David R. Duncan

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Effect of l-aminocyclopropane-l-carboxylic acid, silver nitrate, and norbornadiene on plant regeneration from maize callus cultures

The effect of the ethylene antagonists norbornadiene and silver nitrate and the ethylene precursor l-aminocyclopropane-l-carboxylic acid (ACC) on Zea mays plant regeneration was studied. A 12-fold increase in plant regeneration, as measured by number of plants obtained per gram fresh weight from callus cultures of maize inbreds Pa91 and H99, was obtained by 250 μM norbornadiene and 100 μM silver nitrate treatments. An increase in amout of nonregenerable tissue and a 68% decrease in plant regeneration were associated with callus treated with 1 mM ACC. Ethylene emanation from 1 mM ACC treated callus reached a maximum of 170 nl g−1 h−1 after 3 days compared to 7 nl g−1 h−1 for the control. The free proline content was up to 80% lower in 1 mM ACC treated callus grown for 30 days on medium with or without 12 mM proline, respectively, as compared to each control. These studies indicate that ethylene action inhibitors such as norbornadiene and silver nitrate can be used to increase plant regeneration efficiency from maize callus cultures.

The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus

SummaryFour antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 μ M APM or 10 μ M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 μ M), however, at a higher concentration (5 μM) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations.

Measurements of Viability Suitable for Plant Tissue Cultures

Experiments using plant tissue cultures, such as measuring their tolerance to herbicides or metabolite analogs, elucidating biochemical pathways by exposing the tissues to metabolic inhibitors, or cryopreserving the cultures, require at some point the determination of the tissues viability. Viability is the capacity of a cell or organism to live, and the state of being viable is most commonly recognized by the growth of the studied tissues (1-3). Measurements of growth can include measuring increases in fresh or dry weight of a cultured tissue (2,3) or measuring increases in cell number or size (2-4).

Selection of regenerable maize callus cultures resistant to 5-methyl-DL-tryptophan, S-2-aminoethyl-L-cysteine and high levels of L-lysine plus L-threonine.

Tissues resistant to lethal levels of equimolar L-lysine plus L-threonine (LT), 5-methyl-DL-tryptophan (5MT, a tryptophan analog), or S-2-aminoethyl-L-cysteine (AEC, a lysine analog) were selected from maize callus capable of plant regeneration (H99 and W77-R3019 genotypes).Resistance to LT resulted from resistant calli having a 19 times greater level of free threonine than wild type tissues. The resistance was expressed in roots of whole plants; threonine levels were two to nine times greater in leaves and kernels of resistant plants than in wild type plants. Slightly greater levels of isoleucine, lysine and methionine were also noted, particularly in the kernel. Genetic studies with individual resistant plants did not always produce inheritance ratios typical of simple Mendelian inheritance, but by the third generation after plant regeneration a trend towards homozygosity was apparent and the data suggests that LT resistance is inherited as a single dominant nuclear gene.Resistance to 5MT resulted from resistant calli having a 133 to 161 times greater level of free tryptophan than wild type tissues. Also, phenylalanine was 22 to 30 times as great and histidine, tyrosine and valine were about two times as great as in wild type tissues. Resistance was expressed in roots of whole plants, and tryptophan levels were at least 2000 times greater in resistant than in wild type plants. Phenylalanine was also 32 times greater. All regenerant plants resistant to 5MT were both male and female sterile.Resistance to AFC was caused by decreased AEC uptake by the callus tissue and was not due to increased levels of free lysine. Plants were not regenerated from this callus.

Improved plant regeneration from maize callus cultures using 6-benzylaminopurine.

A new protocol for regenerating plants from cultured type I callus of the maize (Zea mays L.) inbred Pa91 includes growing the callus on medium containing 3.5 mg/l (15.5 μM) of the cytokinin 6-benzylaminopurine (6BA) for 3 to 6 d and then moving the callus to medium containing no growth regulators (H medium) for an additional 15 to 21 d, where the plants actually develop. The number of plants regenerated from the 6BA treated callus was 113% to 148% greater than the number of plants produced from callus placed directly on H medium. This increased plant regeneration induced by 6BA seemed to maximize the number of plants regenerated from a gram of callus and was slightly affected by callus age or prior treatment of callus with AgNO3. Exposure to 6BA for 9 d greatly reduced shoot and root development, and longer exposures totally prevented root formation. This inhibition of root formation could be reversed only slightly by naphthaleneacetic acid. The data indicate that high concentrations of 6BA are effective for increasing plant regeneration from maize callus cultures when short exposure times are used. This procedure has also been effective for regenerating many plants from the inbreds H99 and Mo17.

The effect of gamma radiation and N-ethyl-N-nitrosourea on cultured maize callus growth and plant regeneration

Regenerable maize calli of two inbred lines were exposed to 0 to 100 Gy of gamma rays or treated with 0 to 30 mM of N-ethyl-N-nitrosourea (ENU) to determine their effect on growth and plant regeneration capability. Both growth and plant regeneration capacity decreased with increasing levels of either gamma radiation or ENU; however, plant regeneration capacity was more sensitive to either agent than growth. The 50% inhibition dose (I50) for callus growth (fresh-weight gain) was approximately 100 Gy of gamma radiation and 30 mM ENU. The I50 for plant regeneration capacity of treated callus was approximately 25 Gy of gamma radiation and 2.5 mM ENU. The decrease in plant regeneration capacity correlated with a change in tissue composition of the treated callus from a hard, yellow and opaque tissue to a soft, grayish-yellow and translucent tissue. This change was quantified by measuring the reduction of MnO4- to MnO2 (PR assay) by the callus. These results suggest that the effect of gamma radiation or ENU on plant regeneration capacity must be taken into consideration if these potentially mutagenic agents are to be used on maize callus cultures, for the purpose of producing useful mutations at a whole plant level. The data also suggest that the PR assay may be useful for predicting the actual plant regeneration capacity of maize callus.

Differential response to potassium permanganate of regenerable and of non-regenerable tissue cell walls from maize callus cultures

Type I maize (Zea mays L.) callus typically consists of two kinds of tissue, one type is capable while the other is incapble of regenerating whole plants. Exposing Type I callus to a 20 mM potassium permanganate (KMnO4) solution (pH 7.2) resulted in a brown precipitate forming on intact non-regenerable tissues in the callus. Residue from homogenized callus also reacted with KMnO4. The precipitate could only be dissolved in acid and permanganate could be re-formed from the dissolved precipitate using the strong oxidants potassium periodate (KIO4) or sodium bismuthate. These data suggest that the brown precipitate is manganese dioxide (MnO2). Callus exposed to a 20-mM KMnO4 solution of pH 3.0, produced less MnO2. This callus when exposed to Feulgen reagent developed a red-violet color only on non-regenerable tissue. A similar Feulgen reaction occurred with callus treated first for 2 min with a 34.8-mM acidic solution of KIO4. Even regenerable tissue reacted with Feulgen reagent with prolonged exposure to KIO4. The Feulgen results indicate that a highly oxidizable carbohydrate is associated with non-regenerable tissue but not regenerable tissue and it is the oxidation of this material by KMnO4 that generates MnO2. Residue from homogenized callus still reacted with KMnO4 when washed three times with acetone, 1 N HCl, dimethyl sulfoxide, saturated (0.5 M) EDTA, or when heated in a boiling water bath for 20 min. However, washing the residue with 1 N KOH caused a 6-fold reduction in the formation of MnO2. The results from these residue-washing experiments indicate that the highly oxidizable carbohydrate associated with non-regenerable tissue may be a hemicellulose. Manganese dioxide, formed from KMnO4 by pH adjustment, also was found to bind to homogenized-callus residue. The reaction of non-regenerable tissue with KMnO4 has proven to be a useful method for assessing the quantity of non-regenerable tissue in maize callus and may be useful as a marker for developmental changes in maize cells.

Increased induction of regenerable callus cultures from cultured kernels of the maize inbred FR27rhm

Kernels of the maize inbred FR27rhm were cultured on various media to determine if the treatments would alter the frequency of formation of regenerable callus (induction frequency) by embryos excised from the kernels when they were placed on callus induction medium. The addition of 60 μM dicamba (3,6-dichloro-o-anisic acid) to the kernel culture medium resulted in an induction frequency of 27–38% compared to 0% for controls on standard kernel culture medium. Embryos excised from dicamba-treated kernels also showed in-ovule callus-like tissue proliferation. The increased induction frequency and the callus-like tissue proliferation could also be produced by injecting the ears of field grown FR27rhm plants, 3-d post pollination, with 1.08 μmoles of dicamba. The results indicate that treatment of the developing ear with dicamba, in vivo or the developing kernel in vitro, may be an effective means to increase the frequency of regenerable callus induction from recalcitrant maize genotypes, such as the B73 derivative FR27rhm.

Techniques for selecting mutants from plant tissue cultures.

The theory and goals of mutant selection from plant tissue cultures have been reviewed extensively over the past few years (I-5), and will only be dealt with in a cursory manner in this chapter. For more detail, the reader is encouraged to consult these review articles. The ideal plant tissue culture system is useful for mutation selection because it: