Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR^-K⁺) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR⁺K^-SR⁺). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F₁ plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR^-K⁺) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.     

Arabinogalactan-protein epitope Gal4 is differentially regulated and localized in cell lines of hybrid fir (<Emphasis Type="Italic">Abies alba</Emphasis> × <Emphasis Type="Italic">Abies cephalonica</Emphasis>) with different embryogenic and regeneration potential

Arabinogalactan proteins (AGPs) are important proteoglycans regulating somatic embryogenesis in diverse plant species. Embryogenic cells of somatic embryos are covered by special extracellular cell wall layer called extracellular surface matrix network (ECMSN) at their early developmental stages. Here we show that highly embryogenic cell line AC78 of hybrid fir (Abies alba × Abies cephalonica) differs from very low-embryogenic cell line AC77 in the abundance, subcellular localization and deposition of subset of secreted AGPs. A specific AGP epitope containing Gal residues and reacting to Gal4 antibody is secreted and deposited into ECMSN, which covers the surface of the embryogenic cells showing high embryogenic and regeneration capacity in the cell line AC78. On the other hand, this Gal4 AGP epitope was not secreted and/or found on the surface of meristematic cells showing low embryogenic and regeneration capacity in the cell line AC77, as well as on the surface of non-embryogenic suspensor cells and callus cells in both cell lines AC77 and AC78. As a positive control, we have used another AGP epitope LM2 (containing glucuronic acid) showing no significant differences in these two Abies hybrid lines. This study defines specific AGPs containing β-(1→6)-galactotetraosyl group as a first molecular component of ECMSN covering embryogenic cells in gymnosperms. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

Summary The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

“Arabidobrassica”: Chromosomal recombination and morphogenesis in asymmetric intergeneric hybrid cells

A somatic hybrid cell line, cloned from an individual protoplast-fusion product between Arabidopsis thaliana and Brassica campestris, gave rise to formation of numerous plants differing drastically in morphology. Analysis of these various regenerants, all of which originated from one and the same heterokaryon derived from the fusion of two cells, shows the unspecific elimination of chromosomes of both parental species during the callus growth phase. Whereas the parental cells have so far not been sucessfully regenerated into plants, several of their different asymmetric hybrids are capable of morphogenesis. Furthermore, chromosomal analysis indicates extensive recombination. Most of the plants are predoinantly morphologically regular. Abnormalities are mostly limited to the flowers which tend to undergo phyllody. The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable. The transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Ultrastructural characteristics of an achlorophyllous callus line ofLotus corniculatus

Summary An achlorophyllus isoline originated spontaneously from a green callus line derived from a genotype ofLotus corniculatus L. cv. Leo. This isoline is characterized by significantly faster callus growth than the green line and by differentiating significantly fewer plants per gram of callus when subcultured on a shoot differentiating medium. The isoline does, however, develop loci of meristematic cells when transferred from a medium containing 2,4-D to one containing benzyl adenine. After this transfer, plastids, mitochondria, dictyosomes, and endoplasmic reticulum appear to be more prevalent in many cells. Nuclei and nucleoli become more prominent in these cells. Ingrowths, lined with plasmalemma, develop along the outer tangential cell wall of many cells in contact with the culture medium and along various walls of cells found in the interior of the callus. These cells have characteristics of transfer cells. A few cells at the periphery of meristematic groups of cells elaborate lamellar structures similar to suberin lamellae in their cell walls.     

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

Summary The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E₁ callus and those of the callus arising from it, according to the culture conditions.The peculiar characteristics of E₁ are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells.An embryogenic friable callus (E₂) is formed in time when E₁ is maintained on solid culture medium. The E₂ cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials.Shaking E₁ in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.Abbreviations c cell - cr crystal - cw cell wall - d dictyosome - er endoplasmic reticulum - m mitochondrion - mb microbody - n nucleus - p plastid - s starch - v vacuole     

Somatic cell cryopreservation and protoplast regeneration of important disease-resistant wild rice Oryza meyeriana Baill

Oryza meyeriana Baill is one of the three wild rice species found in Chiia.O. mcyeriana possesses valuable characteristics but is reluctant in cell culturein vitro. In a series of experiments, callus with no regeneration ability was induced from young panicle ofO. meyeriana. The callus was subcultured and propagated. Embryogenic cell clones were obtained after cryopreswation. Suspension cultures were established and protoplasts were isolated and regenerated into plants. Results of artificial inoculation ofXanthomonas campestris pv.Oryzae showed that the strong resistance did not change in the regenerated plants. The development of protoplast-to-plant system is an important progress towards utilization ofO. meyeriana via cellular engineering. The experiments demonstrated that cryopreservation of plant calli was a new way to obtain embryogenic cell line. Project partially supported by the National Natural Science Foundation of China (Grant No. 392704361, Foundation for Outstanding Young Teachers of China, State Key Program of China and Natural Science Foundation of Hubei Province, China.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

珠子参中HMGR基因对皂苷生物合成的影响

该研究以珠子参愈伤组织为材料,通过同源克隆方法获得了珠子参3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因(PjHMGR,登录号:MG712296)的cDNA序列,并对其进行生物信息学分析。基于PjHMGR序列构建了珠子参过表达载体pCAMBIA2300s-PjHMGR,通过根瘤农杆菌转化法将其转化到珠子参细胞中,成功获得7株阳性转PjHMGR基因细胞系;通过实时荧光定量PCR、比色法及皂化法等技术测定阳性细胞系PjHMGR基因的相对表达量、HMGR酶活、皂苷和植物甾醇含量变化。结果显示:(1)与野生型珠子参细胞系相比,转PjHMGR基因珠子参阳性细胞系中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量均有不同程度的提高;在效果最好的阳性细胞中,PjHMGR基因的相对表达量、HMGR酶活和皂苷含量分别为对照的7.15、6.14和3.50倍。(2)在研究过程中,发现转基因细胞系的植物甾醇含量也有所升高。研究认为,将珠子参关键酶基因PjHMGR过表达载体导入珠子参愈伤组织细胞中,可引起相关关键酶基因相对表达量和PjHMGR酶活性的增加,从而调控珠子参总皂苷的合成,对皂苷合成途径中关键酶基因进行调控将可能实现对皂苷生物合成的调节。     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

Culture phenotype affects on regeneration capacity in the monocotHaworthia comptoniana

Summary Haworthia comptoniana specimens were cultured to determine how benzyladenine (BA) level and in vitro selection for shoot and callus production affected regeneration capacity and plant phenotype. Leaf explants were cultured on Murashige and Skoog medium containing 0 to 10 mg·liter⁻¹ of BA. The highest number of shoots was obtained with 0.5 mg·liter⁻¹ of BA.H. comptoniana stock cultures (hc) maintained with 0.5 mg·liter⁻¹ of BA produced clumps of small shoots interspersed with friable, white, tan, and green callus. A clump of very large shoots was isolated and designated cell line Rhc; it differed from the original hc culture in shoot size, the lack of callus growth, and higher water content. A line of green callus (designated Gc), a line of white callus (Wc), and a line of soft tan callus (Tc) were also isolated from hc. Optimal BA levels for shoot regeneration from lines Gc and Wc were 2 and 5 mg·liter⁻¹, respectively. No normal shoots could be regenerated from Tc. The phenotypes of these cell lines remained stable for 24 subculture generations. The hc line that initially required BA for growth became hormone autotrophic whereas the other lines did not. Culturing using Gelrite and sealing vessels with parafilm promoted vitrification of the hc line. Culturing using GIBCO agar and unsealed vessels reduced vitrification. The ex-vitro greenhouse survival rates for hc and Rhc plantlets were 10 and 80%, respectively. The large size of the Rhc shoots apparently resulted in significantly higher survival rates under greenhouse conditions, but did not result in any phenotypic whole plant changes.     

细枝木麻黄离体培养过程中愈伤组织和再生芽的细胞组织学观察

为探讨细枝木麻黄(Casuarina cunninghamianaMiq.)愈伤组织分化过程的细胞组织学,对离体培养条件下的愈伤组织进行扫描电子显微镜和石蜡切片观察,分析愈伤组织的细胞分裂、分化以及芽再生的发生过程。结果表明,新鲜外植体培养于愈伤组织诱导培养基上,伤口处的薄壁细胞开始脱分化,培养1周后形成明显的愈伤组织;继续培养2周后,胚性愈伤组织形成,且表层细胞启动分化形成芽原基;培养4周,可肉眼观察到胚性芽原基,数量增多并逐渐分化形成不定芽;培养至第6周,生成不定芽,并大量增殖和分化。因此,细枝木麻黄是通过愈伤组织分化形成胚状体的途径进行植株再生的,为建立细枝木麻黄组织培养高效再生体系提供了理论依据。     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

A second mutation enhances resistance of a tobacco mutant to sulfonylurea herbicides

Summary Cultures of Nicotiana tabacum cells homozgous for a mutation (S4) at the SuRB locus that confers resistance to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Chaleff and Ray 1984; Chaleff and Bascomb 1987) were used to isolate a doubly mutant cell line (S4 Hra/S4+) resistant to even higher herbicide concentrations. Growth of cells homozygous for both the S4 and Hra mutations (S4 Hra/S4 Hra) was uninhibited by a herbicide concentration 500-fold higher than a concentration by which growth of S4+/S4+ callus was inhibited by 75%. Plants homozygous for both mutations were at least five-fold more resistant to foliar applications of chlorsulfuron than were singly mutant S4+/S4+ plants. This enhanced resistance was inherited as a single, semidominant, nuclear trait that is genetically linked to the S4 mutation. Acetolactate synthase (ALS) activity in extracts of leaves of doubly mutant (S4 Hra/S4 Hra) plants was approximately 20-fold more resistant to inhibition by chlorsulfuron and sulfometuron methyl than was ALS activity in singly mutant (S4+/ S4+) leaf extracts, which was in turn more resistant to inhibition by these compounds than was the normal enzyme. Extracts prepared from plants of these three genotypes possessed the same ALS specific activities. Therefore, Hra represents a second independent mutation at or near the SuRB locus that reduces the sensitivity of tobacco ALS activity to inhibition by sulfonylurea herbicides.