Induction of Indirect Somatic Embryogenesis on Embryonic Axis of TRI 2025 Tea Clone

Tea (Camellia sinensis L.) is classified as cross-pollinated crop and vegetative multiplication becomes commercially the main method of propagation with some limitations such as high heterogeneity and poor in survival rate and also in rooting. A proven tissue culture method, somatic embryogenesis, is the only challenging way to meet the needs of tea seedlings in large quantities. The study was conducted with TRI2025 tea clone selected from Polyclonal garden of PT. Pagilaran (Batang, Central Java). The explants were cultured on MS media supplemented with four concentrations of 2,4-D (0, 1, 2, and 5 mg L) in two incubation conditions; dark and light. The results showed the only concentration of 2,4-D that can induce somatic embryo was 2 mg L 2,4-D in light condition and its percentage was about 5%. Other concentrations of 2,4-D that given for treatments both in two conditions will not induce somatic embryo. This study needs more improvements for getting powerful and efficient of method to get somatic embryo-derived plant and also for futher successful genetic engineering of tea biotechnology.


Introduction
Tea (Camellia sinensis L.) is classified as self-incompatible characteristic of crops (Chen et al., 2012), therefore its propagation using seeds is not desirable because seed derived progenies showed heterogenity (Mondal, Bhattacharya, & Ahuja, 2001).For commercial purposes, vegetative propagation through stem cutting became the main method, but the multiplication is very slow with low survival rate and season depending rooting (Boonerje, Hoque, & Sarker, 2013).The propagation by in vitro culture method is one choice for multiplication from any plant part due totipotency (Thorpe, 2007).
In tea, there are many reports about somatic embryogenesis with various kinds of explants and also of PGRs used.A 100% successful of somatic embryogenesis on embryo axis of tea using MS media supplemented with 2,4-D was reported by Kaviani (2013) and its cotyledon region was successful induced directly of its capability of somatic embryogenesis using MS media supplemented with ABA and osmoticum agent (PEG) (Suganthi et al., (2012).Ghanati and Iskha (2009) also reported the successful of indirectly induction somatic embryogenesis from leaf tea using modified B5 media supplemented with ABA and BA but without shoot formation, while Seran et al., (2006) was successful indirectly induced somatic embryo of about 8.3% from the same explants using MS media supplemented with BAP and NAA.A half strength of MS media supplemented with BAP was used by Tahardi et al., (2000) using cotyledon explants of Yabukita tea clone to induce somatic embryo with embryogenic convertion of about 56.6%.Previously, nodal segment of tea was also 60% successfully induced its capability of somatic embryogenesis when cultured on modified MS media free of PGRs (Akula & Dodd, 1998).In the present this study, cotyledonary stage was abnormal in structure (Figures 4H-4J) that was indicated with fused cotyledon forming.In tea, cotyledonary stage must has two cotyledon, so in this study, failure for gaining a normal cotyledonary stage might be due to stress condition.Another abnormal cotyledonary stage can be also caused by application of auxin transport inhibitor, such as N-1-naphthylphthalamic acid (NPA) (Hakman et al., 2009;Abrahamssons et al., 2012).

Explants c after cultu former inc (GLS) in b
The somatic embryo was in early-cotyledonary stage and germination stage has not occurred yet.This was a challenge to choose best media for germination.Kaviani (2013) reported that tea somatic embryo from early to the end stage was only cultured on MS media supplemented with 2,4-D.This was different from previous reports of any tea clone or kind of tea explants (Akula & Dodd, 1998;Akula, 2000;Tahardi et al., 2003).
Successful induction of somatic embryo in light or dark condition was different among plants.Somatic embryogenesis can be achieved in dark condition (Tahardi et al., 2000;Gomes et al., 2006), dark condition followed by light treatment (Sunandar et al., 2017), or fully in light condition (de-la-Pena et al., 2008;Bakhshaie et al., 2010;Lema-Ruminska & Kulus, 2012).In tea micropropagation, light was also important to induce somatic embryogenesis (Akula et al., 2000, Akula & Dodd, 1998) and were in confirmation of the present results of successful induction of somatic embryogenesis when explants were culture on light condition.The critical role of light capability to induce somatic embryogenesis was related proteins, such as kind of arrestin-like protein, G-protein, and nucleoside diphosphate kinase (NDPK) (Nato et al., 2000).
Previously, Kaviani (2013) successfully induced somatic embryo on explants of embryonic axis of tea seed that are similar to the present study.Akula and Dodd (1998) also successfully induced somatic embryo of tea using different explants; nodal segment on modification of MS media.Therefore, present and previous findings are indicating that successful induction of embryogenesis requires several favorable factors and is highly specific specific to the type of tea clone, explants, culturing media, PGRs, incubation and suitable growing conditions.
General initial response for almost every explants cultured on all media tested was callus production.This phenomenon was observed due to former incision of root meristem to remove its growth point and was a common practice in plant tissue culture (Iwase et al., 2011).Callus was a crumb and yellowish-white in color that formed at about 7 DAC.In contrast, the apical meristem incision was not entered callogenesis stage, except in explants cultured on MS media supplemented with 5 mg L -1 2,4-D.

Conclusion
This study successfully induced somatic embryo indirectly on embryonic axis of TRI2025 tea clone cultured on MS media supplemented with 2 mg L -1 of 2,4-D in light.Globular and elongated embryo was successful produced by culturing on MS media free PGRs, but subsequently abnormal early-cotyledonary was formed.
Figure condition DAC