In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.)

Abstract

An efficient in vitro protocol was developed for callus induction, high frequency plant regeneration through callus cultures derived from cotyledonary leaf and epicotyl explants, rooting of shoots derived from callus and establishment onto the natural conditions in two cultivars of pigeon pea; ICPL 87119 and ICPL 8863. Cotyledonary leaf and epicotyl explants were tested for callus induction across 48 different combinations and concentrations of auxins and cytokinins in MS medium, wherein, higher doses of auxins (15 mg/1 NAA) in combination with lower doses of cytokinins (0.5 mg/l kinetin) induced regenerable callus from leaf explants while lower doses of auxins (0.2 mg/1 NAA) in combination with higher doses of cytokinins (8 mg/1 kinetin) induced regenerable callus from epicotyl explants in both the genotypes. Plantlet regeneration from leaf and epicotyl derived callus was optimized at 0.05 mg/l TDZ in both genotypes. Rooting was optimized on ½ MS + 0.5 mg/1 IBA media in both genotypes. Well-rooted plants were acclimatized and established successfully into natural conditions in potting mixture-containing soil: FYM in 1:1 ratio resulting in 48.01 per cent survivability. Regenerated plants were uniform morphologically with normal leaf shape and growth. This protocol finds its significance in rapid multiplication of transgenic plants.

An improved protocol for efficient regeneration, transformation and gene-editing using CRISPR/Cas9 in pigeonpea (Cajanus cajan(L.) Millsp.)

“…Additionally, the nature of calli impacts its response competency, and is thus, regarded as the most crucial factor for regeneration. Previous report demonstrated shoot initiation in pigeonpea from explants kept on appropriate doses along with several combinations of plant growth regulators (PGRs) such as MS basal media supplemented with 0.5mg/l BAP, 0.1 mg/l NAA [52], 0.05 mg/l thidiazuron (TDZ) [28], 3.0 mg/l BAP, and 0.5 mg/l NAA [26]. Earlier reports established that BAP alone is sufficient for shoot bud differentiation in pigeonpea [17,18,23].…”

“…The establishment of productive tissue culture and reliable transformation protocols plays a major role in the advent of genetic improvement of the crop plants (14) and many times recalcitrant plant system too (15), including pigeonpea [16]. The earlier published reports have established or exhibited the regeneration of shoots from non-meristematic as well as differentiated tissues like leaf [17][18][19][20][21][22] and various seedling explants such as hypocotyls [23], cotyledons [23][24], cotyledonary nodes [25][26], epicotyls [23,27,28] and embryonal axes [29] in pigeonpea.…”