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（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。     

The effect of in planta TIBA and proline treatment on somatic embryogenesis of sugar beet (Beta vulgaris L.)

The effect of in planta TIBA and L-proline onin vitro seedlings and cell culture of sugar beet was investigated. Sterilized seeds were grownin vitro on 1/2 MS medium supplemented with 0 or3 mg/l TIBA. Calli obtained on young leaves cultured on MS medium containing 1 mg/l BAP, were used for the initiation of cell suspension cultures using MS basal composition supplemented with 0 or 50 mM proline. Aliquots of 1 ml from cell suspension culture were inoculated onto the first somatic embryo induction MS medium containing TIBA 0.5 mg/l, BAP 1.0 mg/l, and proline at 0 or 50 mM. After three weeks of culture, embryogenic calli were transferred to the second embryo induction medium supplemented with NAA and BAP at 0.2 and 0.5 mg/l, respectively. The frequency of somatic embryos of calli obtained from in plantaTIBA together with proline treatments on average was20 which was higher than that of the other treatments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intergeneric hybridization between Brassica species and Crambe abyssinica

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N₆-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Indirect Organogenesis through Seedling-Derived Leaf Segments of <Emphasis Type="Italic">Ficus Religiosa</Emphasis> - a Multipurpose Woody Medicinal Plant

The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.     

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     

花生原生质体分离与培养

以花生品种花育20为材料,探讨不同的酶液浓度、酶解时间及渗透压对花生原生质体分离的影响。结果表明:原生质体分离的适宜酶液配比是2%纤维素酶（Cellulase Onozuka RS）、0.2%果胶酶(Pectolyase Y-23),甘露醇渗透压调节剂的浓度为0.7 mol/L,暗处理10 h,叶片原生质体产量为4.86×105/ml,存活率75.6％,愈伤组织原生质体产量为4.97×105/ml,存活率75.4％。将分离的原生质体培养在添加1mg/LNAA和4mg/LBAP的改良MS液体培养基中。培养约2～3d后,细胞开始分裂。然后部分细胞继续分裂,并形成细胞团。培养5～6周后,将培养物转移到添加2mg/L2,4-D和3mg/LBAP的MS液体培养基（pH 5.8）中进行培养。7～8周后,将形成的直径为1～2mm的小愈伤组织转移到添加1mg/LNAA和5mg/LBAP的MS固体培养基上培养,小愈伤组织迅速生长。转移3～4周后,愈伤组织长至7～9mm。     

Production of interspecific hybrids between Glycine max and G. tomentella through embryo culture

Summary Interspecific hybrids have been obtained in an incompatible cross between Glycine max and G. tomentella through the in vitro culture of hybrid embryos. The percentage of successful pod setting in the crosses averaged 12.8% but there were marked differences depending on the soybean cultivar used as the female parent. Hybrid embryos at globular to heart stages were extracted from the embryo sac 15–25 days after pollination and cultured in vitro. Hybrid plants were successfully obtained by culturing the embryos on B5 medium supplemented with 0.1 mg/l IBA followed by culture on B5 medium supplemented with 0.1 mg/l TBA plus 0.25 mg/l 2-iP. The F1 plants resembled the wild male parent in growth form, but had an intermediate leaf shape between that of the parents.     

Plant regeneration from protoplasts of Iris germanica L.

Summary Protoplasts were isolated enzymatically from suspension cultures derived from embryogenic calli induced by leaf base culture of Iris germanica. In protoplast culture, the effects of glucose concentration, different sugars and combinations of 2,4-D and KIN on protoplast division and colony formation were examined. N6 medium supplemented with 0.1–1 mg/l 2,4-D, 1 mg/l KIN, 200mg/l casein hydrolysate, 250 mg/l proline, 0.2 M glucose and 20 g/l agarose was suitable for protoplast division and colony formation. When colonies formed were transferred onto hormone-free MS medium, many plantlets were regenerated through somatic embryogenesis. Thus, we could establish a plant regeneration system from protoplasts of I. germanica.Contribution from the Laboratory of Plant Breeding, Faculty of Agriculture, Miyazaki University, Japan, No. 95.     

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Summary Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N₆) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N₆ medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.     

Resistance to Polymyxa betae in Beta Species of the Section Procumbentes, in Hybrids with B. vulgaris and in Monosomic Chromosome Additions of B. procumbens in B. vulgaris

Plant regeneration from callus cultures of Allium trifoliatum subsp. hirsutum fertile accession F-370, was studied as a means for clonal multiplication and germplasm storage of Allium spp. Callus was induced on in votro-cultured basal leaf explants. Best proliferation was obtained on modified BDS medium supplemented with (mg/1): 0.75 picloram, 2.0 benzyl adenine, and 900 casein hydrolysate. Shoot and root organogenesis were obtained in 3 to 5 month old subcultured calli, on BDS or MS medium supplemented with (mg/1): either 0.03 picloram or no auxin, 2 BA or 2-isopentenyladenine, and 900 casein hydrolysate. Direct bulb formation, without shoot elongation, occurred on BDS medium with 10 mg/1 IBA. Under these conditions, callus formation and organogenesis were not obtained with A. trifoliatum subsp. hirsutum var. sterile, a male-sterile genotype. Most regenerants were phenotypically normal, but some abnormal shoots were also observed, i.e. shoots with vitrified or extremely broad leaves. Isozyme polymorphism analysis of seven proteins in the latter regenerants, and in several callus cultures, revealed significant deviation from the original pattern in esterase, 6-phosphogluconate dehydrogenase and superoxide dismutase. No such deviations were detected in normal regenerated plants.     

High frequency of plant regeneration from anther culture in flax, Linum usitatissimum L.

The effects of induction medium compositions on flax anther culture were investigated in order to improve the efficiency of callus induction and plant regeneration. Anthers were inoculated onto the modified MS medium supplemented with five different combinations of plant growth regulators. The medium containing the combination of 2mg/l 2,4- dichlorophenoxy-acetic acid (2,4-D) and 1 mg/1 6-benzylaminopurine (BAP) produced a significantly higher number of calli forming shoots/100 responded anthers and a significant increase in overall efficiency of regeneration than the same basal medium containing 1 mg/1 a-naphthalene-acetic acid (NAA) and 2 mg/1 BAP (CK). Among the five levels of thiamin hydrochloride tested, the modified MS medium containing 10 mg/1 thiamin hydrochloride significantly increased the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration compared with the same basal medium containing 0.1 mg/1 thiamin hydrochloride. Maltose concentration had a significant effect on the percentage of anthers producing call, the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration. The medium containing 6% or 9% maltose produced the highest overall efficiency of regeneration among the five levels of maltose evaluated. Sucrose concentration significantly affected the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration, and dramatically affected the frequency of microspore-derived plants and the frequency of spontaneous chromosome doubling in microspore-derived plants. The efficiency of doubled haploid line production obtained in this study appears adequate for applied breeding programmes.     

结球甘蓝的离体快繁技术研究

以结球甘蓝无菌苗的胚轴和子叶为材料，几乎不生长愈伤组织，分化出不定芽。不定芽在添加6-BA的MS培养基上增殖很快，适宜的生根培养基为1/2MS+IBA0.5mg/L+ NAA0.1mg/L。通过增加培养基中糖和琼脂粉的用量，提高培养时的光照强度等有效控制了玻璃苗的产生。     

Somaclonal variation of regenerated plants in chili pepper (Capsicum annuum L.)

The morphological and genetic variations in somaclones of chili pepper (Capsicumannuum L.) derived from tissue culture were evaluated. Cotyledonary node explants of cultivars, Shishitou and Takanotsume, were cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA)5 mg/l for shoots regeneration and regenerated shoots were rooted on MS medium supplemented with naphthalene acetic acid(NAA) 0.1 mg/l and indol-3-butyric acid(IBA) 0.05 mg/l. The regenerated plants(R₀) were selfed to obtain seeds for next generation (R₁ lines). Qualitative characters were studied in R₀generation and both qualitative and quantitative characters were studied in R₁ generation. In R₀ generation, variations were noticed in plant growth habit, stem color, flower color and color of unripe fruits, and expression of anthocyanin in unripe fruits. Comparison among the R₁ lines and their parents were made for morphological and agronomic characters. Significant variation among R₁ lines and differences between R₁ lines and their parents were observed. Genetic variations among three somaclones were revealed by random amplified polymorphic DNA (RAPD) analysis. Variation, such as early flowering and increase of yield components, is an indication of the response to selection for any specific character. Occurrence of productive variants among somaclones of established cultivars, like Shishitou and Takanotsume, indicates the possibility of their improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regeneration of dihaploid chicory (Cichorium intybus L. var. foliosum Hegi) via microspore culture

In order to develop fully inbred chicory plants, dihaploid plants were raised from callus derived from microspores of three selected Witloof, Robin and Treviso types. Microspores were isolated from florets containing pollen at the uninuclear state and cultured in a modified MS medium plus 0.5mg/l 2,4-D, 0.5mg/l IAA and 2.0mg/l zeatin. During culture periods of up to 6 months, gametoplasts emerged from pollen grains, divided and started to form colonies and calli. These were subcultured on the same basal medium supplemented with 0.5mg/l BA and 0.5mg/l IAA. Shoot growth was enhanced on a low salt-containing medium supplemented with 0.4mg/l kinetin and 0.2mg/l IAA. Shoots were rooted on a half-strength Lepoivre medium plus 0.2mg/l IBA and finally transferred to soil. Florets were excised from 34 capitula, but only microspores from four of them developed into plants via callus. More than 450 plants were raised in the greenhouse and the field. Leaves from these plants were subjected to DNA fluorescence analysis via flow cytometry: a range of ploidy levels was detected. The cell composition of 44 of these plants was predominantly haploid, with a diploid background. Regenerant plant phenotypes were compared with the parent genotypes. The value of such haploids in commercial chicory breeding is discussed.     

Somaclonal Variation in Tissue Culture of Triticum aestivum×Agropyron desertorum F1 Hybrid

Callus induced from immature inflorescences of the partially self-fertile hybrids (2 n = 35; ABDPP) between Triticum aestivum (2n = 42; AABBDD) and Agropyron desertorum (2n = 28; PPPP) led to the regeneration of 88 plants on MS medium supplemented with 2 mg/l of 2,4-D. These regenerants were used to investigate somaclonal variation and to obtain more selfed derivatives. Immature inflorescences at the stage of developing floral primordia gave the best response in terms of callus induction and plant regeneration. The regenerants exhibited great variability for most morphological traits. Although the regenerants did not exhibit variation in chromosome number, they did show a higher degree of meiotic instability than the initial hybrid. In particular, the regenerants gave much higher selfed seed-set (5.49 %) than the donor hybrid (0.46 %), so that a total of 484 selfed seeds were obtained.     

大豆幼荚子叶原生质体培养及植株再生

本文研究了13个栽培大豆（Glycine max L.）品种原生质体培养的再生能力。从大豆幼荚子叶酶解游离原生质体,用Gellan Gum进行株状包埋,悬浮在含2,4-D 0.1-0.2mg/L,BA0.5-1.0mg/L的改良MS液体培养基中,原生质体培养3天后开始第一次分裂,以后持续分裂。供试基因型间的10天植板率差异显著,变幅为33-67%。30天内形成大量的     

In vitro adventitious shoot regeneration for effective maintenance of male sterile niger Guizotia abyssinica (L.f.) Cass.)

A rapid and efficient method of in vitro plant regeneration for large scale propagation of male sterile plants of niger (Guizotia abyssinica) was developed. Leaf segments from mature plants were cultured on Murashige & Skoog's basal medium (MS) supplemented with N⁶-benzyladenine (BA) and kinetin individually and in combination with low concentrations of indole-3-butyric acid (IBA); and -Naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media tested. The best response in terms of frequency of shoot regeneration and the number of shoots per leaf explant was observed on medium supplemented with 2.22 M BA. Transfer of shoot bud clusters to fresh medium with same composition promoted further multiplication of the shoot buds, while medium with reduced BA concentration (0.89 M) facilitated shoot elongation. Shoots that were rooted on half-strength MS medium gelled with 0.2 or 0.4% agar and supplemented with 4.9 M IBA survived with a frequency of 61.36% on transfer to ex vitrum conditions. Field evaluation of the regenerants revealed the genetic stability of the plantlets and are being used in breeding of experimental hybrids.     

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）     

Plant regeneration from in vitro cultures of cotyledon explants and anthers of Sinapis alba and its implications on breeding of crucifers

Summary Protocols for plant regeneration from cotyledon explant and anther cultures of Sinapis alba have been developed for creating doubled-haploids and somaclonal variation. Among the several cultivars tested in this study, only Arda responded well to in vitro plant regeneration both from anther-as well as cotyledoncultures. Multiple shoot formation in cotyledon explants, which always followed a brief callusing phase, was found to be the best on MS medium with ZEA (1.0mg/l) and NAA (0.1mg/l). Regeneration frequency declined sharply in the absence of auxin or presence of other cytokinins and/or auxin. The frequency of shoot regeneration also declined with reduction in the photoperiod to 16h. On MS + BAP (1.0mg/l) + NAA (1.0mg/l) medium, cotyledonary explants showed profuse callusing, which could regenerate shoots on high ZEA + low NAA/IAA medium. However, it declined with progressing time in culture. Anthers, excised from fresh as well as cold pretreated buds, cultured on 10% sucrose containing MS media with different hormonal constitution, developed calli and/or embryos. Initial culture temperature was important with embryogenesis occurring only in anthers cultured at 30°C for 3 weeks. A high temperature (35°C) treatment was lethal for both callus as well as embryo formation. While BAP + NAA and ZEA + NAA/IAA supported embryogenesis, further plant regeneration from anther-or embryo-callus could be achieved in ZEA + NAA/IAA media. Some of the regenerants flowered already in vitro and had small and sterile flowers. Cytological examination of some of the root differentiating calli indicated the presence of haploid as well as diploid cells. Shoots were rooted during prolonged incubation on the same medium or on transfer to MS (reduced)/ B5 + ZEA + NAA media.