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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 125, Issue 1, pp 177–181 | Cite as

Reducing etiolation-like effect and flowering in an in vitro micropropagated Trifolium resupinatum elite genotype

  • Olívia Campos Costa
  • Daniela Lopes Oliveira
  • Ana Rita Silva
  • Ana Barradas
  • João Paulo Crespo
  • Ana Sofia DuqueEmail author
  • Pedro Fevereiro
Research Note

Abstract

There is an ever-increasing demand for high quality forage legumes, essential to feed livestock. Micropropagation may be useful to preserve elite allelic compositions in allogamous and auto-incompatible forage legume species, as is the case of Trifolium resupinatum L. (Persian clover). Etiolation-like phenotypes in in vitro cultured plants, with long weak stems and sparse leaves, together with flowering, compromises the long term establishment and maintenance of explants and the adequate development of plants when transferred to ex vitro. Aiming to develop a solution to limit the impact of in vitro etiolation-like effects, flowering and plant viability, we used a very prone to etiolate elite T. resupinatum genotype maintained through in vitro propagation for over 1 year. Stem segments were subcultured in Murashige and Skoog (MS) supplemented with 2.22, 4.56 or 9.12 µM of zeatin; with 4.44 or 8.88 µM of benzyladenine or with 2.89 or 14.44 µM of gibberellic acid. Gibberellic acid supplementation was detrimental and ultimately led to the death of the explants. Supplementation with 2.22 µM zeatin produce no significant differences from control; however higher concentrations of zeatin, as well as of benzyladenine, diminished the longer internodes and increased the development of shoots with more stout stems. Although rooting was prevented by cytokinins, it was successfully induced when explants were transferred to MS media without plant growth regulators. The 9.12 µM zeatin condition was the most favorable since it reversed the etiolation-like phenotype, inhibited in vitro flowering and improved acclimation from 28.7 to 90 % in the FTTr07.13 T. resupinatum genotype.

Keywords

Etiolation-like effect In vitro flowering Trifolium resupinatum Elite genotype 

Introduction

Legumes, belonging to the Fabaceae family, are essential to humans, either as food source, raw materials for food and feed industries or as feed for livestock (Araújo et al. 2015). Due to increasing demand in high quality forage legumes, seed and grasslands producers and forage breeders need to multiply selected elite plants and preserve the desired traits. In clovers this is possible when species are essentially autogamous, as the case of Trifolium nigrescens (Abberton 2007) but virtually impossible when species are allogamic annuals and essentially auto-incompatible like various Trifolium species, including T. resupinatum. In these cases in vitro micropropagation is the best approach for the preservation and clonal multiplication of elite genotypes (George et al. 2008; Loberant and Altman 2010). Several elite genotypes of different Trifolium species, selected by Fertiprado (http://www.fertiprado.pt/) based on their biomass productivity and growth characteristics, have been micropropagated in vitro using nodal segments (Duque et al. 2015). Our final goal was to obtain a high number of plants from elite Trifolium spp. genotypes by clonal propagation, to successfully acclimatize these genotypes in greenhouse facilities and to make them available for cross or auto pollination. The elite T. resupinatum genotype (FTTr07.13) maintained in vitro for over 1 year stood out by presenting an extremely etiolated-like phenotype, with long weak stems and sparse leaves, together with high percentages of in vitro flowering; regardless of the light intensity, temperature and photoperiod applied (data not shown). Endogenous growth regulators balances may be responsible for the observed phenotype.

In vitro flowering and shorting of generation time in pea, lentil and faba bean have been accomplished by the application of antigibberellin compounds (Ribalta et al. 2014; Mobini et al. 2015). The reduction of internodes length favors at the same time the in vitro flowering of pea (Ribalta et al. 2014), which is in opposition of what we observed in T. resupinatum FTTr07.13 phenotype (flowering but with very elongated internodes). The effect of gibberellins (GA) on flowering induction is complex as it is species-specific (Goldberg-Moeller et al. 2013). The effects of GA, positive or negative, might be operational at several developmental stages, such as during acquisition of floral competence, floral induction and/or reproductive meristem formation (Mutasa-Gottgens and Hedden 2009).

For T. resupinatum and other allogamic clover species in vitro etiolation-like effect produces thin stems, induces flower transition and compromise survival when plants are acclimated to ex vitro conditions. Plants with elongated internodes, when transferred to ex vitro, are not able to maintain an erect growth habit, leading to a prostate bearing. This prostate habit negatively affects bee pollination when plants are transferred to greenhouse facilities (Barradas A., personal communication). Furthermore, in vitro flowering is detrimental for efficient micropropagation; which primary objective remains the production of a maximum number of genetically identical shoots that can be easily rooted and acclimatized (Aremu et al. 2012).

Ultimately, explant responses to micropropagation and the regulation of organogenesis are associated with the endogenous plant growth regulators balance in combination with the interaction with those exogenously supplied (Aremu et al. 2014). Based on preceding observations, and also in previous work using 1.80 µM benzyladenine for propagation of eight forage allogamous legume species (Duque et al. 2015), we tested several concentrations of GA3 (gibberellic acid) and CKs (using zeatin and benzyladenine). Our aim was to identify the adequate plant growth regulator supplementation able to reverse the etiolation-like effect and flowering induced by in vitro culture in the prone to etiolation elite T. resupinatum genotype FTTr07.13.

Materials and methods

FTTr07.13 plantlets were micropropagated in 66 mm Ø glass vials covered with a sterile polyethylene film (Silvex® wrap, Portugal) containing 20 cm3 of Murashige and Skoog (1962) basal salts and vitamins growth-regulator-free medium supplemented with 1.5 % (w/v) sucrose and solidified with 0.8 % (w/v) microagar (Duchefa, The Netherlands) (MS0) as described in Duque et al. (2015). In vitro 1-month-old grown plantlets were used to produce explants. Each explant consisted of a nodal segment of about 0.5 cm, containing an axillary bud. Shoot development was induced by placing the explants on MS0 medium supplemented with 2.22, 4.56 and 9.12 µM of zeatin (Zea), 4.44 and 8.88 µM of benzyladenine (BA) or 2.89 and 14.44 µM of gibberellic acid (GA3). Explants sub-cultured in growth regulator-free MS0 were used as control for evaluating in vitro growth parameters. Plant growth regulators were filter sterilized through 0.2 µm filters (Whatman, USA) and added to pre-sterilized pH 5.8 MS media (110 °C, 30 min) before plating. Cultures were maintained in a growth chamber (Phytotron EDPA 700, Aralab, Portugal) under a 12 h photoperiod at 100 µmol m−2 s−1 provided by cool white fluorescent light and a day/night temperature of 23 °C/20 °C. After 4 weeks, plants from each experimental condition were transferred to MS0 for assessing the ability to differentiate roots. Acclimation was performed in a greenhouse where plants with well-developed root system were potted in peat (2.5 dm3 pots) using a protocol previously developed for Medicago truncatula (Araújo et al. 2004) and covered with a plastic tunnel. After 1 week, the plastic tunnel was removed and plants were allowed to grow to maturity. Plants were maintained in a conventional greenhouse kept cool by ventilation and/or shielding with average daily temperatures from 15 to 35 °C and natural irradiance.

The following parameters were quantified after 4 weeks in the different media under analysis: explants survival, shoot length, number of internodes, stem thickness, calli formation on stem cutting edges, flowering inhibition and root differentiation. All treatments were repeated three times with 4–6 replicates per treatment (n = 12–18). Statistical analysis was performed by one-way analysis of variance ANOVA using Microsoft Excel for Windows (version 2010). Results are presented as mean ± SD with a p ≤ 0.05 statistical significance.

Results and discussion

Stem thickness and internode length

Supplementation with cytokinins led to a general increase of stem thickness when compared with explants growing on MS0 medium (Table 1; Fig. 1a–c). Such effect was more pronounced with 9.12 µM Zea, where stems developed with a robust, erect and thicker growth (Fig. 1b) clearly distinct of those grown in the control media (Fig. 1a). Shoot length decreased significantly in media with 4.56 and 9.12 µM of Zea and with 4.44 µM of BA in comparison to control conditions (Table 1). A significant increase in the number of internodes in media supplemented with 9.12 µM Zea and 4.44 µM BA was also observed (Table 1). In vitro supplementation of gibberellins was expected to induce stem elongation (Kurepin and Pharis 2014). However, the external addition of relatively high concentrations of GA3 could also decrease the internal GA concentration since the activity of GA response regulates GA biosynthesis via a feedback mechanism (Dill and Sun 2001). The extent of GA-mediated growth is determined by the balance between the amount of the GA signal and the levels and/or activities of its repressors (Greenboim-Wainberg et al. 2005; Fleet and Sun 2005; Weiss and Ori 2007). This effect was not observed in our experiments and the addition of GA3 did not revert the etiolation-like phenotype. Further studies are needed to understand the effect of GAs in T. resupinatum and other Trifolium species.
Table 1

Effect of plant growth regulators supplementation on the response of T. resupinatum FTTr07.13 genotype

Medium supplementation

Survival (%)

Shoot length*

Number of internodes*

Calli formation (%)

Flowering inhibition (%)

Rooting (%)

Stem thickness***

MS0**

50.0

7.4 ± 2.2a

3.3 ± 1.0a

0.0

0.0

83.3

(~0.6)

2.22 µM Zea

62.5

4.4 ± 2.7a,b,d

3.8 ± 0.8a,c

20.0

20.0

40.0

(~0.8)

4.56 µM Zea

50.0

4.1 ± 1.2b

6.2 ± 2.5a,b,c

80.0

40.0

0.0

(~1.0)

9.12 µM Zea

81.8

2.4 ± 1.6c,d

7.7 ± 3.2b,c

85.7

71.4

0.0

(~1.5)

4.44 µM BA

66.7

3.9 ± 2.2b,d

7.0 ± 2.1b

100.0

37.5

0.0

(~1.0)

8.88 µM BA

83.3

4.7 ± 3.5a,b,d

5.0 ± 1.7c

100.0

40.0

0.0

(~1.0)

2.89 µM GA3

50.0

nd

nd

33.3

16.7

33.3

(~0.6)

14.44 µM GA3

41.7

nd

nd

0.0

0.0

0.0

(~0.4)

Values followed by the same letter are not significantly different at p ≤ 0.05 according to ANOVA test. n = 12–18

nd not determined

* Values in cm (mean ± SD)

** MS0: MS basal growth-regulator-free medium supplemented with 1.5 % (w/v) sucrose and solidified with 0.8 % (w/v) microagar

*** Approximated values in mm of the stem thickness measured 1.5 cm from the explant base with a digital caliper

Fig. 1

In vitro de-etiolation of the Trifolium resupinatum FTTr07.13 elite genotype: a T. resupinatum in growth-regulator free MS0 medium (flowers shown by arrows); b increased shoots thickness with 9.12 µM Zea supplementation; c Calli formation (see arrows) with 4.44 µM BA supplementation; d root development after transference from Zea supplemented to growth-regulator free MS0 medium; e Plants in ex vitro, with vigorous growth in the greenhouse conditions

Flower inhibition

All the FTTr07.13 plants differentiate flowers on MS0 medium. Such high incidence is undesirable since it negatively affects clonal propagation (George et al. 2008). Flowering inhibition was observed with increasing concentrations of Zea; from 20.0 % in 2.22 µM to 71.4 % in 9.12 µM (Table 1). Benzyladedine supplementation also reduced flowering, although in a lower percentage (37.5 and 40 %, respectively for 4.44 and 8.88 µM of BA; Table 1). In explants subjected to 2.89 µM GA3 16.7 % of flowering inhibition was observed, however, when the medium was supplemented with 14.44 µM GA3 flowering was not inhibited. As mentioned above the effect of gibberellins (GA) on flowering induction is complex and species-specific (Goldberg-Moeller et al. 2013) and deceptively in our FTTr07.13 T. resupinatum genotype a high GA3 supplementation did not inhibit in vitro flowering.

Calli, shoot and root development

Cytokinins promoted callus development at the base of the explant, surrounding the stem excision cutting edge. Callus proliferation increased with Zea concentrations, from 20.0 % with 2.22 µM to 85.7 % with 9.12 µM Zea (Table 1). When BA was applied a 100 % callus development was observed (Fig. 1c). Supplementation with BA led to increased calli formation as well as an increased explant hyperhydration, stressing the different effects of these two cytokinins in in vitro plant development (Gaspar et al. 1996). Calli formation have been reported as a result of a cytokinin and auxin balance (Gaspar et al. 1996; Thorpe 2007). Calli formation supports the assumption that this Trifolium genotype may have high endogenous auxin levels. Nevertheless, more studies are needed to support this hypothesis.

When CKs were added, more than one shoot developed from one single explant, especially when BA was applied (2–4 shoots with Zea and 6.5 ± 1.9 shoots with BA) (Fig. 1c). These results are consistent with reported effects of cytokinins on plant development since they are associated with increased differentiation, with the trigger of axillary bud development and with the de-etiolation of dark grown plants (Chory et al. 1994; Gaspar et al. 1996; Symons and Reid 2003; Greenboim-Wainberg et al. 2005). Explants subjected to 2.89 µM GA3 developed weaker shoots than those in MS0. In all media supplemented with GA3, the survival was always similar or lower than in MS0 (Table 1) and developing explants had a fragile appearance and extremely weak and etiolated-like shoots.

With the exception of 2.22 µM Zea, all cytokinins concentrations inhibited root development. The observation that CKs supplementation did not allowed root differentiation is in accordance to previous reports (Greenboim-Wainberg et al. 2005; Weiss and Ori 2007). However, all explants grown on media supplemented with CKs differentiated roots upon calli excision and after being transferred to growth-regulator-free MS0 medium (Fig. 1d). This is in accordance with our previous work were we were able to root several Trifolium spp. and Hedysarum coronarium shoots developed in 1.8 µM BA upon transference to solidified MS0 growth-regulator-free medium (Duque et al. 2015). When medium was supplemented with the 2.89 µM GA3 rooting decreased to 33 % and with 14.44 µM GA3 rooting was completely inhibit (Table 1).

Acclimation

The best acclimation conditions were obtained with medium supplemented with 9.12 µM Zea. This concentration increased the explants survival, the shoot thickness and the number of internodes (Table 1; Fig. 1b). These explants were transplanted to ex vitro conditions, with 90 % of successful acclimation rate (Fig. 1e), higher than the observed with explants grown in MS0 (28.7 %). For all the other treatments with cytokinins the acclimation rate ranged from 20 to 40 %. Plants subjected to the GA3 treatments failed to survive the acclimation procedure.

Conclusions

We have shown that the etiolation-like effect and flowering induced by in vitro culture in T. resupinatum can be reversed by appropriate supplementation of growth regulators. In a two-step in vitro scheme, using MS supplemented with 9.12 µM Zea as a propagation media followed by subculture in growth-regulator-free MS media for rooting, a method for the micropropagation of a T. resupinatum genotype prone to etiolation was established. Continuous maintenance and multiplication of T. resupinatum shoots was accomplished by subculture, every 4 weeks, to fresh 9.12 µM Zea supplemented MS medium by propagation of isolated 0.5–2.0 cm steam segments. In vitro plants maintained under such established conditions could be easily acclimated to ex vitro, with vigorous growth in the greenhouse or field conditions. This approach could also be applied on other forage legumes showing the same response to in vitro propagation.

Notes

Acknowledgments

Authors acknowledge financial support from Fundação para a Ciência e a Tecnologia (Lisboa, Portugal) through the research Grant SFRH/BPD/74784/2010 (Duque AS), the Research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013) and PRODER (4.1) Project Micropropelite (No. 020536053572). We acknowledge Dr. Susana Araújo for revising the manuscript.

References

  1. Abberton MT (2007) Interspecific hybridization in the genus Trifolium. Plant Breed 126:337–342CrossRefGoogle Scholar
  2. Araújo SS, Duque AS, Santos DM, Fevereiro P (2004) An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong. Plant Cell, Tissue Organ Cult 78:123–131CrossRefGoogle Scholar
  3. Araújo SS, Beebe S, Crespi M, Delbreil B, González EM, Gruber V, Lejeune-Henaut I, Link W, Monteros MJ, Prats E, Rao I, Vadez V, Vaz Patto MC (2015) Abiotic stress responses in legumes: strategies used to cope with environmental challenges. Crit Rev Plant Sci 34(1–3):237–280CrossRefGoogle Scholar
  4. Aremu AO, Bairu MW, Dolezˇal K, Finnie JF, Van Staden J (2012) Topolins: A panacea to plant tissue culture challenges? Plant Cell, Tissue Organ Cult 108:1–16CrossRefGoogle Scholar
  5. 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 118:245–256CrossRefGoogle Scholar
  6. Chory J, Reinecke D, Sim S, Washburn T, Brenner M (1994) A role for cytokinins in de-etiolation in Arabidopsis (det mutants have an altered response to cytokinins). Plant Physiol 104:339–347PubMedPubMedCentralGoogle Scholar
  7. Dill A, Sun T (2001) Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana. Genetics 159:777–785PubMedPubMedCentralGoogle Scholar
  8. Duque AS, Barradas A, Godinho B, Silva AR, Araújo SS, Crespo JP, Fevereiro P (2015) Development of protocols for micropropagation of elite genotype forage allogamous legume species. Acta Hortic 1083:409–413CrossRefGoogle Scholar
  9. Fleet CM, Sun T (2005) A DELLAcate balance: the role of gibberellin in plant morphogenesis. Curr Opin Plant Biol 8:77–85CrossRefPubMedGoogle Scholar
  10. Gaspar T, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA (1996) Plant hormones and plant growth regulators in plant tissue culture. Vitr Cell Dev Biol Plant 32:272–289CrossRefGoogle Scholar
  11. George EF, Hall MA, De Klerk G-J (eds) (2008) Plant tissue culture procedure - background. In: Plant propagation by tissue culture, vol 1, 3rd edn. Springer, The Netherlands, pp 1–28Google Scholar
  12. Goldberg-Moeller R, Shalom L, Shlizerman L, Samuels S, Zur N, Ophir R, Blumwald E, Sadka A (2013) Effects of gibberellin treatment during flowering induction period on global gene expression and the transcription of flowering-control genes in Citrus buds. Plant Sci 198:46–57CrossRefPubMedGoogle Scholar
  13. Greenboim-Wainberg Y, Maymon I, Borochov R, Alvarez J, Olszewski N, Ori N, Eshed Y, Weiss D (2005) Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling. Plant Cell 17:92–102CrossRefPubMedPubMedCentralGoogle Scholar
  14. Kurepin LV, Pharis RP (2014) Light signaling and the phytohormonal regulation of shoot growth. Plant Sci 229:280–289CrossRefPubMedGoogle Scholar
  15. Loberant B, Altman A (2010) Micropropagation of plants. In: Flickinger MC (ed) Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology. Wiley, New York, pp 1–17Google Scholar
  16. Mobini SH, Lulsdorf M, Warkentin TD, Vandenberg A (2015) Plant growth regulators improve in vitro flowering and rapid generation advancement in lentil and faba bean. Vitr Cell Dev Biol Plant 51:71–79CrossRefGoogle Scholar
  17. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  18. Mutasa-Gottgens E, Hedden P (2009) Gibberellin as a factor in floral regulatory networks. J Exp Bot 60:1979–1989CrossRefPubMedGoogle Scholar
  19. Ribalta FM, Croser JS, Erskine W, Finnegan PM, Lulsdorf MM, Ochatt SJ (2014) Antigibberellin-induced reduction of internode length favors in vitro flowering and seed-set in different pea genotypes. Biol Plant 58(1):39–46CrossRefGoogle Scholar
  20. Symons GM, Reid JB (2003) Interactions between light and plant hormones during de-etiolation. J Plant Growth Regul 22:3–14CrossRefGoogle Scholar
  21. Thorpe TA (2007) History of plant tissue culture. Mol Biotechnol 37(2):169–180CrossRefPubMedGoogle Scholar
  22. Weiss D, Ori N (2007) Mechanisms of cross talk between gibberellin and other hormones. Plant Physiol 144:1240–1246CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Olívia Campos Costa
    • 1
    • 3
  • Daniela Lopes Oliveira
    • 1
  • Ana Rita Silva
    • 3
  • Ana Barradas
    • 3
  • João Paulo Crespo
    • 3
  • Ana Sofia Duque
    • 1
    Email author
  • Pedro Fevereiro
    • 1
    • 2
  1. 1.Plant Cell Biotechnology Laboratory, Green-it UnitInstituto de Tecnologia Química e Biológica António Xavier (ITQB)OeirasPortugal
  2. 2.Departamento de Biologia VegetalFaculdade de Ciências da Universidade de LisboaLisbonPortugal
  3. 3.Fertiprado - Sementes e Nutrientes Lda.VaiamontePortugal

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