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Sep 25, 2013 - Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts. S. Vinoth ...
J Appl Phycol (2014) 26:1527–1537 DOI 10.1007/s10811-013-0151-z

Optimization of somatic embryogenesis protocol in Lycopersicon esculentum L. using plant growth regulators and seaweed extracts S. Vinoth & P. Gurusaravanan & N. Jayabalan

Received: 28 June 2013 / Revised and accepted: 6 September 2013 / Published online: 25 September 2013 # Springer Science+Business Media Dordrecht 2013

Abstract In the present study a simple and efficient somatic embryogenesis system was developed from leaf explants of Lycopersicon esculentum L. The protocol has been developed by using plant growth regulators and seaweed extracts a natural biostimulant. The leaf sections were initially cultured on to leaf embryogenic callus induction medium fortified with various concentration and combinations of 2,4dichlorophenoxy acetic acid (0.2–1.0 mg L−1), picloram (0.2–1.0 mg L−1), and kinetin (0.1–0.5 mg L−1). The best responding concentration in induction of friable embryogenic callus was tested for the proliferation. The friable cultures were detached from the mother culture and inoculated in three different media supplemented with plant growth regulators, plus 0–25 % Caulerpa scalpelliformis or 0–25 % Gracilaria corticata extracts for embryo development. A twofold increase in maturation and germination of somatic embryos was observed in the media containing seaweed extracts (MSMG2 and MSMG3) than the control (MSMG1). The plantlets transferred from plant growth chamber to greenhouse conditions exhibited higher survival rate (90 %) than directly shifted plantlets. Keywords Tomato . Caulerpa scalpelliformis . Gracilaria corticata . Seaweeds . Somatic embryogenesis

Introduction Tomato is a major dietary vegetable crop well known for the nutritional composition and it is cultivated all over the world. S. Vinoth (*) : P. Gurusaravanan : N. Jayabalan Department of Plant Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 24, 620 024 Tamilnadu, India e-mail: [email protected]

At present tomato is consumed at higher rate and it is grown worldwide with an annual production of 150 million tonnes in the year 2011 (Indian Horticulture 2011). Tomato plants are frequently exposed to adverse environmental conditions such as biotic and abiotic stress that leads to the reduction in total production. So there is in need of crop improvement, modern plant biotechnology technique paves the way to overcome the stresses through plant tissue culture and genetic modification systems. The optimization of tissue culture protocol is a major criterion for the mass production of transgenics and for commercialization. Although tomato is one of the genetic models for improving the other dicotyledonous crops, scientists still are focusing on improving the crop by innovative protocols. A mass propagation protocol has been established using leaf, stem, peduncle, inflorescence, hypocotyls, and cotyledon explants (Bhatia et al. 2004). But development of a genotype independent protocol is absolutely necessary for tomato transformation. In order to produce plants from transformed cells, somatic embryogenesis is one of the novel techniques for the mass production of transgenic without chimerics. There are few reports of somatic embryogenesis of tomato (Chen and Adachi 1994; Gill et al. 1995; Kaparakis and Alderson 2002) indicating the classical protocols for achieving embryogenesis have not been successful (Young et al. 1987; Lamproye et al. 1990). There are several factors that influence the optimization of reproducible protocol that depend largely on the genotype, explant, medium composition, pH, carbon source, and concentration of plant growth regulators in the medium (El-Farash et al. 1993). It has been reported that most genotypes of tomato respond uniquely to plant growth regulators during regeneration, so it is necessary to develop a protocol for each genotype individually (Kurtz and Lineberger 1983). Organic elicitors such as hemoglobin, casein hydrolysate, yeast extract, and glutamine enhanced the regeneration of tissues (Baskaran and van Staden 2012), and it has been reported that seaweed extracts

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Ramanathapuram district, Tamilnadu, India, in January 2012. The samples were cleaned with seawater four to five times to remove sand particles and other impurities before transport to the Department of Plant Biotechnology, Bharathidasan University, Tamilnadu. The samples were then again washed five times with tap water and shade dried and finely chopped into fine powder. In order to prepare the seaweed extracts, about 500 g of each sample was boiled in a conical flask containing 500 mL distilled water. Then the extracts were cooled to room temperature and filtered through Whatman no. 41 filter paper two times. One set of extracts were stored at −20 °C and another set was used for the further studies.

have potential activity in stimulating the growth of the plants in vivo, but have been underutilized so far (Khan et al. 2009). Seaweed extracts exhibit growth-stimulating activities enhancing the growth of vegetables, fruits, and other crops (Blunden 1991; Crouch and van Staden 1994; Washington et al. 1999). Plants treated with seaweed extracts show an increase in nutrient uptake, seed germination, and deep root formation (Mancuso et al. 2006; Atzmon and van Staden 1994; Demir et al. 2006). Apart from macro and micro elements, seaweeds also contain natural growth promoters such as auxins, cytokinins, and abscisic acid-like substances leading to enhancement of growth and crop yield (Crouch et al. 1992; Crouch and van Staden 1993; Reitz and Trumble 1996; Durand et al. 2003; Stirk et al. 2003a, b; Ördög et al. 2004). Seaweeds have also been used as a soil conditioner and as a substitute for chemical fertilizers (Crouch and van Staden 1993). The establishment of a somatic embryogenesis protocol for tomato using seaweed extracts would provide an improvement of this crop organically. The aim of this study was to optimize the somatic embryogenesis protocol using extracts of the seaweeds Caulerpa scalpelliformis and Gracilaria corticata and plant growth regulators, as well as to determine the presence of plant-growth-promoting substances in seaweeds by HPLC analysis.

Plant material and explant preparation Tomato seeds, Co-3 cultivar, were obtained from Tamilnadu Agricultural University, Coimbatore, Tamilnadu, India. Seeds of uniform size and shape were soaked in tap water overnight and then placed in a pot containing soil, sand, and organic manure in the ratio of 2:1:1 in a greenhouse. After 2 weeks, the leaf explants were collected and washed with 5× Teepol solution for 1 min and then rinsed with tap water for 10 min. Subsequently, the leaves were rinsed two times with sterile water and then sterilized with 0.1 % mercuric chloride for 5 min followed by three rinses with sterile water under aseptic conditions. The tip and basal portion of the leaves were excised using a sterile blade and the remaining leaf portions were cut into small 2 cm2 pieces which were then abaxially cultured on leaf embryogenic callus induction medium (LECIM).

Materials and methods The seaweeds, Caulerpa scalpelliformis (Chlorophyta) and Gracilaria corticata (Rhodophyta) were collected from the

Table 1 Composition of media for embryogenic friable callus induction, proliferation, different developmental stages, and maturation of somatic embryos Medium composition Plant growth regulators

Leaf embryogenic callus induction medium (LECIM) Somatic embryo proliferation medium (SEPM) Embryo development medium MSPM MSCS MSGC Embryo maturation medium MSMG1 MSMG2 MSMG3

Seaweed extracts

Carbon source

2,4-D (mg L−1)

Pic (mg L−1)

Kin (mg L−1)

TDZ (mg L−1)

GA3 (mg L−1)

ABA (mg L−1)

CS (%)

GC (%)

Sucrose Maltose (%) (%)

0.2–1.0

0.2–1.0

0.1–0.5











3



0.8



0.3

1–0.15











3.0



0.8

– – –

0.3 0.3 0.3

0.15 0.15 0.15

0.5 0.5 0.5

– – –

– – –

– 0–25 –

– – 0–25

1.5 1.5 1.5





– – –

– – –

– – –

– – –

0.3–1.5 – –

0.3–1.5 – –

– 5–25 –

– – 5–25

2.0 2.0 2.0

1.0 1.0 1.0

– – –

All media were supplemented with MS salts and B5 vitamins with the abovementioned concentration and combinations

Agar

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Fig. 1 Embryogenic callus induction, proliferation, and different developmental stages of somatic embryos of L. esculentum L. a Embryogenic callus induction from leaf explants of tomato in disposable Petri plate containing MS salts, B5 vitamins, Pic (0.6 mg L−1), Kin (0.3 mg L−1). b Dispersion of somatic embryos from mother tissue in liquid MS medium supplemented with MSPM+ (0–25 %) seaweed extracts. c Globular stage embryo. d Initiation of conversion from globular to heart shape stage. e Heart shaped embryo. f and g Torpedo stage embryo. h and i Cotyledonary stage embryo under NIKON stereo microscope. Bars=1 cm (a–b), 100 μm (c–i)

Embryogenic callus induction from leaf explants The excised square of leaf was cultured on LECIM medium supplemented with MS salts (Murashige and Skoog 1962), B5 vitamins, 3 % sucrose, and various concentrations and combinations of plant growth regulators (PGR): 2,4-dichlorophenoxy acetic acid (2,4-D; 0.2–1.0 mg L−1), picloram (pic; 0.2–1.0 mg L−1), and kinetin (kin; 0.1–0.5 mg L−1). The medium pH was adjusted to 5.8±0.5 prior to the addition of 0.8 % agar. The media were autoclaved at 121 °C for 5 min in 500-mL conical

flasks (Borosil). Then the media were then poured into disposable Petri plates (Tarson 90 mm) in a laminar flow hood. About four to five excised leaf pieces were cultured in the each plate and incubated at 25 °C in the light provided by white fluorescent lamps (Philips) with a 16-h photoperiod. The callus was subcultured on to the same fresh medium every 10 days. Proliferation and development of somatic embryos in liquid medium The best concentrations of PGRs for callus induction

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Table 2 Effect of different plant growth regulators on induction of somatic embryos in leaf explants of Lycopersicon esculentum L Plant growth regulators (mg L−1) 2,4-D

Pic

Kin

0.2 0.4 0.6 0.8 1.0

0.1 0.2 0.3 0.4 0.5 0.1 0.2 0.3 0.4 0.5

0.2 0.4 0.6 0.8 1.0

Percentage of explant responding (%)

Callus morphology

Induction of somatic embryogenesis (%)

60 90 75 55 40 65 80 90 85 70

GYF YF YF YF GYF YF YF YF YF GYC

0±0.00g 89.3±0.15b 77.7±0.21e 63.5±0.27ef 0±0.00g 63.7±0.15f 79.1±0.10d 97.8±0.13a 88.2±0.13c 0±0.00

The data collected after 20 days of culture in the medium were supplemented with MS salts and B5vitamins; the auxins were tested in combination with Kin for friable embryogenic callus induction. Means followed by the same letter within columns are not significantly different, according to Duncan’s multiple range test (P