Field performance of artificial seed-derived sugarcane plants

This study shows the behaviour of sugarcane plants cv. CP-5243 derived from artificial seed compared with traditional and isolated bud methods. Artificial seed-acclimatised plants were planted in field conditions simultaneously with two-control treatments previously germinated: macropropagated plants derived from stems of three buds and axillary buds isolated from field-grown plants. Plants from artificial seed were taller and had a smaller diameter at 8 months, but these differences disappeared at 12 months. With respect to sugar analysis and yield, no differences in all parameters evaluated were found between artificial seed-derived plants and plants derived from the two other methods.     

Association and heritability of sugarcane smut resistance to races A and B in Hawaii

Summary This study was conducted to estimate the degree of association of smut (Ustilago scitaminea Syd.) resistance in sugarcane (Saccharum spp.) between races A and B in Hawaii and to estimate the heritability of resistance to both races. The estimated degree of association was 0.18. Although statistically significant, the degree of association of resistance between races A and B was near zero and therefore of no practical importance. Selection for resistance to one race would have little effect on the frequency of resistance to the other race in the following sexual generation. Heritabilities in the broad sense estimated from the parent population on a plot mean basis were 0.96 and 0.91 for races A and B, respectively. Selection for smut resistance should be very effective between populations of asexual generations. Heritabilities in the narrow sense estimated from parent-progeny regression analysis on a family mean basis were 0.51 and 0.47 for races A and B, respectively. Selecting and breeding for resistance should result in a fairly rapid increase in the frequency of resistance in the progeny population.Contribution from Department of Genetics and Pathology, Hawaiian Sugar Planters' Assn. Exp. Stn., P.O. Box 1057, Aiea, HI 96701. Published with the approval of the Director as paper no. 652 in the Journal Series of the Exp. Stn., Hawaiian Sugar Planters' Assn. Research funded in part by USDA/ARS Cooperative Agreement No. 58-9AHZ-0-492     

Biochemical characterization of embryogenic and non-embryogenic calluses of sugarcane

Summary With the aim to determine a possible relationship between somatic embryogenesis and some metabolic contents in embryogenic and non-embryogenic calluses of sugarcane (Saccharum sp. var CP-5243), the present study was carried out. Embryogenic callus has more soluble proteins, free proline, proteolytic activity, soluble sugars, and invertase, and lower putreseine/(spermidine + spermine) than non-embryogenic tissue. Non-embryogenic callus has a higher peroxidase and gallic acid level, lower dry matter/fresh matter ratio, and more gross fat compared with embryogenic callus.     

Evaluation of cultivar-testing locations in sugarcane

Summary Selection of test locations, representative of conditions and practices of an area can be a challenging process in a breeding program. Data from two groups of sugarcane (trispecies hybrids of Saccharum sp.) cultivar experiments in Florida were analyzed to determine if relative cultivar response at any two of six current locations was sufficiently similar so that at least one location could be replaced by a location with a different environment. The parameter analyzed was metric tons per ha of sugar (THS). To determine similarity between location pairs for all cultivars within each group of cultivars, an unbiased stability-variance parameter ( ) developed by Shukla was used. After identified similar location pairs, single degree of freedom interactions were calculated for important cultivars to determine which of the location pairs identified by contained the two most similar locations. Use of the above procedure can assist in making optimum location assignments in a breeding program.Cooperative investigation of the U.S. Sugarcane Field Stn., Canal Point, FL; and University of Florida, Everglades. Res. and Educ. Ctr., Belle Glade, FL, USA     

In vitro evaluation of sugarcane resistance to gumming disease and of Xanthomonas campestris pv. vasculorum aggressiveness

Sugarcane plantlets were sectioned halfway between the base and the youngest ligule and then inoculated by soaking the wound in a suspension of Xanthomonas campestris pv. vasculorum. The infection caused rapid necrosis of the inoculated leaves, chlorosis of uninoculated leaves, or death of the inoculated plantlet. New tillers sometimes showed chlorosis or white streaks. The effects of the inoculum concentration, the cultivar, and the bacterial strain on symptom severity were determined. The ranking of cultivars depended on the inoculum concentration, and strains were found to differ with regard to aggressiveness. However, cultivars and strains were more effectively classified in greenhouse trials. The poor expression of leaf resistance appeared to limit the use of the in vitro test.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

Leaf tissue from five sugarcane clones with distinctive markers was cultured on a medium favoring callus growth. Transferred to a differentiation medium, calli produced over 5000 plants. Plants differentiated from two clones with stem markers exhibited a high rate of remission of the marker, but the marker reappeared in the vegetative progeny of these plants, and remission was, therefore, transient. Plants differentiated from callus from two clones with leaf markers showed a low rate of remission (2 or 3 per thousand) of the marker and the vegetative progeny was stable. A clone with variegated leaves produced plants with the majority having green leaves, some were albino, and some variegated, suggesting that plant differentiation may start with more than one cell. Permanent phenotypic change may result from tissue culture, but the results suggest that such changes are not frequent and may be confounded by temporary alterations or by chimeras formed in the process of differentiation.     

Biochemical genetic markers in sugarcane

Summary Isozyme variation was used to identify biochemical markers of potential utility in sugarcane genetics and breeding. Electrophoretic polymorphism was surveyed for nine enzymes among 39 wild and noble sugarcane clones, belonging to the species most closely related to modern varieties. Up to 114 distinct bands showing presence versus absence type of variation were revealed and used for qualitative characterization of the materials. Multivariate analysis of the data isolated the Erianthus clone sampled and separated the Saccharum spontaneum clones from the S. robustum and S. officinarum clones; the latter two were not differentiated from one another. The analysis of self-progenies of a 2n=112 S. spontaneum and of a commercial variety showed examples of mono- and polyfactorial segregations. Within the progeny of the variety, co-segregation of two isozymes frequent in S. spontaneum led to them being assigned to a single chromosome initially contributed by a S. spontaneum donor. This illustrates how combined survey of ancestral species and segregation analysis in modern breeding materials should permit using the lack of interspecific cross-over to establish linkage groups in a sugarcane genome.     

Field performance of temporary immersion bioreactor-derived sugarcane plants

Summary The temporary immersion bioreactor has been found to be an important tool for sugarcane micropropagation, allowing higher shoot formation rates and cost reduction. This research was conducted to demonstrate the agricultural value of temporary immersion bioreactor-derived sugarcane plants. The experiment was carried out for about 2 yr to study the field performance of these plants. Two control treatments were also evaluated representing the conventional forms of micro- and macropropagation. Growth of sugarcane stools, first ratoon and the use of micropropagated plants for macropropagation were recorded. Some botanical and chemical characteristics were evaluated. Differences among propagation systems were only found in the first 6 mo. of field growth, regarding the stem length and diameter. Such differences disappeared with the course of the experiment.     

Effect of various growth regulators on shoot regeneration of sugarcane

Summary Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.     

The frequency of marker changes in sugarcane plants regenerated from callus culture

This study investigates the frequency of apparent and permanent expression of marker change following two types of tissue culture, conventional callus and direct regeneration cultures, and for two markers it relates this frequency to that following breeding. Each clone was used for only one marker. After conventional callus culture, plants of the sugarcane clone Arundoid B, a clone having a growth habit with shortened internodes and leaves, were freed of this marker at a rate of 1 in 172 plants. Marker remission in a second clone with a leaf blotch was enhanced in the presence of a mutagen. Callus culture alone gave a remission rate of 1/280 plants, while treatment of callus with ethyl methanesulfonate gave a remission rate of 1/42 plants. Of two markers subjected to vegetative and sexual transmission, the first, a leaf marker, was stable in callus culture with no remissions; crossing with non-marker parents produced progeny with 54% lacking the marker. The second, a stalk marker (multibud), showed epigenetic effects during two generations of vegetative propagation; plants lacking the multibud marker produced vegetative progeny in which the marker reappeared. Nine crosses to nonmarker parents produced progeny of which an average of 29% had the marker. The use of stalk chimeras as markers demonstrated that passage through conventional callus or direct regeneration culture resulted in the loss of the donor phenotype in all plants regenerated. Phenotypic variation in plants derived from callus culture appears to arise from several sources; chimeral segregants, epigenetic transients, and mutational variants.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

Comparative genetics in sugarcane enables structured map enhancement and validation of marker-trait associations

As sugarcane is a complex polyaneuploid with many chromosomes, large numbers of markers are required to generate genetic maps with reasonable levels of genome coverage. Comparative mapping was investigated as an approach for both quantitative trait loci (QTL) validation and genetic map enhancement in sugarcane. More than 1000 SSR and AFLP markers were scored in a bi-parental Australian sugarcane population (Q3) that was segregating widely for sugar content-related traits. Two maps were constructed, one for each parent. The Q117 (female) and MQ77-340 (male) maps each contained almost 400 markers distributed onto approximately 100 linkage groups (LGs), of which nearly half could be assigned to homology groups (HGs) on the basis of SSRs. Then, using common SSR and AFLP markers, the two Q3 parental maps were aligned with the maps of the French cultivar, R570, and of the Australian cultivar, Q165^A (A denotes variety covered by Australian plant breeding rights). As a result of comparative mapping, all ten HGs in the Q117 map, and all eleven HGs in the MQ77-340 map could be re-assigned to seven of the expected eight sugarcane HGs, revealing that one sugarcane HG was not covered at all in either Q3 parental map, and that other HGs were poorly represented. QTL analysis in the Q3 population identified approximately 75 marker-trait associations (MTAs) from approximately 18 chromosomal regions or putative QTL in each map for three sugar content-related traits. QTL location appeared to be consistent between the 4 maps; two of the eight HGs were observed to contain MTAs for brix in two or three maps, strongly suggesting the location of sugar content-related trait loci in these HGs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-term culture of embryogenic sugarcane callus

Embryogenic calluses of sugarcane capable of regenerating green plants after long-term culture were sought. The largest quantities of embryogenic calluses were produced on Murashige & Skoog medium, but cultures maintained on Chu N6 medium remained embryogenic and totipotent longer. Both media contained 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d). The effect of supplements on somatic embryogenesis was examined. Kinetin (0.5 M) and 10% (v/v) coconut water in callus initiation medium were inhibitory to subsequent embryogenesis. Embryogenic calluses on N6 medium increased in fresh weight with proline concentration up to 90 mM. Maximum fresh weight was achieved with 5% sucrose. Although genotypic differences were observed, embryogenesis occurred in all 17 sugarcane clones tested. Embryogenic calluses of one cultivar regenerated green plants after 16 months, but suspensions were totipotent for only 8 months. Total number of regenerated plants decreased with time in culture, while the number of pale green plants increased starting after 5 months in culture.Published as Paper No. 785 in the journal series of the Experiment Station, HSPA     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

Estimation of N2-fixation by sugarcane,Saccharum sp., and soybean,Glycine max,grown in soil with15N-labelled organic matter

Summary Two varieties of sugarcane, and nodulated and non-nodulated soybean isolines, were planted in a soil previously mixed with¹⁵N-labelled plant material. 45 days was allowed to elapse before planting, to permit initiation of organic matter mineralization. Plants were grown for 60 days, then harvested, dried, weighed and analysed for total N. Analysis of soil samples pre-incubated in the laboratory was carried out to evaluate ammonium and nitrate from added organic matter. Dry weights of the soybean isolines were similar, but total N was higher for the nodulated line. Both sugarcane varieties showed similar weight and total N. Nitrogen derived from applied organic matter (NdfOM) was higher in non-nodulated soybean than in all other plants. Although there is the possibility of different¹⁵N availabilities between species, nitrogen derived from fixation (Nfix) was calculated based on the¹⁵N enrichment of the non-nodulating soybean. Nfix was 72% for nodulating soybean and ranged from 19 to 39% for different parts of sugarcane plants, despite high levels of available-N. Nitrogen derived from soil was calculated by difference. NdfOM was lower in roots than in upper parts (leaves+stalks) of plants. Use of¹⁵N labelled organic matter seems a useful approach to the longer term measurement of N₂-fixation.IAEA Project BRA/5/009-CENA.     

Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration

Summary A cryopreservation process using encapsulation/dehydration was set up for apices sampled on in vitro plantlets of sugarcane. After dissection, apices were cultured for one day on standard medium and then encapsulated in medium with 3% alginate. Optimal conditions comprised preculture for 2 days in liquid medium with 250 g.l^–1 sucrose, desiccation for 6 hours under the laminar flow or for 10–11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid nitrogen ranged between 38 and 91% for the 5 varieties experimented. Cryopreservation did not modify the electrophoretic profiles for aminoleucine peptidases and amylases with plants of the variety Co 6415.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - EDTA ethylenediamine tetracetic acid - AMP aminoleucine peptidases - AMY amylases - RFLP restriction fragment length polymorphism     

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K_m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.