Maintenance of callus-associated endophyte balance to mitigate oxidative browning in plant tissue culture practices

The frequently occurring browning of plant tissue cultures is a considerable problem in causing economic losses. However, the mechanism that causes browning of plant tissues is still controvesial and technologies to effectively prevent browning of plant tissues are still scarce. In the present work, two callus lines derived from grapevine shoot tips (ST-callus) and fruit flesh (F-callus) were used to investigate the relationship between callus-associated endophytes and callus browning. We observed the transfer of browning effects from grapevine brown calli to normal calli, in a contact cocultivation experiment, then detected the emergence of endophytic bacteria from brown calli when the calli were incubated in a microbial culture medium, and the isolates were identified as genus Bacilus. The inoculation of pure cultured B. sp. strain ST-B1 into normal calli at different concentrations caused gradient callus browning, validating the callus browning-causing (CBC) endophyte. In addition, the moderate inhibition of endophytic bacteria in grapevine calli by culturing the calli in ampicillin-containing media reduced the incidence and severity of callus oxidative browning. The experiments were performed separately on two types of grapevine calli, ST-callus (derived from the tip of grapevine shoots), and F-callus (derived from the pulp of grape berries), and gave similar results. The DNA sequence amplicon approach showed that the CBC endophytic bacteria were found in both the normal and brown calli, which differed greatly in their relative abundances. And compared to the normal calli, brown calli greatly reduced the diversity of bacterial endophytes, while the diversity of fungal endophytes between normal and browning calli showed no obvious difference. The work demonstrated that callus-associated endophytes are involved in causing oxidative browning of plant cells, and suggested technologies to minimize the occurrence of the oxidative browning during plant tissue culture practices.