In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus

Summary Daylily plantlets with tetraploid or octaploid chromosome numbers were induced from colchicine-treated diploid callus (2n=22). Callus tissue of the daylily Hemerocallis flava L. was placed on a morphogenetic induction medium, a modified MS formula supplemented with 1 mg 2,4-D and 1 mg kinetin/liter, plus 0,10, 20, or 40 mg/liter colchicine in the dark at 12°C for 3 days and recuperated for 1 week under the same environmental conditions except that colchicine was left out. The calluses were then returned to the normal growth temperature 25°C. Ploidy was identified by chromosome counts of squashed root-tip cells collected from the newly potted plantlets and later by measurements of stomates and pollen grains. Over 50% of the plants initiated from the colchicine-treated calluses were completely tetraploid. All of the plantlets differentiated from untreated callus had a diploid number of 22. Of the 3 colchicine levels applied, the 20 mg/liter treatment appeared to be the most effective in production of tetraploidy.     

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Summary Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N₆) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N₆ medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.     

Effects of growth regulators on callus cell growth,plant regeneration,and somaclonal variation of smooth bromegrass (Bromus inermis Leyss.)

Summary Immature inflorescences of smooth bromegrass were cultured on MS agar media supplemented with varying combinations of 2,4-D and kinetin. Callus was initiated from segments of young inflorescences on each medium. All of the calli were subcultured monthly for 5–6 times and transferred onto hormone-free MS medium for plant regeneration. Addition of kinetin to the basal medium stimulated shoot initiation in the callus cultures. Plantlets were regenerated only from calli grown on media containing 2 and 6 mg I^-1 2,4-D with a supplement of 0.2 mg I^-1 kinetin. No albino plantlets were produced. Morphological characteristics and dry matter yield of ten somaclones and the parental plant (SBG7) were studied in the greenhouse in a randomized complete block experiment with five replications. There was significant variation (P>0.01) among genotypes for all morphological characteristics studied. Although all somaclone heights and leaf widths were lower than those of the parental plant, the somaclone F9A, F10A, and F10B had larger tiller numbers, and leaf/stem ratio by dry weight than the parental plant. Only somaclone F9B gave higher specific leaf area and leaf area ratio than the parental plant. Almost all somaclones had the same leaf length, total dry weight, and specific leaf weight as the parental plant. The variation found in somaclones should permit selection for desirable agronomic traits.     

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)

Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin     

Genetic variation and control of plant regeneration in leek (Allium ampeloprasum L.)

Zygotic embryos of leek (Allium ampeloprasum L.) were isolated from mature seeds of different cultivars, selfings and full-sib families. The embryos were cultured on callus induction and shoot regeneration medium and employed to study several parameters: percentage of embryos forming calluses, percentage of embryos forming compact calluses, callus weight, percentage of regenerating calluses, numbers of shoot primordia and numbers of regenerated shoots. Differences between cultivars and selfings were found for most parameters studied. For all cultivars all parameters, except callus weight, decreased after one generation of selfing. Compact callus types enhanced primordia formation and shoot regeneration. Genetic characteristics of callus development and plant regeneration were studied in a 4 × 4 diallel cross. Analysis of variance revealed significant differences between full-sib families. The diallel analysis showed that additive gene effects were significant for all parameters. The predominance of additive gene effects indicated high narrow-sense heritability. Breeding for an increased number of regenerated shoots was successful.     

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

Summary A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l^-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l^-1 kinetin.Differences between leek genotypes in callus type, callus weight, shoot regeneration and primordia formation were observed. Histological observations showed that plant regeneration took place, both via the pathway of somatic embryogenesis and organogenesis.Abbreviation 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog (1962) medium     

Regeneration in sugarcane via somatic embryogenesis and genomic instability in regenerated plants

In the present study, embryogenic calli of sugarcane variety BL4 were induced using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in different concentrations and combinations. In contrast to earlier studies, embryogenic callus sectors were identified and isolated microscopically within 1–2 weeks. Subsequently, 51 media formulations were used for regeneration of proliferated embryogenic callus, using MS medium supplemented with three different cytokinins [kinetin, 6-Benzylamino purine (BAP), and thidiazuron (TDZ)] and auxins (IAA/NAA and IBA) in different combination and concentrations. After acclimatization, the genomic DNA of regenerated plants was studied to explore the insertion polymorphism of transposable elements in order to ascertain the variation among somaclones. Though low concentration of kinetin with 2,4-D was found supportive to embryogenic callus development, the highest number of regenerated plantlets was observed using BAP (1 μM), however the plantlets had very low fresh weight (2.2 g). Conversely, TDZ alone supported a significant increase in the number of plantlets regenerated (38–40) with higher fresh weight. The somaclones generated during this study showed considerable positional polymorphism of activator-like transposable elements possibly due to the stress associated with in vitro culture. This study provides a procedure to produce regenerated sugarcane plants from embryogenic callus in a relatively short time.     

Callus induction and plant regeneration from anther and inflorescence culture of Sorghum

Summary Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506-5501, USA. Contribution no. 88-566-J.     

Plant regeneration from callus culture in potato

Summary A procedure for plant regeneration from callus culture of potato, Solanum tuberosum L. is described. Calli were induced from 1–2 mm long shoot apices of potato cultivars Cara and A25/19 on half-strength Murashige and Skoog's medium (half-MS) supplemented with 3.2 mg IAA (indole-3-acetic acid), 1.0 mg kinetin (6-furfurylamino)purine], and 0.5 mg 2,4-D [2,4-dichlorophenoxy)acetic acid]/1. Sixty percent explants produced nodular calli on this medium within 30 days. Calli differentiated into shoot-primordia when subcultured on half-MS medium supplemented with 0.5 mg 2,4-D and 1.0 mg zeatin [6-(4-hydroxy-3-methybut-2 enylamino)amino purine]/1. Differentiated calli on half-MS medium without growth hormones produced complete plantlets which were cloned on the same medium and transferred into soil.     

Regeneration and genetic transformation of Spanish rice cultivars using mature embryos

To establish a plant regeneration system from embryogenic callus derived from mature rice embryos, the addition of aminoacids and the effect of two macronutrient solutions MSD and N6D to the basal callus induction medium was tested in three Spanish varieties, Senia, Tebre and Bahia. Aminoacids enhanced the production of embryogenic callus in Tebre and Senia whereas in the case of Bahia, embryogenic callus, which gave rise to a high rate of differentiated shoots, was induced without aminoacids. The macronutrient solution had also to be adjusted for each variety. Pre-regeneration treatment with ABA significantly improved the regeneration rate in all media tested, independently of the media in which the embryogenic callus were induced. In a comparison of growth regulators, BA yielded more shoots than Kin in all varieties whereas the effect of the auxins NAA or IAA was dependent on the variety. Transgenic plants from the three varieties were obtained via an Agrobacterium tumefaciens transformation system, using the optimised culture media. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotypic differences in organogenesis from callus of ten triticale lines

Summary Callus was obtained from immature excised embryos of triticale using MS medium supplemented with 3 mg/l 2,4-D and 1 mg/l kinetin. The presence of 2,4-D was essential for continued callus proliferation. Plantlets were induced from the calli by sub-culturing on medium either devoid of auxin or containing 0.1 mg/l 2,4-D. The capacity to produce callus and to form organs and plantlets differed markedly among the genotypes used. Lines also had distinct response to presence and absence of 2,4-D in the regeneration media. The callus of most triticale lines used differentiated into organs more readily on MS medium supplemented with 0.1 mg/l 2,4-D than on medium without growth regulators. Very high frequencies (up to 75%) of plantlet regeneration were observed in several of the triticale lines studied.     

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.     

Production of Primary Triticale from Calluses of Immature Abnormal Hybrid Embryos between Triticum durum and Secale cereale

Plant regeneration was attempted in callus induced from the immature abnormal hybrid embryos between T. durum and S. cereale, using 4-Huorophenoxyacetic acid as a growth regulator. In particular, the relationship of numerical variation in chromosomes between the callus tissues and the regenerated plants was investigated. Cytological observation revealed that there was no distinctive numerical difference between the shoot-forming (SF) and the non-shoot-forming calluses and also between the SF calluses and the regenerated plants. The root-tips of regenerated plants consisted of cells having various chromosome numbers, including the expected 2 n = 3 ×= 21 (genomes, ABR) of which the frequency was 69.8 %. The regenerated plants showed partial fertility, notwithstanding that the hybrid plants were expected to be sterile. Since the frequency of abnormal embryos was about 90 % in this cross, the utilization of abnormal embryos was demonstrated by use of callus culture.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. I. Double-layer culture technique

Summary Calluses of spring and winter wheats (Triticum aestivum L.) were selected for Fusarium resistance in vitro, using the double-layer culture technique. Potato-dextrose agar medium in vials was inoculated with mycelia of Fusarium graminearum and F. culmorum. After one week, fungal cells were killed by autoclaving and the agar medium containing the thermostable toxic metabolites was overlayered with MS callus-growing medium. Later, wheat calluses were placed on the upper medium for 4–5 weeks, and from the surviving calluses plants were regenerated. R₂ seedling populations from self-fertilized R₁ plants of 4 varieties were tested for Fusarium resistance by artificial infections in the greenhouse, and 3% of the regenerated R₂ plants have been found to be more resistant than the original cultivars.     

Interspecific hybridization inBrassica juncea × Brassica hirta using embryo rescue

Summary Interspecific hybrids have been obtained in an otherwise incompatible cross betweenBrassica juncea × Brassica hirta through the in vitro culture of hybrid ovules and ovaries. The best response was observed from ovules and ovaries cultured 10–15 and 5–7 days after pollination respectively on a basal medium supplemented with indoleacetic acid, kinetin and casein hydrolysate. In some cases the basal cut end of the ovaries proliferated to form callus and shoots. The in vitro-derived hybrid seeds varied in their colour, size and shape, and the F₁ plants in the field showed a large diversity in their morphological traits. The hybrids were sterile, and had an intermediate number of chromosomes (2n=30).     

Investigations on the Anther Culturability of Four German Spring Wheat Cultivars and the Influence of Light on Regeneration of Green vs. Albino Plants

Homozygous doubled-haploid plantlets derived from anther culture of wheat (Triticum aestivum L.) and triticale (×Triticosecale Wittmack) are useful breeding materials. However, efficiency of an-drogenesis needs improvement. We used media (basic components, are the same as 85DI2) each containing one of the seven auxins [2,4,5-trichlorophenoxyacetic acid (2,4,5–T), P-chloraphenoxyacetic acid (pCPA), 3,6-dichloro-o-anisic acid (dicamba), 4-amino-3,5,6-trichloropicolinic acid (picloram), indole-3-butrytic acid (IBA), indole-3-acctic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) as a control] in combination with 6-furturyl-aminopurine (kinetin). In addition, each of the four cytokinins [6-benzylaminopurine (6-HA), 2-isopenlylnyl adenine (2-ip), 6-(4-hydroxy-3-meihylbut-2-enylamino) purinc (zeatin), and kinetin as a control] was tested in combination with 1-naphthalene acetic acid (NAA). Anthers containing microsporcs at miduninucleatc stage from live wheat cultivars (Angus, Centurk, Chris, K.itt, and Pavon 76) and two octoploid trilicale lines (T81, T82) were tested mainly for callus induction and polyhaploid production on each of the 11 media. The cultivar × medium interaction was not significant, When averaged over all growth regulators, Pavon was (he best cultivar which produced 14.4 % calli and 23 % polyhaploid plantlets. Averaged over all cultivars, the medium containing 2, 4-U produced the highest calli (13.9 %). Undifferentiated calli were regenerated on 87T1 medium, which contained IAA (1 mg/1) and kinetin (2 mg/1).     

High frequency regeneration and heritable somaclonal variation in Brassica juncea

Summary Multiple shoot formation in cotyledonary callus of Indian mustard (Brassica juncea cv. Prakash) was induced on modified MS media supplemented with high cytokinin (kinetin or zeatin) and low IAA concentrations. Complete plants were obtained on prolonged incubation of shoots on the same medium. 6-Benzyladenine alone or in combination with IAA or NAA did not support plantlet regeneration. A total of 71 plants were transferred to greenhouse. The seed, however, could be collected from 37 plants only. The seed was sown in the field to evaluate the material for somaclonal variation in R₁ generation. Data were recorded for yield, plant height, number of primary branches, siliqua number, 1,000 seed weight and oil content. Somaclonal lines showed tremendous amount of variation for all the characters studied. A number of plants in this generation showed significantly higher yield and/or other improved agriculturally important characteristics as compared to the control. A line with dwarf plant type was also identified. A number of plants were selected from this generation and carried forward to R₂ generation. Most of these lines bred true in R₂ generation. The material seems to be very promising for future breeding programmes.     

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.     

Effects of Growth Regulators and Genotypes on Callus and Embryoid Induction from Maize Anther Culture *

Fifty genotypes and ten growth-regulator combinations were used in two experiments (I and II) to investigate genotype and hormonal effects on production of callus and embryoids via anther culture in maize (Zea mays L.). Hormonal effects across all genotypes were not significant in either experiment. However, highest callus-induction frequencies in both experiments occurred on YP basal medium plus 2,4-D at 2.0 mg 1^?1, and kinetin at 1.5 mg 1^?1 indicating that this combination was more effective than others. Genotypic differences in callus or embryoid induction across all media were significant. The most responsive genotypes in experiment I were single-cross hybrids Yuanwu × 592 and K727 × K305, which produced 18.3 and 6.7 % calli, respectively, with their appropriate media. The most responsive entry in experiment II was CIMMYT Pool 29, which produced 15.0 % calli on appropriate medium and an average of 10.0 % calli across 10 media. Twenty-three plantlets was regenerated from this study. Most of them developed embryogenically.     

Regeneration potential of CIMMYT durum wheat and triticale varieties from immature embryos

Twenty‐five durum wheat elite advanced lines and released varieties, and five triticale varieties were evaluated for their ability to produce embryogenic callus using three different media. For callus initiation and maintenance there were basal Murashige and Skoog (MS) medium containing double strains of macroelements and 2.5 mg/l 2,4D (DW1), basal MS medium containing 2.0 mg/l 2,4D (DW2), or basal MS medium supplemented with 1.0 mg/l 2,4 D and coconut milk (DW3). Plant regeneration was achieved on basal MS medium with indoleacetic acid and 6‐benzylaminopurine, and plants rooted on MS with 1‐naphthale‐neacetic acid. DW3 medium proved better than the other media tested for embryogenic callus initiation and maintenance. Regeneration rates varied widely with both genotype and initiation medium, with values ranging from no regeneration to 100% regeneration; the plantlets produced per embryo ranged from five to 20. Fourteen of the durum wheat genotypes showed 63–100% regeneration from DW3 callus formation medium, four lines from DW1 medium, and two lines from DW2. Four of the triticale varieties had regeneration of 48–100% from DW3 medium. After six subcultures, over a 6‐month period, genotypes lost their ability to regenerate plants. Only 10 lines retained some plant regeneration potential but regeneration was at reduced levels. Successful regeneration of durum wheat and triticale varieties will be used as an integral part of the transformation process.