates were selected and identified. This showed 85% Gram-positive bacteria including 80% under phylum Firmicutes (55% spore-forming Bacillaceae and 25% Staphylococcaceae) and 5% Actinobacteria, and 15% Gram-negative Proteobacteria. About 50% isolates remained wholly non-obvious upon culturing on PEM while the rest showed slow growth with many displaying growth enhancement upon host tissue extract supplementation. Culturing the isolates on PPM-supplemented NA indicated 90–95% as tolerating 0.05–0.1% PPM and 65% overriding 0.2% PPM. The isolates, however, did not display obvious growth in PPM-supplemented PEM where the spore formers survived. The results indicate the prevalence of diverse PPM™-tolerant endophytic bacteria in papaya most of which survive covertly in MS-based medium and the need for taking this into account while using PPM™ for contamination management.


Carica papaya Culture indexing Microbial contamination Micropropagation Plant tissue culture Viable but non-cultivable bacteria 



The study was funded under the ICAR-Extramural Research Project “Micropropagation and Filed Evaluation of Arka Prabhath Papaya” by the Indian Council of Agricultural Research, New Delhi, India. The supply of explant material from Arka Prabhath field plants by Dr. C. Vagugi, Division of Fruit Crops, ICAR-IIHR is gratefully acknowledged. Thanks to Aparna C. Sekhar, Sadiq Shaik Pasha and Shwetha M.D. for the help during the conduct of some of the trials. This publication bears ICAR-IIHR contribution no. 20/2017.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2017_2185_MOESM1_ESM.jpg (273 kb)
Fig. S1 The extent of diversity of endophytic bacteria from PPM- versus PPM + sources upon dilution plating of the colony growths on NA. Bacterial culture from PPMˉ source applied on NA (a), PPM+ NA (b) and bacterial culture from PPM+ stocks on PPM+ NA (c) at 104 dilution (upper row) or at 105 dilution (lower row) (JPEG 273 kb)
299_2017_2185_MOESM2_ESM.doc (84 kb)
Supplementary material 2 (DOC 84 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Division of Biotechnology, Endophytic and Molecular Microbiology LaboratoryICAR-Indian Institute of Horticultural ResearchBengaluruIndia

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