Production of interspecific hybrids between Glycine max and G. tomentella through embryo culture

Summary Interspecific hybrids have been obtained in an incompatible cross between Glycine max and G. tomentella through the in vitro culture of hybrid embryos. The percentage of successful pod setting in the crosses averaged 12.8% but there were marked differences depending on the soybean cultivar used as the female parent. Hybrid embryos at globular to heart stages were extracted from the embryo sac 15–25 days after pollination and cultured in vitro. Hybrid plants were successfully obtained by culturing the embryos on B5 medium supplemented with 0.1 mg/l IBA followed by culture on B5 medium supplemented with 0.1 mg/l TBA plus 0.25 mg/l 2-iP. The F1 plants resembled the wild male parent in growth form, but had an intermediate leaf shape between that of the parents.     

Efficient In Vitro Shoot-regeneration Systems in Cassava (Manihot esculenta Crantz)

Two different protocols for in vitro regeneration of cassava using zygotic embryos and nodal axillary meristems have been developed. In both cases, buds were regenerated directly from excised explants without an intervening callus phase after a two-step culture procedure. In cotyledonary explants derived from zygotic embryos, prolific shoot formation occurred within 2—3 weeks on MS medium supplemented with 0.5—5 mg/1 BAP alone or in combination with 0.1 mg/1 NAA. Nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on MS medium containing 10 mg/1 BAP. These latter structures, when cultured on MS medium supplemented with 0.1 mg/1 NAA, 1 mg/1 BAP and 0.1 mg/1 GA₃, produced multiple shoots. Somatic embryos isolated at the globular/torpedo stage from zygotic embryo explants were also capable of multiple shoot production on medium with 1 mg/1 BAP. Rooting of regenerated shoots exceeded 95 % in phytohormone-free MS medium. No change in their ploidy levels was observed. Therefore, the protocols developed should be of use in the particle gun and Agrobacterium-mediated genetic transformation of cassava.     

Embryo rescue in Rosa hybrida L.

Summary Two crosses between Rosa hybrida L. cultivars, that fail to produce progenies by conventional means, were carried out. In one of them, total hip abscision occured seven weeks after pollination; in the other one, only 2% of the fertilized hips remained on the plants after eleven weeks. Four- and five-week-old fertilized ovules were isolated in the first cross and six-week-old embryos in the second. The ovules were cultured on six media which differed in their mineral salt and sucrose concentrations while the embryos were cultured on only one of these media and eventually cold treated for one month at 4° C before being placed in a culture room set at 23° C. The embryos that had been isolated in ovulo when they were still not visible under a binocular lens developed atypically. The embryos isolated in ovulo when they were heart-shaped and on average 0.27 mm long performed in ovulo germination on some media and/or enlargement on all of them after two weeks; after another two week culture, once isolated from the ovule, all of them germinated. The cultured isolated embryos, that were exposed to cold, enlarged and germinated more rapidly when placed at 23° C than those not exposed to cold. Furthermore, the plantlets resulting from untreated embryo germination were characterized by large cotyledons which were only partially green. These results are discussed in regard to embryogenesis, precocious germination and dormancy.     

Diploid, tetraploid, and octoploid plants from anther culture of tetraploid orchard grass, Dactylis glomerata L.

This report describes a study of androgenesis in Dactylis glomerata, where the main aim was to find anther culture-responsive clones. Two types of media and two sugars were compared for their effectiveness in anther culture induction and subsequent plantlet production. Embryo formation from the cultured anthers was obtained from 28 of the 108 cloned genotypes using two different substrates, R₂M and FW. Both induction media supported the formation of embryos from the cultured anthers, but around 4.5 times more embryos (0.81 embryos per 100 anthers) were obtained with R₂M compared with FW, and R₂M also gave 5.5 times more green plants (0.054 green plants per 100 anthers) than the FW substrate. In the investigation of a carbohydrate source, responsive clones from the genotype study were tested using maltose as a substitute for sucrose in R₂M. Using maltose instead of sucrose increased embryo formation so that 133 embryos per 100 anthers were obtained compared with 7.1 embryos per 100 anthers obtained with sucrose. The total number of green plants obtained was also improved with maltose compared with sucrose, resulting in 66.3 and 1.9 green plants per 100 cultured anthers, respectively.     

Individual reaction of <Emphasis Type="Italic">Capsicum</Emphasis> F<Subscript>2</Subscript> hybrid genotypes in anther cultures

The present study evaluated the individual plants reaction of F₂ hybrid generation of C. annuum: ATZ1 × PO and ATZ1 × CDT as well as two interspecific hybrids: C. frutescens × C. annuum ATM1 and C. frutescens × C. chinense on androgenesis conditions in in vitro anther cultures. The experiment was carried out following a modified method of Dumas de Vaulx et al. (Agronomie 1:859–864, 1981). There were demonstrated clear differences in the effectiveness of androgenesis both between the pepper hybrid forms as well as among individual plants of all the genotypes tested. The highest effectiveness of androgenic embryos development was observed for the cultivated form of C. annuum: (ATZ1 × PO)F₂. Anthers of most of the plants of this hybrid produced embryos at the level higher than 5%, while in anther cultures of the second C. annuum hybrid (ATZ1 × CDT)F₂ almost 3-fold fewer embryos and plants were produced. Anthers isolated from flower buds of interspecific hybrids formed much lower number of embryos. A positive reaction was recorded for five hybrid plants of (C. frutescens × C. annuum ATM1)F₂, while in case of (C. frutescens × C. chinense)F₂ androgenic embryos were obtained from anthers of two plants. Only in the case of a one of these plants did the effectiveness of androgenesis exceed 5%. The ploidy level of the regenerants was determined by flow cytometry. Among the regenerants there were observed both haploid forms and the plants with the diploid number of chromosomes.     

Efficient hybridization between Lycopersicon esculentum and L. peruvianum via 'embryo rescue' and in vitro propagation

For the transfer of valuable traits from wild species into the cultivated tomato, excised globular-stage embryos 13 and 15 days after pollination (DAP) were cultured in vitro. Plants were regenerated from interspecific crosses of Lycopersicon esculentum cv. ‘Early pink’×L. peruvianum PI270435, and backcrosses of L. esculentum‘Giban (JF) No. 1’× (‘Early pink’× PI270435). Somatic embryos and single cotyledons emerged on hypocotyl sections of the embryos. Five to nine plantlets per embryo were obtained by clonal propagation. The hybrid nature of the plants is confirmed by comparing hybrids and parents in their ability to regenerate shoots from leaf segments in vitro, by comparing plant morphology and characteristics and by chromosome number. This study describes an efficient ‘embryo rescue’ method, as well as somatic embryogenesis by clonal propagation. A novel and simple method for the characterization of the interspecific hybrids is also reported.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF₁ hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 ^°C after being transferred to solid regeneration mediumand an initial period of low temperature (2 ^°C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.     

Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr.

Interspecific F₁ hybrids were obtained from a cross between Cucumis sativus L. (2n = 2x = 14) and C. hystrix Chakr. (2n = 2x = 24). Controlled crossing resulted in fruit containing embryos which were excised and rescued on a Murashige and Skoog solid medium. A total of 59 vigorous plants were obtained from a fruit containing 159 embryos (37.3% regeneration rate). Hybrid plants were morphologically uniform. The multiple branching habit, densely brown hairs (especially on corolla and pistil), orange-yellow collora, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix paternal parent. While appearance of the first pistillate flower was more similar to that of C. sativus maternal parent than to C. hystrix, staminate flower appearance was mid-parent in occurence. The diameter and internode length of stem, shape and size of leaves and flowers were intermediate when compared to the parents. An elongated green, trilobate style/stigma which was not apparent in either parent was observed in staminate flowers of F₁ plants. Similarly, the style/stigma of pistillate flower of F₁ plants were longger when compared to their parents. The brown pubescence observed on pistillate flowers of the F₁ and C. hystrix was not observed on the C. sativus parent. The somatic chromosome number of F₁ plants was 19. Two morphologically distinct groups of chromosomes were observed in the F₁ hybrid; 7 relatively large chromosomes characteristic of C. sativus, and 12 smaller chromosomes characteristic of C. hystrix. Analysis of malate dehydrogenase isozyme banding patterns provided additional comfirmation of hybridity. Reciprocal crossing of F₁ plants to either parent and self-crossing indicated that the hybrids were male and female sterile. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of intergeneric hybrids between a C3-C4 intermediate species Moricandia arvensis and a C3 species Brassica oleracea through ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (a C₃-C₄ intermediate species) and Brassica oleracea (a C₃ species) were obtained through ovary culture. Many hybrid embryos (2.71 per pollination) were produced in the M. arvensis × B. oleracea cross, but none were produced from the reciprocal cross. Though most embryos failed to develop into plantlets directly, plants were obtained by inducing shoots from hypocotyl explants. The hybrid plants were morphologically intermediate between the parents except for the petal color. Cytogenetic observations indicated that partial homology existed between the two genomes. Ovary culture is an efficient technique for gene transfer from M. arvensis to B. oleracea.     

Plant Regeneration from the Hybridization of Brassica juncea and B. napus Through Embryo Culture

Crosses were made to produce interspecific hybrids between Brassica napus × B. juncea and their reciprocals with the aid of embryo culture techniques. A better response of hybrid embryo culture was obtained from two cross combinations of B. juncea × B. napus (Ames 24521 × Huyou 15 and Vittasso × Zheshuang 72) than from their reciprocals. Embryo culture was more effective in terms of plant regeneration when embryos were cultured in vitro at 15 days after pollination (DAP), while more calli were initiated when embryos were excised and cultured at 10 DAP. A better response was observed on the MS medium with 0.3 mg l^?1 naphthylacetic acid (NAA) + 1.5 mg l^?1 6‐benzylaminopurine (BAP) and with 0.3 mg l^?1 NAA + 2.0 mg l^?1 BAP. Callus formation and plant regeneration on these two media reached 55.43 and 26.65 %, and 66.98 and 24.61 %, respectively.     

Production of hybrids between Cajanus acutifolius and C. cajan

There are many wild species of pigeonpea which are endemic to Australia. These wild species are cross incompatible with cultivated species of Indian origin. Cajanus acutifolius is one such species which does not easily cross with cultivated pigeonpea. Interspecific pollinations lead to hybrid seeds which were semi-shrivelled. Very few seeds germinated to give rise to F1 plants. Backcrossing the hybrid plants to C. cajan, the male parent, gave rise to aborting seeds which did not germinate in vivo hence BC1 plants are obtained after saving the aborting embryos in vitro. BC₁ plants showed normal meiotic pairing, but had low pollen fertility. The reasons for embryo abortion and low pollen fertility in spite of normal meiosis could be due to the effect of wild species cytoplasm. This revised version was published online in August 2006 with corrections to the Cover Date.     

Anther Culture Derived Homozygous Lines in Hordeum bulbosum

The production of homozygous doubled haploids via anther culture was investigated in the self-incompatible diploid species Hordeum bulbosum. Anthers from three accessions (GBC77, GBC752 and GBC753) were cultured on FHG media using IAA or three levels (1, 50 and 100 mg/l) of the auxin phenylacetic acid (PAA). Four green plants and 63 albino plants were obtained from a total of 1620 plated anthers (540/accession). The best mean anther response, number of embryogenic calli and regenerated plants were obtained with 50 mg/1 PAA. Three of the four green plants survived to maturity and, based on root-tip squashes stained with feulgen, all had 14 chromosomes like the anther donor parent. These anther culture-derived plants contained only some of the parental DNA bands, as observed by PCR analysis, indicating that they are of gametic origin rather than arising from somatic parental tissue. This is the first report of homozygous lines produced from this self-incompatible species.     

Effect of parental genotypes and colchicine treatment on the androgenic response of wheat F1 hybrids

The effect of the parental genotypes and colchicine treatment on the androgenic response of wheat (Triticum aestivum L.) F1 hybrids was studied. For this, anthers from three F1 hybrids and their parents were cultured on W14 initiation medium and W14 supplemented with 0.03% colchicine. The number of responding anthers, microspore‐derived structures/100 anthers, green plants/embryos cultured, green plants/100 anthers and albino plants/100 anthers were recorded. It was observed that embryo formation and plant regeneration ability were genetically controlled and genotype dependent. In both treatments the variety Kavkaz had a significantly higher percentage of responding anthers, microspore‐derived structures and green plants/100 anthers than the other genotypes. On the other hand, the variety Myconos also demonstrated high microspore‐derived structure production and green plant regeneration when treated with colchicine. The good response observed in these two varieties indicates the importance of colchicine treatment only for certain genotypes. Green plant production capacity of the hybrids was intermediate to that of the parental varieties. As one parent with a high or even an intermediate response to anther culture could lead to the production of sufficient (for breeding purposes) green plants from the F₁ hybrids, it was concluded that screening the inbred lines for the response to anther culture with and without colchicine treatment could contribute to utilization of breeding material with a low response to anther culture via the proper hybrid combinations.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.     

Development and characterization of interspecific hybrids of Trifolium alexandrinum×T. apertum using embryo rescue

This is the first report on the development of interspecific hybrids between Trifolium alexandrinum and T. apertum using embryo rescue and characterization of F₁ plants. T. apertum was used as the male parent and T. alexandrinum as the female parent. Development of interspecific hybrids under natural conditions is not successful and so embryo rescue was attempted. Of the several combinations tried, pollination 2 days after emasculation and embryos rescued 11 days after pollination was found to be the best. For embryo culture, EC3 medium consisting of MS basal supplemented with 2.3 μM kinetin and 3% sucrose was used. Germinated embryos were transferred to LSP3 medium 25 days after inoculation wherein most of the cultures showed multiple shoots that were split and subcultured on RL1 medium for rooting. After hardening, about 75% of hybrids were successfully transferred to the field. The hybrids, in general, showed morphological traits intermediate between the two parents; however, a few hybrids showed better growth than either parent. Some F₁ plants were almost 3 weeks later in flowering than the female parent. Pollen fertility among these plants ranged from 78 to approximately 100%. Chromosomal associations at diakinesis and isozyme banding patterns for acid phosphatase (ACP), superoxide dismutase (SOD) and peroxidase also confirmed the hybrid nature of the plants.     

Production of doubled haploids in triticale (×Triticosecale Wittm.) by means of crosses with maize (Zea mays L.) using picloram and dicamba

The aim of the present study of triticale × maize crosses was to find an appropriate growth regulator treatment to improve the yield of triticale haploids and the subsequent production of doubled haploids. The growth effect in unpollinated ovaries of triticale was examined after treatment with 1000 mg/1 indole-3-acetic acid (IAA) or 100 mg/1 solutions of the following auxin analogues: 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-o-anisic acid (dicamba), 4-chloro-o-tolyloxyacetic acid (MCPA), phenylacetic acid (PAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4,5-trichlorophenoxyacetic acid (2,4,5- T), respectively. Dicamba stimulated growth of the ovaries significantly more than picloram and both stimulated more growth than the other growth regulators tested. Neither dicamba nor picloram induced embryo development in unpollinated pistils. Dicamba and picloram solutions, at concentrations of 25, 50, 75 and 100 mg/1, were subsequently applied to pistils of triticale pollinated with maize. On average, between 17.1 and 21.5 embryos/100 fiorets were excised after treatment with 75 or 100 mg/1 solutions of picloram or dicamba but the concentrations of 20 and 50 mg were less effective. The frequencies of excised embryos did not differ between genotypes. Seventy-six green haploids were obtained from 100 embryos rescued in vitro on the 190–2 and modified B5 media, the first medium being superior. The plants were subjected to colchicine treatment at the 3–4 tiller stage. Out of 68 plants brought to maturity, 25 exhibited fertile sectors. In comparison with previous studies, picloram and dicamba significantly improved the efficiency of the triticale × maize crossing. The low dependence on the mother germplasm makes triticale × maize crossing an efficient alternative to the androgenetic methods of doubled haploid production in triticale.     

Interspecific hybridization inBrassica juncea × Brassica hirta using embryo rescue

Summary Interspecific hybrids have been obtained in an otherwise incompatible cross betweenBrassica juncea × Brassica hirta through the in vitro culture of hybrid ovules and ovaries. The best response was observed from ovules and ovaries cultured 10–15 and 5–7 days after pollination respectively on a basal medium supplemented with indoleacetic acid, kinetin and casein hydrolysate. In some cases the basal cut end of the ovaries proliferated to form callus and shoots. The in vitro-derived hybrid seeds varied in their colour, size and shape, and the F₁ plants in the field showed a large diversity in their morphological traits. The hybrids were sterile, and had an intermediate number of chromosomes (2n=30).