In vitro attempts to overcome the cross-incompatibility between Vaccinium corymbosum L. and V. elliottii Chapm.

Summary Seed was readily obtained from V. corymbosum zygotes using embryo rescue techniques, even when embryos were cultured at proembryonic stages. Best in vitro seed development was obtained when ovules were cultured attached to placental tissues. Successful fruit and seed development in culture occurred only when the fruit was cut longitudinally or when the basal portion of the fruit was removed previous to plating. Addition of various vitamins, amino acids, and growth regulators to the nutrient medium did not increase seed production. Attempts to rescue hybrid embryos from V. croymbosum (tetraploid) x V. elliottii (diploid) crosses by in ovulo and in ovary culture gave a few presumably hybrid seed, but at a rate no greater than when normal crossing procedures are used.Florida Agricultural Experiment Station Journal Series No. 5748     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

4种野生兰花种子特征及离体培养初报

对4种野生兰花--虾脊兰、羊耳蒜、石仙桃及密花石斛的果荚、种子、种胚性状进行初步研究,并对其种子进行离体培养。结果显示,4种野生兰种子为白色、淡绿或黄褐,呈纺锤体或椭球体。羊耳蒜、石仙桃及密花石斛种子具种孔,种胚浓密且清晰可见;种子萌发较快且萌发率高,其中以石仙桃及密花石斛种子萌发最快,仅需36d,萌发率高达100%。虾脊兰种子未见种孔,种胚不甚明显,种子萌发缓慢且萌发率低。4种兰科植物种子均以原球茎方式萌发,通过继代培养,获得了一批无菌苗。     

Minituber production from immature seed suspension culture ofOrchis papilionacea

Summary Ripe and immature seeds ofOrchis papilionacea (method I and II, respectively) cultured on modified double strength Curtis medium were assayed for minituber production. Ripe seed germination both on solid and in liquid medium was low and the protocorms obtained developed into white calluses. Germination increased from, 9 to 33% when immature seed suspension culture was used. Protocorms obtained in suspension culture under light developed into minitubers, whereas those obtained on solid media developed into callus. A 30 s ultrasonication of immature seeds 1 wk after suspension, culture initiation further enhanced germination and minituber production. Minitubers had to be transferred and embedded in solid regeneration medium for normal growth.     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

Induction of vivipary in Arabidopsis by silique culture: implications for seed dormancy and germination

Culture of excised fruits (siliques) of different ages of Arabidopsis thaliana in a solidified mineral salt medium supplemented with vitamins, myo-inositol, and 3% sucrose induces vivipary. Whereas early stage and immature embryos complete their full development before germinating viviparously in seeds enclosed in the silique, mature green embryos enclosed in green ovules germinate without further growth in culture. Vivipary is not observed in cultured siliques enclosing brown ovules with yellowish mature embryos inside. Suggestive of a role for abscisic acid in preventing vivipary on the mother plant, addition of the hormone to the culture medium is found to inhibit vivipary in cultured siliques. Although dried green ovules enclosing mature embryos require a cold treatment for germination, undried ovules of the same age do not germinate even after a cold treatment. This indicates that mature embryos enclosed in green ovules that germinate viviparously are cold resistant and have not become dormant at the time of culture of siliques. The circumvention by silique culture of a cold treatment and light exposure normally required for germination of isolated seeds of A. thaliana provides new possibilities to study the molecular biology of vivipary and seed germination in this model plant.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Recovery of cassava (Manihot esculenta Crantz) plants from culture of immature zygotic embryos

An embryo culture protocol using immature cassava seeds has been developed to enhance successful seed germination and reduce time for population establishment. Embryonic axes were excised from seeds 40 days after pollination and placed on 1/3 MS medium supplemented with growth factors. Fruits were either air-dried at 20 °C to aid dehiscence, or dissected immediately after harvest. Culture of embryonic axes from seeds obtained from mature fruits (90 days after pollination) served as control. Average percent germination and plantlet recovery rate were higher for embryos cultured from non air-dried immature seeds than from air-dried immature seeds. Immature seeds that were air-dried before germination had ≥50% reduction in germination rate and ≥75% reduction in plantlet recovery rate, indicating that cassava immature zygotic embryos are susceptible to osmotic pressure changes. Genotypic effects were observed in shoot elongation, formation of internodes, and vigor of cultures from both mature and immature seeds. The high percentage of plants recovered from immature seeds through embryo culture opens up opportunities for genetic stock development in cassava that has been previously unexplored. This revised version was published online in June 2006 with corrections to the Cover Date.     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997     

An Analysis of Seed Development in Pisum sativum: VIII. DOES ABSCISIC ACID PREVENT PRECOCIOUS GERMINATION AND CONTROL STORAGE PROTEIN SYNTHESIS?

The influence of abscisic acid (ABA) on the precocious germinationand storage protein production of pea seeds has been examinedusing embryo and pod culture. The precocious germination ofembryos in culture could not be inhibited fully by ABA on apermissive medium (2% sucrose) even at 0.1 mol m^–3. However,increasing the sucrose concentration to 5% caused near completeinhibition when ABA was added to the medium. Embryos of differentweights cultured on a high osmoticum (mannitol-containing medium),equivalent to 10% sucrose, did not show any consistent differencein ABA content. When fluridone was added to a non-permissiveculture medium, no decrease in ABA content of the embryos couldbe observed and no precocious germination was induced. In contrast,fluridone was able to prevent the accumulation of ABA in seedspresent in pods cultured in its presence from an early stageof development. These seeds, however, grew normally and reachedmaturity, did not germinate precociously in vivo, were desiccationtolerant and still produced storage protein message whetheror not ABA was included in the culture medium. It does not appear,therefore, that ABA regulates normal development or storageprotein synthesis in pea embryos. Key words: Abscisic acid, peas, Pisum sativum, seed development     

Hyperosmotic stress-induced somatic embryogenesis and its continuous culture in Japanese honewort (Cryptotaenia japonica)

Japanese honewort (Cryptotaenia japonica) is consumed as a traditional vegetable and has medicinal applications. In Japan, C. japonica is mainly produced using hydroponic culture systems; however, damping-off is often caused by the adherence of pathogens to its seeds. Therefore, the use of sterile artificial seeds in hydroponic culture is likely to be effective for preventing disease. In this study, we established methods for stress-induced somatic embryogenesis and artificial seed production in Japanese honewort. Shoot apex explants from seedlings were treated with 0.7 M sucrose as a hyperosmotic stress for 3 or 6 weeks, and then transferred to stress-free conditions. Somatic embryos were formed after culture in stress-free conditions for 7 weeks. Stress-treated shoot apex explants that formed somatic embryos were cultured in Murashige and Skoog liquid medium with shaking. After 2 weeks of culture, approximately 800 somatic embryos were formed from each explant. Somatic embryos were formed continuously during 37 weeks under the same culture conditions. Thus, somatic embryogenesis was effectively induced in Japanese honewort via hyperosmotic stress, and embryogenic competence was maintained under stress- and phytohormone-free conditions. The somatic embryos produced by liquid culture were used to produce artificial seeds by enveloping the embryos in whipped alginate gel to avoid hypoxic conditions. The artificial seeds had a high germination rate (72%). This system is suitable for the sterile, highly productive hydroponic culture of Japanese honewort.     

Beneficial effects of taurine on mouse zygotes developing in protein-free culture medium

The objectives of this study were to determine if mouse zygotes from outbred mice can develop in simple culture medium in the absence of bovine serum albumin (BSA), and if taurine can be used as a medium supplement to improve development. Zygotes from 2 stocks of outbred mice (CD-1 and CF-1) were cultured in simple embryo culture medium (TE medium) lacking BSA and with or without taurine (24 mM), or in regular TE medium with BSA. The presence of BSA had little or no effect on development, but development to post-blastocyst endpoints was enhanced when CD-1 zygotes were cultured in medium containing taurine. In addition, when CD-1 blastocysts were transferred to pseudopregnant animals, embryos cultured in the presence of taurine developed into fetuses more often than those cultured in medium without taurine, and their weights were higher than those of embryos cultured in regular TE medium with BSA. These beneficial effects of taurine do not appear to be the nonspecific effects of a fixed nitrogen source, because the addition of glycine to BSA-free TE medium did not have similar beneficial effects. It was concluded that mouse zygotes from outbred mice do not require BSA for their preimplantation development in culture and that the presence of taurine in preimplantation culture medium is beneficial not only for preimplantation development of the zygotes, but also for their post-blastocyst development.     

An analysis of seed development in Pisum sativum. VII. Embryo development and precocious germination in vitro

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.     

Effects of media on embryo enlargement,germination and plant development in early-ripening genotypes of Prunus grown in vitro

Embryo culture of 5–10 mm long embryos of Prunus was investigated. The effects of various media on embryo enlargement, germination and plant formation were compared. Results show that embryos in all genotypes enlarged during stratification on any tested medium. Beneficial embryo enlargement, germination and plant development of peach and nectarine occurred when cultured on WP medium. The embryos of a plum were more responsive to C₂d medium for enlargement, germination and plant development. All genotypes germinated well with a large number of embryos growing into plants on WP and C₂d media.     

Production of aneuploid melon plants following in vitro culture of seeds from a triploid x diploid cross

Aneuploid melon plants (Cucumis melo L.) were obtained from in vitro cultured seed, which were produced by crossing triploid (3x=36) x diploid (2x=24) plants. Twenty-six fruits were obtained from pollination of 29 bisexual flowers of triploid plants. Seeds were collected from the fruits at 2, 3, 4, 5 and >7 weeks after pollination and germinated in vitro on Murashige & Skoogs (MS) medium. Embryos developed from 0.6 to 1.6% of the cultured seeds after three weeks in culture. Shoots developed from 0 to 47% of embryos after transfer to half-strength MS medium. Some (0 to 50%) of elongated shoots that rooted were subcultured on the same medium. Five rooted plantlets were obtained through culture of 5,353 seeds. Four of the plants were aneuploid, with chromosome numbers of 27, 35, 45 and 46, respectively, and the one was tetraploid (4x=48).     

Embryo culture and taxane production inTaxus spp

Summary We have previously shown (Flores and Sgrignoli, 1991) that immature embryos ofTaxus brevifolia andT. X media are capable of precocious germination and can grow into seedlings in vitro. The cultural and environmental parameters for embryo germination and conversion into seedlings have been optimized and extended toT. baccata andT. cuspidata. A 14-h photoperiod improved embryo germination and growth into seedlings. A pregermination cold treatment of the seeds had a positive effect on both the onset and percentage of germination. Embryos from cold-treated seeds germinated earlier and at a higher frequency than those from control seeds. Boron was necessary for embryo germination, and levels of this micronutrient were established for optimal growth and germination ofT. brevifolia andT. X media cv. Hicksii embryos. Gupta and Durzan’s medium was superior to White’s for embryo germination and root formation. Naphthaleneacetic acid stimulated root formation in embryo-derived seedlings. We also found that immature embryos could be induced to form callus with embryogenic potential. Taxol and related taxanes were detected in embryo- derived seedlings.     

Sucrose effects on in vitro fruiting and seed production of Centaurium pulchellum

The effect of sucrose on fruiting, seed production, and seed germination of lesser centaury [Centaurium pulchellum (Sw.) Druce] was examined using explants of flowers and flower buds. Sucrose concentrations in the culture medium ranged from 0.003 to 0.3 M. It has been shown that the number of auxiliary buds, capsules dimension, number of viable seeds per capsule and seed dimensions increased with the increase of sucrose concentrations. The highest values were recorded at sucrose concentrations higher than 0.03 M, except for seeds size, which were larger at sucrose concentration ranging from 0.003 to 0.1 M. The germination of in vitro produced seeds was affected by previous culture history: a higher germination percentage was obtained in seeds that were raised from explants originally grown on medium with sucrose concentrations higher than 0.003 M.     

Somatic embryo formation and germination from immature embryo-derived suspension-cultured cells of Angelica sinensis (Oliv.) Diels

Embryogenic callus was induced from immature embryos of Angelica sinensis cultured on Murashige and Skoog (MS) basal medium. Embryogenic callus growth was more rapid on MS basal medium than on B5 or White medium. Embryogenic callus was used to establish a suspension culture and somatic embryos and germinating embryos developed during the culture. A shaking speed of 80 rpm was found to be optimal for establishing suspension cultures, while 100 rpm produced more somatic embryos and germinating embryos with an initiation cell density of 0.2 ml packed cell volume/25 ml medium. Adding 0.3% agar to the liquid medium also stimulated the formation of somatic and germinating embryos. While no plant growth regulators were needed for culture initiation and plant regeneration, the addition of 0.5–1 mg/l 2,4-dichlorophenoxyacetic acid was needed to maintain the embryogenic suspension culture by preventing embryo germination. Forty percent of the germinating embryos survived after culturing on filter paper moistened with liquid half-strength MS medium containing 3% sucrose. The plants were successfully transferred into soil. Received: 19 March 1997 / Revision received: 21 November 1997 / Accepted: 19 January 1998     

狭叶薰衣草的离体培养及挥发性成分的分析

为建立新疆狭叶薰衣草(Lavandula angustifolia)的快速繁殖体系,以种子、茎、叶为外植体,对种子萌发、愈伤组织诱导、丛芽分化和生根的最适培养条件进行了研究;用水蒸气蒸馏法提取狭叶薰衣草挥发油,采用气相色谱-质谱法测定挥发油成分。结果表明,种子浸泡的适宜时间为6 h,切开种皮培养,出芽时间最少为6 d;诱导种子出芽的适宜培养基为MS+6-BA2 mg/L;以茎为外植体诱导愈伤组织效果较好,适宜培养基为MS+6-BA 2 mg/L+2,4-D 1 mg/L;诱导分化丛芽的适宜培养基为MS+6-BA 1 mg/L+NAA 0.5 mg/L;生根的适宜培养基为1/2MS+NAA 1 mg/L+6-BA 0.5 mg/L;盆栽薰衣草和无菌苗薰衣草的挥发油主要成分相差较大,离体培养的薰衣草的主要挥发性成分有叶绿醇、丁香油烃、氧化石竹烯等。     

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.