Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

Effects of different combinations of benzyl adenine and indole acetic acid concentrations on in vitro plant regeneration in hexaploid wheat

Development of a reliable in vitro plant regeneration procedure for hexaploid bread wheat (Triticum aestivum L.) is a prerequisite for its improvement by genetic transformation. Here, we report the effects of two growth regulators, benzyl adenine (BA) and indole acetic acid (IAA) on callus induction and plant regeneration from scutellum cultures of two commercial bread wheat cultivars: Giza 164 and Sakha 69. Callus induction was obtained from isolated embryos cultured on modified Murashige and Skoog (MS) basal medium. After four weeks of callus induction, all calli were plated on MS basal medium for regeneration. Wheat genotype and callus induction medium played a dominant role in plantlet regeneration. 2.0 mg/L BA and 0.2 mg/L IAA were the best combinations for inducing callus and let to highest regeneration frequency (81.67%) across the cultivars. Overall, based on our medium conditions, Giza 164 displayed higher regeneration frequency (81.11%) than Sakha 69. These results will facilitate genetic transformation for the economic varieties Giza 164 and Sakha 69.     

Somatic Embryogenesis and Morphogenetic Potential of Spring Barley (Hordeum vulgare L.) in the System of Technological Genetic Transformation

We studied somatic embryogenesis and morphogenetic potential in young embryos of 17 spring barley cultivars. A considerable effect of the genotype as well as of certain biologically active compounds in the growth medium on the frequency of embryogenic callus formation and regenerative capacity was observed. The Murashige and Skoog medium complemented with myoinositol and casein hydrolysate is suitable for embryogenic callus induction. The regeneration medium should be complemented by hormones, 2.5 mg/l auxin 2,4-D and 0.1–0.5 mg/l cytokinin 6-BAP, to increase the frequency of somatic embryo maturation. The presence of cefotaxime and high level of copper are desirable to increase the efficiency of callogenesis, regeneration, and callus quality. Three cultivars with economically valuable characters and high morphogenetic potential were recommended for development of efficient technology for barley transformation.     

大麦成熟胚愈伤组织的诱导和植株再生的研究

以10个大麦优良品种为实验材料,成熟胚为外植体,研究基因型、种子的不同切割方式、培养基、激素等对大麦成熟胚愈伤组织的诱导及植株再生的影响.结果表明,种子纵切后接种出愈率显著高于横切;改良MS培养基能提高出愈率;在愈伤组织诱导过程中,不同品种对激素2,4-D与Dicamba的反应表现不同;初代愈伤组织经过3次继代培养后会转变为两种类型的胚性愈伤组织;不同品种的植株再生在不同浓度有机添加物的分化培养基上表现不同;长时间的继代培养,一些品种在植株再生过程中出现一定数量的白化苗.供试材料均能进行愈伤组织诱导,但是只有部分品种能再生植株.本实验筛选出愈伤组织诱导频率和绿苗分化率均较高,适合于遗传转化的受体材料,如87-3175、87-0053、97-4010、97-6004及208813-509.     

Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

Seasonal effect on tissue culture response and plant regeneration frequency from non-bombarded and bombarded immature scutella of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) harvested from controlled environment

Scutella of immature embryos from two barley cultivars were used for cell culture and transformation. Explants were supplied by continuous growth of donor plants in a 2-week schedule under defined conditions at first plants were grown for 6–7 weeks in a growth chamber, followed by 10–13 weeks in a greenhouse with stringent control of temperature and light round the year. Strong seasonal variation in plant regeneration frequency was observed for both genotypes in non-bombarded (control) as well as bombarded and subsequent selected explants. Scutella from immature embryos of cv. Salome showed increased frequencies of plant regeneration from January to March, reaching highest values in March/April and followed by a continuous and strong decrease from May to December. This tendency was observed in all 3 years studied, although absolute numbers of plant regeneration varied between the years. The same seasonal effect was evident for plant regeneration from immature scutella of cv. Golden Promise. Frequency of embryogenic callus formation was also found to be influenced by season but this effect was not so pronounced as for plant regeneration.     

棉花体细胞胚胎发生的研究进展

经过30多年的发展,已经在多个不同的棉花品种中获得了体细胞胚,并再生成苗。但由于体细胞胚胎发生往往受多种因素影响,如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展,分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述,并对研究中存在的问题进行了讨论。     

棉花体细胞胚胎发生的研究进展

经过30多年的发展, 已经在多个不同的棉花品种中获得了体细胞胚, 并再生成苗。但由于体细胞胚胎发生往往受多种因素影响, 如何从愈伤组织高效率地转化为胚性愈伤组织依然是限制棉花组织培养与遗传转化的关键问题之一。本文概述了棉花体细胞胚胎发生的研究进展, 分别从棉花体细胞胚胎发生的起源、影响棉花体细胞胚胎发生的内外因素以及寻找棉花体细胞胚胎发生特异表达基因等几方面进行综述, 并对研究中存在的问题进行了讨论。     

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.     

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.     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.     

Estimation of the callus formation and regeneration efficiency in spring varieties of barley zoned in Ukraine

Eight commercial varieties of spring barley (Getman, Tabora, Adagio, Galaktik, Europrestige, Korona, Nevada, and Stalker) were used for the in vitro culture establishment and the development of an efficient protocol for the induction of the callus formation and plant regeneration. Mature embroys of the above-mentioned genotypes were used as explants. The induction of callus formation, culture passage, and plant regeneration were carried out using a medium containing MS salts supplemented with casein hydrolysate (1 g/l), L-proline (690 mg/l), thiamine-HCI (1 mg/l), maltose (30 g/l), 2,4-D (2 mg/l), CuSO4 (12.5 mg/l), myoinositol (250 mg/1), and gelrite (3.5 g/l) at pH 5,6–5,8. The ability of callus formation was observed in all studied varieties, and the callus formation’s frequency varied from 65 ± 3.4% to 100%. The highest regeneration potential was revealed for the Korona (88 ± 2,8%), Europrestige (89 ± 6.5%), Tabora (93 ± 3.4%), Getman (99 ± 0.8%), and Nevada (100%) varieties. For all cultivars the generation of plants from a calli occurred via the organogenesis and somatic embryogenesis. The highest total regeneration potential was registered for the Getman variety (50 ± 5%), which was selected for the further development of effective genetic transformation protocols.     

Transformation of fruit trees. Useful breeding tool or continued future prospect?

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genes from the transformed plant as a research priority in coming years     

Adventitious shoot regeneration from seven commercial strawberry cultivars (<Emphasis Type="Italic">Fragaria </Emphasis>×<Emphasis Type="Italic"> ananassa</Emphasis> Duch.) using a range of explant types

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

Shoot apical meristem: A sustainable explant for genetic transformation of cereal crops

Summary Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to rontine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures, of immature embryos or their cell suspensions, and cryoperserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months, of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristerms from seedlings of maize oat, sorghum, millet, wheat, and barley in an effort to develop a less genetype-dependent and efficient cereal regneration system that can be maintained in vitro for long pertiods of time without the need for cryopreservation. Furthermore, apical mesistem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.     

Particle bombardment-mediated transformation of barley with an Arabidopsis NDPK2 cDNA

As barley is recalcitrant to transformation with current methods, a new improved system is required to apply genetic transformation in breeding programs. In a previous study, we defined optimal conditions for plant regeneration (PR) using mature embryos. This study was conducted to establish an improved transformation system employing the previously adjusted regeneration conditions. Optimal DNA delivery condition for the embryogenic calli developed from mature embryos was bombardment pressure of 1,100 psi at the target distance of 6 cm. The feasibility of the regeneration and DNA delivery conditions was confirmed by developing transgenic barley plants transformed with the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) cDNA via particle bombardment of embryogenic calli from mature embryos. Stable integration of AtNDPK2 cDNA into barley genome was confirmed by PCR and Southern blot analysis of AtNDPK2 transgene. Transgenic plants showed about 10% reduction in membrane damage caused by methyl viologen, indicating the expression of AtNDPK2 transgene. The results demonstrated that the transformation system developed in this study employing the PR from mature embryo-derived embryonic callus is applicable in transgenic barley production.     

Comparison of different methods for plant regeneration and transformation of the legume Galega orientalis Lam. (goat's rue)

 For the first time, regeneration and transformation have been achieved from the legume Galega orientalis Lam. (goat's rue). Two different regeneration protocols are described, one based on direct shoot induction from meristems and the other involving callus induction and shoot induction from callus with the plant growth regulator thidiazuron (TDZ). Different media and explants were evaluated. Three different transformation methods were compared: cocultivation with four different Agrobacterium tumefaciens strains, electroporation of embryos and apical meristems and particle bombardment of embryos. TDZ-promoted shoot induction on calli from immature embryos gave the best results. Transformation using this regeneration protocol was most successful with particle bombardment. Stable transformation has yet to be proven. Received: 11August 1997 / Revision received: 6 April 1998 / Accepted: 1 March 1999     

High frequency plant regeneration from immature embryos of an elite barley cultivar (Hordeum vulgare L. cv. Morex)

An efficient plant regeneration system was developed for Hordeum vulgare L. 'Morex' barley, an important United States malting cultivar. The protocol was based on a series of experiments involving the sizes of immature embryos and the culture media. We found that the embryo size is critical for the establishment of embryogenic callus. Smaller embryos (0.5-1.5 mm) showed a much higher ability to produce embryogenic callus capable of regenerating green plants with fewer albinos than did the larger embryos (1.6-3.0 mm). Either 3 mg/l 2,4-dichlorophenoxyacetic acid or dicamba in modified Murashige and Skoog's (MS) medium was optimum for the induction of embryogenic callus. The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium. Efficient shoot regeneration was obtained on modified MS medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BA). Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/l IBA. Plants were successfully transferred to soil and grown to maturity in the greenhouse. This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar.