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.     

Impact of excess zinc on growth parameters, cell division, nutrient accumulation, photosynthetic pigments and oxidative stress of sugarcane (Saccharum spp.)

The present study employed a sand culture experiment with three levels of zinc viz., 0.065 (control), 65.0 and 130 mg l^?1 Zn (excess) as zinc sulfate, respectively, in sugarcane (Saccharum spp.), cultivar CoLk 8102. The results indicated growth depression, dark green leaves, decreased root number and length and sharp depression in mitotic activity of roots due to high doses of Zn (65 and 130 mg l^?1); effects were significant at 130 mg l^?1 Zn supply. The endogenous ion contents measurements revealed roots to be the major sink for excess Zn with lower amounts in leaves of sugarcane plants. High level of Zn decreased total phosphorus in leaves and increased it in roots. Fe and Cu content decreased, while, Mn increased in sugarcane plants due to high Zn in the growing medium. Plants experienced oxidative stress when exposed to higher levels of zinc. Biochemical investigations indicated high level of hydrogen peroxide, malondialdehyde contents with high chlorophyll a, b and carotenoids contents and activity of superoxide dismutase, catalase and peroxidase enzymes under high Zn conditions. These findings confirm suggest that excess Zn adversely affects root growth and mitotic efficiency, enhances chromosomal aberrations and increases growth and nutrient accumulation abnormalities, as well as oxidative stress.     

Gas exchange,chlorophyll fluorescence,and osmotic adjustment in two mango cultivars under drought stress

The responses of photosynthetic gas exchange, chlorophyll fluorescence, content of pigments, main osmolytes, and malondialdehyde (MDA) to water-withholding for 15 days and re-hydration in seedlings of two mango cultivars (Mangifera indica L. var. “Choke Anand’ and var. “Khieo Sawoei”) under 50% sunlight and full sunlight were investigated. For both cultivars, the water-witholding resulted in progressively decreases in leaf relative water content, net photosynthesis (P _n), stomatal conductance (g _s), and increases in the conversion of xanthophyll cycle pigments estimated by an index of leaf spectral reflectance (ΔPRI), carotenoid to chlorophyll ratio, non-photochemical quenching (NPQ), the contents of malondialdehyde (MDA) and compatible solutes (total soluble sugar and proline). The effect of the water stress was more pronounced in full sunlight than 50% sunlight. The maximum photochemistry efficiency measured at dawn was fairly constant during the period of the treatment for both cultivars under both light regimes. The water stress caused less pronounced inhibition of photosynthesis in “Choke Anand” than in “Khieo Sawoei” cultivar under both light regimes. After re-hydration, the recovery was relatively quicker in “Choke Anand” than in “Khieo Sawoei” cultivar. Both cultivars in both 50% and full sunlight showed complete recovery in photochemistry after 5 days of re-watering but photosynthesis did not show a complete recovery as indicated by gas exchange rates. As the results of lower NPQ, ΔPRI and osmotic adjustment in the cultivar “Khieo Sawoei” compared to the cultivar “Choke Anand”, the former cultivar was less tolerant to drought than the latter. Our study further showed that partial shading (e.g., 50% of sunlight) significantly alleviated the harmful effect of drought stress on mango cultivars but in fact stomata of seedlings grown in partial shade was more responsive to water deficit than in full light.     

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.     

Production of transgenic sugarcane (Saccharum officinarum L.) plants by intact cell electroporation

Summary We describe an efficient procedure for genetic transformation of commercial sugarcane varieties POJ 2878 and Ja 60-5. The transformation protocol is based on electroporation of a plasmid conferring GUS activity into cell clusters isolated from embryogenic calli. Six to eight weeks after electroporation, Ja 60-5 plants regenerated from electroporated tissues were tested and confirmed to be transgenic using histochemical glucuronidase and Southern hybridization analysis. Electroporation of intact cells is an efficient and reproducible method for sugarcane transformation and may also be useful for transformation of other plants.Abhrevations GUS -glucuronidase - CAT chloramphenicol acetyl transferase - PCV packed cell volume - PCR polymerase chain reaction - DTT dithiotreitol - Hepes N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NOS nopaline synthase - 4-MUG 4-methylumbelliferyl -D-glucuronide     

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.     

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...     

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.     

Effects of salt stress in relation to osmotic adjustment on sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) callus cultures

In vitro responses of embryogenic sugarcane (Saccharum officinarum L.; cv. CoC-671) calli stressed with different levels of NaCl (0.0, 42.8, 85.6, 128.3, 171.1, 213.9 or 256.7 mM) were studied. The results showed that a significant decrease in callus growth and cell viability occurred with ≥85.6 mM NaCl. Higher amounts of free proline and glycine betaine were accumulated in NaCl-stressed calli. Although the leached and retained Na⁺ contents increased, the retained K⁺ content decreased with increasing levels of NaCl. Such a mechanism implies that sugarcane can be considered as a Na⁺-excluder. The accumulation of salt ions and osmolytes could play an important role in osmotic adjustment in sugarcane cells under salt stress.     

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     

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.     

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.

     

Photosynthesis, antioxidant activities and transcriptional responses in two sugarcane (Saccharum officinarum L.) cultivars under salt stress

This study analyzes changes in gene expression and the biochemical and physiological properties of the antioxidant system in the leaves of two sugarcane cultivars under salt stress. In both salt-stressed cultivars, no alteration in the foliar nitrogen content was observed; however, there was a reduction in the phosphorus and potassium levels and an increase in the sodium and chloride concentrations. There was also a reduction in gas exchange on the third day under salt stress. Although the content of soluble sugars remained stable in both species, there was a decrease in free amino acids. However, only the RB872552 cultivar displayed a lower leaf protein content compared to the control. The salt stress resulted in higher superoxide dismutase and l-ascorbate peroxidase activities, but only for the RB92579 cultivar. On the other hand, both cultivars were able to maintain lower malondialdehyde contents than the control plants. The gene expression analysis revealed down-regulated expression levels, including the levels of those enzymes linked to higher activities under salt stress. Our results showed that gene induction and leaf antioxidative cycle enzyme activity do not occur at the same time. The variations in gene expression and physiological responses are also discussed.     

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.     

渗透胁迫对绿豆叶圆片叶绿素荧光的影响

绿豆叶圆片用0、-0．3、-0．6、-1．2、-1．8、-2．4MPa等6个渗透梯度处理24h后,其叶绿素荧光的反应表明：光系统Ⅱ的潜在量子产量(FD／Fm)和电子传递受体(QA)的库容(pool size)均随胁迫强度的增大而减小,但QA库容的下降明显比Fg／Fm的下降缓和。结合原初荧光Fo和最大荧光Fm的变化,分析认为本实验中渗透胁迫并未导致明显的光系统Ⅱ反应中心的失活或破坏。造成光系统Ⅱ潜在量子效率降低的原因主要是通过光系统Ⅱ反应中心到Ⅱ的电子传递受到阻抑。