Murray W. Nabors

NaCl-tolerant tobacco plants from cultured cells

Summary We have obtained NaCl-tolerant cell lines by exposing tobacco cell suspensions to increasing levels of NaCl. Tolerance to 8.8 g/l NaCl is the maximum obtainable in cell suspensions. Normal lines are tolerant to about 1.6 g/l NaCl. Plants regenerated from resistant cell lines transmit tolerance to two subsequent generations. The level of NaCI-resistance in regenerated plants is higher than that of cell cultures. Few nontolerant F2 plants survive watering with solutions containing more than 15.4 gil NaCI whereas most tolerant plants survive 33.4 g/l NaCl.

Long-duration, high-frequency plant regeneration from cereal tissue cultures

By visual examination of calli derived from germinating seeds of wheat, oats, rice, proso millet, and pearl millet it has been possible to visually select embryogenic (E) callus which, on transfer to a regeneration medium, forms plants an average of 33 times more frequently than non-embryogenic (NE) callus of equal mass. Embryogenic callus consists of small isodiametric cells averaging 31 μm in diameter; NE callus consists of long tubular cells averaging 52 μm in width and 355 μm in length. Production of E callus is in many cases promoted by media containing 2,4-di- or 2,4,5-trichlorophenoxyacetic acid (2,4-D or 2,4,5-T) plus indole-3-acetic acid or tryptophan+kinetin. Production on NE callus is promoted by media containing 2,4-D or 2,4,5-T alone. As a result of initial experiments to optimize both media for E callus production and media for plant regeneration, callus derived in six passages from an average of 26 seeds could produce about 1,000 regenerated plants.

SODIUM CHLORIDE TOLERANT LINES OF TOBACCO CELLS

Abstract Tissue culture may provide a rapid method for producing sodium chloride tolerant crop plants. As a first step toward this goal we have isolated several salt tolerant lines of tobacco cells. Both mutagen-treated and untreated cultures develop salt tolerance. The occurrence of spontaneous or induced variants in somatic cell cultures could provide a source of many agriculturally desirable phenotypes.

High frequency, long term regeneration of rice from callus culture☆

Totipotency in cereal tissue cultures is often lost soon after callus initiation, but embryogenic cultures (producing embryo-like forms which develop into complete plants) derived from various plant parts provide long term totipotent cultures. Rice (Oryza sativa vars. Pokkali, IR-36, Mashuri, and Giza-159) calli containing both embryogenic and non-embryogenic regions were initiated from immature embryos and mature seeds cultured on Linsmaier and Skoog (LS) medium, 4% sucrose, 1% agar and variable 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The most callus was initiated on 1.0 mg/l 2,4,5-T or 2,4-D for Pokkali, on 1.0 mg/l 2,4,5-T for Giza-159, on 2.0 mg/l 2,4,5-T for IR-36 and on 1.0 mg/l 2,4-D for Mashuri. There was not as distinct a visual difference between rice embryogenic and non-embryogenic callus as is found in pearl millet, proso millet and oats. Embryogenic rice callus initiates multiple shoots with roots which develop into fertile plants. Non-embryogenic callus initiates occasional shoots without roots. Twenty percent of embryogenic calli but only 6% of non-embryogenic calli initiated shoots after 50 weeks in culture. Embryogenic calli consistently initiated an average of 9 shoots per 10 cc of callus while non-embryogenic calli initiated 0.4 shoots per 10 cc. Differential shoot initiation indicated that visual selection was accurate in distinguishing between the two callus types.

The growth physics and water relations of red-light-induced germination in lettuce seeds : III. Changes in the osmotic and pressure potential in the embryonic axes of red-and far-red-treated seeds.

Irradiation of Grand Rapids lettuce (Lactuca sativa L.) seeds with red light (R) results in an increase in the growth response in the embryonic axes which enables them to overcome the mechanical restriction of the endosperm or the osmotic restriction of an external solution. When the growth rates of axes from R-and far-red(FR)-treated seeds are equalized with polyethylene-glycol-4000 solutions, a water potential difference of 3.4 bars between the axes is obtained. Measurements of the osmotic potential indicate a 1.0–2.0-bar difference between the treatments, depending upon incubation time. Although the difference in water potential remained constant during the growth of the axes, the osmotic constituents of the axes of R-treated seeds were diluted to a greater extent than those of the axes of FR-treated seeds. The length-to-volume ratio of the growing axes of R-treated seeds increased at a greater rate than that of the axes of FR-treated seeds. These data indicate that changes in the cell-wall properties may account for the remainder of the observed water potential difference. We propose that the phytochrome-mediated growth increase in these embryonic axes is an integrated function of the cells: increased wall loosening may be inextricably linked to simultaneous increases in the osmotic constitutents. The driving force for growth may not involve solely a change in the osmotic potential or the pressure potential, with passive readjustment of the other parameter, but the activation of a cellular component that links changes in the wall properties with the accumulation of osmotic constituents.

Green Spots are Predictors of High Callus Growth Rates and Shoot Formation in Normal and in Salt Stressed Tissue Cultures of Oat (Avena sativa L.)

Summary A general method for rapidly producing callus from the roots of germinating oat seedlings is discussed. Shoot regeneration occurs through the 38th week of culture. The occurrence of green spots in secondary callus is positively correlated with shoot regeneration although only regeneration decreases with time. Callus with green spots grows more rapidly and produces many more new green spots than plain callus. Continued green spot and shoot production requires auxin. A period of high temperature (30°C) is necessary for the establishment of regenerated plants in greenhouse or growth chamber conditions. Several hundred such plants have been produced. Cool temperatures (20°C) are required for seed set. The selection of callus tolerant to as much as 0.15 m NaCl is discussed.

Plant regeneration from cell suspension cultures of Vigna aconitifolia.

Plant regeneration has been achieved routinely from established cell suspension culture lines of Vigna aconitifolia (moth bean), a highly drought tolerant grain legume. The cultures originated from three-week-old leaf callus. Several media including MS, B5, AA, SL, PCM, SH and L-6 were tested for their effects on cell growth. Maximum growth was observed in L-6 medium containing 44.5 μM 2,4-D. After 6 to 8 weeks the suspensions were filtered through 500, 250, 125 and 60 μm sieves, respectively, for four to five subcultures. An embryogenic cell line (VA-686) was obtained from the cell fraction collected below 250 μm. The VA-686 cell line is being maintained on L-6 medium with 4.5 μM 2,4-D and 2.3 μM Zeatin. Somatic embryogenesis was induced by transferring the cells to L-6 medium with 4.6 μM zeatin in which green cell clusters were produced. The somatic embryos developed from most of the cell clusters when plated on L-6 agar medium with 2.3 μM BA.Plantlets were obtained from the embryos on L-6 medium with 10.0 μM IBA. The regenerated plants were grown to maturity in the greenhouse.

The mechanism and regulation of proline accumulation in suspension cell cultures of the halophytic grass Distichlis spicata L.

Summary Proline accumulation in the halophyte Distichlis spicata L. occurred rapidly following initiation of NaCl-induced stress and stabilized at an elevated level within 48 h of exposure to NaCl. Accumulation of proline was inhibited by cycloheximide but not by actinomycin D, suggesting that mRNA translation but not mRNA transcription may be necessary prior to production of proline in stressed cells. Protoplasts and vacuoles were isolated to examine partitioning of proline between protoplast and vacuole. Significant amounts of free proline leaked from the protoplasts or cells into the enzyme solution during isolation. Numerous vacuoles were released from a single protoplast during protoplast lysis. The isolated vacuoles contained a minor fraction of the cellular proline.

Long Term Plant Regeneration, Somatic Embryogenesis and Green Spot Formation in Secondary Oat (Avena sativa) Callus

Summary Calli initiated from mature seeds, mesocotyls, and immature embryos of oat ( Avena sativa ) produced embryogenic and non-embryogenic callus and green spots. Embryogenic callus is white, opaque and often convoluted while non-embryogenic callus is rough and yellow to translucent in appearance. Non-embryogenic callus can produce shoots or roots. Embryogenic callus can produce shoots, roots, or complete plantlets. Shoot and plantlet production occurs at much higher frequencies and for longer durations in embryogenic callus. After 36 weeks in culture, 20 percent of embryogenic calli initiated shoots or plantlets while non-embryogenic calli formed no shoots. Elongated regions called green spots are produced by non-embryogenic callus and have a distinct structure which resembles a root apex. New green spots are formed adjacent to but not in direct contact with existing green spots. Very few green spots are initiated in calli dissected free of green spots. A model which explains the ontological relationship between observed calli types and green spots is given. The use of embryogenic callus offers a significant advance in the control of regeneration in oats.

Somaclonal variant plants of wheat derived from mature embryo explants of three genotypes.

Somaclones regenerated from three wheat (Triticum aestivum L.) cultivars, Glennson, Pavon and PAK-16171 were evaluated for variation in agronomic and morphological characters. Calli were initiated from germinating seeds on Linsmaier and Skoog (LS) medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, 2% sucrose and 1% agar. Calli were isolated and regenerated into whole plants on LS medium containing 0.1 mg/l indole - 3-acetic acid and 0.5 mg/l benzyladenine. Comparisons among the somaclones and their parents were made for plant height, spike length, number of grains per spike, and 100 grain weight. Significant variation was observed in these characters between the somaclones and parents. Genotypic differences were observed among the somaclones for many of these agronomic and morphological characters.