Plant regeneration from protoplasts isolated from primary calli using mature embryos of two Brazilian rice cultivars

A plant regeneration system from rice protoplasts using calli derived from mature embryos was established for the two Brazilian modern rice cultivars IAC-201 and IAC-165. After 30 to 40 days of in vitro culture it was possible to obtain on average 6 million protoplasts per gram of callus. Microscopic selection of embryogenic calli was a key step for protoplast isolation. The production of embryogenic calli increased when L-proline and casein hydrolysate were used in the callus induction medium. The Oc or IR52 nurse cell lines were essential for protoplast division. Different regeneration media were studied and 139 plants were regenerated which set seed. Some of the regenerated plants showed morphological variation such as the presence of awns in spite of the short time of the in vitro culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of somaclonal variation and in vitro selection for chilling tolerance improvement in rice

Embryo-derived calli of four rice varieties cultivated at high altitude in Burundi — Facagro 57, Facagro 76, Kirundo 3 and Kirundo 9 — were submitted to different temperature regimes. The percentage of regenerating calli greatly varied depending on variety, length of culture and callus temperature treatment. The reduction of regeneration percentages induced by low temperature was more pronounced in the more sensitive varieties. Regenerated plants (R0) and their progenies in R1, R2 and R3 were cold-screened together with control plants. In all varieties, significantly higher survival rates were obtained in R3 with in vitro plants than with control plants. Such chilling tolerance improvement was not obtained following a massal selection applied during 3 successive generations onto the control plants. In vitro plants regenerated from calli cultivated either at 25 °C, either at 4 °C, were cultivated at different altitudes in Burundi during two successive generations. For most observed traits, the in vitro plants were characterized by lower means, larger variation and higher maximum values than the control plants. The most chilling-tolerant somaclonal families were most usually characterized by extensive differences in fatty acid composition, chilling-induced electrolyte leakage and chlorophyll fluorescence, compared to the varieties they derived from. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.     

甘薯（Ipomoea batatas (L.) Lam.）及其近缘野生种原生质体的植株再生

对甘薯品种高系14号及其近缘野生种I.triloba L、和I.lacunosa L,进行原生质体植株再生研究。从离体培养植株的叶柄分离出原生质体,将其培养在含有0.05mg/L 2,4-D和0.5mg/L激动素(KT)的MS培养基中,从原生质体获得了高频率的愈伤组织。培养8-12周后,将直径达2—3mm的小愈伤组织转移到添加0.05mg/L 2,4-D的MS培养基上。转移3-6周后,将愈伤组织进一步转移到添加吲哚乙酸(IAA)和6-苄基嘌呤(BAP)的MS培养基上,一些愈伤组织再生出植株。未再生植株的愈伤组织进一步在MS基本培养基上培养,它们也再生出植株。本研究从I.triloba原生质体获得高频率的植株再生;首次从I.lacunosa原生质体再生出植株;从高系14号原生质体也再生出完整植株。     

A comparison of the somaclonal variation level of Rosa hybrida L. cv Meirutral plants regenerated from callus or direct induction from different vegetative and embryonic tissues

Summary Flowering plants of Rosa hybrida L. cv Meirutral have been obtained either from direct regeneration of adventitious shoots on leaf and root fragments, or through organogenesis and somatic embryogenesis on calli derived from anther, ovule, petal, sepal, receptacle, leaf, stem internode, root and zygotic embryo tissues. The calli derived from floral parts exhibited rhizogenesis. In this case direct induction of adventitious shoots from selected roots had to be performed in order to generate plants. A histological study of the morphogenetic calli was carried out. The plants regenerated directly and those regenerated from calli of leaf, stem internode, root and zygotic embryo tissues, together with reference plants propagated by cuttings, were compared on a phenotypic basis by taking into account petal number, form and colour, and plant growth habit. From these observations, it can be concluded that directly regenerated plants are as stable as reference plants while plants regenerated from callus are unstable, especially those derived from zygotic embryo tissues.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA 3-indole-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene acetic acid     

植物激素对石刁柏花药培养中染色体倍性变异的影响

在石刁柏花药培养过程中,2,4-D在引起愈伤组织细胞染色体倍性变异方面起主导作用,高浓度的2,4-D虽然能提高愈伤组织中单倍体细胞的频率,但同时又加剧其多倍化。不同激素配比的分化培养基在诱使再生植株染色体倍性变异方面存在着明显差异。在附加1.0ppmBA和0.2ppmIBA的培养基上,再生植株茎尖单倍体细胞频率最高,达31.17％。比较愈伤组织和再生植株的染色体倍性变异幅度,发现后者明显地大于前者。     

In vitro Selection for Fusaric Acid Resistant Barley Plants

Calli of two genotypes of barley,‘Dissa’and W 193, were used for selection of resistance against fusaric acid, a pathotoxin of Fusarium. Callus was initiated from 7- to 10 days old immature embryos. 1000 calli of the‘Dissa’and 500 of the W 193 genotypes were grown for 4 selection cycles on medium with 0.8 mM fusaric acid. In the first selection cycle, about 80 % of the calli were killed; after the 4 selection cycles, 8 to 11 % resistant calli were obtained and plants were regenerated. Resistant calli maintained on non-toxic medium showed retention of resistance ability after 3 months of sub-culturing. Plants could be regenerated from the surviving calli and testing by leaf bioassay revealed that many were resistant to the same toxin concentration employed for callus selection (100 %), while some were only resistant up to a concentration of 75 %.     

甘薯胚性细胞悬浮培养系的建立

地甘薯胚性细胞悬浮增减系的进行了研究。将１２个基因的长约０．５ｍｍ的茎尖培养在含有０．２ｍｇ／Ｌ或２．０ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基上，形成了胚性愈伤组织。胚性愈伤组织的形成率因基因型和２，４－Ｄ深度不同而很大差异，为０－７５．７％。一方面，将胚性愈伤组织继续增减在含有２，４－Ｄ的ＭＳ培养基上，它们形成了处于各发育时期的体细胞胚。将具有体细胞胚的胚性愈伤组织转移到ＭＳ基本培养基上，体细胞胚发育成     

Increased regeneration from NaCl-tolerant callus in rice

Summary NaCl-tolerant calli were selected from two Japonica and two Indica rice (Oryza sativa L.) cultivars on basal media containing 6,000, 9,000, 12,000 or 15,000 ppm NaCl. Frequency of callus formation decreased with the increase of NaCl in the medium, especially in Indica. About half of the calli of Japonica cultivars selected on NaCl-ammended media survived 20,000 ppm NaCl but none of the Indica callus survived. In Japonica, more plants were regenerated from calli selected on all concentrations of NaCl media than from NaCl-free medium. Concentration of Cl^- in callus increased dramatically with increased NaCl content but peroxidase activity decreased.     

In vitro selection and plant regeneration of copper-tolerant plants from leaf explants of Nicotiana tabacum L. cv. 'Xanthi'

Copper tolerance of Nicotiana tabacum L. var. Xanthi in vitro was achieved through plant regeneration from leaf explants on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/l BA, 0.1–0.25 mg/l IAA and 60  μ m Cu. Tolerant organogenic calli showed more vigorous growth in medium containing 60  μ m Cu than the non-tolerant calli. Standard growth parameters such as fresh and dry weight of organogenic callus, growth tolerance index (GTI), enzyme activity (peroxidase and catalase) and copper accumulation were used as indicators of copper tolerance. The activities of peroxidase and catalase as well as estimation of protein, total amino acid and chlorophyll were greater in tolerant calli than non-tolerant ones. The GTI in the 4 weeks after the beginning of treatments yielded significant differences among the tolerant and non-tolerant organogenic callus cultures. The accumulation of copper in the tolerant calli increased significantly with an increase in copper concentration in the medium. Shoot bud regeneration was achieved in both tolerant and non-tolerant organogenic calli on MS medium containing 0.5 mg/l BA and 0.1 mg/l IAA. The tolerant regenerated shoots were rooted on half-strength basal MS medium with 60  μ m Cu for selection of tolerant clones. This study may help in the selection and characterization of Cu-tolerant lines of N. tabacum cv. 'Xanthi' for building conservation strategies and also for phytoremediation programmes.     

甘薯和Ipomoea lacunosa的种间体细胞杂种植株再生及鉴定

用ＰＥＧ融合法融合甘薯品种高系１４号和近缘野生种Ｉｐｏｍｏｅａ ｌａｃｕｎｏｓａ的原生质体，将融合原生质体培养在含有０．０５ｍｇ／Ｌ２，４－Ｄ和０．５ｍｇ／Ｌ ＫＴ的ＭＳ培养基上，愈伤组织迅速增殖。将其中的７０个愈伤组织培养在添加３．０ｍｇ／Ｌ ＢＡＰ的ＭＳ培养基上，并进一步培养在ＭＳ基本培养基上，获得９株再生植株。过氧化物酶同工酶、酯酶同工酶和ＲＡＰＤ分析表明，其中２株再生植株（ＫＬ１和ＫＬ３）     

谷子不育系及杂交种再生植株性状的表现

从谷子不育系及杂交种服性愈伤组织产生的再生植株当代和二代调查中,发现再生植株及后代普遍出现了株高降低、穗长缩短、穗型紧密和分蘖、分枝性增强的趋势。其中产生一些特殊表型株及株型好、抗虫性强的植株,再生不育株不育度提高,再生杂交种R_2代出现了三个饱满度好的大粒性穗行。     

商陆抗病毒蛋白基因在麝香百合中的转化和表达

以麝香百合叶片愈伤组织为受体,利用根瘤农杆菌介导法将美洲商陆蛋白（PAP）基因和抗卡那霉素筛选基因以共转化的方式转入百合叶片愈伤组织中,然后在含有MS培养基中筛选愈伤组织并得到再生植株,在建立的农杆菌转化百合的遗传转化体系中,获得49株再生植株,经PCR检测表明PAP基因已经转移到有抗性愈伤组织再生出百合植株中。     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

Methods for Selection of Low-Temperature Tolerant Mutants of Chrysanthemum morifolium Ramat. Using Irradiated Cell Suspension Cultures III. Comparison of Mutants Selected with or without Preselection in vitro at Low-Temperature

One hundred variants of Chrysanthemum morifolium cv. ‘Parliament’, selected on a single plant basis for early flowering at low temperature, were tested on a clonal basis. These variants originated from irradiated or non-irradiated microcalli subjected to a one-step greenhouse selection procedure, or, a two-step selection procedure (preselection in vitro at 6°C followed by selection in the greenhouse). Clones originating from irradiated calli flowered significantly earlier than the controls. Most of the low-temperature tolerant (LTT) mutants were found among plants obtained by the one-step selection procedure. Variants originating from slowly regenerating calli yielded more LTT mutants as compared to those of fast(er) regenerating calli. We therefore conclude that, irradiation and subsequent selection of plants from slowly regenerating calli considerably increases the efficiency of selecting LTT mutants. The expected advantage of a preselection in vitro at low-temperature could not be confirmed.     

通过基因枪和农杆菌介导用BADH基因转化小麦

土壤盐碱是一种严重障碍作物生产的环境因子,甜菜碱醛脱氢酶BADH基因是一种重要的可赋予植物渗透调节抗性的基因。本研究用基因枪法及农杆菌介导法向小麦幼胚和成熟胚愈伤组织导入了BADH基因。用PDS-1000／HE基因枪轰击2933块幼胚愈伤组织,分化出了45株再生植株,分化率为1．53％。PCR分析表明,其中的5株为BADH基因转化植株。用PPT涂抹其叶片,进一步证实了PCR的结果。以小麦成熟胚愈伤组织为受体,用农杆菌介导转化1968块愈伤,仅再生出了5株绿苗,PCR检测结果均为阴性。但对其转化愈伤组织的PCR检测表明,外源基因已在受体细胞中实现了整合。以幼胚愈伤组织为受体,用农杆菌介导转化2933块愈伤,共再生出了21株绿苗。对其进行PCR检测,仅有5株为BADH基因转化植株。转化处理过的幼胚愈伤组织的绿苗再生率（0．72％）高于成熟胚愈伤（0．25％）。与对照相比,所有的转化植株均能够在0．5％NaCl（w／w）条件下正常生长,表明外源BADH基因已经整合并表达。     

Plant regeneration from embryo-callus culture in barley

Summary Plants were regenerated from callus cultures initiated from immature embryos of barley, Hordeum vulgare L. Immature embryos from seven diverse genotypes were cultured on modified Murashige and Skoog (MS) medium supplemented with 1.5 mg 2,4-D and 6.5 mg IAA/l. Of the 249 embryos cultured, 30% initiated callus within 8 days. Subculture of callus for 80 to 100 days on half-MS medium supplemented with 0.5 mg/l 2,4-D and 1.0 mg/l zeatin resulted in organogenesis. Culture of organogenic calli for 30 days on half-MS medium without growth regulators produced plants which originated mostly via multiple shoot formation. Callusing response of the tested genotypes ranged from zero to 44%; however, only 23% of the calli were regenerative. Regenerated plants included variants for chlorophyll deficiency, plant height, stem thickness, spike shape, pollen fertility, seed set and ploidy.     

Increased frost tolerance and amino acid content in leaves,tubers and leaf callus of regenerated hydroxyproline resistant potato clones

Summary A number of previously selected hydroxyproline (hyp) resistant cell lines of a diploid potato (Solanum tuberosum L., clone H²578, 2n=2x=24) could be regenerated into plants which were further analysed. Hyp resistance, although lower than in the originally selected calli, was still present in regenerated shoots and in callus initiated from these shoots and it was not lost upon (mini)tuber propagation. Regenerated shoots showed a wide range of phenotypic variation. The chromosome number, analysed in 4 clones, appeared to be hypotetraploid (44 or 45). Tuber-propagated regenerants generally showed increased frost tolerance both at the plant and the cell level. In leaves this seemed to be associated with increased levels of both proline and total amino acid content. However, in callus only the total amino acid content but not proline was still elevated. In the wild type the frost tolerance of the tubers appeared to be lower than that of the leaves. Between tubers of wild type and the hyp resistant regenerants no differences in frost tolerance were found, although proline and total amino acid content tended to be higher in tubers from the regenerants.     

大豆花药愈伤组织的分化及其内源激素分析

选用３１个栽培大豆基因型进行花药培养。愈伤组织诱导率２．２％￣３６．６％,８个基因型的出愈率在２５％以上,６个基因型产生了芽或胚状体,只有丰收黄、鲁豆１０呈二个基因型既产生了芽,又产生了胚状体,具有相对高的培养力。３年内共产生了１４个再生芽、９个胚状体、６个芽状物和一个根、芽齐全的小再生植株。虽然获得花粉植株属于１５年来的第一例,但愈伤组织分化率仍然很低,这与愈伤组织的状态和质量较差有很大关系。愈