Recurrent secondary embryogenesis in androgenic embryos and clonal fidelity assessment of haploid plants of Tea, Camellia assamica ssp. assamica and Camellia assamica ssp. lasiocaylx

Abstract

Globular androgenic haploid embryos of TV21 and TV19 cultivars of Camellia ssp., obtained on embryo induction medium (EIM), Murashige and Skoog medium with 10 µM 6-benzylaminopurine (BAP), 3 µM gibberellic acid (GA3), 800 mg l−1 L-glutamine and 200 mg l−1 L-serine, were pretreated with 18 µM abscisic acid (ABA) and 25 g l−1 mannitol for 20 days in liquid medium to attain maturation. The pre-treatment favoured the primary globular embryos to grow and pass through all the developmental stages from globular, heart-shape, torpedo shape to fully matured docotyledonous embryos. Fivefold and threefold increase in secondary embryogenesis (SE) was attained in Camellia assamica ssp. assamica (TV21; Assam Type) and Camellia assamica ssp. lasiocaylx (TV19; Cambod Type), respectively, when pre-treated primary embryos were transferred to the liquid embryo development (ED) medium, supplemented with tenfold reduced concentration of growth regulators that were present in EIM. Embryos were germinated well on 10 µM BAP, 0.5 µM GA3, 1 µM indole-3-butyric acid (IBA), 80 mg l−1 L-glutamine and 20 mgl−1 L-serine, following liquid-to-semi-solid transition method. Well developed shoots and roots were formed when embryos were initially kept for four weeks in liquid germination medium, followed by their transfer to semi-solid medium on similar medium composition. The method promoted recurrent secondary embryogenesis in lesser time with highest germination rate, 66.6% and 30.3% in TV21 and TV19 cultivars, respectively. Histology and field emission scanning electron microscopy (FESEM) were performed to determine ontological events during embryo development. Clonal fidelity of haploid plantlets was assessed using random amplified polymorphic DNA (RAPD) to ensure no somaclonal variations occurred during the multistep process of secondary embryogenesis, embryo maturation and germination. A two-step transition protocol from liquid-semi-solid medium is beneficial for embryos to overcome hyperhydricity. Secondary embryogenesis is the best mode to obtain genetically uniform true-to-type haploid plantlets.