Aloe vera L. (Asphodelaceae): Supplementation of in-vitro culture medium with Aloe vera gel for production of genetically stable plants
As in vitro culture environments can be mutagenic, it's challenging to produce true-to-true type plants from meristems. Therefore, the present study aimed to devlop an efficient protocol for micropropagation from shoot tip explants of medicinally important Aloe vera using different concentrations of A. vera leaf gel (AvG) as organic supplement to the culture media. Assessment of the genetic stability of A. vera in vitro multiplied plants compared to the donor plant is one of the most important pre-requisites especially before its use for commercial and medicinal purposes. For shoot multiplication, the highest number of axillary shoots recorded was 13.27 ±5.11 with an average length of 2.21 ±0.84 cm on M3 medium (MS + 0.2 mg L−1 of β-indole butyric acid (IBA) + 3 mg L−1 benzylamino-purine (BA) + 5% AvG). All three strength of MS medium (1, 1/2, 1/4) with different concentrations of AvG resulted in 100% root induction after 4 weeks of culture. All the rooted microshoots (100%) were successfully acclimatized. To provide a reliable conclusion on its nutritive supply to MS medium, the total phenolic (TPC), condensed tannins (TTC), flavonoid (TFC), and flavonol (TFlC) content as well as the antioxydant activity of AvG were investigated. TPC and TTC values were found to be 31.02 ± 0.46 μg gallic acid equivalent mg−1 of extract and 4.00 ± 0.96 μg catechin equivalent mg−1 of extract, respectively while TFC and TFlC were 13.58 ± 0.84 and 9.75 ± 0.58 μg rutin equivalent mg−1 of extract, respectively. The results of antioxydant activity of AvG following DPPH, FRAP and ABTS assays were equal to 12.50 ± 0.89; 49.59 ± 2.07 and 64.93 ± 3.23 µmol Trolox equivalent mg−1 of extract, respectively which may explain the positive role of AvG as supplement.
The genetic stability of in vitro propagated A. vera plants was evaluated using 10 inter simple sequence repeat (ISSR), 10 start codon targeted (SCoT) and 10 simple sequence repeat (SSR) markers. Out of these, 5 ISSR, 3 SCoT and 6 SSR primers produced resolvable, reproducible and scorable bands. ISSR primers generated 32 amplification products ranging from 200 to 1040 bp in size with a mean number of 6.4 bands while SCoT primers generated 33 amplicons with an average of 11 bands in the range of 160–2410 bp. In the SSR products, 6 bands were observed, each primer produced one band ranging from 410 to 720 bp. All banding profiles were monomorphic proving that the stability of the micropropagated A. vera plants was maintained. This study proved the suitability of supplementing in vitro culture media with AvG to enhance direct regeneration through shoot tip explants of A. vera for large scale micropropagation applications.