text: Syria's future We're not giving up Give now Four years on and the civil war in Syria shows no signs of ending. Sadly, nor does the suffering of more than 12 million ordinary Syrians who are in need, including 5 million children. Many are homeless, have no income and no certainty over what lies ahead. Please support our work in the Middle East. As people continue to seek a safe place to escape from the fighting, Tearfund's partners are providing food, help with shelter, clothing, stoves and hygiene goods. Eventually, we hope to help refugees return to their homes. Across the Middle East, millions of people have been forced to abandon their homes and flee for safety. Please use this prayer guide to pray for them as a church, small group or individual. Another grim milestone for Syria Syria's civil war has notched up another grim milestone – the number of refugees it has created has now topped 3 million. Read More Providing help and hope in Syria's war zone Despite the continuing war inside Syria, Tearfund is bringing aid and hope to those who remain within the country. Read More Syria - How do you heal a nation? Some 900 Syrians have received psychosocial care from Tearfund, giving peace a chance to grow in an atmosphere of forgiveness and wholeness, building foundations for the future even as the war continues to tear their homeland apart. Read More Posted by Prayer team'Light for Syria': Prayers for the three-year anniversary of the conflict 15 March 2014 marks the third anniversary of the ongoing conflict in Syria. Please use these prayer materials, developed with the help of our partner organisations in the region, to ask God to shine the light of his love and hope... Read More Posted by Caroline MaxwellStop their hope from fading away - why we need to unite in prayer for Syria It's hard to imagine what that feels like to lose your home and loved ones, and to become a stranger in a new country. But that's exactly what thousands of Syrians do every day as they cross the border and try to find sanctuary in neighbouring countries. Read More Overlooked and overcrowded Tearfund reports examines life for Syrian refugees outside official camps. Read More Posted by Robert SchofieldHow can I pray for Syria? How do we pray for a crisis as complex as Syria? It's the biggest humanitarian crisis of our time, there don't seem to be any obvious good guys and there doesn't seem to be any end in sight. So, how do we pray? Read More Syria crisis resources Thousands of Syrians have been forced to flee their country, seeking safety from conflict. Tearfund partners are working to help people find food and shelter. Read More


text:
Your cart is empty!
If you have diabetes or poor circulation, consult your physician or podiatrist before use.
My spouse des not go anywhere without these packed. She uses them on travels to the Caribbean, Disney, long walks. Great product to have,
Flents Baseball Finger Splint - Large
Our Price:$2.46
Gaviscon Extra Strength Liquid Relieves Heartburn - 12 Oz
Our Price:$8.95
3M Nexcare Coban Self Adhesive Wrap, 3 Inches X 5 Yards, #H1583 - 1 Ea
Our Price:$3.81
Cortizone 10 Hydrocortisone Anti-Itch Creme, Maximum Strength - 2 Oz
Our Price:$5.98
Multi-Day (Essential One A Day) Multivitamin Dietary Supplement Tablets - 100 Ea
Our Price:$3.08
Dentek Temparin Max Lost Filling And Loose Cap Repair - 3 Applicators
Our Price:$3.79

subset: commoncrawl


text:
What's On This Week
A daily updated summary of the week in TV.
Wednesday, October 7, 2015
Alternate Name: Taylor Edwin Hackford, Taylor Edwin Hackford
A daily updated summary of the week in TV.
From All Movie Guide: American producer/director Taylor Hackford was hired by a Los Angeles TV station after his two-year hitch in the Peace Corps. On his own, he created New Visions Productions, which he eventually merged into the New Century Company before giving up producing to concentrate on directing. He won an Academy Award in 1978 for Teenage Father, a short-subject elaboration of a TV news story on which he'd previously worked. Hackford's first feature was The Idolmaker (1980), a jaundiced recreation of the "Philadelphia school" of '50s rock & roll; he later returned to the rarefied world of vintage rock in his Ritchie Valens biopic La Bamba and his revelatory documentary Chuck Berry: Hail! Hail! Rock 'n' Roll (both 1987). He also directed Dolores Claiborne (1995), the Al Pacino vehicle The Devil's Advocate (1997), and edited the boxing documentary When We Were Kings (1996). Though Hackford has toted up some impressive credits over his career, few of his films have matched the audience appeal or box-office bankability of his biggest hit, 1982's An Officer and A Gentleman. Hackford married British actress Helen Mirren in 1997, whom he had lived with since 1986.


text:
A traditional yet sophisticated sculpted design that is perfect for everyday or special occasions. The Austen Serving Set is made from made from stainless steel, a highly eco-friendly material manufactured from a high percentage of recycled steel and is itself 100% recyclable.
3 Piece Serving Set includes: Serving Fork, Slotted Serving Spoon and Butter Knife.


text:
- Show Prep
- Listen to FTL
- FTL Info
- Community
- Support FTL
- Learn More
This Saturday, the DEA wants people around the country to raid their medicine cabinets and bring their unwanted drugs to more than 3,400 collecting sites around the country. Bring us your unused prescription drugs, DEA saysFlushing drugs down the toilet could contaminate the water supply. And tossing them in the trash can make them easy to find for misuse.
LIVE 7 Days a Week!
Mon-Sun: 7pm-10pm Eastern
Call toll-free:
Welcome Free Talk Live 2.5! Now, with Reddit!
You'll need a Reddit account before you can vote on and submit show prep. (That is different from your FTL/AMP account.)
The content here is the same as you will see on the FTL Subreddit.
Enjoy the site and thanks for submitting your content!


text:
Home →
→ News & Tips
June 11, 2014
News & Tips
Skating dates are projected and may change pending weather conditions. We recommend calling ahead to confirm skating hours before visiting any of these locations.
LakesideProspect Park718-462-0010Hours: Oct. 25 through spring (weather pending): Wednesday and Thursday 12-6pm; Friday2-8pm; Saturday 10am-10pm; Sunday 10am-6:30pm.Price: Prices were not available at press time. Call for updated information.McCarren Rink Site of McCarren Park Pool, 776 Lorimer St. (between Bayard Street and Driggs Avenue), Greenpoint 347-482-7798 Hours: Opens Nov. 15, closing date is weather pending. Daily, 11am-10pm (including all holidays). Price: $8; $5 students and seniors; $4 children. $5 skate rental.
Aviator Sports and Event Center Floyd Bennett Field, 3159 Flatbush Ave., Marine Park/Mill Basin 718-758-7500 Hours: Daily, year-round: Monday 11am-3:50pm; Tuesday 11am-4:20pm; Wednesday-Thursday 11am-3:50pm; Friday 11am-4:20pm and 7:30-11pm; Saturday 12-6pm and 7-11pm; Sunday 12-3pm and 3:30-6:30pm. Call for holiday schedule. Price: $10; $8 children. $5 skate rental. Free lockers; bring your own lock.
Steeplechase Figure Skating at Abe Stark RinkAbe Stark Rink, Coney Island Boardwalk, Broadway and West 19th Street, Coney Island 718-253-8919 or 718-946-6536; nycgovparks.org/facilities/iceskating/1Hours: Nov. 1–March 29, 2015: Saturday and Sunday 1-3:30pm. Price: $9. $5 skate rental.Persons younger than 18 must have written consent form signed by parent or guardian. Agreement forms are available at the rink and a parent must sign the form in the presence of a rink employee or the parent's signature must be notarized. All information is subject to change.
Bank of America Winter Village at Bryant Park 40th-42nd streets between Fifth and Sixth avenues, Midtown 212-661-6640 Hours: Oct. 21–March 1, 2015; Monday-Sunday 8am-10pm. See website for holiday hours. All hours weather permitting. Price: Free to skate. $15 skate rental. Prices are subject to change; check website for details. Lasker Skating Rink North end of Central Park, near 110th Street and Lenox Avenue, Harlem 917-492-3856 Hours: Late October to end of March: Monday-Thursday 10am-4pm; Friday 10am-4:50pm and 6-11pm; Saturday 1-11pm; Sunday 12:30-4:30pm. Price: $7.50; $2.25 seniors; $4 children ages 12 and younger. $6.50 skate rental.
The Rink at Brookfield PlaceWinter Garden, upper plaza between the Winter Garden and the North Cove Marina, Battery Park City646-656-1384 Hours: Mid-November through mid-March: 10am-10pm daily. Price: $12.50. $5 skate rental; $15 skate sharpening.
The Rink at Rockefeller Center Fifth Avenue between 49th and 50th streets, Rockefeller Center 212-332-7654 or 212-332-7655 for reservations Hours: Oct. 11 through April 2015: 8:30am-midnight daily, with occasional half-hour cleaning sessions throughout the day. Group rates and skate lessons available. Price: $27-30; $15 children younger than 11 and seniors. $12 skate rental. Riverbank State Park 679 Riverside Drive, Hamilton Heights 212-694-3642 This season's information was not available at press time. Call for updates. Trump Rink/ Formerly Wollman Rink Central Park (enter at 59th Street and Sixth Avenue) 212-439-6900 Hours: Open late October through early April (weather permitting). The rink opens daily at 10am and closes at the following times: Monday-Tuesday at 2:30pm; Wednesday-Thursday at 10pm; Friday-Saturday at 11pm; Sunday at 9pm. Price: Monday-Thursday: $11.25; $6 children ages 11 and younger. Friday-Sunday: $18; $6 children ages 11 and younger. $8 skate rentals. No credit cards.
Sky Rink at Chelsea Piers Chelsea Piers, West 23rd Street and Hudson River Park, Chelsea 212-336-6100 Hours: Sept. 15–Dec. 14: Monday 1:30-5pm; Tuesday and Thursday 3-5pm; Friday 1:30-5:20pm; Saturday-Sunday 1:30-3:50pm. Price: $10. $5 skate rental; $4.25 helmet rental. Speed skates, double-bladed skates, cell phones, bags, music devices, and cameras are not permitted on the ice.
City Ice Pavilion 47-32 32nd Place (between 47th and 48th avenues), Long Island City 718-706-6667 Hours: Year-round: Wednesday 12-3:50pm; Saturday-Sunday 12-2:20pm. Price: $6 Wednesday; $9 weekends and holidays. $5 skate rental; $2 helmet rental. World Ice Arena 131-35 Avery Ave., Flushing Meadows Corona Park, Flushing 718-760-9001 Hours: Year-round: Monday-Thursday 9am-5:15pm; Friday 9am-5:15pm and 7-9:50pm; Saturday 12-4:45pm and 8-9:50pm; Sunday 12-4:45pm. Opens at 11am on summer weekdays. Price: $6 weekdays; $9 weekends and holidays, including school vacation weeks. $5 skate rental.
Staten Island War Memorial Ice Skating Rink Clove Lakes Park, Victory Boulevard (west of Clove Road)718-720-1010 or 718-720-1014 Hours: Oct. 25–March 29, 2015: Friday 4-10:30pm; Saturday 12-4:30pm and 7-10:30pm; Sunday 12-7pm. Holiday hours: 12-4pm; Christmas recess 12-5pm and 7-10:30pm. Closed Thanksgiving Day. Open Black Friday 12-10:30pm. Price: $9. $5 skate rental.
Staten Island Skating Pavilion Ice Skating Rink 3080 Arthur Kill Road, Staten Island 718-948-4800 Hours: Open year-round (including all holidays): Friday 8-11pm; Saturday 2:30-4:30pm; Sunday 2-3:50pm. Check website for holiday schedule. Skate with Elsa and Ana from Frozen Dec. 30 at 11am; check the website for details. Price: $11; $10 children. $5 skate rental.
Christopher Morley Park 500 Searingtown Road, Roslyn 516-571-8113 Hours: Nov. 29 through mid-March: Monday-Tuesday 10-1:15-3:15pm and 4-6pm; Wednesday and Saturday-Sunday 10:30am-12:30pm, 1:15-3:15pm, and 4-6pm; Friday 4-6pm and 7:30-9:30pm. Price: Non-residents: $20; $14 children. Residents with Leisure Pass: $7; $5 children. $5 skate rental. Grant Park Skating Rink Broadway and Sheridan Avenue, Hewlett 516-571-7821 Hours: November through mid-March: Wednesday-Thursday 10:30am-12:30pm and 4-6pm; Friday 4-6pm and 7-9pm; Saturday 10:30-12:30pm, 1:15-3:15pm, 4-6pm, and 7-9 pm; Sunday 10:30am-12:30pm, 1:15-3:15pm, and 4-6pm. Price: $20; $14 children. With Leisure Pass: $7; $5 children (children ages 12 and younger must be accompanied by an adult); $4 seniors. $5 skate rental.
Marjorie R. Post Community Park Ice Rink 451 Unqua Road (at Merrick Road), Massapequa 516-797-7990 Hours: Updated hours were not available at press time; call or check the website for updates. Price: Admission per two-hour session. Residents: $5; $4 children ages 5-17; $2 children ages 2-4 and seniors ages 60 and older. Non-residents: $8; $7 children ages 5-17; $2 children ages 2-4 and seniors ages 60 and older. $3 skate rental.
Syosset-Woodbury Community Park Ice Rink 7800 Jericho Turnpike, Syosset 516-677-5990 Hours: Opens Nov. 28 for the season. Skating hours were not available at press time; call or check the website for updates. Price: Admission per two-hour session. Residents: $5; $4 children ages 5-17; $2 children ages 2-4 and seniors ages 60 and older. Non-residents: $8; $7 children ages 5-17; $2 children ages 2-4 and seniors. $3 skate rental.
Andrew Stergiopoulos Ice Rink/Parkwood Ice Skating Rink Parkwood Sports Complex, 65 Arrandale Ave., Great Neck 516-487-2975 x3 Hours: September–May: Monday 12:30-2:30pm; Tuesday 10am-12pm and 12:30-2:30pm; Wednesday 10am-12pm, 12:30-2:30pm, 2:45-5:30pm, and 8:45-10:30pm; Thursday 10am-12pm and 12:30-2:30pm; Friday 12:30-2:30pm, 3:30-5:30pm, and 8-10pm; Saturday 3-5pm; Sunday 10am-12pm, 2:30-4:30pm, and adults only 7-9pm. Price: Residents or customers with Park Card: $7; $5 children and seniors. Non-residents or customers without Park Card: $10; $6 children. $3.50 skate rental.
Cantiague Park Ice Skating Rink 480 W. John St., Hicksville 516-571-7056 or 516-571-7058 Hours: September–June, 1-3pm daily; additional sessions on Fridays from 3:30-5:30pm and 9-11pm. Hours vary by month; call to confirm. Price: $20; $14 children (with accompanying adult). With Leisure Pass: $7; $5 children ages12 and younger with adult; $4 seniors, veterans, disabled, and civil volunteers; free for children ages 3 and younger with paying adult. $5 skate rental.
Freeport Recreation Center130 E. Merrick Road, Freeport 516-377-2314 Hours: Opens Oct. 18 for public skating: Thursday 4-6pm; Friday 7:45-9:30pm; Saturday 1-3pm; Sunday 1:30-3pm and 3:15-4:45pm. Check the website for school recess hours. Price: Residents: $6; $5 children; $4 seniors/handicapped. Non-residents: $8; $6 children; $5 seniors/handicapped. $4 skate rental.
Iceland Ice Skating 3345 Hillside Ave., New Hyde Park 516-746-1100, x1 Hours: Year-round: Friday 3:30-5:30pm and 8:30-10:30pm (the later session is Teen Night for ages 11 and older only); Saturday 12-1:30pm (children's session for ages 10 and younger only) and 1:45-3:45pm; Sunday 11:15am-12:45pm (children's session for ages 10 and younger only) and 1-3pm. Price: $8; $7 children ages 10 and younger. $4 skate rental. Children's session: $8 parents; $7 children. Teen Night: $10. Prices and times are subject to change.
Newbridge Arena 2600 Newbridge Road, Bellmore 516-783-6181 Hours: Open year-round. Sept. 14–March 2015: Monday and Wednesday 4:15-6:15pm; Thursday 8-10am; Friday 8:30-10:30am, 4:30-6:30, and 8:30-10:30pm; Saturday-Sunday 12:30-2:30pm and 2:45-4:45pm. Call or check the website for holiday hours and April–August hours. Price: $8 non-residents; $7 Town of Hempstead residents; $6 Town of Hempstead Park District residents (proof of residency required); $4.50 non-resident seniors; $3.50 resident seniors and handicapped individuals; free for children younger than 5 on Mondays and Fridays until 6pm (with paid adult). $4 skate rental.
Port Washington Skating Center70 Seaview Blvd., Port Washington 516-484-6800Hours: Open year-round. November–March hours: Monday-Wednesday 1-2:30pm; Thursday and Friday 1-4pm; Saturday-Sunday 2:15-3:45pm. Hours subject to change in December. See website for more information and special holiday hours. Skating and hockey instruction are offered for children. Price: $8. $4 skate rental.
Town of Oyster Bay Ice Skating Center Bethpage Community Park, 1001 Stewart Ave. (at Cherry Avenue), Bethpage 516-433-7465 x1 Hours: Sept. 14 – March 2015: Monday 4-6pm; Tuesday 10am-12pm; Wednesday 4-6pm; Thursday 10am-12pm; Friday 4-6pm and 8-10pm; Saturday-Sunday 1-3pm and 3:30-5:30pm. Schedule is subject to change. Price: Town of Oyster Bay residents: $6; $3 children ages 2-4; $5 children ages 5-17; $4 seniors ages 60 and older, veterans, and firefighters. $4 skate rental. Freestyle sessions: $12; $10 children ages 5-7. Non-residents: $10; $4 children ages 2-4; $8 children ages 5-17; $5 seniors ages 60 and older, veterans, and volunteer firefighters. $4 skate rental.
Mitchell Park Ice RinkMitchell Park on Front Street, Greenport 631-477-2200 Hours: Open while the weather permits: Monday 3-6pm; Tuesday-Friday 3-5pm; Saturday 11am-6pm; Sunday 11am-5:30pm. Holiday hours vary. Price: Weekdays: $5; $3 children and seniors. Weekends and school holidays: $10; $7 children and seniors. $5 skate rental.
The Rinx at Harborfront ParkPort Jefferson Village Center, 101-A E. Broadway, Port Jefferson 631-403-4357Hours: Nov. 28 through mid-March: Monday-Thursday 12-1:30pm, 2-3:30pm, 4-5:30pm, and 6:30-8pm; Friday 12-1:30pm, 2-3:30pm, 4-5:30pm, 6:30-7:30pm, and 8-10pm; Saturday 10-11:30am, 12:30-2pm, 2:30-4pm, 4:30-6pm, 6:30-7:30pm, and 8-10pm; Sunday 10-11:30am, 12:30-2pm, 2:30-4pm, 4:30-6pm, and 6:30-7:30pm. On holiday and school vacations, call for additional public admission times. Price: Weekdays: $9.50 ($11 Friday and Saturday night); $7 children ages 11 and younger; $4.50 seniors. Weekends and school holidays: $10.50; $8 children ages 11 and younger; $5.50 seniors. $5 skate rental.
Tanger Outlets at the Arches152 The Arches Circle, Deer Park 631-242-0239 Hours: Open late November through mid-March. Nov. 23–Dec. 31: Monday-Thursday 3-9pm; Friday 3-10pm; Saturday 11am-10pm; Sunday 11am-9pm. Jan. 1–March 15: Monday-Thursday 3-9pm; Friday 3-9pm; Saturday 11am-9pm; Sunday 11am-8pm. Hours are subject to change due to weatherPrice: Monday-Tuesday: $10 admission and skate rental. Wednesday-Sunday $10 admission; $4 skate rental.
Dix Hills Ice Rink575 Vanderbilt Parkway, Dix Hills 631-462-5883 Hours: Open year-round. Fall/winter schedule: Monday 12:15-2:15; Monday-Wednesday 10am-12pm; Thursday 10am-12pm and 3:45-5:45pm; Friday 10am-12pm, 12:15-2:15pm, 3:45-5:45pm, and 8:15-10:15pm (DJ night); Saturday 11:30am-1:30pm, 2-4pm, and 9-10:30pm; Sunday 11:30am-1:30pm and 2-4pm. Price: $12; $9 children and seniors. With Rec Card: $7.50; $6 children; $5 seniors. $4 skate rental.
The Rinx660 Terry Road, Hauppauge 631-232-3222Hours: Year-round: Monday-Friday 12:30-2:30pm; Tuesday-Friday 4-5:45pm; Friday 8:15-10:15pm (Rock & Roll Night); Saturday-Sunday 11am-3pm. On holiday and school vacation days, call for additional public admission times. Price: Weekdays: $10.50 ($13 on Friday nights); $8 children ages 11 and younger; $4.50 seniors. Weekends and school holidays: $11.50; $9 children ages 11 and younger; $5.50 seniors. Islip residents receive $1 off adult admission with recreation card. $5.50 skate rental.
Superior Ice Rink 270 Indian Head Road, Kings Park 631-269-3900 Hours: Year-round: Wednesday 12:30-2:30pm and 4:30-6pm; Friday 8:30-10:30pm (with DJ); Saturday-Sunday 12:30-2:30pm. Price: $8; $6 children ages 11 and younger; $5 seniors and children ages 3 and younger. $3-$5 skate rental.
Bear Mountain State Park Ice RinkPalisades Parkway/Route 9W North, Bear Mountain 845-786-2701 Hours: Scheduled to open Nov. 1. Monday-Tuesday 10-11:30am and 12-1:30pm; Friday 8-9:30pm; Saturday-Sunday 10-11:30am, 12-1:30pm, 2-3:30pm, 4-5:30pm, 6-7:30pm, and 8-9:30pm (last session is on Saturdays only). See website for additional holiday schedule information. Price: $5; $4 children ages 4-11; $2 non-skaters and seniors; free for children ages 3 and younger. $4 skate rental. $5 skate sharpening. $8 parking on weekends and holidays.
Palisades Center Ice Rink 4900 Palisades Center Drive, West Nyack 845-353-4855 Hours: Daily, year-round. Call or check the website for public skating times. Price: $9; $7 seniors and children younger than 10. $4 skate rental. Ten-visit punch cards and season passes available.
Sport-o-Rama20 College Road, Monsey 845-356-3919 Hours: Wednesday skate sessions: 3:30-5pm through Nov. 7; 4:10-5:10pm Nov. 14–Feb. 25, 2015. Saturday skate sessions: 9-11pm Nov. 1–April 25, 2015. Sunday skate sessions: 2:55-4:30pm Nov. 2–March 15, 2015. Holidays including Veterans Day, Christmas week, Presidents Day week, Easter, and Spring Break: 2-4:30pm. Time subject to change; call to verify. Price: $6.50 Wednesdays; $8.50 weekends; $9 holidays. $3 skate rental.
Ebersole Ice RinkDelfino Park, 110 Lake St., White Plains914-422-1390 or 914-422-1348Hours: Oct. 17–March: Wednesday 12-2pm; Thursday 12-2pm and 3-5pm; Friday 10am-2pm, 3-5pm, and 8-10pm; Saturday 12-3pm; Sunday 8:30-10:15am (adults only) and 1:15-3:30pm. Contact rink for holiday week specials.Price: $5 skate rental. Non-resident or resident without recreation ID: $11; $9 children; $8 seniors. Residents with recreation ID: $7 adult; $6 children; $5 seniors. Still accepting registration for group lessons, which start Nov. 7. Group packages available.
EJ Murray Memorial Skating Ice and Roller Skating Rink348 Tuckahoe Road, Yonkers914-377-6469Hours: Tuesday-Thursday 10am-12pm; Friday 10am-12pm and 8-10pm; Saturday 11am-1pm, 2-4pm, and 8-10pm; Sunday 11am-1pm, 2-4pm, and 5:15-7:15pm. Extended hours during holiday periods; call for schedule.Price: $8. $4 skate rental.
Hommocks Park Ice RinkBoston Post Road-Weaver Street, Larchmont914-834-1069Hours: Oct. 5–June 21, 2015: Monday 12:30-2:30pm; Tuesday and Thursday 1-5:15pm; Friday 1-5:15pm and 7:15-9:15pm; Saturday 1-5:15pm; Sunday 11am-5:15pm.Price: $8; $7 children younger than 12; $6 seniors. $4 skate rental. Discount cards (for 12 sessions) and season passes available.
The Ice Hutch655 Garden Ave., Mount Vernon914-699-6787 or 914-668-1165Hours: Year-round. Call for weekly schedule. Price: $8; $5 seniors. $4 skate rental.
Westchester Skating Academy91 Fairview Park Drive, Elmsford914-347-8232Hours: Year-round: Monday 11:40am-1:10pm and 1:30-4:30pm; Tuesday 9:30-11:30am and 1:30-3:30pm (Mondays and Tuesdays are subject to change during the winter season); Wednesday 9:30am-11:30am, 11:40am-1:10pm, and 1:30-5pm; Thursday 9:30-11:30am; Friday 9:30-11:30am, 12-1:20pm, 1:30-4:30pm, and 7:30-10pm; Saturday-Sunday 1:30-4pm. Extended hours during holiday periods. Hours subject to change. Check website.Price: $10.75; $9.75 children ages 4-10; $6.25 seniors and children ages 3 and younger. $4.50 skate rental.
Danbury Ice Arena1 Independence Way, Danbury203-794-1704Hours: Year-round: Monday, Tuesday, and Thursday 11am-2pm; Wednesday 11am-2pm and 5:40-7:40; Friday 11am-2pm and 8:30-10:30pm; Saturday 1:10-3:10pm; Sunday 2:30-4:30pm. Schedule is subject to change.Price: $8; $6 children ages 12 and younger. $4 skate rental; $4 helmet rental. DJ Skate on Fridays: $9.
Darien Ice Rink55 Old Kings Highway N., Darien203-655-8251Hours: Year-round: Monday, Tuesday, and Thursday 9:50-11:50am and 12-2pm; Wednesday and Friday 12-2pm. Schedule subject to change.Price: $7; $6 children. $4 skate rental.
Dorothy Hamill Skating RinkSue Merz Way off Sherman Avenue, Greenwich203-531-8560Hours: Limited public skating opens Sept. 20, full public skating Oct. 13 through March 14, 2015: Monday 12-1:30pm; Tuesday 12-1:30pm, 6:30-7:45pm, and adults only skate 7:45-9pm; Wednesday 12-1:30pm and 3:30-5pm; Thursday 12-1:30pm; Friday 12-1:30pm, 3:30-5pm, and 7:30-9pm; Saturday-Sunday 2-4pm.Price: Proof of Greenwich residency is required for admission. A Greenwich resident may bring a non-resident as a guest. Resident: $7; $5 children ages 5-15; free for children ages 4 and younger when accompanied by a paying adult; free for seniors ages 65 and older using senior resident pass. Non-resident guest: $9; $7 children ages 5-15; free for children ages 4 and younger when accompanied by a paying adult. $4 skate rental.
Fairfield Ice Academy85 Mill Plain Road (Building J), Fairfield203-254-8399Hours: Year-round. Hours vary, check the website.Price: $7. $5 skate rental; $5 helmet rental.
Sports Center of Connecticut784 River Road, Shelton203-929-6500; sportscenterct.comHours: Year-round: Hours vary, check the website.Price: $9.75; $7.75 children ages 12 and younger. $5.50 skate rental. $7 skate aids for children ages 6 and younger ($5 deposit required)
Stamford Twin Rinks1063 Hope St., Stamford203-968-9000 or 203-321-1516Hours: Year-round: Monday 9am-2pm; Tuesday 10:30am-2pm; Wednesday 9am-1:40pm; Thursday 9-11:40am; Friday 9am-1:50pm and 7:45-10pm; Saturday 12:20-2:20pm; Sunday 1-3pm.Price: $9; $10 Friday night skating. $4.50 skate rental. Preferred customer discount cards available.
Terry Conners Ice RinkCove Island Park, 1125 Cove Road, Stamford203-977-4728; tcrink.comHours: Year-round: Monday and Friday 10:30am-2pm; Tuesday-Thursday 11:30am-2pm; Saturday-Sunday 12:20-1:50pm. Check website for changes to the schedule.Price: $8; $6 children and students; free for seniors. $4 skate rental. $4 training aids. Multi-use discount card available for residents only.
Winter Garden Arena111 Prospect Ridge, Ridgefield203-438-4423Hours: September through May: Monday-Friday 10am-2pm.Price: $8; $5 children; free for children younger than 3. $4 skate rental; $5 helmet rental.
Wonderland of Ice123 Glenwood Ave., Bridgeport203-576-8110; wonderlandofice.comHours: Year-round: Monday-Friday 11am-1pm; Saturday 2:30-4:30pm; Sunday 2-4pm. Schedule is subject to change. See website for monthly updates.Price: $6; $4 children ages 11 and younger. $4 skate rental; $5 helmet rental.
When to Visit the NY Transit Museum
Your Guide to Visiting Central Park
First-Ever Thomas Land to Open in August
2015 Rockland Boulders Home Games, Giveaways, and Promotions
Party Planning
After-School Activities
Camp Guide
Sports for Kids
Child Care Options
Education Resources
Family Health Guide
Special Needs Guide
Oct 07, 2015
Oct 06, 2015
Oct 02, 2015
Oct 09, 2015
Oct 08, 2015
Sep 21, 2015
Sep 17, 2015
Sep 10, 2015
Fairfield County, CT
Kids will love these fun educational apps that will keep them learning even during the summer months. Dr. Levy's 5 picks include apps for kids ages 4-16 that help build reading and math skills.
Copyright 2015 | All Rights Reserved | Davler Media


text:
Pea has lagged behind other model legumes in the molecular study of nodulation and mycorrhizae-formation because of the difficulty to transform its roots and its poor growth on agar plates. Here we describe for pea 1) a transformation technique which permits the complementation of two known non-nodulating pea mutants, 2) a rhizobial inoculation method which allows the study of early cellular events giving rise to nodule primordia, and 3) a targeted fungal inoculation method which allows us to study short segments of mycorrhizal roots assured to be infected. These tools are certain to advance our knowledge of pea root symbioses.
Keywords:Composite plants; gene complementation; mycorrhizae; nodulation; pea mutants; targeted spot-inoculation; transformation
Pisum sativum (pea) has been a model organism in plant research for more than a century; however, it has lagged behind other plants in becoming the model organism to study root symbioses, i.e., those resulting in nodulation and mycorrhiza-formation. Reasons for this stem from the fact that pea is a large plant which requires a large area for growth and has a relatively long life-cycle (~ 90 days). It has a large genome (about 4 × 109 base pairs [1]) still to be sequenced, and many molecular techniques, especially those of transformation, are difficult to use [1] to study the function of the genes involved in the nodulation process. Yet, the agricultural and economic importance of pea is significant, especially in Canada, so despite the difficulty in manipulating this species, it is relevant to study its beneficial root symbioses and to capitalize on its large collection of symbiotic mutants [2]. Pea production is expected to increase further as farming practises are focusing on agricultural sustainability, with farmers once again utilizing the benefits of crop rotation to decrease fertilizer applications.
Two legumes, Lotus japonicus and Medicago truncatula (barrel medic), have been used as model plants to study the plant-rhizobial and the plant-fungal relationships that lead to the fixation of nitrogen and the increased uptake of phosphorus, respectively [e.g., [3,4]]. These model organisms have helped advance our understanding of the molecular dialogue between plants, rhizobia, and fungi, and the signalling pathway which ensues. The early symbiotic events occurring in the root and leading to nodule organogenesis or to mycorrhiza formation have been a subject of great interest but are difficult to study [e.g., [5,6]]. Complications arise in locating in time and space specific root events, such as those that occur before the microsymbionts have entered the root and those that take place within non-translucent and thick roots after the microsymbiont entry [5,6]. Many techniques have been developed and refined to advance the study of symbioses in these model species.
The powerful technique of transformation has been widely used to study gene function and it has served as an important crop improvement tool [e.g., [7]]. Pea, like most other large legume plants, has been considered to be recalcitrant to transformation [8,9], although, as summarized in a recent paper [10], several successful methods have been developed by infecting explants with Agrobacterium tumefaciens. Unfortunately, most of these procedures are plagued by long shoot regeneration periods; indeed, up to 9 months [e.g., [11-13]] may be required before rooting can take place. Bean et al. [11] reported additional drawbacks including low fertility, phenotypic abnormalities, altered ploidy, and loss of transgene activity in subsequent generations. Despite the difficulties, one group has been successful at introducing in pea, via A. tumefaciens, a construct comprising the nodulation reporter ENOD12A gene [14]. The success rate was, however, low, and the transformed population not homogeneous as transformants varied in the number of gene copies inserted. The pitfalls associated with the above-mentioned methods make them labour-intensive and not efficient for research applications. If the gene of interest is involved in root biology, e.g., in rhizobial and mycorrhizal symbioses, a convenient way to avoid the problems encountered with A. tumefaciens transformation techniques is to use Agrobacterium rhizogenes, the agent responsible for hairy root disease. Pea is susceptible to this type of transformation, and isolated root-organ cultures have been obtained [15]. When studying nodulation, however, those cultures are not useful because nodule development requires shoot photosynthates [16]. To address this problem, techniques have been developed to create composite plants that have transformed roots but a wild-type shoot [17]. These chimeric plants have proven to be useful in studies of root nutrient uptake, hormone transport, and the nodulation and mycorrhizal symbiotic pathways [e.g., [7]]. Likely because of the difficulty encountered in pea transformation, there has been only one report of successful nodule formation on composite wild-type/transgenic pea plants [18], and gene complementation in pea mutants deficient in nodulation (i.e., Nod- mutants) has not been previously reported. This has forced researchers studying pea to resort to the transformation of pea genes into close relatives, such as Trifolium repens (white clover) [19] or barrel medic [2,20].
Spot-inoculation is a technique that has been exploited to induce the formation of a nodule at a known location and has facilitated the identification of the first stages of nodule organogenesis, including pre-infection events [e.g., [5]]. This technique also enables one to know precisely the age of a developing or mature nodule, allowing a linkage between gene expression and a specific developmental stage. Spot-inoculation is now a common practice in Lotus and Medicago whereby plants are grown on agar plates with varying nutrients [e.g., [21,22]]. A similar technique has been employed in the study of mycorrhiza formation [23] and its adaptation for continuous observation with a confocal microscope has proven to be a powerful tool to gain insight into the events leading to root infection [6]. Yet spot-inoculation of larger plants such as pea has not been widely performed, likely because these are difficult to grow on agar plates.
Here, we present a protocol for the rapid development of transformed hairy roots on composite plants of Pisum sativum using A. rhizogenes. We show that the transformed roots of two non-nodulating pea mutants complemented with the appropriate wild-type gene are capable of nodulating with Rhizobium leguminosarum. Furthermore, we describe a technique of spot-inoculation refined for our species of interest, pea. We report that this technique is valuable for studying not only nodule organogenesis but also mycorrhizae. We trust that these techniques will be of great use to the many scientists worldwide interested in the study of pea root symbioses.
Pea Transformation
In our hands, the best method to produce transformed composite pea plants (Figures 1 and 2) relied on the infection of 10 day-old pea seedlings using Fibrgro® cubes imbibed with A. rhizogenes suspension as in Collier et al. [24]. After a short wilting period (Figure 2D), the treated plants were incubated in high humidity conditions (Figures 1 and 2F) until roots developed ~10 days after infection (Figure 2G). To assess the validity of our technique, we used Frisson and its mutants P5 [25] and P56 [26] as a tool to score for transformation efficiency. As described in [27], P5 and P56 are sym10 mutants that are defective in nod factor perception and thus are non-nodulating. Our rationale was that the complementation of these mutants with the wild-type SYM10 gene should reverse their nodulation phenotype and the presence of nodules should serve as the ultimate scoring for transformation success. A. rhizogenes strains AR12 and AR1193 [[17] and [28], respectively] carrying the sym10 genes were both used to transform the mutant plants. Using the described protocol (see Materials and methods, and Figures 1 and 2), we complemented successfully the sym10 mutants P5 and P56 because roots of composite plants challenged with Rhizobium leguminosarum had nodules 21 days after inoculation (Figure 3). Furthermore, no nodulation was observed on P5 or P56 mutant plants (n = 8 and 7, respectively) transformed with AR1193 carrying no construct (Figure 3), while nodulation occurred on wild type plants (n = 4) transformed with AR1193 (data not shown).
Figure 1. Schematic representation of the time-line required for the generation of composite pea plants, with an emphasis on the parameters which must be optimized.
Figure 2. Illustrations depicting the main steps for the production of composite pea plants. A. Plants grown for 10 days in vermiculite (7 days in dark followed by 3 in light). B. Fibrgro® cubes saturated with A. rhizogenes. C. A shoot of a 10 day-old plant cut above node 2 and inserted into Fibrgro® cube. D. A wilted pea plant about to receive water for recovery. E. Plants placed in growth tray under a plastic dome. F. High humidity is provided by plastic lid with closed ventilation holes and metal tray with water. G. A root (arrow) protruding through a Fibrgro® cube 10 days after A. rhizogenes infection. H. Callus-forming plants, the regenerated roots of which have been excised, placed in an upright position in vermiculite before being inoculated with R. leguminosarum 3 days later.
Figure 3. Macro-photography of developing nodules on transformed roots. Non-nodulation mutants P56 (A) and P5 (B) had their nodulation capabilities restored through A. rhizogenes transformation. Each inset shows a magnification of the area framed in the main photograph. Root nodules (arrows) that have emerged from the transformed roots should not be confused with vermiculite pieces (arrowheads). C. P56 mutant transformed with AR1193 lacking the sym10 construct did not exhibit any nodules.
This high-throughput root transformation system is the first to show complementation of a nodulation gene in pea. We defined complementation efficiency as the ratio of Agrobacterium-infected plants which displayed at least one nodulated root (Table 1). In our experiments, strain AR12 was less efficient than strain AR1193 at producing transformed roots as less nodules were observed on the roots of plants transformed with AR12 (data not shown). To improve complementation efficiency using AR12, we applied antibiotic counter-selection to inhibit the growth of non-transformed roots in favor of transformed roots. A range of kanamycin concentrations (2 - 15 μg/mL) was tested but produced no satisfactory results, as treatments caused significant plant growth retardation. Because AR1193 was more efficient than AR12 at transforming pea, we pursued our optimization experiments using it solely.
Table 1. Success rate of sym10 complementation in pea mutants P5 and P56
Induction of root formation
To trigger the growth of transformed roots [29], we excised roots which had protruded from the callus (Figure 2G) as most of the roots that emerged were adventitious, i.e., arising from the stem. We also treated plants two days after AR1193 infection with IAA (0.1 - 10 μM) for 48 hours because IAA had been commonly used in rooting media [e.g., [30]]. To determine treatment impact, we compared treated plants to plants receiving only water (Table 1). Plants that received exogenous IAA had in general higher complementation efficiencies than plants receiving only water. The highest complementation efficiency, 70% for P56 and 73% for P5, was however obtained when all of the roots appearing through the Fibrgro® cube were excised (Table 1). This treatment ultimately resulted in an average number of nodules per root of 9.6 and 7.3 for P5 and P56, respectively. The pruning seemingly promoted root growth from the callus while it limited further adventitious root development.
Spot-inoculation protocols have been developed for a variety of legumes but these techniques have not been easily transferred to pea. We developed a highly reliable rhizobial spot-inoculation technique by growing seedlings in growth pouches (see Materials and Methods) and placing a drop of the rhizobial inoculum on a lateral root located in the zone that is most susceptible to infection. We found that the substrate and the method of delivery of the inoculum were keys in the success of pea spot-inoculation. With the procedure presented in Material and Methods and in Figure 4, a nodule formed within ~0.5 cm from the intended location in 95-100% of the cases (Figure 5). Using this technique, it becomes possible to determine the exact age of the nodule from the time of inoculation to 42 days thereafter. We were able to apply what we learned with the rhizobial spot-inoculation and we targeted mycorrhizal infection.
Figure 4. Schematic representation of the important steps for spot-inoculation. A. Imbibed seeds placed in vermiculite. B. Seedling radicle placed in trough of trimmed pouch. C. Pea cotyledons and epicotyls protruding from tightly-closed pouches. D. Pots, with pouches inside, filled with vermiculite to prevent light exposure. E. Inoculation 5 days later with R. leguminosarum bv. vicieae placed as a minute drop (seen as a black dot drawn with a marker on plastic) at the most susceptible zone of infection of several lateral roots. F. Pouches later returned to pots. G. Healthy plants allowed to grow until inspection.
Figure 5. Macro-photography of nodules developing on roots of plants grown in pouches. A. An overview of an 8 day-old spot-inoculated plant. B. A close-up focussed on the root hairs of the seedling 5 DAI. The unfocussed black dots are located on the surface of the pouch and correspond to the different inoculation sites. In C to F, two nodules (arrowheads) can be seen developing over time. The nodules are five-day old (C), seven-day old (D), 10 day-old (E) and 14 day-old (F). In E and F, two additional nodules (black circles) appeared later at the spot-inoculation site; they cannot be given a specific age. One (asterisks in F) of the nodules appears much larger than the others; it is evident that this large nodule has multiple meristems. In D, two roots (arrows) which did not grow in contact with the filter paper of the pouch dried out. White arrowheads indicate pieces of vermiculite.
Substrate Choice
Pea has a large root system, making it difficult to be cultivated over a long period of time on agar slides as Fåhraeus did with white clover seedlings [31] and impractical to use it for studying nodule development in mutants with delayed nodulation. Yet, a few researchers have been successful at spot-inoculating pea utilizing either the Fåhraeus technique [32] or agar-filled plates [33]. In our hands, unfortunately, healthy nodulating pea plants are not obtained on agar plates, and other labs have reported similar problems in the past [e.g., [34]]. With filter paper within a pouch as a substrate, the size of a plant is less of an issue for growth. Turgeon and Bauer [35] grew Glycine max (soybean) in germination pouches, held in an upright position in plastic trays with high walls, and spot-inoculated them with one to three nL drop of inoculum, which they placed in the root zone that is most susceptible to infection. In our attempts to replicate this technique for pea, the roots either tended to dry rapidly or became easily water-logged, thus stunting the growth of the plant and preventing the formation of nodules. To palliate this problem, we have cut the bottom part of the pouches which we then placed in pots filled with vermiculite (Figure 4B). The paper of the pouch is capable of acting as a wick and drawing water when needed from the wet vermiculite. Apparently, not all pouches are similar because recently a group of researchers obtained healthy-looking nodulated plants in such a system [36]. The paper in the pouch is, however, noticeably distinct from the one we used; the different result may also lie with the pea cultivars used, the method of pre-germination or the method of inoculation (flood-inoculation versus spot-inoculation) [36].
Rhizobial Inoculum Delivery
Diaz et al. [37] reported successful nodulation on the primary root of pea spot-inoculated with a drop of an inoculum made of a mixture of rhizobia and 0.15% agar. Employing this technique, van Brussel et al. [32] were in fact able to observe the first described pre-infection threads. However, when we tried this method, the agar-mixed rhizobia impeded root growth (drying the root in some cases) in the area of application and no nodules were observed. Therefore, we tested different means of delivering the inoculum. First, a small strip of Parafilm™ was used to hold the drop of inoculum in place at the desired location [38]. Second, a rhizobia-india ink suspension was applied [38]. Finally, glass micro-carriers which were successful in spot-inoculating alder with Frankia [39] were used to transfer rhizobia to the root and prevent the inoculum drop from moving away from the chosen location. The above techniques not only were tedious and time-consuming, but they failed to produce nodules locally. We found that two details were important to obtain nodules where we wanted them to develop (Figure 6). First, the bacterial micro-drop had to be delivered on a root system growing on a paper just damp; if the paper was too moist, the drop was quickly lost. Second, the drop should be left for a few seconds on the root before the pouch can be closed. Furthermore, roots which do not touch the paper of the pouch, or may not touch the paper once grown, should not be inoculated as they have an increased chance to dry out (Figure 5D).
Figure 6. Microscopy of spot-inoculated lateral roots. A. An unstained longitudinal section of a spot-inoculated lateral root 24 hours after rhizobial inoculation showing anticlinal divisions in its inner cortex (arrows), and in its pericycle (asterisk), located adjacent to a xylem pole (X). Bar: 100 μm. B. A close-up on the area framed in Figure A. Metabolically active cells, one with a large nucleus (arrowhead), exhibit anticlinal divisions. Bar: 10 μm. C. A longitudinal section of a root inoculated 3 days earlier with rhizobia and stained with toluidine blue. The nodule meristem (NM) has passed the mid-point of the root cortical region. Bar: 100 μm. Note that for all the photographs, there was no indication of nodule emergence and it was only because of the spot-inoculation that these events were easily captured. All sections were made with a vibratome.
Fungal Inoculum Delivery
To investigate mycorrhizal infection in pea, we adapted the method of delivery used in the rhizobial spot-inoculation, i.e., we brought a small amount of fungal propagules to plants grown in pouches. Fungal spores obtained from root-organ cultures [40] were transferred to the plant within an agar plug (Figure 7A, inset, and 7B). The plug, adhering to the paper of the pouch with melted agar, was placed below a growing lateral root. Root segments expected to be infected were excised at 21 and 35 days after inoculation and stained to visualize infection. Three of the eight plants harvested and processed at 21 days after inoculation (DAI) showed evidence of fungal infection, including extraradical hyphae and hyphopodia (Figure 7C), at the spot-inoculation site. At 35 DAI, success was rated at 78%, i.e., 11 infected roots out of 14 displayed infection units, which were comprised of intraradical hyphae, arbuscules (Figure 7D), or vesicles (Figure 7E).
Figure 7. Fungal spot-inoculation of plants grown in a pouch. A. An overview of the root system of a 40-day old plant grown in a pouch. The plant was challenged when it was 5 day-old with fungal spores located in an agar plug. Inset: A close-up of the area indicated by a white square in A. The plug (delineated by arrows) was placed below the lateral roots. B. Close up on the roots which were pushed gently into the plug containing the fungal spores. Bar: 0.5 mm. C. An extra-radical hypha and some hyphopodia (asterisks) on the surface of a cleared root inoculated 21 days earlier. D. Arbuscules located within inner cortical cells of a cleared root inoculated 35 days earlier. E. Intraradical hyphae and a vesicle in the cortex of a root 35 DAI. F. An infection unit with extensive hyphal branches; note the hyphopodium (asterisk), the main intraradical hypha (arrowhead), an arbuscule (arrow), and a vesicle (V). C - F: Bar: 20 μm; ink-vinegar staining.
Here we focus on the wide applications of the three techniques presented in this paper. A. rhizogenes root transformation of legumes once thought to be recalcitrant, such as soybean [e.g., [29]], Phaseolus vulgaris L. [41], Aeschynomene indica [42], and now pea, should bring new insight into the roles of genes involved in nodulation and mycorrhizae formation. Model legumes have opened the avenue to molecular studies on root symbioses and to transfer the acquired knowledge to transformation and molecular understanding of the pulses is essential. With the transformation technique described here, the number of nodulated roots per pea plant was between 1 and 4, with average complementation efficiency (at least one nodulated root per plant) of 0.70. The transformation efficiency with our system should be at least as high as the observed complementation efficiency because it is likely that some transformed roots did not nodulate. These numbers should be compared to those obtained by Collier et al. [24] for soybean who reported between 2 and 4 transformed roots per transformed plants with a transformation efficiency of ~ 80%. It is of interest to note that although not equivalent in their efficiency, both agrobacterial strains used (AR12 and AR1193) were successful in transforming pea. Future directions should include a reliable marker, such as pHairyRed [43], for an easy and convenient selection of transformed roots.
The spot-inoculation technique, whether rhizobial or fungal, should be useful in a number of different studies. The infected roots can be cut 0.5 cm on either side of the spot at any time after inoculation and sectioned (fresh or fixed) or cleared to study specific ages of nodules (Figure 6) or mycorrhizae (Figure 7), respectively. These anatomical studies may be complemented with molecular studies whereby gene expression is assessed in response to microbial challenges. With mock-inoculated roots used as a control, expression of specific genes can be correlated to specific symbiotic events (Clemow and Guinel, unpublished). Furthermore, at all times, the right side of the plastic pouch can be cut so that the plastic cover becomes a flap which can be lifted and replaced at the researcher's convenience. This permits to follow the infection, especially that resulting in nodulation, over time by macro-photography (Figure 5), so that the dynamic growth of lateral roots with their nodules can be followed. In the case where rhizobia transformed with either a lacZ gene or another visual marker such as green fluorescent protein are used to perform spot-inoculation, rhizobial progression can be tracked.
We see some great advantages for our spot-inoculation methods. For the rhizobial delivery, in contrast to prior techniques which have relied on the primary root of pea for spot-inoculation [37], our method utilizes its lateral roots which are where this species develops the majority of its nodules [44]. The fact that pea primary and lateral roots have different anatomy [45] could have an unforeseen effect on nodule development. For both symbioses, in most cases, spot-inoculation guarantees infection events close to the spot. Needless to say, this shortens tremendously the time needed to look for symbiotic structures. The technique not only allows for early detection of events but also permits an easier characterization of nodulation (Macdonald and Guinel, unpublished) and mycorrhizae-formation mutants.
One minor inconvenience with our technique is that it does not limit nodule production to one specific location as nodules occasionally developed elsewhere on the root system where rhizobia were not applied. However, since we are interested in knowing the precise age of a developing/nodule, we do not take these "straggling" nodules into account. Also, occasionally spot-inoculated roots develop more than one nodule in the inoculated area.
The techniques described here should open many doors to researchers working on pea root symbioses. Root transformation will allow not only nodulation gene complementation in pea but also the alteration of expression of specific genes of interest. Furthermore, the spot-inoculation technique should increase greatly the probability of finding infection events; it will especially permit the study of pre-infection events in the roots of plants grown in pouches challenged with either rhizobia or mycorrhizal fungi.
Materials and methods
Plant materials, bacterial strains and vectors, and fungal inoculum
Seeds of Pisum sativum L. cv. Sparkle and cv. Frisson, and seeds of its mutants P5 and P56 [generous gift from Gérard Duc (Dijon, France)], were surface-sterilized, imbibed, and planted in sterile vermiculite to germinate (see below for specifics). Unless otherwise mentioned, P. sativum plants were grown under incandescent (OGE 600 hour, 60 - 120 W, 120 Volts, General Electric) and cool white fluorescent lights (Watt-Miser GE, F96TIZ-CW-HO-WM) in a growth-room with a 23°C/18°C, 16 h/8 h light/dark regime. The total light intensity received by the plants was 280 μmol·m-2·s-1.
Agrobacterium rhizogenes AR12 and AR1193 [[17] and [28], respectively] containing SYM10 [27] were cultured in 100 mL of Luria Bertani (LB) broth with 100 μg/mL rifampicin and 100 μg/mL spectinomycin (all from Bioshop, Burlington, ON). As a control, AR1193 with no SYM10 construct was grown in LB with 100 μg/mL rifampicin. Cultures were grown at 28°C for 24 hours on an orbital shaker (New Brunswick Scientific, Edison, NJ) at 200 rpm until the culture reached an absorbance between 0.6 and 0.8 at 600 nm on a spectrophotometer (Varian Inc., Mississauga, ON). The bacterial culture was then transferred to sterile centrifuge tubes and spun at 6000 × g for 15 min, 4°C. The pellet was re-suspended in ice-cold, 1/4 × Murashige and Skoog basal medium (Sigma-Aldrich M5524, Oakville, ON), pH 5.8, until an optical density of 0.3 at 600 nm was reached.
Rhizobium leguminosarum bv. viciae 128C53K (gift from Dr. S. Smith, Milwaukee, WI) was kept on slants at -20°C and cultured in 20 mL of yeast-mannitol broth, consisting of (g/L) mannitol 10.0, K2HPO4 0.5, MgSO4 0.2, NaCl 0.1, yeast extract 0.4 and of pH 6.8. The medium inoculated with a loop-full of stock culture was grown at 25°C in Erlenmeyer flasks on an orbital shaker at 100 rpm. Cells were grown to late log phase (48 hours) at which time the viable cell density was 1.2 × 106 colony forming units (CFUs)/mL.
Phytagel™media plugs containing spores of the AM fungus Glomus irregulare (DAOM 197919) were obtained from Agriculture and Agri-Food Canada's Glomeromycota in vitro collection. The fungus was propagated using root-organ cultures [40] of transformed carrot (Daucus carota) roots (Culture Collection, Mycothéque de l'Université Catholique de Louvain, Belgium).
Pea transformation
Figures 1 and 2 highlight the important steps of the protocol with which we obtained the highest transformation efficiencies.
1| Surface-sterilize seeds in 8% bleach for 5 minutes to eliminate microbial and fungal contamination during germination. Wash seeds in three rinses of sterile water for 1 minute each and store them in sterile water in darkness for 12 to 15 hours to imbibe.
2| Plant in vermiculite imbibed seeds at a depth of 1-2 cm and let grow in the dark until the seedlings are ~ 8 cm tall [about 7 days after planting (DAP)].
NOTE: The dark treatment promotes etiolation and thus facilitates the shoot excision at a later step.
3| Set plants into a controlled growth-room for 3 days to provide time for the leaves to develop (Figure 2A).
4| Cut Fibrgro® cubes (Homegrown Hydroponics Inc., Breslau, ON) into smaller cubes (~ 2.5 cm3). Use a dissecting probe to make a small starter hole, a quarter to half way through the cube, into which the stem of the plant will be inserted (step 7). Sterilize the cubes (pre-vacuum cycle for 20 min) in autoclave.
NOTE: Without this hole the stems of the plants broke easily when inserted.
5| Prepare the culture of A. rhizogenes 9 DAP as described above.
6| Place 2 to 4 sterile Fibrgro® cubes, with the holes facing up, in 12 cm sterile Petri plates. Inoculate the cubes with the diluted A. rhizogenes culture until complete saturation; 4 to 7 mL of inoculum is usually sufficient per cube (Figure 2B).
7| Cut off the shoot of 10-day old plants above node 2 with a clean razor blade and place their stem gently in the hole of the Fibrgro® cube (Figure 2C).
NOTE: When the distance between node 2 and 3 is too short, the plant is cut above node 1 instead so that the leaves are never in contact with the inoculated cube.
8| Place the Petri plates containing the plant/cubes in a plastic tray with drainage holes and cover with a transparent dome with ventilation holes closed (Homegrown Hydroponics Inc., Breslau, ON). Leave tray on lab bench for 24 hours (temperature of 21°C and light intensity of about 10 μmol·m-2·s-1). Remove the lid on the following day and leave the plants on the bench for an additional 24 hours to promote wilting of the shoots (Figure 2D).
NOTE: The wilting period is required for a callus to develop as it likely draws the inoculum into the plant [24]).
9| Add sterile water to the Petri plates when the stems are wilted, and replace the dome for an additional 24 hours (Figure 2E). Place the plastic tray into a metal watering tray and set the plants in the growth room (Figure 2F). Add water to the metal tray to cover the bottom of the plastic tray.
NOTE: Keep the plastic dome snug to the plastic tray and the ventilation holes closed to enhance humidity, which increases transgenic yield [29].
10| Dissect carefully with a pair of tweezers the Fibrgro® cube away from the roots when the roots are visible (~10DAI) (Figure 2G). Excise all roots with a sterile blade.
NOTE: At this stage most of the roots are adventitious and their removal promotes root growth from the callus.
11| Transfer the callus-forming shoot with its epicotyl to a pot filled with sterile vermiculite; tightly pack the vermiculite around the rootless callus so that the shoot is kept upright (Figure 2H).
12| Two days before inoculation, grow a culture of R. leguminosarum as explained above. Inoculate the plants with 5 mL of a 5% bacterial solution 3 days after moving them to vermiculite.
13| Inspect plants 21 DAI and check for nodules.
Rhizobial Spot-Inoculation of Pea
Figure 4 highlights the important steps in the protocol for successful spot-inoculation.
1| As 1| above.
2| Plant seeds in 7.5 cm pots filled with sterile vermiculite and let grow for 3 days (Figure 4A).
NOTE: Growing seeds in vermiculite is crucial to obtain plants with straight roots for easy transfer to pouches. It is important to allow the pea seedlings to develop for 3 days as the length of the radicle is optimal at this time.
3| Three days later, prepare to transfer one seedling per germination pouch (cyg™ seed germination pouch, Mega International, St-Paul, MN). Make a hole with a clean razor blade in the top centre of the pouch filter paper, so that the root enters the pouch but the cotyledons do not fall through the hole.
4| Wet the pouch with sterile low-nitrogen nutrient solution [as in [44]] until the entire surface of the filter paper has absorbed liquid (~ 7 mL).
NOTE: Micro-nutrients such as boron are required for nodules to develop [46]. It is essential to supply these nutrients instead of water alone as the seedling will not have access to them in a soil-free environment.
5| Remove gently the seedlings from the pots, and the seed-coat from the seedling, and place the primary root through the hole previously made (Figure 4B). The root must be in contact with the moist filter paper of the growth pouch.
6| Tape the top of the pouch closed, leaving a hole for the shoot to emerge (Figure 4B). Taping the pouch helps to maintain a moist inner environment preventing the roots from drying out. It also prevents vermiculite from entering the pouch (see below).
7| Cut a piece of the growth pouch 2.5 cm from its bottom with a pair of scissors (Figure 4B). The filter paper within the pouch will thus be in contact with the moist vermiculite (see below); this will ensure a constant supply of water to the developing roots while at the same time prevent any water-logging. The sides of the pouch can be trimmed for a better fitting when the pouches are inserted into the pots.
8| Place the growth pouches with the seedlings in 15 cm diameter pots (Figure 4C) and fill those with sterile vermiculite to cover completely the pouches (Figure 4D).
NOTE: Covering the pouch with vermiculite prevents light from reaching the roots; this is important as exposure to light inhibits pea nodulation [47].
9| Moisten the vermiculite with water and place the pots in trays in a controlled growth-room. Water the plants by adding water to the tray. The vermiculite maintains a humid environment for the pouch and filter paper.
10| Two days before inoculation, grow a rhizobial culture as above.
NOTE: Do not water the plants during this time to reduce the amount of residual water in the pouch. If the pouches are too wet, the inoculum drop will not remain on the root. It is of prime importance to time the earlier watering so that at this stage the pouches are only damp.
11| After 5 days of growth in the pouches, lateral roots will be of an optimal length for spot-inoculation (Figure 4E). Remove the pouches from the pots and find under the dissecting microscope the zone that is the most susceptible to infection, i.e., the region where roots hairs are starting to emerge from the root. This location is usually 0.5-1 cm away from the root tip.
NOTE: Condensation on the inside of the plastic pouch can impede the viewing of roots. Gently lift the top layer of the pouch from the bottom and use a sterile filter paper to remove the water droplets.
12| Mark the zone of the most susceptibility on the outside of the pouch with a water-proof marker (Figure 4E). Do not damage the root when applying the marker; if needed, hold the top surface of the pouch away from the root and apply the mark to the desired location.
13| Lift the top plastic sheet at the bottom end of the pouch and apply with a micro-pipette onto the root a 0.5 μL drop of rhizobial suspension adjusted to the viable cell density of 6.4 × 104 CFUs/mL.
NOTE: Do not place the top of the pouch back onto the root right away; let the drop sink in the root and paper before closing the pouch.
14| Return the pouches to the pots and cover with vermiculite (Figure 4F). Water the plants when needed; a good indication to do so is given when the colour of the pouch changes as it starts to dry. Harvest at the desired time (Figure 4G).
15| In our experiments carried out under the described conditions, we observed nodulation events, such as divisions of pericycle cells, as early as 24 hours after spot-inoculation; pink nodules are seen at 10 DAI.
Fungal Spot-Inoculation of Pea
The protocol used in the fungal spot inoculation of pea begins with steps 1-9 of the rhizobial spot-inoculation protocol as stated above, except for step 4 where a low-phosphate nutrient solution [as in [48]] is used instead of a low-nitrogen one.
10| After 5 days of growth in pouches, prepare enough freshly autoclaved Phytagel™ (Sigma P8961) medium [0.3% in minimal medium [23,49]] so that there is approximately 1 mL of liquid per plant to be inoculated.
NOTE: The medium should be kept warm (60°C) to maintain its liquid form until the time of inoculation.
11| Remove each pouch from the pots; under axenic conditions, choose a location along one or more lateral roots for inoculation to occur. Mark this location on the top of the plastic sheet with a waterproof marker.
12| Lift the top plastic sheet at the bottom end and gently lift the lateral root chosen using tweezers. Apply 1 mL of liquid Phytagel™ to the filter paper of the growth pouch in the desired location below the selected lateral root. Using another set of tweezers, place a media plug containing the fungal inoculum onto the Phytagel™ and lower the root into place over the top of the plug, gently pushing the root into the medium of the plug.
13| Allow a minute or so for the plug to set in place and for the Phytagel-™ to solidify before lowering the plastic sheet. Return the pouches to the pots and cover them with fresh vermiculite. Water the plants when needed; however, be mindful of overwatering which can encourage fungal contamination of the pouches. Harvest plants at the desired time.
14| When harvesting the plants, remove the pouch from pots with special care so that none of the roots within the pouch are disturbed. Note the location of the plug and make cuts in the plastic to expose the root system. Remove the fragment of lateral root that was placed on top of the plug using a scalpel blade. Clear the root fragment and stain it with ink-vinegar [50] in order to observe fungal infection.
List of Abbreviations
CFU: colony forming unit; DAI: day after inoculation; DAP: day after planting; IAA: indole-3-acetic acid; LB: Luria Bertoni; rpm: rotations per minute.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SRC conceived, designed, and carried out the experiments of transformation and rhizobial spot-inoculation. LC conceived and performed the experiments of fungal inoculation. LHM made the SYM10 construct and discussed experimental approaches with SRC. SRC wrote the first draft of the manuscript. FCG directed the experiments and wrote the final draft of this manuscript. All authors have read and approved the final manuscript.
The work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery grant to FCG and by Wilfrid Laurier University. We would like to acknowledge Dr. Gérard Duc (INRA, Dijon, France) and Dr. Stewart Smith (EMD Crop Bioscience, Milwaukee, WI) for the sym10 pea mutants and the Rhizobium inoculum, respectively. We would like to thank Dr. Karel Novak (Institute of Microbiology, Prague, Czech Republic) and Dr. Elena Dolgikh (ARRIAM, St-Petersburg, Russia) for their comments on many forms of this manuscript. Finally, we would like to thank Dr. David Barker (INRA/CNRS, Toulouse, France) for welcoming FCG in his lab to initiate these experiments, and Emily Macdonald for her help with the macro-photography.
Ellis THN, Poyser SJ: An integrated and comparative view of pea genetic and cytogenetic maps.
New Phytol 2002, 153:17-25. Publisher Full Text
Dolgikh EA, Leppyanen IV, Osipova MA, Savelyeva NV, Borisov AY, Tsyganov VE, Geurts R, Tikhonovich IA: Genetic dissection of Rhizobium-induced infection and nodule organogenesis in pea based on ENOD12A and ENOD5 expression analysis.
Plant Biol 2011, 13:285-296. PubMed Abstract | Publisher Full Text
Szczyglowski K, Stougaard J: Lotus genome: pod of gold for legume research.
Young ND, Udvardi M: Translating Medicago truncatula genomics to crop legumes.
Curr Opin Plant Biol 2009, 12:193-201. PubMed Abstract | Publisher Full Text
Timmers ACJ, Auriac M-C, Truchet G: Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements.
Development 1999, 126:3617-3628. PubMed Abstract | Publisher Full Text
Genre A, Chabaud M, Timmers T, Bonfante P, Barker DG: Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermis cells before infection.
Plant Cell 2005, 17:3489-3499. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Somers DA, Samac DA, Olhoft PM: Recent advances in legume transformation.
Plant Physiol 2003, 131:892-899. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
de Kathen A, Jacobsen H: Cell competence for Agrobacterium-mediated DNA transfer in Pisum sativum L.
Transgenic Res 1995, 4:184-191. Publisher Full Text
Švábová L, Griga M: The effect of cocultivation treatments on transformation efficiency in pea (Pisum sativum L).
Plant Cell Tiss Org 2008, 95:293-304. Publisher Full Text
Krejčí P, Matušková P, Hanáček P, Reinöhl V, Procházka S: The transformation of pea (Pisum sativum L.): applicable methods of Agrobacterium tumefaciens-mediated gene transfer.
Acta Physiol Plant 2007, 29:157-163. Publisher Full Text
Bean SJ, Gooding PS, Mullineaux PM, Davies DR: A simple system for pea transformation.
Polowick PL, Quandt J, Mahon JD: The ability of pea transformation technology to transfer genes into peas adapted to western Canadian growing conditions.
Plant Sci 2000, 153:161-170. PubMed Abstract | Publisher Full Text
Grant JE, Cooper PA, McAra AE, Frew TJ: Transformation of pea (Pisum sativum L) using immature cotyledons.
Schneider A, Walker SA, Poyser S, Sagan M, Ellis THN, Downie JA: Genetic mapping and functional analysis of a nodulation-defective mutant (sym19) of pea (Pisum sativum L.).
Mol Gen Genet 1999, 262:1-11. PubMed Abstract | Publisher Full Text
Wen F, Zhu Y, Hawes MC: Effect of pectin methylesterase gene expression on pea root development.
Plant Cell 1999, 11:1129-1140. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ding Y, Oldroyd GED: Positioning the nodule, the hormone dictum.
Plant Signaling & Behavior 2009, 4:89-93. PubMed Abstract | Publisher Full Text
Hansen J, Jørgensen J-E, Stougaard J, Marcker KA: Hairy roots - a short cut to transgenic root nodules.
Plant Cell Rep 1989, 8:12-15. Publisher Full Text
Hohnjec N, Perlick AM, Pühler A, Küster H: The Medicago truncatula sucrose synthase gene MtSucS1 is activated both in the infected region of root nodules and in the cortex of roots colonized by arbuscular mycorrhizal fungi.
MPMI 2003, 16:903-915. PubMed Abstract | Publisher Full Text
Díaz CL, Melchers LS, Hooykaas PJJ, Lugtenberg BJJ, Kijne JW: Root lectin as a determinant of host-plant specificity in the Rhizobium-legume symbiosis.
Nature 1989, 338:579-581. Publisher Full Text
Edwards A, Heckmann AB, Yousafzai F, Duc G, Downie JA: Structural implications of mutations in the pea SYM8 symbiosis gene, the DMI1 ortholog, encoding a predicted ion channel.
MPMI 2007, 20:1183-1191. PubMed Abstract | Publisher Full Text
Niwa S, Kawaguchi M, Imaizumi-Anraku H, Chechetka SA, Ishizaka M, Ikuta A, Kouchi H: Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501.
MPMI 2001, 14:848-856. PubMed Abstract | Publisher Full Text
van Noorden GE, Kerim T, Goffard N, Wiblin R, Pellerone FI, Rolfe BG, Mathesius U: Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti.
Plant Physiol 2007, 144:1115-1131. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chabaud M, Venard C, Defaux-Petras A, Bécard G, Barker DG: Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi.
Collier R, Fuchs B, Walter N, Lutke WK, Taylor CG: Ex vitro composite plants: an inexpensive, rapid method for root biology.
Plant J 2005, 43:449-457. PubMed Abstract | Publisher Full Text
Duc G, Messager A: Mutagenesis of pea (Pisum sativum L.) and the isolation of mutants for nodulation and nitrogen fixation.
Plant Sci 1989, 60:207-213. Publisher Full Text
Sagan M, Huguet T, Duc G: Phenotypic characterization and classification of nodulation mutants of pea (Pisum sativum L.).
Plant Sci 1994, 100:59-70. Publisher Full Text
Madsen EB, Madsen LH, Radutoiu S, Olbryt M, Rakwalska M, Szczglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J: A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals.
Nature 2003, 425:637-640. PubMed Abstract | Publisher Full Text
Stougaard J, Abildsten D, Marcker KA: The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants.
Mol Gen Genet 1987, 207:251-255. Publisher Full Text
Kereszt A, Li D, Indrasumunar I, Nguyen CD, Nontachaiyapoom S, Kinkema M, Gresshoff PM: Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.
Nature Protoc 2007, 2:948-952. Publisher Full Text
Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R: RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.
J Exp Bot 2004, 55:983-992. PubMed Abstract | Publisher Full Text
Fåhraeus G: The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique.
J Gen Microbiol 1957, 16:374-381. PubMed Abstract
van Brussel AAN, Bakhuizen R, Van Spronsen PC, Spaink HP, Tak T, Lugtenberg BJJ, Kijne JW: Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium.
Science 1992, 257:70-72. PubMed Abstract | Publisher Full Text
Albrecht C, Geurts R, Lapeyrie F, Bisseling T: Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A.
Plant J 1998, 15:605-614. Publisher Full Text
Fred EB, Haas ARC: The etching of marble by roots in the presence and absence of bacteria.
J Gen Physiol 1919, 1:631-638. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Turgeon BG, Bauer WD: Spot inoculation of soybean roots with Rhizobium japonicum.
Protoplasma 1983, 115:122-128. Publisher Full Text
Bourion V, Rizvi SMH, Fournier S, de Larambergue H, Galmiche F, Marget P, Duc G, Burstin J: Genetic dissection of nitrogen nutrition in pea through a QTL approach of root, nodule, and shoot variability.
Theor Appl Genet 2010, 121:71-86. PubMed Abstract | Publisher Full Text
Díaz CL, van Spronsen PC, Bakhuizen R, Logman GJJ, Lugtenberg EJJ, Kijne JW: Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots.
Planta 1986, 168:350-359. Publisher Full Text
Bhuvaneswari TV, Turgeon BG, Bauer WD: Early events in the infection of soybean (Glycine max L. Merr) by Rhizobium japonicum. I. Localization of infectible root cells.
Plant Physiol 1980, 66:1027-1031. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vergnaud L, Chaboud A, Prin Y, Rougier M: Preinfection events in the establishment of Alnus-Frankia symbiosis: Development of a spot inoculation technique.
Plant Soil 1985, 87:67-78. Publisher Full Text
Fortin JA, Bécard G, Declerck S, Dalpé Y, St-Arnaud M, Coughlan AP, Piché Y: Arbuscular mycorrhizae on root-organ cultures.
Can J Bot 2002, 80:1-20. Publisher Full Text
Estrada-Navarrete G, Alvarado-Affantranger X, Olivares J, Guillen G, Diaz-Camino C, Quinto C, Gresshoff PM, Sanchez F: Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes.
Nature Protoc 2007, 2:1819-1824. Publisher Full Text
Bondali K, Gherbi H, Franche C, Bastien G, Fardoux J, Barker D, Giraud E, Cartieaux F: The nod factor-independent symbiotic signaling pathway: Development of Agrobacterium rhizogenes-mediated transformation for the legume Aeschynomene indica.
MPMI 2010, 23:1537-1544. PubMed Abstract | Publisher Full Text
Lin M-H, Gresshoff PM, Indrasumunar A, Ferguson BJ: pHairyRed: A novel binary vector containing the DsRed2 reporter gene for visual selection of transgenic hairy roots.
Mol Plant 2011, 4:537-545. PubMed Abstract | Publisher Full Text
Guinel FC, LaRue TA: Light microscopy study of nodule initiation in Pisum sativum L. cv Sparkle and in its low-nodulating mutant E2 (sym5).
Pepper AN, Morse AP, Guinel FC: Abnormal root and nodule vasculature in R50 (sym16), a pea nodulation mutant which accumulates cytokinins.
Ann Bot 2007, 99:765-776. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Brenchley WE, Thornton HG: The relation between the development, structure and functioning of the nodules on Vicia faba, as influenced by the presence or absence of boron in the nutrient medium.
Proc Roy Soc London B 1925, 98:373-399. Publisher Full Text
Lee KH, LaRue TA: Ethylene as a possible mediator of light- and nitrate-induced inhibition of nodulation of Pisum sativum L. cv Sparkle.
Plant Physiol 1992, 100:1334-1338. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Resendes CM, Geil RD, Guinel FC: Mycorrhizal development in a low nodulating pea mutant.
New Phytol 2001, 150:563-572. Publisher Full Text
Bécard G, Fortin JA: Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots.
New Phytol 1988, 108:211-218. Publisher Full Text
Vierheilig H, Coughlan AP, Wyss U, Piché Y: Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi.


text:
Browse our large selection of reflective paracord. We have the best prices anywhere on reflective paracord. In addition to the best prices, we have the largested selection of reflective colors.
The braid within this paracord consists of a reflective strand. This allows light to bounce off and make visibility very easy at night. It works great for bracelets, dog collars and leashes and even motorcyclists use this paracord.


text:
MBB Dailies
Going For the Golden Glow
February 22nd, 2013
A $6.99 Fresh Glow
While Hollywood, celebrities and announcers are prepping, spraying and smearing on potions and formulas to get their skin glowing, I am excited to share a $6.99 product: NYX Born To Glow Liquid Illuminator.
First, I was impressed with the whole NYX cosmetic line, beautiful shades for eyes, lips and face; in fact, I am still evaluating it now and plan to mention many more fabulous and affordable products.
But, I was really impressed with Born To Glow Liquid Illuminator. Don't get scared over the word "illuminator", I'll admit I was, and I am well aware that certain shimmers women over 40 should stay away from. This liquid is different, it offers a subtle gleam that gives you that candlelight glow or blur enhancing your skin and the peach/bronze tone lights up the face with freshness.
This product gets thumbs up, and in fact I'm on my way to reordering it now at ulta.com.


text:
Water resources strategy supports industry rejig
ENDS Report 411, April 2009, pp. 40-41
The Environment Agency's water resources strategy for England paints an alarming picture of a water-stressed future. It advocates restructuring the water industry to abolish financial incentives on firms to supply more water and suggests unbundling firms' supply and retail arms may allow more integrated resource management.
Please sign in or register to continue.
Please sign in or register to continue.
New to the ENDS Report? Please register for free limited access.
Already subscribe but don't have a password?
Activate your web account here


text:
The defending Super Bowl-champion New Orleans Saints had the luxury of playing in their own backyard during last year's NFC Playoffs en route to the franchise's first NFL title.
Now they'll have to go on the road to open the 2010 postseason, which begins with Saturday's matchup against the NFC West-champion Seattle Seahawks from Qwest Field.
The Saints were 6-2 on the road this season, giving them consecutive winning records away from the Big Easy for the first time since the 1991-92 campaigns, so it shouldn't be an issue competing in the Emerald City.
"We're going to have to have a good week of practice. We recognize that [the week] is a little bit shorter and we'll be traveling out there for the first game Saturday," said Saints head coach Sean Payton in regards to preparing for the Seahawks. "We're looking forward to the challenge and we know it's going to be a tough place to play."
Since Payton took over as head coach in New Orleans, his teams are 4-1 in the playoffs with a pair of NFC title game appearances and of course, last year's win over Peyton Manning and the Indianapolis Colts in Super Bowl XLIV.
Saints Pro Bowl quarterback Drew Brees just joined Manning as the only signal- callers in league history to pass for more than 4,000 yards in five consecutive seasons, and finished the regular season ranked first in the NFL in completion percentage, second in completions, tied for second in touchdown passes and ranked third in passing yardage. Brees originally broke the NFL record for completions in a season with 448, but Manning took over in the final week of 2010 to finish with 450.
The Saints will be counting on Brees to get them their first-ever playoff win on the road against a team he lit up back in Week 11 at the Superdome in leading New Orleans to a 34-19 victory. Even though the Seahawks racked up 424 yards of offense in that game, New Orleans outdid them with 494 total yards, thanks to Brees' 383 yards through the air and four touchdown passes. Wide receiver Marques Colston had a great day as well for the Saints, coming up with 113 yards and a pair of scores on eight receptions.
Colston, Lance Moore and Robert Meachem will see plenty of chances to make a play on Saturday as New Orleans looks to get its third-ranked passing attack in gear in one of the loudest stadiums in the league. The team won't have leading rusher Chris Ivory due to a season-ending foot injury, however. Ivory had 99 yards and a score in the Saints' rout of the Seahawks on Nov. 21 and compiled 716 rushing yards in his rookie season.
New Orleans is 5-6 overall in the postseason and is a heavy favorite to even that mark on Saturday, though the franchise has never won in three lifetime playoff games at enemy venues.
Seattle entered the record books by becoming the first team in league history to win a division with a sub-.500 record. It completed the 2010 season with a 7-9 mark and wrestled the NFC West away from the St. Louis Rams with this past Sunday's 16-6 triumph at Qwest Field.
To the victor go the spoils was the motto for that matchup, as the Seahawks earned their fifth division title in the past seven years and registered their sixth playoff appearance over the previous eight seasons by topping the Rams. They hadn't been to the playoffs since the 2007 season and made a Super Bowl appearance after going 13-3 in the 2005 campaign.
Head coach Pete Carroll was able to get the franchise back into the postseason in his first year at the helm, but is currently faced with a dilemma at quarterback.
Since Matt Hasselbeck was sidelined for last weekend's division-clinching win with a hip injury suffered a week earlier against Tampa Bay, Carroll handed the job to backup Charlie Whitehurst, who did just enough to get the win by completing 22-of-36 passes for 192 yards with a touchdown and no interceptions. Hasselbeck and Whitehurst split the reps in Seattle's first practice in preparation for the Saints, and Carroll said the two will share snaps throughout the week before a decision is made on who starts.
"We'll figure it out, we don't know yet," Carroll was quoted as saying in the Seattle Post Intelligencer. "Right now our concerns are about health, making sure everyone's OK. The way the game worked out, it really helped us. Charlie did a great job to survive that game. The rush was on him, he was running for his life at times."
Hasselbeck is a semi-mobile quarterback, but would have struggled avoiding the rush against the Rams with an ailing hip. With an extra week of rest, he may get the nod for Saturday's game just by reputation. Hasselbeck is one start away from tying former offensive tackle Walter Jones for most postseason starts in franchise history, and he holds club playoff records for pass attempts (325), completions (189), yards (2,211) and touchdowns (11).
Seattle has a tough task at hand with the defending Super Bowl champions in town and hasn't played all that well in the past few weeks. Sunday's 10-point triumph over the Rams was just the team's third victory in its past 10 games.
The Saints took a 6-5 edge in their all-time regular-season series with the Seahawks by virtue of the above-mentioned 15-point win at the Superdome back in Week 11. New Orleans also posted a 28-17 victory in its last visit to Qwest Field, which took place in 2007 and halted a three-game losing streak in the set, and is 3-3 in six overall trips to Seattle. These teams have never faced one another in postseason play.
Three of Payton's four playoff wins with the Saints occurred during their run to the Super Bowl last year. Carroll is 1-2 in the playoffs, with each of those contests coming during his three-year tenure with New England from 1997-99.
Carroll is 0-2 lifetime against the Saints, including a 1998 loss while with the Patriots. Payton owns a 2-0 career mark against Seattle, with November's meeting his only head-to-head encounter with the former USC head coach.
The Saints' claim to fame is their rabid pass attack, which finished third in the NFL with an average of 277.6 passing yards per game in the regular season. In order for them to keep moving on in the playoffs, however, Brees (4,620 passing yards, 33 TD, 22 INT) will have to be on the same page with all of his receivers and the offensive line must offer the gold standard in protection. Brees has been sacked seven times in the past three games, including a trio of times in last week's 23-13 loss to Tampa Bay. The front line was able to keep Brees' jersey clean on a run to the Super Bowl last season, which helps explain why he has an NFL-best 66.7 completion percentage in the postseason. Brees' 103.7 passer rating in the playoffs is second-highest in league annals — only behind Green Bay legend Bart Starr (104.8). Brees, who is hoping to at least duplicate the type of performance he had against Seattle more than a month ago, threw for a season-low 196 yards versus Tampa Bay on Sunday, and his 22 interceptions this season are a career high. Since Ivory was placed on injured reserve this week, Pierre Thomas, Julius Jones and Reggie Bush will carry the load for the 28th-ranked ground attack. Bush (150 rushing yards) compiled a team-high 70 yards on nine carries versus the Bucs, while Thomas (269 rushing yards, 2 TD) is second behind Ivory in rushing yards but is considered questionable due to an ankle injury.
Not known for having a stout defense, which was a problem area during the team's stretch of seven losses over the last 10 games of the regular season, the Seahawks did enough to defeat the Rams by holding them to 184 total yards. Defensive end Raheem Brock (32 tackles, 9 sacks) recorded 2 1/2 sacks on Rams rookie Sam Bradford, while Chris Clemons (49 tackles) led the Seahawks in that category with a career-high 11 for the season. Safety Jordan Babineaux (46 tackles, 2 sacks, 2 INT) had two of the team's seven pass breakups this past weekend, while outside linebacker Will Herring (36 tackles, 1 sack) had the lone interception of Bradford — the first of his career. The Seahawks not only took Bradford's targets out of the picture, but also held Pro Bowl running back Steven Jackson to 45 rushing yards and the Rams to 47 total on the ground. Seattle is 21st against the rush and has a solid corps of linebackers led by Lofa Tatupu (88 tackles, 1 sack) in the middle. Outside linebacker Aaron Curry (73 tackles, 4 sacks) has established himself as one of the best and quickest linebackers in the league, but had no tackles against the Rams. That should change this week with Bush and a possibly Thomas coming out of the flats. Veteran safety Lawyer Milloy (88 tackles) is grateful for having a chance to be back in the playoffs and finished tied with Curry for second on the team with four sacks. Rookie safety Earl Thomas (75 tackles) had one of two interceptions of Brees in the first meeting with New Orleans.
Hasselbeck (3,001 passing yards, 12 TD, 17 INT) seemed to have no trouble moving the football against the Saints last November. If the long-time starter does get the nod over Whitehurst, the Saints must be cautious of his smarts and experience. Hasselbeck has the ability to improvise when the play breaks down and tried convincing Carroll to play against the Rams. The head coach opted for Whitehurst (507 passing yards, 2 TD, 3 INT), who had trouble getting the offense to find success on third down, as the Seahawks converted 4-of-15 tries in those situations against the Rams. St. Louis was no better, however, managing a 2-for-14 clip. The Seahawks finished 19th in passing during the regular season and got a breakout performance from wideout Mike Williams (65 reception, 2 TD). Prior to the season, Williams was signed to a one-year contract despite having been out of the league the past two seasons, and worked his way to starting status while a three-year contract extension after leading the Seahawks in catches and receiving yards (751) and hauling in Whitehurst's only touchdown pass last Sunday. Ben Obomanu (30 receptions) finished tied for the team lead with four touchdown catches with four and racked up a game-high five receptions for 39 yards against St. Louis. Wide receiver Ruvell Martin (7 receptions, 1 TD) even got into the act with a game-best 85 yards on three receptions. Marshawn Lynch and Justin Forsett have anchored Seattle's 31st rushing attack.
The Saints were dead last this season with just nine interceptions, with safety Malcolm Jenkins (64 tackles, 1 sack) and corner Jabari Greer (61 tackles) leading the way with two apiece. New Orleans has the fourth-ranked defense in the NFL and is second against the pass, however. Seattle will try to get the ball to Williams as often as possible and the former first-round pick wants to prove his new contract was deserving. Bucs quarterback Josh Freeman threw for 255 yards and two scores against a New Orleans defense that registered four pass breakups last week, while Hasselbeck was able to throw for 366 yards in the first meeting with the Saints, who only allowed one touchdown pass with no interceptions that day. Defensive coordinator Gregg Williams will dial up the blitz in his relentless play-calling style, possibly going with a few corner attacks or with Jenkins or fellow safety Roman Harper (98 tackles, 3 sacks, 1 INT). New Orleans had no sacks in the first meeting with the Seahawks, but if Carroll goes with a banged-up Hasselbeck with limited mobility in this one, the defense certainly has a chance to improve on that total. Defensive end Will Smith (39 tackles, 6 sacks) has recorded a sack in each of the last two games, while counterpart Alex Brown (39 tackles, 2 sacks) recorded just his second of the season versus Tampa. Tackle Sedrick Ellis (44 tackles, 6 sacks) is tied with Smith for the team lead in sacks. Saints linebacker and leading tackler Jonathan Vilma (105 tackles, 4 sacks) was just added to the NFC Pro Bowl roster on Tuesday.
Courtney Roby was the Saints' primary kickoff returner for much of this season, averaging 23.8 yards on 33 tries, but was placed on injured reserve prior to the Tampa Bay game. Devery Henderson took over those duties last week and will be trying to set up Brees and the offense in good territory. Bush and Moore had 14 and 11 punt returns, respectively, this season. Bush missed several weeks dealing with a broken leg and returned one punt for seven yards last week. Kicker Garrett Hartley bounced back from an ugly start to finish with an 81.4 percent field goal percentage. He made 6-of-7 tries between 40-to-49 yards. Thomas Morstead is the team's punter and averaged 45.9 yards on 57 attempts.
Leon Washington is Seattle's main return man on both kickoffs and punts. He had 57 returns for 1,461 yards this season, averaging 25.6 yards per try. On 22 punt returns, Washington tallied 249 yards for an average of 11.3 per runback, and Carroll will be hoping the Saints don't decide to kick away from one of his best weapons. Rookie wide receiver Golden Tate has experience returning kicks too. Kicker Olindo Mare had an up-and-down campaign in the Emerald City this season, making 79.5 percent of his field goal attempts. Punter Jon Ryan averaged 41.7 yards on 78 punts for the year.
The Saints are aware they could be headed for a trap, since they're so heavily favored to dominate this postseason showdown with the Seahawks. The defending champions proved they can win on the road during the regular season, but must get that first playoff road victory out of the way. Who else besides Brees would New Orleans want under center to achieve that feat? A strong effort from the running backs will take the pressure off of Brees in a decisive triumph at Qwest Field, where opponents tend to struggle because of the noise. The Saints will turn that boisterousness into a Mardi Gras remix.
Tags: Coach Sean Payton, Completion Percentage, Completions, Consecutive Seasons, drew brees, Emerald City, first game, Indianapolis Colts, League History, New Orleans Saints, Nfc Playoffs, Nfc West, nfl record, Nfl Title, Peyton Manning, SEATTLE SEAHAWKS, Signal Callers, Super Bowl Xliv, title game
Fair Use Notice This website may at times present copyrighted material, the use of which might not always been specifically authorized by the copyright owner. Such material is made available in an effort to advance understandings of democratic, economic, environmental, human rights, political, scientific, and social justice issues, among others. The author believes that this constitutes a fair use of any such copyrighted material as provided for in section 107 of the U. S. Copyright Law. In accordance with Title 17 U.S.C. Section 107, the articles published on this website are distributed without profit for research and informational purposes. In most instances a link is placed to originator of Article and it is never expressly mentioned as written by, we use published by certain entities who write or publish for this said Blog..


text:
Did Lois Lerner Violate IRS Policy on Email Preservation?
Perhaps the crashed hard drive is not the problem.
June 17, 2014 - 11:24 am
Lois Lerner told the IT technician who was unable to recover the data from her crashed hard drive, "Sometimes stuff just happens."
A lot is being made of both the alleged crash of Lois Lerner's hard drive and the lack of backup tapes for the missing time period in the congressional investigation into the IRS's targeting of conservative groups. The IRS told congressional investigators last week that two years' worth of emails sent and received by Lerner, the former IRS official at the center of the investigation, have been destroyed because of a computer crash in mid-2011. And now, we learn, the hard drives of six other IRS employees also crashed and the emails have gone missing. But some of the "stuff" that happened to Lois Lerner's email may be the direct result of her failure to follow IRS policy.
The IRS had policies in place in 2011 to prevent destruction of federal records, including emails. At the time, they also had a policy that limited employee mailboxes to 500 MB, so when employees approached their limit they were required to delete emails from the server to make room.
The Secure Enterprise Messaging system (SEMS) establishes a standard size of 500 MB (500 megabytes) for individual mailboxes. The system mails you daily warning messages that the limit is being approached when your mailbox reaches a size of 475 MB. When it exceeds the 500 MB limit, you will receive the following warning each time you attempt to send a message:
- "You have exceeded your storage limit on your mailbox " .
- Delete some mail from your mailbox or contact your system administrator to adjust your storage limit. (Consider whether any of the items you want to delete may be a federal record. IRM 18.104.22.168.2 above.)
When an email is sent or received it goes to the email server, called an exchange. From there, the email client (Microsoft Outlook, your phone, your home computer) can access the email. It is then stored on the server, sometimes remaining there well beyond the moment the user actually deletes the email from the client. (There are also multiple logs that record all of the actions related to the emails and the servers.) Emails can also be saved in a file on a local computer, so if they're deleted from the server, the individual still has a record of the correspondence.
The IRS is apparently alleging two things: that Lerner's personal computer crashed, rendering emails stored on her computer unrecoverable and also that there is no backup of what was on the email servers in 2011 because the backup tapes are overwritten every six months. Many IT professionals, including a former IRS employee, have called into question the IRS's assertion that the agency has no backup of data stored on the servers for the time period in question. In fact, IRS commissioner Koskinen testified in March that IRS emails, including Lois Lerner's, "get taken off and stored in servers." The IRS also says it was able to recover 24,000 emails that related to Lerner, some of them from an "earlier 2011 data collection of Ms. Lerner's email." This would seem to confirm Commissioner Koskinen's statement that the emails are "stored in servers" since the IRS says they were unable to recover any emails from Lerner's hard drive.
In a letter explaining the missing emails to Congress the IRS said, "Any of Ms. Lerner's email that was only stored on that computer's hard drive would have been lost when the computer crashed and could not be recovered." IT professionals are also scoffing at the notion that the data was not recoverable.
But before we even get to the problem of a possible backup failure or the computer crash, we must consider how Lerner got to the point of having "email that was only stored on the computer's hard drive."


text:
Please enter your current email address to receive instructions on resetting your password.
Your password has been reset. Please check your email for further instructions.
You are sending a link to the page. Please completely fill out the form
Add an additional message below.
The URL for the page will automatically be added to your message.
Quote from the book by Paulette Davis Horton, titled "Death in 60 Days, Who Silenced Booker T. Washington? A Nurse's View":
"In praising the works of Booker T. Washington, S.G. McCormick once asked Julius Rosenwald, 'How did he get his amazing common sense; whence came his keenness and accuracy of intuition; who taught him the meaning of beauty, of order, of system, of the relationship of little things to big? How did he learn to conceive of duties and rights as he did and never to speak of the one without emphasizing the other? What other dreamer among the stars ever walked so firmly upon the earth and saw realities and put them into all of his life as did Booker T. Washington?
This facility meets ADA Accessibility Guidelines for wheelchair accessibility, including an accessible entrance,
access to goods and services, usability of rest rooms and accessible parking and drop-off areas. For additional
details, please see the ADA website.


text:
The Collection
Use the field below to browse the Currier & Ives Collection at Springfield Museums
Bust portrait sketch of Charles Dickens looking off to his left. Black and white. From the Inventory Listing of Sidney A. Alpert "Boz/ (Charles Dickens), N. Currier - S "
Date: 1839
Artists: Nathaniel Currier
Medium: Hand-colored lithograph
Measurements: 14 1/2 x 18 1/4" inches
Access Number: 2004-D03-612
Gift of Lenore B. and Sidney A. Alpert supplemented with Museum Acquistions Funds
Users, log in to add this piece to your personal lightboxReturn To Search Results


text: For a very long time it's been way too difficult to set up Wing to debug Chandler. For my 20% project I decided to improve this. So I just checked in new "pre-built" Wing project (debug and release) for each of the platforms that "just work" -- well almost. There are a few minor snags, which I've documented in the wiki: http://wiki.osafoundation.org/bin/view/Projects/DebuggingChandler under Debugging Chandler. I've also included instructions on how to easily debug functional tests and included some important wing lore. I suspect I may have missed one or two points, so please give me your feedback for improvement. John P.S. Bear has not yet included the new project files in the developer builds, but they should appear shortly. In the mean time you'll find them in SVN in the Chandler directory.