Development of embryogenic cell suspension cultures and efficient plant regeneration of Carica papaya L.

Abstract:【Objective】Embryogenic callus from different papaya explants can be induced to generate somatic embryos, which develop into plants. However, there are some problems in this pathway, such as genotype dependence, limited number and asynchronous development of somatic embryos, poor rooting quality and low plant regeneration rate caused due to callus formation at the base of somatic embryos. This study was designed to overcome these obstacles, and to establish an efficient plant regeneration technology for large-scale rapid propagation of papaya seedlings, cell engineering and molecular breeding.【Methods】Hybrid Carica papaya L.‘Zihui’was used as the experimental materials. Immature zy-gotic embryos (IZE) excised from fruit 110 to 120 days post-anthesis were used as the explants to induce embryogenic callus on the medium containing half-strength MS (Murashige and Skoog, 1962) basal salts and vitamins, 400 mg·L^-1 glutamine and 60 g·L^-1 sucrose supplemented with 0-5.0 mg·L^-1 2, 4- dichlorophenoxyacetic acid (2, 4-D) for 60 days. Light yellow and fragile embryogenic calluses were allowed to proliferate on the same medium at 28-30 days intervals for 3-4 months to get enough embryogenic calluses. In the process of liquid culture, 900 μm mesh sieves in the first subculture and 154 μm mesh sieves subsequently were used to filter out large particle cultures in the medium, until homogeneous embryogenic cell suspension was obtained. Embryogenic suspension cells were transferred to 100 mL conical flasks with 30 ml liquid somatic embryo induction medium (MSI) containing halfstrength MS basal salts and full-strength vitamins, 50 mg·L^-1 myo-inositol, 400 mg·L^-1 glutamine and 30 g·L^-1 sucrose, and agitated at 110 r·min^-1 in a gyratory shaker for 21 days. The induced somatic embryos were transferred to semi-solid somatic embryo mature medium [MSM: MSI + 5 g · L^{- 1} activated carbon (AC) + 4.0 g·L^{- 1} gel] allowed to further develop into cotyledonary somatic embryos, and then budded and rooted into plantlets on germinating medium. The effects of different concentrations of 6- benzyl amino purine (6-BA), naphthalene acetic acid (NAA) and AC on the germination and rooting of cotyledonary somatic embryos were investigated.【Results】Complete immature zygotic embryos can be easily and rapidly separated by extrusion method without the assistance of stereoscope. 4 mg·L^-1 was the optimum 2, 4-D concentration for embryogenic callus induction of IZE from Zihui cultivar, and the maximum embryogenic callus induction rate was 62.86%. After 5 subculture cycles of sieving culture of embryogenic callus in liquid medium, homogeneous embryogenic cell suspensions with a large number of single cells and small cell groups were established. A large number of spherical embryos were induced by liquid culture for 21 days, and then developed into cotyledonary somatic embryos synchronously on MSM in 30-45 days. About 97.58% of the cotyledonary somatic embryos germinated on MS medium supplemented with 0.4 mg · L^{- 1} 6-BA and 0.02 mg · L^{- 1} NAA, 95.48% of which showed callus formation at the base of somatic embryos, the rooting rate was 18.18%, and the quality of roots was poor. The supplementation of 5 g · L^{- 1} AC generated 92.62% cotyledonary somatic embryos with synchronous germination and rooting, and significantly reduced the callus rate to 33.10%. Synchronous germination and rooting were also achieved in MSM medium without any plant hormone, with a synchronous germination and rooting rate of 88.33%, and the callus rate was reduced to 11.67%. The plant regeneration rate was 81.36% when these plantlets were transferred onto the regeneration medium with 0.1 mg·L-1 6-BA, 2 mg·L-1 IBA and 10 g·L-1 AC. Plants with well-developed shoots and roots were subsequently hardened to seedlings in a potting mixture with 2/3 peaty soil and 1/3 vermiculite.【Conclusion】The optimal embryogenic callus induction medium was 1/2 MS medium +4 mg ·L- 1 2, 4-D+400 mg · L^{- 1} glutamine + 60 g·L^-1 sucrose with immature zygote embryos of Zihui at 110-120 days as explants. Spherical embryos can be efficiently induced from ECS with liquid culture, and subsequent synchronous germination and rooting of somatic embryos in the medium containing 1/2 MS salt + MS vitamin + 50 mg ·L^-1 inositol + 400 mg ·L^-1 glutamine + 30 g ·L^-1 sucrose + 5 g ·L^-1 AC. This regeneration protocol established in C. papaya L.‘Zihui’achieves a high frequency of somatic embryogenesis with good synchronization and plant regeneration.