Science Behind “Anthesis”

…

and …

2015 Grower Results

Matthew DeBacco

Anthesis

Cucurbit Maxus

About “Mad Scientist,” Matt D.

• Master’s in Agronomy from University of Connecticut • Thesis on Organic Suppression of Powdery Mildew on Cucurbit & Assessing

Decomposition of Fiber Pots

• Worked for UConn Extension Service with their Nutrient Management Program and was their in-field diagnostician for vegetable growers

http://assets.sbnation.com/assets/2459443/UConnHusky-Logo.jpg

About Mad Scientist, Matt D. • Growing pumpkins for last 16 years

• Has grown pumpkins continuously for 2,367 days

• Two-time CT State Record Holder – Personal Best: 1,766.5 pounds

Making the best of a ‘bad’ situation

• 2012 my patch was infected with Mosaic Virus

http://news.bbcimg.co.uk/media/images/44014000/jpg/_44014297_gloc.jpg

Cucumber Mosaic Virus

Zucchini Yellow Mosaic Virus

Matt DeBacco’s patch, 2012 http://fatalii.net/chile/kuvat/kirvasika3532532.jpg

What the Virus in my patch taught me

• Pumpkin cells, after 20 days post pollination, are no longer actively dividing.

• All younger pumpkins (under 20 days) developed viral mosaic patterns, but the one older pumpkin in the patch never showed visual symptoms even though the entire plant was severely infected.

http://schoolworkhelper.net/wp-content/uploads/2010/07/Viral-replication.gif http://www.biology.iupui.edu/biocourses/N100/images/8mitosiscropped.jpg

This led to two years of research into the scientific literature.

• Mostly, it led to dead ends.

http://sellingsimplified.com/main/wp-content/uploads/Dead-End-Good-Ways-To-Make-Money1.jpg

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https://aidanmkeith.files.wordpress.com/2012/11/piles.jpg

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Putting the pieces together was a journey

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Two Key Articles

Tomato Fruit Growth

…

Integrating Cell Division, Cell Growth and

Endoreduplication by Experimentation and

Modeling

• 145 pg Doctoral Thesis by Julienne Fanwoua

• Early Application of Ethrel Extends Tomato Fruit Cell Division and

Increases Fruit Size and Yield with Ripening

Delay

Atta-Aly, M.A., G.S. Riad, Z.S. El-Lacheene, and A.S. El-Beltagy. 1999. Early application of ethrel extends tomato fruit cell division and increases fruit size and yield with ripening delay. J. Plant Growth Regulat.18:15–24.

• Cell division, cell growth, and cell endoreduplication are three fundamental cellular processes responsible for fruit growth.

http://www.biology.iupui.edu/biocourses/N100/images/8mitosiscropped.jpg http://www.biw.kuleuven.be/biosyst/mebios/OT2012_MAP/WP1

http://revivechurch.org/revivechurchorg/wp-content/uploads/2013/10/what-i-learned-11.png

http://plantsinaction.science.uq.edu.au/book/export/html/70

Cell cycle

http://www2.le.ac.uk/departments/genetics/vgec/diagrams/22-Cell-cycle.gif

Mitosis

http://www2.le.ac.uk/departments/genetics/vgec/diagrams/38%20mitotis%20phases.jpg

Mitosis

http://media1.shmoop.com/images/biology/biobook_dna_graphik_26.png

Cell Growth = Cell Expansion

• Same number of cells, but each cell is increasing in size (swelling).

http://openi.nlm.nih.gov/imgs/512/151/2530862/2530862_gb-2008-9-7-226-1.png

Cell Expansion • Contributes by far the most to fruit growth

http://postharvest.tfrec.wsu.edu/pages/N4I1B

Cell Expansion (alone)

• Can increase cell size up to 11,000 fold (of initial volume)

Fruit Size is determined by…

• Number of cells and

• Size of individual cells

http://img.webmd.com/dtmcms/live/webmd/consumer_assets/site_images/articles/health_tools/fat_fighting_foods_slideshow/webmd_rf_photo_of_grapes_and_raisins.jpg http://images.calorieking.com.au/branding/dc/runtime/portionsense/140.jpg

https://m1.behance.net/rendition/modules/45655451/disp/bbaa72c9b1867efdfd1e3504eb55f657.jpg

Cell Division Mitosis

• *Genetic variation in final fruit size (large vs. small

fruited melons) was mainly accounted for by variation in final pericarp cell number caused by differences in cell division duration

http://openi.nlm.nih.gov/detailedresult.php?img=2685393_1471-2229-9-49-6&req=4 Figure 6: Pericarp growth following anthesis.

Final Pericarp Mass

• The duration of cell division had the largest influence on final cell number and final pericarp mass.

http://upload.wikimedia.org/wikipedia/commons/thumb/1/11/Drupe_fruit_diagram-en.svg/2000px-Drupe_fruit_diagram-en.svg.png http://www.fruitsinfo.com/images/fruit3.gif

Limited Time for Cell Division

• Duration of cell division after anthesis ranges from 1-2 weeks in cucurbits and tomatoes

– Age at which cells cannot divide = 16 days

• Under normal conditions

http://rbcweb.shopfactory.com/contents/media/limited_time.gif http://3.bp.blogspot.com/-DxHXoZ70QWw/TfaPndvpj5I/AAAAAAAAAOQ/GcFuJjwxTtw/s1600/16DaysLogo-1.png

http://static.flickr.com/50/159047357_4cba94d98f.jpg

Summary

• * Cell division is of great importance in the variation of final fruit size.

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http://1.bp.blogspot.com/-V-hSF9LKAVE/TzYhENKokKI/AAAAAAAAAGk/8YGbf4xjdHc/s1600/Elodea.jpg

http://www.mbg.com.my/MBG/images/stories/promotion/mango-size01.jpg

So….. Now what?

• How can I apply this knowledge?

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Plant Hormones Influence Cell Signaling

http://www.mhhe.com/biosci/genbio/enger/student/olc/art_quizzes/genbiomedia/0486.jpg

Ethylene • Often thought of as just a ripening hormone…

http://labs.bio.unc.edu/Kieber/Figures/Ethylene.jpg http://upload.wikimedia.org/wikipedia/commons/thumb/3/3b/Ethylene-CRC-MW-dimensions-2D.png/160px-Ethylene-CRC-MW-dimensions-2D.png

http://i.ytimg.com/vi/A2qoQgrkE_U/hqdefault.jpg

Why not Gibberellic acid (GA)?

http://www.extension.org/sites/default/files/w/e/e1/GA_cluster.jpg

http://5e.plantphys.net/images/ch20/wt2002b_s.jpg

http://www.sciencephoto.com/image/442065/350wm/C0118720-Effect_of_Gibberellic_Acid_on_Cabbage-SPL.jpg

Why Ethylene and not Gibberellin? -Direction of cell growth is influenced by effects on cellulose microfibril orientation. -Giberrellins lead to growth mainly to the longitudinal direction (elongation) -Ethylene promotes a more random orientation and growth in all three dimensions (swelling)

(Based on Raven et al. 1992)

http://plantsinaction.science.uq.edu.au/edition1/?q=figure_view/290

Timing is Very Important • Applying the same

product at different stages of plant development results in very different outcomes

http://img.pandawhale.com/post-8936-Woman-at-beach-Perfect-timing-N0Ig.jpeg http://www.macforensicslab.com/ProductsAndServices/images/icon_Timing.jpg

Ethylene = Ripening*

• *When applied later in development

http://cen.acs.org/content/cen/articles/92/web/2014/11/Engineered-Bacteria-Ripen-Fruit-Belching/_jcr_content/articlebody/subpar/articlemedia_0.img.jpg/1416932919785.jpg

http://ripening-fruit.com/yahoo_site_admin/assets/images/tomato_ripening_cherries_big.121132938_std.jpg

http://www.scielo.br/img/revistas/bjpp/v17n3/a02fig01.jpg

http://www.californiaavocado.com/blog/wp-content/uploads/2014/06/ripen-avocado-in-bag.png

Ethylene = Causes plants to only produce female flowers* (when applied at the 1-5 leaf stage)

• *For cucumbers, apply 250 to 500 ppm when plants have 1,3 and 5 leaves

• Induces gynoecious female line for hybrid seed production

http://www.harvesttotable.com/wp-content/uploads/2009/05/Cucumber-flower1.jpg

*Anthesis*

• Anthesis = the period of blooming in flowers

http://www.bigpumpkins.com/DiaryImages/2014/Lrg/219464.jpg

https://media.licdn.com/media/p/6/005/00f/151/07d5b5d.png

Anthesis = [an-thee-sis] • The period or act of expansion in flowers,

especially the maturing of the stamens

• The action or period of opening of a flower • Origin of Anthesis

• New Latin, from Greek anthēsis bloom, from anthein to flower, from anthos

– First Known Use: circa 1823

http://dictionary.reference.com/browse/anthesis http://www.merriam-webster.com/dictionary/anthesis

http://www.scielo.br/img/revistas/aabc/2011nahead/aop4411fig02.jpg

Early Ethrel Application Extends Tomato Fruit Cell Division and Increases Fruit Size and Yield with Ripening Delay

• This document provides specific materials and methods

• Ethrel (2, chloroethyl phosphonic acid) is an ethylene-releasing substance

http://www.press.uchicago.edu/dms/ucp/books/jacket/978/02/26/31/9780226316567.jpeg

http://images.sciencedaily.com/2008/06/080628065632-large.jpg

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Ethrel® Application Extends Tomato Fruit

Cell Division

• Flowers were sprayed in the early morning two days after anthesis with 100ppm Ethrel®

– Tomato fruits produce their highest ethylene level at the stage of fruit set (48 hrs. after anthesis)

http://www.bigpumpkins.com/Diary/DiaryViewOne.asp?eid=221669 http://tvbythenumbers.zap2it.com/wp-content/uploads/2010/06/48-Hours_mystery.png

Ethrel® Application Extends Tomato Fruit Cell Division

• 100ppm applied two days after anthesis

• Cell Division was extended up to 16-18 days

– 10 -12 days = just water (control)

http://wid.wisc.edu/content/uploads/2012/09/cells-web-550x367.jpg

Ethrel® Application Increases Fruit Size and Yield with Ripening Delay

• Weight was significantly increased by 8% to 34%

– and ripened 10 days later than the (water) control.

http://bonnieplants.com/wp-content/uploads/bonnie-original-tomato.jpg http://i.bnet.com/blogs/srqpix.jpg

Trial #2

• When same treatment was applied to the entire plant one week after anthesis of the 1st flower cluster, yield was increased by 15%

http://grandpacliff.com/Plants/Img-Plants/plant-whole-pt.jpg

https://s-media-cache-ak0.pinimg.com/236x/6d/7d/c8/6d7dc84c3366eeb29fd59e283d7ab0e2.jpg

Terms Defined

• ACC = 1-Aminocyclopropane-1-carboxylic acid • 1-Aminocyclopropane-1-carboxylic acid (ACC) is a disubstituted cyclic alpha-

amino acid in which a three-membered cyclopropane ring is fused to the C(alpha)-atom of the amino acid.

• ACC plays an important role in the biosynthesis of the plant hormone ethylene. It is synthesized by the enzyme ACC synthase from methionine and converted to ethylene by ACC oxidase

• AOA = Aminooxyacetic acid • Aminooxyacetic acid (AOA) can also inhibit 1-aminocyclopropane-1-

carboxylate synthase preventing ethylene synthesis, which can increase the vase life of cut flowers.

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What about Cytokinins?

• Increase fruit size (in apples) by increasing number of cell layers

– not cell expansion

• Play an important role in determining the eventual cell number of the fruit

http://education-portal.com/cimages/multimages/16/Key_Concept.png http://www.efoodsdirect.com/media/catalog/product/cache/1/image/9df78eab33525d08d6e5fb8d27136e95/a/p/apples2.jpg

Ethylene and Cytokinins

• Ethylene production was stimulated by submicromolar concentrations of cytokinins in seedlings

http://www.barnaor.com/images/NewsImages/strips.jpg

http://bcs.whfreeman.com/thelifewire8e/content/cat_010/f37018.jpg

Site of application is important

• Localized applications of cytokinins to tomatoes is known to increase…

• Sink strength

• Fruit size and

• Fruit dry matter at harvest

– How? By enabling them to attract photoassimilates and nutrients from foliage

https://media.licdn.com/mpr/mpr/p/7/005/092/256/3a950e9.jpg

Source : Sink Relationship

http://www.frontiersin.org/files/Articles/73624/fpls-05-00011-HTML/image_m/fpls-05-00011-g001.jpg http://cnx.org/resources/cd8839949717d6b04f4fade2e6b0f7cf/Figure_30_05_07.jpg

Cytokinins are partially miscible in water -Slightly soluble in hot water

-Soluble in dilute acid

• Ethrel is an acid that could aid in the incorporation of cytokinis to water for even application to fruit

http://www.chem1.com/acad/webtext/solut/solut-images/miscibility.png http://www.uiowa.edu/~c004131a/f8_18.gif

So what does this all mean?

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http://upload.wikimedia.org/wikipedia/commons/2/21/Mosaicr_seagull.jpg

Research Conclusions

• Ethylene, when applied 48 hrs. post pollination, can extend cellular division about 6 more days

• End of season weight can increase by 15% (range of 8-34%)

• The pumpkin should ripen 10 days later

• Cytokinins can increase fruit sink strength, size and dry matter

http://image.slidesharecdn.com/researchconclusions5-111020040315-phpapp02/95/research-conclusions-1-728.jpg?cb=1319083428

The Directions are Simple (Supporting science is complex)

• Apply Ethylene + Cytokinins 48 hours after pollination

*directly to the fruit*

http://www.keepcalmandposters.com/posters/2413221.png http://biznology.com/wp-content/uploads/2014/05/artworks-000058593031-9xetyb-original.jpg

Anthesis Specific Observations

• Pumpkins should be smaller than untreated controls up to about day 35 – This indicates a potential delay in cell expansion due to the

increased time of cell division

• Anthesis-treated pumpkins should have improved late season growth rates – The theory is there are more cells present in the pumpkin

and also a ripening delay

https://7373-presscdn-0-43-pagely.netdna-ssl.com/wp-content/uploads/2013/06/binoculars1.jpg

2015 Grower Reported Data

“I will say the Anthesis-applied pumpkin was still growing, where the other fruits slowed way down.”

-Andy Box

“The Anthesis-treated pumpkins that I had going grew better than anything I ever had”

-Allen Bradly

http://www.gannett-cdn.com/-mm-/382e7a583110dfb611e22bc73f3f8339b9df2203/c=0-0-1629-1224&r=x404&c=534x401/local/-/media/GreatFalls/2014/08/11/leadphotogroupchat.jpg http://earlylearningcentral.ca/wp-content/uploads/2012/10/pumpkin-measuring.jpg

R² = 0.9965

R² = 0.9902

R² = 0.9983

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Anthesis and Untreated (Control) Pumpkins in Andy Box's 2015 Patch

Anthesis Treated

Untreated 1

Untreated 2

Graph 1 Summary

1. Figure 1: It is clearly evident that the Anthesis-treated pumpkin was of comparable size up to the critical day 35.

2. After day 35, the Anthesis-treated pumpkin had an increased growth rate that was sustained for the remainder of the season, resulting in his top producing pumpkin for the year.

3. Both of the untreated control pumpkins have a very similar growth curve for the season and ended up within 4.9% of each other

4. The Anthesis-treated is noticeably greater past day 40 and finished 13.0% greater than the average of the untreated pumpkins.

5. The graph supports the grower’s comments that the Anthesis-treated pumpkin continued to grow while the other fruits decreased in growth rate.

6. Trend lines use the Logarithmic Regression with R2 values provided.

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Growth Curves for Anthesis and Non-Treated Pumpkins Grown in 150sq.ft.

Anthesis Treated '15

Untreated '14

Untreated '13

Graph 2 Summary

1. Growth curves for Anthesis-treated and untreated (control) pumpkins grown in the same 150 sq.ft. location by Bart Toftness during three different years with almost daily measurements taken.

2. The growth curve shows that the Anthesis-treated pumpkin (diamond shape) was smaller up to day 37 than the untreated ones, suggesting a delay in cell expansion.

3. After day 37, the growth rate is accelerated and continues for an extended period of time, leading to an improvement in late season growth that increases final size of the pumpkin.

4. Day 60 and beyond, there is a continual upward trend of growth with no indication of a plateau.

5. Even though the Anthesis-treated pumpkin is the oldest, the delay in harvest was grower’s choice, as it was still showing positive gains and was allowed to remain on the vine until a later weigh-off.

6. The Anthesis-applied pumpkin grew the biggest pumpkin out of this space even though, up to day 30, it was the smallest.

Anthesis 2015 Harvest Data Summary

Top 10 Average for Anthesis-Treated Pumpkins 1819.85 lbs.

Top 20 Average for Anthesis-Treated Pumpkins 1643.25 lbs. … …

Total Number of Personal Best

for growers using Anthesis 8

Total Number of Top 2015 pumpkins

using Anthesis

(not counting personal bests)

6

… …

Pumpkins over 2,000 pounds 2

Pumpkins 1,900-2,000 pounds 2

… …

Tomatoes grown over 5 pounds 4

Improvements for 2016

• Ethrel liquid will be replaced with a powder

– Due to the small quantity of Ethrel needed, some growers reported evaporated samples.

• Small tubes will be of improved quality

– Small blue tubes last year had inconsistent quality

• Only pre-orders will be taken (Jan. 1st-March 15th)

– Makes it easier to know how much I need to prepare

• Orders will be mailed out in mid-April

– This will ensure growers have fresh product that will fit the 4 month self-life.

http://www.ddubcustomsportswear.com/wp-content/uploads/2014/08/improve.jpg

Anthesis Specifics

• The directions are not hard, but must be followed for effectiveness.

http://images3.cliqueclack.com/p/files/2014/07/INSTRUCTIONS-e1404879204784.jpg

Cucurbit (Part A)

• This is a fine grain powder – (very small quantity)

• Will easily dissolve in distilled water

• *Store in the refrigerator

• *Keep away from water until ready to use

• Does come in a sealed container

• Shelf-life of about 2 years @ refrigeration temps.

http://www.polyvore.com/cgi/img-thing?.out=jpg&size=l&tid=57680386

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Cost of Cucurbit

• $3.00 per application

•*Do not allow mixed solution to stand overnight.

http://lghttp.41526.nexcesscdn.net/8015C4D/magento/media/catalog/product/cache/1/image/1400x/9df78eab33525d08d6e5fb8d27136e95/G/S/GS-111700-2.jpg

http://lghttp.41526.nexcesscdn.net/8015C4D/magento/media/catalog/product/cache/1/image/1400x/9df78eab33525d08d6e5fb8d27136e95/4/2/4216r.jpg

http://thumbs.dreamstime.com/x/three-dollars-13693976.jpg

Maxus (Part B) • This is a clear liquid suspension

• *Store in freezer*

• Comes in a brown light block container to reduce breakdown

• Frozen has a 5 month shelf life

– (In refrigerator it has only a 3 week shelf-life)

• Requires cold shipping from supplier

http://www.cleanfax.com/wp-content/uploads/2015/07/Tech-tip-frozen-gauge-website.jpg

http://facultyfiles.deanza.edu/images/gawrychjeff/Frozencar.jpg

http://i.ebayimg.com/images/g/~H4AAOxy6MBSN1uG/s-l300.jpg

https://s-media-cache-ak0.pinimg.com/736x/c7/1c/60/c71c60967e20d353144af1748355f549.jpg

Cost of Maxus

• $5.00 per application

• Must be placed immediately in the freezer upon arrival

• Take out of the freezer and place in the refrigerator the day of pollination to provide two days to defrost

http://img0135.psstatic.com/186784127_eppendorf-022363514-polypropylene-flex-tubes-.jpg https://upload.wikimedia.org/wikipedia/commons/thumb/6/69/US-Series-2006G-$5-obverse.jpg/1024px-US-Series-2006G-$5-obverse.jpg

Buyer Beware of other hormone suppliers

• There are slightly different types of hormones that have different modes of action. (Example: there are at least three different types of cytokinins)

• Hormones have a shelf life, and other suppliers do not provide shelf life information or state proper storage conditions, which will reduce the effectiveness.

http://jacobsinjurylaw.com/wp-content/uploads/It%E2%80%99s-Buyer-Beware.png

Spray bottle + Mixing container $5.00

http://files.hgsitebuilder.com/hostgator884163/image/1550mlnesttubes.jpg

The only ingredient not included… Distilled Water

• Only need ~29 mL per application

https://s3.amazonaws.com/mws-assets-production/app/public/spree/products/5142/large/distilled-water-case-of-6-one-gallon-bottles.jpg?1332783919 https://www.aquagrade.com/wp-content/uploads/2013/12/Arrowhead-Distilled-Water.jpg

http://canigivemybaby.com/wp-content/uploads/2011/09/can-i-give-my-baby-distilled-water.jpg

http://www.distilleddeionizedwater.com/wp-content/uploads/2012/09/Distilled-Water1.jpg

Basic Instructions

• Keep brown vial in freezer

• Keep blue-capped vial in refrigerator

• Mix both with distilled water and immediately apply directly to the pumpkin 48 hrs. after pollination

• Mist entire pumpkin, do not apply to petals or inner flower parts (stigmas/lobes)

• Discard what you do not use the same day

http://cultivateprayer.com/wp-content/uploads/2011/12/Basic-Instructions.jpg

• Contains plant hormones known to increase cell division

• Duration of cell division has the greatest influence on final mass.

• Must be applied 48 hours post pollination

• Requires only one application

• Get yours today!

Anthesis