Hemoglobin Promotes Somatic Embryogenesis in Peanut Cultures

Abstract

Critical parameters influencing somatic embryogenesis include growth regulators and oxygen supply. Consequently, the present investigation has focused on optimization of a somatic embryogenic system for peanut (Arachis hypogaea L.) through media supplementation with the auxin, picloram. The latter at 30 mg L⁻¹ was optimal for inducing regeneration of somatic embryos from cultured explants of zygotic embryos. In contrast, somatic embryogenesis did not occur in the absence of this growth regulator. An assessment has also been made of the beneficial effect on somatic embryogenesis and plant regeneration of the commercial hemoglobin (Hb) solution, Erythrogen^TM. Hemoglobin at 1:50 and 1:100 (v:v) stimulated increases in mean fresh weight (up to a maximum of 57% over control), mean number of explants producing somatic embryos (15%) and mean number of somatic embryos per explant (29%).

• Somatic embryogenesis
• Hemoglobin
• Peanut
• Arachis hypogaea

Introduction

Peanut is an important protein and high quality oil-yielding species in the sub-tropics and has been the focus of breeding strategies for crop improvement. Innovative somatic cell techniques can also be exploited to underpin conventional approaches to introduce agronomically-useful traits into peanut. For example, plant genetic manipulation by transformation necessitates reliable regeneration from cultured cells and/or explants, which, in peanut, can be achieved through organogenesis (shoot formation) or somatic embryogenesis. The latter process is, in general, more efficient than organogenesis, since it generates large populations of embryos from somatic cells. Such embryos can be exploited directly for the mass propagation of plants or, following further multiplication often under bioreactor conditions, via “artificial seed” technology. Protocols have been reported for plant regeneration in peanut through somatic embryogenesis using immature zygotic embryos, seedling cotyledons, hypocotyls and epicotyls, or leaflets (Little et al., [[2000]]; Radhakrishnan et al., [[2001]]), as sources of explants.

One crucial factor for the successful induction and differentiation of somatic embryos is the provision of the correct balance and concentrations of exogenous growth regulators in the culture medium, coupled with an adequate and sustained supply of oxygen. For example, concerning growth regulators, 4-amino-3,5,6-trichloropicolinic acid (picloram) and thidiazuron both induce somatic embryogenesis in peanut (McKently, [[1995]]; Murthy et al., [[1995]]). In the case of somatic embryos cultured under bioreactor conditions, the dissolved oxygen (DO) concentration is a critical factor influencing cell growth and differentiation, as reflected by studies using carrot (Shimazu and Kurata, [[1999]]) and banana (Kovsky et al., [[2002]]). Oxygen enrichment (20–40%) through gaseous bubbling enhanced the later stages of somatic embryo development in carrot. It is timely to evaluate other, novel, approaches for enhancing respiratory gas supply to cultured somatic embryos. In this respect, chemically-modified Hb solutions or inert, respiratory gas-dissolving perfluorocarbon (PFC) liquids are effective in facilitating the supply of oxygen to cultured eucaryotic cells and tissues, including those recovered from cryostorage (Garratt et al., [[2001]]; Lowe et al., [[1998]], [[2001]]). However, there have been no reports of the effectiveness of these novel strategies for enhancing oxygen supply in cultures of somatic embryos. Consequently, the present investigation has optimized the culture conditions, in terms of medium supplementation with picloram, and evaluated the additional beneficial effects on the growth and differentiation of somatic embryos of peanut of supplementing agar-solidified culture medium with a commercial Hb solution (Erythrogen^TM).

Materials and Methods

Plant Materials and Seed Sterilization

Seeds of peanut (Arachis hypogaea L.) cv. TMV-7 were obtained from the Oil Seeds Research Station, Tamil Nadu Agricultural University, India. Seeds were wrapped in 2 layers of muslin, washed in running water for 1 h, followed by 5% (v:v) Triton X-100 for 10 min and rinsed five times in distilled water. Seeds were placed in 70% (v:v) ethanol for 2 min, rinsed three times in sterile water, immersed in 0.1% (w/v) mercuric chloride for 5 min and washed three times with distilled water.

Medium for Tissue Culture

Mature zygotic embryos were excised from sterilized seeds and the radicles of the embryos removed and discarded. Explants were placed vertically in a MS-based callus induction medium (Murashige and Skoog, [[1962]]) with B5 vitamins (Gamborg et al., [[1968]]) and 15–35 mg L⁻¹ of picloram (designated MS-I medium). Cultures were maintained in the dark at 25 ± 2°C. After 28 days, callus that produced somatic embryos was sub-cultured to medium of the same composition to permit embryo maturation.

Supplementation of Medium with Hemoglobin

Following optimization of the concentration of picloram in MS-I medium, the latter was supplemented with Erythrogen^TM (Biorelease Corporation, Salem, USA), a sterile stabilized, bovine Hb solution (103 g L⁻¹, pH 7.42), to give final concentrations of 1:50, 1:100, 1:500, and 1:1000 (v:v), which represented 2.06, 1.03, 0.206, and 0.103 g Hb L⁻¹, respectively. After addition of Hb to the medium, the pH of the latter was adjusted to 5.8 with 1.0 M NaOH; each treatment was repeated five times and each experiment replicated three times. Data were collected on the increase in fresh weight (g) after 42 days of culture, the percentage of explants responding to Hb and the number of somatic embryos induced per explant.

Plant Regeneration from Somatic Embryos

Mature somatic embryos were excised from the parent callus and placed on a plant regeneration medium consisting of MS salts, B5 vitamins and 0.5 mg L⁻¹ of benzylaminopurine (designated MS-II medium). Cultures were maintained in the light (16 h photoperiod; 85 µmol m² s⁻¹; Cool White fluorescent tubes) at 25 ± 2°C. Shoots were transferred, when 10 cm in height, to a rooting medium consisting of MS basal salts with 1 mg L⁻¹ α-naphthaleneacetic acid, 30 g L⁻¹ sucrose, and 8 g L⁻¹ agar. After rooting, the regenerated plants were transferred to 5 cm diameter plastic pots, each containing equal volumes of sterile soil, sand and vermiculite (Parrys Ltd., Chennai, India). Plants were covered with plastic bags and, after 14–21 days, the bags were removed progressively and the plants transferred to the glasshouse under natural daylight and photoperiod. Acclimated plants were transferred to field conditions.

Statistical Methods

Means and standard deviations (s.d.) were used throughout. Statistical significance between mean values was assessed using conventional ANOVA (Snedecor and Cochran, [[1989]]). A probability of P < 0.05 was considered significant.

Results

Effect of Picloram on Somatic Embryogenesis of Peanut

Picloram at 30 mg L⁻¹ was most effective for the regeneration of shoots by somatic embryogenesis from embryogenic callus initiated from explants (Table 1), where both the mean number of explants regenerating somatic embryos and the mean number of embryos produced per explant were maximal. In contrast, somatic embryos were not induced in medium lacking picloram.

Table 1. Effect of picloram concentrations on somatic embryogenesis in peanut

Picloram concentration (mg L^−1)	Mean number of explants regenerating somatic embryos (%)	Mean number of somatic embryos regenerated per explant
Control	0	0
15	74.3 ± 0.42	3.59 ± 0.47
20	72.6 ± 0.46	4.13 ± 0.34
25	80.0 ± 0.54	4.39 ± 0.13
30	85.3 ± 0.63	4.46 ± 1.21
35	70.5 ± 0.43	3.45 ± 0.45

Values are mean s.d., n = 15 throughout.

Effect of Erythrogen^TM on Somatic Embryogenesis of Peanut

Hb solution (Erythrogen^TM) at 1:50 and 1:100 (v:v) stimulated callus initiation from zygotic embryos, as reflected by up to a 57% increase (P < 0.05) in fresh weight over control, after 42 days of culture (Table 2). Hemoglobin at 1:50 and 1:100 (v:v) also stimulated the production of embryogenic callus. Thus, both the mean number of explants regenerating somatic embryos and the mean number of somatic embryos induced per explant were increased, compared to control explants cultured on medium lacking Hb. For example, Hb at 1:50 (v:v) induced the maximum response in the percentage (98%) of explants of zygotic embryos producing somatic embryos, as compared to control and other treatments (Table 2). The induction of somatic embryos with Hb at 1:500 and 1:1000 (v:v) was comparable to control.

Table 2. Effect of Hb (Erythrogen^TM) concentration with 30 mg L⁻¹ picloram on somatic embryogenesis in peanut

Erythrogen^TM concentration (v:v)	Callus fresh weight (g)	Mean number of explants regenerating somatic embryos (%)	Mean number of somatic embryos regenerated per explant
Control	0.14 ± 0.11	85.3 ± 1.41	4.5 ± 1.2
1:50	0.22 ± 0.08a	98.3 ± 0.41a	5.8 ± 1.5a
1:100	0.17 ± 0.06a	96.6 ± 0.62a	5.2 ± 1.3a
1:500	0.15 ± 0.08	84.0 ± 0.83	4.8 ± 1.1a
1:1000	0.13 ± 0.04	80.6 ± 0.80	3.9 ± 1.1

Values are mean s.d., n = 15 throughout.

^aP < 0.05 compared to control mean value.

Discussion

The present investigation demonstrates, for the first time, that medium supplementation with Hb, in the presence of the auxin, picloram, enhances somatic embryogenesis in peanut. The optimum concentration of picloram identified (30 mg L⁻¹) was consistent with that reported in an earlier study, also in peanut, by McKently ([[1995]]). The results reported here re-inforce the critical need for the addition to culture medium of appropriate concentrations of auxins to induce this plant regeneration response (Little et al., [[2000]]; Ozias-Akins et al., [[1992]]).

Maximum induction of somatic embryos and conversion to plants occurred when picloram containing medium was further supplemented with Erythrogen^TM at 1 : 50 (v : v). The beneficial effect of this concentration of Hb, in terms of increase in mitotic division and plant regeneration efficacy, was consistent with earlier studies using, for example, protoplasts (naked cells) of Petunia hybrida (Anthony et al., [[1997]]), rice (Azhakanandam et al., [[1997]]), and Passiflora giberti (Anthony et al., [[1997]]). However, in subsequent investigations with cotton callus, maximum stimulation of growth occurred with Erythrogen^TM at 1:750 (v:v), probably reflecting the de-differentiated status of the tissue (Garratt et al., [[2001]]). There is speculation that these beneficial effects of Hb, observed in several plant species, is due to the trapping of oxygen from air-medium interfaces, facilitating the delivery of this gas to cultured cells (Anthony et al., [[1997]]).

It is well established that oxygen deprivation reduces both growth and mitotic division of plant cells in vitro through inhibition of the production of respiratory ATP, whilst high oxygen availability preferentially enhances the growth of cells of some species, such as those of non-embryogenic calli of wheat (Carman, [[1988]]). Oxygen supply has also been demonstrated to affect the differentiation of somatic embryos from suspensions. In this respect, Shimazu and Kurata ([[1999]]) reported a DO concentration of 7% (v:v) inhibited production of cotyledonary-stage somatic embryos of carrot by up to 65%, compared to cultures maintained with a DO of 30–40% (v:v). It is recognized that there are inconsistencies in the literature concerning the influence of oxygenation regimes on induction and subsequent differentiation of somatic embryos. Species variation, medium composition, and cellular responsiveness at different stages of development may all be contributing factors (Ibaraki and Kurata, [[2001]]).

The present finding that, with the highest concentration of Erythrogen^TM tested (1:50 v:v), there was maximum somatic embryo production in peanut, but not at the expense of biomass, contradicted the report of Archambault et al. ([[1994]]). The latter authors found that a high DO concentration favored biomass production and inhibited somatic embryogenesis in Californian poppy (Eschscholtzia californica). This may be explained by a preferential stimulation of the growth of undifferentiated cells (Kovsky et al., [[2002]]).

In earlier studies with cultured cotton callus, medium supplementation with Erythrogen^TM modified cellular antioxidant status, with elevations in superoxide dismutase activity and hydrogen peroxide concentration that paralleled the increasing addition of Hb (Garratt et al., [[2001]]). In this respect, there is evidence that antioxidant status influences morphogenesis and cellular differentiation in callus cultures (Benson, [[2000]]; Benson et al., [[1997]]). Consequently, the possibility exists that the beneficial effects of medium supplementation with Hb observed in the present study were due, not only to any alterations in oxygen availability, but also to modified antioxidant status favoring the scavenging of reactive oxygen species. Clearly, this is an area for further investigation. Other extensions of this work should focus on identifying any synergistic effects of nonionic surfactants (e.g., Pluronic® F-68), that can enhance cellular differentiation in vitro (Lowe et al., [[2001]]), with further comparisons between Hb and PFC options for enhancing oxygen supply. Indeed, it has been emphasized that additional detailed studies are required to evaluate the effects of oxygen and carbon dioxide on somatic embryogenesis (El Meakaoui and Tremblay, [[1999]]). The use of Hb and PFC technologies will facilitate such evaluations.

Acknowledgments

NJ is grateful to the British Council, New Delhi and Department of Science and Technology, Government of India, New Delhi for providing grants from the UK-India Science and Technology Fund.