Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

Improved Regeneration Efficiency from Mature Embryos of Barley Cultivars

A reliable protocol for plant regeneration from mature embryo derived calli of nine barley (Hordeum vulgare) cultivars has been developed. The auxins 2,4-dichlorophenoxyacetic acid, picloram and dicamba proved effective in inducing callus from mature embryos of most of the barley cultivars. The induced primary callus was loose, friable and translucent. It ultimately yielded creamy white and compact callus after 2 - 3 transfers on fresh medium of the same composition. Callus induction and regeneration capacity were highly cultivar dependent. Addition of a high concentration of picloram (4 mg dm^-3) promoted regeneration in 3 cultivars (Tallon, Grimmett and Sloop). In cv. Arapiles, abscisic acid and betaine were crucial in generating morphogenic callus from the mature embryos. Plants regenerated from these calli were hardy and developed roots readily when transferred to hormone free medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

CHARACTERIZATION OF TISSUE CULTURE INITIATION AND PLANT REGENERATION IN SPOROBOLUS VIRGINICUS (GRAMINEAE)

Tissue cultures of the halophytic saltmarsh grass Sporobolus virginicus were initiated from unemerged immature inflorescence tissue. Typical graminaceous embryogenic and nonembryogenic callus and cell types were noted. Embryogenic callus was compact golden yellow. Histological evidence indicated that proliferation of the ovary tissue of the immature pistil was the source for embryogenic callus. Plants regenerated after first reducing and then eliminating auxin from the culture medium. Regeneration was observed both through the concerted development of bipolar meristems from somatic embryos and by the formation of multiple shoot meristems that were either connected through callus tissue to root meristems or which later adventitiously rooted. The main mode of regeneration appeared to be somatic embryogenesis with additional multiple shoot formation probably due to precocious germination of somatic embryos. Plants recovered from culture were acclimated to soil, grown up in a greenhouse, and planted in field plots with saline irrigation to ensure stability of salt tolerance.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM TISSUE CULTURES OF PENNISETUM AMERICANUM,AND P. AMERICANUM x P. PURPUREUM HYBRID

Immature embryos as well as explants obtained from young inflorescences of Pennisetum americanum (pearl millet) give rise to callus tissues on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). A compact and pale-yellow callus that arises from the peripheral cells of the scutellum, and from the young inflorescences, undergoes further organized growth. When transferred to a 2,4-D-free medium, supplemented with indole-acetic acid or kinetin, or both, embryoids are formed in the organized areas of the callus. Embryoids show a bipolar organization with a shoot-coleorhiza (root) axis and have a coleoptile-like structure surrounded at the base by a cup-shaped structure that resembles the scutellum in texture and morphology. Embryoids show bilateral or radial symmetry and “germinate” in vitro to form plants that have been grown to maturity in soil. Similar embryogenic callus cultures have been produced from young inflorescence tissues of hybrid Pennisetum, a triploid sexually sterile hybrid of P. americanum x P. purpureum. Plants derived from these have also been transferred to soil. The regenerated plants showed normal chromosome numbers.     

Factors influencing <Emphasis Type="Italic">in vitro</Emphasis> regeneration from protoplasts of wild cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> A) and RAPD analysis of regenerated plantlets

Plants of a diploid wild cotton species (G. klotzschianum A.) were efficiently regenerated from protoplasts isolated from immature somatic embryos and suspension cultures by studying various factors affecting regeneration. Purified protoplasts were cultured with the density of 2–10×10⁵ ml⁻¹, and the medium was k₃ inorganic salts with modified KM₈P organic compositions, supplemented with several combinations of PGRs. Calluses were formed from protoplasts of suspension cultures and immature somatic embryos. The influences of carbon sources and GA₃ on callus differentiation and somatic embryo germination were analyzed. Somatic embryos germinated normally and formed regenerated plantlets. Regenerated plantlets were transferred to the soil and seeds were obtained. Random amplified polymorphic DNA (RAPD) analysis using 80 arbitrary oligonucleotide 10-mers showed 23 primers that gave 74 clear reproducible bands, with amplification products being monomorphic for 14 tested plantlets. A total of 1036 bands obtained exhibited no aberration in RAPD banding patterns in the 14 plants. Plants regenerated via somatic embryogenesis from the diploid cotton protoplasts have genetic homogeneity.     

不同组织培养途径对小麦再生能力的研究

从现在推广的小麦优良品种和有苗头的新品系中选用10个小麦基因型品种进行组织培养,从愈伤组织诱导率、绿苗分化率等方面比较了幼穗培养、花药培养、幼胚培养三种培养方式的培养效果。结果表明,幼胚培养效果最好,基因型间差异小,都能获得足够数量的再生植株。幼穗的培养效果最差,愈伤组织分化生根和绿芽十分容易,但分化成完整植株则较为困难。花药培养在基因型间差异非常明显而且有较多白化苗。此外,本研究还分析了影响小麦再生能力的因素,建立了一套高效、可靠的小麦组培再生系统,为小麦的转基因技术提供优良的受体材料。     

Induction and characterization of embryonic callus types for the initiation of suspension cultures of leek (Allium ampeloprasum L.)

In this paper the development and characterization of a friable, embroyonic callus culture of leek is described. This callus type was initiated on immature embryos and differed in appearance from formerly induced compact, embryogenic callus [4]. The friable callus was comprised of numerous globular embryoids, embedded in a mucilaginous substance. The genotype of the donor plant and the embryo size were important parameters in the initiation of this callus type. Embryos of 0.5–2.5 mm gave the highest frequency of friable callus production. The basal media and inclusion of -proline into the media did not influence the friable callus production. Light microscopic comparison of compact and friable callus showed striking differences. Compact callus consisted of a meristematic zone and contained vascular elements. Friable callus was less differentiated and contained aggregates of embryonic cells, separated by intercellular spaces, and somatic embryos. Ten independently induced friable callus cultures were tested for their amenability to form suspension cultures. From one of these, two highly embryonic suspension cultures were selected.     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

Induction of morphogenically competent callus and suspension cell cultures from leaf explants and mature seeds ofDigitaria sanguinalis

Summary Digitaria sanguinalis (crabgrass) has recently been introduced as a high quality forage crop. We report here a tissue culture system showing a high level of regeneration developed to aid in a breeding program. Two morphologically distinct types of callus, compact opaque and friable translucent, were induced from leaf blade explants and mature seeds when cultured on MS medium containing 0.9 μM 2,4-dichlorophenoxyacetic acid. Proline (25 mM) inhibited induction of callus but was required for continued maintenance. Plants were readily regenerated from the compact opaque callus. Selectively subcultured friable translucent callus continued to produce colony sectors of the morphogenically competent compact opaque callus when transferred to regeneration medium. Suspension cell cultures derived from callus or directly from leaf blade explants also produced regenerable callus.     

A comparative study of somatic embryogenesis in Secale vavilovii

Summary The ability of immature embryos, inflorescences and leaves of Secale vavilovii to form embryogenic callus was tested on Murashige and Skoog (1962) medium supplemented with different concentrations of 2,4-D. All cultured immature embryos formed calluses. The highest percentage of embryogenic callus production was from 1–2 mm embryos. Young leaves also formed calluses, mainly from the 10–15 mm basal segment, the percentages of embryogenic calluses being higher when cultures were maintained in darkness. Embryogenic calluses were obtained also from all the cultured immature inflorescences, in the three cases, rooted green plants were obtained and grown in soil. Comparison of the responses of the three explants used indicates that immature inflorescence is the most useful explant for obtaining regenerated plants in Secale vavilovii.     

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

Summary Immature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as aged callus. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.Florida Agricultural Experiment Station Journal Series No. R-00494     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

小麦不同生理状态的幼穗和幼胚盾片与诱导分化能力关系的研究

用植物组织培养的方法,研究了冬小麦品种鄂恩1号和品系鄂55072不同生理状态的幼穗和幼胚盾片与诱导分化的关系。结果表明,长度在0.4-2.0cm间的幼穗和直径在0.4-1.5mm间的盾片随生理状态不同,其诱导和分化频率有明显的差异,幼嫩的材料再生频率较高。经SAS统计分析,发现0.5-1.0cm长的幼穗和直径为0.4-1.1mm的盾片是处于诱导分化的最佳生理时期的实验材料,平均每个胚性愈伤组织的植株再生分别达到3.17和5.63株。通过比较幼穗和幼胚盾片的植物组织培养结果,发现幼胚盾片比幼穗愈伤组织出现早,生长快,植株再生绿苗率高,这表明小麦幼胚盾片是较好的植物组织培养的材料。     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

节节麦×普通小麦杂种的胚援救和胚愈伤组织再生植株

通过活体-离体胚培养和胚愈伤组织培养有效地克服了节节麦(Aegilops tauschii Cosson.)×小麦(Triticum aestivum L.)杂种幼胚的败育,产生了大量的杂种植株。采用活体-离体胚培技术,节节麦×小麦三个组合杂种幼胚的成苗率为55%,是前人所用传统胚培方法成功率的5—20倍。杂种幼胚在添加有2 mg/L 2,4-D的MS培养基上诱导为愈伤组织,经继代产生全能性愈伤组织,继而分化出再生植株。愈伤组织经继代保存150天仍不丧失分化能力。本文还对两种产生杂种的组织培养方法进行了比较研究。     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

PROLIFERATION OF AND PLANT REGENERATION FROM THE EPIBLAST OF TRITICUM AESTIVUM (WHEAT; GRAMINEAE) EMBRYOS

Cultured immature embryos of wheat, approximately 1 mm in length, often showed proliferation of the epiblast. Murashige and Skoog's medium with 0.2 or 0.4 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l benzylaminopurine supported epiblast proliferation in 20–90% of the embryos depending upon their orientation. Epiblast growth occurred more frequently when the scutellum of the embryo was placed in contact with the medium. The compact tissue derived from the epiblast resembled scutellar callus morphologically and in its developmental potential. Differentiation took place by the formation of a leafy structure along one side of the epiblast proliferation, followed by the initiation of shoot primordia. The pathway through which rooted plants were formed has been interpreted as precocious germination of somatic embryos. The relevance of these observations to the interpretation of the homology of the epiblast is discussed.     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone