Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

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     

A novel method for increasing the frequency of somatic embryogenesis in wheat tissue culture by NaCl and KCl supplementation

The effect of NaCl, KCl and LiCl on the growth and morphogeneis of tissue cultures originating from immature embryos of four wheat (Triticum aestivum L.) and one triticale (Triticosecale)varieties was investigated. The morphogenetic pathway to plant regeneration in Chinese Spring wheat was determined as incomplete somatic embryogenesis because the differentiation and subsequent germination of the shoot apices happened in the early phase of embryo development. Culture medium supplemented by NaCl suppressed the differentiation of shoot apices resulting in the development of more typical somatic embryoids. Forty mM concentrations of both NaCl or KCl increased the formation of somatic embryos in Chinese Spring. Arthur and GK Kincso wheat varieties while Lasko triticale regenerated well without the addition. The salts inhibited plantlet formation from somatic embryoids so the salts supplement should be omitted. Forty mM LiCl inhibited growth while 10mM LiCl had no effect on growth or embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

Efficient plant regeneration of Malaysian <Emphasis Type="Italic">indica</Emphasis> rice MR 219 and 232 via somatic embryogenesis system

Improvement on rice plant regeneration system from an embryogenic callus using two Malaysian indica rice MR 219 and MR 232 was developed in this study. Different stages of rice explants (zygotic embryos) were tested for callus induction and regeneration using various carbon sources and amino acids. The present study shows that dough stage of zygotic embryos was the best stage of explants for the embryogenic callus induction and regeneration of both rice cultivars. The embryogenic callus nature with the compact structure was confirmed by scanning electron microscopy (SEM) analysis. Inclusion of maltose at the concentration between 10 and 20 mg/L shown higher responsive for the development of green somatic embryos and initiation of rice regeneration with an average of 29–37 (87–91%) regenerated plantlets, respectively. All in vitro regenerated rice plantlets were green, morphological and physiologically healthy condition. Rice plantlets were hardened in acclimatization room for 3 weeks and later transferred into soil with 95% survival in both varieties were recorded. Hence, regeneration system from zygotic rice embryos via somatic embryogenesis system was carried out successfully by using 10 g/L of maltose and combinations of glutamine, asparagine and arginine amino acids.     

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     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

Development of haploid cell lines from immature barley,Hordeum vulgare,embryos

Callus and suspension cell lines were derived from haploid barley embryos produced by the Bulbosum method. Embryos 1 to 2 mm long callused on medium containing a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast-growing nodular, beige callus (Type 1), slow-growing, light brown, watery callus (Type 2) and a dense, light yellow, nodular callus (Type 3) were recovered. Type 3 callus was embryogenic and was produced on embryos 1 to 2 mm in length. Although callus cultures gradually became polyploid, a small proportion of haploid cells was retained and the majority of regenerated plantlets were haploid. The organogenic potential of long-term (Type 1) callus cultures was generally low and decreased with time. Attempts to inducede novo shoot formation in Type 1 cultures were not successful.     

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.     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

Somatic cell genetic studies inBrassica species

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.     

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×10⁶/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl^–1 GA₃, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.Abbreviations BAP Benzylaminopurine - GA₃ Gibberellic acid - MT Murashige and Tucker medium (1969) - FDA Fluorescein diacetate     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

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)     

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.     

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.     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.