PLURONIC F-68 ENHANCED SHOOT REGENERATION IN A POTENTIALLY NOVEL CITRUS ROOTSTOCK

Abstract

The effects have been studied in vitro of the non-ionic, co-polymer surfactant, Pluronic F-68, on shoot regeneration and bud induction in epicotyl and cotyledon explants of Citrus depressa, a potential alternative rootstock to C. jambhiri for commercial Citrus. Supplementation of Murashige and Skoog (1962)-based, agar-solidified shoot regeneration/bud induction (SRBI) medium with 1.0 mg l⁻¹ 6-benzylaminopurine and 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) increased mean fresh weight by a maximum of 60%, the proportion of explants exhibiting shoot/bud regeneration by 25% and the mean number of shoots per epicotyl explant by 184%, compared to untreated controls. Similarly, 0.5% (w/v) Pluronic F-68 significantly (P < 0.05) enhanced the mean percentage bud induction (91%) and the number of buds regenerated (>4-fold) per cotyledon explant. Interestingly, the mean fresh weight gain for both explants was unaffected across the range of concentrations (0.001–0.1% w/v) of Pluronic F-68 evaluated. Regenerated plants from epicotyl explants were transferred and acclimatized to glasshouse conditions.

ABBREVIATIONS
BAP	=	6-benzylaminopurine
g. f. wt.	=	gram fresh weight
NAA	=	α-naphthaleneacetic acid
SRBI	=	MS-based shoot regeneration/bud induction medium.

INTRODUCTION

The genus Citrus, grown widely throughout tropical and sub-tropical regions, is of considerable economic importance, containing edible fruit species and, as importantly, germplasms for intrageneric rootstocks [[1]]. In India, for example, Citrus ranks third, after banana and mango, with an estimated production of > 3 × 10⁶ metric tonnes per year over an area of > 4 × 10⁵ hectares [[2]].

C. jambhiri Lush is used commercially as rootstock for Sweet orange (C. sinensis L. Osb.), one of the most important sub-groups of oranges. A serious decline in yield and fruit quality in Citrus has been attributed to bud union crease disease, especially after 10 years of growth [[3]]. Tree health declines, with pitting of the wood and gum impregnation of bark at, or close to, the graft union. Such effects are linked to scion/rootstock interactions and also involve grafttransmissible pathogens. In contrast, C. depressa does not exhibit crease disease and is thus more attractive as a viable alternative rootstock, especially for sweet orange.

Clonal propagation strategies based on tissue culture have been described for Citrus and allied genera [4-8], but have focused on the synergistic effects of phytohormones for adventitious shoot regeneration. Previous work has demonstrated that supplementation of culture medium with the commercial and relatively inexpensive non-ionic, co-polymer surfactant, Pluronic F-68 (poloxamer 188), stimulates growth and differentiation of tissues and organs from several plant species in vitro [9-13]. However, to date, there have been no corresponding studies exploiting novel biotechnological developments in tissue culture technology involving members of a top-fruit tree rootstock genus of such importance as Citrus. Thus, the present investigation has evaluated the beneficial effects of Pluronic F-68 to promote adventitious shoot regeneration and bud induction in both epicotyl and cotyledon explants of C. depressa, a species that is seen as forming the basis of the next generation of commercial Citrus rootstocks. The availability of an efficient micropropagation system, giving readily rooted stock plants, will provide the basis for a rapid clonal propagation system per se and, in a commercial context, the opportunity for the rapid incorporation of C. depressa as a long-term replacement rootstock for orange plantings.

MATERIALS AND METHODS

Plant Material and Explant Preparation

Seeds of C. depressa Hayata were collected from a single ripe fruit (9 months post-flowering), from an individual mature (10 year-old) tree supplied by the Department of Horticulture, Punjab University, India. Seeds were surface sterilised with 0.1% (w/v) of freshly prepared mercuric chloride solution (10 min) before rinsing 3 times with sterile, reverse-osmosis water. The testas were removed and seeds were germinated on 50 ml aliquots of agar-solidified (0.8% w/v) MS medium [[14]] lacking growth regulators (designated MS0), pH 5.8, in 175 ml capacity screw-capped glass jars (3–4 seeds/jar) at 25 ± 2°C (16 h photoperiod, 25 μ mol m ² s ¹, Cool-White fluorescent tubes “Pluslux 4000”; Thorn, UK).

Culture Media, Incubation Conditions

Epicotyl and cotyledon explants from 21–28 d-old uniform nucellar-derived plants [[15]], were cultured in 9 cm diam. Petri dishes (10 explants/dish), each containing 25 ml a liquots of agar-solidified (0.8% w/v) SRBI medium, with 1 mg l⁻¹ BAP [[15]]. The effects were evaluated of supplementing SRBI medium with different concentrations [0.001, 0.01, 0.1, 0.5 % (w/v)] of commercial grade Pluronic F-68 (Sigma, Poole, UK) on in vitro shoot regeneration and bud induction from cotyledon and epicotyl explants. Each experiment was repeated 3 times (150 samples per explant type/treatment) with each individual treatment replicated 3 times within an experiment. The appropriate volume of a 10% (w/v) filtersterilised (0.2 μM pore size, Minisart filter, Sartorious, UK) stock solution of Pluronic F-68, in SRBI liquid medium, was added to molten agar medium prior to pouring into the Petri dishes. SRBI medium, without supplements, was used as control. Cultures were maintained as described earlier.

Measurement of Growth

Increases in biomass (g. f. wt), percentage of explants exhibiting bud induction/shoot regeneration, mean number of shoot buds/shoots per explant were recorded after 56 d of culture on SRBI medium, for both explant sources. For rooting, samples only of epicotyl-derived regenerants (5 per jar; n = 100) were taken and were rooted on previously standardised RM medium (MS-based medium with 1 mg l ¹ NAA) [[15]]; cultures were maintained as before, with a random sample of 100 shoots evaluated for rooting.

Establishment of Plants Ex Vitro

A random sample (n = 20) of in vitro-rooted plants were removed (after 21–28 d) from culture jars, washed free of agar under running tap water and transferred to 9 cm diam. plastic pots containing a mixture (6:6:1:1 by volume) of Levington M3 Compost (Fisons Ltd, Ipswich, UK), John Innes No. 3 Compost (John Innes Manufacturers Association, Lincoln, UK), Silvaperl and Vermiperl (William Sinclair, Horticultural Ltd., Lincoln, UK). Plants were watered and enclosed in polythene bags. The latter were perforated sequentially from day 10 and removed after 14–21 d. Plants were grown at a maximum day temperature of 25 ± 2°C and a minimum night temperature of 18 ± 2°C in the glasshouse under natural daylight supplemented with a 16 h photoperiod (180 μ mol m⁻² s⁻¹, Cool-White fluorescent tubes).

Cytological Assessments

Samples (n = 10) of plants regenerated from epicotyl explants cultured with 0.5% (w/v) Pluronic F-68 and controls were analysed for their somatic (root) chromosome status following the protocol of Andras et al. [[16]].

Statistical Analyses

Statistical analyses were performed according to Snedecor and Cochran [[17]]. Means and standard errors (s.e.m.) were used throughout. ANOVA tests were performed using the commercial Minitab® statistical software package (Version 9.2; Minitab Inc., USA). A probability of P < 0.05 was considered significant.

RESULTS

Bud and Shoot Regeneration

For epicotyl explant-derived cultures, supplementation of SRBI medium with 0.5 % (w/v) Pluronic F-68 significantly (P < 0.05) increased (4.0 ± 0.2, g. f. wt) the mean fresh weight gain of shoots/buds associated with the explants, compared to control (2.5 ± 0.0 g. f. wt.; Table 1). Lower concentrations [0.001, 0.01, 0.1 % (w/v)] of Pluronic F-68 had no significant effects (2.0 ± 0.1, 1.9 ± 0.1, 2.3 ± 0.1 g. f. wt.) on the mean weights when compared with the control on SRBI medium (2.5 ± 0.1 g. f. wt.). SRBI medium supplemented with 0.5 % (w/v) Pluronic F-68 also significantly improved the mean percentage of explants undergoing shoot regeneration (89 ± 3%) and the mean number of shoots (8.3 ± 0.3) per explant, compared to values for explants cultured on unsupplemented SRBI medium (71 ± 1% and 4.5 ± 0.2, respectively).

Table 1. Mean Increase in Biomass (g.f.wt.), Mean Percentage of Shoot/Buds Regenerated, and Mean Number of Shoots/Buds Regenerated per Explant for Epicotyl and Cotyledon Explants of Citrus depressa Cultured for 56 Days in Semi-solidified Medium Alone (Control) or Medium Supplemented with 0.5% (w/v) Pluronic F-68

	Explant Source
	Epicotyl	Cotyledon
Increase in biomass (mean g.f.wt.)
Control	2.5 ± 0.1	2.3 ± 0.1
Pluronic (0.5% w/v)	4.0 ± 0.2^*	2.4 ± 0.1
Proportion of explants exhibiting shoot/bud regeneration (mean %)
Control	71 ± 1	34 ± 4
Pluronic (0.5% w/v)	89 ± 3^*	65 ± 5^*
Mean number of shoot/buds per explant
Control	4.5 ± 0.2	1.1 ± 0.1
Pluronic (0.5% w/v)	8.3 ± 0.3^*	4.7 ± 0.3^*

Values are mean ± s.e.m., n = 3 throughout.

^* P < 0.05 compared to control [0% (w/v) Pluronic F-68] mean values.

For cotyledonary explants, Pluronic F-68 significantly (P < 0.05) enhanced both shoot/bud regeneration and the mean number of shoot buds per explant (Table 1). However, unlike epicotyl-derived regenerants, the mean biomass increase in fresh weight of cotyledon explants was not significantly different for all concentrations of Pluronic F-68 tested, compared to controls. The mean percentage of explants exhibiting bud induction (65 ± 5%) and the number of buds per cotyledon explant (4.7 ± 0.3) in SRBI medium supplemented with 0.5% (w/v) Pluronic F-68 was significantly (P < 0.05) higher than with SRBI medium alone (34 ± 4% and 1.1 ± 0.1, respectively). In contrast, no corresponding differences in bud induction capacity were observed for cotyledon explants cultured in the presence of the lower concentrations [0.001, 0.01, 0.1 % (w/v)] of Pluronic F-68.

Cytology of Regenerated Plants

The survival of rooted plants ex vitro was > 95%. Cytological analyses confirmed that there were no differences in the somatic chromosome complements (2n = 2x = 18) of randomly selected plants regenerated from epicotyl explants using media with or without Pluronic F-68 (n = 10 for each concentration); all regenerants were thus normal diploids.

DISCUSSION

The results of these experiments show that supplementation of culture medium with 0.5% (w/v) Pluronic F-68 significantly improved the number of potentially deliverable micropropagated plants from epicotyl and/or cotyledonary explants of C. depressa. In this study maternal cell-derived clones, arising as a result of polyembryony, were used for the first time for shoot regeneration studies with Pluronic F-68. This ensured genetic uniformity of the plant material, thereby eliminating inconsistencies in response between treatments. The most dramatic improvements in growth were seen with cotyledon explants, where a doubling was observed of the mean number of induced buds/shoots per explant, coupled with a 3-fold increase in the mean number of shoots recovered per explant. Overall, the results of this study demonstrate for the first time, that supplementation of culture medium with a relatively inexpensive surfactant enhances growth and differentiation in a commercially-valuable rootstock of Citrus. This work forms a baseline for the future large-scale micropropagation of this novel Citrus rootstock which, in turn, may serve to reverse the current decline in yields of quality fruit, thereby restoring economic returns to growers.

The present findings are generally consistent with previous observations of the stimulatory effects on growth and shoot regeneration of Pluronic F-68 [[9]] but, in the present study, for one of the more important fruit tree genera. The beneficial effects of Pluronic F-68, albeit dependent on concentration, in plant cell and tissue culture systems may be driven, in part, through increases in plasma membrane permeability to nutrients, growth regulators and/or respiratory gases [[9]]. It is noteworthy that the lower concentrations of Pluronic F-68 [0.001 – 0.1%(w/v)] tested in the present study did not significantly influence shoot regeneration. This was consistent with some earlier studies with, for example, Hypericum perforatum, where similarly low concentrations of the surfactant did not affect the mean number of shoots per explant or the total biomass of leaf explants with regenerated shoots [[11]]. In contrast, in related studies with Corchorus capsularis (Jute) [[10]], Solanum dulcamara (Woody Nightshade) [[18]] and Dendranthema grandiflora (Chrysanthemum) [[12]], supplementation of culture media with concentrations of Pluronic F-68 in the range 0.001 – 0.1% (w/v) stimulated growth and subsequent differentiation in vitro. Indeed, it is noteworthy that in the study using C. capsularis, cotyledonary tissue was the most responsive to Pluronic at 0.5% (w/v) [[10]] which is consistent with the present observations.

Thus, the present study re-inforces earlier conclusions that the responses of cultured plant material to Pluronic F-68 depends not only on concentration but, crucially, on the source tissue and species of origin [[9]]. This is further supported by results from a related investigation, also involving the woody plant, Cassava (Manihot esculenta), in which the addition of 2.0% (w/v) of Pluronic F-68 to the culture medium raised the percentage of nodal explants forming at least 5 shoots from 0 to 20–60% [[19]]. It is therefore reasonable to conclude that, for plant culture systems that respond to the surfactant, there is an optimum concentration which produces maximum growth stimulation.

ACKNOWLEDGMENTS

Financial support was provided to G.O.C. by Colciencias (Government of Colombia) and to M.S.I.G. by the British Council under the auspices of an Association of Commonwealth Universities Fellowship Award. The authors thank Dr C. Andras for assistance with cytological analyses.