Frequency Of Embryo Formation Research Articles

Androgenesis in Hot Pepper by Chilli Anther Culture

In vitro androgenesis techniques play a key role in the quick generation of fully homozygous lines. This work's goal was to put the procedure for producing androgenic embryos in hot pepper populations into practice. A number of variables can affect androgenetic success, including the types and quantities of carbohydrate sources and the nature of the nutritional medium. Different sugar types were tested at different doses in anther culture of pepper to elucidate their influence on the yield of androgenesis. In this study, sucrose was used as a source of sugar, Murashige & Skoog (1962-MS) medium, silver nitrate (AgNO3), naphthaleneacetic acid (NAA), BAP and one breeding lines of pepper were used to know the genotypic effects on haploid embryo formation. It was found that one of the key elements influencing pepper anther and microspore culture success is genotype. Also, microspore stage and the cold treatment of flower bud responsible for the success of embryo formation. This embryo formation frequency has been calculated according to the results that were generated in one month and the frequency of embryo formation is 0.7%.

Use of nurse endosperm for the culture of haploid embryos produced by durum wheat x maize crosses

The use of doubled-haploids in plant breeding programs enables accelerating the release of new varieties adapted to climate change. The durum wheat x maize crosses technique is a method of choice for producing durum wheat haploid plants. The haploid embryos produced by this method develop without albumen and their survival is ensured by post-pollination hormonal treatments. In this study, nine post-pollination treatments with 2,4-Dichlorophenoxyacetic acid (2,4-D), Picloram and Dicamba at the concentrations of 10, 50 and 100 mg.L-1 were applied to 7 durum wheat genotypes. The effects of genotype and post-pollination treatment on durum wheat haploid embryos produced by durum wheat x maize crosses and the use of the endosperm nursing technique for haploid plantlets regeneration were investigated. The haploid induction parameters varied with the durum wheat genotypes as well as the post-pollination treatments. The phenomenon of polyembryony resulting from durum wheat x maize crosses is reported for the first time in this article. The durum wheat genotypes showed different abilities to produce monoembryo and polyembryos. The post-pollination treatments with 2,4-D (10 mg.L-1) and Picloram (10 and 100 mg.L-1) gave a higher embryo formation frequency than the treatments with Dicamba. The embryo conversion to plantlet was greatly improved, especially in recalcitrant genotypes using the durum wheat endosperm as supplemental nourishment in combination with the Gamborg B5 regeneration medium.
 Keywords: Durum wheat; Haploid embryo; Maize; Nurse Endosperm Technique; Polyembryony

Production of Doubled Haploid through Anther Culture of the Male-Fertile Lines in the CMS System of Eggplant with the Cytoplasm of <i>Solanum grandifolium</i>

To produce doubled haploid homozygous male-sterile line of eggplant with the cytoplasm of Solanum grandifolium, anther culture of the male-fertile (MF) BC5 in the S. grandifolium induced CMS system was done. Anthers were cultured in an embryo induction medium and were heated to 35℃ for 3 d in the dark and then transferred to 25℃ under a 16 h photoperiod. After 20 d the anthers were transferred to a germination medium. The presence of pollen in the anther, number of chromosomes in the root tip cells, size of the stomatal guard cells and pollen fertility of the regenerated plants were investigated. The frequency of embryo formation in the MF line was 1.02%. From the embryos, one regenerated plant showed pollen non-formation type male sterility. Root tip chromosome number and stomatal guard cell size confirmed that the regenerated male-sterile plant is a microspore originated doubled haploid through spontaneous chromosome doubling. The present study demonstrates the first successful production of a doubled haploid homozygous male-sterile line in a CMS system of eggplant with the cytoplasm of S. grandifolium. Therefore, this material could be a useful breeding material for eggplant and improve the speed of conventional breeding.

In vivo haploid induction potential of Himalayan maize (Zea mays) and cogon grass (Imperata cylindrica) gene pools in different segregational cycles of intra and inter-generic crosses of wheat

AbstractMaize and Imperata cylindrica have been utilized globally as a pollen source for induction of haploids in wheat through chromosome elimination technique. Pollen parents with a higher haploid induction rate are desired for recovering the high frequency of haploids in wheat and related species. The present investigation was carried out with the aim to assess haploid induction efficiency of diverse germplasm of maize and I. cylindrica in different generations of intra and intergeneric crosses of hexaploid and tetraploid wheat and triticale-wheat derivatives. Crosses of twenty-six lines (female) with each of two I. cylindrica and twenty-one maize genotypes (testers) were evaluated for four haploid induction parameters viz., pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF) and haploid formation frequency (HFF). I. cylindrica outperformed maize in haploid induction rate with a frequency of embryos formed with I. cylindrica (18.39%) were significantly higher as compared to maize (4.08%). In the case of I. cylindrica genotype Ic-ye identified best with mean EFF of 30.55, 14.48 and 25.43% for hexaploids, tetraploids and triticale × wheat derivatives, respectively whereas in the case of maize genotype HPMC-60 performed best with EFF of 12.61% for hexaploids, HPMC-58 (12.58%) for tetraploids and HPMC-16 for triticale × wheat derivatives with EFF of 8.91%. I. cylindrica genotype Ic-ye and maize genotypes HPMC-14, HPMC-53, HPMC-60, HPMC-64 with significantly positive GCA for haploid induction parameters may be utilized as efficient pollen parents for recovering higher frequency of haploids in wheat.

Micropropagation of Tulip via Somatic Embryogenesis

An effective method of tulip regeneration via somatic embryogenesis (SE) was developed. Explants, flower stem slices excised from cooled bulbs were incubated in darkness on MS modified media containing auxins alone (2,4-dichlorophenoxyacetic acid—2,4-D, 1-naphthalene acetic acid—NAA and 4-amino-3,5,6-trichloro-2-pyridine carboxylic acid—picloram) or combined with thidiazuron (TDZ) at 0.1 and 0.5 mg L−1. Yellowish-white callus with a granular structure was developed in the presence of all auxins on the cut surface from the tissues of the vascular bundles. From this, lines of embryogenic calli were derived. The addition of TDZ to the medium with auxins significantly stimulated somatic embryo formation. Cyclic and the most intensive proliferation of embryogenic callus as well as embryo formation was obtained in the presence of 2,4-D at 0.1 mg L−1 combined with TDZ at 0.5 mg L−1. Addition of proline enhanced either callus proliferation rate or frequency of embryo formation. The best quality embryos with cotyledons longer than 10 mm able to form bulbs were recorded when TDZ was replaced with 6-benzylaminopurine (BAP) at the concentration of 0.1 mg L−1. Histomorphology showed that the development of somatic embryos could have either external or internal origins. Embryos of external origin were initiated by cell division on the edge of embryogenic calli. Embryos of internal origin resulted from the division of parenchyma cells inside the tissue. Embryonic cells were characterized by their small volume, regular shape, dense cytoplasm and large nuclei. The globular embryos were covered by a distinct layer of periderm. Then, the embryos developed into structures having leaf-shaped cotyledons with a procambial strand and a sideward-orientated meristem of the vegetative apex (stolon). Cotyledon embryos did not show vascular connections with the parent tissue, and they did not develop embryonic roots.

Auxin Induced Haploid Induction in Wide Crosses of Durum Wheat

Doubled haploidy breeding via wide hybridization has been used in durum wheat haploid production for creating homozygosity in the shortest possible time. Post pollination treatment with hormones is a key factor for inducing haploid embryos in durum wheat wide crosses. An intergeneric hybridization experiment was carried out in seven durum wheat genotypes using Imperata cylindrica and two composites of Maize viz., Bajaura Makka and Early Composite, as pollen sources. The pollinated spikes were injected with 2,4-Dichlorophenoxyacetic acid (2,4-D) in five different concentrations i.e., 100, 150, 200, 250 and 300 ppm, for three consecutive days at 24, 48 and 72 hrs after pollination. Analysis of variance revealed that the five concentrations of 2,4-D significantly differed in their ability to induce haploid embryos and 2,4-D at 250 ppm was found to be most effective in durum wheat haploid production through wide hybridization. The highest frequency of embryo carrying seeds was recorded to be 65.75 and 36.73 percent, at 250 ppm with I. cylindrica and Bajaura Makka, respectively in first cropping season. During second season, embryo formation frequency was observed to be maximum, 70.69, 32.84 and 27.59 percent, at 250 ppm 2,4-D with all three pollen sources, viz., I. cylindrica, Bajaura Makka and Early Composite, respectively.

Explant type and hormone regime influences somatic embryogenesis and regeneration of cassava

Cassava (Manihot esculenta Crantz) is a tropical root crop that serves as a food staple and a vital income source to resource deprived farmers in the tropics. Despite its importance, cassava production and consumption is faced by a myriad of biotic/abiotic constraints. Genetic transformation which can be applied to mitigate these challenges however has as a prerequisite the availability of robust regeneration systems. This study evaluated the effect of explant type and hormone regime on somatic embryogenesis and regeneration of Kenyan cassava cultivars. The embryogenic competence of immature leaf lobe and stem explants of three cassava cultivars was determined by culturing them in MS medium supplemented with picloram (4, 6, 8 and 10 mg/l). The optimum media composition for embryo maturation, germination and plant recovery was assessed by culturing embryos in media supplemented with varying concentrations of 6-Benzylaminopurine (BAP), α–Naphthalene acetic acid (NAA) and Gibberellic acid (GA3). Somatic embryo formation frequencies in leaf explants were significantly higher (P≤0.05) than in stem explants. Embryo formation rates were found to increase with increasing concentrations of picloram. Cultivar 08/080 gave the least response to embryogenesis while embryogenesis rates for cultivar 08/274 were similar to the control cultivar TMS 60444. Maturation and plant recovery rates differed significantly with regard to the ratios of BAP, NAA and GA3 in the maturation media. The optimal medium for embryo germination and plant recovery was supplemented with 1 mg/l BAP, 0.02 mg/l NAA and 1.5 mg/l GA3. This optimized regeneration protocol can be coupled with mutation breeding or genetic transformation to improve the cassava germplasm. Key words: Cassava, explant, regeneration, somatic embryogenesis, plant growth regulator.

Effect of hormonal treatments on haploid formation and in vitro haploid regeneration in wheat x maize system

Haploids are useful for basic studies on inter-genomic relationship, molecular studies and in practical breeding. In the present investigation post pollination treatment with different growth regulators and their combinations were analyzed. Twenty nine wheat F 1 ’s and three maize parents were utilized to obtained haploid embryos. Three different hormonal treatments were designed to observe their effect on different parameters in wheat-maize haploid system. Out of three treatments, combination of GA3 and 2,4-D showed highly significant effect on caryopsis formation frequency (64.40), embryo formation frequency (18.84), embryo germination frequency (55.82) and haploid regeneration frequency (36.51). The growth pattern of haploid embryos on nutrient medium was quite different. The differential response of haploid embryos on same nutrient medium might be because of residual effect of different growth regulators.

Dihaploid induction in tetraploid durum wheat (Triticum durum L.) using pollen of Imperata cylindrica

Doubled haploidy breeding protocols have revolutionized the varietal development programmes in bread wheat, however, the protocols have not proved much efficient in durum wheat. Presently, the durum wheat × maize system is being widely followed for haploid induction but the frequency of haploid development is very poor which has limited its application in durum wheat improvement programmes. In order to formulate an efficient wide hybridization-mediated approach for haploid induction in durum wheat, different genotypes of durum wheat were subjected to hybridization with Imperata cylindrica, a wild perennial grass for the first time in this laboratory. The investigation was carried out for two seasons. During the 1st year, the investigation was carried out on one genotype only, Langdon, in order to notice the development of haploid embryos and the factors that influence the haploid induction frequency. The most important factor influencing the embryo formation frequency was found to be the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Upon pollinating the emasculated spikes of durum wheat genotypes, various concentrations of 2,4-D were injected into the uppermost internode of wheat culm for three consecutive days after pollination to find out the most responding concentration for haploid induction. During the next year, the protocol was applied to eight durum wheat genotypes. The frequency of haploid induction parameters varied with the durum wheat genotypes as well as 2,4-D concentration used. The mean pseudoseed and embryo formation frequency over all the genotypes ranged from 30.2 to 56.3% and 1.2 to 18.4%, respectively. The average embryo formation frequency over all the genotypes was found to be highest (18.4%) at 250 mg/l 2,4-D whereas it was superior for the genotypes WH 896 and Langdon over all the 2,4-D concentrations. At the most responding 2,4-D concentration (250 mg/l), the genotype A-9-30-1 exhibited the highest embryo formation frequency (32.1%). The ploidy status of the developed embryos was identified using cytological analysis carried out on the rootlets of the tissue culture generated plantlets.

The D-genome plays a critical role in the formation of haploid Aegilops tauschii through Imperata cylindrica mediated uniparental chromosome elimination

Doubled haploid (DH) breeding eventually offers unique advantages for rapid genetic improvement of wheat in a single generation by achieving absolute homozygosity and enhanced selection efficiency. A new perennial invasive weed i.e., Imperata cylindrica has already proved its importance in haploid wheat induction through Imperata-mediated chromosome elimination process. Whereas, cytological evidences and the actual mechanism of haploid induction through chromosome elimination in wheat x I. cylindrica hybrids are well known, but which genome(s) of wheat (AA/BB/DD) are actually involved in such haploid formation has remained elusive. Therefore, in order to identify the genome-specific triggering of Imperata-mediated chromosome elimination in development of wheat haploids, 11 wheat genotypes including Ae. tauschii and D-genome chromosome substitution lines of the durum wheat variety ‘Langdon’ were crossed with I. cylindrica and examined with the prime aim of producing haploids of Ae. tauschii. The crosses suggest that only bread wheat, Ae. tauschii and Triticale (containing D genome) were successful to develop haploid plants through chromosome elimination process. Higher frequencies of embryo formation were obtained primarily in substitution lines with chromosomes 7D. The detailed anatomical and cytological analyses suggest that : (i) genome/genotypic specificity plays a key role in haploid induction through Imperata-mediated chromosome elimination process, (ii) D-genome triggers the chromosome elimination and haploid production in wheat x Imperata, (iii) some D-genome chromosomes (primarily 7D) substituted lines in wheat genetic background may enhance crossability with Imperata in the formation of haploid wheat. To the best of our knowledge, this is the first report of haploid induction in Ae. tauschii.

An insight into wheat haploid production using wheat x maize wide hybridization

The present study was carried out with six wheat and four maize genotypes from Mahyco Research Centre, Dawalwadi, Jalna, MH to develop wheat double haploid lines by wheat x maize crossing system. The following steps were followed while conducting the experiment: emasculation of wheat spikes, crossing or pollination of emasculated spikes with maize pollens, treating pollinated spikes with 100 ppm 2,4-D solution, excision of embryo and subsequent culture on half strength MS media. In total, 296 spikes were emasculated and pollinated with randomly collected maize pollens after 24 hrs. Success was obtained in rescuing a total of 27 embryos from 575 caryopses among two genotypes i.e. MW2 and MW3. Percentage of embryo formation varied in the range of 0-10 per cent owing to non-standard conditions. However, the frequency of embryo formation could be increased by focusing on the most important of all culture conditions i.e. temperature (18-21 C).

High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.)

Gherkin (Cucumis anguria L.) is an important commercial vegetable crop. An efficient protocol for plantlet regeneration from hypocotyl and leaf explants through direct somatic embryogenesis was developed. High frequency of somatic embryos (21.6 and 34.0) were obtained from hypocotyl and leaf explants when cultured on Murashige and Skoog (MS) salts plus B5 vitamins (MSB5) supplemented with 5% sucrose, 1.5mgL−1 2,4-D, 0.5mgL−1 BAP and 150μM l-glutamine. Initial culturing of embryos in the dark conditions for two weeks, followed by four weeks under light resulted in a higher frequency of embryo formation when compared to continuous light conditions. Histological observation showed that the somatic embryos originated from single cells of the epidermal layer. Histological evidence on formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. Maturation of somatic embryos occurred on plant growth regulator-free MSB5 semi-solid medium containing 3% sucrose and 0.5% (w/v) activated charcoal (AC). Conversion of embryos into plantlets was achieved on MSB5 medium supplemented with 3% sucrose, 0.3% AC and 0.5mgL−1 gibberellic acid (GA3). Sucrose was found to be the best carbon source for SE induction, maturation and germination. Ninety percent of embryos were converted into normal plantlets. The plantlets were successfully acclimatized in the greenhouse with 95% survival rate and transferred to ex vitro conditions which developed with normal phenotypes. This regeneration protocol assured successful embryo induction and plantlet conversion. This is the first report for the induction of direct somatic embryogenesis from hypocotyl and leaf explants of C. anguria. The result of this study is beneficial for genetic transformation and mass clonal propagation.

Stable Production of Wheat Haploid and Doubled Haploid by Wheat × Maize Cross

One of the most effective ways to produce wheat haploid is the method of crossing wheat with maize. However, the problem of unstable frequencies in haploid and doubled haploid production affects its application. In this study, 13 wheat F1hybrids crossed with maize to explore the effects of wheat growth condition, hormone treatment, culture medium, and strong seedling treatment on production of haploid and doubled haploid. The frequency of embryo formation was 23.9% and the number of embryos per spike was 6.8 for the wheat plants grown in filed and then detached before pollination and hydroponically cultured after pollination. The efficiency of this method was three times higher than that of the method with plants transplanted into pot after green recovering in early spring and then grown in the unheated greenhouse condition. Embryo formation frequencies were obviously different among wheat crosses. There was no significant difference in the frequency of embryo formation between treatments of dipping spike with Dicamba(21.5%) and 2,4-D(21.1%). However, wheat cross had significant effect on embryo formation. The mean frequency of embryo germination on B5medium was 82.0%, ranging from 70.9% to 88.3%, whereas averaged frequency of embryo germination was 76.6% on 1/2 MS medium, ranging from 70.0% to 86.0%. There was no significant difference between the two culture media in embryo germination. The efficiency of doubled haploid production for the seedlings treated with culture medium and transplanted into pot was 67.6% and 8.6%, respectively. Most of seedlings transplanted into pot had no tiller and grew weakly; this is the reason for low frequency of survival plant and very low frequency of doubling after colchicine treatment.

Somatic embryogenesis and plant regeneration from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L−1 GA3. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.

Efficiency of SSR markers for determining the origin of melon plantlets derived through unfertilized ovary culture

The effects of temperature pre-treatment, thidiazuron, naphthaleneacetic acid, and 6-benzylaminopurine on in vitro gynogenic plant production from un-pollinated melon (Cucumis melo L.) ovaries were investigated. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. The temperature pre-treatment (4°C) for 4 days increased embryo formation frequency (63.3%) significantly. Addition of thidiazuron (0.04 and 0.02 mg/l) in the induction medium significantly increased the number of responding ovaries (46.6%, 65.83%), respectively. The maximum number of plantlet regeneration (22.5%) was achieved by culturing the ovary derived embryos on Murashigue and Skoog medium (MS medium) supplement with 0.6 mg/l 6-benzylaminopurine. Spontaneous doubled haploids originated directly through embryogenesis were subjected to genetic analysis using SSR molecular marker with 23 primers pair for homozygosity. SSR markers with microsatellite CMGA172, confirmed that the alleles in the parental material were also present in the gynogenic plantlets, but amplified only two alleles as compared to four alleles of the heterozygous parent material at same locus. Therefore these regenerated plantlets were consider homozygous and produced through a process of gametophytic embryogenesis.

In vitro culture of unfertilized ovules in carrot (Daucus carota L.)

To induce development of isolated carrot ovules, flowers were pollinated with pollen from different species. Effects of pollen origin, medium composition, and light conditions were evaluated for 13 carrot cultivars. The highest efficiency of development was recorded following pollination of carrot flowers with parsley pollen. The highest frequency of embryo formation (0.84%) was obtained for cv. ‘Flamanka’; whereas the highest frequency of callus formation (1.56%) was obtained for cv. ‘Karlena’. Medium supplemented with indole-3-acetic acid (IAA) promoted embryo development (0.63%), while supplementation with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) promoted callus development (1.34%). Placement of isolated ovules in the dark stimulated embryo development. Embryos and calli thus obtained were regenerated into plants. Flow cytometry revealed that 97.7% regenerants were diploid. Based on a phosphoglucoisomerase (PGI) isozyme assay, 45.9% diploid regenerants were homozygous. Pollen tube growth of parsley, parsnip, celery, and cabbage in carrot pistils were studied using the aniline blue fluorescent method. The pollen tube growth was inhibited in transmitting tissues of the pistils.

Embryogenic callus culture of Tribulus terrestris L. a potential source of harmaline, harmine and diosgenin

In the present study, a simple one medium formulation protocol for callus culture, somatic embryogenesis and in vitro production of β-carboline alkaloids and diosgenin in Tribulus terrestris L. was developed. Extensive callus induction and proliferation was obtained in leaf explant on Murashige and Skoog (MS) medium supplemented with 5.0 μM 6 benzyl adenine (BA) and 2.5 μM α-naphthaleneacetic acid (NAA). The embryogenic callus was maintained on subculture to fresh parental medium at 4-week intervals over a period of 28 months. The frequency of embryo formation was at a maximum (18.1 ± 0.9 per g of callus) on MS medium containing 5.0 μM BA and 2.5 μM NAA together with 75 mg l−1 casein hydrolysate. Globular embryo developed into torpedo stage embryo under the influence of starvation. The accumulation of β-carboline alkaloids (harmaline and harmine) and steroidal saponin (diosgenin) in non-embryogenic and embryogenic callus culture derived from leaf explant was compared with root, leaf, stem, and fruit of the mother plant. The embryogenic callus accumulated equivalent amounts of harmaline (66.4 ± 0.5 μg/g dry weight), harmine (82.7 ± 0.6 μg/g dry weight), and diosgenin (170.7 ± 1.0 μg/g dry weight) to that of the fruit of T. terrestris. The embryogenic callus culture of this species might offer a potential source for production of important pharmaceuticals.

Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using SSR markers

Several factors, i.e. the duration of thermal shock pretreatment at 35 °C, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in a variety of cucumber ( Cucumis sativus L) ovary culture. The results showed that a thermal shock for 3 days at 35 °C at the start of the culture resulted in higher frequency of embryo formation than 2 or 4 days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO 3 to induction medium had no significant effect on frequency of embryo formation but shortened embryo sprouting period and improved number of embryos formed in each ovary slice. All the experiment materials responded well to ovary culture, and there were no difference among genotypes used in this study. Among the forty regenerated plants obtained, two were identified as haploid plants (2 n = x = 7), five were tetraploid plants (2 n = 4 x = 28), and the rest were diploid plants. Microsatellite markers (SSR) were used to analyze the homozygosity of the diploid plants, the putative chromosome-doubled haploids. Of the 33 diploid plants, 17 (51.5%) were identified as double haploids. Based on the above results, we have established a useful protocol for production of cucumber doubled haploids with ovary culture.

Chromosome doubling effects of selected antimitotic agents in Brassica napus microspore culture

Effects of microspore culture treatment with antimitotic agents colchicine, trifluralin and oryzalin on the frequency of embryo formation, embryo development, plant regeneration and diploidization rate in three F<sub>1</sub> hybrids of winter rapeseed cultivars were compared. The ploidy level analysis of 1709 flowering microspore-derived plants showed that in vitro applications of all antimitotic drugs increased the rate of doubled haploid (DH) plants significantly. The mean rate of DH plants from the trifluralin treatment was 85.7%, from colchicine 74.1% and 66.5% in the case of oryzalin, while only 42.3% in the untreated control variant whereas in vivo additional application of colchicine at the plantlet stage did not significantly increase the mean rate of DH plants (55.6%). Although there were no significant differences in diploidization efficiency between the in vitro applications of particular antimitotic agents, trifluralin showed to be the most suitable because of its positive effect on embryo development and conversion into whole plants. In addition, the diploidization rate was sufficient and stable in all genotypes tested. The results indicate that the trifluralin treatment of microspore cultures could provide efficient chromosome doubling for the production of doubled haploid lines from winter oilseed rape breeding materials.

Embryogenesis in Seedles Grapes and Hybrid Combinations of (Vitis Vinifera L.)

ABSTRACTThe possibility for induction of in vitro embryogenesis on solid culture medium from seedless grape cultivars and hybrid combinations, giving an account of the effect of explant age, genotype and growth regulators was investigated. We established that in all studied treatments and genotypes explants isolated 3 days after pollination formed only callus. Comparing the capability for embryo induction, seed buds isolated 10 and 30 days after pollination showed increased regeneration capacity with specifics toward development of somatic or zygotic embryo. The frequency of zygotic embryo formation was higher in the inbreed treatments while the hybrid genotypes produced more somatic embryos. Converting into green plants suitable for micropropagation was observed only from explants isolated 10 and 30 days after pollination. The best embryogenic ability was established for Rusalka3-inbred and the hybrid combination Russalka X Alicante Buchet. Regarding the effect of the culture media the higher concentration of growth regulators in ICI did not promote better frequency of embryo formation than I3 medium, but resulted in obtaining of more green plants with normal development and growth.