Phenotypic characterization of an Arabidopsis T-DNA insertion line SALK_063500

In this article we report the identification of a homozygous lethal T-DNA (transfer DNA) line within the coding region of the At1G05290 gene in the genome of Arabidopsis thaliana (Arabidopsis) line, SALK_063500. The T-DNA insertion is found within exon one of the AT1G05290 gene, however a homozygous T-DNA allele is unattainable. In the heterozygous T-DNA allele the expression levels of AT1G05290 were compared to wild type Arabidopsis (Col-0, Columbia). Further analyses revealed an aberrant silique phenotype found in the heterozygous SALK_063500 plants that is attributed to the reduced rate of pollen tube germination. These data are original and have not been published elsewhere.

Arabidopsis T-DNA SALK_063500 Silique Pollen Phenotype AT1G05290 a b s t r a c t In this article we report the identification of a homozygous lethal T-DNA (transfer DNA) line within the coding region of the At1G05290 gene in the genome of Arabidopsis thaliana (Arabidopsis) line, SALK_063500. The T-DNA insertion is found within exon one of the AT1G05290 gene, however a homozygous T-DNA allele is unattainable. In the heterozygous T-DNA allele the expression levels of AT1G05290 were compared to wild type Arabidopsis (Col-0, Columbia). Further analyses revealed an aberrant silique phenotype found in the heterozygous SALK_063500 plants that is attributed to the reduced rate of pollen tube germination. These data are original  Value of the data T-DNA insertion lines provide an important resource for genetic analyses in plant research, and SALK lines are the most commonly used T-DNA insertion lines. Therefore assessments of phenotypes observed in SALK lines are valuable assets for advancing our understanding of basic plant biology.
Documentation of the phenotype of the SALK_063500 line will make the plant community aware of the role AT1G05290 plays in pollen development, thereby furthering research in this field.
The data presented could provide insights into understanding the molecular mechanisms of male sterility in plants.  Table 1.

Data
The data presented here provide information on the phenotype of a T-DNA insertion line, SALK_063500. Fig. 1A-B shows the T-DNA insertion within AT1G05290 and the wild type and heterozygous T-DNA alleles amplified. No homozygous T-DNA alleles were found after screening over a thousand SALK_063500 progeny. Fig. 2 shows the level of AT1G05290 expression in seedlings under normal growth conditions. Figs. 3 and 4 show the silique phenotype observed in both Col-0 and heterozygous SALK_063500 plants. Figs. 5 and 6 show the pollen tube germination rates in vitro and in vivo of Col-0 and heterozygous SALK_063500 plants. Table 1 provides the sequences of the primers used in Figs. 1 and 2.

Experimental design, materials and methods
Arabidopsis seeds of wild type Columbia-0 (Col-0) and SALK_063500 were obtained from the Arabidopsis Biological Resource Center [1]. Plants were grown in the growth chambers on soil at 22°C under  SALK_063500 seeds were grown on 0.5% Phytagel TM plates (0.5× Murashige and Skoog (MS) salts [Invitrogen] containing 0.5% (w/v) sucrose, was adjusted to pH 5.72 with 1N KOH and solidified with 0.5% (w/v) Phytagel TM [Sigma]) at 22°C under constant light condition [2]. The 12 day old plants were harvested from Phytagel plates and directly preserved in RNALater [Ambion] until ready for RNA extraction. Total RNA was extracted using the RNAeasy kits [Qiagen] according to the manufacturer's instructions. CDNA was transcribed from total RNA, using the High Capacity RNA to cDNA kit [Applied Biosystems] and RT-qPCR analysis was conducted with SYBR Green master mix [Applied Biosystems]. The 2 −ΔΔCt method was used to calculate the relative expression of At1g05290 using UBQ11 (AT4G0505) as an internal control. A total of 6 replicates were used for each sample. For silique phenotypes, 30 siliques from three individuals representing the genotype were collected, imaged under the Olympus stereo dissecting microscope (SZH10) and measured using image J [3,4]. For in vitro pollen germination studies, mature pollen was harvested from flowers left at room temperate for 2 h, and then grown overnight on slides coated with solid pollen germinating media (18% Sucrose, 0.01% Boric Acid, 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4 and 0.5% Noble agar [Difco] was adjusted to pH 7.0 [5]. Pollen germination studies done in vivo were performed by collecting pollinated pistils 6 h after pollination and briefly fixed in ethanol:acetic acid (3:1) for 2 h at room temperature. Fixed pistils were then washed with distilled water three times and further treated in 8 M NaOH overnight. Pistil tissues were then washed in distilled water and stained with aniline blue solution (0.1% aniline blue in 0.1 M K 2 HPO 4 -KOH buffer, pH 11) for 5 h in the dark before being viewed under the Olympus BX51 compound light microscope [6].