Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Aneuploid plants derived from crosses with triploid grapes through immature seed culture and subsequent embryo culture

Through immature seed culture and subsequent embryo culture, aneuploid plants were derived from various crosses among 184 different triploid hybrid grape vines. In self-pollinations of the 184 vines, 0 to 1.6% of flowers produced immature seeds. In 16 reciprocal crosses between diploid and triploid and between tetraploid and triploid grapes, 0 to 23.0% of flowers produced immature seeds. The immature seeds excised 30–50 days after pollination were cultured for three months on Nitsch and Nitsch medium supplemented with L-glutamine, L-serine, L-cysteine and casein hydrolysate. Embryos developed within the cultured immature seeds were subcultured onto germination medium consisting of MS medium with 1 μM BA. Thirty-four of 137 embryos from 458 immature seeds germinated. Five of the 34 embryos grew normally. The five recovered plants were aneuploids with chromosome numbers from 51 to 59. The rates of embryo and plant recovery were different in different crosses with triploid grapes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Durum wheat as a candidate for the unknown female progenitor of bread wheat: an empirical study with a highly fertile F<Subscript>1</Subscript> hybrid with <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss.

Hexaploid bread wheat was derived from a hybrid cross between a cultivated form of tetraploid Triticum wheat (female progenitor) and a wild diploid species, Aegilops tauschii Coss. (male progenitor). This cross produced a fertile triploid F₁ hybrid that set hexaploid seeds. The identity of the female progenitor is unknown, but various cultivated tetraploid Triticum wheats exist today. Genetic and archaeological evidence suggests that durum wheat (T. turgidum ssp. durum) may be the female progenitor. In previous studies, however, F₁ hybrids of durum wheat crossed with Ae. tauschii consistently had low levels of fertility. To establish an empirical basis for the theory of durum wheat being the female progenitor of bread wheat, we crossed a durum wheat cultivar that carries a gene for meiotic restitution with a line of Ae. tauschii. F₁ hybrids were produced without using embryo rescue techniques. These triploid F₁ hybrids were highly fertile and spontaneously set hexaploid F₂ seeds at the average selfed seedset rate of 51.5%. To the best of our knowledge, this is the first example of the production of highly fertile F₁ hybrids between durum wheat and Ae. tauschii. The F₁ and F₂ hybrids are both similar morphologically to bread wheat and have vigorous growth habits. Cytological analyses of F₁ male gametogenesis showed that meiotic restitution is responsible for the high fertility of the triploid F₁ hybrids. The implications of these findings for the origin of bread wheat are discussed.     

Extensive citrus triploid hybrid production by 2x?×?4x sexual hybridizations and parent-effect on the length of the juvenile phase

The citrus fresh market demands the production of seedless citrus fruits, as seedy fruits are not accepted by consumers. The recovery of triploid plants has proven to be the most promising approach to achieve this goal, since triploids have very low fertility, are generally seedless and do not induce seeds in other cultivars by cross pollination. Triploid plants can be recovered by 2x?×?4x sexual hybridization. In this work, we present an effective methodology to recover triploid plants from 2x?×?4x hybridizations based on in vitro embryo rescue, ploidy level analysis by flow cytometry and genetic origin of triploid plants. The pollen viability of diploid and tetraploid citrus genotypes was analyzed by comparing the pollen germination rate in vitro. The pollen viability of tetraploid (doubled-diploid) genotypes is generally reduced but sufficient for successful pollination. Triploid embryos were identified in normal and undeveloped seeds that did not germinate under greenhouse conditions. The influence of parents and environmental conditions on obtaining triploid plants was analyzed and a strong interaction was noted between the parents and environmental conditions. The parental effect on the length of the juvenile phase was also demonstrated through observations of a large number of progeny over the last 15?years. The juvenile phase length of the triploid hybrids obtained with 'Fortune' mandarin as female parent and tetraploid 'Orlando' tangelo as male parent was shorter than the juvenile phase obtained with a clementine as female parent and tetraploids of 'Nova', 'W. Leaf' and 'Pineapple' male parents. Key message Effective methodology to recover citrus triploid plants from 2x?×?4x sexual hybridizations and the parental effect on the length of the juvenile phase.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

Micropropagation and production of arbutin in oriental strawberry tree (<Emphasis Type="Italic">Arbutus andrachne</Emphasis> L.)

A protocol for micropropagation of Arbutus andrachne from seeds was developed. Results indicated that none of the seeds cultured on Murashige and Skoog (MS) medium, with or without plant growth regulators (PGRs), germinated. Seeds soaked in 250 mg l⁻¹ gibberellic acid (GA₃) at 4°C for 3 days, then cultured on water-agar medium containing 2.0 mg l⁻¹ GA₃ exhibited 80–100% germination and developed into usable seedlings. Shoot proliferation was tested on MS or B5 medium containing different concentrations of cytokinin. No shoot proliferation was observed on PGR-free medium. Proliferation was more successful on MS than on B5 medium. On both media, the most successful proliferation was obtained using zeatin as a cytokinin type. Rooting was tested on MS medium containing different concentrations of auxin. Rooting failed on PGR-free medium and on medium containing indole-3-acetic acid (IAA), 0.25 or 0.5 mg l⁻¹ indole-3-butyric acid (IBA), or 0.25, 0.5 or 2.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooting (40%) was most successful with 1.0 mg l⁻¹ NAA. Rooted plantlets exhibited 80% survival in all mixtures of peatmoss and perlite, and acclimatized plants were successfully grown in the greenhouse. Quantitative analysis of arbutin performed on in vivo and in vitro leaves using high-performance liquid chromatography (HPLC) revealed that in vivo leaves contained higher arbutin content (0.3–0.81% w/w) than in vitro leaves (0.09% w/w). The highest yield of arbutin in vivo was detected in leaves collected in August, and the lowest yield in leaves collected in December.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

三倍体罗汉果及其亲本的同工酶比较研究

为了加快三倍体罗汉果育种的进程和效率,进一步提高其亲本选择性,该文对F1代3x罗汉果与其4x和2x亲本的过氧化物酶(POD)同工酶和酯酶(EST)同工酶进行了比较。结果表明:F1代3x罗汉果与其4x和2x亲本的POD和EST同工酶均有一定差异,3x和4x的POD和EST同工酶不同迁移率的平均酶带数目较2x多,酶带活性也较强;4x的POD同工酶数目较3x的多,但EST同工酶两者差异较小;F1代3x罗汉果的同工酶均出现了与其4x和2x亲本不同的新酶带,预示着其可能具有的杂种优势;聚类分析结果显示,F1代3x罗汉果与其4x母本的遗传距离更近,并且当4x母本间亲缘遗传关系较近时,其F1代3x在亲缘遗传上也相聚较近,说明F1代3x在亲缘遗传上更倾向于其4x母本。由于三倍体罗汉果育种亲本间存在杂种优势,且更倾向于其母本遗传,因此通过该研究可以初步总结出三倍体罗汉果及其父本、母本的遗传规律,并提出罗汉果三倍体良种选育的建议,即在3x罗汉果育种时需要更加关注4x母本的优良性状表现,并以亲本的遗传背景为基础,选择遗传差异较为显著的父本和母本。     

Ovule rescue efficiency of Gossypium hirsutum × G. arboreum progeny from field-grown fruit is affected by media composition and antimicrobial compounds

Upland cotton (Gossypium hirsutum L.) is reproductively isolated from G. arboreum L. via post-zygotic breeding barriers. Literature on somatic embryogenesis of cotton suggests a number of media modifications that might also prove useful for ovule rescue of interspecific crosses. Additionally, endogenous microbes are common in field grown cotton and these potential contaminants must be controlled if interspecific progeny are to be obtained via large-scale field crossing followed by ovule or embryo culture. This study compared nine tissue culture media and two antimicrobial overlay treatments in a factorial design. The overlay treatments were: a 2 ml overlay of 250 mg l⁻¹ cefotaxime, 50 mg l⁻¹ tetracycline HCl, 2.5 mg l⁻¹ amphotericin B, and 50 mg l⁻¹ benomyl applied when the ovules were plated, and no overlay. All of the media in the factorial also contained 250 mg l⁻¹ cefotaxime. Crosses were made in a field at Stoneville, MS between the upland cultivar DeltaPine 90 and the G. arboreum accession A₂-190. Antimicrobial compounds greatly improved the efficiency of obtaining interspecific cotton progenies from field-grown fruit. Germination was not affected by the overlay nor did overlay treatment interact with media. Media significantly affected germination. Of the media studied, the highest frequency of germination was observed for MSB with 1.9 g l⁻¹ additional KNO₃. The addition of 0.5 g l⁻¹ asparagine and 1 g l⁻¹ glutamine did not affect the number of seedlings obtained. A filter paper growing surface or the addition of 0.5 mg l⁻¹ NAA and 0.05 mg l⁻¹ kinetin were disadvantageous.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Use of 2n pollen-producing triploid hybrids to introduce tetraploid Mexican wild species germ plasm to cultivated tetraploid potato gene pool

Summary Tetraploid (2n=4x=48) 2EBN Mexican wild species in the series Longipedicellata, which consists of Solanum fendleri, S. hjertingii, S. papita, S. polytrichon, and S. stoloniferum, were crossed with two 2EBN cultivated diploid (2n=2x=24) clones. The resulting triploid hybrids (2n=3x=36) produced 2n pollen (triplandroids) by the mechanism of parallel orientation of anaphase II spindles. The percentage of stainable pollen in 520 triploids ranged between 0 and 23.5%, with a mean of 2.7%. Triploids producing between 13.0 and 23.5% stainable pollen were crossed as staminate parents to the tetraploid cultivars, resulting in abundant pentaploid (2n=5x=60) and near-pentaploid hybrid progeny. Crosses of triploids with lower percentage of stainable pollen as pollen parent to the tetraploid cultivars did not yield fruit, unless rescue pollen from a tetraploid cultivar was added 2 days later. Pentaploid hybrids were selected among selfed tetraploid progenies using morphological and isoxyme markers transmitted from their cultivated diploid parents. These pentaploid hybrids were vigorous and had uniformly sterile pollen. They were female fertile and were crossed with tetraploid cultivars, yielding an average of 19 seeds per fruit. Triplandroids provide the opportunity of transferring 2EBN tetraploid Mexican wild species in the series Longipedicellata germ plasm into the 4EBN cultivated potatoes.Cooperative investigations of the ARS, USDA, and the Washington State University Agricultural Research Center, Prosser, WA 99350, USA. H/LA Paper No. 90-03, College of Agriculture and Home Economies Research Center, Washington State University, Pullman, WA 99164, USA     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Influence of agar and activated charcoal on uptake of gibberellin and plant morphogenesis In vitro

Summary Agar and activated charcoal (AC) are commonly used in tissue culture. However, their deeper actions and functions are largely unknown. This experiment investigated the effect of agar and AC, singly and jointly, on gibberellin (GA) uptake by corn shoots. Corn seeds were germinated on Murashige and Skoog medium (MS). Shoot excised from 1-wk-old seedlings were cultured on liquid (0.0 g l⁻¹ agar) or solid (8 g l⁻¹ agar) MS containing 3 μM indole-3-acetic acid, 13.3 μM N⁶-benzyladenine, and 6000 CPM ml⁻¹ [³H]GA₄ as tracer. Both liquid and solid media had two treatments, one without AC and one supplemented with 5 g l⁻¹AC. Uptake of [³H]GA₄ and morphogenesis of corn shoots were recorded after 2 wk of culture. Corn explants cultured in AC-free media acquired high levels of [³H]GA₄, while explants from AC-containing media showed only traces of [³H]GA₄. Explants cultured in AC-free liquid medium contained about twice the amount of [³H]GA₄ as those from AC-free solid medium. Addition of agar reduced shoot length, while addition of AC increased both shool and root length. It is concluded that: (1) agar reduced the uptake of GA₄; and (2) GA₄ was irreversibly adsorbed by AC, and thus became unavailable to corn explants.     

Factors influencing efficiency of somatic embryogenesis of Gentiana kurroo (Royle) cell suspension

In this paper, we would like to show unexpected morphogenic potential of cell suspensions derived from seedling explants of Gentiana kurroo (Royle). Suspension cultures were established with the use of embryogenic callus derived from seedling explants (root, hypocotyl and cotyledons). Proembryogenic mass proliferated in liquid MS medium supplemented with 0.5 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ Kin. The highest growth coefficient was achieved for root derived cell suspensions. The microscopic analysis showed differences in aggregate structure depending on their size. To assess the embryogenic capability of the particular culture, 100 mg of cell aggregates was implanted on MS agar medium supplemented with Kin (0.0–2.0 mg l⁻¹), GA₃ (0.0–2.0 mg l⁻¹) and AS (80.0 mg l⁻¹). The highest number of somatic embryos was obtained for cotyledon-derived cell suspension on GA₃-free medium, but the best morphological quality of embryos was observed in the presence of 0.5–1.0 mg l⁻¹ Kin, 0.5 mg l⁻¹ GA₃ and 80.0 mg l⁻¹ AS. The morphogenic competence of cultures also depended on the size of the aggregate fraction and was lower when size of aggregates decreased. Flow cytometry analysis reveled luck of uniformity of regenerants derived from hypocotyl suspension and 100% of uniformity for cotyledon suspension.     

Implementation of extensive citrus triploid breeding programs based on 4x?×?2x sexual hybridisations

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x?×?2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x?×?2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3?%), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x?×?2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations.     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

In vitro propagation of the endangered orchid Laelia speciosa

Laelia speciosa is an endangered epiphytic orchid. The effects of various media components on germination of L. speciosa were evaluated. Pods were collected at 4, 7, and 9 months following hand-pollination, and seeds were germinated on Murashige and Skoog (MS) media with 30 g l⁻¹ sucrose and five concentrations (0.0, 0.04, 0.22, 0.44, and 2.22 μM) of benzyladenine (BA) under light and dark conditions. Gibberellic acid (GA₃; 0.0, 0.29, 1.44, 2.89, 14. 43 and 28.87 μM) with naphthaleneacetic acid (NAA; 0.0, 0.54, 1.34, 2.69, and 5.37 μM) were evaluated for in vitro subcultivation. MS medium with 30 g l⁻¹ sucrose was effective for germination. The effects of BA and light on germination of L. speciosa seeds differed with pod maturity. All mature seeds germinated using 0.44 μM BA and light. The highest frequency of germinated seedlings (60%) was obtained using mature seeds grown on MS medium without BA and under light conditions. For subculture, MS with 30 g l⁻¹ sucrose, 2.69 μM NAA, and 0.29 μM GA₃ was effective. Plantlets of 5 cm in length were transplanted to the greenhouse, and a 77.5% of survival rate was obtained. A successful protocol for micropropagation by seed germination will contribute to the development of a sustainable management program for L. speciosa.     

Evaluation of key factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of somatic embryos of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.)

Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l⁻¹ kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l⁻¹ partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l⁻¹ kanamycin and 250 mg l⁻¹ timentin for 2 months. Then, kanamycin level was increased to 100 mg l⁻¹ for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA₃ for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l⁻¹ agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.