Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

The use of mutagens to increase the efficiency of the androgenic progeny production in <Emphasis Type="Italic">Solanum nigrum</Emphasis>

Pollen embryogenesis was successfully induced in Solanum nigrum L. (2n=6×=72). Stimulation of androgenesis expressed as the frequency of androgenic responsive anthers was observed after 10 and 20 mM ethyl methanesulphonate (EMS), 10 and 20 mM sodium azide (NaN₃) and 0.2 mM N-nitroso-N-methylurea (MNU) treatment applied on seeds for 24 h. The frequency of androgenesis on the medium with sucrose was higher than on the medium with maltose. Androgenic regenerants originated also in the anthers collected from donor plants where survival after mutagenic treatment was lower than 50 %. Green haploid (3x), aneuploid (to 8x) and dihaploid (6x) plants were obtained. The high frequency of aneuploids among androgenic plants is explained by cell division irregularities in microsporial calli.     

Induction of haploid and diploid plants though in vitro anther culture of haploid wheat (n=3x=21)

Summary Five haploid plants of wheat were used for anther culture. Embryos were formed and six plants were regenerated. Of these, two were haploid (n=3x=21) and two diploid (2n=6x=42). The two diploids derived from the anthers of the same haploid wheat plant gave seeds, but the fertility was reduced in one of them showing, abnormalities at meiosis.     

Cytological and Embryological Studies on Haploid Plant Production from Cultured Unpollinated Ovaries of Nicotiana tabacum L.

We obtained mature haploid (n = 24) ovary plants from in vitro cultured unpollinated young ovaries. These ovaries were induced to form embryoids which then developed into plants. The results obtained are summarized as follows: 1. The origin of development of the ovary haploid plants has been followed by light microscopy. Embryological abservations revealed that there are two ways of plantlet production: (1) Ovary haploid plant was derived from the macrospore without an intervening callus phase. (2) Ovary haploid plant was derived directly from the egg cell of mature embryo sac. In addition, Callus derived haploid plant was also obtained from the base and the tip of a bud of the above mentioned haploid plantlet. In same medium embryoids was derived from callus. Finally, plantlet was developed. 2. The exogenous hormones are necessary for high induction frequency of embryoid from unpollinated isolated young ovary, but these are not definitely necessary for induction of embryonic callus to form embryoids which then developed into plant. 3. The induction frequency of embryoid from in vitro cultured ovary and embryonic callus significantly increased when the concentration of thiamine, pyridoxine, ascorbic acid, nicotinic acid, inositol and folic acid was raised.     

Chromosome elimination and in vivo haploid production induced by Stock 6-derived inducer line in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

In vivo haploid production induced by inducer lines derived from Stock 6 is widely used in breeding program of maize (Zea mays L.), but the mechanisms behind have not yet been fully understood. In this study, average frequency of haploid induction in four inbred lines by Stock 6-derived inducer line HZI1 was above 10%. About 0.2% kernels from the cross Hua24 x HZI1 had mosaic endosperm showing yellow shrunken parts from Hua24 to normal parts with purple aleurone from HZI1. Individual lagged chromosomes and micronuclei were observed in mitotic cells of ovules pollinated by HZI1. Above 56.4% of the radicles from the kernels with purple aleurone and colorless embryos were mixoploid (2n = 9-21), and more than 45.22% cells were haploid cells (2n = 10) in three crosses. More than 62.5% of the radicles from the kernels with purple aleurone and purple embryos were mixoploid (2n = 9-21) having 54.27% cells with 2n = 20. SSR analysis showed that all haploids from the cross Hua24 x HZI1 shared the same genomic compositions as Hua24 except for plants Nos. 862 and 857 with some polymorphic DNA bands. The results revealed that chromosome elimination after fertilization caused the haploid production in maize.     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Alloplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis> with <Emphasis Type="Italic">Enarthrocarpus lyratus</Emphasis> cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species

A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.     

Construction and characterization of a bacterial artificial chromosome library of banana (<Emphasis Type="Italic">Musa acuminata</Emphasis> Colla)

A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.     

Recent advances in anther culture of Hevea brasiliensis (Muell.-Arg.)

Summary The yield of pollen embryoids from cultured Hevea anthers was increased 4 fold by optimizing the proportion of ammonium nitrate to potassium nitrate in the dedifferentiation medium. For optimal differentiation of pollen embryoids, kinetin, 2,4-D and -naphtalene acetic acid are required. Anther culture for 50 days on the dedifferentiation medium is a prerequisite for the selective development of calli and embryoids from microspores.The determination of chromosome numbers in embryoids, plantlets and regenerated trees reveals that they originate from (poly)haploid pollen grains (n=2x=18). Aneuploid, triploid (3x=27) and tetraploid (4x=36) cells were encountered in increasing frequencies as the embryoids and plants developed. A few haploid cells with 9 chromosomes were consistently observed. Buds from shoots with mixoploid chromosome numbers can be grafted and the change in the chromosome constitution of the developing new shoots followed.     

Phenotypic and cytological variation among plants derived from anther cultures ofLilium longiflorum

Summary Anther culture of the Easter Lily (Lilium longiflorum; 2n=2x=24) was attempted in order to evaluate its potential in generating haploids for the production of hybrid cultivars. The effects of genotype, temperature (low temperature treatment of buds and high temperature treatment of cultures), sucrose concentration and growth regulators were tested. The most important factors for callus induction were the genotype and the presence of 2,4-dichlorophenoxyacetic acid. Pre-treatments at low or high temperature had no apparent effect, while high sucrose concentration was inhibitory. Callus was derived from 28 of the 108 genotypes tested and plants were regenerated. Phenotypic variations were observed among these regenerants. Somatic chromosome numbers were determined in 42 plants derived from 10 donor genotypes. Thirteen plants were diploid and 29 were mixoploid with chromosome numbers ranging from 11 to 26. Four of the mixoploid plants had a high proportion of cells with haploid chromosome numbers, particularly at early stages of development. Meiosis was examined in plants with flower buds. Most plants had 12 bivalents at Metaphase I, but also aneuploids were observed. Other irregularities included bridges and laggards at Anaphase I. The occurrence of high frequencies of haploid cells (up to 80%) in root tips suggests that some plants may be of gametic origin. Research was supported by the Easter Lily Research Foundation, the Ohio Floriculture Foundation, the Gloeckner Foundation and the Oregon Agricultural Experiment Station (technical paper no. 8398).     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

In vitro development of plants from microspores of rice

Summary Rice (Oryza sativa L., 2n=24) anthers containing microspores in the early-uninucleate to first-mitosis stages were induced successfully to develop into plants in vitro through an intermediary step of callus formation. Callus initiation occurred with highest frequency in anthers containing mid-uninucleate microspores. The callus derived from different stages of microspore development differed in the potential to differentiate into plants. The plants regenerated from pollen callus were predominantly haploid or diploid; polyploid and aneuploid plants were relatively infrequent. The first division of the uninucleate microspores was asymmetrical, resulting in the formation of large vegetative and small generative nuclei. The vegetative nucleus divided repeatedly and assumed the major role in the formation of callus, whereas the generative nucleus degenerated rapidly. Simultaneous division of the two nuclei was observed in a few pollen grains. Nuclear fusion during the very initial stages of pollen development was postulated to account for the occurrence of the diploid and polyploid plants. This work was supported by the National Science Council, Republic of China.     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Induction of Haploid Durum Wheat Plants Through Pollination of Maize Pollen

When tetraploid wheat (Triticum durum Desf. ) variety DR147 was crossed with maize (Zea mays L. ) variety suppersweet ss 7700, pollen readily germinated on the stigma and one or more pollen tubes reached the embryo sac in 83.4% of wheat florets. The frequency of fertilization and embryo formation was 44.5% and 42. 6% respectively. The hybrids were karyotypically unstable and the maize chromosomes were eliminated early in the development. Thus haploid wheat embryos were form. Although the double fertilization frequency of durum wheat X maize was high (32.7%) to form embryos and endosperms, yet the endosperms were highly abnormal. It was very difficult to produce viable mature seeds from the mother durum wheat plants. The survival of hybrid embryos produced by durum wheat X maize could be improved or prolonged by treatment with 100 ppm 2, 4-D (either by dipping inflorescences in solution or injecting 0.3 to 0.5 mL 2, 4-D solution into the uppermost internodes of the wheat stem). 9 to 13 days after pollination, caryopsis were excised from the pollinated spikes and surface sterilized for peeling of the embryos in different developing stages. The embryos were plated on MS solid medium containing 3% sucrose, 200 mg/L casein hydrolysate for embryo rescue. The experimental results revealed that the well developed embryos (larger than 0. 5 mm with scutellum structure) were easy to produce calli by callus induction or produce haploid wheat plants by embryo rescue, whereas the poorly developed embryos (globular, pear or torpedo-shaped embryos smaller than 0.3 mm) responsed very poorly. The germination frequencies of well and poorly developed embryos were 83.3 % and 12.5 %, respectively. Chromosome counts of root tip cells of the rescued plants proved their haploid nature (2n= 2x= 14).     

A complete set of maize individual chromosome additions to the oat genome

All 10 chromosomes of maize (Zea mays, 2n = 2x = 20) were recovered as single additions to the haploid complement of oat (Avena sativa, 2n = 6x = 42) among F(1) plants generated from crosses involving three different lines of maize to eight different lines of oat. In vitro rescue culture of more than 4,300 immature F(1) embryos resulted in a germination frequency of 11% with recovery of 379 F(1) plantlets (8.7%) of moderately vigorous growth. Some F(1) plants were sectored with distinct chromosome constitutions among tillers of the same plant and also between root and shoot cells. Meiotic restitution facilitated development of un-reduced gametes in the F(1). Self-pollination of these partially fertile F(1) plants resulted in disomic additions (2n = 6x + 2 = 44) for maize chromosomes 1, 2, 3, 4, 6, 7, and 9. Maize chromosome 8 was recovered as a monosomic addition (2n = 6x + 1 = 43). Monosomic additions for maize chromosomes 5 and 10 to a haploid complement of oat (n = 3x + 1 = 22) were recovered several times among the F(1) plants. Although partially fertile, these chromosome 5 and 10 addition plants have not yet transmitted the added maize chromosome to F(2) offspring. We discuss the development and general utility of this set of oat-maize addition lines as a novel tool for maize genomics and genetics.     

Anther culture with different genotypes of opium poppy (Papaver somniferum L.): effect of cold treatment

This study concerns anther culture and the production of microspore-derived calluses and plants of the opium poppy (Papaver somniferum L.). It was confirmed that growth regulators were necessary for microspore callus production. Cold treatment (7 d at 7°C) of the buds prior to culture lead to a twofold increase in the frequency of responsive anthers and in the number of calluses per 100 anthers plated. Callus was produced from cultured anthers of several genotypes, covering a wide genetic background. Step by step removal of growth regulators from the culture medium promoted organogenesis and plant regeneration. Most regenerated plants were diploid. The overall process of microspore embryogenesis closely resembled that described in previous reports on somatic callus production and plant regeneration from poppy hypocotyls in vitro.     

Callus Induction,Plant Regeneration and an Assessment of Cytological Variation in Regenerated Plants of Lolium multiflorum L.

Callus cultures were initiated from roots, apical meristem tips and leaf explants of several genotypes of Lolium multiflorum L. (Italian Ryegrass). Genotypes were selected which showed a high frequency of callus initiation and from which plants could be regenerated. Plants could be routinely produced from root-derived callus of only one of the genotypes tested. The selected genotypes were still amenable if the temperature and concentration of 2,4-D in the medium were altered. Increase in temperature caused callus from one genotype to give rise to more albino regenerants. Callus formation and plant regeneration occurred at a higher frequency from diploid than tetraploid explants. All regenerants from the diploid cultures had the 2n = 2x = 14 chromosome number whereas plants regenerated from callus derived from tetraploid cultures lost up to 3 chromosomes.     

Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

Two pathways of plant regeneration in wheat anther culture

The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n=3x=21 were the most frequent.     

Evaluation of the resistance to two nematodes: Radopholus similis and Meloidogyne spp. in four banana genotypes in Morocco

Radopholus similis and Meloidogyne spp. are the main nematode parasites of banana plants grown under plastic shelters in Morocco. A test was made in pots to evaluate the resistance of four genotypes of banana to these nematodes. Infection by Meloidogyne spp. brought about an increase in root weight in all banana plants tested because of gall formation. The inoculation of R. similis produced a reduction in length and diameter of the pseudo-trunk as well as in root and aerial mass in all genotypes. Pisang jari buaya showed the significantly lowest number of Meloidogyne nematodes per 10 g of roots, whereas for R. similis, the significantly smallest numbers were obtained in Pisang berlin and Pisang jari buaya. Therefore, Pisang jari buaya was the only banana genotype studied to show some degree of resistance to both nematodes.