Formation Of Embryo-like Structures Research Articles

P–195 The influence of hormones and initial cell number on the size of self-assembled embryo-like structures

Abstract Study question Do hormonal treatments and initial cell number influence the formation of embryo-like structures (ELS) during their development in regard to size? Summary answer The chosen initial cell number for ELS-assembly seems to influence the ELS size only until day 4, while hormones affect embryo size throughout their development. What is known already The initial cell number is an important parameter for the development of ELS, which might help to better understand how embryos regulate their size. Previous studies on differently sized natural murine embryos revealed that an initial difference in size at the early stage is compensated until E6.75. Normal-size embryos experience an increased mitotic activity before E6.75, whereas larger sized embryos show an increased apoptotic activity, indicating an important control point of cell turnover by adapting mitotic activity and cell survival. Embryo development is strongly dependent on appropriate β-estradiol and progesterone levels. Study design, size, duration The first set of experiments interrogated the influence of initial cell number (two conditions) on the size of formed ELS during the first 3 days (D1–3). The second set included two different hormonal treatments and the two conditions of initial cell number (the same as in the first experiments) generating four different groups. For each day one Aggrewell (generating 1200 ELS/well) per condition was harvested. Experiments were repeated at least three times. Participants/materials, setting, methods ELS are generated by self-assembly in microwell-chamber plates combining embryonic stem cells, trophoblast stem cells and extraembryonic endoderm stem cells. Cells were cultured with and without addition of β-estradiol and progesterone, starting with different initial cell numbers (106 vs. 42 cells/ELS). ELS were harvested, stained, and at least 40 randomly picked ELS per condition were measured and statistically analyzed with Two-way ANOVA and Tukey’s multiple comparison test. Results show the average area ± SD. Main results and the role of chance The results show a continuous increase in the size of ELS during the first three days of cultivation, with significant lower values (on D1-D3) when ELS were assembled from 42 initial cells (D1: 224.1±87.7 μm²; D3: 674.0± 84.4 μm²) compared to ELS formed with 106 initial cells (D1: 467.1±224.1 μm²; D3: 1275.0±348.0 μm²). Onward on the course of self-assembly, ELS with 42 initial cells were still smaller on D4 (1465.7±657.6 μm²) compared to ELS formed with 106 initial cells (2028.6±522.4 μm²). However, these differences could not be measured on D5 (106 initial cells: 1892.2±603.7 μm²; 42 initial cells: 1855±448.5 μm²), D6 (106 initial cells: 2143.3±622.1 μm²; 42 initial cells: 1788.4±585.5 μm²) and D7 (106 initial cells: 2146.7±628.1 μm²; 42 initial cells: 2319.5±778.8 μm²). Differences between the conditions with and without hormonal treatments (HT) could also be detected especially when ELS were generated with 42 cells: on D4 ELS with HT (1730.4±852.4 μm²) were significantly larger than without hormones (1201.2±462.9 μm²). In contrast, on D7 HT influenced the size of ELS distinctly depending on the initial cell number (42 cells: 1989.2±558.3 μm² with HT vs. 2649.7±999.4 μm² without HT; 106 cells: 2334.9±770.2μm² with HT vs. 1958.6±486.1 μm² without HT). Limitations, reasons for caution An even cell distribution is crucial for reproducible ELS-formation. Unfortunately, the used techniques for cell seeding led to an uneven distribution within the microwells. Moreover, different orientation of ELS during the size assessment might be an additional reason for the high variance of ELS size within one condition. Wider implications of the findings: Even if the results seem to be in accordance with the observations made with natural embryos regarding compensation of size until E6.75, additional experiments need to be conducted. Further investigations should be carried out by testing different culture formats to obtain a more even cell distribution during the cultivation. Trial registration number Not applicable

The Effect of Zinc, Copper, and Silver Ions on Oat (Avena sativa L.) Androgenesis.

Oat (Avena sativa L.) cultivars ‘Bingo’ and ‘Chwat’ were used to compare the embryogenesis competence of another culture. Despite the embryo-like structures obtained from both tested cultivars, only ‘Chwat’ produced green plantlets, which confirmed the cultivar dependency. ‘Chwat’ produced the highest number of embryo-like structures and green plantlets (0.7/100 anthers and 0.1/100 anthers, respectively). The embryo-like structure formation also depended on cold pretreatment combined with Cu2+, Zn2+, or Ag+ ion supplementation, which was applied during the tiller pretreatment or added to the induction media. The highest number of embryo-like structures (2.1/100 anthers) were observed on anthers derived from the tillers kept in a 50% Hoagland medium with the addition of 10 µM of CuSO4. In turn, the induction media supplemented with the ions Cu2+, Zn2+, or Ag+ increased neither the number of embryo-like structures nor the green plantlet production compared to the control conditions. However, such ion applications turned out to be most effective when the induction medium was enriched with 25 µM of AgNO3 and left to obtain the highest number of embryo-like structures and green plantlets (0.8/100 anthers and 0.2/100 anthers, respectively). Therefore, more attention should be paid to the possibilities of adjusting the media nutrient composition, as this may be the only way to significantly increase the efficiency of this method.

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.

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm3 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm3 kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

Effects of thidiazuron on in vitro morphogenic response of Rhododendron sichotense Pojark. and Rhododendron catawbiense cv. Grandiflorum leaf explants

Thidiazuron (TDZ)-induced shoot organogenesis from in vitro-derived leaf explants of winter-hardy Rhododendron sichotense Pojark. and Rhododendron catawbiense cv. Grandiflorum was studied using two TDZ application methods: adding various concentrations to Anderson nutrient medium and a liquid pulse treatment. The highest frequency of shoot regeneration (93%) and maximum number of shoots per explant (24.6) from R. sichotense was obtained after a two-stage culture procedure that included exposure of leaf explants to 1.0 μМ TDZ followed by TDZ-free cultivation. TDZ at concentrations of 0.5 and 1.0 μМ was effective for leaf explants of R. catawbiense cv. Grandiflorum, inducing 60 and 85% regeneration frequencies, and 36.0 and 25.0 shoots per explant, respectively. The liquid-pulse treatment of leaf explants with 30 μM TDZ for 4 h was effective only for R. catawbiense cv. Grandiflorum. Histological analysis revealed that epidermal cells of the adaxial side at the leaf-blade base were involved in de novo shoot regeneration in both genotypes. However, the adventitious shoot buds of R. sichotense developed from protuberances, whereas shoot organogenesis of R. catawbiense cv. Grandiflorum occurred through the formation of embryo-like structures. Optimum root formation for both genotypes was achieved using indole-3-butyric acid liquid-pulse treatment of microshoots followed by ex vitro rooting. An efficient system was developed for multiplication of R. sichotense and R. catawbiense cv. Grandiflorum, which are cold-hardy genotypes promising for future genetic transformation.

Effects of Exogenous Application of Plant Growth Regulators on Growth, Yield, and In Vitro Gynogenesis in Cucumber

The effects of exogenous application of plant growth regulators (PGRs) were evaluated on both field performance and in vitro gynogenesis of ‘Chai Lai’ and ‘Big C’ cucumber (Cucumis sativus L.). Plants were sprayed with two concentrations of N6-furfuryladenine [kinetin (KIN); 2 and 20 ppm], 2,3,5-triiodobenzoic acid (TIBA; 1 and 10 ppm), naphthaleneacetic acid (NAA; 10 and 100 ppm), abscisic acid (ABA; 2 and 20 ppm), thidiazuron (TDZ; 1 and 10 ppm), and maleic hydrazide (MH; 10 and 100 ppm) to assess their effects on vegetative growth and floral and yield related traits in the Winter of 2013 and in the Summer of 2014 compared with distilled water control. Meanwhile, the effects of two PGRs (KIN and TIBA) on cucumber gynogenesis were also investigated in vitro. Growth parameters and floral and yield-related traits were significantly affected by the various PGRs in both cultivars during both seasons. In both cultivars, the highest yield was obtained with the application of 10 ppm NAA during the Winter of 2013 (1.5- to 1.8-fold over control) and with 1 ppm TIBA during the Summer of 2014 (2.1- to 2.2-fold over control). With regard to the ovary culture response, exogenous application of KIN and TIBA on floral buds tended to enhance callus formation in ‘Chai Lai’ cultured on I7 medium, whereas no effect was observed in ‘Big C’. The embryo-like structure (ELS) formation efficiencies also tended to increase with 2 and 20 ppm KIN and 1 ppm TIBA application in ‘Chai Lai’ and with 20 ppm KIN and 1 ppm TIBA application in ‘Big C’ when cultured on I7 medium. Nevertheless, TIBA at high concentrations (10 ppm) decreased the percentages of ELS formation and the number of ELSs/piece in both cultivars. These results suggest that the polar auxin transport may play a major role on growth, floral and yield-related traits, yield as well as in vitro gynogenesis in cucumber. However, the success of exogenous applications of these PGRs depended on several factors including plant genotypes, growing seasons, types and concentrations of PGRs, and for ovary culture, the responses also varied according to the induction media used. Chemical names: abscisic acid (ABA); maleic hydrazide (MH); naphthaleneacetic acid (NAA); N6-furfuryladenine (kinetin; KIN); thidiazuron (TDZ); 2,3,5-triiodobenzoic acid (TIBA).

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Cefotaxime (100mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production. Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F1 hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.

Regeneration of oat androgenic plants in relation to induction media and culture conditions of embryo-like structures

The effect of C17 and W14 induction media on the formation of embryo-like structures (ELS) from F3 generation of nine hexaploid oat hybrids was investigated in the study. In all genotypes, the highest number of ELS (0.6 - 12.1/100 anthers) was obtained on C17 medium. The efficiency of plant regeneration on medium 190-2 was tested, in relation to different ELS culture conditions. The highest rate of green plants per 100 ELS (3.3 - 42.4) was produced by incubation at 22<sup>o</sup>C in the dark for the first two weeks. Among 36 green regenerants, 28 (77.8%) were haploid and 8 (22.2%) were spontaneous doubled haploids, fully fertile. After colchicine treatment of haploid plants, 19 were partially fertile and set from 1 to 15 seed per panicle.

Effects of exogenous application of polyamines on wheat anther cultures

Androgenesis of wheat genotypes was evaluated by pretreating anthers or embryo-like structures (ELS) with polyamines. Anthers of the genotype DH were pretreated with different concentrations of putrescine, spermidine, and spermine for 1, 3, and 6 h, and those of drought-tolerant International Center for Agricultural Research in the Dry Areas (ICARDA) wheat accessions were treated for 1 and 3 h. ELS of two genotypes were also treated for 30 and 60 min with the same polyamines and evaluated for green plant regeneration. The pretreatment of anthers with polyamines enhanced the development of ELS in all genotypes. The formation of ELS varied significantly with genotype. Pretreated anthers showed that four treatments improved significantly green plant regeneration with the genotype ICR 17. However, two treatments (1 mM putrescine or spermine for 1 h) significantly improved green plant regeneration per 100 ELS of only two ICARDA genotypes. ELS treated with polyamines for 30 min were greener and formed more adventitious roots. The chloroplasts of these greener ELS examined with a transmission electron microscope had agranal to grana thylakoids, while those of the control had plastids with mostly starch globules. Although exogenous application of polyamines to anthers improved the production of ELS and green plants, the effects of putrescine, spermidine, and spermine was dependent on genotype and the duration of pretreatment of anthers with the polyamines.

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l−1 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.

Thidiazuron-induced rapid shoot regeneration via embryo-like structure formation from shoot tip-derived callus culture of sugarcane

Rapid shoot regeneration system from callus cultures of a sugarcane (Saccharum officinarum L.) cultivar NiF8 was established. Apical meristematic tissues harvested from young sugarcane shoots were cultured on modified Murashige and Skoog medium containing 2 mg l−1 of 2,4-dichlorophenoxyacetic acid for callus induction. The sugarcane callus was then transferred onto media with different concentrations of thidiazuron (TDZ) (0.5, 1, 2 and 3 mg l−1) with or without 0.1 mg l−1 of 1-naphthaleneacetic acid (NAA) for shoot regeneration. The highest regeneration frequency (80.0%) was observed after three weeks of culture on medium containing 1 mg l−1 TDZ and 0.1 mg l−1 NAA. Histological observation showed that differentiation of proembryoid-like structure with pro-vascular strands were observed 3 days after transfer onto the optimum medium followed by formation of apical meristematic tissue and leaf-like structures after 5 to 7 days, suggesting that TDZ induced rapid shoot regeneration via a process similar to somatic embryogenesis. This study demonstrated a rapid, reproducible and efficient regeneration procedure of sugarcane, which is suitable for biotechnological application including genetic transformation.

The Arabidopsis Histone Deacetylases HDA6 and HDA19 Contribute to the Repression of Embryonic Properties after Germination

Histone deacetylase (HDAC) is a chromatin-remodeling factor that contributes to transcriptional repression in eukaryotes. In Arabidopsis (Arabidopsis thaliana), the transcription factors LEAFY COTYLEDON1 (LEC1), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3) play key roles in embryogenesis. Although the repression of embryogenesis-related genes during germination has been proposed to occur, the role of HDAC in this process has not been elucidated. To address this question, the effects of an HDAC inhibitor and suppression of the Arabidopsis HDAC genes on this process were investigated. Here, we show that treatment of an HDA6 repression line with the HDAC inhibitor trichostatin A resulted in growth arrest and elevated transcription of LEC1, FUS3, and ABI3 during germination. The growth-arrest phenotype of the repression line was suppressed by lec1, fus3, and abi3. An HDA6/HDA19 double-repression line displayed arrested growth after germination and the formation of embryo-like structures on the true leaves of 6-week-old plants even without trichostatin A. The growth-arrest phenotype of this line was rescued by lec1. These results suggest that during germination in Arabidopsis, HDA6 and HDA19 redundantly regulate the repression of embryonic properties directly or indirectly via repression of embryo-specific gene function.

The Effect of Various Culture Media on the Formation of Embryo-like Structures in Cultures Derived from Explants Taken from Mature Larix decidua

The effect of various collection dates and nine different culture media on the formation of ‘embryo-like structures’ (ELS) in cultures derived from explants taken from a 42-year-old Larix decidua tree was studied. The best medium was AFC, a medium low in NH4−, NO3 +, Mg2+ and SO42− but high in PO43− compared with the concentration of these elements in the other media. On AFC, ELS production reached a peak with collections made in late summer during the period when needle primordia are being initiated. For the other media, collection date had a lesser effect on ELS initiation.

Micropropagation of Plantlets through Callus in Kihada (Phellodendron amurense Rupr.)

Hypocotyl and internodal explants were collected from in vitro grown seedlings and in vitro proliferated shoots, respectively. Callus induction and subsequent plant regeneration were established from those explants. Friable callus with somatic embryo-like structure (ELS) developed from hypocotyl and internodal explants on Murashige and Skoog (MS) medium supplemented with 0.89-4.44μM 6-benzylaminopurine (BAP) and 2.26-9.05μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.69-10.74μM α-naphthaleneacetic acid (NAA). The maximal frequency of callus and ELS formation were obtained when the MS medium was amended with 0.89μM BAP and 4.52μM 2,4-D. After callus induction, they were cultured on the MS medium supplemented with 0.98-4.44μM BAP and 0.54-2.69μM NAA or 0.49-2.46μM IBA for the regeneration of the shoots. Among different hormonal conditions, a combination of 2.22μM BAP and 1.07μM NAA produced the highest percentage of shoot differentiation from calli. In vitro grown shoots were rooted on the MS medium with either of 0.5-4.0μM indole-3-butyric acid (IBA), NAA, or indole-3-acetic acid (IAA). Regenerants were transferred to Kanuma soil and successfully established under ex vitro environment.

Comparison of in vitro androgenic response and F1 seed production in wheat (Triticum aestivum L.)

Nine parental lines along with their F1 progenies were investigated for general combining ability (GCA) effects on embryo-like-structure (ELS) production in anther culture and seed production. One hundred and twenty anthers were plated each in 60 mm Petri dish in 4 replications. Significant differences were found among the cultivars in respect to the production of ELS, while among the parents no significant differences in seed production were observed. Evaluating the combining ability effects, among the female parent lines GK Mura and GK Favorit showed the best results in ELS production and in hybrid seed production. Among the male parent varieties similar positive result was performed in the ELS- and Fl seed production by GK Kalasz and GK Zugoly. Hybrid seed- and ELS production in anther culture are genetically independent characters. But, in our experiment both of them showed similar regulation. This might be due to a hybrid effect. ELS formation is found to be controlled mostly by additive gene effects.

Interaction of environment and ABA and GA treatments on the maize anther culture response

Treatments designed to influence abscisic acid (ABA) or gibberellin (GA) concentrations were applied to developing tassels of maize (Zea mays L.) plants in different environments or to anthers in culture to determine the effect on formation of embryo-like structures (ELS). Production of ELS was significantly affected in certain environments when ABA, GA3, ancymidol, or fluridone solutions were pipetted into whorls of field-grown plants approximately 3 days before tassel harvest. In 1996 anthers from 10 μM ancymidol-treated plants were most responsive, producing 35 ELS/100 anthers and 50 μM GA3-treated plants were least responsive, producing 12 ELS/100 anthers. In 1997 under hotter, drier conditions, anthers from 50 μM GA3-treated plants were most responsive, producing 20 ELS/100 anthers and those from 50 μM ABA-treated plants were least responsive, producing 2.4 ELS/100 anthers. Anthers from growth chamber plants were significantly more responsive when grown in a 16-h than a 12-h photoperiod. With the 16-h photoperiod the response was significantly greater with a 250 μM ABA whorl treatment. With the 12-h photoperiod there was no significant effect from whorl treatments. Modification of the culture medium with added ABA, GA3, ancymidol, or fluridone was generally ineffective, except in 1997 when the response was significantly higher with 1 μM ABA added to the culture medium. The results suggest that the maize anther culture response may be influenced by environmental conditions that interact with ABA and GA treatments to donor plants during tassel development.

Arabidopsis LEAFY COTYLEDON1 Is Sufficient to Induce Embryo Development in Vegetative Cells

The Arabidopsis LEAFY COTYLEDON1 ( LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box–binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

The effect of collection date and frozen storage on the formation of embryo-like structures and elongating shoots from explants from mature Larix decidua and L. x eurolepis

Branches were collected in the winter and early spring from a 37-year-old Larix decidua and a L. x eurolepis tree. Part of these were used immediately and the rest stored at −5 °C and −10 °C for 3 months. Primordial shoot explants were excised from stored and non-stored material and cultured on half-strength Litvay medium free of growth regulators. Storage at −5 °C promoted formation of embryo-like structures which formed shoots with elongated stems (long-shoots). The highest number of these was obtained with material collected shortly before bud break. De-needled stem sections, cut from elongating stems in early summer, produced axillary short-shoots instead of embryo-like structures.

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5-1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1-7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1-3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.

RFLP analysis to identify putative chromosomal regions involved in the anther culture response and callus formation of maize

RFLP analysis was performed with anther culture-derived callus lines developed from the maize F1 hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.