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Optimizing shoot culture media for Rubus germplasm: the effects of NH4 +, NO3 , and total nitrogen


A mixture component design was applied to testing the Murashige and Skoog (MS) medium nitrogen salts (NH4NO3 and KNO3) in order to optimize growth medium nitrogen for shoot cultures of red raspberries and a range of Rubus germplasm. All other components were those of MS medium with 2.5× MS mesos (CaCl2, MgSO4, KH2PO4) nutrients developed in an earlier study. Eight red raspberry (Rubus idaeus L.) cultivars and one accession of Rubus odoratus L. were evaluated for overall quality, shoot length, proliferation, leaf characteristics, and physiological disorders after 9-wk growth on each treatment. Twelve Rubus genotypes were later tested with the five best treatments. The concentration of NO3 in the medium was the most significant factor for improved shoot quality for most cultivars. The effects of the two-component mixture (NH4 + and K+) and interactions between the mixture components and NO3 were found in many growth responses. Combinations of intermediate or high NO3 (40–60 mM), and intermediate to high NH4 + (30–45 mM) produced the best growth in most cultivars. The 2.5× mesos MS medium with standard MS nitrogen (20 mM NH4 + and 40 mM NO3 ) produced significantly better growth for most genotypes than unmodified MS. Three other nitrogen treatments also improved shoot quality for many genotypes: lower NH4 + with lower total nitrogen (10 mM NH4 + and 40 mM NO3 ) or intermediate or high concentrations of both nitrogen components and higher total nitrogen (30 mM NH4 + and 60 mM NO3 ; 45 mM NH4 + and 60 mM NO3 ) than that of MS medium.

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Funding for this study was from the US Department of Agriculture, Agricultural Research Service CRIS project 5358-21000-033D. Sukalya Poothong acknowledges the scholarship from the Ministry of Science and Technology (Thailand) for her Ph.D. studies.

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Correspondence to Barbara M. Reed.

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Editor: David Duncan

Electronic supplementary material

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Supplement 1

Overall quality graphs (rated 1=poor [dark blue] to 3=good [red]) showing the growth appearance on standard MS medium compared to three levels of NO3 in raspberry shoots of ‘Canby’ (a), ‘Chilliwack’ (b), ‘Indian Summer’ (c), ‘Malling Exploit’ (d), ‘Scepter’ (e), ‘Trailblazer’ (f), ‘Tulameen’ (g), ‘Willamette’ (h), and R. odoratus (i) (PPTX 8113 kb)

Supplement 2

The effects of ammonium and potassium proportion (NH4 +:K+) and total NO3 on the number of shoots, leaf color (SPAD reading), and leaf area (cm2) in additional raspberry cultivars. (PPTX 555 kb)

Supplement 3

The effects of the two mixture components (NH4 + and K+) and the numeric factor (NO3 ) on physiological disorders in nine raspberry cultivars grown on 2.5× MS mesos. (DOCX 29 kb)

Supplement 4

The effects of ammonium and potassium proportion (NH4 +:K+) and total NO3 on leaf spot elimination (rated 1=presence of leaf spots to 3=absent), callus elimination (1=callus ≥5 mm to 3=absent), and hyperhydricity elimination (1=presence of hyperhydricity to 3=absent) for red raspberry shoot cultures. (PPTX 429 kb)

Supplement 5

Best treatments (ammonium and potassium proportions (NH4 +:K+) and total NO3 ) based on the general linear model (GLM) in nine raspberry cultivars. (DOCX 30 kb)

Supplement 6

The effects of the best nitrogen treatments on overall quality and plant appearance of 12 Rubus cultivars and species when analyzed with or without shoots grown on the standard MS medium. (DOCX 34 kb)

Supplement 7

The effects of modified nitrogen levels with 2.5× MS mesos medium on leaf color (SPAD) (a), leaf area (cm2) (b), leaf spot elimination (1=leaf spots present on many leaves, 2=less than 2 leaves, 3=absence) (c), and callus elimination (1=callus ≥5 mm, 2=callus <5 mm, and 3=absence) (d). The mixture component is listed first followed by the total N (mM) and the treatment (e.g., Trt. 3) based on Table 1. Vertical bars indicate standard errors. Means followed by the same letter are not significantly different (p<0.05). Red points represent the responses of shoots grown on standard MS (Trt. 10) but not included in statistical analysis. (PPTX 355 kb)

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Poothong, S., Reed, B.M. Optimizing shoot culture media for Rubus germplasm: the effects of NH4 +, NO3 , and total nitrogen. In Vitro Cell.Dev.Biol.-Plant 52, 265–275 (2016).

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  • Medium optimization
  • Mixture component design
  • Nitrogen source
  • Potassium