Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.)

A protocol for plant regeneration in Indian cultivars of barley (Hordeum vulgare L.) has been developed using mature embryo culture. The influence of various auxins 2,4-D (2,4-dichlorophenoxyacetic acid), Dicamba (3,6-dichloro-o-anisic acid) and Picloram (4-amino-3,5,6-trichloropicolinic acid) on the callus induction and subsequent plant regeneration revealed highest percent of callus induction form cultivar (cv) BL 2 on MSB₅ medium (MS salts + B₅ vitamins) supplemented with 6 mg l^?1 Picloram, but maximum number of shoot buds (6–13) were regenerated on MSB₅ medium containing 0.5 mg l^?1 Picloram. Regenerated shoots were rooted on half-strength MSB₅ medium. Plantlets were successfully transferred to soil and grown to maturity in greenhouse. The effect of copper sulphate revealed significant improvement in callus induction and plant regeneration when the concentration of CuSO₄ was increased to 3 μM (30 times higher than normal MS medium) for cv BL 2. Regeneration potential differed for different cultivars of barley used, with highest for cv BL 2 and lowest for cv BH 924. We conclude that the Indian barley genotypes exhibit plant regeneration from mature embryo cultures. The protocol has potential application in barley improvement through genetic engineering.     

Long term regeneration by somatic embryogenesis in barley (Hordeum vulgare L.) tissue cultures derived from apical meristem explants

Callus cultures were initiated from apical meristem explants of one to four-week-old aseptically-grown barley (Hordeum vulgare L. cv. Atlas 57) plants. Embryogenic callus and plants were produced in three separate experiments; the cultures have retained regenerative capacity for three years after initiation. Our results demonstrate that explants other than immature embryos are embryogenically competent in barley and that regeneration occurs by both somatic embryogenesis and organogenesis.     

Influence of Mechanical Stress on Auxin-Stimulated Growth of Excised Pea Stem Sections

The auxin to cytokinin ratios are described for promoting growth in the in vitro cultures of soybean (Glycine max (L.) Merr. cv. Bragg) and perennial clover (Trifolium repens L. cv. Regal Ladinc). Callus growth was induced on somatic tissue with 50:1 auxin to cytokinin (w/w) ratio. A 5:1 ratio served for initiation of cell suspensions from callus and for subsequent growth of callus from cells in suspension. A 1:2 ratio served for regeneration of buds and plantlets from the callus grown from cells. Although (2,4-dichlorophenoxy) acetic acid was the auxin for suspension and regenerative cultures, (2,4,5-trichlorophenoxy)acetic acid was the more effective auxin for initiation of callus on somatic tissue. All cultures were grown with 6-furfurylaminopurine as the cytokinin. The phytohormones strongly influenced the rates of culture growth, but determination of culture type was augmented by dl-alpha tocopherol acetate and iron. Tocopherol and a relatively high complement of iron promoted growth of juvenile cultures, whereas low level of iron and absence of tocopherol favored growth to comparatively more differentiated cultures. Without tocopherol, no callus formed on somatic tissue during the allotted period of incubation. Tocopherol plus a complement of low iron enabled growth of callus on rapidly growing somatic tissue. A high level of iron enabled comparatively more callus growth but suppressed growth of somatic tissue. In suspension cultures tocopherol and a high iron level enhanced dispersion of cells. A low iron complement in the absence of tocopherol induced growth of callus from cells and subsequent regeneration of buds and plantlets from the callus.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Callus Induction and Plant Regeneration from Barley Mature Embryos

Callus cultures were induced starting from excised mature embryosin spring barley, Hordeum vulgare cv Maxima On a medium containinga high level of auxin, a first primary callus was induced whichwas friable, unorganized and capable of direct plant regenerationin the tested conditions This callus type was characterizedby fast growth and high variability in chromosome number Subsequently,a secondary callus type arose from the primary calli subculturedon the same medium in the light This callus type was white andcompact and consisted predominantly of diploid cells When transferredto hormone-free medium it gave rise to green shoots Completerooting of the shoots was achieved on half-strength basal mediumfollowed by exposure to higher light intensity Regenerated plantletscould then be transferred directly into soil without sufferingany loss in vitality Although showing different degrees in morphologicalvariability, they all maintained the diploid chromosome number Hordeum vulgare L, spring barley, morphogenic calli, organogenesis     

Thidiazuron promotes in vitro regeneration of wheat and barley

Summary Thidiazuron (TDZ) is a substituted phenylurea which has been shown to be an efficacious regulator of in vitro morphogenesis of many dicot plant species. However, information regarding the effect of TDZ on in vitro regeneration of monocot species is limited. We investigated the effects of TDZ on in vitro regeneration of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) and found that it promoted shoot regeneration from callus in these two important cereal species. Plant regeneration from calluses derived from immature embryo culture of barley and wheat was observed in regeneration media with a wide range of TDZ concentrations (0.045–45 μM). Shoot regeneration from barley calluses was the highest (38.3% for cv. Golden Promise) at 4.5 μM (1 mg l⁻¹) TDZ, while the optimal TDZ level for wheat regeneration seemed to be 0.9 μM (0.2 mg l⁻¹) (87% for cv. Bob White and 49.4% for cv. Hi Line). Roots developed normally when the regenerated wheat and barley shoots from TDZ-containing media were transferred to the rooting medium. Comparison with other plant growth regulators commonly used in wheat and barley regeneration media suggested that TDZ was among the best for in vitro regeneration of wheat and barley. Both authors contributed equally     

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     

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.     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

Electrostimulated regeneration of plantlets from protoplasts derived from cell suspensions of barley (Hordeum vulgare)

For transformation and somatic hybridisation of barley ( Hordeum vulgare L.), it is necessary to develop an efficient and reliable system for routne plant regeneration from protoplasts. Freshly-isolated cell suspension-derived protoplasts were treated with both rectangular and exponential electric pulses with the aim of increasing plating efficiency as well as to stimulate regenerative capacity. Suspensions were initiated from callus from immature embryos of barley (cv. Dissa). Increasing field strength, capicitance, or number of applied pulses resulted in a decreased protoplast viability and plating efficency. However, the regeneration of albino leaves and albino plantlets from electro-treated protoplasts was stimulated in comparison with controls.     

The effect of genotype on a barley scutella culture. Histological aspects

Cereals are known to be recalcitrant to the induction of morphogenesis in vitro and the majority of the methods used are callus-mediated and species/genotype-dependent. In the present investigation, a method of morphogenesis induction from immature scutella of selected barley cultivars was used, and particular attention was paid to histology in the initial stages of the regeneration process in order to confirm whether it occurs directly or indirectly (via callus formation). The length of the period from inoculating scutella on the medium to obtaining plantlets depended on the cultivar and the individual scutellum of the barley and varied between 2.5–4 months. The regeneration efficiency and viability of barley scutella was revealed to be highly genotype dependent. The average number of regenerated plants per regenerating scutellum was highest in the case of cv Granal (3.7). A histological analysis of the cultured explants showed both non-morphogenic and morphogenic callus formation. Two types of indirect morphogenetic response were observed: organogenesis (shoot bud formation) and somatic embryogenesis. This is the first report concerning an analysis of in vitro regeneration from immature scutella of barley cultivars (Stratus, Ryton, Granal and Binal).     

Tissue culture and plant regeneration from immature embryo explants of Barley,Hordeum vulgare

Summary Immature embryo explants taken 8 days after anthesis were used to establish callus cultures of spring barley. Two types of calli were observed. A soft, watery callus produced a limited number of shoots and a harder, more compact, yellowish callus gave rise to numerous green primordia and shoots. Gamborg's B5 basal medium supplemented with either 2,4-D (2,4-dichlorophenoxyacetic acid) or Cl₃ POP (2,4,5-trichlorophenoxypropionic acid) was found to give good callus growth and shoot initiation. Media containing 2,4-D at 1.0 mg L^–1 or Cl₃ POP at 5.0 mg L^–1 produced numerous cultures resulting in regeneration of plants. Plantlets developed roots on basal medium with Cl₃ POP at 1.0 mg L^–1 or on auxin-free medium. Twenty genetically diverse genotypes were screened to determine if these techniques were suitable for a wide range of spring barley cultivars. Regeneration of plantlets was obtained for 19 of the 20 genotypes approximately 4 months after culture initiation. Lines differed in the ability to develop vigorously growing calli and in the ability of calli to develop large numbers of shoots and regenerated plantlets.Contribution from Department of Crop Science, Oregon State University, Corvallis, OR 97331. Oregon Agric. Exp. Stn. No. 7582     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

A novel approach for efficient plant regeneration from long-term suspension culture of wheat

Summary Hexaploid wheat plants were easily regenerated from young embryo-derived callus for twelve genotypes tested. After a 2.5 years culture period, however, most of the callus cells lost their ability to regenerate into shoots, but not into roots.A novel approach was used to regenerate shoots from the long-term suspension cultured cells. In general, instead of selecting embryogenic callus as source material, this approach requires the inoculation of unselected callus into liquid medium followed by removing the free floating cell portion, selecting out non-root forming cell clumps from the root forming primary suspension culture, and growing the putative shoot-competent clumps in liquid medium with reduced auxin concentrations. We have successfully established shoot-competent wheat suspension cultures for cv. Mustang. High (>80%) frequencies of plant regeneration were observed from plating of 2.5 year suspension cultures. The suspension cultures established by this approach have been utilized to select for heat tolerant variants and will be an ideal source material for protoplast culture and transformation studies. This approach can also be applied to other cereal crops which form roots easily but are unstable in maintaining long term regenerable cultures and which are not easily adaptable to suspension culture.     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Factors affecting plant regeneration from immature inflorescence of two winter wheat cultivars

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kızıltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kızıltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kızıltan-91 and Bezostaja-01, respectively. In Kızıltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kızıltan-91 donor plants. In contrast to Kızıltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kızıltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.     

Partial desiccation augments plant regeneration from irradiated embryogenic cultures of sugarcane

Partial desiccation treatment was applied to improve plant regeneration response in irradiated in vitro cultures. Embryogenic callus cultures of sugarcane cv. Co-671 were exposed to different doses of gamma radiation (0–80 Gy) and radiation effect was evaluated in terms of post-irradiation callus recovery, growth and regeneration of plants. Proliferative capacity of cultures was inversely correlated with radiation dose as the percentage surviving cultures or white proliferating clumps (WPC) decreased as the radiation dose increased up to 80 Gy. LD50 was found to be around 20–30 Gy and at higher doses, poor regeneration frequency was observed after 4–6 weeks of post-irradiation culture. To stimulate regeneration response, irradiated cultures were subjected to partial desiccation for 6 h and the treatment resulted in enhanced plant regeneration response. The study suggests that partial desiccation treatment can be useful in stimulating regeneration response of irradiated in vitro cultures.     

Mature embryo axis-based high frequency somatic embryogenesis and plant regeneration from multiple cultivars of barley (Hordeum vulgare L.)

A highly reproducible regeneration system through somatic embryogenesis from the excised mature embryos (MEs) of dry seeds of a range of European barley cultivars was developed. By minimizing the germination of plated MEs, primary callus could be obtained with high frequency which permitted efficient embryogenesis and regeneration of a large number of green plants. Different approaches were tested to reduce or prevent normal germination: (i) the use of a well defined balance of maltose and 2,4-D in the induction medium, (ii) soaking of seeds in water containing 2,4-D solution, (iii) direct culture of excised embryonic axes, (iv) longitudinally bisected MEs giving two halves, and (v) complete removal of the elongated main shoot including any roots within a week of culture initiation. Culturing of bisected MEs and whole embryonic axes gave the best responses with respect to large amounts of callus combined with minimal germination. The incorporation of BAP at low levels in the medium was found to be most effective for embryogenesis and the maintenance of long-term morphogenic capacity (more than 11 months up to now). This procedure allows the complete regeneration of plants in 16-20 weeks, from the initial isolation of MEs through all the steps to the development of plants ready to be transferred to the soil. The protocol was first developed for cv. Golden Promise and successfully applied to commercial cultivars. All cultivars tested formed embryogenic callus, with overall rates ranging from 22-55% and an average number of green plants per embryogenic callus from 1.5 to 7.5 across the genotypes.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O₂ uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.