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。     

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     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)

Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin     

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.     

Intra-varietal variation for in vitro plant regeneration in the genus Trifolium

Summary Seedlings of Trifolium repens showed considerable variation with regard to the morphology and growth of their calli, and their ability for in vitro differentiation of shoots. One of the lines selected for regeneration in primary callus cultures also showed shoot formation from protoplasts. Somatic embryogenesis in callus cultures of T. pratense and T. arvense occurred only in selected seedling lines. This paper highlights the importance of screening a large number of plants within a cultivar of outbreeding species to achieve reproducible plant regeneration from tissue culture.     

Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz)

Disorganised embryogenic tissues have been utilised as target tissues for transgene insertion and transgenic plant regeneration in cassava (Manihot esculenta). The production of friable embryogenic callus in fourteen geographically diverse cassava cultivars, from which eleven were established as embryogenic suspension cultures, is reported. Embryogenic tissues were similar in nature in all cultivars tested although there was variation in the time required to generate friable callus and the growth rates of suspension cultures. Regeneration of plants has been achieved from eight cultivars but varied significantly in efficiency, with cv. TMS 60444and Line 2 from Zimbabwe being the most responsive. Tissues from the remaining eight cultivars became arrested at globular and torpedo stages of regeneration indicating that they most likely process an inherent ability to produce plants but require further research to allow this to be realised. Significant numbers of transgenic plants containing transgenes for putative resistance to important viral diseases of cassava in addition to visual marker genes have been regenerated. Transgenic plants from three the cultivars TMS 60444, Bonoua Rouge and M.Col 1505 were recovered after particle bombardment of embryogenic suspension cultures. Correlation's have been made between abnormal leaf morphology and plant vigour with the use of embryogenic suspension cultures for transgene insertion. As an result friable embryogenic callus is now being successfully utilsed as the target tissue for genetic transformation and plant regeneration at ILTAB. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Effect of genotype and light intensity on somatic embryogenesis and plant regeneration in melon (Cucumis melo L.)

The efficiency of 14 commercial cultivars of melon (Cucumis melo L.) for callus induction, plant regeneration and somatic embryogenesis under different photosynthetic photon flux densities (PPFDs) (150 or 50μmol m^?2 s^?1) was investigated. Cotyledonal explants were cultured on a Murashige and Skoog (MS) medium supplemented either with 9.0 μM 2,4-dichlorophenoxyacetic acid and 23.2μM kinetin or with 0.05 μM 2,4-dichlorophenoxyacetic acid and 0.26 μM 6-benzyladenine for the induction of somatic embryogenesis and shoots, respectively. For embryo maturation and root induction, growing callus tissues were transferred on growth regulator-free MS medium. Both genotype and the intensity of light significantly affected the rate of somatic embryo-genesis, embryo maturation and plant regeneration. On average, 12–47 primary globular-stage embryos were produced per mm² of explant surface. Fully developed, cotyledonary-stage somatic embryos were obtained from only three cultivars. Relatively high root induction rates were observed both on the shoot induction medium (11 cultivars) and on growth regulator-free medium (seven cultivars). In contrast, only six cultivars responded positively to the shoot induction treatment. Callus growth and somatic embryogenesis were significantly improved if cultures were incubated under higher PPFD values, although plant regeneration from all cultivars was significantly reduced under the same conditions.     

Regeneration and transformation of cassava

A prerequisite for the development of a successful transformation system is the availability of efficient regeneration systems. Up to 1995 the only available regeneration system in cassava was an organized type of somatic embryogenesis. Transformation of these organized somatic embryogenic cultures with particle bombardment or Agrobacterium tumefaciens resulted in chimeric transformed embryos. However, the transformed sector was lost after repeated cycles of secondary somatic embryogenesis. After 1995 a less organized system of somatic embryogenesis was developed, so called friable embryogenic callus (FEC) and a system of adventitious shoot regeneration. The FEC regeneration system was combined successfully with particle bombardment. Selection of transgenic plants was based on either luciferase activity, or resistance to the aminoglycoside paromomycin or the herbicide phosphinothricin. Furthermore, protoplasts of FEC are able to regenerate into plants and can be transformed by electroporation. The adventitious shoot regeneration system was combined successfully with Agrobacterium tumefaciens. For this mature somatic embryos were cocultivated with Agrobacterium and cultured for adventitious shoot development. After selection based on the aminoglycoside geneticin or on hygromycin transgenic plants were formed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation and control of plant regeneration in leek (Allium ampeloprasum L.)

Zygotic embryos of leek (Allium ampeloprasum L.) were isolated from mature seeds of different cultivars, selfings and full-sib families. The embryos were cultured on callus induction and shoot regeneration medium and employed to study several parameters: percentage of embryos forming calluses, percentage of embryos forming compact calluses, callus weight, percentage of regenerating calluses, numbers of shoot primordia and numbers of regenerated shoots. Differences between cultivars and selfings were found for most parameters studied. For all cultivars all parameters, except callus weight, decreased after one generation of selfing. Compact callus types enhanced primordia formation and shoot regeneration. Genetic characteristics of callus development and plant regeneration were studied in a 4 × 4 diallel cross. Analysis of variance revealed significant differences between full-sib families. The diallel analysis showed that additive gene effects were significant for all parameters. The predominance of additive gene effects indicated high narrow-sense heritability. Breeding for an increased number of regenerated shoots was successful.     

Callus Production and Plant Regeneration from Mature Embryos of Poa pratensis L.

Plant regeneration from callus cultures may provide a source ot somaclonal variation for the improvement ot the apomictic grass Poa pratensis L. It is first necessary to be able to induce callus and regenerate plants in this species at a high frequency. Variation was observed between 50 cutivars of Poa pratensis for callus induction and plant regeneration. Using the cultivars ‘Merion’ and ‘Victa’, three basal media were tested along with various media additives. Murashige and Skoog's basal medium with 0.2 mg 1^?1 2,4-dichlorophenoxyacetic acid, 0.1 mg 1^?1 6-benzylamanopurine, 100 mg 1^?1 casein hydrolysate and 25 g 1^?1 sucrose is considered to be a good medium for callus growth and plant regeneration. Embryo-like structures were observed in the callus of some cultivars but plant regeneration appeared to be predominantly from shoot meristems on the callus surface. The majority of regenerated shoots were green, but chlorophyll deficient shoots were obtained from media containing coconut milk. Green plantlets could be transferred to soil without difficulty.     

ABA对水稻愈伤组织、不定胚发育及其植株再生的影响

以长期培养的水稻愈伤组织为材料，用不同浓度的ABA对其进行了预处理，从愈伤组织的结构变化、植株再分化率、不定胚和器官分化的形成进行了研究。结果表明：经10 mg/L ABA预处理的愈伤组织外缘部分表现出禾本科类不定胚形成前期的形态结构，10 mg/L ABA预处理不仅能使分化时间缩短一周，而且使植株再生率明显提高，说明ABA对     

长江和黄河流域棉区棉花品种体细胞胚胎发生和植株再生比较研究

选用我国长江和黄河流域棉区各10个棉花品种以及珂字201和YZ1共22个基因型,研究和优化棉花体细胞胚胎发生的相关条件参数。在此基础上,比较了两生态区棉花品种的体细胞再生能力。结果表明, 在IBA+KT的激素组合下,多数基因型有较强再生能力;在愈伤组织诱导时期,铵态氮是必需的,而在愈伤组织继代分化时期,一定浓度的硝态氮则能促进分化;没有铁盐不能诱导出愈伤组织,而铁盐浓度为56 mg L^-1时有利于胚分化;长江流域和黄河流域棉花品种的植株再生潜力没有明显差别,但长江流域的品种体细胞胚胎发生所需的时间长。本文首次获得了鄂抗棉3号、5号、鄂棉20、鄂棉23、豫棉9号、豫早73、豫棉12、豫棉1221等8个品种的体细胞胚胎发生和植株再生。     

Somatic Embryogenesis in Maize and Comparison of Genetic Variability Induced by Gamma Radiation and Tissue Culture Techniques

Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F₁ and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with ⁶⁰Co gamma radiation in situ. Mature caryopses were harvested and assigned as M₁ material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R₁ generation, while regenerants from irradiated explants were considered as M₁R₁ generation. The genetic variability was evaluated in the M₂, R₂ and M₂R₂ generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M₂ populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M₂R₂ generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R₃ and M₃R₃ generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.     

A Regeneration System for Cotton Variety Xinluzao 45 via Somatic Embryogenesis

[Objective] The purpose of this study was to expand upland cotton genotypes suitable for regeneration via somatic embryogenesis, so as to provide excellent receptor materials for the genetic transformation of cotton. [Method] Six different hormone combinations were studied to establish a somatic embryo regeneration system for hypocotyl explants of Xinluzao 45, a widely planted variety in Xinjiang Province. [Result] Faint yellow callus was induced on a medium consisting of hormone combinations C1 (0.1 mg·L^－1 acetic acid dichlorobenzene oxide [2, 4-D] + 0.1 mg·L^－1 kinetin [KT]) or C5 (0.1 mg·L^－1 2, 4-D + 0.5 mg·L^－1 indo-3-butyric acid + 0.1 mg·L^－1 KT). The generated callus was able to successfully induce embryogenic callus on hormone-free medium containing doubled KNO3. The embryogenic callus then developed into somatic embryos and regenerated plants. Maximum rates of callus induction and regeneration were 92.2% and 40.3%, respectively. Although no significant difference was observed in the rate of callus induction of Xinluzao 45 hypocotyls between hormone combinations C1 and C5, the incidence of induced embryonic callus was higher on C1 than on C5. [Conclusion] The somatic embryo regeneration system developed in this study provides technical support that will facilitate Agrobacterium-mediated transformation of upland cotton Xinluzao 45.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察

 MSB培养基添加两种常用植物激素组合，Indole-3-butyric acid (IBA) + kinetin (KT)和2, 4-dichlorophenoxyacetic acid (2, 4-D) + KT，分别命名为IK和DK处理用来诱导陆地棉体细胞胚胎发生。陆地棉品系YZ1下胚轴切段作为外植体在诱导培养基上培养4周后，继代到分化培养基上促进体细胞胚胎发生。结果表明，两种处理均有体细胞胚胎发生，其中IK处理上外植体嫁接33 d后就可见体细胞胚出现，分化率高达97.6%，大多数外植体表现为体细胞胚较胚性愈伤早出现。DK处理中体细胞胚胎发生慢，体细胞胚都是经过明显的胚性愈伤发育而来，胚性愈伤分化率明显低于IK处理，仅为28.6%。此外，IK处理中的外植体在培养过程中大部分都出现了须根，而DK处理中则没有。两种处理中出现了两种不同的体细胞胚胎发生过程，组织学观察结果表明DK和IK处理中的体细胞胚分别主要起源于初生形成层和皮层。