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     

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

Somatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.     

大麦原生质体培养再生胚性愈伤组织和白化苗

从来自春大麦品种“如车”成熟胚的愈伤组织中,挑选出适于悬浮培养的松脆型胚性愈伤组织,在短期内建立胚性细胞悬浮系。此系酶解后分离出的原生质体在修改的MS培养基上能够持续分裂形成愈伤组织。将其直接转至分化培养基上获得结构紧密的胚性愈伤组织并再生白化苗.     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

Transformation of Medicago sativa L. Using a Ti Plasmid Derived Vector

A cultivar of Medicago sativa was transformed with Agrobacterium tumefaciens strain At81 binary-vector carrying the plasmid pKan3A, with the neomycin phosphotransferase gene under the control of the mannopine biosynthesis promoter. Stem segments were infected with the bacterium and kanamycin resistant calli were obtained; plant regeneration via somatic embryogenesis and polyembryogenesis was achieved only in media devoid of the antibiotic. Genetic transformation was confirmed by the presence of the structural gene through DNA-DNA hybridization and the enzymatic assay showed its functional expression. Mesophyll protoplasts, leaf calli and S1 progeny of transformed plants grew in the presence of kanamycin (100 μgml^-1). Results are discussed in relation to the use of kanamycin-resistant plants in somatic hybridization in the genus Medicago.     

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.     

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.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Towards genetic transformation in the monocot Alstroemeria L.

The successful application of plant biotechnology to Alstroemeria improvement will largely depend on the availability of an efficient regeneration/transformation system. Regeneration in Alstroemeria is accomplished from nodular embryogenic callus initiated from zygotic embryos. Histological studies of embryogenic callus initiation from 4-weeks old cultured ovules revealed that the outermost layers of the protoderm of the embryogenic nodules divided to form either a new nodule or aproembryo. Transient gene expression after particle bombardment of nodular embryogenic callus was optimized using DNA of pAHC25. The highest β-glucuronidase expression was found when the GUS gene was under control of the maize ubiquitin promoter, the target tissue was placed 5 cm below the microcarrier launch assembly and when the rupture disc-breakage point was between 650–900 psi. Kanamycin blocked regeneration of somatic embryos, however, did not block growth of nodular embryogenic callus. With phosphinothricin both callus growth and regeneration were blocked. Bombardment of nodular embryogenic callus with DNA of pAHC25 combined with selection on medium containing phosphinothricin resulted in putative transgenic chimeric. Friable calli were selected from nodular embryogenic callus and used to initiate suspensions. These cell suspensions were subjected to transformation by particle bombardment using DNA of pAHC25 and resulted in a stable transformed friable callus line after selection based on luciferase activity. Even after 2 years of maintenance this callus line was luciferase positive and the Polymerase Chain Reaction analysis demonstrated the presence of the introduced gene in this friable callus line. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Electrophoretic patterns of acid soluble proteins and active isoforms of peroxidase and polyphenoloxidase typifying calli and somatic embryos of two reputed date palm cultivars in Morocco

Summary When subjected to micropropagation by tissue culture, the two reputed cultivars of date palm (Phoenix dactylifera L.); Bou-Sthammi noire, resistant to Bayoud disease and Bou-Feggous, of high fruit quality, give rise to three types of calli, called white and root-forming callus, hyperhydric and degenerating callus and friable and embryogenic callus. All explant sources, calli and germinated embryos were analysed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) for acid soluble protein composition. Phenol-oxidizing enzymes; peroxidase and polyphenoloxidase, were also, evaluated and the isoforms separated by polyacrylamide gel electrophoresis. When compared with the explant and germinated embryos, embryogenic calli of the two date palm cultivars could be identified by a concentrated polypeptide of molecular weight 27 500 and polypeptides of molecular weights 70 000 and 11 500. Hyperhydric and degenerating callus contained the polypeptide exhibiting the molecular weight 32 000. Embryogenic calli showed high levels of soluble, ionically and covalently bound peroxidases. The soluble acidic isoperoxidase of R _f 0.60, revealed in these calli and germinated embryos could be a marker of the two tissues. White and root-forming calli of Bou-Feggous cultivar were typified by soluble acidic isoperoxidases with high mobility (R _f 0.75) and anodic ionically wall-bound polyphenoloxidases similar to those of the explant sources. Polyphemoloxidase activities detected in calli and embryos were very low when compared with those of explants. Used as an early test to screen embryogenic calli of date palm, acid soluble proteins, peroxidase and polyphenoloxidase data could lead to introduce lightening and economy in the tissue culture technique.     

Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae)

The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

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.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.     

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.     

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.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.