Saccharum Species Hybrid Research Articles

Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds.

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing beta-glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l(-1)) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l(-1) 6-benzyladenine (BA) and 5.0 mg l(-1) PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l(-1) BA, 1.0 mg l(-1) kinetin (Kin), 0.5 mg l(-1) alpha-napthaleneacetic acid (NAA) and 5.0 mg l(-1) PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l(-1) NAA and 5.0 mg l(-1) PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical beta-glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 micro M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50-60% of the transgenic plants sprayed with BASTA (60 g l(-1) glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.

Optimizing nutrient and irrigation requirement of sugarcane (Saccharum species hybrid complex) and French bean (Phaseolus vulgaris) intercropping system

A field experiment was conducted during 201013, on a sandy-loam soil at Pusa, Bihar to find out optimum nu- trient and irrigation requirement of French bean (Phaseolus vulgaris L.) when intercropped with autumn sugarcane (Saccharum spp. hybrid complex). Fifteen treatment combinations consisted of 5 fertility levels [sugarcane + French bean (1: 2) with 100% recommended dose of fertilizer (RDF) to both the crops and residue incorporation, sugarcane + French bean (1: 2) with 100% RDF to sugarcane and 50% RDF to French bean and residue incorpo- ration, sugarcane + French bean (1: 2) with 100% RDF to sugarcane and 100% N to French bean and residue re- moval (existing recommendation), sugarcane + French bean (1: 2) with 100% RDF to sugarcane and 100% N and P to French bean and residue incorporation, sugarcane + French bean (1: 2) with 100% RDF to sugarcane and 100% N and K to French bean and residue incorporation) and 3 irrigation schedules [no irrigation, 1 irrigation at 45 days after sowing (DAS) and 2 irrigations at 45 and 75 DAS] were evaluated in factorial randomized block design with 2 additional treatments as sole sugarcane and sole French bean. Application of 100% RDF to sugarcane and French bean with residue incorporation produced significantly higher grain (1.15 t/ha) and haulm yield (1.90 t/ha) of French bean. The treatments of sugarcane + French bean with 100% RDF to both the crops and residue incor- poration, sugarcane + French bean with 100% RDF to sugarcane and 100% N and K to French bean and residue incorporation and sugarcane + French bean with 100% RDF to sugarcane and 100% N and P to French bean and residue incorporation being similar noticed significantly enhanced cane-equivalent yield (100.0, 95.8 and 94.8 t/ ha), gross returns ( 2,29,400; 2,19,600 and 2,17,300/ ha), net returns ( 1,41,900; 1,33,100 and 1,30,100/ha) and benefit: cost ratio (2.62, 2.53 and 2.49), respectively, over rest of the treatments. The same treatment also ac- counted for higher land-equivalent ratio, area-time equivalent ratio and N, P and K uptake of sugarcane and French bean with greater available N, P and K status in post-harvest soil. Among the irrigation schedules, the highest cane yield (89.0 t/ha), French bean grain (1.20 t/ha), haulm yield (1.98 t/ha) and cane equivalent yield (106.0 t/ha) were recorded in treatment that received 2 irrigations at 45 and 75 DAS. Similarly, the maximum land- equivalent ratio, area time-equivalent ratio, gross returns, net returns, benefit: cost ratio and N, P and K uptake was noted from the enhanced levels of irrigation. The sugar yield was significantly increased with each successive increase in level of irrigation. Considering the total productivity and economics, sugarcane + French bean (1: 2) with 100% RDF to sugarcane and 100% N and K to French bean and residue incorporation, could be adopted un- der similar agroecological situations with twice irrigation up to the period of French bean at 45 and 75 DAS and normal irrigation for sugarcane after harvest of French bean.

Yield and soil organic carbon pool in relation to soil fertility of sugarcane (Saccharum species hybrid complex) plant-ratoon system under integrated nutrient management

A field experiment was conducted for consecutive 6 years ( from 200809 to 201314) at Pusa, Bihar, to evalu- ate the integrated effect of organic and inorganic fertilizer on soil organic carbon pool, nutrient status and produc- tivity of sugarcane (BO 137) plantratoon system in Typic Ustochrept. The application of organics, viz. farmyard manure (FYM), bio-compost and bio-methanated distillery effluent (BMDE), along with inorganic fertilizer (100% NPK) in canes plant and its residual effect in ratoon significantly increased number of millable cane, cane yield and sugar yield over the control. The highest increment in yield in plant (24.95%) and ratoon (16.05%) was ob- served in plots treated with BMDE @ 150 m3/ha. The mean uptake of nutrients (N, P, K and S) and sugar yield varied and followed the similar trends as cane yield. The soil organic carbon pool, viz. carbon storage (180.9 235.2 kg/m2), water-soluble carbon (52.882.4 mg/g), CO evolution (48.570.1 mg/100 g soil) and soil microbial 2 biomass carbon (88.7121 mg/g) varied significantly due to different treatments. The highest amount of soil or- ganic pools were recorded in treatments receiving BMDE (150 m3/ha) followed by bio-compost (20 t/ha) and the lowest being in the control. Integrated use of organics with inorganic fertilizers facilitated the accumulation of or- ganic carbon which in turn had significant increment effect on the soil carbon pool and fertility status (N, P, K and S) of soil with reduction in bulk density beneficial for sustaining productivity of sugarcane plant - ratoon system. Thus, it is imperative that application of either 100% NPK recommended dose of fertilizer along with bio-compost 20 t/ha or 100% NP along with bio-methanated distillery effluent 150 m3/ha (supplying 100% K) improved the fertil- ity of soil which were reflected in increased productivity of sugarcane plant-ratoon system.

Sucrose phosphate synthase and sucrose synthase activity during maturation of internodal tissue in sugarcane

Sucrose accumulation rates, sucrose-phosphate synthase (SPS, EC 2.4.1.14) and soluble sucrose synthase (SuSy, EC 2.4.1.13) activities were measured in internodal tissue from a sugarcane (Saccharum species hybrids) variety N19. The sucrose accumulation rate sharply increases between internodes 3 to 11. In the older internodes SPS activity was at least three times higher than the SuSy activity. A highly significant positive correlation was found between SPS activity and sucrose content. In contrast, no significant correlation was observed between SuSy and sucrose content. In agreement, when radiolabelled glucose was fed to internodes with a high sucrose accumulation rate, label was equally distributed in the hexose moieties of sucrose. This clearly indicates that SPS is the major sucrose synthesis activity in the culm of sugarcane. Different kinetic forms of SPS apparently exist in the internodal tissue at different stages of development.

Sucrose phosphate synthase and sucrose synthase activity during maturation of internodal tissue in sugarcane

Sucrose accumulation rates, sucrose-phosphate synthase (SPS, EC 2.4.1.14) and soluble sucrose synthase (SuSy, EC 2.4.1.13) activities were measured in internodal tissue from a sugarcane (Saccharum species hybrids) variety N19. The sucrose accumulation rate sharply increases between internodes 3 to 11. In the older internodes SPS activity was at least three times higher than the SuSy activity. A highly significant positive correlation was found between SPS activity and sucrose content. In contrast, no significant correlation was observed between SuSy and sucrose content. In agreement, when radiolabelled glucose was fed to internodes with a high sucrose accumulation rate, label was equally distributed in the hexose moieties of sucrose. This clearly indicates that SPS is the major sucrose synthesis activity in the culm of sugarcane. Different kinetic forms of SPS apparently exist in the internodal tissue at different stages of development.

Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.

Morphologic, Cytogenetic, and Enzymatic Variation in Saccharum Species Hybrid Clones Derived from Callus Tissue

Two populations derived respectively from callus tissue of sugarcane clones H 37–1933 and H 50–7209 were studied for differences in morphology, chromosome number, and four enzyme systems. Variations observed in morphological characteristics were not directly correlated with differences in enzyme systems. Greater variation in both respects was observed in plants from the H 50–7209 (a chromosomal mosaic) population than in plants from the H 37–1933 (chromosomally stable) population. Among 37 plants of H 50–7209, all but one had cell-to-cell variation in chromosome number. In all but one of these 36 variable plants, the overall range was 2n = 94–120. The exceptional plant had a range of 2n = 17–118. Among 8 plants of H 37–1933, all had 2n = ca. 106.

Chromosome Numbers of Some Saccharum Species Hybrids and Their Cell Suspension Cultures

Chromosome numbers of five Saccharum species hybrids and their cell suspension cultures were determined. The chromosome number was stable in four parental clones (2n = 122, 114, 114, and 112), but was variable in another (2n = 108-128). The cell suspension cultures have been maintained in a yeast extract-enriched nutrient medium for more than 6 years. These cultures were variable in chromosome number for all clones, with a partial aneuploid series at the haploid and/or polyploid level. Each clone had different chromosomal population modes after 6 years of culture. The loss of chromosomes over a period of time would have an effect on the genetic makeup of a cell population. This has implications in the use and interpretation of data from pathological, cytogenetical, biochemical, and physiological studies using cell cultures and is probably a partial explanation for the loss of totipotency in 6-year-old sugarcane tissue and suspension cultures.

CHROMOSOME NUMBERS OF SOME SACCHARUM SPECIES HYBRIDS AND THEIR CELL SUSPENSION CULTURES

Chromosome numbers of five Saccharum species hybrids and their cell suspension cultures were determined. The chromosome number was stable in four parental clones (2n = 122, 114, 114, and 112), but was variable in another (2n = 108‐128). The cell suspension cultures have been maintained in a yeast extract‐enriched nutrient medium for more than 6 years. These cultures were variable in chromosome number for all clones, with a partial aneuploid series at the haploid and/or polyploid level. Each clone had different chromosomal population modes after 6 years of culture. The loss of chromosomes over a period of time would have an effect on the genetic makeup of a cell population. This has implications in the use and interpretation of data from pathological, cytogenetical, biochemical, and physiological studies using cell cultures and is probably a partial explanation for the loss of totipotency in 6‐year‐old sugarcane tissue and suspension cultures.