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Christopher S. Cramer

Determination of ploidy is an essential plant breeding technique. Laboratory exercises for teaching students how to determine ploidy in plant tissues using various techniques are described for geranium and onion. The different methods include root tip squashes, pollen mother cell squashes, pollen grain size and germinal pore counts, stomata size and density determination, and gross morphology.

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Jason D. Lattier, Hsuan Chen, and Ryan N. Contreras

positive correlation with stomata size and a negative correlation with stomata density across a wide range of angiosperms ( Beaulieu et al., 2008 ). Stomata have proven useful anatomical characters to differentiate ploidy levels in woody plant species

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Jiro Matsushima, Keizo Yonemori, and Kenzo Iwao


Transpiration rate, stem diameter, and leaf temperature of satsuma mandarin (Citrus unshiu Marc.) were found to be sensitive indicators of stomatal response to ozone exposure. Time of reaction of each to 1.2 ppm ozone was 3 min. Stem diameter showed marked oscillation and leaf temperature slight oscillation, when ozone was applied. Correlation coefficiennt between ozone concentration and transpiration rate at the equilibrium status after the beginning of exposure was r = 0.718, and increasing the concentration of ozone decreased stomata size. Thus, it was determined that low sensitivity of satsuma mandarin to ozone is due to rapid stomatal closure by ozone.

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Masaki Yahata, Hisato Kunitake, Tsutomu Yabuya, Kensuke Yamashita, Yukiko Kashihara, and Haruki Komatsu

To produce the homozygous strain of a haploid plant derived from small seed-derived seedlings of `Banpeiyu' pummelo (Citrus grandis Osbeck), we carried out colchicine treatment to axillary shoot buds of the haploid. Many shoots with cytochimeras (X+2X and 2X+4X) arose from the colchicine-treated axillary buds. When cytochimeric buds of 2X+4X were top-grafted onto trifoliate orange [Poncirus trifoliata (L.) Raf.], a complete diploid shoot with 18 chromosomes was obtained from the cytochimera. This diploid strain showed vigorous growth compared with the original haploid. The leaf weight per unit area and the stomata size in this diploid were significantly larger than those of the original haploid plant, and were almost equal to those of `Banpeiyu' pummelo. The diploid strain was confirmed to be a doubled haploid of a haploid from `Banpeiyu' pummelo, based on random amplified polymorphic DNA (RAPD) analysis and chromosome composition analysis by chromomycin A3 (CMA) staining.

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Md. Mizanur Rahim Khan, Mst. Hasnunnahar, and S. Isshiki

To restore fertility of the F1 between Solanum macrocarpon and eggplant, amphidiploids were produced through doubling of chromosomes by colchicine treatment. Shoot tips and axillary buds of F1 plants were kept for 2 and 4 days in liquid Murashige and Skoog (MS) media with 0.05% colchicine. As a result of colchicine treatment, two amphidiploids were identified by observing root tip cell chromosome number, stomatal guard cell size, and pollen characteristics. The amphidiploids contained 48 chromosomes, twice the normal diploid number of 24. Stomata size and pollen diameter were significantly larger in amphidiploids than F1. Flower diameter and length and width of anther, petal, and sepal were significantly larger in the amphidiploids than in F1. Pollen stainability was 40% in amphidiploids but only 0.86% in the diploid F1. The amphidiploids, after selfing and backcrossing with S. macrocarpon, set fruits with healthy seeds. Therefore, production of amphidiploids by colchicine treatment restored the pollen and seed fertility of F1 between S. macrocarpon and eggplant. The amphidiploids produced in this study would be very useful in future breeding programs of eggplant.

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Carole L. Bassett, D. Michael Glenn, Philip L. Forsline, Michael E. Wisniewski, and Robert E. Farrell Jr

Reduced availability of water for agricultural use has been forecast for much of the planet as a result of global warming and greater urban demand for water in large metropolitan areas. Strategic improvement of water use efficiency (WUE) and drought tolerance in perennial crops, like fruit trees, could reduce water use without compromising yield or quality. We studied water use in apple trees using ‘Royal Gala’, a relatively water use-efficient cultivar, as a standard. To examine whether genes useful for improving WUE are represented in a wild relative genetically close to M. ×domestica, we surveyed Malus sieversii for traits associated with WUE and drought resistance using material collected from xeric sites in Kazakhstan. This collection has been maintained in Geneva, NY, and surveyed for various phenotypes and has been genetically characterized using simple sequence repeats (SSRs). These data suggest that most of the diversity in this population is contained within a subpopulation of 34 individuals. Analysis of this subpopulation for morphological traits traditionally associated with WUE or drought resistance, e.g., leaf size and stomata size and arrangement, indicated that these traits were not substantially different. These results imply that some of the genetic diversity may be associated with changes in the biochemistry, uptake, and/or transport of water, carbon, or oxygen that have allowed these trees to survive in water-limited environments. Furthermore, genes responding to drought treatment were isolated from ‘Royal Gala’ and categorized according to the biological processes with which they are associated. A large fraction of upregulated genes from roots were identified as stress-responsive, whereas genes from leaves were for the most part associated with photosynthesis. We plan to examine expression of these genes in the M. sieversii population during water deficit in future studies to compare their patterns of expression with ‘Royal Gala’.

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Seenivasan Natarajan and Jeff S. Kuehny

photosynthetic photon flux of 500 μmol·m −2 ·s −1 on recently matured leaves of 8-week-old plants; two measurements were recorded per plant on separate leaves. Stomata size, stomatal number, and leaf thickness. Two recently matured leaf samples from each

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Guo-Gui Ning, Xue-Ping Shi, Hui-Rong Hu, Yan Yan, and Man-Zhu Bao

with various ploidy levels. Fig. 1. Development of polyploid plants from double-flower petunia lines. ( A1–A4) Plant morphological characters, stomata size, chromosome number, and flow cytometry results from young diploid plants of double

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Xiaohua Du, Mengye Wang, Aneta Słomka, and Huichao Liu

closed plastic chamber from Oct. 2015 to Mar. 2016 and then under insect netting from Apr. to June 2016 to avoid high temperatures and insect pollinators. Five plants of each line were selected for detailed morpho-anatomic analyses. The leaf stomata size

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Qingling Li, Yuesheng Yang, and Hong Wu

tetraploid and octoploid plantlets in shoot tip multiplication culture. Morphology and cichoric acid content differences among diploid, tetraploid, and octoploid plants. Stomata size and frequency differed significantly among diploid, tetraploid, and