Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of Robinia pseudoacacia L.

Abstract

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 9 10 5 protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 lM NAA and 1 lM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7-10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 9 10 5 protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 lM NAA and 1 lM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 lM NAA and 5 lM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 lM NAA and 1 lM BAP within 25-30 days and multiplied on MS medium with 1.25 lM BAP. Excised microshoots were dipped in 1-2 ml of 2.0 lM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.

Comparison of in vitro regeneration pathways in Punica granatum L.

“…In the present studies we found that roots developed on charcoal medium without any plant growth regulators. This promotive effect of a growth regulator free medium on in vitro root induction has been recorded in other trees such as Robinia pseudoacacia (Kanwar et al 2009) and Morus alba (Agarwal and Kanwar 2007). Plant Cell Tiss Organ Cult (2010) 100:199-207 205 Acclimatization of in vitro raised plantlets…”

Effect of Different Cellulase and Pectinase Enzyme Treatments on Protoplast Isolation and Viability in Lilium ledebeourii Bioss.

“…Analysis of variance on protoplast viability (%; n = 3) in Lilium ledeoborii treatment had eventually reduced the number of viable protoplasts in the protoplast isolation of Crocus sativus L. (Darvishi et al, 2006). In contrast, a digestion period of up to 20 h resulted in the best yield of protoplasts (9.45 × 10 5 protoplasts/g FW) from the callus tissue of the nitrogen fixing woody plant, Robinia pseudoacacia (Kanwar et al, 2009). It's concluded that, the best treatment for isolation of Lilium protoplast was 4% cellulase and 1% pectinase for 24 h.…”

Protoplast isolation and culture from Kalanchoë species: optimization of plant growth regulator concentration for efficient callus production

“…For plant regeneration from protoplast-derived callus, we used media supplemented with combinations of either NAA and BAP, or NAA and zeatin. NAA and BAP induce shoot regeneration from protoplast cultures of K. blossfeldiana (Castelblanque et al 2010) and Robinia pseudoacacia (Kanwar et al 2009). Zeatin is frequently combined with NAA and used in protoplast regeneration protocols for shoot induction e.g.…”