A framework for date palm (Phoenix dactylifera L.) tissue regeneration and stable transformation.

The date palm is a resilient, socioeconomically valuable desert fruit tree renowned for its heat, drought, and salinity tolerance. Date palm fruits are rich in nutrients and antioxidants, and their beneficial health properties can mitigate current and future food security challenges. However, it is challenging to improve date palm production through conventional breeding methods due to its slow growth. Date palm seeds do not produce true-to-type progeny, and commercial propagation relies on direct organogenesis from maternal tissue. Consequently, numerous economically important and valuable cultivars are lost due to tissue recalcitrance and challenges in inducing cell dedifferentiation and regeneration. Moreover, genetic engineering of date palms is currently impossible due to the lack of a stable genetic transformation protocol. This hampers the development of genetic resources in date palms. This study established a tissue culture pipeline and a genetic transformation protocol for various commercially important date palm cultivars. We used the non-invasive visual reporter RUBY and four morphogenic regulators to validate and improve date palm transformation potential. We found that the date palm BABY-BOOM (PdBBM) and the WOUND INDUCED DEDIFFERENTIATION (PdWIND1) enhanced transformation efficacy. We show that PdBBM can induce embryogenesis in hormone-free media and regenerate roots and shoots in recalcitrant varieties. On the other hand, PdWIND1 maintained embryogenic cells in their undifferentiated state. Our study provides a foundation for genetically improving date palms and a potential solution for preserving economically valuable varieties.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including ß-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and ß-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, ß-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial ß-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of ß-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.