Colchine-induced aneuploids from cell culture of sugarcane

Summary Sugarcane (Saccharum spp.) clones are amenable to gross chromosome manipulation due to their high polyploid nature (2n=100–120). This study was conducted to analyze the effects on plant morphology of altering chomosome number via callus culture. Callus cultures from clone H69-9092 were established, and plants were regenerated following colchicine treatment of cultured cells. Cytological analysis showed that variant somaclones were aneuploids with a wide range in chromosome numbers (2n=66–196). Some 22 visually distinct somaclones were planted in 1.35 m² plots with five replications to compare morphological and quality characteristics with H69-9092 at 8 months of growth. Extreme morphological variation was observed between somaclones, but coefficients of variation for quality factors-fibers %, refractometer solids %, pol %, and juice purity-and stomatal length were smaller than those for morphological traits associated with stalk volume and leaf area. Significant negative correlations were found between chromosome number and most morphological traits, e.g., stalk length (r=-0.58), number (r=-0.69), diameter (r=-0.54) and volume (r=-0.65); internode length (r=-0.57); and leaf area (r=-0.48). A positive correlation was found between chromosome number and stomatal length (r=-0.66). No significant correlations were found between chromosome number and quality factors. Aneuploids with higher than parental chromosome number had reduced growth. However, depression in growth was generally not observed in somaclones lower in chromosome number than the parent.Published with the approval of the Director as Paper No. 598 in the Journal Series of the Experiment Station, Hawaiian Sugar Planters' Association.     

Tissue and cell culture as AIDS to sugarcane breeding. I. Creation of genetic variation through callus culture

Summary Four-hundred and seventeen plants from 8 sugarcane varieties were selected among the 4600 plants that had been generated from calluses of shoot-apices, rolled young leaves and young inflorescences of 58 varieties. They were planted in the field (Field Test-II stage) in December, 1971 and examined for morphology, sugar content and chromosome number in the summer of 1972 and thereafter. The callus derivatives of the 8 varieties differed from their donors in the frequency of morphological changes: F146 (1.8%), N:Co310 (2.4%), F161 (4.8%), F164 (6.7%), F162 (7.4%), 56–2080 (15.8%), F170 (27.6%), and F156 (34.0%). Auricle length showed the greatest frequency of difference (8.6%); dewlap shape ranked next (6.5%); followed by hair group (6.2%); attitude of top leaves (1.9%). The sucrose content of callus derivative was increased over their donors by 2% to 12%. Chromosome number (2n) varied from 86 to 126 in the F156 derivatives in contrast with the donor's 114 whereas those of F164 varied from 88 to 108 as compared with the donor's 108. The derivatives of F146 generally centred around the original chromosome number of 110 and it is considered to be the most genetically stable cultivar. The cause of the occurrence of chromosome mosaicism in sugarcane is discussed. There was no apparent correlation between morphological modifications and changes in chromosome number.     

Application of cell and tissue culture and in vitro selection for disease resistance breeding — a review

Summary Somaclonal variation, i.e. the variation induced by cell and tissue culture, offers an opportunity to broaden the genetic variation of crops. As a result of somaclonal variation a wide range of plant characteristics can be altered. However, the selection of agronomically important traits, e.g. disease resistance, has many limitations. The efficiency of selection can be increased by the application of in vitro selection procedures. Selection strategies that may be applied to obtain disease resistant somaclonal variants are described. Their merits and limitations, in relation to the efficiency of the procedures, the frequency of disease resistant variants and the genetics of the resistance obtained, are discussed.     

Heritable tissue culture induced variation in Zinnia marylandica

Summary Adventitious shoots of Zinnia marylandica, an amphidiploid with limited genetic segregation, were regenerated from cotyledonary tissue on Murashige-Skoog (MS) media containing 0.2 or 22.2 M thidiazuron (TDZ) and grown through flowering. Fisher's Test for Equal Variance indicated tissue culture induced plants had more variation than seed-derived control plants. Twelve of 149 (8%) plants derived from 0.2 M TDZ and three of 23 (13%) plants from 22.2 M TDZ had variant characters. Aberrant characteristics in self-pollinated variants included plant height, fertility, flower color and morphology, and were sexually transmitted, indicating genetic change had occurred. Aberrant characteristics not observed in regenerated plants arose in progeny.Abbreviation TDZ thidiazuron     

植物组织培养中体细胞无性系变异的研究

综述了植物组织培养中体细胞无性系变异概念,变异来源、因素,变异表现和检测方法,指出了无性系变异研究中的不足及今后研究方向。     

Somaclonal variation in sugarcane through tissue culture and evaluation for quantitative and quality traits

Eight genotypes of Saccharum officinarum were crossed with Saccharum spontaneum and 14 genotypes of S. officinarum were crossed with Erianthus arundinaceus. A total of 39 hybrids were evolved. These 39 hybrids were raised in the field and used as donor clones for in vitro culture studies. Plantlets were regenerated from 1-month-old callus. The grown up plants were transplanted to well prepared field, to study the variations generated for the biometric as well as for biochemical characters. There were significant differences between the donor clones and their sub clones for all the character of interest. The somatic segregation was gradual and wider, showing a range of divergence from the mean towards the end of the scale. Fifty-one sub clones were selected with commercial potential which have 13% fibre, 200 cm stalk length, 10 cm internode length and pure obtainable cane sugar per cent of 10.     

Screening for and heritability of flood-tolerance in the Florida (CP) sugarcane breeding population

Summary An experiment screening for flood-tolerance and estimating the heritability of flood-tolerance was conducted on 160 sugarcane clones in the Canal Point, Florida, USA sugarcane breeding population. Clones were grown in two environments, a drained control and a continuous, five-month flood. The test ran for two crop years, plant-cane and first ratoon. A wide range of production was observed, with some clones dying out after the first year. Several clones had two-year mean cane production in flood that was 70% of their production in control. Heritabilities calculated by parent-offspring regression ranged from 0.29 to 0.51.     

Isolation,fusion and multiplication of sugarcane protoplasts and comparison of sexual and parasexual hybridization

Summary The problems associated with the isolation of leaf protoplasts in sugarcanes have been overcome by the choice of spindle tissue as source material. It is now possible to isolate, fuse, regenerate and multiply sugarcane protoplasts. However, the usefulness is limited due to the shortlived chromosomal stability during the callus stage. Nevertheless, preliminary results are promising and the method should prove feasible with refinement in technique. Two breeding systems for using parasexual hybridization are discussed.     

Relationships among ancestral species of sugarcane revealed with RFLP using single copy maize nuclear probes

Summary DNA restriction fragment length polymorphism (RFLP) analysis was performed on 50 wild and old cultivated sugarcane accessions. Ninety-four maize low copy nuclear DNA sequences of known chromosomal position were screened for hybridization to digested sugarcane genomic DNA blots. Seventy-five (80%) gave very strong hybridization signals and usually yielded many bands and detected profuse polymorphism. Twenty-nine probes and 36 probe/enzyme combinations were selected on the basis of the scorability of the banding profiles. A total of 1110 fragments were separately identified among the 50 genotypes. Multivariate analyses of the data allowed the separation of the three basic species, Saccharum spontaneum, S. robustum and S. officinarum, showed that S. spontaneum had structure which could be related to the geographic origin of the clones and supported current hypotheses on the origin of secondary species S. barberi and S. sinense. The use of more probes did not improve the resolution between the various species examined but identified a few key polymorphisms which were not accounted for by current phylogenetic hypotheses and can guide future analyses. RFLPs in sugarcane will be useful essentially for depicting the genomic constitution of modern varieties of interspecific origin.     

Stability and potential use of RAPD markers in a sugarcane genealogy

Summary A complete ancestral history of the recently developed and closely related South African commercial sugarcane varieties N11 and NCo376, which differ markedly in their response to sugarcane mosaic virus (SCMV), was elucidated from archival records. The genealogy spans seven generations, starting with early intraspecific crosses between varieties of Saccharum officinarum and interspecific crosses between S. officinarum and either S. spontaneum or S. barberi. In total, the genealogy comprises 38 different varieties. Genomic DNA samples from N11 and NCo376 respectively were screened for polymorphisms using the PCR-RAPD technique. Ten polymorphic fragments ranging in molecular size from 317 to 1263bp were identified from a total of 1159 loci amplified with 100 random decamer primers. Two of the 10 polymorphic fragments were shown to be consistently present in N11 (resistant) and absent in NCo376 (susceptible), while 8 showed the reverse occurrence. The primers producing the polymorphisms were used to screen genomic DNA samples from all 19 varieties representing the genealogy. Results have indicated that (1) specific PCR-RAPD generated polymorphic fragments can indeed be identified across the seven generations; (2) certain fragments are sufficiently definitive to be used as markers to trace parentage; (3) the validity of documented crosses and/or the authenticity of germplasm material may be questioned using this technique, and (4) there is the potential to subject the markers to linkage analysis once a full and accurate assessment of the SCMV resistance phenotype is obtained.     

Genetic diversity among wild sugarcane germplasm from Laos revealed with markers

A group of 35 wild Saccharum complex clones, collected in Laos(ECL), was studied for its nuclear genetic diversity by RFLP revealed by 10dispersed low copy probes. Representatives of the main germplasmdiversity, regularly used in introgression, were also included. A preliminarycharacterisation of the new clones was performed by Erianthus specificprimers. A set of the 11 ECL clones most outstanding for their diseaseresistance and vigor and basic germplasm members were also characterisedby 9 cytoplasmic probes. Nuclear diversity evidenced that ECL clonesrepresented an independent genetic pool consisting at least of 3 differentgroups. A group of bands was exclusively exhibited by ECL clones. Theoccurrence of genetic recombination among them was suggested by thepresence of the new bands in 2 or 3 clonal groups. Erianthus specificamplification was positive for 7 ECL clones from different nuclear groups.Dendrogram analysis based on cytoplasmic diversity divided genotypes into2 major cytoplasmic types comprising 5 groups. The ECL clones separatedin 2 distinct groups: one located in the same cytoplasmic type as Erianthus clones; the other one, in an intermediate position, closer to Saccharum species. Basic clones groups were consistent with theirtaxonomic classification. The diversity revealed by nuclear singlecopy/nuclear repetitive/cytoplasmic patterns of the ECL germplasmcontrasts with what is usually observed and points to an active populationevolution. Further studies needed to confirmed this hypothesis arediscussed.     

风蜡花‘Snow Flake’外植体消毒方法的研究

本文研究了风蜡花‘Snow Flake’组织培养快速繁殖中,外植体消毒的影响因素,分别从灭菌剂及灭菌时间,外植体取材时间、取材部位,以及抗菌素几个方面进行研究。结果表明：在10月到翌年3月取材,以顶芽为外植体,在75%的酒精中浸泡30s后用0.1% HgCl2处理8min,消毒效果较好。而在培养基中添加抗菌素,对‘Snow Flake’ 内生菌污染控制效果不理想。     

Plant regeneration from embryo-callus culture in barley

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

Modification of oilseed rape to produce oils for industrial use by means of applied tissue culture methodology

Summary A programme of research was designed to investigate methods for the modification of the fatty acid profiles of high performance lines of oilseed rape (Brassica napus L.) in an attempt to produce lines with enhanced levels of industrially useful fatty acids. The methodology employed to achieve these objectives was based on the exploitation of somaclonal or protoclonal variation, and targeted somatic hybridization using wild cruciferous germplasm as fusion partners.A range of somaclonal lines was produced from shoot regeneration protocols. These lines underwent replicated, randomised glasshouse trials for morphological assessment followed by gas chromatographic analysis to monitor any changes in fatty acid profile. It was found that a small number of lines exhibited potentially useful changes in oleic acid and polyunsaturated fatty acid content. Protoplast regeneration and electrofusion protocols for a range of winter oilseed rape lines were developed, and methods for the isolation and fusion of protoplasts of the wild crucifer Lunaria annua (chosen for its high nervonic acid content) established.Abbreviations GC Gas Chromatography     

Wheat somaclonal variants showing earliness, improved spot blotch resistance and higher yield

Somaclones (R₂, R₃ and R₄generations) were regenerated from immature embryos of two spring wheat varieties,HUW-206 and HUW-234. Many somaclones displayed improved earliness, enhanced resistance to spot blotch disease and increased yield over the parent. The superiority of variants for yield traits and disease resistance was established in R₄ generation, confirming the possibility of wheat improvement through somaclonal variation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tissue and cell culture as aids to sugarcane breeding. II. Performance and yield potential of callus derived lines

Summary Thirteen callus derivatives from the sugarcane (Saccharum sp. hybrid) varieties F156 and F164 were studied to investigate the potential of tissue culture methods to generate superior germplasm for plant breeding in a field trail conducted at a single location. Comparisons between callus derivatives and donors were made for 9 characters, viz. cane yield, sugar yield, stalk number, length, diameter, volume, density, weight/stalk, and percent fiber. Statistical analyses were run, which involved stepwise multiple regression, broad-sense heritability (H), genetic advance (G). Esterase zymogram of superior callus derivatives was examined. 70–6132 was 32, 34 and 6% higher than its donor (F164) in cane yield, sugar yield and stalk number, respectively. The differences between them for the first two characters reached the 5% probability level. 70–6132 was also higher for these 3 characters than F160, the No. 1 commercial variety, by 20, 16 and 8%, respectively. 70–6136 ranked next only to 70–6132. Its grand means for these 3 characters outdid the parental (F164) means by 21, 24 and 3%, respectively. 70–6069 was significantly higher in stalk diameter and volume than most of the callus derivatives but not higher than its donor (F156). The performances of other derivatives were either similar or inferior to both their donors and F160. Stalk number, stalk length and weight/stalk accounted for 43, 29 and 18% of the variation of cane yield, respectively. H values for the 7 characters (stalk density and percent fiber were not included) ranged from 31 (stalk length) to 66% (stalk volume). G/\-x 100 for the 7 characters ranged from 3.3 (stalk length) to 18.6% (sugar yield). 70–6132 had two bands missing, 70–6069 had two bands more whereas those of 70–6136 showed no visible change, in esterase zymogram as compared with the donors. Due to the improved yield and the genetic changes revealed by the isoenzyme patterns in callus derived lines, tissue culture methods can be used to create superior varieties for agricultural use.     

Analysis of Chinese Spring regenerants obtained from short- and long-term wheat somatic embryogenesis

Summary Somatic embryogenesis was initiated from immature embryos on Murashige-Skoog (MS) medium plus 2 mg.l^-1 2,4-dichlorophenoxyacetic acid, 2% sucrose and 0.6% agarose. Somatic embryos were isolated and regenerated into whole green plants on MS medium devoid of 2,4-D. These regenerants were previously demonstrated to differ in their mitochondrial DNA organization. In order to estimate their characteristics three progenies of short-term culture regenerants and three progenies of long-term culture regenerants were analyzed and compared to the parental line. These somaclones obtained from the wheat variety Chinese Spring were evaluated for variation of 13 agronomic and morphological quantitative characters in comparison to the parental line. Significant variation was observed for plant height, spike length, main tiller diameter, between the somaclones regenerated from long-term culture and their parent. Differences were observed to increase with the duration of culture, leading to a significant modification of the structure of the plants. Several changes occurred during the somatic tissue cultures, but to a lesser extent than has previously been described in the literature.     

Is somaclonal variation a reliable tool for spring wheat improvement?

Summary Seed progeny of tissue culture regenerants of a spring wheat (Triticum aestivum L. cv. HY320) was evaluated for key agronomic traits for three years under field conditions. Initially, 27 regenerant families were tested in hill plots. Among-family and within-family variation was generally highly significant (p < 0.01) and nonsignificant, respectively. The variation observed among regenerants on the basis of hill plot testing was not duplicated in subsequent four-row plot experiments. On average, regenerant families yielded 28 and 5% less than the control in dryland and irrigated tests, respectively. Low yielding regenerants tended to produce fewer, lighter kernels per spike. Higher grain protein levels among regenerants were associated with low yields (r=0.85). This study demonstrated that putative somaclonal variation arising from tissue culture failed to produce genotypes agronomically superior to the parental cultivar, HY 320.NRCC Publication No. 33533     

Analysis of tissue culture-derived variation in pea (Pisum sativum L.)-preliminary results

Summary The possibility of producing agronomically-useful somaclones via organogenesis and somatic embryogenesis from callus cultures of pea (Pisum sativum L.) was studied. Organogenic calli were induced from immature leaflets on MSB medium with NAA and BAP. Embryogenic calli were derived either from immature zygotic embryos (using 2,4-D) or from shoot apices (using picloram) of aseptically-germinated seedlings.The seed progenies (T₁ to T₃-generation) of primary regenerants were grown in field conditions and their phenotypic variation was evaluated and compared with control, non-tissue culture-derived plant material. In addition, electrophoretic analyses of selected isoenzyme systems and total proteins have been done. The results do not show dramatic changes in qualitative and quantitative traits. The evaluation of at least two future generations (T₄, T₅) is planned.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - MSB medium (mineral salts after Murashige & Skoog, 1962, vitamins after Gamborg et al., 1968) - NAA -naphthalene-acetic acid, picloram-4-amino-3,5,6-trichloro picolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - ORG organogenesis - SE somatic embryogenesis     

Inheritance of male sterility mutations induced in haploid sorghum tissue culture

Summary A high frequency of male sterile mutants regeneration was shown in callus cultures derived from leaves and panicles of haploid sorghum (Ms_c1, A1 cytoplasm) and a spontaneous autodiploid obtained from this haploid. The cultures derived from the embryos of this autodiploid yielded significantly fewer mutants. Absolutely or partially male sterile mutants appeared among the regenerants or in the progeny of fertile regenerants. In the self-fertilized progenies of partially male sterile mutants and in the hybrids of sterile mutants with autodiploid line (i.e. under one and the same nuclear genome) male sterility mutations were inherited as cytoplasmic. Non-Mendelian segregation of sterile, partially male sterile and fertile plants was observed in these progenies. Partially male sterile plants were characterized by somatic segregation of male sterility genetic factors. In test-crosses with some CMS A1 fertility restorers, mutations were manifested as nuclear recessive while with others as nuclear dominant. These differences are supposed to be the result of interaction of fertility restorer genes of these testers with the novel cytoplasm. Male sterility mutations accompanied with female sterility were inherited as nuclear recessives.Abbreviations f fertile - ps partially male sterile - s male sterile plants