Optimization of mature embryo-based high frequency callus induction and plant regeneration from elite wheat cultivars grown in China

We have developed an efficient procedure for plant regeneration of elite wheat cultivars using mature embryos. Firstly, we established the optimal combination of basal media, inoculation method and pretreatment method using biostatistical methods. The results indicated that the combination of MS medium and longitudinally bisected mature embryos showed the highest culture efficiency, whereas the pretreatment method had no significant effects on callus induction or plant regeneration. A 70% primary callus induction rate was achieved on MS medium containing 2 mg/l 2,4‐d for all tested cultivars. Primary calli were then transferred onto the subculture medium to initiate embryogenic calli. Supplementation of the subculture medium with the appropriate combination of phytohormones (2.0 mg/l 2,4‐d , 0.5 mg/l BA and 0.1 mg/l NAA) significantly enhanced embryogenic callus production. The addition of AgNO₃ (10 mg/l) in regeneration medium promoted plant regeneration, whereas CuSO₄ stimulated root formation. The use of this protocol achieved successful plant regeneration in eight tested cultivars. The culture efficiency ranged from 15.3% to 36.8%, suggesting this regeneration system may be an effective alternative for wheat genetic transformation.     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

玉米成熟胚胚性愈伤组织的诱导、高频再生及转化的研究

以玉米自交系CML295、CML304和18-599R的成熟胚为外植体, 结合幼胚离体培养方法, 探讨并优化了成熟胚来源的胚性愈伤组织诱导及继代培养方法。对其愈伤组织的形态和组织切片的研究结果显示, 继代过程可产生良好的Ⅱ型胚性愈伤组织。在分化培养中分别获得68.6%、75.4%和84.8%的高频再生率, 每愈伤组织块成苗数分别为2.45、2.43和2.75。利用基因枪法转化pCAMBIA1301质粒后的GUS瞬时表达效率分别为57.9%、62.5%和73.1%, 转化pCAMBIA1303质粒后检测GFP的瞬时表达效率分别为23.3%、40%和45.5%。以上3种基因型成熟胚来源的愈伤组织转化率与其对应的幼胚来源的胚性愈伤组织转化率相似。这一技术体系为玉米的遗传改良和功能基因组研究提供了重要的技术平台。     

Influence of genotype and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. cultivars

Twelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog‐based media. Each contained 9.05 μM 2,4‐dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1‐naphthaleneacetic acid and 2.22 μ 6‐benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.     

Barley lines showing prominent high green shoot regeneration in cultures derived from immature embryos

Eighty‐four cultivars of barley (Hordeum vulgare) and 95 wild strains (82 of H. spontaneum and 13 of H. agriocrithon) were surveyed for the production of callus, callus growth, and shoot regeneration in cultures derived from immature embryos. All cultivars except for ‘Turkey 381′, induced calli from more than 90% of embryos. On the other hand, the wild lines showed a large variation in the percentage of callus induction from 0 to 100%. Among the cultivars, those with the brittle rachis genotype, bt Bt₂, on chromosome 3H, regenerated shoots with a significantly higher percentage than the cultivars with the Bt bt₂ genotype. Green shoots were produced in a higher ratio (0.84) in the cultivars than in the wild lines (0.52). Among the lines examined,‘Lenins’ regenerated shoots efficiently (90.4%) and produced the highest number of calli with green shoots per embryo (4.77) followed by ‘Golden Promise’ (3.15). Examination of callus growth and shoot regeneration from embryos at different developmental stages revealed that scutellum development affected the quantity and quality of callus and shoot regeneration.     

Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos

Summary Experiments upon in vitro culture of immature durum wheat embryos, harvested at different growth stages, were made in two consecutive years. Callus formation and plantlet regeneration were obtained. The ability to form callus and the degree of morphogenetic processes varied with the different hormonal treatments used and with the age of the embryos. In the first year the best response for callus growth was observed with 2,4-D 2 mg l^-1 plus adenine 50 mg l^-1 or 2,4-D 5 mg l^-1 alone in the more mature embryos (15 and 20 days after anthesis). On the contrary, NAA 5 mg l^-1 had a greater shoot regeneration effect. In the next year, at all 2,4-D concentrations and for the two different ages of the embryos tested, all embryos formed callus. Regeneration of plantlets was obtained in higher percentage in calli originated from the more developed embryos. The effect of changed media upon plantlet regeneration was studied after callus transplant.Investigation by cytophotometry and chromosome counts on different calli showed, practically in all cells, a diploid condition. A histological analysis demonstrated embryogenic somatic characteristics in many samples of callus. The pattern of organogenesis seemed to be via adventitious bud formation but structures resembling embryoids were also observed in the callus.     

Correlation of isoenzyme polymorphism and Bayoud-disease resistance in date palm cultivars and progeny

Summary Seedlings of date palm (Phoenix dactylifera L.), obtained from seven cultivars crossed with two males, were analyzed by polyacrylamide gel electrophoresis for esterase (EST), glutamate oxaloacetate transaminase (GOT), endopeptidase (ENP) and alcohol dehydrogenase (ADH) polymorphisms. Eleven, eight, five and two phenotypes were revealed for the enzymes tested, respectively. Seedlings of F₁ populations derived from Bayoud (Fusarium)-resistant and low fruit quality cultivars were characterized by a high electrophoretic polymorphism, when compared with progenies of Bayoud-susceptible and high fruit quality cultivars. In almost all cases, the most frequent electrophoretic phenotypes scored for each enzyme in different F₁ populations, were similar to those of the corresponding parent cultivars. Heterozygous phenotypes have been found for, at least, 3 loci; Got-2, Est-1 and Enp, indicating that such loci could be used to screen for hybrid seedlings. The expected Mendelian segregation of allozymes has been observed for these 3 loci, in many F₁ populations. It seems that progenies of Bayoud-resistant cultivars are characterized by a high level of electrophoretic polymorphism. The estimation of this index and the search for genetic linkage with segregating allozymes, may be biochemical criteria useful as an aid in distinguishing date palm seedling populations derived from Bayoud-resistant cultivars and suitable for breeding programs.     

Towards genetic transformation in the monocot Alstroemeria L.

The successful application of plant biotechnology to Alstroemeria improvement will largely depend on the availability of an efficient regeneration/transformation system. Regeneration in Alstroemeria is accomplished from nodular embryogenic callus initiated from zygotic embryos. Histological studies of embryogenic callus initiation from 4-weeks old cultured ovules revealed that the outermost layers of the protoderm of the embryogenic nodules divided to form either a new nodule or aproembryo. Transient gene expression after particle bombardment of nodular embryogenic callus was optimized using DNA of pAHC25. The highest β-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tissue was placed 5 cm below the microcarrier launch assembly and when the rupture disc-breakage point was between 650–900 psi. Kanamycin blocked regeneration of somatic embryos, however, did not block growth of nodular embryogenic callus. With phosphinothricin both callus growth and regeneration were blocked. Bombardment of nodular embryogenic callus with DNA of pAHC25 combined with selection on medium containing phosphinothricin resulted in putative transgenic chimeric. Friable calli were selected from nodular embryogenic callus and used to initiate suspensions. These cell suspensions were subjected to transformation by particle bombardment using DNA of pAHC25 and resulted in a stable transformed friable callus line after selection based on luciferase activity. Even after 2 years of maintenance this callus line was luciferase positive and the Polymerase Chain Reaction analysis demonstrated the presence of the introduced gene in this friable callus line. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro selection of wheat callus tolerant to high levels of salt and plant regeneration

Summary Embryogenic calli isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The effect of NaCl on the selected and unselected cell lines has been investigated. The results indicated that the relative growth rate of callus decreased as the concentration of NaCl increased in both callus lines. The selected callus line gave a higher growth weight in the presence of NaCl in the medium and was highly significant as compared with unselected callus line across medium protocols in all wheat cultivars. The dry weight of both kinds of callus lines of all wheat cultivars increased markedly with increasing NaCl concentration in most cases. The Na⁺ and Cl^- contents of both callus lines were increased with increasing salinity levels while K⁺ content was decreased. The selected callus line of each cultivar at the same salinity level produced significant amounts of Na⁺, K⁺ and Cl^- higher than the unselected callus line in most salinity levels. However, the unselected callus lines of the cultivars Giza-157 and Sakha-90 at the same salinity level produced significant amounts of K⁺ higher than the selected callus line in most salinity levels. The proline content of both kinds of callus lines for all wheat cultivars was increased with increasing salinity level. However, the selected callus line gave a significantly higher proline content than the unselected callus line in all wheat cultivars at the same Salinity level. Results from the in vitro selection for NaCl tolerance showed that the stepwise method of increasing NaCl in the medium was more effective for plant regeneration than other methods.     

Evaluation of carbon sources,gelling agents,growth hormones and additives for efficient callus induction and plant regeneration in Indian wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotypes using mature embryos

Various factors affecting in vitro regeneration like different carbon sources, different gelling agents, and growth additives were assessed comprehensively for callus induction and plant regeneration for five Indian wheat cultivars using mature embryos as the explants for the first time. The tissue culture responses of cultivars WH-1105, HD-2967, and PBW-343 have not been reported earlier. Besides, the effect of different concentrations of the cytokinin, zeatin has also been optimized. Using the optimized factors, the efficiency of five different varieties, i.e., HD 2967, C 306, RAJ 3765, WH 1105, and PBW 343 was evaluated for regeneration. Modified MS basal medium containing dicamba reduced precocious germination of the embryo and induced embryogenic callus more efficiently. Removal of embryogenic calli from non-regenerable structures during early callus phase improved plant regeneration. These calli on zeatin (1.0 mgl^-1) and dicamba (0.1 mgl^-1) containing medium showed the highest regeneration frequency (98%) with a maximum of 8-9 shoots per calli. Maltose had the maximum callusing and regeneration percentage than other carbon sources. Various gelling agents did not have any significant difference on the regeneration. Of all the varieties, C-306 and HD-2967 were found to be more regenerative and can be used in transformation experiments.     

High-efficiency transformation of Gossypium hirsutum embryogenic calli mediated by Agrobacterium tumefaciens and regeneration of insect-resistant plants

A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis‐active Cry1Ac and API‐B chimeric gene. Infected embryogenic calli were co‐cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7‐8 weeks. Then, the kanamycin‐resistant calli (Km¹) subcultured in proliferation medium would re‐differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100‐ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty‐five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API‐B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium‐mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.     

Increased regeneration from NaCl-tolerant callus in rice

Summary NaCl-tolerant calli were selected from two Japonica and two Indica rice (Oryza sativa L.) cultivars on basal media containing 6,000, 9,000, 12,000 or 15,000 ppm NaCl. Frequency of callus formation decreased with the increase of NaCl in the medium, especially in Indica. About half of the calli of Japonica cultivars selected on NaCl-ammended media survived 20,000 ppm NaCl but none of the Indica callus survived. In Japonica, more plants were regenerated from calli selected on all concentrations of NaCl media than from NaCl-free medium. Concentration of Cl^- in callus increased dramatically with increased NaCl content but peroxidase activity decreased.     

绿色棉新彩棉7号体细胞胚胎发生及其植株再生

以绿色棉新彩棉7号的子叶、下胚轴为外植体,MSB(MS培养基附加B5维生素)基本培养基附加不同激素组合,诱导愈伤组织及调控分化,通过体细胞胚胎发生方式获得再生植株.结果表明:0.1 mg· L-1 KT(Kinetin,激动素)+ 0.1 mg·L-12,4-D(2,4-dichlorophenoxyacetic,2,4-二氯苯氧乙酸)为诱导愈伤组织的最适植物激素组合,不同外植体处理出愈率均达到100％,但下胚轴纵切面背向培养基放置培养更有利于诱导愈伤组织形成;分化调控阶段的最佳植物激素组合为0.15 mg·L-1 KT+ 0.3 mg·L-1 IBA(Indole-3-butyric acid,吲哚丁酸),胚性愈伤分化率可达23.33％; MSB中去除NH4NO3同时KNO3加倍,附加0.5 g·L-1Asn(Asparagine,天冬酰胺)和lg· L-1 Gln(Glutamine,谷氨酰胺),胚性愈伤可进一步分化获得体细胞胚,将成熟的子叶胚接种于1/2MS获得完整的再生植株. 本研究通过体细胞胚发生途径获得了新彩棉7号的再生植株,为天然彩色棉基因工程研究奠定了一定基础.     

大麦成熟胚胚性愈伤组织的高频诱导和植株再生

大麦成熟胚的初代愈伤组织是水质、柔软的非胚性愈伤组织，在继代培养中能以一定频率出现胚性愈伤组织。本文研究了基因型、有机添加物和胚切割等因素对大麦胚性愈伤组织诱导频率的影响。结果表明：供试的１０个大麦品种在胚性愈伤组织诱导率和植株再生率上有显著差异；培养基中添加水解酷蛋白、Ｌ－脯氨酸以及提高维生素Ｂ１和肌醇的浓度可以促进胚性愈伤组织的形成；胚芽段比全胚或胚根段更易形成胚性愈伤组织。胚性愈伤组织转入无     

Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of temperate grasses. Brachypodium as a model plant has many favorable features, such as small size, small genome, short life cycle, self-fertility, and simple growth requirements. The genome sequence of the standard line Bd21 has been released and genomic resources have been developed.It is imperative to develop a method for efficient Agrobacterium-mediated Brachypodium transformation. Yellowish and compact embryogenic calli derived from immature embryos of the Bd21 were transformed with the Agrobacterium strain AGL1. Seven- and nine-week-old calli were used for transformation with Agrobacterium carrying either pCAMBIA 1301 and pCAUGH. Transformation efficiencies were assessed through histochemical GUS assay. The efficiency of transformation with pCAMBIA 1301 (based on the number of callus lines producing GUS-detected plantlets and the number of calli used for transformation) reached 20.1% (7-week-old calli) and 1.7% (9-week-old calli), and with pCAUGH (based on the number of GUS-detected plantlets and the number of regenerants) 90 and 87% for 7- and 9-week-old calli, respectively. High selection pressure was obtained by using pCAUGH, which is preferred for saving labor and time consumption during the callus selection.     

Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.     

Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variation

Somaclonal variations, induced in vitro, were used to enhance tolerance to aluminium (Al) toxicity in rice. Tolerant plants were developed through in vitro screening of embryogenic calli. The calli were derived from mature seed embryos and cultured on medium stressed with different concentrations of Al₂(SO₄)₃⋅18H₂O. Seed germination, callus induction, plantlet regeneration and callus health declined with increased concentration of Al. At higher Al concentrations, callus health deteriorated drastically with partial to total necrosis. Plantlet regeneration varied largely among varieties and treatments. The variety IR72 produced maximum plantlets among all genotypes tested. An amount of 60 ppm or more Al was highly toxic, which greatly reduced plantlet regeneration from callus. R₀ plantlets were grown under glasshouse. Based on the appearance of bronzing symptoms on leaves, the tolerant R₁ plants were selected. R₁ and R₂ lines derived from putative tolerant somaclones, were evaluated in fiberglass tanks filled with Al-toxic soil. R₃ population was evaluated in the field. A few lines derived from IR72 showed high yield and good plant type. The progenies at R₃ showed normal root growth under stressed environment in sand culture. The study revealed that in vitro screening would be an appropriate alternative to conventional breeding in evolving Al-tolerant lines as observed in case of other abiotic stresses. The technique was useful in creating de novo synthesized Al-tolerance character in rice.     

In vitro culture of tissues,cells and protoplasts of Trifolium alexandrinum L. (Egyptian clover)

Summary Callus induction derived from root, hypocotyl and cotyledon explants were investigated for four cultivars of Trifolium alexandrinum L. The Murashige & Skoog (1962) medium containing 2.0 mg/L NAA and 0.5 mg/L BAP was able to induce callus from different explants. A wide range of culture media were tested to determine the morphogenetic potential of different callus types derived from different explant types. Shoot morphogenetic development was observed with the cultivars Sakha 4 and Giza 10. Cell suspension cultures were established from hypocotyl derived callus. Methods for isolation and culture of protoplasts from cotyledons are described.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.