An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Purification and Characterization of ß-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii.

The demand for food-grade ß-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for ß-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of ß-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (w/v) methanol, 0.56% (w/v) sorbitol, and 0.48% (w/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications.