Micropropagation of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) and assessment of genetic fidelity of micropropagated plants using RAPD markers

Abstract

A micropropagation protocol was developed for multiplication of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) using nodal explants. The maximum shoot regeneration was observed on low level of BAP (0.1 mg l⁻¹) or kinetin (0.5 mg l⁻¹). BA was recorded to be better than kinetin in terms of number of days taken to bud break. The maximum number of shoots per explant was observed on 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ kinetin. Shoot proliferation decreased with increasing concentration of BA alone, but in case of a combination of BA and NAA (0.1 mg l⁻¹ each), it increased with increasing concentration of BA up to 10.0 mg l⁻¹. None of the treatments including BA or kinetin alone or BA in combination with NAA produced significantly more shoots for commercial exploitation. In the case of a combination of BA + kinetin + IBA, the maximum (5.5 shoots per explants) proliferation was observed on MS medium containing 1.0 mg l⁻¹ BA + 0.5 mg l⁻¹ kinetin + 0.5 mg l⁻¹ IBA or 0.25 mg l⁻¹ BA + 1.0 mg l⁻¹ kinetin + 1.0 mg l⁻¹ IBA. Regenerated shoots showed root induction on MS basal medium or on MS medium containing 1.0 mg l⁻¹ IBA. It is concluded that a five-fold increase (1.0 mg l⁻¹ BA + 0.5 mg l⁻¹ kinetin + 0.5 mg l⁻¹ IBA) in axillary shoot proliferation, while seven-fold increase (0.25 mg/l mg l⁻¹ BA + 1.0 mg l⁻¹ kinetin + 1.0 mg l⁻¹ IBA) during the second cycle of multiplication could be obtained using the two plant growth regulator combinations. PCR amplification with 14 different random primers confirmed no somaclonal variant up to two cycles of shoot multiplication.