Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4 degrees C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9 degrees C and thereafter at 28 degrees C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 muM zeatin and 5 muM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.

Cytoplasmic male sterility in plants is associated with mitochondrial dysfunction. We have proposed that a nuclear-encoded chimeric peptide formed by mitochondrial sequences when imported into the mitochondria may impair organelle function and induce male sterility in plants. A model developed to test this hypothesis is reported here. Assuning that the editing process in higher plant mitochondria reflects a requirement for producing active proteins, we have used edited and unedited coding sequences of wheat ATP synthase subunit 9 (atp9) fused to the coding sequence of a yeast coxlV transit peptide. Transgenic plants containing unedited atp9 exhibited either fertile, semifertile, or male-sterile phenotypes; controls containing edited atp9 or only the selectable marker gave fertile plants. Pollen fertility ranged from 31% to 75% in fertile plants, 10% to 20% in semifertile plants, and <2% in malesterile plants. Genetic and molecular data showed that the chimeric plasmid containing the transgene is inherited as a Mendelian trait. The transgenic protein is imported into the mitochondria. The production and frequency of semifertile or male-sterile transgenic plants conform to the proposed hypothesis.Male sterility in plants leads to pollen abortion. This phenomenon is often observed in alloplasmic plants combining cytoplasmic and nuclear genetic material from two different species (1). Maternally inherited cytoplasmic male sterility (CMS) has been recently reviewed (2). Molecular studies on CMS have revealed the existence of modifications in mitochondrial DNA. In a number of CMS plants, the sterile phenotype is associated with the production of proteins arising from chimeric genes (3, 4); their incorporation into the mitochondrial membrane or into multiprotein enzyme complexes may lead to the impairment of mitochondrial function.The hypothesis developed in this report is that the impairment of mitochondrial function may be obtained by introducing an altered subunit in the ATP synthase complex. The protein ATP9 represents a good candidate because it is involved in the proton channel of the ATP synthase. ATP9 is mitochondrially encoded in plants but nuclear-encoded in Neurospora (5) and mammals (6). A mitochondrially encoded protein can be introduced into nuclei and imported into mitochondria when fused to a targeting sequence (7,8). An approach similar to the one described here has been used to introduce herbicide resistance into tobacco chloroplasts (9).We have shown a discrepancy between the amino acid sequence of the ATP9 protein and the predicted sequence deduced from the gene.

We have previously shown that the expression of an unedited atp9 chimeric gene correlated with malesterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the Fl progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.The development of strategies to improve crop plants by the production of hybrid varieties is a major goal in plant breeding. Hybrid progeny often have a higher yield, increased resistance to disease, and an enhanced performance in different environments compared with the parental lines (1). Male sterility mutations that guarantee the outcrossing of naturally autogamic plant lines (2) have proven to be useful for the production of hybrid lines with increased crop productivity. However, hybrid production is limited to those species of plants in which male sterility mutations have been detected. Moreover, in certain plants, such as corn, wheat, rice, or tomato, where seeds and fruits are harvested products, a male fertility restorer system is required.Many male sterility mutations interfere with tapetal cell differentiation and/or function, indicating that this tissue is essential for the production of functional pollen grains (3-7).The tapetal cells are the target to produce engineered malesterile plants. In view of this, several authors have reported the production of transgenic male-sterile plants by the expression of degradative enzymes in tapetal cells (8)(9)(10) or by the inhibition of particular enzymes by antisense strategies (11). One interesting, naturally-occurring phenotype is cytoplasmic male sterility. Molecular analysis of cytoplasmic male sterility plants has shown the presence of chimeric genes arising from rearrangement of the mtDNA. The production of proteins encoded by the chimeric genes has been correlated with male-sterile phenotype, and it has been proposed that it affects mitochondrial function (12-14).The different nature of the genes involved in cytoplasmic male sterility plants reported so far suggests that mitochondrial impairment may be the common consequence of the different chimeric gene products. If this is true, then a mitochondrial dysfunction could also be produced by other means and male-sterile plants could be obtained. Recently, we have reported the production of male-sterile tobacco plants by expression of the unedited coding region of mitochondrial ...

Cultivation of the biofuel plant Jatropha (Jatropha curcas L.) has spread around the world because of its drought resistance, high seed oil content, and adaptability to di erent environmental conditions. Because of these attributes, Jatropha has the potential to be one of the main resources for next-generation biodiesel fuel. To improve the productivity of Jatropha biomass, it is important to understand the molecular functions of key Jatropha genes, and to modify various agronomic traits of Jatropha via molecular breeding. A reliable and e cient protocol for genetic transformation of Jatropha is a prerequisite for molecular biology research and breeding on this plant. Here, we developed a system in which the herbicide bispyribac sodium salt, which inhibits acetolactate synthase, was used as the selection agent, and a two-point-mutated acetolactate synthase gene (mALS) was used to confer resistance upon transformants. Application of this system signi cantly improved the e ciency of Agrobacterium tumefaciens-mediated stable transformation of the high-yielding elite Jatropha population, IP-2P. e bispyribac-mALS system was also successfully applied in the Agrobacterium rhizogenes-mediated hairy roots system, which allowed integration of a foreign gene and expression in Jatropha root tissues within 2 weeks. e new protocols described here are powerful tools not only for functional studies on endogenous genes, but also for the molecular breeding of Jatropha to develop elite varieties.

Flowers of tobacco transformed with an unedited copy of the mitochondrial atp9 gene sequence fused to the yeast coxIV mitochondrial targeting presequence, showed several anther abnormalities leading to pollen abortion. The gene was expressed in vegetative and reproductive tissues of the plant. Cytological analysis revealed that tapetum development was impaired. Mitochondria of the tapetum cells were severely affected showing characteristic signs of degeneration: loss of cristae and swelling. These mitochondrial modifications were correlated with the presence of the transcript and translated product of the 'unedited' atp9 and a significant decrease in oxygen consumption in non-photosynthetic tissues. The main effect of the unedited atp9 expression in transgenic plants was male sterility.