Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.     

Characterization of ethylene/gibberellic acid control of germination in Lactuca sativa L.

Ethylene production during germination of lettuce seeds (Lactucasativa L., cv. Premier Great Lakes) occurred at two distinctlydifferent rates. A very low rate of ethylene release was observedprior to the 12th hour of incubation at 22?C. The rate of ethyleneproduction, however, increased 100 fold between the 12th and16th hour of incubation. This high rate of ethylene productiononly occurred in the presence of seeds which exhibited a visibleprotrusion of the radicle. The duration of exposure to a supraoptimaltemperature (32?C) was inversely proportional to the percentgermination at 32?C. Ethylene production and growth were notblocked by incubating visibly germinated seeds at 32?C. Exogenous ethylene partially restored germination at 32?C, butonly in the light. Gibberellic acid partially substituted forthe induced light requirement but not for ethylene. It was concludedthat the supraoptimal temperature raised the threshold concentrationof ethylene required for germination. This threshold requirementwas satisfied in the presence of exogenous ethylene. Germinationat 32?C was abo dependent upon the presence of GA. With exogenousethylene present, the GA-mediated system was presumably reinstatedor bypassed by exposing the seeds to either light or GA. Theinitial low rate of ethylene production apparently regulatessubsequent germination but only when present at a minimum thresholdconcentration. Those events initiating germination have obviouslyoccurred prior to the time of radicle emergence. Post-germinationethylene production, therefore, did not break thermodormancy,but occurred simultaneously with radicle emergence. (Received November 29, 1976; )     

Inhibitory effects of full daylight on the germination of Lactuca sativa L.

Using glass filters that transmit various spectral bands and different intensities of natural daylight, experiments with achenes of lettuce cv. Vanquard were performed. Germination during prolonged treatment depended both on the far red/red radiation ratio and on the irradiance. The promotive effect of red radiation present in natural light prevailed at low irradiances, the inhibitory effect of far red radiation at high irradiances. The dormancy imposed by prolonged white light of high intensity can be cancelled by transferring the achenes to darkness or to diffuse weak white light. The effects are obviously of the high irradiance response type; they are exerted by the same mechanism that causes seed dormancy under leaf canopies. Some considerations on the ecological significance of seed behaviour are given.Abbreviations FR far red radiation - R red radiation - HIR high irradiance response - P_fr the far red absorbing form of phytochrome - P_r the red absorbing form of phytochrome     

Blue and Green Light-Induced Phototropism in Arabidopsis thaliana and Lactuca sativa L. Seedlings

Exposure time-response curves for blue and green light-induced phototropic bending in hypocotyls of Arabidopsis thaliana (L.) Heynh. and Lactuca sativa L. seedlings are presented. These seedlings show significant phototropic sensitivity up to 540 to 550 nanometers. Since wave-lengths longer than 560 nanometers do not induce phototropic bending, it is suggested that the response to 510 to 550 nanometers light is mediated by the specific blue light photoreceptor of phototropism. We advise care in the use of green `safelights' for studies of phototropism.     

Cell-Wall Autohydrolysis in Isolated Endosperms of Lettuce (Lactuca sativa L.)

Cell walls prepared from the endosperm tissue of hydrated lettuce (Lactuca sativa L.) seeds undergo autohydrolysis. Release of carbohydrates is most rapid (0.4-0.6 [mu]g per endosperm) within the 1st h of incubation in buffer, but substantial autolysis is sustained for at least 10 h. Autolysis is temperature sensitive, and the optimum rate occurs at pH 5. The rate of autolysis increases markedly in the period just prior to radicle emergence. The cell-wall polysaccharide composition in micropylar and lateral endosperm regions differs significantly; the micropylar walls are rich in arabinose and glucose with substantially lower amounts of mannose. Although walls prepared from both micropylar and lateral regions undergo autolysis, micropylar walls release carbohydrates at a higher rate than lateral walls. Autolysis products elute as large polymers when subjected to size-exclusion chromatography, suggesting that endo-enzyme activity is responsible for release of fragments containing arabinose, galactose, mannose, and uronic acids. Arabinose, galactose, mannose, and glucose are also released as monomers. As a function of time, the ratio of polymers to monomers decreases, indicating that exo-enzyme activity is also present. Thermoinhibition or treatment with abscisic acid suppresses germination and reduces the rates of autolysis of walls isolated from the endosperm by about 25%. Treatments that alleviate thermoinhibition (kinetin and gibberellic acid) increase the rates of autolysis by 20 to 30% when compared to thermoinhibited controls.     

Regulation of Root Growth in Lactuca sativa L. Seedlings by the Ent-Kaurane Diterpenoid Epinodosin

Epinodosin, an ent-kaurane diterpenoid isolated from Isodon japonica var. galaucocalyx, had a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in Lactuca sativa L. seedlings. Lower levels of epinodosin (25–100 μM) significantly promoted root growth, but higher concentrations (150–200 μM), by contrast, had inhibitory effects. In addition, all of the tested concentrations (20–80 μM) inhibited root hair development of lettuce seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of epinodosin (25–100 μM) resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in lettuce seedling root tips. On the other hand, epinodosin at higher concentrations inhibited root growth by strongly affecting both the cell length in the mature region and the division of meristematic cells. Comet assay analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced by higher levels of epinodosin.     

Development of EST-SSR markers for the study of population structure in lettuce (Lactuca sativa L.)

A set of 61 simple sequence repeat (SSR) markers was developed from the 19,523 Lactuca sativa and Lactuca serriola unigenes. Approximately 4.5% of the unigenes contained a perfect SSR at least 20 bp long, corresponding to roughly 1 perfect SSR per 14.7 kb. Marker polymorphism was tested on a set comprising 96 accessions representing all major horticultural types and 3 wild species (L. serriola, Lactuca saligna, and Lactuca virosa). Both the average marker heterozygosity (UHe = 0.32) and the number of different alleles per locus (Na = 3.56) were significantly reduced in expressed sequence tag (EST)-SSRs as compared with anonymous SSRs (UHe = 0.59, Na = 5.53). Marker transfer rate to the wild species corresponded to the decreasing sexual compatibility with L. sativa and was higher for EST-SSRs (100% L. serriola, 87% L. saligna, and 75% L. virosa) than for anonymous SSRs (93%, 66%, and 42%, respectively). Assessment of population structure among 90 L. sativa cultivars with SSRs was in good agreement with classification into the horticultural types. The average marker heterozygosity was smallest in iceberg (0.097), Latin (0.140), and romaine-type (0.151) cultivars while highest in leaf (green leaf 0.208 and red leaf 0.240) lettuces. The level of marker heterozygosity is in accord with morphological variability observed in different horticultural types.     

二氢血根碱对莴苣幼苗根生长及其细胞分裂的影响

以莴苣幼苗为受体,用培养皿法检测从细果角茴香中分离得到的二氢血根碱对莴苣幼苗根生长和根毛发育的影响,并采用根尖细胞有丝分裂检测和单细胞凝胶电泳法对其可能的作用机制进行了初步研究.结果显示:较低浓度(25、50μmol/L)二氢血根碱能显著促进莴苣根的生长,较高浓度(200、300μmol/L)二氢血根碱显著抑制根的生长;二氢血根碱(10、20、30、40、50μmol/L)对莴苣幼苗根毛发育有极显著的抑制作用,且两者均表现了浓度依赖性.较低浓度(25、50μmol/L)二氢血根碱使根尖细胞有丝分裂指数显著增加,而对根尖细胞DNA没有显著影响;较高浓度(200、300μmol/L)二氢血根碱使根尖细胞有丝分裂指数显著下降,同时根尖细胞DNA受到显著性损伤.研究发现,低浓度的二氢血根碱对莴苣幼苗根生长的促进作用主要是由于根尖细胞有丝分裂活力增加所致;而高浓度二氢血根碱对莴苣幼苗根的抑制作用极可能是由于根尖细胞DNA受到损伤,使得细胞分裂活力降低,分裂期细胞数目减少,从而导致根生长受到抑制.     

Microsatellite fingerprinting in lettuce (Lactuca sativa L.) and wild relatives

Southern hybridisation with a single microsatellite probe, (TCT)₁₀, sufficed to discriminate between a representative set of cultivars/accessions of lettuce, Lactuca sativa L., and its wild relatives L. serriola, L. saligna and L. virosa. Variability within cultivars was tested in a relatively modern cultivar (Hector), where no variation was found, and in an older and morphologically more variable cultivar (Madrilene), where heterogeneity was observed in the TCT fingerprint. (TCT)₁₀ fingerprinting should be useful for variety identification and homogeneity testing in lettuce. Received: 25 July 1997 / Revision received: 5 August 1997 / Accepted: 30 August 1997     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. Grand Rapids

The timing of mobilisation of lipid, sucrose, raffinose and phytate in lettuce seeds (achenes) (cv. Grand Rapids) has been examined. These reserves (33%, 1.5%, 0.7%, 1.4% of achene dry weight, respectively) are stored mostly in the cotyledons. Except for a slight degradation of raffinose and increase in sucrose, there is no detectable reserve mobilisation during germination. The endosperm (8% of seed dry weight), which has thick, mannan-containing cell walls (carbohydrate, 3,4% of seed dry weight), is completely degraded within about 15h following germination. Mannanase activity increases about 100-fold during the same period and arises in all regions of the endosperm. Also during this period sucrose and raffinose are degraded and fructose and glucose accumulate in the embryo. The endosperm hydrolysis products are taken up by the embryo, and are probably used as an additional reserve to support early seedling growth. However, endosperm cell-wall carbohydrates, such as mannose, are not found as free sugars. Lipid and phytate are degraded in a later, second phase of mobilisation. Low levels of sucrose are present in the embryo, mostly in the cotyledons, and large amounts of fractose and glucose (14% of seedling dry weight at 3 days after sowing) accumulate in the hypocotyl and radicle. It is suggested that sucrose, produced in the cotyledons by gluco-neogenesis, is translocated to the axis and converted there to fructose and glucose.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Morphology of Head Formation of Cos Lettuce (Lactuca sativa L. cv. Romana) 2. The Development of Spiral-leaved Heads

The morphogenesis of a malformation occurring in cos lettuceheads (Lactuca sativa L. cv. romana) during and at the end ofthe heading period, is described. Abnormal leaf growth resultedin twisting of the midrib of the affected leaf around its ownaxis, giving the leaf a spiral-like look. Twisting began atthe outer leaves and then, mainly by affecting successive orsometimes alternating leaves, spread inwards in the directionto the younger leaves. Accelerated midrib elongation accompaniedleaf twisting. The external appearance of this malformationwas influenced by the location and number of the twisted leaves,and by the degree of twisting of the individual leaves.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

莴苣大孢子发生过程中钙的分布动态

莴苣胚囊发育为蓼型,减数分裂形成的4个大孢子中只有合点端的一个大孢子可继续发育,其余3个大孢子从珠孔端依次退化.大孢子母细胞中的钙沉淀颗粒很少,减数分裂后的四分体中的钙沉淀颗粒稍有增加.以后,4个大孢子中的钙沉淀颗粒在数量上有明显差异:即将退化的大孢子中钙明显减少,而未退化大孢子细胞质中则保持有较多的细小钙沉淀颗粒.大孢子的退化是一种细胞程序死亡现象,细胞中的钙浓度降低时可能启动了大孢子细胞的程序性死亡过程,而细胞中的钙浓度高时则保持大孢子细胞的继续发育.文章首次揭示了大孢子发生过程中钙的分布特征.     

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

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

Light and molybdenum requirements for substrate induction of nitrate reductase in cotyledons of lettuce,Lactuca sativa L.

Light was required for induction of nitrate reductase (NR, E.C. 1.6.6.1) in intact cotyledons of 2-day old seedlings ofLactuca sativa L. Molybdate strongly enhanced efficiency of induction. Benzyladenine (BA), gibberellin, and succinic acid-2,2-dimethylhydrazide reduced the enzyme activity. BA thrice enhanced incorporation of labelled leucine to the protein fraction. (2-chloroethyl)trimethylammonium chloride did not affect NR activity and markedly inhibited greening and protein synthesis. KNO₃ stimulated protein synthesis as well as growth of the cotyledons.