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Biochemical and histological changes during in vitro rooting of microcuttings of Bacopa monnieri (L.) Wettst.

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Abstract

The present study was conducted to investigate the biochemical changes vis-à-vis histological changes during adventitious rooting of microcuttings of Bacopa monnieri (L.) Wettst. The rooting in these microcuttings was induced on basal MS medium and medium supplemented with different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA). Presence of lower auxin concentration (1.0 µM) in the medium enhanced rooting and significantly improved number of roots per shoot but maximum root length was observed on basal MS medium. Histological studies were conducted to identify different phases of rooting in these microcuttings. The root meristemoids with distinct polarity become visible after 3 days and mark the beginning of in vitro root initiation phase. It was followed by primordia elongation, root emergence and visible rooting on the 5th day of culture on medium supplemented with auxins. Biochemical studies were also conducted from basal portions of microcuttings cultured on MS medium supplemented with 1.0 µM IBA and control (basal MS medium) from 0 to 7 days. Total carbohydrate content was lower during initial periods (up to day 1) and was found to increase during root initiation and primordia development, which reflects high energy demands for active cell divisions. A significantly higher level of phenols was recorded in microcuttings on medium supplemented with IBA. Polyphenol oxidase, peroxidase (POX), ascorbate peroxidase activities were also found to vary during different phases of rhizogenesis. Early phases were also marked with the lower activities of POX and IAAO. This study revealed significant role of enzymes, sugars and phenols during different phases of rooting.

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Abbreviations

IBA:

Indole butyric acid

POX:

Peroxidase

APx:

Ascorbate peroxidases

D.P.X.:

Distyrene plasticizer and xylene

PPO:

Polyphenol oxidase

TSS:

Total soluble sugar

RS:

Reducing sugar

IAAO:

Indole-3-acetic acid oxidase

IAA:

Indole-3-acetic acid

TBA:

t-Butyl alcohol

References

  • Ahkami AM, Lischewski S, Haensch KT, Porfirova S, Hofmann J, Rolletschek H, Melzer M, Franken P, Hause B, Druege U, Hajirezaei MR (2009) Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism. New Phytol 181:613–625

    Article  CAS  PubMed  Google Scholar 

  • Altamura MM (1996) Root histogenesis in herbaceous and woody explants cultured in vitro. A critical review. Agronomie 16:589–602

    Article  Google Scholar 

  • Alvarez R, Nissen SJ, Sutter EG (1989) Relationship between indole-3-acetic acid levels in apple (Malus pumilla Mill) rootstocks cultured in vitro and adventitious root formation in the presence of indole-3-butyric acid. Plant Physiol 89:439–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bansal M, Reddy MS, Kumar A (2016) Seasonal variations in harvest index and bacoside A contents amongst accessions of Bacopa monnieri (L.) Wettst. Collected from wild populations. Physiol Mol Biol Plants 22:407–413

    Article  PubMed  PubMed Central  Google Scholar 

  • Barghchi M (1988) Micropropagation of Alnus cordata (Loisel.) Loisel. Plant Cell, Tissue Organ Cult 15:233–244

    Article  CAS  Google Scholar 

  • Bisbis C, Kevers C, Crevecouer M, Dommes J, Gasper T (2003) Restart of lignifications in micropropagated walnut shoots coincides with rooting induction. Biol Plant 47:1–5

    Article  CAS  Google Scholar 

  • Bojanovic JJ, Jevtovic AD, Pantic VS, Dugandzic SM, Javonovic DS (1970) Thymus histones in young and old rats. Gerontologia 16:304–312

    Article  CAS  PubMed  Google Scholar 

  • Camellia N, Thohirah NA, Abdullah NAP, Mohd. Khidir O (2009) Improvement on rooting quality of Jatropha curcas using indole butyric acid (IBA). Res J Agric Biol Sci 5:338–343

    Google Scholar 

  • Cheniany M, Ebrahimzadeh H, Maoudi-nejad A, Vahdati K, Leslie C (2010) Effect of endogenous phenol and some antioxidant enzyme activities on rooting of Persian walnut (Juglans regia L.). Afr J Plant Sci 4:479–487

    CAS  Google Scholar 

  • De Klerk GJ (1996) Marker of adventitious root formation. Agronomie 16:609–616

    Article  Google Scholar 

  • De Klerk GJ, Arnholdt-Schmitt B, Lieberei R, Neumann KH (1997) Regeneration of roots, shoots and embryos: physiological, biochemical and molecular aspects. Biol Plant 39:53–66

    Article  CAS  Google Scholar 

  • De Klerk GJ, Kreiken WVD, Jong JC (1999) The formation of adventitious roots: new concepts, new possibilities. In vitro Cell Dev Biol Plant 35:189–199

    Article  Google Scholar 

  • De Klerk G-J, Guan H, Huisman P, Marinova S (2011) Effects of phenolic compounds on adventitious root formation and oxidative decarboxylation of applied indoleacetic acid in Malus ‘Jork9’. Plant Growth Regul 63:175–185

    Article  Google Scholar 

  • Diaz-Sala C, Hutchison KW, Goldfarb B, Greenwood MS (1996) Maturation-related loss in rooting competence by loblolly pine stem cuttings: the role of auxin transport, metabolism and tissue sensitivity. Physiol Plant 97:481–490

    Article  CAS  Google Scholar 

  • Dubois M, Gilles KA, Hamilton JK, Roberts PA, Smith F (1956) Calorimetric methods for determination of sugar and related substances. Determination of concentration of pure sugar solution. Annu Chem 28:350–360

    Article  CAS  Google Scholar 

  • Eliasson L (1978) Effects of nutrient and light on growth and root formation in Pisum sativum cuttings. Physiol Plant 43:13–18

    Article  CAS  Google Scholar 

  • Foong TW, Barnes MF (1981) Levels of reserve metabolites and oxidative enzymes in cutting of easy to root and difficult to root Rhododendrons. Biochem Physiol Pflanzen 176:206–216

    Article  Google Scholar 

  • Garcia-Gomez MR, Sanchez-Romero C, Barcelo-Munoz A, Heredia A, Pliego-Alfaro F (1995) Peroxidase activity during adventitious root formation in avocado microcuttings. Can J Bot 73:1522–1526

    Article  CAS  Google Scholar 

  • Gaspar T, Kevers C, Hausman JF, Berthon JY, Ripetti V (1992) Practical uses of peroxidase activity as a predictive marker of rooting performance of micropropagated shoots. Agronomic 12:757–765

    Article  Google Scholar 

  • Gasper T, Kevers C, Huasman JF (1997) Indissociable chief factors in the inductive phase of adventitious rooting. In: Altman A, Waisel M (eds) Biology of root formation and development, vol 65. Plenum Press, New York, pp 55–63

    Chapter  Google Scholar 

  • Habib N, Akram MS, Javed MT, Azeem M, Ali Q, Shaheen HL, Ashraf M (2016) Nitric oxide regulated improvement in growth and yield of rice plants grown under salinity stress: antioxidant defense system. Appl Ecol Environ Res 14:91–105

    Article  Google Scholar 

  • Haissig BE (1984) Carbohydrate accumulation and partitioning in Pinus banksiana seedlings and seedling cuttings. Physiol Plant 61:13–19

    Article  CAS  Google Scholar 

  • Haissig BE (1986) Metabolic processes in adventitious rooting of cuttings. In: Jackson MB (ed) New root formation in plants and cuttings. Martinus Nijhoff Publisher, Dordrecht, pp 141–189

    Chapter  Google Scholar 

  • Han H, Zhang S, Sun X (2009) A review on the molecular mechanism of plants modulated by auxin. Afr J Biotechnol 8:348–353

    CAS  Google Scholar 

  • Hartmann HT, Kester DE, Davies FT (1993) Plant propagation—principles and practices, 5th edn. Prentice Hall, Englewood Cliffs, pp 199–255

    Google Scholar 

  • Heloir MC, Kevers C, Hausman JF, Gaspar T (1996) Changes in the concentrations of auxins and polyamines during rooting of in vitro propagated walnut shoots. Tree Physiol 16:515–519

    Article  CAS  PubMed  Google Scholar 

  • Hicks GS (1987) Adventitious rooting of apple microcuttings in vitro, an anatomical study. Can J Bot 65:1913–1920

    Article  Google Scholar 

  • Hunt MA, Trueman SJ, Rasmussen A (2011) Indole-3-butyric acid accelerates adventitious root formation and impedes shoot growth of Pinus elliottii var. elliottii × P. caribaea var. Hondurensis cuttings. New For 41:349–360

    Article  Google Scholar 

  • Inze D, Van Montagu M (1995) Oxidative stress in plants. Curr Opin Biotechnol 6:153–158

    Article  CAS  Google Scholar 

  • Jaleel CA, Gopi R, Sankar B, Manivannan P, Kishorekumar A, Sridharan R, Panneerselvam R (2007) Studies on germination, seedling vigour, lipid peroxidation and proline metabolism in Catharanthus roseus seedlings under salt stress. S Afr J Bot 73:190–195

    Article  Google Scholar 

  • Kar M, Mishra D (1976) Catalase, peroxidase and polyphenol oxidase activities during rice leaf senescence. Plant Physiol 57:315–319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kibber H, Johnston ME, William RR (2004) Adventitious root formation in cuttings of Backhousia citriodora F. Muell 1. Plant genotype, juvenility and characteristics of cuttings. Sci Hortic 102:133–143

    Article  Google Scholar 

  • Kumar A (1996) Studies on in vitro propagation, biochemistry and field evaluation of two economically important plants: Rosa damascene Mill. and Gladiolus spp., Ph.D. Thesis. Kumaun University, Nainital

  • Kumar A, Sood A, Palni LMS, Gupta AK (1999) In vitro propagation of Gladiolus hybridus Hort. Synergistic effect of heat shock and sucrose on morphogenesis. Plant Cell, Tissue Organ Cult 57:105–112

    Article  CAS  Google Scholar 

  • Kumar A, Sood A, Palni LMS, Palni UT, Gupta AK (2001) Micropropagation of Rosa damascene from selected bushes. J Hortic Sci Biotechnol 76:30–34

    Article  CAS  Google Scholar 

  • Leaky RRB (2004) Physiology of vegetative reproduction. In: Burley J, Evans J, Youngquist JA (eds) Encyclopaedia of forest sciences. Academic Press, London, pp 1655–1668

    Chapter  Google Scholar 

  • Lee TT, Starrat AN, Jevnikar JJ (1982) Regulation of enzymic oxidation of indole-3-acetic acid by phenols: structure-activity relationship. Phytochemistry 21:517–523

    Article  CAS  Google Scholar 

  • Liu JH, Reid DM (1992) Adventitious rooting in hypocotyls of sunflower (Helianthus annus) seedlings IV. The role of changes in endogenous free and conjugated indole-3-acetic acid. Physiol Plant 86:285–292

    Article  CAS  Google Scholar 

  • Loach K, Whalley DN (1978) Water and carbohydrate relationships during rooting of cuttings. Acta Hortic 79:161–168

    Article  Google Scholar 

  • Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Mahadevan A, Sridhar R (1986) Methods in physiological plant pathology, 3rd edn. Sivakami Publicattion, Madras, p 316

    Google Scholar 

  • Mark A, Bernard D, Kristen S, Fawzi AR (2004) Oxidases, peroxidases and nitrogen peroxide: the suberin connection. Phytochem Rev 3:113–126

    Article  Google Scholar 

  • McEwen CM Jr (1971) Monoamine oxidase. Methods Enzymol 17B:686–692

    Article  Google Scholar 

  • Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410

    Article  CAS  PubMed  Google Scholar 

  • Molassiotis AN, Dimassi K, Diamantidis G, Therios I (2004) Changes in peroxidases and catalase activity during in vitro rooting. Biol Plant 48:1–5

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nag S, Saha K, Choudhuri MA (2001) Role of auxin and polyamines in adventitious root formation in relation to changes in compounds involved in rooting. J Plant Growth Regul 20:182–194

    Article  CAS  Google Scholar 

  • Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  • Nanda KK, Purohit AN, Mchrotra K (1968) Effect of sucrose, auxin and gibberellic acid on rooting of stem segments of Populus nigra under varying light conditions. Plant Cell Physiol 9:735–743

    CAS  Google Scholar 

  • Patrick JW, Wareing PF (1978) Auxin promoted transport of metabolites in stems of Phaseolus vulgaris L. J Exp Bot 29:359–366

    Article  CAS  Google Scholar 

  • Pellinen RI, Minna-Sisko K, Tauriainen AA, Palva ET, Kangasja RVI (2002) Hydrogen peroxide activates cell death and defense gene expression in birch. Plant Physiol 130:549–560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qaddoury A, Amssa M (2003) Endogenous phenolic contents, peroxidase and polyphenol oxidase activities in date palm (Phoenix dactylifera L.) off shoots related to rooting ability. Acta Physiol Plant 25:417–421

    Article  CAS  Google Scholar 

  • Quoirin G, Boxus P, Gaspar T (1974) Root initiation and isoperoxidases of stem tip cuttings from mature Prunus plants. Physiol Veg 12:165–174

    CAS  Google Scholar 

  • Rios DG, Gea MA, Revilla MA, Rodriguez R, Sanchenz-Olate ME, Rodriguez R (1999) Rooting responses in relation with PO, PPO and IAAO activities on walnut (Juglans regia L.) explants. Acta Hort 442:241–249

    Google Scholar 

  • Rout GR (2006) Effect of auxins on adventitious root development from single node cuttings of Camellia sinensis L. Kuntze and associated biochemical changes. Plant Growth Regul 48:111–117

    Article  CAS  Google Scholar 

  • San Jose MC, Vidal N, Ballester A (1992) Anatomical and biochemical changes during root formation in oak and apple shoots cultured in vitro. Agronomie 12:767–774

    Article  Google Scholar 

  • San Jose MC, Romero L, Janeiro LV (2012) Effect of indole-3-butyric acid on root formation in Alnus glutinosa microcuttings. Silva Fenn 46:643–654

    Article  Google Scholar 

  • Sato Y, Sugiyama M, Gorecki RJ, Fukuda H, Komamine A (1993) Interrelationship between lignin deposition and the activities of peroxidase isozymes in differentiating tracheary elements of Zinnia. Planta 189:584–589

    Article  CAS  Google Scholar 

  • Saxena C, Samantaray S, Rout GR, Das P (2000) Effect of auxins on in vitro rooting of Plumbago zeylanica: peroxidase activity as a marker for rooting induction. Biol Plant 43:121–124

    Article  CAS  Google Scholar 

  • Schnieder EA, Wightman F (1974) Metabolism of auxin in higher plants. Annu Rev Plant Physiol 25:487–513

    Article  Google Scholar 

  • Sharma S, Millam S (2004) Soamtic embryogenesis in Solanum tuberosum L.: a histological examination of key developmental stages. Plant Cell Rep 23:115–119

    CAS  PubMed  Google Scholar 

  • Sivaci A, Yalcim I (2007) Determination of physiological changes in relation to effect of exogenous indole-butyric acid and callus formation in some kinds of apple (Malus sylvestris M.) stem cuttings. Fen Bilimleri Dergisi 27:1–12

    Google Scholar 

  • Steffens B, Wang J, Sauter M (2006) Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice. Planta 223:604–612

    Article  CAS  PubMed  Google Scholar 

  • Sumner JB (1935) A more specific reagent for determination of sugar in urine. J Biol Chem 69:393

    Google Scholar 

  • Swain T, Hillis WE (1959) The phenolic constituents of Prunus domestica: 1. The quantitative analysis of phenolic constituents. J Sci Food Agric 10:63–68

    Article  CAS  Google Scholar 

  • Synkova H, Pospisilova J (2002) In vitro precultivation of tobacco affects the response of antioxidative enzymes to ex vitro acclimation. J Plant Physiol 159:781–789

    Article  CAS  Google Scholar 

  • Tchinda ND, Messi HJCM, Nzweundji FG, Oumar D, Dongmo B, Sanonne Agbor GA, Ndoumou DO (2013) Biochemical aspects of single-node cuttings of Ricinodendron heudelotii (Baill.) in relation with rooting. Afr J Biotechnol 12:1049–1056

    CAS  Google Scholar 

  • Tchoundjeu Z, Avana ML, Leakey RRB, Simons AJ, Asaah E, Duguma B, Bell M (2002) Vegetative propagation of Prunus africana: effects of rooting medium, auxin concentration and leaf area. Agrofor Syst 54:183–192

    Article  Google Scholar 

  • Tremblay FM, Lalonde M (1984) Requirements for in vitro propagation of seven nitrogen-fixing Alnus species. Plant Cell, Tissue Organ Cult 3:189–199

    Article  Google Scholar 

  • Tremblay FM, Perinet P, Lalonde M (1986) Tissue culture of Alnus spp. With regard to symbiosis. In: Bajaj YPS (eds) Biotechnology in agriculture and forestry, vol 1. Trees 1. Springer, Berlin, pp 87–100

  • Tworkoski T, Takeda F (2007) Rooting response of shoot cuttings from three peach growth habits. Sci Hortic 115:98–100

    Article  CAS  Google Scholar 

  • Tyburski J, Jasionowicz P, Tretyn A (2006) The effects of ascorbate on root regeneration in seedling cuttings of tomato. Plant Growth Regul 48:157–173

    Article  CAS  Google Scholar 

  • Vaughn KC, Lax AR, Duke SO (1988) Polyphenol oxidase: the chloroplast oxidase with no established function. Physiol Plant 72:659–665

    Article  CAS  Google Scholar 

  • Vidal N, Arellano G, San Jose MC, Vieitez AM, Ballester A (2003) Developmental stages during the rooting of in vitro-cultured Quercus robur shoots from material of juvenile and mature origin. Tree Physiol 23:1247–1254

    Article  CAS  PubMed  Google Scholar 

  • Wiesmann Z, Riov J, Epstein E (1988) Comparison of movement and metabolism of indole-3-butyric acid in mung bean cuttings. Physiol Plant 74:556–560

    Article  Google Scholar 

  • Wilson PJ, Van Staden J (1990) Rhizocaline, rooting co-factors, and the concept of promoters and inhibitors of adventitious rooting—a review. Ann Bot 66:479–490

    Article  CAS  Google Scholar 

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Acknowledgements

Authors are thankful to TIFAC-CORE, Thapar University, for facilities to carry out this research. The kind help of Prof M. S. Reddy in statistical analysis is thankfully acknowledged.

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  1. Department of Biotechnology, TIFAC-Centre of Relevance and Excellence in Agro and Industrial Biotechnology (CORE), Thapar University, Patiala, 147001, India

    Aanchal Goel, Amanpreet Kaur & Anil Kumar

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  1. Aanchal Goel
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  2. Amanpreet Kaur
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Correspondence to Anil Kumar.

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Communicated by H. Peng.

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Goel, A., Kaur, A. & Kumar, A. Biochemical and histological changes during in vitro rooting of microcuttings of Bacopa monnieri (L.) Wettst.. Acta Physiol Plant 40, 64 (2018). https://doi.org/10.1007/s11738-018-2641-8

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