Functional analysis of CyclinD2;1-type genes expressed in transgenic banana plants

Abstract

Early maturity is one of the most important aerial growth traits next to bunch size, in determining banana productivity. However, low seed fertility in banana and the lack of breeding lines limit the application of conventional breeding for this trait. Genetic transformation with a CyclinD2-type gene, responsible for the CyclinD2 protein sub unit, which modulates the cell cycle progression at the G1/S phase, enhanced growth of tobacco plants. On this basis, investigations were carried out on the possibility of increasing the growth rate of banana plants through transformation with and expression of a CyclinD2- type coding sequence. Arabidopsis thaliana;CyclinD2;1 (Arath;CycD2;1) gene and its banana ortholog were over-expressed in banana to test their growth enhancement potential. The banana CyclinD2;1 (Musac;CycD2;1) was isolated from an East African highland cooking banana (AAA) cultivar “Nakasabira” and a cDNA was created by PCR using degenerated primers which was followed by genome walking. Characterization of the banana cyclin protein revealed an IWKVHAHY motif that was found to be conserved across the Musaceae family. Phylogenic analysis revealed a higher protein sequence identity of this banana cyclin to CyclinD2;1 of monocot plants than that of Arabidopsis. This cyclin was also found to be expressed highly in meristematic tissue which linked it to the cell cycle. The coding sequence was submitted to the GeneBank under accession number HQ839770. Arabidopsis and banana CyclinD2;1 gene coding sequences under the control of a constitutive promoter were used to transform embryogenic cells of the banana cultivar “Sukalindiizi” (AAB) using the Agrobacterium transformation system. A higher relative expression of Arath;CyclinD2;1 was found in the shoot than in the root apices and expression reduced transcript amounts of the endogenous banana CyclinD2;1. Plants of transformed banana line D2-41 had the highest Arath;CyclinD2;1 transcript amount and exhibited a significantly faster leaf elongation rate, better root growth, faster first leaf opening and a bigger lamina composed of bigger epidermal cells than non-transformed control plants. Banana plants transformed with Musac;CyclinD2;1 had a higher transcript amount of the transgene in the root apices when compared to the shoot apices. The higher transcript amount in the roots of plants of transformed line NKS-30 was related to faster root growth and development of an extensive root system. Overall, this study has provided evidence that expression of cyclin coding sequences in transformed banana is related to growth promotion. Specifically, Arath;CyclinD2;1 promoted shoot growth while the Musa homolog promoted root growth. Shoot and root growth phenotypes obtained in this study might have the potential to improve banana productivity in terms of short plant growth cycle, increased bunch weight, improved plant anchorage and increased plant resistance to root nematode damage. Future work should assess the produced plants in the field to allow transformed plants to exhibit their full potential and to be able to fully evaluate the vegetative and flowering phases.