Zea mays (maize) is an important model organism for studying monocot growth and development. Genotyping maize in the greenhouse or field can be time consuming and costly. Here, we describe a method to remove or chip a small amount of the endosperm from a maize kernel to genotype the kernel prior to planting. The seed chip is removed with a razor blade for DNA extraction and subsequent genotyping. When done correctly, seeds germinate normally and the kernel genotype can be determined before planting thus saving time, money, and field space.

Cell division requires spatial coordination to properly position the division plane. How division plane positioning contributes to plant growth remains unknown. Two unrelated microtubule binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9), are together required for normal Arabidopsis growth and division. tan1 air9 double mutants have synthetic growth and division plane orientation defects while single mutants lack obvious defects. We show that the first 132 amino acids of TAN1 (TAN11-132) rescue the tan1 air9 double mutant and localize to the division site during telophase. Loss of both rescue and division-site localization occurred when interaction between TAN1 and PHRAGMOPLAST ORIENTING KINESIN1 (POK1) was disrupted by replacing six amino acid residues with alanines in TAN11-132. However, full-length TAN1 with the same alanine substitutions significantly rescued the tan1 air9 double mutant and remained at the division site throughout mitosis, although its accumulation was reduced and phragmoplast positioning defects occurred. POK1 often fails to accumulate at the division site in tan1 air9 mutants, suggesting that both TAN1 and AIR9 stabilize POK1 there. Finally, a mitosis specific promoter driving TAN1 rescued the tan1 air9 double mutant phenotypes indicating that defects seen in the root differentiation zone reflect the loss of mitotic-specific TAN1 activity.One sentence summarySpecific amino acids within TAN1 are required for its correct localization and function partially through interaction with POK1; both TAN1 and AIR9 mediate POK1 division site localization.

Proper plant growth and development requires spatial coordination of cell divisions. Two unrelated microtubule-binding proteins, TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES9 (AIR9), are together required for normal growth and division-plane orientation in Arabidopsis (Arabidopsis thaliana). The tan1 air9 double mutant has synthetic growth and division-plane orientation defects while single mutants lack obvious defects. Here we show that the division site–localized protein, PHRAGMOPLAST ORIENTING KINESIN1 (POK1), was aberrantly lost from the division site during metaphase and telophase in the tan1 air9 mutant. Since TAN1 and POK1 interact via the first 132 amino acids of TAN1 (TAN11-132), we assessed the localization and function of TAN11-132 in the tan1 air9 double mutant. TAN11-132 rescued tan1 air9 mutant phenotypes and localized to the division site during telophase. However, replacing six amino-acid residues within TAN11-132, which disrupted the POK1–TAN1 interaction in the yeast-two-hybrid system, caused loss of both rescue and division-site localization of TAN11-132 in the tan1 air9 mutant. Full-length TAN1 with the same alanine substitutions had defects in phragmoplast guidance and reduced TAN1 and POK1 localization at the division site but rescued most tan1 air9 mutant phenotypes. Together, these data suggest that TAN1 and AIR9 are required for POK1 localization, and yet unknown proteins may stabilize TAN1–POK1 interactions.

Cell division plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXIN-INDUCED-IN-ROOT-CULTURES9 (AIR9) are division-site localized microtubule-binding proteins required for division plane positioning. tan1 and air9 Arabidopsis thaliana single mutants have minor or no noticeable phenotypes but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions, and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in nondividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/M-specific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division plane defects, pKN:TAN1-YFP rescued root growth and the root differentiation zone cell file rotation defects in the tan1 air9 double mutant. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells.

Gene-editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene-editing technology as well as develop their soft skills in data management and scientific communication.