In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum): accumulation of proline, decrease in osmotic potential, and increase in frost tolerance

Five hundred hydroxyproline-resistant cell lines were selected from cell cultures of wheat ( Triticum aestivum L. cv. Koga II) after plating on 10 to 30 m M hydroxyproline (Hyp) containing solid Gamborg B 5 medium. All selected cell lines from 30 m M Hyp-medium contained increased (up to 17-fold) levels of free proline. Seventy-four cell lines were transferred to Hyp-free medium and subcultivated 25 times, for 12 months altogether, after which 80% still had increased proline levels. Fourteen cell lines with increased proline levels were further investigated in liquid media with regard to their frost tolerance, which was measured by means of electrolyte leakage. Ten of them showed increased fros tolerance, with LT 50 values as low as 2.7°C below that of the wild type (-4.7°C). Besides increased proline levels and increased percentage dry weight, the Hyp-resistant cell lines had lower osmotic potentials. Osmotic potentials correlated better than levels of free proline with the increase in frost tolerance.     

Characterization and regeneration of salt- and water-stress mutants from protoplast culture of Nicotiana plumbaginifolia (Viviani)

Summary Protoplast-derived colonies of haploid N. plumbaginifolia leaves were used to select for resistance to NaCl, KCl and polyethylene glycol 6000 (PEG). Salt-and PEG-tolerant cell lines were isolated on the basis of growth in a culture medium containing inhibitory concentrations of either NaCl or KCl (200 mM) or PEG (25%). The frequency of resistant lines ranged from 10^-5 to 10^-6. One resistant line from each treatment was regenerated into plants. All resistant lines produced 10–25 times more proline than the wild type when grown on a non-selective medium. Similar values were also observed in the leaves of resistant progeny plants. In each mutant line, salt or PEG resistance was transmitted as a single dominant nuclear gene as shown by segregation ratios in progenies of crosses between resistant and wild-type plants. The latter observation demonstrates clearly the existence of a genetic basis for increased salt tolerance.     

Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

Production of sodium-chloride-tolerant Brassica juncea plants by in vitro selection at the somatic embryo level

Summary Somatic embryos, developed from hypocotyl segments of light-grown seedlings of Brassica juncea cv RLM198, were subjected to selection at varying concentrations of sodium chloride (NaCl). Plants were developed from proliferated somatic embryos selected on NaCl-containing medium. The selections were characterized for salt tolerance, esterase isozyme pattern, and proline accumulation. It has been found that: (i) selected tolerant lines showed better root growth, shoot growth, and fresh weight accumulation on salt-containing medium when compared to the control; (ii) salt tolerance was transmitted to the next generation in seed progeny of tolerant plants grown in the absence of exposure to salt; (iii) both the starting material and the tolerant selections accumulated proline, even when grown in salt-free medium. On salt-containing medium, however, the differences in accumulated proline between the control and tolerant lines became more pronounced, and (iv) the patterns of esterase isozymes of two tolerant selections were similar but distinctly different from that of the parental control.     

松花菜种质资源评价及根肿病、黑斑病兼抗材料筛选

松花菜是目前我国花椰菜消费的主要类型。根肿病和黑斑病是直接影响松花菜产量及品质的重要病害。选育商品性好、高抗多抗品种是松花菜的主要育种目标之一。本研究首次建立了松花菜种质资源花球性状评价体系,从花球紧实度、球形、球面光滑度、球面颜色、球面蕾粒大小、二级侧枝长度、花梗颜色、球面长毛情况8个方面对66份松花菜自交系进行评价,并对不同性状赋值以便统计、比较。利用上述体系共筛选出11份花球性状优良的材料。采用苗期人工接种技术分别对松花菜根肿病和黑斑病抗性进行鉴定评价,结果显示无免疫根肿病材料,有2份高抗材料,9份抗病,16份中抗;无免疫和高抗黑斑病材料,有4份抗病材料,10份中抗。另外,兼抗两种病害的材料较少,其中GY-40对两种病害均表现抗病,GY-21对根肿病表现抗病,对黑斑病表现中抗,GY-39对两种病害均表现中抗。值得一提的是,GY-40性状优良且同时对根肿病和黑斑病具有较高抗性,是选育商品性好、抗病性强松花菜品种的理想亲本。本研究结果为评价松花菜种质资源花球相关性状提供了参考,对于规范资源的收集、整理和保存具有重要意义,同时为开展抗根肿病和黑斑病松花菜品种选育提供了优异材料。     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

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.     

Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance

The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25?weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants.     

Induction of Multiple Bud Clumps from Inflorescence Tips and Regeneration of Salt-tolerant Plantlets in Beta vulgaris

We developed an efficient method for sugar beet multiplication in vitro from excised immature inflorescence tips. On Murashige & Skoog medium supplemented with 4.4 μM 6-benzylamino- purine and 1.3 μM naphthaleneacetic acid, multiple bud clumps were induced from segments of inflorescence tips. The clumps proliferated rapidly. By radiation of small bud clumps at an appropriate dose and by directional selection for NaCl tolerance, we obtained salt-tolerant bud clumps and regenerated plantlets. The plants were vernalized and self-pollinated. The seeds of the regenerated plants were sown in pots of sand and irrigated every day with a solution of 342 mM NaCl. Some of the seeds germinated and grew normally in the 342 mM NaCl solution, exhibiting higher salt-tolerance than the control ones; such seedlings after the saline selection were transplanted to soil and the plants grew normally and produced plump root tuber similar to controls. The seeds from two selected lines germinated and grew for a few weeks in 513 mM NaCl solution before the seedlings withered. In saline soil where the salt concentration was about 154 mM, the yields of tuber from the plants of three salt-tolerant lines were about 45–50 tons ha⁻¹, approximately 2.6–2.9 times of the controls. It is concluded that we have got salt-tolerant materials with good agronomic traits for sugar beet breeding via selection in vitro.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana

Summary Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

Partial suppression of gene encoding proline dehydrogenase enhances plant tolerance to various abiotic stresses

The role of gene of proline dehydrogenase (PDH) in the maintenance of stress tolerance was investigated using the model transgenic plants of tobacco (Nicotiana tabacum L.) carrying an antisense suppressor of PDH gene (a fragment of Arabidopsis PDH gene under the control of cauliflower mosaic virus 35S promoter in antisense orientation) and notable for a low activity of PDH and elevated content of proline. The progeny of transgenic plants belonging to the 5th generation (T₅) with partially suppressed PDH activity was more resistant to various types of stress as compared with the control plants of tobacco, cv. Petit Havana SR-1 (SR1). The seedlings of transgenic lines cultured in Petri dishes on agar media supplemented with stress agents were resistant to high NaCl concentrations (200–300 mM) and water deficit simulated by an increased agar content in the medium (14 g/l) as compared to the control seedlings of cv. SR1. Juvenile plants of transgenic lines grown in pots filled with a mixture of vermiculite and perlite also manifested the higher resistance to water deficit and low temperatures (2°C and −2°C) than the control plants. Thus, the partial PDH suppression correlated with an increase in nonspecific resistance to different types of abiotic stress: salinity, water deficit, and low temperatures. Such transgenic lines of tobacco are promising genetic models for thorough investigation of molecular mechanisms of stress resistance in plants.     

NaCl-tolerant plants of Poncirus trifoliata regenerated from tolerant cell lines

Summary Salt-tolerant cell lines of citrus rootstock (Poncirus trifoliata cv Pomeroy) were selected by subculturing embryo-derived calli on media containing sublethal concentrations of NaCl (5 and 10 g/l). Selected lines showed a normal growth in the presence of salt at the concentrations used for selection, and salt tolerance persisted after a passage on a salt-free media. Their K⁺ and Ca²⁺ content remained higher than in control cells for increasing NaCl concentration in the medium, suggesting a modification of cell membrane permeability as the main cause of NaCl tolerance. Shoots and plants regenerated from selected cell lines showed improved growth and salt tolerance. Calli induced from these plants tolerated a salt concentration of 10 g/l, indicating the persistance of the selected trait.     

Improved protoplast culture and stability of cytoplasmic traits in plants regenerated from leaf protoplasts of cauliflower (Brassica oteracea ssp.botrytis)

Nearly 1000 plants have been regenerated from leaf protoplasts of two cauliflower (Brassica oleracea ssp.botrytis) alloplasmic inbred lines. One line (7642A) carried the Ogura (R1) cms cytoplasm derived from radish; the other line (7642B) carried a normalBrassica cytoplasm and was the fertile maintainer for the cms line. The majority of regenerated plants displayed normal vegetative morphology; they formed normal cauliflower heads and retained the floral characteristics of seed-grown plants from which they were derived. We found no change in either male sterility or in the low temperature-induced chlorosis associated with the 7642A line. Mitochondrial DNA analysis by hybridization with five cloned mtDNA probes revealed no apparent alteration in 75 regenerated plants of both lines. These results indicate that cytoplasmic traits inBrassica oleracea are stable after one cycle of in vitro culture and regeneration.     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

In vitro selection of mung bean and tomato for improving tolerance to NaCl

Mung bean and tomato were in vitro selected from cotyledons on MS medium for improved tolerance to NaCl. The growth responses; the Na, K, proline and anthocyanin contents; and activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5), tyrosine ammonia lyase (TAL, EC 4.3.1) and chalcone isomerase (CI, EC 5.5.1.5) of the selected plants were characterised and compared with those of the original plants in relation to treatment with NaCl. The treatments significantly reduced fresh and dry weights of shoots and roots; the reduction was least pronounced in selected plants. Meanwhile, Na content was significantly increased; however, K was decreased, a trend that was obvious in original plants but withdrawn following in vitro selection with a consequent lowering in Na/K ratio. In addition, proline was greatly induced by NaCl; the induction was most pronounced in selected plants. Moreover, NaCl significantly increased anthocyanin and activities of PAL, TAL and CI in shoots and roots of both species; the increase was lesser in the selected than in the original plants. These findings indicated that selection of mung bean and tomato resulted in a recovery of growth, overproduction of proline and K and withdrawal of Na and secondary metabolism parameters relative to original plants pointing out to an improved tolerance to NaCl following in vitro selection.