Establishment of a low-temperature flower bud storage system for continuous anther regeneration and transformation in Hevea brasiliensis

This study evaluated the factors influencing low-temperature storage of flower buds and anther regeneration in Hevea brasiliensis . Comparative analyses were conducted with developmental stages of flower buds (microspore mother, tetrad, middle uninucleate, and late uninucleate), durations (15, 30, and 45 days), and storage temperatures (5, 10, and 15°C). Among the stages, middle uninucleate buds showed the best regeneration, followed by tetrad and late uninucleate buds; microspore mother buds showed the lowest regeneration. Preservation at 15°C yielded the best results; 10°C was moderate; and 5°C was the least. The regeneration at 15 days was slightly higher than that at 30 days and significantly higher than that at 45 days. The best results were obtained when 40 middle uninucleate buds were preserved at 15 °C for 30 days, yielding 12.33 embryogenic calli, which produced 28.67 cotyledonary embryos with a 77.78% plantlet rate. Microspore developmental stage, temperature, and preservation duration strongly affected bud regeneration potential. Buds were preserved on M1 medium at 15 °C for 15–45 days, followed by anther culture on M2 medium for recovery and callus induction. Recovery occurred within 15–45 days, while globular embryos formed after 45–75 days. Embryogenic calli transferred to M3 medium produced cotyledonary embryos within 40–50 days, which developed into plantlets on M4 medium within 50–70 days, extending embryogenic callus availability by 18–65 days of the flowering season. Cell viability was positively correlated with callus capacity, while SOD (Superoxide dismutase) and CAT (Catalase) activities were negatively correlated. The regenerated plants derived from preserved buds were phenotypically normal, diploid, and exhibited low levels of ISSR and Indel variation, with non-significant genomic differences. The transient transformation efficiency of callus from preserved buds did not differ significantly from that of the controls. A low-temperature bud preservation and anther regeneration system was established, extending explant usability by 18–65 days for one season.