Enhanced Plantlet Regeneration from Cultured Meristems in Sprouting Buds of Saffron Corms

K. Majourhat,
Department of Biology
Faculty of Sciences-Semlalia-
University Cadi-Ayyad of Marrakech.
Morocco / P. Martínez-Gómez,
Departamento de Mejora Vegetal
CEBAS-CSIC. PoBox 164.
Murcia
Spain
J.A. Fernandez
Departamento de Biotecnologia, IRD (UCLM) Campus Universitario de Albacete s/n. 02071
Albacete
Spain / A. Piqueras,
Departamento de Mejora Vegetal
CEBAS-CSIC. PoBox 164.
Murcia
Spain

Keywords:Crocus sativus, micropropagation, axillary bud, cytokinins

Abstract

When meristems dissected from dormant corms were used for micropropagation of saffron, low and heterogeneous morphogenic potential as well as high contamination levels have been found (Piqueras and Hernandez, 2004). With the aim to prevent the above mentioned inconvenient, shoot cultures have been initiated from meristems in sprouted saffron corms before flowering. These explants showed a significant lower contamination rate (less than 30 %) when cultured in an culture medium composed of QL salts, MS vitamins and 30 g/l sucrose solidified with 7.5 g/l of agar. To study the morphogenic response induced by different cytokinin types and concentrations (BAP, 2iP and TDZ) the explants were cultivated in medium supplemented with them for sex weeks. After this period, the number of new shoots per initial explant, their length and quality were recorded and used as parameters for the selection of the most effective cytokinin level for multiplication. By using this new explant for the initiation of axillary shoot cultures in saffron a new micropropagation procedure has been developed suitable for the clonal propagation or ex situ germplasm conservation of selected genotypes.

Introduction

Crocus sativus L., a perennial bulbous plant, is native to southern Europe and Asia. It has been grown for economic purpose since ancient times (Stephens, 2003). The stigmas that produce, named saffron, are of great commercial value cause of its odoriferous, colouring and medicinal properties (Dauria et al., 2006, Sheng et al., 2006, Abdullaev and Espinosa-Aguirre, 2004). This triploid (2n=3X=24), sterile geophyte is propagated solely by vegetative way using the annual renewal corms. Thanks to this storage organ, as all other crocus species, the saffron plant is adapted to overcome a dry dormant period (Mathew, 1982; Negbi et al., 1989). The development of an efficient micropropagation procedure is needed to produce high quality pathogen free corms and to preserve the genetic resurces of this valuable spice (Piqueras et al., 1999).

In this work, we have tried to develop an efficient in vitro protocol for over year mass propagation of saffron plantlets to overcome the absence of saffron plants during the dormancy phase. The qualitative and quantitative effect of cytokinins on offshoot growth multiplication and quality was also investigated.

MATERIALS AMD METHODS

The shoots initiated from the apical and lateral dormant buds on the corms maintained at room temperature were excised and used as explants. Culture medium was composed of Quorin and Lepoivre salts, Murashige and Skoog vitamins and 3 % sucrose solidified with 7.5 g/l of agar. Several cytokinins (BAP, 2iP and TDZ) at different concentrations (0, 0.2, 1 and 5 mg/l) were tested to study their morphogenic effect incorporated to the culture medium. After six weeks of culture the newly formed shoots were scored for their number, their length and their quality to select the most effective type and level of cytokinin. The quality of shoot was appreciated visually.

For the histological study, samples of the explant basal zone were sampled at different dates of culture and processed for light microscopy as described by Piqueras and Fernandez, 2004).To compare the effect of cytokinin type and concentration a multi-way ANOVA analysis was run using SPSS software

RESULTS AND DISCUSSION

Histological sections of the basal region in the sprouts have shown the presence of meristems intercalated between the expanding leaves of the sprouts (Fig. 1a). The meristems showed a progressive elongation and multiplication in the media supplemented with citokinins (Fig. 1b).

Using the shoots initiated from the apical and lateral dormant buds of the corms, the contamination percentage was decreased largely. It was less than 30%. Such a method could be very useful for this kind of material (corms, bulbs) that is difficult to free from contamination (Piqueras and Fernandez, 2004, Hussey, 1975).The effect of cytokinin type, concentration and their interaction, was found to be significant (P<0.05) for shoot number and length (Table1).Table1 shows the effect of cytokinin type and concentration on the number of offshoot produced.

Although 2iP stimulated the elongation of the newly formed offshoots, it remained as the least efficient of the three citoquinins tested. In fact, the number of sprout was the lowest and their quality is worst (Table 2). On the contrary Crocus sativus explants were more sensitive to the effect of the BAP and the TDZ. Indeed, the multiplication rate increases as more as the concentration of these two phytohormones increase. Nevertheless, the elongation of the formed shoot is inhibited by highest concentrations (Table 2). In the presence of BAP at 5 mg/l, Crocus sativus explants multiply with good rate and best quality than other concentrations (Fig. 2). Using meristems isolated from dormant corms as explants, Piqueras and Fernandez, (2004) reported that BAP was the most efficient cytokinin for shoot proliferation from saffron meristems. Other work reported such effect of cytokinin type on the quality and quantity of saffron leaves and corms grown in vitro (Plessner et al., 1990).Also, the offshoots produced in presence of TDZ at 5 mg/l were relatively hyperhydrated. This physiological disorder that affects all kind of plant could be due to the imbalance of growth regulators. This phenomenon, in some cases, was associated with cytokinins concentration and type (Kataeva et al., 1991).

The use of clean stock plants for in vitro propagation is a prerequisite to a successful multiplication protocol and to increase healthy plant production. The preparation of explants that we have used in this work seems to be a very easy and efficient way to over come the contamination problem. Under these conditions it can be produced until four good quality plantlets after 6 weeks from each explant using BAP as growth regulator. This in vitro propagation protocol can be used for an efficient clonal multiplication or for ex situ germplasm conservation of pest free selected genotypes that can response to the norms European and Mediterranean Plant Protection Organization (EPPO Bulletin, 2002).

Literature Cited

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EPPO Bulletin 2002. Classification scheme for crocus. EPPO Bulletin 32:123-128.

Kataeva N.V., Alexandrova I.G.,Butenko R.G. andDragavtceva E.V. 1991. Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro. Plant cell, Tissue and Organ culture. 27(2):149-154.

Mathew B. 1982. The Crocus, A revision of the genus Crocus (Iridaceae). B.T. Batsford. London.pp 11-12.

Negbi M., Dagan B., Ada D. and Basker D. 1989. Growth, flowering, vegetative reproduction and dormancy in the saffron Crocus (Crocus sativus L.). Israel Journal of Botany, 38: 95-113.

Piqueras A., Han B.H., Escribano, J., Rubio C., Hellin, E. and Fernandez J.A. 1999. Development of cormogenic nodules and microcorms by tissue culture, a new tool for the multiplication and genetic improvement of saffron. Agronomie, 19:603-610.

Piqueras A. and Fernandez J.A. 2004. Phase change from dormancy to continuous shoot proliferation in cultured meristems of saffron corm. In Vitro Cellular & Develop. Biol. 40:74A.

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Table1. Multifactor ANOVA analysis for effect of Cytokinin type and concentration on offshoot number and length

Source / Dependent variable / df / Mean Square / F / Significance
Cytokinin / Offshoot / 2 / 2.896 / 13.031 / 0.000
Length / 2 / 66,099 / 14,127 / 0.000
Concentration / Offshoot / 3 / 9.806 / 44.125 / 0.000
Length / 3 / 47.464 / 10.144 / 0.000
Cytokinin X Concentration / Offshoot / 6 / 0.451 / 2.031 / 0.087
Length / 6 / 20.016 / 4.278 / 0.002
Error / Offshoot / 36 / 0.222
Length / 36 / 4.679

Table2. Effect of the cytokinin type and concentration on in vitro sprouting of Crocus sativus explants

Cytokinin
(concentration in mg/l) / Offshoot
Number (± sd)* / Offshoot
Length (± sd) / Offshoot
Quality (± sd)
Control
BAP (0.2)
BAP (1)
BAP (5)
2iP (0.2)
2iP (1)
2iP (5)
TDZ (0.2)
TDZ (1)
TDZ (5) / 1,00 (± 0.0)e
2,00 (± 0.0)bcde
3,00 (± 0.7)ab
3.50 (± 0.5)a
1.25 (± 0.5)de
1.75 (± 0.5)cde
2.25 (± 0.5)bcd
2.25 (± 0.5)cde
2.50 (± 0.5)abc
3.50 (± 0.5)a / 8.75 (± 0.9)abcd
4.12 (± 0.8)d
12.75 (± 2.6)ab
6.50 (± 1.7)cd
12.00 (± 2.1)abc
13.50 (± 3.0)a
9.25 (± 2.9)abcd
6.75 (± 3.7)cd
7.75 (± 2.0)bcd
4.50 (± 1.2)d / Good
Good
Good
Good
Thin
Thin
Thin
Good
Good
hyperhydrated

* Values in the same column with different letters are significantly different at the 0.05 level.

Fig. 1. Histological section of the basal region of saffron sprouts in control (A) and medium supplemented with 5 mg /l of BAP (B) after three weeks in culture (x100). Arrows indicate intercalar meristems

Fig. 2. Shoots regenerated from meristems of Crocus sativus after 6 weeks of culture in phytohormone free medium (control) and supplemented with the cytokinins, 2ip, TDZ or BAP