RFLP analysis to identify putative chromosomal regions involved in the anther culture response and callus formation of maize

Summary RFLP analysis was performed with anther culture-derived callus lines developed from the maize F₁ hybrids Pa91 x FR16 (PF), H99 x Pa91 (HP) and H99 x FR16 (HF). Relatively evenly spaced RFLP markers were selected to cover the maize genome with 52, 58 and 35 RFLP markers for the PF, HP and HF callus lines, respectively. The results from populations PF and HP combined with limited information from HF showed that six chromosomal regions on chromosomes 1, 2 (two regions), 3, 6 and 8 appear to be associated with the formation of embryo-like structures (ELSs) from microspores or the subsequent formation of regenerable callus from the ELSs. Regions at the end of the long arm of chromosome 2 and on the long arm of chromosome 8 appear to be associated with ELS formation, and the other regions appear to be associated with either ELS or regenerable callus formation or both. Certain regions that we have identified are the same as those found in other studies to be important for friable, embryogenic callus formation (chromosomes 1 and 3 and near the centromere of 2) and for ESL formation (chromosomes 1 and 3). This study has provided evidence for the genetic basis of the maize anther culture response and callus formation.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Optimization of Callus Induction Conditions from Immature Embryos in Maize and Plant Regeneration

This research uses the immature embryos of inbred maize lines (GSH9901, Hi01, Hi02, and Chang 7-2) as receptor materials to establish the callus induction system. These inbred lines provide the receptor materials for the genetic regeneration of maize and the verification of the genetic functions of maize. The factor experiment and orthogonal experiments were used to investigate the impacts of different genotypes, immature embryo size, shield orientation, 2, 4-D concentration, proline concentration, and folic acid concentration on the induction rate of embryogenic callus tissue. A sensitivity experiment testing glyphosate (Bar) and an antibiotic (Cefotaxime sodium) were also conducted. The results indicate that the immature embryos of inbred maize line GSH9901 were the most effective for callus tissue induction, and the immature embryos with a length of 1.6-2.0 mm produce the best result. The upward shield face is more successful for the formation of induced callus. Using orthogonal analysis, we found that the optimal combination for the induction system was A₃ (2,4-D concentration 0.25 mg mL^-1 ), B₁C₃ (proline concentration 0.8 mg mL^-1 ), and D₂ (folate Concentration 0.5 mg mL^-1) and the induction rate reached 84%. We found that cold storage at 4 °C for 1 d is more conducive for the formation of embryogenic callus than the other treatments tested. The sensitivity experiment for callus tissue screening revealed the critical concentration of glyphosate to be 10 mg ml^-1 , and the critical concentration of antibiotic is 250 mg ml^-1 . Using this combination of glyphosate and antibiotic resulted in regenerated plants. This study established the optimal conditions for immature embryo callus tissue induction in maize.     

Genetic Variability in the Progeny of Androgenic Dihaploid Plants and Selection of High Agronomic Performing Lines in Brassica Juncea

Androgenic lines of Brassica juncea cv. PR-45 raised by anther culture, were screen for genetic variation. 393 androgenic plants were transferred to pots to study the R₀ generation. These plants showed substantial variation for different characters. Seed progenies of 27 lines of R₀ plants were sown in the field to study R₁ generation. Androgenic plants within lines were significantly homogeneous for the various agronomic characters studied. Two lines were shorter (18 – 20 %) than the control plants, with a remarkable feature of early maturation. Three lines showed 27 – 31 % higher yield than the parent cultivar.     

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×10⁶/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R₁ plants were self-pollinated and immature R₂ embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R₂-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.Abbreviations BC backcross - BMS Black Mexican Sweetcorn - 2,4-D 2,4-dichlorophenoxyacetic acid - PWS protoplast wash solution (0.2 M mannitol, 80 mM CaCl₂) - FDA fluorescein diacetate - ABA abscisic acid     

Type I callus as a bombardment target for generating fertile transgenic maize (Zea mays L.)

To develop a less genotype-dependent maize-transformation procedure, we used 10-month-old Type I callus as target tissue for microprojectile bombardment. Twelve transgenic callus lines were obtained from two of the three anther-culture-derived callus cultures representing different gentic backgrounds. Multiple fertile transgenic plants (T₀) were regenerated from each transgenic callus line. Transgenic leaves treated with the herbicide Basta showed no symptoms, indicating that one of the two introduced genes, bar, was functionally expressing. Data from DNA hybridization analysis confirmed that the introduced genes (bar and uidA) were integrated into the plant genome and that all lines derived from independent transformation events. Transmission of the introduced genes and the functional expression of bar in T₁ progeny was also confirmed. Germination of T₁ immature embryos in the presence of bialaphos was used as a screen for functional expression of bar; however, leaf painting of T₁ plants proved a more accurate predictor of bar expression in plants. This study suggests that maize Type I callus can be transformed efficiently through microprojectile bombardment and that fertile transgenic plants can be recovered. This system should facilitate the direct introduction of agronomically important genes in to commercial genotypes.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Heterozygous diploid plants regenerated from anther culture of F1 rice plants

Summary Plants derived from anther culture are theoretically haploid, but diploid plants are also known to arise. Anther culture-derived diploid plants are usually homozygous and are believed to be due to spontaneous doubling of chromosomes in either microsporocytes or callus cells during the culture process. However, heterozygous diploid regenerants may also arise from a) regeneration from cultured somatic cells, b) mutation occurring during or after a spontaneous doubling event, c) fusion of unlike haploid cells in chimeric callus, and d) regeneration from diploid microsporocytes resulting from aberrant meioses. This study was designed to elucidate the frequency and origin of diploid regenerants from rice anther culture. Regenerants were obtained from 11 F₁ genotypes. Progeny testing detected heterozygosity in 7 out of 211 regenerants. Each of the heterozygous regenerants were from ‘Calrose 76’/waxy ‘M-101’, Half of the diploid regenerants from this cross were heterozygous. No heterozygous regenerants arose from the other 10 F₁ genotypes. Progeny testing indicated that two of the heterozygous regenerants were as heterozygous as the F₁ plants for three parental characters. The other five regenerants exhibited decreased levels of heterozygosity. One of the heterozygous regenerants exhibited evidence of mutation for a non-parental character. However, mutation is an unlikely cause of the observed high levels of parental-type heterozygosity. No evidence for the occurrence of chimeric callus was detected, making this an unlikely cause as well. The most likely origin of the observed partial heterozygosity is regeneration from diploid microspores, which could also produce plants exhibiting complete parental-type heterozygosity.     

Selection for increased anther culture response in maize

Summary Anther culture of a three-way cross, (H99 × FR16) × Pa91, resulted in the regeneration of two anther-derived plants which were crossed to produce an F₁ progeny. Fourteen S₁ families derived from this cross were evaluated for their anther culturability. Dramatic increases in the level of androgenesis, expressed as the percentage of cultured anthers which produced embryo-like structures, were observed. An overall mean response frequency of 23.4% was observed for the S₁ families. This was compared to a 3.5% response in the original three-way cross. These results demonstrate that genetic improvement of in vitro androgenesis in maize is possible and that anther culture per se constitutes a procedure for selecting genes which favor increased levels of response.     

Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize

We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R₁ kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R₁ kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R₃ seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R₃ seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.     

ESTABLISHMENT OF FRIABLE EMBRYOGENIC (TYPE II) CALLUS FROM IMMATURE TASSELS OF ZEA MAYS (POACEAE)

Type II callus cultures were initiated from immature tassels of a maize genotype with an A188/B73 genetic background using N6 medium containing 1.0 mg/liter 2,4-D, 100 mg/liter casamino acids, 25 mM proline, and 0.2% phytagel™. Inclusion of 10 μM AgNO₃ in this medium significantly increased the frequency and vigor of the type II callus response. Friable calli emerged from these explants after two consecutive 2-week subculture intervals. Tassels from 10 to 30 mm long were capable of producing type II cultures. The plants regenerated from these cultures were green and indistinguishable from plants regenerated from immature embryo-derived calli.     

Genetic studies of corn (Zea mays L.) anther culture response

Summary Anthers of two maize (Zea mays L.) inbred lines, DBTS (P₁) and B73 (P₂), their F₁, F₂ and first backcross generations — F₁ x DBTS (B₁), and F₁ x B73 (B₂) — were float cultured in YP medium to study the inheritance of corn anther culturability using generation mean analysis. Significant effects of generation were observed for the three traits measured: anther response (%), frequency of embryos (%) and anther productivity. Variation among the generations was similar for anther response and frequency of embryos: no significant differences were found among the P₁, F₁, F₂ and B₁ means, but the means of P₂ and B₂ were significantly lower than those of the other generations. For anther productivity, the F₂ generation tended to have a slightly higher tendency for multiple embryo formation. A simple additive-dominance model was adequate in explaining the inheritance of anther response and frequency of embryos, but digenic epistasis (additive x dominance) was involved in the inheritance of anther productivity. Additive genetic variance was higher than non-additive genetic variance for all the traits; however, only environmental variance was significant. Narrow-sense heritability estimates were 65% and 75% for anther response and frequency of embryos, respectively. Significant inter-plant variation was observed within generations, even for the inbred line DBTS, but isozymic analysis involving five enzyme loci did not reveal any genotypic variability within the inbred lines DBTS and B73.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

Summary Production of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R₀ transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.     

Agronomic performance of lines derived from anther culture, maize pollination and single-seed descent in a spring wheat cross

Anther culture and maize hybridization are two frequently used techniques for doubled haploid production in wheat (Triticum aestivum L.). Information on the field performance of lines derived from these techniques is limited. This study was conducted to compare the performance of F_4:6 lines obtained by single-seed descent with lines obtained by anther culture and maize (Zea mays L.) pollination from the same cross of spring wheat, ’Chris’/MN 7529. Thirty-three lines derived from each of those techniques were evaluated in six environments for grain yield, protein content, test weight, heading date, kernel weight and plant height. Mean performance of the single-seed descent lines exceeded performance of the anther culture lines for grain yield, kernel weight and plant height with no apparent differences for grain protein content, test weight and heading date. No differences between trait means for the single-seed descent and maize pollination lines were found except for plant height. The best 5 lines from each method for grain yield, protein content and test weight were similar in performance except that the protein content was higher for the maize pollination lines than for the single-seed descent lines. Acceptable levels of agronomic performance could be found among lines from each method. Wide acceptance of the doubled haploid technique for pure line production in breeding programs may, however, be limited by the often poor efficiency of doubled haploid line production, resulting in smaller population sizes for selection of desirable traits in comparison to the single-seed descent method. Received: 31 July 1998 / Accepted: 28 November 1998     

Genetic studies of rice (Oryza sativa L.) anther culture response

Anthers of two rice (Oryza sativa L.) varieties, Lunhui 422 (P₁) and Jinzao 5 (P₂), their F₁, F₂ and first backcross generation-F₁ x Lunhui 422 (B₁), and F₁ x Jinzao 5 (B₂)-were cultured in L8 medium to study the inheritance of rice anther culturability using generation mean analysis. Significant effects of generation were observed for the four traits measured: anther response (%), frequency of callus induction (%), frequency of callus differentiation (%) and culture efficiency (%). Variation among the generations was similar for all traits: significant differences were found among six generations and the means of the P₂ and B₂ were significantly lower than those of the other generations. The frequency of callus differentiation showed a nonsignificant difference among the P₁, F₁, F₂ and B₁ generations which had slightly high values than the P₂ and B₂. Additive genetic variance (V_A) was higher than non-additive genetic variance (V_D) for anther response and frequency of callus induction. However, A_V was lower than V_D for frequence of callus differentiation and culture efficiency, V_A was significant for all traits except for the culture efficiency, and V_D was nonsignificant for all traits except for the frequency of callus differentiation. On the other hand, environmental variation (V_E) was significant for the 4 traits. Narrow-sense heritability estimates were 95.52%, 82.19% and 13.54% for anther response, frequency of callus induction and culture efficiency, respectively.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of <Emphasis Type="Italic">Withania somnifera</Emphasis>

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the T_L T-DNA, and 90% of them carried the T_R T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the T_L and the T_R-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the T_R T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the T_L T-DNA genes, while all the callusing lines induced by strain A4 contained the T_L DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.     

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.