Plant regeneration in eggplant ( Solanum melongena L.): A review

Eggplant is highly responsive to various tissue culture techniques. Somatic embryogenesis and direct organogenesis are widely studied protocols in this crop, but potential of regeneration varies with genotype, explant and culture media supplemented with different combination and concentration of growth hormones. The genotype is the most important factor affecting somatic embryogenesis and organogenesis. Embryogenic competence occurs even within explant segments. Among growth regulators, auxins and cytokinins are of more significance as their ratio determines callogenesis, rhizogenesis, embryogenesis and regeneration in eggplant. Organogenesis and somatic embryogenesis related gene expression has been studied and transcripts have been analyzed through molecular studies. Efficient plant regeneration protocols would make a platform for exploitation of useful somaclonal variations, mutation breeding, induction of di-haploids, and genetic transformation with economically important genes for the improvement of eggplant.


INTRODUCTION
Eggplant (Solanum melongena L., 2n = 2x = 24) is a widely adaptive and highly productive vegetable crop of tropical and subtropical regions world, which suffers from various abiotic and biotic stresses particularly insect-pests (Singh et al., 2000;Kaur et al., 2004).To control the pests, various biological and biochemical control measures have been recommended, but cryptic nature of the pest is a big hindrance in efficient management.Consequently, growers use excessive and un-recommended pesticides, which is a matter of concern for food safety, environmental degradation, pest resistance and economics of the crop.The non-availability of resistance in cultivated, cross-incompatibility with wild relatives (Solanum mammosum, Solanum incanum and Solanum grandiflorum) and inadvertent linkage drag of undesirable genes (Baksh and Iqbal, 1979) are problems in developing intrinsic plant resistance through conventional breeding approach.Thus, use of biotechnological techniques can be an alternative approach to tackle such issues.
In eggplant, somatic embryogenesis was first reported from immature seed embryos of two different cultivars by culturing on MS (Murashige and Skoog, 1962) medium with supplementation of indole-3-acetic acid (IAA) (Yamada et al., 1967).Although, this crop is most amenable to in vitro culture, still its genetic make-up, explant and culture media affect its regeneration potential (Kantharajah and Golegaonkar, 2004).Genotype and explant are the most important factor affecting somatic embryogenesis and its further regeneration (Afele et al., 1996;Sharma and Rajam, 1995(a or b?); Dobariya and Kachhadiya, 2004;Franklin et al., 2004;Huda et al., 2007;Mir et al., 2008).The response of growth hormones in the culture media is also variable within genotype and explant for somatic embryogenesis and organogenesis (Slater et al., 2003).
The plant tissue culture methods also provide base for the improvement of crop.To induce somaclonal variations, in vitro mutations, herbicide tolerance, di-haploid induction, genetic transformation of economically important genes and development of somatic hybrids, efficient plant regeneration protocol is required.Such advance techniques in combination with conventional breeding give a momentum to the improvement of a crop.Thus, realizing the prospects for future research, relevant literature to "Plant regeneration in eggplant (Solanum melongena L.)" has been reviewed.

Somatic embryogenesis
Somatic embryogenesis is the process of a single cell or a group of cells initiating the developmental pathway.It was first reported in eggplant from immature seed embryos cultured on MS medium supplemented with IAA (Yamada et al., 1967).In general, it is independent or inversely related to organogenesis (Matsuoka and Hinata, 1979).The different factors such as genotype, explant, combination of growth hormones and some other factors affect somatic embryogenesis in eggplant (Kantharajah and Golegaonkar, 2004).
The genotype is the most important factor affecting somatic embryogenesis and significant quantitative differences in their capacity to form embroids among different species like S. melongena, S. melongena var.insanum, Solanum gilo, Solanum integrifolium and their F1 hybrids, cultivars, and inbred lines (Alicchio et al., 1982;Gleddie et al., 1983;Ali et al., 1991;Rao, 1992;Anisuzzaman et al., 1993;Huda et al., 2007;Mir et al., 2008, Zayova et al., 2008;Chakravarthi et al., 2010;Kaur et al., 2011a and2013).The differential responses for regeneration of adventitious shoots and somatic embryos, number of days to shoot initiation and mean number of shoots per callus (Sharma and Rajam, 1995a;Afele et al., 1996;Dobariya and Kachhadiya, 2004) are also there among cultivars.The molecular investigation using polymerase chain reaction (PCR) of different cultivars for the induction of somatic embryos indicated that embryogenic response is due to differences in mRNA expression and consequently gene expression patterns (Afele et al., 1996).
Gene expression during initial stage of somatic embryogenesis in eggplant revealed that 2,4-D induces specific alteration in gene expression due to differential display of RNA (Momiyama et al., 1995).In spite of this, an antioncogen homolog and the activation of retrotransposon were described during early stages of somatic embryogenesis (Momiyama et al., 1996).Differential display and restriction fragment length polymorphism (RFLP) analysis resulted in the identification of one organogenesis and two somatic embryogenesis related transcripts (Bucherna et al., 2001).
The frequency of embryogenesis depended on optimal ratio of NO 3 -: NH 4 + (2:1) in the medium.The optimal su-crose concentration of the medium was 0.06 M, whereas, elevated or reduced level inhibited the embryo-genesis in eggplant (Gleddie et al., 1983).Sucrose concentrations of 0.2 -0.5% induced somatic embryo-genesis, 1% led to embryogenesis and shoot regene-ration and 2% provoked maximum shoot regeneration, whereas, increased sucrose levels from 3 to 5% decreased the regenerating ability.The lowered sucrose concen-tration from 2 to 0.2% also caused complete bleaching, which can be used for selection of herbicide-resistant mutants (Farooqui et al., 1997).The pesticides like Endlosulfan, Rogor and Kitazin in relation to their concentrations also affected callus induction and multiple shoot formation in eggplant.The callus growth decreased with increasing level of pesticides in medium.Some concentrations (50 -500 ppm) of pesticides in the medium also formed abnormal callus growth and shoot induction.Among pesticides, Rogor (25 ppm) induced maximum callus (76.0%) and shoots (11.0), whereas, Kitazin 45% EC showed more inhibitory effect than the Endosulfan and Rogor (Sammaiah et al., 2011a(Sammaiah et al., , 2011b)).Plant regeneration from tissue culture of S. melongena L. can be achieved via embryogenesis (Ammirato, 1983) and organogenesis (Flick et al., 1983).Light could help the development of adventitious rooted shoots from callus (Macchia et al., 1983;Salih and Al-Mallah, 2000).High concentration of 2ip and low concentration of IAA led to differentiation of leaflets with morphogenetic variation in leaves and cytological studies of plants indicated them genetically aberrant (Fassuliotis, 1975).LS medium without hormones also regenerated plant from callus (Alicchio et al., 1982).Also, MS medium supplemented with different concentrations and combinations of cytokinins and auxins (Table 1) produced more shoot primordial and rooted shoots in calli derived from cotyledon, hypocotyls, leaf and root explants (Macchia et al., 1983;Anwar et al., 2002;Yu et al., 2003;Franklin et al., 2004;Rahman et al., 2006;Chakravarthi et al., 2010).Plants regenerated through somatic embryogenesis had somaclonal variations.Frequencies of somaclonal variations in leaf shape, plant height, fruit shape and pollen fertility was higher with NAA than that of 2, 4-D (Hitomi et al., 1998).Therefore, the future research would determine the importance of new somaclonal lines for genetic variability of eggplant (Zayova et al., 2010(Zayova et al., , 2012)).

Organogenesis
Organogenesis is the morphogenesis of plantlets directly from explants without the intervention of callus in the culture.This omits the callus and embryoid phases, reduces use of auxin from the in vitro culture and leads to direct formation of new shoots from the explants.Anatomically and histolosically, longitudinal sections of leaf explants formed numerous meristematic zones within the tissue, that subsequently converted into shoot buds (Mukherjee et al., 1991).The formation of shoot buds was characterized  (2004) Cotyledon and young leaf explant  by the appearance of shoot apex with the developing leaf primordial (Sarker et al., 2006).Genotype played important role in organogenesis of the shoots directly from the explants.Different varieties and species such as Solanum aethiopicum, Solanum macrocarpon showed different potential in direct plant regeneration, where, 70 -100% explants with a mean of two to seven shoots per explant were obtained (Gisbert et al., 2006;Sarker et al., 2006;Shivraj and Srinath, 2011).
Low sugar concentrations enhanced shoot regeneration, where, higher concentration of glucose and lower of sucrose showed better effects (Mukherjee et al., 1991;Polisetty et al., 1994).Shoot regeneration process had also been affected by the gelling agents and agar was found superior to gerlite (Perrone et al., 1992).Peptone had no effect on reducing hyperhydric shoots of S. melongena and S. integrifolium.Culture vessels with gas-permeability by membrane filter reduce the percentage of hyperhydric shoots and increased survival rate than sealed vessels (Takamura et al., 2006).

Hardening and field establishment
Most of the species grown in vitro require acclimatization process in order to ensure that sufficient number of plants survive and grow vigorously on transferring to the soil.It took 3-4 months from initiation to establishment in pots ex vitro for 99% survival rate (Polisetty et al., 1994), however, rooted plants can be acclimatized in 14 days with 80% success (Salih and Al-Mallah, 2000;Taha and Tizan, 2002;Chakravarthi et al., 2010;Kanna and Jayabalan, 2010;Shivraj and Srinath, 2011;Kaur 2011a, b).Rooted shoots were transferred for establishment in polythene bags filled with a potting mixture of sand, soil and FYM in 1:2:1 ratio (Dobariya and Kachhadiya, 2004).The plantlets were successfully established in polycarbonated polyhouse with 100% survival rate (Bhat et al., 2013).When root system was developed well, plants were hardened in the glass house and transferred to the field for flowering, fruiting and seeding (Kamat and Rao, 1978;Gleddie et al., 1983;Jahan and Syed, 1998;Magioli et al., 1998;Franklin et al., 2004;Sarker et al., 2006).

CONCLUSIONS
Research work has mainly been focused on the development of regeneration protocol, somaclonal variations and their physiological as well as morphological aspects in eggplant.An efficient plant regeneration protocol is a prerequisite for the exploitation of various biotechnological techniques.However, practical utility of the basic protocol is still far away.It can serve as a platform for the transfer of economically important traits through genetic engineering, inducing somaclonal variations, in vitro mutations, double-haploids induction, development and utilizetion of somatic hybrids, determining herbicide or pesticide tolerance limits in eggplant.Therefore, a remark-able progress can be made in eggplant improvement through the combination of conventional and biotechnological approaches.