Micropropagation of sugarcane (Saccharum spp.)

A method for callus induction, adventitious bud regeneration, shoot multiplication and rooting of in vitro formed shoots of Helianthus annuus L. var. Argentario is described. Hypocotyl and cotyledon explants formed callus on medium containing 2 mgl^–1 naphthalene acetic acid and 0.5 mgl^–1 benzyladenine. Adventitious buds were formed on hypocotyl segments on medium containing 0.5–2 mgl^–1 benzyladenine. The optimal level of sucrose concentration for shoot regeneration from hypocotyls was 1.5%. Multiplication from shoot apices was promoted by kinetin (2 mgl^–1) plus gibberellic acid (5 mgl^–1), benzyladenine (2 mgl^–1) plus gibberellic acid (10 mgl^–1) or at lower frequency by benzyladenine (1 mgl^–1). A general feature of the plantlets formed in vitro was the precocious flowering.     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

甘蔗抗旱性生理生化鉴定指标

利用因子分析和灰色关联度分析方法研究了甘蔗叶片相对含水量、膜脂过氧化代谢、活性氧代谢、光合参数及蔗茎产量性状等指标与抗旱性的关系.结果表明,干旱胁迫下甘蔗叶片的MDA含量和PMP明显提高,而RWC、SOD活性、Chl含量、F_v/F_m、F_v/F_o、△F_v/F_t、△F_v/F_o和蔗茎单茎重(SSW)8个抗旱性指标均显著降低.SSW与其它9个生理生化指标的相关性大小依次为PMP＞SOD活性＞MDA含量＞RWC＞F_v/F_o＞F_v/F_m＞Chl含量＞F_v/F_o＞F_v/F_t,其中,SSW与F_v/F_o和ΔF_v/F_t相关性不显著.通过因子分析将10个甘蔗抗旱性指标用4个公共因子表示,累加方差贡献率达到92.08%.因子l主要是反映光合作用特性指标对甘蔗品种抗旱性起支配作用,因子2主要是反映叶片相对含水量及活性氧代谢指标对甘蔗品种抗旱性起支配作用,因子3和因子4分别只有SSW和Chl含量有较大载荷.灰色关联度分析表明,各抗旱性生理生化指标与SSW关联密切程度依次为F_v/F_m＞PMP＞F_v/F_o＞RWC＞MDA含量＞SOD活性＞ΔF_v/F_t＞Chl含量＞F_v/F_o.     

Structure and expression of a sugarcane gene encoding a housekeeping phosphoenolpyruvate carboxylase

A gene (SCPEPCD1) encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from the C-4 monocot sugarcane (Saccharum hybrid var. H32-8560). SCPEPCD1 is ca. 6800 bp long, with 10 exons. The entire gene sequence from –1561 to 262 bp downstream of the putative poly(A) addition signal is reported. A low-level, essentially constitutive pattern of expression, amino acid sequence similarities to other housekeeping PEPC enzymes, and the absence of DNA sequence elements conserved in the upstream region of maize and sorghum C-4-specific PEPC genes indicate that SCPEPCD1 encodes a housekeeping PEPC. Despite this, a motif proposed to act as a phosphorylation site in light-mediated activation of photosynthetic PEPC enzymes [10] is present in the SCPEPCD1 protein; evidence is presented for the presence of this site in other housekeeping PEPC proteins.     

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.     

An in vitro screening system to assess aluminum toxicity in sugarcane (Saccharum spp.) cultivars

In Vitro Cellular & Developmental Biology - Plant - Mutagenic breeding is an approach being developed especially for those characteristics that are not inherited through conventional genetic...     

Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars (Saccharum spp.)

Summary The genomic stability of tissue culture regenerants of sugarcane (Saccharum spp. hybrids, cvs CP721210, CP68-1067 and B43-62) was analyzed by DNA restriction fragment length polymorphism (RFLP). Plants regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts were used. Total DNA isolated from 19 different sources was digested with EcoRI, HindIII, BamHI, BamHI, EcoRI and PstI and probed with six known maize mitochondrial genes (coxI, coxII, atpA, atp6, atp9 and rrn18-rrn5), three random maize mitochondrial cosmid clones, two random maize chloroplast cosmid clones and a wheat Nor locus clone. Hybridization patterns indicated that the variation observed was minor and appeared only in the secondcycle regenerants. No differences were observed among the three cultivars and the regenerants from calli, suspension culture, cryopreserved suspension culture and protoplasts. Mitochondrial DNA (mtDNA) isolated from CP72-1210 plants and its embryogenic cell suspensions, and bulk samples from all CP72-1210 regenerants pooled together were digested with EcoRI, HindIII, PstI, BamHI and SalI and probed with three recombinationally active wheat mtDNA clones, K, K3 and X2. No variation in the mtDNA restriction patterns was observed between the CP72-1210 plants and its regenerants. However, restriction pattern variation was observed only from EcoRI digestion, and hybridization patterns of K3, K and X2 revealed minor variations in the mtDNA of cell suspensions when compared with the DNA of the CP72-1210 plant. Except for a qualitative variation detected by the X2 probe and minor stoichiometric variations detected by the K3 probe, sugarcane DNAs were found to be stable after plant regeneration.Florida Agriculture Experiment Station Journal Series No. R-02703     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens

This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants/number of cell clusters) were between 9.4 × 10–3 and 1.15 × 10–2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for sugarcane transformation. We found that three main factors: (1) the use of young regenerable calluses as target explants; (2) induction and/or improvement of the A. tumefaciens virulence system with sugarcane cell cultures and (3) pre-induction of organogenesis or somatic-embryogenesis-like sexual embryos, seem to be crucial in order to increase the cells competence for T-DNA transfer process. Patterns generated by Southern hybridization confirmed that T-DNAs were randomly integrated into sugarcane genome without th e persistence of A. tumefaciens in the transgenic plants     

Use of bioreactors for large-scale multiplication of sugarcane (Saccharum spp.), energy cane (Saccharum spp.), and related species

The objective of this study was to set up a plant micropropagation facility to mass propagate sugarcane, energy cane, and related clonally propagated species. An efficient methodology for micropropagation of energy cane and perennial grasses using temporary immersion bioreactors was developed. Several different methods of tissue culture initiation, multiplication, and rooting were evaluated for several varieties of sugarcane (Saccharum officinarum L.) and sugarcane-related species such as Erianthus spp., Miscanthus spp., and Sorghum spp. × sugarcane hybrids, all from a germplasm collection. Apical meristem cultures were initiated for all genotypes that were micropropagated, when liquid or semisolid Murashige and Skoog (MS) medium was used, which was supplemented with 0.1–0.2 mg L⁻¹ BAP, 0.1 mg L⁻¹ kinetin, 0–0.1 mg L⁻¹ NAA, and 0–0.2 μg L⁻¹ giberellic acid. These cultures produced shoots between 4 and 8 wk after initiation. Shoot regeneration from leaf rolls or immature inflorescences was observed as early as 4 wk after initiation. Shoot multiplication was successful for all genotypes cultured in MS medium with 0.2 mg L⁻¹ BAP and 0.1 mg L⁻¹ kinetin. Energy cane had a significantly higher combined multiplication rate when grown under four or five LED lamps than when grown under three LED lamps, or under fluorescent lights in a growth chamber. The addition of 2 mg L⁻¹ NAA produced faster and better rooting in all of the genotypes tested. Shoots produced well-developed roots after one cycle of 15–21 d in the bioreactors. The maximum number of plantlets produced per bioreactor was 1080. Plantlets developed a vigorous root system and were ready to be transplanted into the field after 2 mo. A protocol was standardized for different energy cane clones that were recommended for their biomass production and cell wall composition. Different tissues were used to speed up or facilitate tissue culture initiation. Visual assessment of micropropagated plants in the field did not show any off-types, based on gross morphological changes of plant morphology or disease reaction, compared to plants of the same genotype derived from a traditional propagation method (stem cuttings). This is the first report of energy cane and Miscanthus spp. micropropagation using the SETIS bioreactor.

     

Meiotic abnormalities in sugarcane (Saccharum spp.) and parental species: Evidence for peri- and paracentric inversions

The modern cultivars of sugarcane (Saccharum spp.) are highly polyploid and accumulate aneuploidies due to their history of domestication, genetic improvement and interspecific hybrid origin involving the domesticated sweet species Saccharum officinarum (‘noble cane’) and the wild Saccharum spontaneum, both with an evolutionary history of polyploidy. The first hybrids were backcrossed with S. officinarum, and selection from progenies in subsequent generations established the genetic basis of modern cultivars. Saccharum genome complexity has inspired several molecular studies that have elucidated aspects of sugarcane genome constitution, architecture and cytogenetics. Herein, we conducted a comparative analysis of the meiotic behaviour of representatives of the parentals S. officinarum and S. spontaneum, and the commercial variety, SP80-3280. S. officinarum, an octoploid species, exhibited regular meiotic behaviour. In contrast, S. spontaneum and SP80-3280 exhibited several abnormalities from metaphase I to the end of division. We reported and typified, for the first time, the occurrence of peri- and paracentric inversions. Using in-situ hybridisation techniques, we were able to determine how pairing association occurred at diakinesis, the origin of lagging chromosomes and, in particular, the mitotic chromosome composition of SP80-3280. Interestingly, S. spontaneum and recombinant chromosomes showed the most marked tendency to produce laggards in both divisions. Future attempts to advance knowledge on sugarcane genetics and genomics should take meiotic chromosome behaviour information into account.     

5-Azacytidine as a tool to induce somaclonal variants with useful traits in sugarcane (Saccharum spp.)

A possible strategy to produce variant sugarcane plants with beneficial traits was tested by promoting somaclonal variation in vitro through the action of the hypomethylation and mutagenic agent 5-Azacytidine (Azac). Treatment of calli in liquid medium caused high levels of necrosis. Consequently, 6- to 8-week-old calli of cultivar NCo376 were exposed to 50 and 100 μM Azac in semi-solid callus induction medium (CIM) (MS salts and vitamins, sucrose, casein hydrolysate, agar, with or without 3 mg l^?1 2,4-D) for 1 week. They were then transferred to fresh CIM with 2,4-D and to CIM without 2,4-D, for 2 and 8–10 weeks, respectively. The highest callus necrosis (>60 %) and reduced recovery (<40 %) were recorded for calli treated with 100 μM Azac without 2,4-D, which also resulted in lower plant yield (12 plantlets/0.2 g calli) than the control (18 plantlets/0.2 g calli). From methylation-sensitive amplified fragment length polymorphism analyses, the highest polymorphisms (4.2 %) were also obtained from plants derived from the 100 μM Azac treatment without 2,4-D. After 9 months of field growth, Azac-derived plants exhibited phenotypic differences compared with the controls. Ex vitro screening resulted in the identification of one plant from the 100 μM Azac with 2,4-D treatment putatively tolerant to smut, and three plants from the 100 μM Azac with 2,4-D and one from the 50 μM Azac with 2,4-D treatments, potentially tolerant to the herbicide imazapyr.     

Inorganic nitrogen uptake kinetics of sugarcane (Saccharum spp.) varieties under in vitro conditions with varying N supply

An in vitro system was established for the characterisation of inorganic nitrogen uptake by sugarcane plantlets of variety NCo376. After multiplication and rooting, plantlets (0.27–0.3 g fresh mass) were placed on N-free medium for 4 days, and then supplied with 2–20 mM N as NO₃ ^?-N only, NH₄ ⁺-N only or NO₃ ^?-N + NH₄ ⁺-N (as 1:1). With few exceptions, on all the tested N media, the in vitro plants always had a higher V_max for NH₄ ⁺-N (28.69–66.51 μmol g^?1 h^?1) than for NO₃ ^?-N uptake (10.24–30.19 μmol g^?1 h^?1) and the K_m indicated a higher affinity for NO₃ ^?-N (0.02–7.38 mM) than for NH₄ ⁺-N (0.06–9.15 mM). When N was applied as 4 and 20 mM to varieties N12, N19 and N36, the interaction between variety, N form and concentration resulted in differences in the V_max and K_m. The high N-use efficient varieties (N12 and N19), as determined in previous pot and field trials, behaved similarly under all tested conditions and displayed a lower V_max and K_m than the low N-use efficient ones (NCo376 and N36). Based on this finding, it was suggested that the N-use efficient designation (from pot and field trials) may not be ascribed solely to N uptake. Assessment of the relative preference index (RPI) for NO₃ ^?-N and NH₄ ⁺-N uptake revealed that, at present, the RPI has no application in sugarcane due to its preferential uptake of NH₄ ⁺-N.     

Sorghum phosphoenolpyruvate carboxylase gene family: structure,function and molecular evolution

Although housekeeping functions have been shown for the phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in plants and in prokaryotes, PEPC is mainly known for its specific role in the primary photosynthetic CO₂ fixation in C4 and CAM plants. We have shown that in Sorghum, a monocotyledonous C4 plant, the enzyme is encoded in the nucleus by a small multigene family. Here we report the entire nucleotide sequence (7.5 kb) of the third member (CP21) that completes the structure of the Sorghum PEPC gene family. Nucleotide composition, CpG islands and GC content of the three Sorghum PEPC genes are analysed with respect to their possible implications in the regulation of expression. A study of structure/function and phylogenetic relationships based on the compilation of all PEPC sequences known so far is presented. Data demonstrate that (1) the different forms of plant PEPC have very similar primary structures, functional and regulatory properties, (2) neither apparent amino acid sequences nor phylogenetic relationships are specific for the C4 and CAM PEPCs and (3) expression of the different genes coding for the Sorghum PEPC isoenzymes is differently regulated (i.e. by light, nitrogen source) in a spatial and temporal manner. These results suggest that the main distinguishing feature between plant PEPCs is to be found at the level of genes expression rather than in their primary structure.     

Efficacy of pyrethroid and neonicotinoid insecticides for Melanotus communis (Gyll.) (Coleoptera: Elateridae) control in Florida sugarcane (Saccharum spp.)

Wireworms are important pests in newly planted sugarcane fields in Florida. Current management involves an integrated programme of cultural and chemical controls. Current chemical controls used are the organophosphates phorate and ethoprop. As federal regulations tighten the uses of organophosphates, effective chemicals from alternative classes of chemistry need to be found. The organophosphates were the only insecticides that effectively protected the emerging shoots from damage in our study. Phorate consistently reduced the number of wireworms. While the pyrethroids and neonicotinoids were able to protect the seed piece and non‐sprouted buds, they did not consistently protect shoots and tillers. Neither pyrethroids nor neonicotinoids caused wireworm mortality in excess of that which occurred naturally.     

Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics

Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the speciesS. officinarum andS. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n+n transmission of the parental chromosomes instead of the peculiar 2n+n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n=107–115) about 10% were identified as originating fromS. spontaneum and about 10% were identified as recombinant chromosomes between the two speciesS. officinarum andS. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.     

Herbicide-resistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium-mediated transformation

The presence of undesirable plants in sugarcane (Saccharum officinarum L.) plantations reduces crop yields. Using genetic engineering as a complement for traditional breeding methods it is possible to introduce herbicide-resistant traits into Saccharum germplasm. Transgenic sugarcane plants resistant to phosphinothricine (PPT), the active compound of the commercial herbicide BASTA were generated by Agrobacterium tumefaciens-mediated transformation. Meristematic sections of sugarcane were treated with anti-necrotic compounds to minimize oxidative bursts and used as explants. Four transformation protocols were assessed and the transformation frequencies reached 10–35%. The regeneration rate was high and did not appear to be affected by the transformation procedure. Southern blot analysis of several transformed plants indicated the integration per genome of one or two intact copies of the bar gene which encodes PPT acetyltransferase and confers resistance to BASTA. The levels of BASTA resistance were evaluated under greenhouse and small-plot conditions. Received: 8 November 1997 / Accepted: 22 November 1997     

A vigorous mutant sugarcane (Saccharum sp.) clone Co 527

Summary A vigorous fast growing mutant which ends vegetative growth sixty days earlier than the parent variety Co 527 was isolated from gamma irradiated vegetative buds. The mutant initially segregated but stabilized in the vM₄ generation. Its growth rate was almost fifty per cent higher than Co 527 beginning in the early stages of growth. It produced a significantly higher early shoot population which enabled it to yield a higher number of millable canes at maturity. Economic characters like sucrose content and juice purity remained unaffected. This mutant had a chromosomal basis in that the number was 2 to 3 lower than in the parent variety.Research carried out under the FAO/IAEA Co-ordinated Research Programme on the Improvement of Vegetatively Propagated Plants and Tree Crops through Induced mutations (Research Contract No. 1680)