Embryogenesis and plant regeneration of hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) through isolated microspore culture

We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with an optimal plating density of 8 × 10⁴–10 × 10⁴/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when 10 × 10⁴ microspores were grown on an individual plate.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Improvement of isolated microspore culture of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.     

An improved method for isolating RNA from dehydrated and nondehydrated chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plant tissues

High-quality RNA is important in studying gene expression. This report describes an improved method for isolating intact purified RNA from dehydrated organs of chili pepper plants. Common RNA extraction protocols have produced poor yields because dehydrated leaves accumulate polysaccharides and RNases. Our protocol is based on a guanidine thiocyanate extraction combined with additional purification steps using butanol and the ionic detergent CTAB (cetyltrimethylammonium bromide). Using this protocol, RNA yields ranged from 40–70 μg of total RNA per 200 mg of fresh tissue. This method can be adapted to large-scale isolations, allowing the recovery of larger amounts of intact RNA (up to 250 μg per gram of fresh tissue).     

<Emphasis Type="Italic">In vitro</Emphasis> fruiting and seed set of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. CV. Sweet banana

Summary Plantlets of Capsicum annuum L. ev. Sweet Banana regenerated via somatic embryogenesis from immature zygotic embryos were capable of producing flower, fruit, and seed when cultured in small tissue culture containers. In vitro floral buds were first formed on plantlets that grew on plantlet development medium [agar-gelled Murashige and Skoog (MS) basal medium containing 1 mgl⁻¹ (5.3 μM) α-naphthaleneacetic acid (NAA)] in a growth room at 22°C and continuous illumination. However, floral buds rarely developed further into mature flowers. This problem was overcome using the vented autoclavable plant tissue culture containers. In vitro fruit formation and ripening was observed when liquid half-strength MS basal medium supplemented with 5 μg ml⁻¹ silver thiosulfate, 1 mg l⁻¹ (5.3 μM) NAA, and 3% sucrose was added to the surface of the plantlet development medium. Hand-pollination improved fruit set. Further research in needed to determine why the pepper seeds formed in vitro failed to germinate.     

A <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>-induced esterase gene of nonclimacteric pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) fruit during ripening plays a role in resistance against fungal infection

Ripe fruits of pepper (Capsicum annuum) are resistant to the anthracnose fungus, Colletotrichum gloeosporioides, whereas unripe-mature fruits are susceptible. A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between the ripe fruit of pepper and C. gloeosporioides was previously cloned. Deduced amino acid sequence of PepEST cDNA showed homology to both esterases and lipases, and contained -HGGGF- and -GXSXG- motifs and a catalytic triad. Inhibition of PepEST activity by a specific inhibitor of serine hydrolase demonstrated that a serine residue is critical for the enzyme activity. Expression of PepEST gene was fruit-specific in response to C. gloeosporioides inoculation, and up-regulated by wounding or jasmonic acid treatment during ripening. PepEST mRNA and protein was differentially accumulated in ripe vs. unripe fruit from 24 h after inoculation when C. gloeosporioides isinvading into fruits. Immunochemical examination revealed that PepEST accumulation was localized inepidermal and cortical cell layers in infected ripe fruit, but rarely even in epidermal cells in infected unripe one. Over-expression of PepEST in transgenic Arabidopsis plants caused restriction of Alternaria brassicicola colonization by inhibition of spore production, resulting in enhanced resistance against A.brassicicola. These results suggest that PepEST is involved in the resistance of ripe fruit against C.gloeosporioides infection.^†These authors contributed equally to the work     

Suitability of the antioxidative system as marker of magnesium deficiencyin <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. plants under controlled conditions

To explore the viability of using enzyme activities and their substrates as an alternative tool for the determination of mineral (i.e., Mg) critical values, a detailed characterization of the response of the antioxidative system of Capsicum annuum L. leaves under Mg deficiency was carried out. The response of each selected enzyme activity and substrate [i.e., superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR); ascorbate and glutathione pool; protein and chlorophyll concentration] was subjected to mathematical modelling in order to calculate Mg critical values (x_c). Our x_c values ranged from 0.70 to 0.14%, on a dry weight (DW) basis, for GR activity and total glutathione concentration, respectively. Our results suggest that, under Mg deficiency, cells enhance their antioxidative defence system by initially increasing their SOD and GR activities. Subsequently, higher GSH/GSSG ratios were observed, probably due to a greater increase in GR activity (x_c = 0.70% DW) than in total glutathione concentration (x_c = 0.14% DW). In contrast, x_c values for total ascorbate concentrations (x_c = 0.29% DW) were higher than those for DHAR activities (x_c = 0.19% DW). In an attempt to study the limitations regarding the utilization of these enzymes and substrates as markers of Mg critical values in pepper, the x_c values here obtained were compared to those based on growth parameters that have been reported in the literature. Overall, the results indicate that some enzymes and substrates, such as total ascorbate concentration, 1/protein ratio, and DHAR activity, might be suitable markers for the determination of Mg critical values in pepper plants under controlled conditions.     

A CAPS marker associated with the partial restoration of cytoplasmic male sterility in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS), an economically important trait for hybrid seed production in many crops, is a maternally inherited trait in which a plant fails to produce functional anthers, pollen grains, or male gametes. It has long been reported that the restoration of CMS in chili pepper is controlled by a major nuclear gene termed restorer-of-fertility (Rf), along with several modifiers and some environmental factors. In this study, we identified the partial restoration (pr) locus related to the fertility restoration of CMS, demonstrated the inheritance of the trait, and developed a CAPS marker closely linked to the locus. The partially restored plant had normal anthers that produced a mix of normal and aborted pollen grains that stuck tightly to the anther wall, even after dehiscence. This trait was expressed only when the pepper plant had the sterile (S) cytoplasm and homozygous recessive pr alleles. A total of 768 AFLP primer combinations were screened, and bulked segregant analysis (BSA) was performed by preparing two pools of eight Pr/Pr (fully fertile) and eight pr/pr (partially fertile) plants, respectively, selected from the 87 individuals of the F₂ segregating population. Of the eight Pr-linked AFLP markers that were identified, E-AGC/M-GCA₁₂₂ and E-TCT/M-CCG₁₁₆ were the closest to the locus, estimated at about 1.8 cM in genetic distance. E-AGC/M-GCA₁₂₂ was converted into a CAPS marker, PR-CAPS, based on the sequences of the internal and flanking regions of the AFLP fragment. This PR-CAPS marker could be useful in selecting fully fertile lines (Pr/Pr) and eliminating partially fertile (pr/pr) and potential (Pr/pr) lines in segregant populations during the development of new inbred restorer lines.     

Induced androgenesis of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> L.

The aim of the research was to make a preliminary determination of the effectiveness of the induction of haploids in Capsicum frutescens L. In order to induce androgenesis red and yellow fruit forms of species were used, each bred by the researchers on their own. The experiment was performed in October. Anther cultures were conducted according to a modified method developed by Dumas et al. (1981) for C. annuum L. The anthers were laid on CP medium containing 0.01 mg dm⁻³ 2.4-D and 0.01 mg dm⁻³ kinetin, with the addition of 0.5 g dm⁻³ of activated carbon and 5 mg×dm⁻³ of silver nitrate, solidified with 8 g dm⁻³ of agar. The cultures were incubated in the dark at 35 deg C for 8 days. Next they were transferred to 25 deg C under a 12-hour photoperiod. After 14 days of induction, anthers were transferred to R₁ medium supplemented with 0.1 mg dm⁻³ kinetin. Obtained embryos were subsequently transplanted onto V₃ hormone-free medium and well growing plants were planted in greenhouses. The efficiency of androgenesis for both C. frutescens L. forms was relatively low and it did not exceed 5%. The ploidy level of the resulting plants was determined by flow-cytometric analysis. The regenerants consisted of about equal numbers of haploids and diploids. Additionally, among plants regenerated from anthers of yellow fruit forms, two mixoploids were observed.     

Genetic Variability in Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) Estimated by Morphological Data and Amplified Fragment Length Polymorphism Markers

Data on genetic similarity among crop cultivars is of vital importance for the plant breeder. The objectives of this study were to group pepper (Capsicum annuum L.) genotypes into clusters according to their distances as estimated by morphological traits and amplified fragment length polymorphism (AFLP) markers and to assess the relationships between the two. Thirty-nine pepper genotypes obtained from different countries were grown in the greenhouse at University of the Free State, South Africa, during 2001 and 2002 in a randomized complete block design with three replications. A total of 20 different morphological traits were measured and six AFLP primer pairs were used to estimate pairwise genetic distances. Both datasets showed high genetic distances among the different genotypes, indicating high genetic diversity among them. The mean genetic distance among Ethiopian pungent elongated-fruit genotypes, was lower than that between them and the introduced ones. Morphological and AFLP distance estimations generally clustered together genotypes with similar fruit sizes. Significant, positive correlation was observed between morphological and AFLP diversity estimations. The narrow genetic basis among the Ethiopian pungent elongated-fruit cultivars suggests that the pepper breeding program of Ethiopia should focus on enriching its germplasm through local collection and introductions from other parts of the world.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.     

Pepper (<Emphasis Type="Italic">capsicum annuum</Emphasis> L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot

Summary Three auxin-type herbicides, namely 2.4-dichlorophenoxyacetic acid (2,4-D), (4-chlorophenoxy)acetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and quinolinecarboxylic acid (quinclorac) induced direct somatic embryogenesis in seed-derived zygotic embryo explants of sweet pepper (Capsicum annuum L.) when added to Murashige and Skoog medium with 200 mM sucrose. Optimum concentrations for embryogenesis induction were 0.40–0.45 mM and 1.15–1.30 μM for 2.4-D and centrophenoxine, respectively (in the presence of 5.0 gl⁻¹ activated charcoal), or 40 μM for quinclorac (in medium without activated charcoal). Somatic embryos emerged from the epidermal and subepidermal tissues and developed on the surface of the explant. Centrophenoxine- or 2.4-D-mediated embryogenesis was accomplished from 95% of the explants in about 3 wk and, on average, six embryos were formed per explant. Induction efficieney was lower for quinelorac. Centrophenoxine-mediated embryognesis was possible in 10 pepper cultivars, the extent of the reponse-being genotype-dependent. embryos detached from the explant and transplanted onto a growth regulator-free medium germinated; however, the recovered regenerants were without a shoot, and some of them bore a single deformed cotyledon while others had no cotyledons. Regenerants lacking a shoot were generated irrespective of the auxin type applied and across all responsive genotypes investigated. Absence of a shoot, resulting from a failure in the establishment of a normal functioning apical shoot meristem, was the principal developmental disorder that precluded regeneration of normal plants via direct somatic embryogenesis. Since stem cells of the shoot meristem become established in globular and heart-stage embryos, we deduce that the absence of a shoot in germinating embryos could orginate from deviant differentiation at these early stages of embryogeny.     

Regeneration and<Emphasis Type="Italic"> Agrobacterium-</Emphasis>mediated transformation of hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz     

Ectopic expression of <Emphasis Type="Italic">Capsicum</Emphasis>-specific cell wall protein <Emphasis Type="Italic">Capsicum annuum</Emphasis> senescence-delaying 1 (CaSD1) delays senescence and induces trichome formation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of gypsophila (<Emphasis Type="Italic">Gypsophila paniculata</Emphasis> L.)

As a major contributor to the flower market, Gypsophila paniculata is an important target for the breeding of new varieties. However, gypsophila breeding is strongly hampered by the sterility of this species’ genotypes and the lack of a genetic-transformation procedure for this genus. Here we describe the establishment of a transformation procedure for gypsophila (Gypsophila paniculata L.) based on Agrobacterium inoculation of highly regenerative stem segments. The transformation procedure employs stem explants derived from GA₃-pretreated mother plants and a two-step selection scheme. The GA₃ treatment was crucial for obtaining high gene-transfer frequencies (75–90% GUS-expressing explants out of total inoculated explants), as shown using three different gypsophila varieties. An overall transformation efficiency of five GUS-expressing shoots per 100 stem explants was demonstrated for cv. Arbel. The applicability of the transformation system to gypsophila was further reinforced by the generation of transgenic plants expressing Agrobacterium rhizogenes rolC driven by a CaMV 35S promoter. Transgenic gypsophila plantlets exhibited extensive rooting and branching, traits that could be beneficial to the ornamental industry.