Douglas A. Johnson

SEED COATS: STRUCTURE, DEVELOPMENT, COMPOSITION, AND BIOTECHNOLOGY

SummaryAlthough seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be examined from the perspective of molecular genetics and control of development. The seed coat, plays a vital role in the life cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the seed coat are a number of unique tissues that undergo differentiation to serve specific functions in the seed. A large number of genes are known to be specifically expressed within the seed coat tissues; however, very few of them are understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse ways, including defense and control of development. Many of the compounds are sources of industrial products and are components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat development and composition. In this review, we will examine the recent advances in seed coat, biology from the perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities for seed coat modifications in the future.

Ecological correlates of RAPD DNA diversity of wild barley, Hordeum spontaneum, in the Fertile Crescent

Genetic variability in RAPDs (Randomly Amplified Polymorphic DNA) was studied in 104 genotypes of wild barley, Hordeum spontaneum from 21 populations sampled in Israel, Turkey and Iran, seven population from each country. The band (= loci) frequencies were calculated for each population and correlated with ecogeographical variables. In general, high RAPD genetic diversity indices were associated with stressful environments, either with hot or cold steppes and deserts. Interpopulational genetic distances showed no association with the geographic distance between the populations provenance. Significant Spearman rank correlations between RAPD band frequencies and ecogeographical parameters of provenance occured. Frequencies of RAPD bands were significantly correlated with the principal component factors of allozymes. The correlation data indirectly suggest that natural selection appears to be the major determinant of both RAPD and allozyme diversities both being correlated with environmental stress.

Genetic diversity in wild barley (Hordeum spontaneum C. Koch) in the Near East: a molecular analysis using Random Amplified Polymorphic DNA (RAPD) markers

We analyzed the genetic diversity in 88 genotypes from 20 populations of wild barley (Hordeum spontaneum C. Koch) from Israel, Turkey and Iran, by randomly amplified polymorphic DNA (RAPD). Twenty two of the 33 primers used yielded scorable products with 1–11 polymorphic bands. No duplicate patterns were found except for four haplotypes.When the total genetic diversity was estimated, 75% of the variation detected was partitioned within the 88 genotypes and 25% among the populations. When variation between countries was assessed, no substantial differences were found, because most of the variation detected (97%) was partioned within the 20 populations and the remainder among countries. The results of this limited survey indicate that the extensive genetic diversity is present in natural stands of wild barley throughout the Fertile Crescent.

SCB1, a BURP-domain protein gene, from developing soybean seed coats

Abstract. We describe a gene, SCB1 (Seed Coat BURP-domain proteinxa01), that is expressed specifically within the soybean (Glycine max [L.] Merrill) seed coat early in its development. Northern blot analysis and mRNA in situ hybridization revealed novel patterns of gene expression during seed development. SCB1 mRNA accumulated first within the developing thick-walled parenchyma cells of the inner integument and later in the thick- and thin-walled parenchyma cells of the outer integument. This occurred prior to the period of seed coat maturation and seed filling and before either of the layers started to degrade. SCB1 may therefore play a role in the differentiation of the seed coat parenchyma cells. In addition, the protein product appears to be located within cell walls. The SCB1 gene codes for a new member of a class of modular proteins that possess a carboxy-terminal BURP domain and a variety of different repeated sequences. The sequence of the genomic clone revealed the insertion of a Tgm transposable element in the upstream promoter region but it is not certain whether it contributes to the tissue-specific pattern of SCB1 expression.

Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR, with an example from barley

Although the increasing number of expressed sequence tags (ESTs) from the public domain has facilitated the detection of single nucleotide polymorphisms (SNPs), further validation is needed before they can be used as markers. For SNP validation, we have compared 2 independent methods: (1) the primer extension method followed by capillary electrophoresis on an ABI PRISM 3100 Genetic Analyzer and (2) nested PCR followed by agarose-based visualization. We present an assessment of the efficiency and costs associated with these methods, based on a sample of barley cultivars.

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean

Abstract. A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12u2009days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells.

Strategies to mitigate transgene–promoter interactions

The expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters.

DIRECT SHOOT REGENERATION FROM LEAF SEGMENTS OF MATURE PLANTS OF ECHINACEA PURPUREA (L.) MOENCH

SummaryThis is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l−1 (6μM) BA in 30 d. Callus was induced using 0.5 mgl−1 (1μM) α-naphthaleneacetic acid and 2.5 mgl−1 (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl−1 (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.

Bnm1, a Brassica pollen-specific gene

AbstractcDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

Genetic diversity among barley cultivars assessed by sequence-specific amplification polymorphism

We analyzed the genetic structure and relationships among barley cultivars (Hordeum vulgare L.) with sequence-specific amplification polymorphisms (S-SAPs). Polymorphisms were identified in 824 individual barley plants representing 103 cultivars (eight plants per cultivar) widely grown in Canada and the United States, using PCR primers designed from the long terminal repeat of the barley retrotransposon BARE-1 and a subset of four selective MseI primers. From the 404 bands scored, 150 were polymorphic either within or between cultivars. Genetic structure assessed with analysis of molecular variance attributed the largest component of variation to the within groups of cultivars (69–86%). Within-cultivar genetic variation was estimated as average gene diversity over loci and ranged from 0 (completely homogenous) to 0.076 (most heterogeneous cultivar). Only 17 out of 103 cultivars (16%) were judged to be homogenous by this criterion. Relationships among cultivars were analyzed by cluster analysis using unweighted pair-groups using arithmetic averages and found groups similar to those determined by agriculturally significant phenotypic traits such as spike morphology (two-rowed or six-rowed), cultivar type (malting or feed), seed characteristic (hull-less or hulled), and growth habit (winter or spring), with minor overlaps. Discriminant analysis of groups determined by these phenotypic traits fully supported the different groups with minor overlaps between the malting/feed. S-SAP markers generated from retrotransposons such as BARE-1 are invaluable tools for the study of genetic diversity in organisms with a narrow genetic base such as barley. In this study, S-SAP analysis revealed significant amounts of cryptic variation in closely related cultivars including somaclonal variation, which could not be inferred by the pedigree analysis.