Development of a cell suspension protocol for abaca (Musa textilis Nee 'Inosa')

A protocol for the establishment of embryogenic cell suspension in abaca was developed based on the International Network for the Improvement of Banana and Plantain (INIBAP) procedure for Musa. Meristematic buds from shoot cultures were exercised and cultured on P5 medium for several cycles until 'scalps' formed. These scalps were then used to produced embryogenic complexes (ECs) in 2,4-D containing media. Discrete primary somatic embryos (SEs) were observed on ECs 3-4 wk after initial culture. After 6 mo, 6% of the 150 inoculated scalps formed into ECs that had at least 10 SEs. These were then used to commence cell suspensions. Nine liquid media formulations were tested, and only M2 medium produced cell lines that had a characteristics bright yellow suspension indicative of embryogenic potential. However, doubling time during the initiation phase with M2 medium was lower than the doubling time in the rest of the media tested. In several months, cell lines in M2 medium stabilized wiyh an average doubling time of about 4 wk. The old medium was replenished with fresh medium every 4-7 d, replacing at least 80% of the old medium. Maintenance of fine homogenous suspension was done at monthly intervals by transferring 2 mL aliquots with a settled cell volume (SCV) of 0.2-0.3 mL from a 1-mo-old suspension culture to an 8 mL fresh medium in 125-mL Erlenmeyer flasks. As for embryo development, regeneration media trials showed that Murashige and Skoog (MS) and M2 media promoted the formation of yellow, nodular calli, or pro-embryogenic masses (PEMs). MS and M2 medium supplemented with 1 ppm AgNO3 (with or without ascorbic acid) could enhance PEM formation, especially if the cells were pre-induced to form PEMs by gradual reduction of 2,4-D while at the cell suspension stage. Sucrose or glutamine does not seem to have any promotive effect on PEM induction during the regeneration phase. These treatments, however, were not favorable for the conversion of PEMs into mature somatic embryos. Likewise, M3 regeneration media promoted PEM formation but failed to induce somatic embryogenesis in a separate experiment. PEMs derived from the 2,4-D reduction experiment were proliferated in M2 medium and transferred in P5 for embryo development. PEMs bearing mature somatic embryos in P5 medium were transferred to abaca shoot proliferation medium where fully developed shoots were obtained in 6 mo. Although the protocol requires validation, our results clearly demonstrate that abaca could undergo in vitro somatic embryogenesis (SE) and offer a transformation platform for modern genetic improvement work.