Somatic Embryos and Plantlets from an Ancient Clone of the Grapevine (cv. Cabernet-Sauvignon) by Apomixis in vitro

Plantlets were produced by induction of nucellar embryony (apomixis)in isolated unfertilized ovules of a non-apomictic plant—thegrapevine (Vitis vinifera L. cv. Cabernet-Sauvignon). Ovulesgrown in liquid culture with benzyladenine (5–10 µM)and ß-naphthoxyacetic acid (5–25 µM) formeda nucellar callus which gave rise to somatic embryos. For growthof plantlets the embryos were transferred to an agar mediumcontaining gibberellic acid (1 µM) and 2-isopentenyladenine(5 µM). This is the first report of somatic embryo formationin a cultivar of a temperate fruit.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Androgenic potential in coconut (Cocos nucifera L.)

Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38°C) or cold (4°C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38°C for 6 days. Modified Eeuwens Y₃ liquid medium supplemented with 100 μM 2,4-dichlorophenoxyacetic acid (2,4-d), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y₃ medium containing 66 μM 2,4-d, followed by transfer to Y₃ medium without plant growth regulators and finally to Y₃ medium containing 5 μM 6-benzyladenine (BA) and 0.35 μM gibberellic acid (GA₃), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.     

Temperature-stress Pretreatment in Barley Anther Culture

Methods of pretreating anthers at different temperatures priorto culture have been tested, with respect to pollen-callus productionand plant regeneration, in Hordeum vulgare cv. Sabarlis. For callus production, pretreatment of excised spikes (in sealedPetri dishes) was more effective than pretreatment of excisedtillers (in water or in polythene) at both 4 and 25 °C.Pretreatment of individual anthers at these temperatures wasdeleterious. Greater callus yields resulted from pretreatmentat 4 than at 25 °C, both for spikes and tillers, 3–5weeks being required for maximal yields at 4 °C and 3–5days at 25 °C. At 4 °C, a shorter pretreatment was requiredfor spikes than for tillers. Pretreatment of spikes was alsomore effective at 4 than at 7, 14 or 20 °C. Pretreatmentof individual spikelets at 4 °C was as effective as thatof whole spikes. For plant regeneration, calluses derived from pretreatment ofspikes were more effective than those derived from pretreatmentof tillers. More plants resulted from pretreatment at 4 thanat 25 °C, both for spikes and tillers. Maximal pretreatmenttimes for plant regeneration generally exceeded those for callusproduction. Following spike pretreatment at 4 °C the maximumfor plant regeneration exceeded that for callus production byabout 2 weeks. With this optimal pretreatment approximately60 per cent of the calluses gave rise to plantlets. Among this60 per cent, for every three calluses giving albinos, two gavegreen plantlets, equivalent to five green plantlets on averagefor every 100 anthers (= two spikes) cultured. The ratio ofgreen to albino plantlets was lower for all other pretreatments. Hordeum vulgare L., barley, anther culture, pollen callus, pollen plant-production, temperature stress     

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration     

Influence of Genotype, Plant Growth Temperature and Anther Incubation Temperature on Microspore Embryo Production in Brassica napus ssp. oleifera

Eleven F₁ hybrid genotypes of winter rape (Brassica napus ssp.oleifera) were used in a study of induction and growth of microspore-derivedembryos. Plants of each genotype were grown in controlled environmentsat either a constant 15°C or a constant 20°C, both witha 16 h photoperiod. Equal numbers of buds, approximately 2.5mm in length, containing uninucleate microspores were harvestedfrom each genotype and either pretreated (14 d at 4°C) ordissected immediately after harvest. Anthers were cultured onliquid medium based upon that of Murashige and Skoog (1962)and containing 8% sucrose, 0.5 mg dm^–3 naphthylaceticacid and 0.05 mg dm^–3 benzylaminopurine. Anthers fromequal samples of buds were incubated at 35°C for 0, 1, 2or 3 d before transfer to 30°C (21 d) and then 25°C.After a total of 42 d incubation, cultures were scored for thepresence of macroscopic embryos (1–2 mm in length) andfor the presence of anthers containing aborted embryoids whichhad not developed further. The results showed first that bud pretreatment completely inhibitedinduction and secondly that anthers of all genotypes had anabsolute requirement for a 35°C treatment (optimal duration2 d) in order to induce embryoid formation. In the great majorityof genotypes plants grown at 15°C provided more productiveanthers than plants grown at 20°C. However, within eachtreatment there were great differences both in the frequencyof anthers showing induced embryoids and of the final yieldof embryos. There was evidence that hybrids with a common parentresponded similarly under certain treatments. This confirmedthe importance of genotypic control for some components of embryoyield. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Plantlet regeneration via somatic embryogenesis in anther callus of Vitis latifolia L.

Anthers of Vitis latifolia L. (wild grape) cultured on Nitsch and Nitsch medium supplemented with 20 μM 2,4-D and 9 μM BAP produced callus after 4–6 weeks. Subculture of callus onto Nitsch and Nitsch medium containing 10 μM NAA produced somatic embryos within 6 weeks. On growth regulator-free Nitsch and Nitsch basal medium somatic embryos converted to plantlets in 6–8 weeks. One gram of callus produced more than 400 somatic embryos with 13.7% being converted to complete plantlets, which were subsequently established in soil. Regenerated plants were found to have mixoploid populations of cells, 2n = 38 and n = 19. Received: 23 May 1998 / Revision received: 21 September 1998 / Accepted: 10 October 1998     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

HaploidCapsicum through experimental androgenesis

Summary Haploid embryos and plantlets in yet another solanaceous member viz.,Capsicum annuum L. var.grossum Sendt, have been successfully rearedin vitro through culture of young immature anthers. Exogenous auxin (IAA) was essential for the initiation of cell division in pollen oriented towards a haploid morphogenesis. Of the several hundred anthers cultured, not more than 0.1% produced viable plantlets.     

Androgenic Stimulating Factors in the Anther and Isolated Pollen Grain Culture of Datura innoxia Mill

Flower buds, anthers, and/or pollen grains collected at thetime of first haploid mitosis and 1–2 d before or afterthis division were submitted to different treatments beforeculturing anthers or isolated pollen grains. In the case ofanther culture, the percentages of androgenic anthem were notedat the end of 2, 3, 4, and 5 weeks of culture. Statistical studiesof the results thus obtained showed that some factors were highlyeffective in favouring androgenesis. The best results were obtained1–2 d after the first haploid mitosis with anther's centrifugedat 40 g for 5 min after cold treatment of the flower buds (48h at 3 °C); these treatments increased the percentage ofandrogenic pollen grains 12-fold. In case of isolated pollengrains, a system of culture particularly favourable for inductionand development of androgenic embryos was found. This systemincluded a cold treatment of the flower buds (48 h at 3 °C),the centrifugation of the isolated pollen grains (120 g for15 min), and culturing them for 20 d in the dark and then incontinuous light.     

Callus Induction and Plant Regeneration in Alstroemeria

Callus was induced from mature embryos of Alstroemeria cv. ‘Butterfly’cultured on MS medium supplemented with 2·0 or 4·0mg dm^–3 2,4–D or picloram and incubated at 25°Cin the dark. The effect of auxin concentration and precultureof embryos was studied. Callus was capable of regeneration aftertransfer to MS medium containing 4.0 mg dm°3 BAP. Shootsand whole plantlets were regenerated. The effect of growth regulators,used in the callus induction medium and the regeneration medium,on plant regeneration was studied Key words: Alstroemeria, callus, plant regeneration.     

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     

Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

‘Touriga Nacional’ is the most important Portuguese grapevine cultivar used for Port wine, table wine and varietal wine production. In order to obtain a reproducible plant regeneration system that allows the application of biotechnological tools to grapevine breeding, embryogenic cultures were induced from immature flowers of three Touriga Nacional selected clones. Gynoecia and anthers were cultured on Nitsch and Nitsch (Science 163:85–87, 1969) basal medium supplemented with four combinations of the growth regulators 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetyl-l-aspartic acid (IASP), at 28°C, in the dark. Primary callus, observed on anthers and gynoecia in all media, produced embryogenic callus when cultured on differentiation medium, at 24°C under light. The efficiency on induction of embryogenic callus ranged from 1.2 ± 4.7% to 7.9 ± 13.8% in anthers, and from 17.9 ± 24.9% to 25.3 ± 22.9% in gynoecia. Seven lines of embryogenic cultures were established from the three clones. Multiplication of embryogenic calluses was successfully obtained in maintenance medium, at 26°C, in the dark. These embryogenic calluses produced somatic embryos when subcultured on differentiation medium, under a 16 h photoperiod. Somatic embryos were isolated and cultured on germination medium to achieve conversion which ranged from 35.3 ± 48.5% to 72.7 ± 45.6%. The plantlets obtained were cultured in medium without growth regulators. Secondary embryogenesis was also frequently observed in the hypocotyl-root transition region of somatic embryos. Although some morphological variation occurred between somatic embryos, the regenerated plantlets had a normal phenotype. Maintenance of embryogenic cultures has been achieved since 2002.     

Production of in vitro haploid plants from in situ induced haploid embryos in winter squash (<Emphasis Type="Italic">Cucurbita maxima</Emphasis> Duchesne ex Lam.) via irradiated pollen

The influence of pollen irradiation on the production of in vitro haploid plants from in situ induced haploid embryos was investigated in winter squash (Cucurbita maxima Duchesne ex Lam.). Pollen were irradiated at different gamma-ray doses (50, 100, 200 and 300 Gray) and durations (9, 11, 15, 21, and 28 July). Production of in vitro haploid plantlets was influenced by irradiation dose, irradiation duration, genotype, and embryo type and embryo stage. Embryos were only obtained from lower irradiation doses (50 Gray and 100 Gray) and earlier irradiation durations (9, 11, and 15 July). The greatest embryo number per fruit was procured from “G14” and “55SI06” genotypes at 50 Gray gamma-ray dose. Necrotic embryos were higher than normal embryos at delayed harvest times (5 and 6 weeks after the pollination). The convenient harvest time for embryo rescue was observed about 4 weeks (between 25 and 30 days) after pollination. All cotyledon and amorphous embryos had only diploid plants while late-torpedo, arrow-tip, and pro-cotyledon embryos produced 33.3, 50.0, and 66.7% haploid plant. The frequency of haploid plantlets was 0.11, 1.17, 10.96 and 0.28 per 100 seeds, 100 embryos, 100 plantlets and a fruit at 50 Gray gamma-ray dose, respectively.     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

The Effects of Temperature and Light Intensity on Embryo Numbers in Wheat Doubled Haploid Production through WheatxMaize Crosses

Triticum aestivumxZea mayscrosses are now widely used in theproduction of wheat doubled haploids to produce homozygous lines.Seasonal effects are known to influence the number of haploidembryos produced through wheatxmaize crosses, but the effectsof temperature and light have not been quantified. This studyinvestigated the effect of temperature and light intensity onhaploid embryo production. New Zealand wheat cultivars weregrown in a glasshouse until booting when they were transferredto growth cabinets at three temperatures (day/night; 17/12,22/17 or 27/22 °C at an irradiance of 250 µmol m^-2s^-1PAR).In another experiment, wheat lines were transferred to a growthcabinet at one of three light intensities (300, 500 or 1000µmol m^-2s^-1PAR at 22/17 °C day/night, with a photoperiodof 16 h). The temperature and light intensity at which pollinationswere made and subsequent fertilisation and embryo developmentoccurred, significantly (P<0.01) influenced the frequencyof haploid embryo production. The optimal temperature for embryorecovery was 22/17 °C. The greatest number of embryos wasproduced at a light intensity of 1000 µmol m^-2s^-1. Thesefindings will result in improvements in the overall efficiencyof the wheatxmaize system for wheat doubled haploid production.Copyright1998 Annals of Botany Company Intergeneric crossing, temperature, light intensity,Triticum aestivum,wheat,Zea mays,maize.