Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer

Summary Five diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.     

不同能源柳无性系对NaCl胁迫的生理响应

为了探索不同能源柳无性系的耐盐潜力,在盆栽条件下,对1年生能源柳无性系2、4、C、E扦插苗设置NaCl盐分梯度,使其土壤含盐量保持在0、0.2%、0.4%、0.6%、0.8%,并以乡土旱柳为对照,胁迫45d后,对5个无性系的气体交换参数、保护酶活性和有机渗透调节物质进行测定。研究发现:(1)在低浓度盐胁迫下(土壤含盐量0 0.4%),导致各能源柳光合速率降低的是气孔因素;在高浓度盐胁迫下(土壤含盐量大于0.4%),导致各能源柳无性系光合速率下降的主要是非气孔因素。(2)随着土壤盐浓度的增加,各能源柳无性系的过氧化氢酶(CAT)活性逐渐减小,过氧化物酶(POD)活性先增加后减小,而超氧化物歧化酶(SOD)活性呈现增加的趋势,并且4个能源柳无性系在盐胁迫条件下保护酶活性强于旱柳。(3)在土壤含盐量小于0.6%时,4个能源柳无性系的可溶性蛋白含量始终大于旱柳;在整个胁迫过程中,能源柳C的脯氨酸含量始终大于旱柳。(4)与旱柳相比,4个能源柳无性系在土壤含盐量0 0.4%的土壤中都能良好生长,其中能源柳C的耐盐潜力更大,可在土壤含盐量为0.6%的土壤中正常生长。这说明,4个能源柳无性系均可在天津轻、中度盐渍化地区栽培。     

Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stress

Stable callus cultures tolerant to NaCl (68 mM) were developed from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. The accumulation of both inorganic (Na⁺, Cl⁻ and K⁺) and organic (proline and soluble sugars) solutes was determined in selected and non-selected calli after a NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Both salt-tolerant and non-selected calli showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant calli while a significant reduction about 32% was observed in nonselected ones when both were cultivated on 68 mM NaCl. Accumulation of Na⁺ was similar in both salt-tolerant and non-selected calli in the presence of NaCl. Accumulation of Cl⁻ was lower in NaCl-tolerant than in non-selected calli while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected calli when both were exposed to salt. K⁺ level decreased more severely in non-selected calli than in NaCl-tolerant ones after NaCl shock. The results indicated that K⁺ and Cl⁻ may play a key role in in vitro salt-tolerance in sugarcance cell lines obtained by in vitro selection and that organic solutes could contribute mainly to counteract the negative water potential of the outside medium.     

Quantitative trait locus analysis of tuber dormancy in diploid potato (Solanum spp.)

Quantitative trait locus (QTL) analysis for tuber dormancy was performed in a diploid potato population (TRP133) consisting of 110 individuals. The female parent was a hybrid between haploid S. tuberosum (2x) and S. chacoense, while the male parent was a S. phureja clone. The population was characterized for ten isozyme loci, 44 restriction fragment length polymorphisms (RFLPs) and 63 random amplified polymorphic DNAs (RAPDs). Eighty-seven of these loci segregating from the female parent were utilized to develop a linkage map that comprised 10 of the 12 chromosomes in the genome. Dormancy, as measured by days-to-sprouting after harvest, ranged from 10 to 90 days, with a mean of 19 days. QTLs were mapped by conducting one-way analyses of variance for each marker locus by dormancy combination. Twenty-two markers had a significant association with dormancy, identifying six putative QTLs localized on each of chromosomes 2, 3, 4, 5, 7 and 8. The QTL with the strongest effect on dormancy was detected on chromosome 7. A multilocus model was developed using the locus with highest R² value in each QTL. This model explained 57.5% of the phenotypic variation for dormancy. Seven percent of possible epistatic interactions among significant markers were significant when tested through two-way analyses of variance. When these were included in the main-effects model, it explained 72.1% of the phenotypic variation for dormancy. QTL analysis in potato, the methodology to transfer traits and interactions into the 4x level, and QTLs of value for marker-assisted selection, are discussed.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比

研究干旱胁迫和盐胁迫对“芦选一号”。日‘薯愈伤组织可溶性蛋白、可溶性糖、脯氨酸含量、SOD活性等的影响,从而在细胞水平上探讨甘薯抵御渗透胁迫的生理机制。并分析甘薯细胞对干旱处理（PEG-6000）和盐处理（NaCl）的反应差异。结果表明,可溶性蛋白质含量在干旱胁迫下缓慢升高,在轻度和中度盐胁迫的生长前期和中期有较大幅度的上升。但后期下降,表明短时间盐胁迫下,Na^＋可能促进可溶性蛋白的合成;MDA在重度干旱胁迫下的含量显著低于重度盐胁迫,而SOD活性显著高于盐胁迫。表明在盐胁迫下细胞膜透性增加的主要原网是膜脂过氧化作用。干旱处理则是PEG-6000脱水的直接结果;重度干旱胁迫下,可溶性糖含量在短期内迅速升高,然后下降,而脯氨酸含量则在胁迫中后期迅速上升。脯氨酸可能有补偿可溶性糖含量降低的作用。     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

Differential responses of two rice varieties to salt stress

Two rice varieties, viz. Nonabokra and Pokkali, have been evaluated for their responses to salinity in terms of some physiological and biochemical attributes. During the exposure to salinity (200 mM concentration of sodium chloride for 24, 48, and 72 h), a significant increase in sodium was recorded which was also concomitant with the changes of other metabolic profiles like proline, phenol, polyamine, etc. The protein oxidation was significantly increased and also varied between the two cultivars. The changes in activities of anti-oxidative enzymes under stress were significantly different to the control. The detrimental effects of salinity were also evident in terms of lipid peroxidation, chlorophyll content, protein profiles, and generation of free radicals; and these were more pronounced in Pokkali than in Nonabokra. The assessment and analysis of these physiological characters under salinity could unravel the mechanism of salt responses revealed in this present study and thus might be useful for selection of tolerant plant types under the above conditions of salinity.     

Physiological responses to NaCl stress in three wild species of potato in vitro

In this study, responses of wild species of potato to NaCl stress were investigated in vitro. In S. stoloniferum and S. bulbosum, length of the shoot, fresh and dry weight, photosynthetic pigments, K⁺ concentration, K⁺/Na⁺ ratio, ascorbate pool, anthocyanin, and phenolic and flavonoid compounds were decreased in response to salinity. In these species, salinity increased the level of Na⁺, lipid peroxidation, proline and ion leakage percentage. In S. acaule, the length of the shoot, and fresh and dry weight were not affected by salinity. Photosynthetic pigments, Na⁺ concentration, proline, flavonoid and phenolic compounds quantities were increased and K⁺/Na⁺ ratio were decreased. K⁺ concentration, lipid peroxidation, ascorbate pool, anthocyanin and ion leakage were not changed by NaCl stress. Superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase activities were increased in all species. The results suggest that the non-enzymatic antioxidant capacity in S. acaule (salt tolerant) is more important than the enzymatic antioxidant capacity in comparison with the other species.     

Microtuberization in potato (Solanum tuberosum L.)

Twenty-two genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under six in vitro culture conditions. Cultures maintained under a short photoperiod (10 h of 6–12 μmol m^–2 s^–1) and low temperatures (day 20°±2°C and night 18°±2°C) had both a higher yield (255 mg/plantlet) and a greater number (2/plantlet) of microtubers than those maintained under long days (16 h of 38–50 μmol m^–2 s^–1) combined with high temperatures (day 28°±2°C and night 25°±2°C) (yield 207 mg/plantlet; microtuber number, 0.9/plantlet), over a wide range of genotypes. After the plantlets had been cultured under long days for an initial period of 60 days, continuous darkness advanced microtuberization by 2–3 months in various genotypes. Under short-day and low-temperature conditions the addition of 6-benzylaminopurine increased microtuber yield from 255 mg/plantlet to 645 mg/plantlet and average microtuber weight from 115 mg to 364 mg. A similar pattern was observed under conditions of long days and high temperature, and continuous darkness and low-temperature. Microtubers produced under light had a greater number of eyes (maximum average: 5.96/microtuber) than those produced in the dark (maximum average: 3.50/plantlet). The genotype × cultural conditions interactions were significant indicating the importance of developing genotype-specific protocols to maximize microtuberization. Received: 17 September 1997 / Revision received: 12 December 1997 / Accepted: 1 January 1998     

Differential responses of maize MIP genes to salt stress and ABA

Salt stress is known to reduce root hydraulic conductivity and growth. To examine a concomitant regulation of aquaporins, the expression of the maize MIP gene family in response to NaCl was analysed by DNA array hybridization. Plants responded differentially to 100 versus 200 mM NaCl treatments. Leaf water content was reduced rapidly and persistently after the application of 200 mM NaCl in contrast to 100 mM NaCl. Endogenous ABA strongly accumulated in roots after 2 h; it remained at a highly elevated level for 48 h after the addition of 200 mM NaCl, but rapidly declined in plants treated with 100 mM NaCl, indicating an early recovery from water deficit. Interestingly, 2 h after the addition of 100 mM NaCl, when maize regained the osmotic potential allowing water uptake, three highly expressed, specific isoforms ZmPIP1;1, ZmPIP1;5, and ZmPIP2;4 were transiently induced. They were preferentially transcribed in the outer root tissue suggesting a role in cellular water transport. None of the ZmTIP genes was altered. By contrast, after the addition of 200 mM NaCl these responses were missing. Instead, multiple ZmPIP and ZmTIP genes were repressed by 200 mM NaCl after 24 h. After 48 h, deregulations were overridden in both cases indicating homeostasis. ABA (1 muM) exogenously applied to the roots transiently induced ZmPIP2;4 similar to 100 mM NaCl as well as ZmPIP1;2. Thus, the early induction of ZmPIP2;4 by NaCl may be mediated by ABA. Previously, an increase in root hydraulic conductivity had been observed upon ABA application. By contrast, 100 muM ABA led to a complete, possibly non-specific repression of all detected ZmPIP and ZmTIP genes after 24 h.     

Pattern of dry matter accumulation in developing fruit parts of early- and late-maturing pigeon peas (Cajanus cajan (L.)Millsp.)

Pigeon pea cultivars AL 15 (early-) and T₂₁ (late-maturing) were compared for dry matter accumulation in fruit parts pod wall (PW), seed coats (SC) and seed at various seed developmental stages. Significant water loss and dry matter accumulation in the fruit parts commenced much earlier in cv. AL 15 as compared with cv. T₂₁. The pod wall accumulated starch, reducing sugars and N-substances up to 21 and 28 days after anthesis (DAA) in cultivars AL 15 and T₂₁, respectively, which was later distributed to the seed. Growth of pod wall and seed was sequential, not concurrent, as the pod wall lost significant dry matter when the seeds within them reached their maximum dry matter. The fruit parts of cv. AL 15 accumulated more photosynthates than cv. T₂₁ at all comparable stages.     

Physiological and ultrastructural responses of sour orange (Citrus aurantium L.) clones to water stress

Water stress is a major abiotic constraint leading to serious crop losses. Recently, in the Mediterranean region, water stress has become markedly sensed, especially in Citrus orchards. This study investigated the physiological responses of local sour orange (Citrus aurantium L.) clones to severe water stress. Water stress was applied by withholding irrigation during weeks, followed by a rewatering phase during three months. Under water stress, sour orange clones decreased their stomatal conductance, net photosynthetic rate, and transpiration rate. On the contrary, biomass was stable, especially in the Kliaa clone. In addition, reduced leaf water potentials (-3 MPa) and water contents were measured in most of the clones, except Kliaa which kept the highest water potential (-2.5 MPa). After rewatering, all clones recovered except of the Ghars Mrad (GM) clone. Ultrastructural observations of leaf sections by transmission electron microscopy did not reveal marked alterations in the mesophyll cells and chloroplast structure of Kliaa in comparison to the sensitive clone GM, in which palisade parenchyma cells and chloroplasts were disorganized. This contrasting behavior was mainly attributed to genetic differences as attested by molecular analysis. This study highlighted GM as the drought-sensitive clone and Kliaa as the tolerant clone able to develop an avoidance strategy based on an efficient stomatal regulation. Although a high percentage of polyembryony characterizes C. aurantium and justifies its multiplication by seeds, heterogeneous water-stress responses could be observed within sour orange plants in young orchards.     

Chloroplast DNA evolution in potato (Solanum tuberosum L.)

Summary A deletion specific to chloroplast (ct) DNA of potato (Solanum tuberosum ssp. tuberosum) was determined by comparative sequence analysis. The deletion was 241 bp in size, and was not flanked by direct repeats. Five small, open reading frames were found in the corresponding regions of ctDNAs from wild potato (S. tuberosum ssp. andigena) and tomato (Lycopersicon esculentum). Comparison of the sequences of 1.35-kbp HaeIII ctDNA fragments from potato, tomato, and tobacco (Nicotiana tabacum) revealed the following: the locations of the 5 ends of both rubisco large subunit (rbcL) and ATPase beta subunit (atp) mRNAs were probably the same as those of spinach (Spinacia oleracea); the promoter regions of the two genes were highly conserved among the four species; and the 5 untranslated regions diverged at high rates. A phylogenetic tree for the three potato cultivars, one tomato cultivar, and one tobacco cultivar has been constructed by the maximum parsimony method from DNA sequence data, demonstrating that the rate of nucleotide substitution in potato ctDNA is much slower than that in tomato ctDNA. This fact might be due to the differences in the method of propagation between the two crops.     

In-season heat stress compromises postharvest quality and low-temperature sweetening resistance in potato (Solanum tuberosum L.)

Key message

High soil temperature during bulking and maturation of potatoes alters postharvest carbohydrate metabolism to attenuate genotypic resistance to cold-induced sweetening and accelerate loss of process quality.

Abstract

The effects of soil temperature during tuber development on physiological processes affecting retention of postharvest quality in low-temperature sweetening (LTS) resistant and susceptible potato cultivars were investigated. ‘Premier Russet’ (LTS resistant), AO02183-2 (LTS resistant) and ‘Ranger Russet’ (LTS susceptible) tubers were grown at 16 (ambient), 23 and 29 °C during bulking (111–164 DAP) and maturation (151–180 DAP). Bulking at 29 °C virtually eliminated yield despite vigorous vine growth. Tuber specific gravity decreased as soil temperature increased during bulking, but was not affected by temperature during maturation. Bulking at 23 °C and maturation at 29 °C induced higher reducing sugar levels in the proximal (basal) ends of tubers, resulting in non-uniform fry color at harvest, and abolished the LTS-resistant phenotype of ‘Premier Russet’ tubers. AO02183-2 tubers were more tolerant of heat for retention of LTS resistance. Higher bulking and maturation temperatures also accelerated LTS and loss of process quality of ‘Ranger Russet’ tubers, consistent with increased invertase and lower invertase inhibitor activities. During LTS, tuber respiration fell rapidly to a minimum as temperature decreased from 9 to 4 °C, followed by an increase to a maximum as tubers acclimated to 4 °C; respiration then declined over the remaining storage period. The magnitude of this cold-induced acclimation response correlated directly with the extent of buildup in sugars over the 24-day LTS period and thus reflected the effects of in-season heat stress on propensity of tubers to sweeten and lose process quality at 4 °C. While morphologically indistinguishable from control tubers, tubers grown at elevated temperature had different basal metabolic (respiration) rates at harvest and during cold acclimation, reduced dormancy during storage, greater increases in sucrose and reducing sugars and associated loss of process quality during LTS, and reduced ability to improve process quality through reconditioning. Breeding for retention of postharvest quality and LTS resistance should consider strategies for incorporating more robust tolerance to in-season heat stress.     

Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato (Solanum tuberosum L.)

Planta - Tolerance to heat stress for retention of low-temperature sweetening-resistant phenotype in potato is conferred by insensitivity of acid invertase activity to cold induction. Heat stress...     

Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.)

The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.