Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize

Six media compositions and different transfer times were compared in order to improve the quality of microspore derived structures and their regeneration capacity. The production of regenerated plantlets was increased by a factor of 10 by an early transfer of anthers to medium with low sugar concentration and the use of kinetin-like growth regulators. The best transfer date was 3 weeks after initial plating, onto medium containing 25 gl^–1 sucrose and 1 mg l^–1 kinetin. This study demonstrates that anther transfer at an early developmental stage, before macroscopic appearance of embryo-like structures, improves embryo quality and further regeneration. This production of androgenetic structures showing normal morphological features is the first step to obtain an improvement of androgenetic plant regeneration yield.     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.     

Production of transgenic maize from bombarded type II callus: Effect of gold particle size and callus morphology on transformation efficiency

Summary Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and callus morphology on transformation efficiency were investigated. Reducing gold particle size from 1.0 μm or 0.6 μm resulted in a significant increase in the rate of recovery of bialaphos-resistant clones from Type II callus. The average transformation efficiency of pre-embryogenic, early embryogenic and late embryogenic callus did not vary significantly. Rates of transformation, regeneration and fertility achieved for Type II callus are summarized and compared to those achieved for greenhouse- and field-derived immature zygotic embryos.     

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Influence of 2,4-D,TIBA and 3,5-D on the growth response of cultured maize embryos

Induction of embryogenic calli from immature zygotic embryos of maize requires the presence of 2,4-D or similar auxin-like growth regulators in the culture medium. Pulse-chase experiments with 2,4-D, using various concentrations of 2,4-D in the induction medium were tested in relation to induction of callus in the embryogenic inbred line A188 and the non-embryogenic inbred A632. Interactions of 2,4-D, 3,5-D and the auxin transport inhibitor TIBA were also studied. Pulse-chase experiments showed that exposure to 2,4-D influenced the culture response from 0.5 h onwards. After a pulse of 0.5 h, shoot and root elongation of the embryo was stimulated. A pulse of 16 h or longer induced outgrowths and callus formation at the basal side of the scutellum. Pulses of 7 days and longer resulted in the induction of friable embryogenic Type II callus in A188. Embryos were cultured at 2,4-D concentrations ranging from 0.002 to 2000 mg l⁻¹ and optimal concentration for the induction of embryogenic callus in A188 was 2 mg l⁻¹. At lower concentrations there was a transition between callus formation and germination; at increasing concentrations, callus induction was reduced and finally growth responses became blocked. When TIBA was added to medium without 2,4-D, root elongation decreased in a dose-dependent way suggesting the need of polar transport of endogenous auxins for root elongation. When added to medium with 2,4-D, TIBA caused suppression of callus formation, again pointing to the necessity of polar transport of 2,4-D. In combination with 2,4-D, cultures with 3,5-D resembled cultures at lower 2,4-D concentrations, pointing to a competitive interaction between 3,5-D and 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

In vitro plant regeneration from quality protein maize (QPM)

Breeding efforts to obtain more nutritious maize materials aimed at alleviating dietary deficiencies in developing countries have resulted in an improved maize germplasm known as quality protein maize (QPM). Quality protein maize has higher contents of tryptophan, lysine, and leucine than common maize, but suffers from some major agronomic drawbacks found in common inbred maize lines, such as susceptibility to insect pests and fungal and bacterial diseases and herbicide sensitivity. The development of a reproducible and efficient protocol for tissue culture of QPM is expected to solve some of these deficiencies. In this work, we have evaluated different formulations for in vitro induction of morphogenic responses in three QPM lines developed by the International Maize and Wheat Improvement Center (CIMMYT): CML (CIMMYT maize line)-145, CML-176, and CML-186. Only CML-176 and CML-186 have proven to be responsive to the in vitro conditions considered in this work, with CML-176 showing the highest efficiency in regenerable callus formation and growth. N6C1 medium was found to be efficient for in vitro culture of QPM, whereas no plants could be regenerated by using MPC medium. From CML-176 embyogenic calli cultured on N6C1 medium, we were able to regenerate up to 0.3 plants per 500 mg fresh weight (FW) callus. Further modifications in this experimental protocol, including the replacement of 3,6-dichloro-o-anisic acid with 2,4-dichlorophenoxyacetic acid and modification of the N6C1 vitamin balance, significantly increased the regeneration response of the induced calli, with up to 16.8 and 9.3 plants recovered per 500 mg FW callus for CML-176 and CML-186, respectively.     

Identification of genomic regions affecting plant height in sorghum and maize

The objective of this study was to use restriction fragment length polymorphisms (RFLPs) to determine the genetic location and effects of genomic regions controlling plant height in sorghum. F₂ plants (152) from the cross CK60 x PI229828 were used. Genomic and cDNA clones (106) identified 111 loci distributed among ten linkage groups covering 1299 cM. Interval mapping identified four regions, each in a separate linkage group. These regions may correspond to loci (dw) previously identified by alleles with qualitative effects. Also, these regions identified in sorghum may be orthologous to those previously reported for plant height in maize. Gene effects and gene action varied among genomic regions. In each region, PI229828 alleles resulted in increased plant height. Each region accounted for 9.2–28.7% of the phenotypic variation. Positive, additive effects ranged from 15 to 32cm. Tallness was dominant or overdominant and conferred by alleles from PI229828 for three quantitative trait loci (QTL). At the fourth QTL, PI229828 contributed to increased plant height, but short stature was partially dominant. One digenic interaction was significant. The presence of a PI229828 allele at one region diminished the effects of the other region. A multiple model indicated that these four regions collectively accounted for 63.4% of the total phenotypic variation. The utility of this information for germplasm conversion through backcross breeding is discussed.Journal Paper No. J. 15649 of the Iowa Agriculture and Home Economic Experiment Station, Ames, Iowa. Project No. 3134     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.     

The effect of carbon source on callus induction and regeneration ability in Pharbitis nil

Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of 7-day-old sterile seedlings were cut into 25 mm² squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L⁻¹) alone or BA (22 μM·L⁻¹) and NAA (0.55 μM·L⁻¹), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots, whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis on all explants used.     

Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply

Summary The population size of diazotrophic bacteria naturally associated with the maize rhizosphere can be affected by biotic and environmental factors. In this work we have evaluated the effect of two genotypes of maize, with and without nitrogen fertilization, on the population dynamics and distribution of diazotrophic bacteria associated with maize plants over different plant ontogenic stages. The study was carried out in a field experiment with and without nitrogen fertilization, using two maize cultivars (Santa Helena 8447 and Santa Rosa 3063) previously selected from 32 maize cultivars for the lowest and highest response to nitrogen fertilization, respectively. Microbiological and molecular approaches were used to characterize the diazotrophic bacterial population structure. Bacterial population was assessed by the most probable number using semi-solid N-free media, and by DNA isolation from soil and plant tissue followed by amplification of nifH gene fragments using nested PCR, and by RFLP analysis using the restriction endonucleases TaqI and HaeIII. The dynamics of the diazotrophic bacterial population were affected by the ontogenic stage of the maize plants, but not by the cultivar type. Roots were the preferred site of colonization, independent of cultivar type and growth stage. During the first stage of maize growth, addition of nitrogen fertilizer negatively affected the diazotrophic bacterial population.     

High-frequency plant regeneration through callus initiation from mature embryos of maize (<Emphasis Type="Italic">Zea Mays</Emphasis> L.)

An efficient maize regeneration system was developed using mature embryos. Embryos were removed from surface-sterilized mature seeds and sliced into halves. They were used as explants to initiate callus on induction medium supplemented with 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was over 90% for all inbred lines tested. The primary calli were then transferred onto subculture medium supplemented with 2.0 mg l^–1 2,4-D. Following two biweekly subcultures, embryogenic calli were formed. Inclusion of a low concentration (0.2 mg l^–1) of 6-benzylaminopurine (BA) in the subculture medium significantly promoted the formation of embryogenic callus. The addition of silver nitrate (10 mg l^–1) also supported an increased frequency of embryogenesis. The embryogenic callus readily formed plantlets on regeneration medium supplemented with 0.5 mg l^–1 BA. The regenerated plantlets were transferred to half-strength Murashige and Skoog medium supplemented with 0.6 mg l^–1 indole-3-butyric acid to develop healthy roots. The regenerated plantlets were successful on transfer to soil and set seed. Using this system, plantlets were regenerated from seven elite maize inbred lines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficient regeneration system provides a solid basis for genetic transformation of maize.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indole-3-butyric acid - KT KinetinCommunicated by M.C. Jordan