Late Uninucleate Microspores Research Articles

SlMKK4 is responsible for pollen development in tomato

The development of viable pollen is a determinant of male fertility and plays an essential role in the reproductive process of angiosperms. Mitogen-activated protein kinase (MAPK) cascades modulate diverse aspects of plant growth, but their involvement in post-meiotic pollen development is unclear. In this study, SlMKK4 was identified as a crucial regulator in overseeing pollen development in tomatoes (Solanum lycopersicum). Utilizing CRISPR-associated protein 9 to disrupt SlMKK4 resulted in an obvious decrease in pollen viability. The results of cell biology and transcriptomic analyses demonstrated that SlMKK4 specifically regulates auxin and sugar metabolism as well as signal transduction during post-meiotic pollen development. This is supported by the finding that protein–protein interaction assays and in vitro phosphorylation assays indicate that SlMKK4 serves as the upstream MAPKK for SlMPK20, which exhibits a distinct function in regulating the uninucleate (UN) to binucleate (BN) transition during microgametogenesis in tomatoes. Moreover, pollen from transgenic plants experienced significant arrest predominantly at the BN stage, accompanied by subcellular abnormalities manifesting during the late UN microspore phase. Furthermore, transcriptomic analyses indicated that SlMKK4 knockout remarkably downregulated the expression of numerous genes regulating auxin and sugar metabolism as well as signal transduction in anthers. Therefore, our findings suggest that SlMKK4 may serve as one of the upstream SlMAPKKs of SlMPK20 and also play a pivotal role in modulating post-meiotic pollen development in tomato plants.

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses

Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

Gametic embryogenesis and callogenesis in Isolated microspore culture of Jatropha curcas L. a recalcitrant bioenergy crop

Jatropha curcas is an undomesticated crop and its plantations did not meet commercial expectation due to absence of high yielding commercial line with desired agronomic characters. Earlier, breeding efforts did not pay much attention on yield and disease improving traits due to lack of desired genetic variability. Isolated microspore culture is one of the most widely used in vitro techniques to induce gametic embryogenesis with wide degree of genetic variability for the development of haploids and doubled haploids. In this study, an efficient isolated microspore culture system was established for different genotypes with the optimization of various factors that affect microspore embryogenesis. After 15–20 days of culture at 25 °C, tetrads, mid, early un-vacuolated and vacuolated late stage uninucleate microspores, which were preincubated at 4 °C for 7 days under shaking conditions, induced the formation of embryo like structures (ELSs) in a modified MS medium with 2.0 mg/l, 2,4-d, 0.1 mg/l kinetin, 300 mg/l casein hydrolysate, 1 g/l glutamine, 0.5 mg/l folic acid, 0.05 mg/l biotin and 5% sucrose. It is observed that the shock at 4 °C for 7 days, subsequently incubation of microspore cultures at 25 °C for 15 days and then at 15 °C for 10 days played a significant role in induction and accelerated creation of ELSs in three different genotypes. Microscopic analyses confirmed the different developmental stages of microspore embryogenesis including cell division and multicellular embryo like structures. Additionally, flow cytometric analyses verified the calli and ELSs were haploids in nature and strengthened that the origin was from microspores. This study supports the evidence of gametic embryogenesis in Jatropha microspore culture. Development of haploid and doubled haploids in Jatropha Microspore culture.

Overexpression of Two CCCH-type Zinc-Finger Protein Genes Leads to Pollen Abortion in Brassica campestris ssp. chinensis.

The pollen grains produced by flowering plants are vital for sexual reproduction. Previous studies have shown that two CCCH-type zinc-finger protein genes in Brassica campestris, BcMF30a and BcMF30c, are involved in pollen development. Due to their possible functional redundancy, gain-of-function analysis is helpful to reveal their respective biological functions. Here, we found that the phenotypes of BcMF30a and BcMF30c overexpression transgenic plants driven by their native promoters were similar, suggesting their functional redundancy. The results showed that the vegetative growth was not affected in both transgenic plants, but male fertility was reduced. Further analysis found that the abortion of transgenic pollen was caused by the degradation of pollen contents from the late uninucleate microspore stage. Subcellular localization analysis demonstrated that BcMF30a and BcMF30c could localize in cytoplasmic foci. Combined with the studies of other CCCH-type genes, we speculated that the overexpression of these genes can induce the continuous assembly of abnormal cytoplasmic foci, thus resulting in defective plant growth and development, which, in this study, led to pollen abortion. Both the overexpression and knockout of BcMF30a and BcMF30c lead to abnormal pollen development, indicating that the appropriate expression levels of these two genes are critical for the maintenance of normal pollen development.

Induction of Microspore Embryogenesis of Eggplant (<i>Solanum melongena</i> L.) ‘Gelatik’

The haploid or double haploid plant of eggplants could be produced from microspore culture (embryogenesis of microspores). In the breeding programs, microspore can be developed into an embryo directly after exposure to stress treatment during cultured. Stress (temperature and starvation medium) is an important factor in the induction of embryogenesis microspore. This study aims to induced embryogenic microspores from eggplant CV. Gelatik. The stage late-uninucleate microspore (Vacuolate Microspore/VM) and early binucleate (Young Bicellular Pollen/YBP) are the suitable stages to induce multinucleate structure. There are 3 methods used in this research; 1) Determination of the stage development of microspore based on flower buds length and anther length. 2) Induction of embryogenic microspore on the pre-treatment and starvation medium. 3) After giving pre-treatment for 4 days, micropores were transferred to culture medium A2 at 28oC in dark conditions to induce the multicellular structures. This study reported that 50-68.51% of the VM+YBP stage obtained in the range of flower bud lengths of 10-17 mm, and 5.0-6.9 mm, the range of anther length containing VM+YBP of 50-77.48%. The pre-treatment heat shock at 33oC in the medium B for 2 days, produced embryogenic microspores with a high percentage, that is about 50.19%, while microspores at 25oC and 4oC respectively 46.17% and 49.28%. Pre-treatment for 4 days at 4 oC, 25 oC, and 33oC with the percentage of embryogenic microspores apiece 32.87%, 27.45%, and 37.34%. The multicellular (starlike) structure begins forming on the fifth day of incubation in culture medium (A2) after pre-treatment in B medium at 33oC.

Transcriptome Analysis Reveals Important Transcription Factor Families and Reproductive Biological Processes of Flower Development in Celery (Apium graveolens L.)

There are few reports on the reproductive biology of celery, which produces small flowers in a long flowering period. Anther development was analyzed by paraffin sectioning and related genes were examined by transcriptome sequencing and qPCR. The development process was divided into nine stages based on the significant changes in the cell and tissue morphologies. These stages included: archesporial stage, sporogenous cell stage, microspore mother cell stage, dyad and tetrad stage, mononuclear microspore stage, late uninucleate microspore stage, binuclear cell stage, mature pollen stage, and dehiscence stage. A total of 1074 differentially expressed genes were identified by transcriptome sequencing in the early flower bud, middle flower bud, and early flowering period. Functional annotation indicated that these genes were involved in physiological and biochemical processes such as ribosomes metabolism, sugar metabolism, and amino acid metabolism. Transcription factors such as C2H2, AP2/ERF, bZIP, WRKY, and MYB played key regulatory roles in anther development and had different regulatory capabilities at various stages. The expression patterns based on qPCR and transcriptome data of the selected transcription factor genes showed consistency, suggesting that these genes played an important role in different flower development stages. These results provide a theoretical basis for molecular breeding of new celery varieties with pollen abortion. Furthermore, they have enriched research on the reproductive biology of celery and the Apiaceae family.

The anther-specific CYP704B is potentially responsible for MSG26 male sterility in barley.

Plant male sterility is a valuable trait in breeding and hybrid seed production. The barley male-sterility gene msg26 was mapped to a 0.02-cM region that anchors to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence (CAPS) markers, SP1M14 and SP1M49. The barley gene HORVU4Hr1G074840, which encodes a putative cytochrome P450 CYP704B protein, appears to be a strong candidate for the MSG26 trait. Barley (Hordeum vulgare L.) is an important cereal crop worldwide. Traditional breeding in barley is time-consuming and labor-intensive. The use of male-sterile genotypes may significantly improve the efficacy of hybrid breeding and seed production. The barley accession 'GSHO745' is a spontaneous male-sterile mutant from the barley variety, 'Unitan'. The male sterility in 'GSHO745' is controlled by the recessive gene, msg26 (originally named as ms-u). We revealed that the barley plants homozygous for msg26 proceeded normally through Meiosis II until the tetrad stage, but became fully defective in the late uninucleate microspores and developed pollen-less anthers. Using seven barley F2 populations, we mapped MSG26 to a 0.02-cM region that anchored to a 506-kb low-quality assembly between two cleaved amplified polymorphic sequence markers, SP1M14 and SP1M49. The HORVU4Hr1G074840 gene that encodes a putative cytochrome P450 protein (CYP704B) was identified as the most plausible candidate for MSG26. First, HORVU4Hr1G074840 is located in a collinear region of the rice CYP704B2 and the maize CYP704B1. Both of these genes are essential for male gamete production. Second, the male-sterile allele of HORVU4Hr1G074840 in GSHO745 contained a 4-bp deletion in the last exon. The resulting frame shift causes a Gly436Gln substitution, scrambles the sequence of the remainder of the protein, and forms a new termination site at the 70th triplet of the shifted reading frame. We thus called the variant protein CYP704B:p.G436Qfs*70. Third, the barley HORVU4Hr1G074840 gene was specifically expressed in anthers. Altogether, HORVU4Hr1G074840 represents a strong candidate for MSG26 in barley.

Modeling callus induction and regeneration in an anther culture of tomato (Lycopersicon esculentum L.) using image processing and artificial neural network method.

Doubled haploids, subsequent to haploid induction, have wide range of applications in basic and applied plant studies. Various parameters can affect the efficiency of haploid induction through an anther culture of tomato. The hybrid system of image processing-artificial neural network (ANN) was used to better understand callus induction and regeneration in an anther culture of tomato. The effect of parameters such as plant genotype, the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) plant growth regulators, the concentration of gum arabic (GA) additive, the cold pretreatment duration, and flower length on callus induction percentage and number of regenerated calli in an anther culture of tomato were studied using multiple linear regression (MLR) and ANN models. The precise flower bud length was measured using an image processing technique. The 4',6-diamidino-2-phenylindole (DAPI) analysis showed that the flowers with 5-6.9mm length had the highest percentage of the mid- to late-uninucleate microspore stage. The best ANN model for both callus induction percentage and number of regenerated calli was a model with one hidden layer, 12-15 neurons in the first hidden layer, Levenberg-Marquardt learning algorithm, and Tan-Sigmoid transfer function in hidden layer, based on the root mean square error (RMSE), mean absolute error (MAE), and coefficient of determination (R2) statistics. The scatter plot of measured values versus the predicted values showed the superiority of the ANN to MLR model to predict the callus induction percentage in an anther culture of tomato. The sensitivity analysis of MLR and ANN models revealed the plant genotype and 2,4-D concentration as the most important factors affecting both callus induction percentage and number of regenerated calli. Since tomato is a recalcitrant plant to androgenesis-based pathway of haploid induction, therefore the results of the present study can be helpful to develop an efficient haploid induction protocol in tomato through an anther culture pathway.

SELECTION OF SOYBEAN CULTIVARS: PREPARATION THE PLANT BREEDING WITH MICROSPORE CULTURE METHOD

Microspore culture method can be used as plant breeding program. The preparation of cultivars selection is an important step. The research starts with cultivated five cultivars of soybean i.e: Argomulyo, Grobogan, Wilis, Anjasmoro and Black Malika. The appearence of soybean plant was observed until flowering. Selection of plants based on: sum of flower bud every plant, anther midline, total and diameter of microspore every flower bud was measured by the ‘Optilab’ software. The development of microspore done with grouping of flower bud according long 2.02.5 mm, 2.6-3.0 mm, 3.1- 3.6 mm and 3.7-4.1 mm for chooses flower bud with the most late uninucleate microspore stadium. Result of the research shows that long of flower bud 2.6-3.6 mm contain 1847-2010 late uninucleate microspores, diameter 20 µm for 5 cultivars can be used for material of microspore culture. Anjasmoro cultivar, tall of plant gain 68 cm, sum of rame 7-9, anther midline 354.67±59.67 µm, number of microspores each flower bud 2003±216. Result of responsive qulity test with anther incubation on 340C temperature for 4 days represent the most of total viable microspore, 3.371±45 on Anjasmoro cultivar. Plant breeding by Anjasmoro cultivar is the most appropriate for microspore culture treatment.

Effect of the 1BL.1RS wheat-rye translocation on the androgenic response in spring bread wheat

The study was conducted to investigate the effect of the 1BL.1RS wheat-rye-translocation on the androgenic response in spring bread wheat. Therefore, four bread wheat cultivars carrying the translocation, four Greek and three Canadian bread wheat cultivars without the translocation were used. An equal number of anthers from each cultivar, containing microspores in the mid (MU) to late uninucleate (LU) microspore developmental stage, were cultured after cold pre-treatment for seven days at 4°C. W14, 190-2 and the basic MS were used as induction, regeneration, and rooting media respectively. The best androgenic response was recorded in two cultivars carrying the translocation. Only two cultivars lacking the translocation responded to anther culture. It is concluded that the positive effect of the 1BL.1RS translocation on anther culture response of bread wheat cultivars cannot be attributed entirely to its presence because the genetic background of the cultivars carrying the translocation could be also important.

A cytological indicator allows rapid assessment of microspore maturity, leading to improved in vitro anther response in Indica rice (Oryza sativa L.)

The inadequate response of Indica rice (Oryza sativa L.) to anther culture hinders the utility of this technique for rice crop improvement. This study focused on intensifying the in vitro anther response of selected varieties from Sri Lankan germplasm by precisely targeting the most effective maturity stage of microspores for culture. Analysis of five varieties responsive to anther culture (“At 303”, “Bw 272-6B”, “Dahanala”, “Dikwee”, and “Herath Banda”), revealed that the microspore development stage at the time of culture was crucial for fine-tuning the callus induction response. The external guide used to determine microspore maturity which was the internode distance between the last two leaves of the panicle, could not be applied equally for all rice varieties. Anther response could be improved by, at least, four- to sixfold by precisely targeting anthers with late uninucleate microspores for culture. Close correlation was demonstrated between the stage of nuclear division in the microspores and the degree of starch accumulation. Therefore, the degree of starch deposition in the microspores can be used as an effective cytological marker, which can be applied more rapidly and reliably to assess microspore maturity, in place of the internode distance.

Micropropagation and Androgenesis in coconut: an assessment of Sri Lankan implication

The coconut palm is an important cash crop mostly in the sub-tropical countries which provides almost all the necessities of life to the mankind. Genetic improvement for increased productivity is a prioritized research area of coconut palm. In vitro cloning of elite palms via somatic embryogenesis seems to be a promising method due to its potential for massive propagation. Based on the Sri Lankan experience, this paper summarizes the notable achievements towards developing a reliable protocol for clonal propagation of coconut and also dihaploid plant production. The newel explants described by Coconut Research Institute Sri Lanka (CRISL), unfertilized ovaries dissected from immature inflorescences collected from -4 stage (inflorescence to be opened in 4 months time), is a somatic tissue which causes minimal damage to the mother palm while collecting. Limited number of female flowers in an inflorescence which limits large scale culturing could overcome by callus multiplication phase to produce higher number of plants. Somatic embryogenesis and germination of somatic embryos was achieved by reduction of auxin concentration in the culture medium followed by an addition of cytokinin. AINTEGUMENTA-LIKE (ANT-like) gene isolated from coconut genome which shows higher conserved domain sequence similarities with BABYBOOM gene will assist development of suitable molecular markers to assess the morphogenic potential of in vitro cultured coconut tissues and callus. Successful plant regeneration from cultured anthers was reported by culturing anthers at three weeks before splitting (3-WBS) inflorescence stage which contains late uninucleate microspores. Heat pretreatment of anthers for six days at 38°C induced androgenesis in coconut upon culturing in 2, 4-D and 1-naphthyl acetic acid (NAA) added medium. Current clonal propagation protocol is efficient when responsive genotype is in use.Further improvement to current protocol will enable commercially viable protocol for coconut micropropagation.

MORFOLOGI BUNGA YANG SESUAI BAGI KULTUR MIKROSPORA PADA TANAMAN JARAK PAGAR (Jatropha curcas L.)

An experiment aimed in gaining information on morphological characteristics of Jatropha curcas (L.) containing high frequency of uninucleate microspores has been done. This was important due to the successful application of experiment in haploid technique through anther or microspore culture is strongly determined by the availability of information from basic research such as appropriate morphology of the flowers. This scientific article, in our knowledge, was an early explanation of the initial steps of whole procedures in the microspore culture of Jatropha curcas (L.). Results of the research showed that the morphological features of flower that contained high frequency of mid- and late uninucleate microspores was that the flowers must be in the range of 2 to 3 mm in length. Such flowers contained yellowish anthers.Key words: Flowers length, mid- and late-uninucleate, morphology, physic nut, microspore

DETERMINATION OF SOYBEAN (Glycine max L.[Merrill]) MICROSPORES DEVELOPMENT STAGE BASED ON THE LENGTH OF FLOWER BUDS

The number of published reports of flower buds, anthers and on microspore culture of horticultural plants, especially in soybean is very low. The purpose of this research is to determine soybean microspores development stage based on the length of flower buds. Five local cultivars such as Argomulyo, Grobogan,Wilis, Anjasmoro and the black soybean Malika were used. Flower buds were the main material for microspore culture. Anjasmoro cultivar produces the highest number of flower bud. Anjasmoro soybean cultivar at third stage development, 3.1-3.6 mm length, was selected for microspore culture, based on plant height, length of anther, total of microspore per bud. Anjasmoro cultivar has the most late-uninucleate microspore and the total of shed of Anjasmoro microspore in the B medium with 340C for 4 day is 3820. Results revealed that Anjasmoro soybean cultivars had 354.67±59.67 μm length of anther, 2003±216 microspore per bud and the most mid and late-uninucleat microspore on 2.6-3.6 mm length flower bud. Anjasmoro soybean cultivar can be used for microspore culture.

Microspore-derived Embryo Formation and Morphological Changes during the Isolated Microspore Culture of Radish (Raphanus sativus L.)

Raphanus sativus L. cv. Taebaek, a efficiently microspore-derived embryo (MDE)-forming cultivar, and ‘Chungwoon’, a non-MDE-forming cultivar were selected as donor plants for isolated microspore culture. Radish flower bud of 2.0 (small, S), 4.0 (medium, M), and 6.0 (large, L) ± 0.5 ㎜ in length were isolated to determine the temporal relationship between flower bud size and MED yield. Anatomical observations revealed no difference in the structure of the flower buds between the two cultivars. In both cultivars, the stigmas were much longer than the floral leaf in M-sized flower buds. The MDE yields for ‘Taebaek’ per petri dish were 6.6 and 1.3 for M- and L-sized of flower buds, respectively, but MDE formation was not induced in the S flower buds. On the other hand, ‘Chungwoon’ failed to form MDEs in all flower buds. The microspore density of ‘Taebaek’ was 1.3 times more than that of ‘Chungwoon’ for M sized flower buds. Of the M-sized buds from ‘Taebaek’ and ‘Chungwoon’, 92.1 and 81.6%, respectively, were in the late uninucleate microspore stage, which is characterized by the highest frequency of MDE formation. Anatomical observations of MDE formation revealed that the microspores were able to divide to form a primordium from which cell division took place continuously in the ‘Teabeak’ cultivar. However, the microspores of ‘Chungwoon’ failed to progress beyond the primodium stage, resulting in lack of MDE formation. By contrast, after the formation of the primordium, various developmental stages of embyos from microspore were observed in the ‘Taebaek’ cultivar. These results can be used to determine MDE forming potentials of radish cultivars.

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana.

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy. In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat. Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively. This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.

Initiation and development of microspore embryogenesis and plant regeneration of Brassica nigra

Brassica nigra is generally regarded as a recalcitrant species for microspore culture among Brassica crops. Conditions for reliable induction of microspore embryogenesis of B. nigra were studied in this context. Flower bud length and microspore developmental stage were correlated with further embryogenesis. The optimal bud size range was 2.0–2.5 mm for the highest proportion of totipotent, late uninucleate microspore and the highest frequency of microspore embryogenesis. Treatment of a short heat shock by incubating the microspore culture at 32°C for 24 h was suitable for the microspore survival, sustained cell divisions, and further induced embryogenesis. Subsequently, the use of NLN medium with the addition of 13% sucrose and 0.1% activated charcoal (AC) provided the optimal conditions for the development of microspore-derived embryos (MDEs). The early cotyledonary (EC) stage embryos cultured on MS medium fortified with 4.6 μM zeatin (ZT) and 0.12 μM indole-3-acetic acid (IAA) resulted in the most efficient rates of plantlet regeneration. The ploidy levels of regenerated plants of B. nigra were determined by flow cytometry, revealing that 50.6% were diploid. The results enable the advancement of breeding programs and genetic studies in B. nigra.

Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development.

Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu(2+) and Fe(2+)) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4',6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in cassava. This is the first ever detailed report of microspore reprogramming at the tetrad stage and the first report of microspore embryogenesis induction in cassava with detailed evidence.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens

Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-009-0696-0) contains supplementary material, which is available to authorized users.