Abstract
Wild cotton species can contribute to a valuable gene pool for genetic improvement, such as genes related to salt tolerance. However, reproductive isolation of different species poses an obstacle to produce hybrids through conventional breeding. Protoplast fusion technology for somatic cell hybridization provides an opportunity for genetic manipulation and targeting of agronomic traits. Transcriptome sequencing analysis of callus under salt stress is conducive to study salt tolerance genes. In this study, calli were induced to provide materials for extracting protoplasts and also for screening salt tolerance genes. Calli were successfully induced from leaves of Gossypium sturtianum (C1 genome) and hypocotyls of G. raimondii (D5 genome), and embryogenic calli of G. sturtianum and G. raimondii were induced on a differentiation medium with different concentrations of 2, 4-D, KT, and IBA, respectively. In addition, embryogenic calli were also induced successfully from G. raimondii through suspension cultivation. Transcriptome sequencing analysis was performed on the calli of G. raimondii and G. sturtianum, which were treated with 200 mM NaCl at 0, 6, 12, 24, and 48 h, and a total of 12,524 genes were detected with different expression patterns under salt stress. Functional analysis showed that 3,482 genes, which were differentially expressed in calli of G. raimondii and G. sturtianum, were associated with biological processes of nucleic acid binding, plant hormone (such as ABA) biosynthesis, and signal transduction. We demonstrated that DEGs or TFs which related to ABA metabolism were involved in the response to salt stress, including xanthoxin dehydrogenase genes (ABA2), sucrose non-fermenting 1-related protein kinases (SnRK2), NAM, ATAT1/2, and CUC2 transcription factors (NAC), and WRKY class of zinc-finger proteins (WRKY). This research has successfully induced calli from two diploid cotton species and revealed new genes responding to salt stress in callus tissue, which will lay the foundation for protoplast fusion for further understanding of salt stress responses in cotton.
Highlights
Cotton is an agronomically important crop plant consisting of four independently domesticated species (i.e., G. hirsutum, G. barbadense, G. herbaceum, and G. arboreum) that collectively dominate the natural fiber market
Calli from G. sturtianum were light yellow and loose under N or C starvation conditions, and these were selected for subculture on media lacking either carbon or nitrogen sources to induce embryogenic callus formation
G. sturtianum calli were gray under nitrogen deficiency, they grew rapidly, in N1 and N3 media (Supplementary Table 1); when grown in media lacking a carbon source, calli were green and slow to proliferate, indicating that embryogenic callus induction was more successful under nitrogen deficiency
Summary
Cotton (genus Gossypium) is an agronomically important crop plant consisting of four independently domesticated species (i.e., G. hirsutum, G. barbadense, G. herbaceum, and G. arboreum) that collectively dominate the natural fiber market. In addition to these important crop species, the genus include over 50 additional species, most of which produce some degree of fiber (Wang et al, 2018). Protoplasts can be isolated from different explants, such as leaves, hypocotyls, young roots, embryogenic callus, etc. Our ability to induce and to successfully develop embryonic calluses for a diversity of species comprises key steps in generating interspecific hybrids through protoplast fusion/plant regeneration (Sun et al, 2005)
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