Lettuce Cotyledons Research Articles

Etr1-1 Gene Expression Alters Regeneration Patterns in Transgenic Lettuce Stimulating Root Formation

We have evaluated the transformation efficiency of two lettuce (Lactuca sativa L.) cultivars, LE126 and Seagreen, using Agrobacterium tumefaciens-mediated gene transfer. Six-day-old cotyledons were co-cultivated with Agrobacterium cultures carrying binary vectors with two different genetic constructs. The first construct contained the β-glucuronidase gene (GUS) under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S), while the second construct contained the ethylene mutant receptor etr1-1, which confers ethylene insensitivity, under the control of a leaf senescence-specific promoter (sag12). Tissues co-cultivated with the GUS construct showed strong regeneration potential with over 90% of explants developing callus masses and 85% of the calli developing shoots. Histochemical GUS assays showed that 85.7% of the plants recovered were transgenic. Very different results were observed when cotyledon explants were co-cultivated with Agrobacteria carrying the etr1-1 gene. There was a dramatic effect on the regeneration properties of the cultured explants with root formation taking place directly from the cotyledon tissue in 34% of the explants and no callus or shoots observed initially. Eventually callus formed in 10% of cotyledons and some organogenic shoots were obtained (2.86%). These results indicate that the ethylene insensitivity conferred by the etr1-1 gene alters the normal pattern of regeneration in lettuce cotyledons, inhibiting the formation of shoots and stimulating root formation during regeneration.

Sporulation of Bremia lactucae Affected by Temperature, Relative Humidity, and Wind in Controlled Conditions

ABSTRACT The effects of temperature (5 to 25 degrees C), relative humidity (81 to 100%), wind speed (0 to 1.0 m s(-1)), and their interactions on sporulation of Bremia lactucae on lettuce cotyledons were investigated in controlled conditions. Sporulation was affected significantly (P < 0.0001) by temperature, with an optimum at 15 degrees C, and by relative humidity (RH), with sporulation increasing markedly at RH >/= 90%. There was a significant effect of exposure time in relation to temperature (P = 0.0007) but not to RH. In separate experiments, both RH and wind speed significantly (P < 0.0001) affected the number of cotyledons with sporulation and the number of sporangia produced per cotyledon. No sporulation was observed at wind speeds of >0.5 m s(-1), regardless of RH. In still air, the number of sporangiophores produced per cotyledon increased linearly with RH from 81 to 100% (P = 0.0001, r = 0.98). Histological observations indicated that sporulation may be affected by stomatal aperture in response to RH, as more closed stomata and correspondingly fewer sporangiophores were present at lower RH. These results are important for understanding the mechanism of RH effects on sporulation and for predicting conditions conducive to downy mildew development.

Genotypic response of lettuce cotyledons to regeneration in vitro

Twenty-two lettuce (Lactuca sativa) genotypes belonging to different morphological groups were screened for their response to regeneration in tissue culture. A shoot regeneration medium, consisting of SH salts and vitamins, supplemented with 0.1 mg 1−1 IAA, 0.5 mg 1−1 kinetin, 0.05 mg 1−1 zeatin, 30 g 1−1 sucrose and 7 g 1−1 bacteriological agar at pH 5.8 was used. Response in tissue culture measured by callus production, root and shoot regeneration was generally found to be statistically dependent on the genotype of lettuce used. Callus and shoot indices were generated for all genotypes as indicators of performance. The use of Bronze Mignonette as a standard genotype enabled batches of genotypes to be compared in successive experiments. This approach detected minor variations among experiments and allowed the culture performance of all genotypes to be ranked. No correlation was observed between callus index and shoot index, and the variation in performance of genotypes was not statistically linked to their morphological groups. Genotypes that showed good shoot regeneration in tissue culture included: Bambino and Iceberg (crisphead types); Cobham Green and Sweet Butter (butterhead types), Simpson Elite (leaf type), and Rosalita and Paris White (cos types).

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

We determined peroxidase activity and its isozyme pattern in the course of the formation of calluses in an attempt to relate the combined effect of 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA) on the induction of somatic embryos and the relatioship between morphogenesis and changes of peroxidase activity

Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region

The expression of a stress- and salicylic acidinducible protein gene from tobacco, PR1a protein gene, was determined after its Introduction to lettuce (Lactuca sativa L.) plants. The 5' flanking 2.4 Kb fragment from PR1a gene was joined to the bacterial β -glucuronidase (GUS) gene (PR-GUS) and introduced into lettuce cotyledons by Agrobacterium-mediated gene transfer using a binary vector containing a kanamycin-resistance gene as a selectable marker. As a control with constitutive expression, the chimeric gene consisting of CaMV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-resistant regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the lettuce genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These results showed that the promoter of the stress-inducible tobacco PR1a protein gene was introduced into lettuce plants, and the introduced chimeric gene was expressed normally under the regulated control of the PRla promoter.

A cyto-histological study on shoot differentiation and mesophyll morphology of the excised lettuce (Lactuca sativa L.) cotyledons as affected by light conditions during seedling growth.

プロトプラストの単離に使用するレタス子葉組織について, その育成光条件と葉組織の細胞学的特徴および不定芽形成との関係につき研究した。その結果, (1) 蛍光灯と陽光ランプでは芽生えの生長が異なった。蛍光灯150 lx条件の子葉は未展開であったが, 5,000 lx以上の区と陽光ランプ区ではよく展開し正常に生長した。(2) 蛍光灯と陽光ランプで波長別エネルギー分布が異なっていた。(3) 子葉組織の細胞学的観察では, 150 lxの蛍光灯下で育成した子葉細胞で細胞質が多く, 5,000 lx以下で細胞質が少なかった。陽光ランプ下で育成した子葉は, 何れの区でも細胞質が多かった。(4) 子葉の育成条件 (照度) と芽生えのエイジは, 不定芽形成頻度に大きく影響した。以上の結果から, プロトプラストを単離する材料組織の育成方法は, プロトプラストを単離培養の効率化に関係すると考えられた。

A search for elicitors of the hypersensitive reaction in lettuce downy mildew disease

Abstract Attempts were made to isolate elicitors of the hypersensitive reaction (HR) from various infection structures produced by Bremia lactucae including isolated mycelium, primary and secondary vesicles and haustoria. Potential elicitors were assayed by injection into lettuce cotyledons and also following their addition to lettuce protoplasts. The protoplast assay was designed to ensure contact between elicitors and putative receptor sites in the plant cell membrane. Scanning electron microscopy showed that enzymic digestion of cotyledons for 12 h released protoplasts free of residual wall fragments and that the wall reformed slowly following protoplast isolation. Conidiosporangia germinated poorly in the protoplast osmoticum. Protoplast viability (assessed with fluorescein diacetate) was reduced by 20–45% in the presence of germinating spores but no cultivar or isolate specific effects were observed. Prior germination of conidiosporangia in water or use of protoplasts recovered following enzymic digestion for 3, 5, 7, or 9 h did not affect the response of protoplasts. Frozen, thawed and ultrasonicated suspensions of conidiosporangia did not kill protoplasts rapidly but caused non-specific agglutination. Intercellular fluids recovered from infected lettuce did not kill lettuce or cabbage protoplasts. Mycelium of B. lactucae was recovered following enzymic digestion of infected cotyledons. Intercellular hyphae and haustoria recovered appeared to be packed with cytoplasm but were dead as indicated by their failure to exclude Evans blue stain. Primary and secondary vesicles were recovered by homogenization of epidermal strips and purified by combinations of sieving and application to sucrose density gradients. Very few infection structures were viable following extraction and all were dead within 10 min of isolation. The presence of cultivar specific elicitors of the HR was indicated in certain assays of primary vesicles of isolate NW3 recovered 6 h after inoculation of the susceptible cv. Cobham Green. Repeated experiments showed that the occurrence of differential activity was very inconsistent and may have been associated with the presence of many viable vesicles in the active extracts. Difficulties associated with the detection of elicitors of the HR produced by obligate parasites are discussed.

Effect of Aliette on Bremia lactucae on lettuce

Aliette is a systemic fungicide effective against diseases caused by lower fungi but there is some question whether it acts directly on pathogens or indirectly via host defence. Downy mildew development on lettuce cotyledons was markedly decreased by application of 100–500 μg Aliette, active ingredient (a.i.), ml −1 to roots. Sporangial germination on glass and cotyledons was prevented by direct application of 10 and 25 μg Aliette ml −1 respectively. Germ-tube growth and penetration into cotyledons were markedly decreased when 25 μg Aliette ml −1 was applied directly 2 h after sporangial inoculation. The size of secondary vesicles and growth of intercellular hyphae in cotyledons were markedly affected when 250 μg Aliette ml −1 was applied 4 h after sporangial inoculation. Hyphal growth was seen to be affected 24 h later; a low incidence of necrosis of host cells was observed after a further 24 h. The findings gave reason to believe that Aliette acted directly on the pathogen in this system.

Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

Abstract The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH 4 ) 2 SO 4 , chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M , of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K m with all three substrates remained constant during the purification procedure, V max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe 3+ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu 2+ inhibited the activity.

Multifunctional cyclic nucleotide phosphodiesterase activity in lettuce cotyledons

Multifunctional cyclic nucleotide phosphodiesterase activity in lettuce cotyledons

Transfer of label from 3H-glucose in Digitaria eriantha leaves to the rust fungus Puccinia digitariae Pole Evans.

Digitaria eriantha pentzii was fed 3H-glucose prior to inoculation with uredospores of Puccinia digitariae Pole Evans. Twenty-one hours after inoculation, uptake of label from 3H-glucose by the primary infection structures of P. digitariae was demonstrated employing autoradiography. These results indicate that an exchange of nutrients between host and pathogen occurs very early on in the infection process, during the formation of the primary infection structures. Despite contrary reports that obligate parasites receive no nutrition before establishment of haustoria, this study supports the work of Andrews (Can J Bot 53:1103, 1975), who demonstrated uptake of 3H-glucose label from lettuce cotyledons into the primary and secondary infection vesicles, appressoria, and germ tubes of Bremia lactucae.

Comparative ultrastructure of lettuce cotyledons infected with Botrytis cinerea and Bremia lactucae and the histochemical localization of acid phosphatase

The ultrastructure of the compatible interactions between lettuce cotyledons and Botrytis cinerea and Bremia lactucae were compared. Infection by B. cinerea was achieved, following direct penetration through the cuticle, by growth of intramural and intracellular hyphae and was associated with rapid and widespread destruction of host tissue. Using a modified Gomori lead-salt precipitation method acid phosphatase activity was localized in the modified epidermal cell wall surrounding the penetration site of B. cinerea , at various vesicular locations in infected plant cells and dispersed throughout the disorganized cytoplasm of cells at an advanced stage of necrosis. Infection by B. lactucae caused no apparent adverse reaction in the cytoplasm of infected plant cells and acid phosphatase activity was largely confined to those sites, such as the dictyosomes, which were typical of healthy tissue. However, intense enzyme activity was localized within the walls of intruding haustoria and with the collar of apposition material deposited by the host around the necks of these fungus structures. Acid phosphatase was also a conspicuous feature of the apposition material deposited by lettuce in response to infection by B. cinerea .

Interactions of growth regulators, explant age, and culture environment controlling organogenesis from lettuce cotyledons in vitro

Organogenesis in cultured Lactuca sativa L. cv. Grand Rapids cotyledons was controlled by growth regulators and influenced by explant age as well as culture environment. Control explants formed roots with a low frequency. Exposure to indoleacetic acid (IAA) greatly stimulated root initiation. Kinetin (K) applied singly or in combination with IAA induced bud formation. Benzylaminopurine (BA) also caused bud initiation. Root and shoot initiation was optimal with 4-day-old explants. Organogenesis declined with older explants. More buds formed in light than in darkness. Other environmental variables tested had no marked effect on K-induced bud initiation. Control explants on medium containing no growth regulator never formed buds.

Evidence that determinants of race specificity in lettuce downy mildew disease are highly localized

Abstract Attempts were made to detect the production of diffusible specific elicitors or suppressors of the hypersensitive reaction (HR) by the lettuce downy mildew fungus Bremia lactucae. In cv. Mildura prior inoculation with the virulent isolate TV did not induce susceptibility to the avirulent isolate IM43. Resistance (HR) was expressed by cells adjacent to compatible intercellular hyphae or even cells containing compatible haustoria or infection structures. Intercellular fluids recovered from cotyledons heavily infected with a virulent isolate of B. lactucae were examined as a potential source of race-specific elicitors of the HR. Irrespective of the isolate/cultivar combination employed, no symptoms developed in lettuce cotyledons injected with intercellular fluids. However, the non-hosts cabbage and broad bean developed macroscopically visible symptoms by 8 days after leaf injection. Intercellular fluids from uninfected tissue caused no necrosis. A sandwich or transplant technique was adopted in order to maximise the exposure of resistant tissues to compounds diffusing from intercellular hyphae of B. lactucae. No necrosis was observed in resistant lettuce cells unless there were signs of penetration and the development of rudimentary haustoria from invading incompatible hyphae. Necrosis was more widespread following transplants to cabbage. The apparent absence of diffusible elicitors or suppressors of race-specific resistance indicates that recognition in lettuce downy mildew disease probably involves intracellular interactions occurring between the plant plasmalemma and the primary vesicle, secondary vesicle or haustorium of the fungus.

Viability of Bremia lactucae oospores and stimulation of their germination by lettuce seedlings

Oospores of Bremia lactucae were capable of germination in vitro from 23 days to 12 months after formation in lettuce cotyledons inoculated with a mixture of conidia of isolates of compatible mating-type. Rotted lettuce cotyledons containing oospores were stored in tap water at 24 °C. Periodically, over the course of a year, viable oospores were teased from tissue by micromanipulation and plated on 1% water agar close to germinating lettuce seed. This treatment stimulated between 23 and 33% of the spores to germinate. Oospore germination was stimulated also, to some extent, by seed of Lactuca virosa, but not by seed killed by heat treatment or by old, dead seed. The stimulant of oospore germination can be extracted by diethyl ether from water in which lettuce seed has germinated. The stimulant activity of such an extract was lost after heating at 107° and 121° but not after a short exposure to 100°.

Effects of Cytokinin and Several Inorganic Cations on the Polyamine Content of Lettuce Cotyledons

Kinetin (4.7 × 10−5M) and 6-benzyladenine (2.22 × 10−5M) were found to increase ca. 2-fold the putrescine content in cotyledons of lettuce (Lactuca sativa L.) seedlings grown for 3 days under fluorescent light. On the other hand, several inorganic ions (K+, Na+, Ga++, Mg++) at a concentration of 3 × 10−2M reduced the putrescine content. The combination of kinetin with one of several inorganic ions at the same level markedly increased the spermine content, but the putrescine content decreased; calcium and magnesium ions were less effective. The physiological significance of these findings is discussed.

Origin of gametangia in heterothallic isolates of Bremia lactucae

The formation of gametangia by heterothallic isolates of Bremia lactucae was investigated by light microscopy of fresh infected lettuce cotyledons and by scanning electron microscopy of chemically fixed and cryofixed cotyledons. Techniques were developed to produce paradermal fracture planes in the infected tissue which was then examined in the scanning electron microscope. These investigations revealed that gametangia were formed as a result of a complex interaction between two or more vegetative hyphae in close physical association, followed by proliferation of sexual hyphae. Antheridia were attached paragynously to oogonia. The observations support the experimental demonstration of heterothallism in B. lactucae.

Recovery of progeny following sexual reproduction of Bremia lactucae

Heterothallic isolates of Bremia lactucae of different virulence phenotypes were crossed by inoculation together on to lettuce cotyledons, resulting in the production of oospores. Progeny were obtained by inoculating seedlings with suspensions of these oospores which had been prepared by sonication in the presence of glass beads. The progeny exhibited novel virulence phenotypes consistent with the hypothesis that B. lactucae is not haploid and that virulence is recessive. Neither long periods of time nor leaching appeared to be prerequisites for oospore germination.

Catecholamine stimulation of the gibberellin action that induces lettuce hypocotyl elongation

Effects of catecholamines and their derivatives on gibberellic acid (GA)-induced lettuce hypocotyl elongation was studied, because catecholamines have a chemical structure similar to the dihydroconiferyl alcohol that has been isolated from lettuce cotyledons as a GA synergist. Epinephrine, norepinephrine, dopamine and 3,4-dihydroxymandelic acid synergistically enhanced the promoting effect of GA on hypocotyl elongation. In contrast, metanephrine, normetanephrine, DOPA and 3-methoxy-4- hydroxymandelic acid did not enhance the GA effect. The action of catecholamines was inhibited by trans-cinnamic acid which competitively inhibited the action of dihydroconiferyl alcohol; this suggests that the receptor site for catecholamines is the same as that for dihydroconiferyl alcohol. The basic ethyl acetate fraction from lettuce seedlings synergistically enhanced the GA effect. TLC analyses of this basic ethyl acetate fraction revealed that the chromatographic area corresponding to authentic catecholamines could enhance the GA effect. From these results, a possible role for catecholamines in the regulation of lettuce hypocotyl elongation caused by GA was posited, and is discussed here.

Distribution of label from 3H-glucose and 3H-leucine in lettuce cotyledons during the early stages of infection with Bremia lactucae

Light and electron microscope autoradiography were used to study the distribution of label from 3H-glucose and 3H-leucine in lettuce cotyledons that had been supplied either labelled compound through their petioles before inoculation with Bremia lactucae Regel. The distribution and intensity of label in the fungus and host have been analyzed statistically. The pathogen accumulated label from 3H-glucose before penetration and thereafter. During the early stages of infection there was virtually no label from 3H-leucine in fungal structures. However, at 14 and 16 h after inoculation, intercellular hyphae and haustoria were heavily labelled. More glucose than leucine was exuded onto epidermal surfaces and inoculum droplets on cotyledons supplied with 3H-glucose were about 10-fold more radioactive than those on cotyledons supplied with 3H-leucine. When either labelled compound was added in the inoculum drop to non-radioactive cotyledons, the fungus but not the host accumulated tritium. Both labelled substances were metabolized by the spores when germinated in isolation.