Callus induction in the field and establishment of a bud regeneration system for pomegranate

Abstract:【Objective】Pomegranate (Punica granatum L.) is one of the excellent small sundry fruits and has developed quickly in recent years in China, where pomegranate has been cultivated for more than two thousand years for its high economic, nutrition, medicinal and ornamental values. Soft-seeded pomegranate is highly admired by the consumers for its big fruit, sweetness, juiciness, and delicious soft seeds, and has a great development potential in China. However, most of the new soft-seeded cultivars have been obtained from bud mutation, radiation and hybrid breeding. Breeding for soft-seeded cultivars is difficult by conventional crossing and time-consuming. Biotechnology breeding is helpful to solve these problems. Bud regeneration is the precondition and key point of plant regeneration via in vivo callus induction. The goal of this study is to construct an in vivo system for bud regeneration from callus in order to provide solid foundation for expanding propagation, genetic transformation and mutation breeding of pomegranate.【Methods】The effects of different hormone combinations, including TDZ+NAA+KT, TDZ+NAA+KT+6-BA, TDZ+NAA+KT+IBA, TDZ+NAA+KT+IBA+6-BA, TDZ+GA3+KT+6-BA and TDZ+GA3+KT+IBA+6-BA at different concentrations, AgNO3 at different concentrations and branch diameter on callus induction and adventitious bud regeneration in vivo in‘Tunisa'soft-seeded pomegranate cultivar were investigated. Strong branches were randomly selected and cut with a branch shear or hand saw. Then the cut end of the branches was treated with 1.5 mL callus inducing agents, such as 2.0 mg· L^-1 TDZ+1.5 mg· L^-1 AgNO3 for 24 h. Wetted mud was used to seal the cut end and then covered with a white plastic bag to keep moisture. The wetted mud was replaced with fresh mud every 7 days. After the adventitious buds formed from the callus grew up to 2 cm long, themud was removed allowing the buds to grow naturally. The callus morphology formed from the cut end of the branches was observed 30 days after branch cutting. The ability of bud regeneration was represented by the average bud number of each branch. The effects of branch diameter and hormone combinations on in vivo bud regeneration were analyzed.【Results】The morphological characters of pomegranate callus were analyzed. Green callus was formed 10-20 d after cutting. The callus enlarged, growing vigorously. The callus underwent obvious changes into a slightly loose texture and from light green to milky white, or from the white transparent to green granular, and large buds appeared after 30-45 d. The adventitious buds had formed from the callus by day 60. The adventitious buds formed from the callus reached 2 cm long at day 75, and reached 3-6 cm in length and mostly lignified at day 90. The effects of different hormone combinations on bud regeneration were analyzed. The results indicated that there was no significant difference in the effect on the number of buds among the hormone combinations of TDZ, NAA and KT. The treatments of 4 mg· L^-1 TDZ+2 mg· L^-1 AgNO3+0.3 mg· L^-1 NAA+1 mg· L^-1 KT+3 mg· L^-1 BA, 4 mg· L^-1 TDZ+2 mg· L^-1 AgNO3+0.3 mg· L^-1 NAA+1 mg· L^-1 KT+0.3 mg· L^-1 IBA, 4 mg· L^-1 TDZ+ 2 mg· L^-1 AgNO3+0.3 mg· L^-1 NAA+1 mg· L^-1 KT+0.3 mg· L^-1 IBA+3 mg· L^-1 BA, 4 mg·L^-1 TDZ+2 mg· L^-1 AgNO3+ 0.5 mg· L^-1 GA3+1 mg· L^-1 KT+3 mg· L^-1 BA and 4 mg· L^-1 TDZ+2 mg· L^-1 AgNO3+0.5 mg·L^-1 GA3+1 mg· L^-1 KT+ 3 mg· L^-1 BA+0.3 mg· L^-1 IBA improved the average bud number. Moreover, compared with the other treatments, the treatment of 4 mg· L^-1 TDZ+2 mg· L-1 AgNO3+0.5 mg· L^-1 GA3+1 mg· L^-1 KT+3 mg· L^-1 BA+0.3 mg· L^-1 IBA induced the earliest adventitious buds that appeared 30 days after treatment. But with the increase in the concentration of the hormone, the adventitious buds rate reduced. Higher concentrations of hormone promoted callus cell division, but inhibited the adventitious bud differentiation. The number of the regenerated buds had no significant difference among different branch diameters. Moreover, to balance minimizing number of branches to cut and minimizing the damage to trees, we suggested that 1.0-2.0 cm branches were suitable for adventitious bud induction. In addition, AgNO3 had an impact on the formation of callus and the average bud number, and the best concentration was 2.0 mg· L^-1.【Conclusion】A callus induction and bud regeneration system in pomegranate was successfully constructed in vivo, which will promote pomegranate expanding and gene transformation.