Somatic embryogenesis has been accomplished from tender leaf base explant of date palm (Phoenix dactylifera L.). Three to five mm long tender leaf base explants derived from the meristamatic region of 2-3 year old offshoots of date palm cv. Kheneizi were cultured on Murashige and Skoog (MS) basal medium supplemented with 10, 50, 100 and 150 mg l -1 2,4-dichlorophenoxy acetic acid (2,4-D) and incubated in dark for 6 weeks to initiate callus. Callogenesis was obtained in all 2,4-D concentrations tested; however, callus growth was most significant in media supplemented with 100 mg l -1 2,4-D. The leaf explants with callus were transferred to hormone-free MS medium for 4 weeks and then further sub-cultured to a medium supplemented with 0.5 mg l -1 α-naphthalene acetic acid (NAA) and 0.25 mg l -1 6-benzyl amino purine (BAP) which was effective in inducing shoot and root primordia within 10 weeks. In another 12 weeks, two more sub-culturing of shoot clumps in the same medium resulted in the development of shoot with roots and gave whole plants by 8 weeks. The plantlets were hardened and acclimatized to the ambient conditions and planted in pots, containing 1:1:1 peat, sand and dehydrated cow manure, which resulted in over 60% ex vitro plant survival. Early plant regeneration was achieved by this technique.
Background
High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications.
Results
Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A260/A280 and A260/A230 ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 oC. Pre-warming of the buffer for 5–10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol’s broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds.
Conclusions
The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.