Hemoglobin (Erythrogen™)-enhanced mitotic division and plant regeneration from cultured rice protoplasts (Oryza sativa L.)

Supplementation of culture medium with hemoglobin solution (Erythrogen™) promoted mitotic division, cell colony formation, and plant regeneration from rice (Oryza sativa L. cv. Taipei 309) protoplasts. The mean (± s.e.m., N = 5) final protoplast plating efficiency (FPE) at 28 days following exposure to 1:50 (v:v) Erythrogen™ (0.55 ± 0.06%) was significantly greater (P < 0.01) than in untreated controls (0.26 ± 0.02%). A similar, but less pronounced effect (P < 0.01) also occurred with 1:100 (v:v) (PE 0.53 ± 0.04%) and 1:500 (v:v) (PE 0.46 ± 0.05%) Erythrogen™, respectively. In contrast, there was no corresponding increase in plating efficiency with Erythrogen™ at 1:1,000 (v:v). These beneficial effects were sustained throughout culture. This leads to a 44% increase (P < 0.01) in protoplast-derived calli producing shoots compared to controls. Cytological analyses confirmed the diploid status of plants regenerated from Erythrogen™-treated and control protoplasts.     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Molecular mapping of two semidwarf genes in an indica rice variety Aitaiyin3 (Oryza sativa L.)

Genetic analysis established that Aitaiyin3, a dwarf rice variety derived from a semidwarf cultivar Taiyin1, carries two recessive semidwarf genes. By using simple sequence repeat (SSR) markers, we mapped the two semidwarf genes, sd-1 and sd-t2 on chromosomes 1 and 4, respectively. Sd-t2 was thus named because the semidrawf gene sd-t has already been identified from Aitaiyin 2 whose origin could be traced back to Taiyin1. The result of the molecular mapping of sd-1 gene revealed it is linked to four SSR markers found on chromosome 1. These markers are: RM297, RM302, RM212, and OSR3 spaced at 4.7 cM, 0 cM, 0.8cM and 0 cM, respectively. Sd-t2 was found to be located on chromosome 4 using five SSR markers: two markers, SSR332 and RM1305 located proximal to sd-t2 are spaced 11.6 cM, 3.8 cM, respectively, while the three distally located primers, RM5633, RM307, and RM401 are separated by distances of 0.4 cM, 0.0 cM, and 0.4 cM, respectively. __________ Translated from Acta Genetica Sinica, 2005, 32 (2) [译自: 遗传学报, 2005,32(2)]     

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m⁻³ NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na⁺) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K⁺ and Ca²⁺ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.     

Nitrate reductase induction and molecular characterization in rice (Oryza sativa L.)

Summary Barley nitrate reductase cDNA clone bNRp10 was used as a hybridization probe to screen a genomic DNA library of rice (Oryza sativa L.) cultivar M201. Two different lambda clones were isolated, subcloned to plasmids, and partially characterized. The subclone pHBH1 was tentatively identified as encoding a NADH nitrate reductase. Southern and dot blot analysis suggest that, in rice, nitrate reductase is encoded by a small gene family. Regulation of NADH nitrate reductase was investigated in rice cultivars Labelle and M201 representing the subspecies indica and japonica, respectively. In the absence of nitrate, only trace levels of nitrate reductase activity and mRNA were detected in seedling leaves. Upon addition of nitrate to seedling roots, nitrate reductase activity and mRNA increased rapidly in leaves. Nitrate reductase activity continued to increase over a 24 h period, but the mRNA accumulation peaked at about 6 h and then declined. Western blot analysis with a barley NADH nitrate reductase antiserum showed the presence of two bands of approximately 115 and 105 kDa. These protein bands were not detected in extracts of tissue grown in the absence of nitrate.     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura)

 Somatic embryogenesis was observed in ray-floret explants of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu on Murashige and Skoog medium containing high concentrations of 3-indoleacetic acid (IAA) and kinetin. 1-Naphthaleneacetic acid also induced somatic embryogenesis but indole-3-butyric acid or 2,4-dichlorophenoxy acetic acid did not. Other cytokinins, such as 6-benzylaminopurine (BAP) and thidiazuron, were also not effective. No embryos were seen at lower IAA concentrations with kinetin and various concentrations of BAP, although higher BAP concentrations yielded many adventitious shoots. In contrast, no somatic embryogenesis was observed from leaves using any combination of plant growth regulators. Histologically, primordia showed a typical embryo shape with a well-developed vascular bundle between the shoot and the root primordia. Embryos had both stomata cells and a root system with polarity. Plants were efficiently regenerated from ray floret-derived embryos subcultured in the appropriate medium. Received: 30 April 1999 / Revision received: 7 October 1999 / Accepted: 27 January 2000     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.Abbreviations ABA Abscisic acid - PEG Polyethylene glycol 4000 - SM1 Smith Standard Embryonic Tissue Capture Medium - SM2 Smith Standard Embryogenesis Medium - SM3 Smith Embryo Develop Medium     

A rapid screening technique for salt tolerance in rice (Oryza sativa L.)

An effecient, reproducible and simple mass screening technique for the selection of salt tolerant rice lines has been developed. Fourteen-day old seedlings raised in silica gravel culture were transplanted to foam-plugged holes in polystyrene (thermopal) sheets floated over 100 dm³ of nutrient solution in painted galvanised-iron growth tanks lined with plastic (120×90×30cm). Three days after transplanting, NaCl was added to salinize the medium in increments, at the rate of 25 mol m^-3 per 24 hours, up to the desired salinity levels which ranged from 50–200 mol m^-3 NaCl. Six plants of each line were transplanted and allowed to grow for 15 days after the maximum desired stress level was achieved in each case. Absolute shoot fresh and dry weights, as well as percent mortality, were used as criteria for assessing relative salt tolerance. Related studies were also conducted to standardize the technique. The validity of this technique was tested by conducting experiments in salinised soil (pot culture) and in salt-affected field where 9 rice lines were grown up to maturity and absolute paddy yield was considered as the criterion for salt tolerance. Salt tolerance behaviour of cultivars based on different selection criteria was compared. Good reproducibility of results among the three solution culture experiments and their close association with the results of pot culture and of salt-affected field study, authenticated the validity of this technique for practical purposes.     

Plantlet regeneration from somatic hybrids of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola Vasing)

Summary Somatic hybridization of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola), a close relative of barnyard millet, was attempted using electrofusion and a new culture method developed for rice protoplasts (Kyozuka et al. 1987) to incorporate some of the agronomically important characters of the latter species into rice. Selection of hybrids was based on inactivation of rice protoplasts by iodoacetamide and the inability of barnyard grass protoplasts to divide. A total of 166 calli were identified as hybrids by isozyme and chromosome analyses. Hybrid calli were highly morphogenic, and 44 shoots were obtained. Most of them, however, were abnormal, and nine grew to plantlets whose morphology was distinct from that of either parent. Our study clearly demonstrates the totipotency of protoplasts in graminaceous monocots.     

An efficient androgenic embryogenesis and plant regeneration method through isolated microspore culture in timothy (Phleum pratense L.)

A method employing isolated microspore culture was established for the androgenic embryogenesis of timothy (Phleum pratense L). Embryos/calli were obtained and green plants regenerated. The induction medium was PG-96 (1.0 mg l⁻¹ 2,4-D, 0.1 mg l⁻¹ Kinetin) supplemented with 6% maltose monohydrate. Timothy microspore culture was genotype-dependent, among 12 genotypes, 6 produced embryos/calli and 4 produced green plants. Macerating the spikes with a blender and purifying the microspores at a mannitol/maltose monohydrate interface gave a relatively high percentage of cell vitality. The optimum microspore developmental stage was from the very late uninucleate stage to the binucleate stage. Heat shock promoted the initiation of microspore culture. Over 150 regenerated green plants were obtained; in a random sample of 32 of these 65.6% were doubled haploids (6n=42). Albinism was a problem in plant regeneration (9.3–22%). This paper is the first to describe timothy androgenic embryogenesis by isolated microspore culture. Received: 9 September 1999 / Revision received: 6 December 1999 / Accepted: 13 December 1999     

Identification of salt-stress responsive genes in rice (Oryza sativa L.) by cDNA array

To identify salt stress-responsive genes, we constructed a cDNA library with the salttolerant rice cultivar, Lansheng. About 15000 plasmids were extracted and dotted on filters with Biomeck 2000 HDRT system or by hand. Thirty genes were identified to display altered expression levels responding to 150 mmol/L NaCl. Among them eighteen genes were up-regulated and the remainders downregulated. Twenty-seven genes have their homologous genes in GenBank Databases. The expression of twelve genes was studied by Northern analysis. Based on the functions, these genes can be classified into five categories, including photosynthesis-related gene, transportrelated gene, metabolismrelated gene, stress-or resistancerelated gene and the others with various functions. The results showed that salt stress influenced many aspects of rice growth. Some of these genes may play important roles in plant salt tolerance.     

Effect of air desiccation and salt stress factors on in vitro regeneration of rice (Oryza sativa L.)

Enhancement of callus induction and its regeneration efficiency through in vitro techniques has been optimized for 2 abiotic stresses (salt and air desiccation) using 3 rice genotypes viz. BR10, BRRI dhan32 and BRRI dhan47. The highest frequency of callus induction was obtained for BRRI dhan32 (64.44%) in MS medium supplemented with 2, 4-D (2.5 mgL⁻¹) and Kin (1.0 mgL⁻¹). Different concentrations of NaCl (2.9, 5.9, 8.8 and 11.7 gL⁻¹) were used and its effect was recorded on the basis of viability of calli (VC), relative growth rate (RGR), tolerance index (TI) and relative water content (RWC). It was observed that in all cases BRRI dhan47 showed highest performance on tolerance to VC (45.33%), RGR (1.03%), TI (0.20%) and RWC (10.23%) with 11.7 gL⁻¹ NaCl. Plant regeneration capability was recorded after partial air desiccation pretreatment to calli for 15, 30, 45 and 60 h. In this case BRRI dhan32 gave maximum number of regeneration (76.19%) when 4 weeks old calli were desiccated for 45 h. It was observed that air desiccation was 2-3 folds more effective for enhancing green plantlet regeneration compared to controls. Furthermore, desiccated calli also showed the better capability to survive in NaCl induced abiotic stress; and gave 1.9 fold (88.80%) increased regeneration in 11.7 gL⁻¹ salt level for BRRI dhan47. Analysis of variance (ANOVA) showed that the genotypes, air desiccation and NaCl had significant effect on plant regeneration at P < 0.01.     

The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm

Summary Embryogenic callus consisting of both Type 1, firm, compact, translucent, relatively slow growing callus and Type 2, highly friable, rapidly growing callus with well-formed somatic embryos, were observed in elite maize germplasm, notably B73 and hybrids with B73. Parental genotype is very important in the ability to identify and isolate embryogenic callus after 14 and 28 days in culture. A partial diallel analysis revealed that a large proportion of the genotypic variation was of the additive type although heterosis did positively increase culture response in most cases. A significant negative maternal effect for culture response was noted for inbred B73 from Reid-type germplasm while four lines sampled from Lancaster germplasm showed similar response whether used as male or female. Although significant media differences were observed in some genotypes, culture media did not preclude observation of Type 1 or Type 2 embryogenic cultures in this study after 14 and 28 days. Plants could be regenerated from all genotypes in this study after 14-days of culture, but not all genotypes were capable of sustained subculture and plant regeneration. Plant regeneration from Type 2 cultures of B73 and B73 hybrids has been obtained up to a year after initiation.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets

In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.     

水稻黄绿叶突变体ygl-63的特征和基因定位

叶绿体的正常发育对于植物至关重要,突变体研究是探明叶绿体发育过程中基因功能的有效途径。叶色突变体已引起人们广泛的关注,通过对各种植物材料的研究,叶色突变的分子机制已取得一定进展,但远未被阐明,尤其在水稻当中。目前,已报道的水稻叶色突变体,主要表现为黄化、白化、亮绿、条斑条纹、温敏变色、转绿和转紫等。该研究使用甲基磺酸乙酯( EMS)处理粳稻日本晴,获得一份遗传稳定的突变体ygl-63,其整个生育期叶片均表现为黄绿色。通过测定ygl-63和野生型苗期叶片的叶绿素含量发现,ygl-63中叶绿素a、叶绿素b和总叶绿素含量与野生型相比分别下降了31.9％、42.2％和34.1％,同时叶绿素a/b值较野生型增加。这表明叶绿素含量的降低是导致ygl-63黄绿叶突变性状的主要原因,并且叶绿素b的降幅大于叶绿素a。在成熟后调查主要农艺性状发现ygl-63单株有效穗数和结实率分别减少8.9％和8.5％;千粒重增加10.4％;而株高,穗长和每穗着粒数和野生型相比差异并不显著。通过测量微量元素发现,ygl-63种子中的铁和锌含量较野生型显著降低,分别减少85.7％和64.8％。将ygl-63与正常绿色品种明恢63杂交获得F1和F2群体,进行遗传分析发现,ygl-63突变性状受1对隐性基因控制,通过基因定位,将该基因定位到水稻第11染色体长臂的分子标记InDel-3和InDel-5之间约2.4 cM范围内。该基因被认为是一个新的水稻叶色突变基因,暂命名为ygl-63( g)。所得结果为今后对ygl-63( g)基因的进一步研究奠定了基础。     

Effect of sorbitol concentration on regeneration of embryogenic calli in upland rice varieties (Oryza sativa L.)

This study describes the impact of sorbitol on plantlets regeneration frequency (PRF) of four rice cultivars (japonica, Oryza sativa L.) both of which mature and immature embryo-derived calli were investigated. The variance analysis results showed that PRF of the three elite upland rice cultivars, Handao297 (HD297), Handao502 (HD502), Handao65 (HD65) and one lowland rice cultivar Zhongzuo93 (ZZ93) were significantly increased with addition of appropriate amount of sorbitol in culture media. Supplementing appropriate sorbitol in the media of a continous culture from induction and maintenance to regeneration for mature embryo-derived calli could improve PRF dramatically, originally from 27.6% up to a maximum of 71.8%. Especially to low regenerative capacity (LRC) cultivar HD65, the PRF was increased over 7-fold (from 9.7% to 74.0%). The optimum concentrations of sorbitol for calli induction, subculture and differentiation were 5, 20 and 40 g/l, respectively. Adding sorbitol, only in maintenance media at concentration of 20 g/l, also enhanced the PRF greatly in all the cultivars from 27.6% to 43.3%. Similar results were observed when incorporating with maltose in regenerating media both in immature and mature embryo-derived calli. The optimal concentration was 25 g/l sorbitol + 20 g/l maltose and 20 g/l sorbitol + 25 g/l maltose, respectively. HD297 appeared to be the most responsive genotype compared to other cultivars in PRF, 99.2% in immature embryo-derived calli and 76.8% in mature embryo-derived calli. The results and relevant conclusions might be valuable to establish an efficient plant regeneration system from somatic embryogenesis culture in upland rice.