Abstract
Lachenalia species are endemic southern African plants with narrow geographical distribution, and are well-traded as ornamental plants in the international floriculture industry. In an attempt to have a better understanding of their growth and hormonal physiology, we evaluated the effects of different plant growth regulators (PGRs) during the clonal regeneration of Lachenalia montana. An auxin (α-naphthaleneacetic acid = NAA) and three cytokinin (CK) types (benzyladenine = BA, meta-topolin riboside = mTR and isopentenyladenine = iP), each at three concentrations (1, 5 and 10 µM), were tested and the effect of these PGRs on the accumulation of endogenous CK metabolites was evaluated to provide clues on the observed morphological responses. As the most efficient PGR, 10 µM mTR treatment produced the highest number of shoots (approximately five shoots per explant) while 1 µM BA-treated plants had more bulbs (approximately three bulbs per plantlet). Rooting was generally lower with increasing concentration of PGRs especially with the aromatic-type CKs. Based on the concentrations of endogenous CKs, 10 µM mTR regenerants also had the highest CKs (40 142.5 pmol g−1 DW) which were mainly of the aromatic-type (98%). In terms of the functional role of the CKs, O-glucosides (which are reversible CK storage forms) were the most dominant CK-type in the regenerants from 10 µM mTR treatment. On the other hand, the poor rooting, mostly prominent in regenerants from BA treatments was closely related to the high accumulation of N 9-glucosides (well-known CK metabolites directly involved in rooting inhibition) when compared to regenerants from other treatments. Overall, the current findings provide evidence on the interrelationship existing among the exogenous PGRs, phenotypic responses and the endogenous CKs in the in vitro regenerants.
Similar content being viewed by others
Abbreviations
- BA:
-
N 6-Benzyladenine
- BA9G:
-
N 6-Benzyladenine-9-glucoside
- BAR:
-
N 6-Benzyladenine-9-riboside
- BAR5′MP:
-
N 6-Benzyladenine-9-riboside-5′-monophosphate
- CK:
-
Cytokinin
- cZ:
-
cis-Zeatin
- cZOG:
-
cis-Zeatin-O-glucoside
- cZR:
-
cis-Zeatin-9-riboside
- cZR5′MP:
-
cis-Zeatin-9-riboside-5′-monophosphate
- cZROG:
-
cis-Zeatin-O-glucoside riboside
- DHZ:
-
Dihydrozeatin
- DHZOG:
-
Dihydrozeatin-O-glucoside
- DHZR:
-
Dihydrozeatin-9-riboside
- DHZROG:
-
Dihydrozeatin-O-glucoside riboside
- ESI:
-
Electro-spray interface
- iP:
-
N 6-Isopentenyladenine
- iPR:
-
N 6-Isopentenyladenine-9-riboside
- iPR5′MP:
-
N 6-Isopentenyladenine-9-riboside-5′-monophosphate
- MS:
-
Murashige and Skoog medium
- mT:
-
meta-Topolin
- mT9G:
-
meta-Topolin-9-glucoside
- mTOG:
-
meta-Topolin-O-glucoside
- mTR:
-
meta-Topolin-9-riboside
- mTR5′MP:
-
meta-Topolin-9-riboside-5′-monophosphate
- mTROG:
-
meta-Topolin-O-glucoside riboside
- NAA:
-
α-Naphthaleneacetic acid
- oT:
-
ortho-Topolin
- oTR:
-
ortho-Topolin-9-riboside
- PPF:
-
Photosynthetic photon flux density
- pT:
-
para-Topolin
- pTR:
-
para-Topolin-9-riboside
- pTROG:
-
para-Topolin-O-glucoside riboside
- tZ:
-
trans-Zeatin
- tZOG:
-
trans-Zeatin-O-glucoside
- tZR:
-
trans-Zeatin-9-riboside
- tZR5′MP:
-
trans-Zeatin-9-riboside-5′-monophosphate
- tZROG:
-
trans-Zeatin-O-glucoside riboside
- UHPLC® :
-
Ultra-high performance liquid chromatography
References
Aremu AO, Bairu MW, Doležal K, Finnie JF, Van Staden J (2012) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tissue Organ Cult 108:1–16
Aremu AO, Plačková L, Bairu MW, Novák O, Plíhalová L, Doležal K, Finnie JF, Van Staden J (2014) How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea? Plant Cell Tissue Organ Cult 118:245–256
Aremu AO, Masondo NA, Rengasamy KRR, Amoo SO, Gruz J, Bíba O, Šubrtová M, Pěnčík A, Novák O, Doležal K, Van Staden J (2015) Physiological role of phenolic biostimulants isolated from brown seaweed Ecklonia maxima on plant growth and development. Planta 241:1313–1324
Aremu AO, Plačková L, Pěnčík A, Novák O, Doležal K, Van Staden J (2016) Auxin-cytokinin interaction and variations in their metabolic products in the regulation of organogenesis in two Eucomis species. New Biotechnol 33:883–890
Ault JR (1995) In vitro rooting and greenhouse acclimatization of Lachenalia shoots. HortScience 30:1304–1305
Bairu MW, Kane ME (2011) Physiological and developmental problems encountered by in vitro cultured plants. Plant Growth Regul 63:101–103
Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63:147–173
Beyl CA (2005) Getting started with tissue culture: media preparation, sterile technique, and laboratory equipment. In: Trigiano RN, Gray DJ (eds) Plant development and biotechnology. CRC Press, Florida, pp 19–38
Ċosiċ T, Motyka V, Raspor M, Savić J, Cingel A, Vinterhalter B, Vinterhalter D, Trávníčková A, Dobrev P, Bohanec B, Ninković S (2015) In vitro shoot organogenesis and comparative analysis of endogenous phytohormones in kohlrabi (Brassica oleracea var. gongylodes): effects of genotype, explant type and applied cytokinins. Plant Cell Tissue Organ Cult 121:741–760
Doležal K, Popa I, Hauserová E, Spíchal L, Chakrabarty K, Novák O, Kryštof V, Voller J, Holub J, Strnad M (2007) Preparation, biological activity and endogenous occurrence of N 6-benzyladenosines. Bioorg Med Chem 15:3737–3747
du Toit ES, Robbertse PJ, Niederwieser JG (2001) An evaluation of bulb growth and structure of Lachenalia cv. Ronina bulbs. S Afr J Bot 67:667–670
du Toit ES, Robbertse PJ, Niederwieser JG (2002) Effects of growth and storage temperature on Lachenalia cv. Ronina bulb morphology. Sci Hortic 94:117–123
du Toit ES, Robbertse PJ, Niederwieser JG (2004) Plant carbohydrate partitioning of Lachenalia cv. Ronina during bulb production. Sci Hortic 102:433–440
Duncan G (2012) The genus Lachenalia. Botanical magazine monograph. Kew Publishing, Royal Botanic Gardens, Kew
Fennell CW, Van Staden J (2004) Biotechnology of southern African bulbs. S Afr J Bot 70:37–46
Gajdošová S, Spíchal L, Kamínek M, Hoyerová K, Novák O, Dobrev PI, Galuszka P, Klíma P, Gaudinová A, Žižková E, Hanuš J, Dančák M, Trávníček B, Pešek B, Krupička M, Vaňková R, Strnad M, Motyka V (2011) Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J Exp Bot 62:2827–2840
Grace OM, Van Staden J (2003) A horticultural history of Lachenalia (Hyacinthaceae). S Afr J Sci 99:526–531
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil, vol 347, 2nd edn. Circular California Agricultural Experiment Station, Berkeley, College of Agriculture, University of California
Holub J, Hanuš J, Hanke DE, Strnad M (1998) Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul 26:109–115
Jameson PE (2017) Cytokinins. In: Murray BG, Murphy DJ (eds) Encyclopedia of applied plant sciences, 2nd edn. Academic Press, Oxford, pp 391–402. doi:10.1016/B978-0-12-394807-6.00102-7
Kamínek M, Vaněk T, Motyka V (1987) Cytokinin activities of N6-benzyladenosine derivatives hydroxylated on the side-chain phenyl ring. J Plant Growth Regul 6:113–120
Kapczńska A (2009) Lachenalia - breeding, culture and usage of a new ornamental bulbous plant. Hortic Landscape Archit 30:5–10
Kapczyńska A (2013) Effect of plant spacing on the growth, flowering and bulb production of four lachenalia cultivars. S Afr J Bot 88:164–169
Kiran NS, Polanská L, Fohlerová R, Mazura P, Válková M, Šmeral M, Zouhar J, Malbeck J, Dobrev PI, Macháčková I, Brzobohatý B (2006) Ectopic over-expression of the maize β-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. J Exp Bot 57:985–996
Kleynhans R, Niederwieser JG, Hancke FL (2002) Lachenalia: Development and commercialization of a new flower bulb crop. Acta Hortic 570:81–85
Kumar V, Moyo M, Van Staden J (2016) Enhancing plant regeneration of Lachenalia viridiflora, a critically endangered ornamental geophyte with high floricultural potential. Sci Hortic 211:263–268
Malá J, Máchová P, Cvrčková H, Karady M, Novák O, Mikulík J, Hauserová E, Greplová J, Strnad M, Doležal K (2009) Micropropagation of wild service tree (Sorbus torminalis [L.] Crantz): the regulative role of different aromatic cytokinins during organogenesis. J Plant Growth Regul 28:341–348
McCartan SA, Van Staden J (1999) Micropropagation of members of the Hyacinthaceae with medicinal and ornamental potential—a review. S Afr J Bot 65:361–369
Montalbán IA, Novák O, Rolčik J, Strnad M, Moncaleán P (2013) Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees. Physiol Plant 148:214–231
Motte H, Vereecke D, Geelen D, Werbrouck S (2014) The molecular path to in vitro shoot regeneration. Biotechnol Adv 32:107–121
Moyo M, Bairu MW, Amoo SO, Van Staden J (2011) Plant biotechnology in South Africa: micropropagation research endeavours, prospects and challenges. S Afr J Bot 77:996–1011
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Nel DD (1983) Rapid propagation of Lachenalia hybrids in vitro. S Afr J Bot 2:245–246
Niederwieser JG (2004) Role of biotechnology in the development and production of Lachenalia and Ornithogalum cultivars in South Africa. S Afr J Bot 70:47–51
Niederwieser JG, Van Staden J (1990) The relationship between genotype, tissue age and endogenous cytokinin levels on adventitious bud formation on leaves of Lachenalia. Plant Cell Tissue Organ Cult 22:223–228
Niederwieser JG, Van Staden J, Upfold SJ, Drewes FE (1992) Metabolism of 6-benzyladenine by leaf explants of Lachenalia during adventitious bud formation. S Afr J Bot 58:236–238
Novák O, Hauserová E, Amakorová P, Doležal K, Strnad M (2008) Cytokinin profiling in plant tissues using ultra-performance liquid chromatography—electrospray tandem mass spectrometry. Phytochemistry 69:2214–2224
Pérez-Jiménez M, Cantero-Navarro E, Pérez-Alfocea F, Cos-Terrer J (2014) Endogenous hormones response to cytokinins with regard to organogenesis in explants of peach (Prunus persica L. Batsch) cultivars and rootstocks (P. persica × Prunus dulcis). Plant Physiol Biochem 84:197–202
Plačková L, Hrdlička J, Smýkalová I, Cvečková M, Novák O, Griga M, Doležal K (2015) Cytokinin profiling of long-term in vitro pea (Pisum sativum L.) shoot cultures. Plant Growth Regul 77:125–132
Plíhalová L, Vylíčilová H, Doležal K, Zahajská L, Zatloukal M, Strnad M (2016) Synthesis of aromatic cytokinins for plant biotechnology. New Biotechnol 33:614–624
Raimondo D, von Staden L, Foden W, Victor JE, Helme NA, Turner RC, Kamundi DA, Manyama PA (2009) Red list of south african plants. Strelitzia 25. South African National Biodiversity Institute, Pretoria
Reinten EY, Coetzee JH, van Wyk BE (2011) The potential of South African indigenous plants for the international cut flower trade. S Afr J Bot 77:934–946
Ruffoni B, Savona M (2013) Physiological and biochemical analysis of growth abnormalities associated with plant tissue culture. Hortic Environ Biotechnol 54:191–205
Sakakibara H (2006) Cytokinins: activity, biosynthesis, and translocation. Ann Rev Plant Biol 57:431–449
Schäfer M, Brütting C, Meza-Canales ID, Großkinsky DK, Vankova R, Baldwin IT, Meldau S (2015) The role of cis-zeatin-type cytokinins in plant growth regulation and mediating responses to environmental interactions. J Exp Bot 66:4873–4884
Slabbert MM, Niederwieser GJ (1999) In vitro bulblet production of Lachenalia. Plant Cell Rep 18:620–624
Acknowledgements
We thank Prof G. D. Duncan for the generous gift of bulbs used for the study. This work was financially supported by the University of KwaZulu-Natal and National Research Foundation (Green Economy Fellowship—U98028), South Africa. Additional finance was provided by the Ministry of Education, Youth and Sport of the Czech Republic (the Program “Návrat” for Research, Development, and Innovations, no. LK21306), National Program for Sustainability (Grant LO1204) and the Czech Science Foundation (Grant 14-34792S). We thank Mrs Alison Young (UKZN Botanical Garden, Pietermaritzburg, South Africa) and her staff for maintaining the mother plants in the greenhouse. We acknowledge the contribution of the Southern African Systems Analysis Centre, the National Research Foundation and the Department of Science and Technology in South Africa as well as the International Institute of Applied Systems Analysis in Austria.
Author contributions
AOA conceived the research idea and designed the experiments. Micropropagation experiments and data collection were conducted by AOA, NAM, MM and SOA. LP and ON conducted and (together with KD) analysed the CK data. AOA prepared the draft manuscript with help of all the other authors. KD prepared mTR, KD and JVS contributed research facilities/reagents/materials and supervised the research. All authors read and edited the final manuscript.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Aremu, A.O., Plačková, L., Masondo, N.A. et al. Regulating the regulators: responses of four plant growth regulators during clonal propagation of Lachenalia montana . Plant Growth Regul 82, 305–315 (2017). https://doi.org/10.1007/s10725-017-0260-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10725-017-0260-9