Growing a Bigger Pumpkin by Increasing Cell Division

Matthew DeBacco

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About “Mad Scientist” Matt D.

• Masters 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 as 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 15 years

• Growing pumpkins continuously for the last 2,100+ days

• Two time and current CT State Record Holder @ 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 and 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 lead to two years of research into the scientific literature

• Mostly it lead to dead ends

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Then there was a break through

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Two Key Articles

Tomato Fruit Growth

…

Integrating Cell Division, Cell Growth and

Endoreduplication by Experimentation and

Modelling

• 145 pg Doctor 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

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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

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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)

Endoreduplication

• The replication of DNA during the S phase of the cell cycle without the subsequent completion of mitosis and/or cytokinesis.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endoreplication.html

Endoreduplication compared to Mitosis

Endoreduplication- Does not need reorganization and patterning of the tissue.

http://www.rv-orchidworks.com/orchidtalk/content/172-scientific-behind-how-orchid-flowers-keep-growing-even-after-buds-open.html

Endoreduplication

Grand Gala apples, at right, undergo endoreduplication, which causes cells to grow larger than in the regular Gala apples, at left.

(Purdue University photo/Peter Hirst)

http://www.purdue.edu/newsroom/research/2010/100630HirstGala.html

Regular Gala

apples

*Grand Gala apples undergo

endoreduplication

Endoreduplication Benefits

1. Allows an organ to enlarge in size even when it has lost the capacity for multiplication.

2. Does not need reorganization and patterning of the tissue.

3. Is much more energy efficient.

– It involves much less protein synthesis and energy expenditure on the part of the cells.

4. The resulting cells are polyploidy – (Study on chickpeas does not show a decrease in seed yield)

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

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Summary

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

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So….. Now what

• How can I apply this knowledge?

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Plant Hormones Influence Cell Signaling

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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

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

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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 result in very different outcomes

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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

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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

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

• This document provides specific materials and methods

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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 (2 days 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 Increase 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 flowers anthesis of the 1st cluster yield was increased by 15%

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

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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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So what does this all mean?

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The Directions are Simple (supporting science complex)

• Apply 100ppm of Ethrel® 2 days after pollination

*directly to the fruit*

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Warning very small tolerance of error • To much = negative effects (deformities, no seeds…)

• To little = not effective

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Ethrel® (Ethephon)

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

• Aqueous solution of ethrel is stable below pH 3.5

• Above pH 3.5 the hydrolysis of ethrel begins with the release of free ethylene along with chloride and phosphate ions

• Ethrel, can enter into the fruits cells

http://www.cropscience.bayer.ca/~/media/Bayer%20CropScience/Country-Canada-Internet/Products/Ethrel/Ethrel.ashx

What should happen after Ethrel® application

• Cell Division should be extended up to 16-18 days

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

• The pumpkin should ripen 10 days later

Pumpkin Weight (lbs) 15% increase

100 115

500 575

1000 1150

1500 1725

1650 1897.5

1900 2185

2009 2310

2323.7 2672

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

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Ethylene and Cytokinins

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

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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

Tank mix of Ethrel® + Cytokinin

• One study looking at apples and thinning related to applying both of these

• Note applied at different times in this study

https://lh6.ggpht.com/R9iSRrVH1sfVzJoZJRhFD6bIYIsG1JMmPg4Uxw_mILIjW5993qrg98-BctWdVdO8Hr4=w300

http://nwdistrict.ifas.ufl.edu/phag/files/2012/08/Sprayer-for-Web.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

Now the cost…

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Estimated cost of Ethrel®

• About $1.00 per application • Easy procedure

– (It is like someone circled Waldo)

•*Mix only the amount of spray you expect to use each day.

•*Do not allow mixed solution to stand overnight.

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Cytokinins are more difficult

• Often need to be stored at very low temperatures and require special “Cold shipping”

• About $5.00 per application

– *So, when you see them online the odds are you are not getting a properly stored product

http://www.nortechlabs.com/images/cold-shipping-supplies-index.jpg http://i.dailymail.co.uk/i/pix/2014/01/06/article-2534477-1A74C4E100000578-311_470x532.jpg

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Conclusion

• Ethrel has the potential to be very effective

• Cytokinins may be well worth their special requirements – *There are different cytokinins so it is important to select the “best” match

• Cost to benefit ratio is the greatest that I have seen

• If you are growing giant tomatoes this is worth a look

• Contains plant hormones known to increase cell division

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

• Must be applied 2 days post pollination

• Requires only one application

• Get yours today!

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