2017 Planting Experiment - Amount to Plant - Baptisia Portion

2017 Planting Experiment - Amount to Plant

2/13/2017

For the 2017 planting I have to decide how much I will plant. First I needed to start with shelf space. For the Portulaca experiment, I was using one shelf for experimentation. This did keep me busy, but I wanted to do more. I added another unit with 3 additional shelves. For this planting I will use only the new shelves and keep my old shelve vacant for random plants. With the number of shelves determined, 3, I can focus on what is on the shelves.

I need to decide how many units per shelf. For the Portulaca experiment, the shelf could hold 98 glass jars. I discussed the matter with a colleague who suggested I use plastic cups. I evaluated the idea. Plastic cups fit 72 per shelf. This is a 27% decrease in the number of units on a shelf. For every 4 shelves of jars, it is as if I get a bonus shelf when compared to the cups. Jars are about $10.00 per 12 units that would total $245.00 for 3 shelves. Cups are about $3.00 per 24 units that would total $27.00 for 3 shelves. Cups are nearly 11% of the cost of jars. Once the experiment is complete, I will have to clean the jars. If I clean jars at the speed I have the Portulaca experiment, it may take a year. I’m not lazy (although some will argue), just busy. With the plastic cups I can dump the dirt then recycle them, they are #2 plastic, within a day. Jars take up space. I have to keep them boxed up before use and when the experiment is completed. The plastic cups stack on each other, and although seems wasteful, are cheap and disposable. I could clean and reuse the cups, but that will be based on their condition and my cost/benefit ratio that won’t be discussed. The pros for the cups out weight that of the jars (Table 1). I tried the jars before with good results. It is worth testing cups as they are similar but have low costs. The 3 shelves, 72 cups per shelf, in total can hold 216 cups. With cups selected, I can plan how many and what kind of seeds I will plant.

Table 1. Pros and Cons of Jars and Cups.

	Jars	Cups
Units per shelf	27% more per shelf	less per shelf
Cost	$245.00	$27.00
Amount of work	clean when done	dispose recycle
Storage	must house	collapsible
Pro: Color		Cups Win
Con: Color

To start, determining the species I will plant is based on different factors. Half depends on how many cups do I want to appropriate to a species. The other half depends on how many seeds I have available. Then I must combine those findings to determine how many seeds per how many cups. A literature search can help justify numbers and provide an idea on what type of germination rates to expect. Rather than no previous background, I have decided a non-intensive literature search to cite a few germination percentages. This will provide a base background and justification, yet no doubt will lack the full benefit on an intense search. If germination rates are poor, then a deeper investigation of the literature will be warranted. Until then, the few examples I find will suffice my curiosity on the subject.

I plan to devote one shelf to Baptisia species that includes B. alba, B. perfoliata, and B. lanceolate. I wish only to examine germination, growth, and compare those characteristics among species. I will have 24 replicates (cups) for each species. I will maintain one plant per cup. If more seeds germinate than one, the more vigorous plant will be allowed to grow and I will remove the others.

Based on literature about the genus germination, I must decide how many seeds I will place within each cup. I searched for literature online and developed a table taking note of overall percent germination for each experiment (Table 2). I then averaged the percent germination to determine 42% of seeds germinate. I understand this is a rough estimate as each experiment contained different conditions, yet it gives me a baseline that I will use as justification. With the baseline 42% germination, I can estimate how many seeds I need per cup.

Table 2. Baptisia germination literature search.

Species	%	Citation	Type
B. arachnifera	90%	Pattavina within Estep 2011	Cold
B. australis	90%	Dirr 1987	Scarring
B. australis	90%	Boyle 2005	Scarring
B. australis	80%	Bratcher 1993	Cold 10 weekd
B. australis	80%	Bratcher 1990	Cold 10 weeks
B. australis	70%	Bratcher 1993	None
B. australis	70%	Bratcher 1990	No Stratification
B. australis	70%	Bratcher 1990	Cold 8 weeks
B. arachnifera	60%	Estep 2011	No Stratification
B. australis	60%	Bratcher 1990	Cold 2 weeks
B. australis	60%	Bratcher 1990	Cold 6 weeks
B. australis	45%	Boyle 2005	None
B. australis	45%	Boyle 2005	Water soak
B. australis	40%	Bratcher 1990	Cold 4 weeks
B. alba	30%	Ksiazek 2014	Ground level
B. tinctoria	22%	Wennerberg 2005	Cold 3 weeks + acid, in Field
B. lanceolata	15%	Simons 2010	Less than in all field treatments
B. tinctoria	13%	Wennerberg 2005	Cold 3 weeks + acid, in Greenhouse
B. lanceolata	11%	Estes 2007	Lab
B. australis	10%	Boyle 2005	Scarring and water soak
B. lanceolata	6%	Estes 2007	Lab
B. leucantha	2%	Voigt 1977	Cold 8 weeks moist
B. lanceolata	2%	Estes 2007	Nursery
B. leucantha	0%	Voigt 1977	None
B. alba	0%	Ksiazek 2014	on green roofs
Average	42%	From 25 different experiments

At the time I was disappointed in my research on the understanding of statistical power but I am pleased to put it to use in determining the number of seeds per cup. Statistical power is used to estimate the probability you will determine something based on numeral factors. It can also be used to estimate how many samples you need to increase the probability you will obtain a result. The philosophy and application of statistical power is debatable on many levels, yet if used wisely can provide justification to experimental design. Although many equations for statistical power and sample size are complex, when much is unknown rough estimate equations are developed. I find these rough estimate equations useful in that a simpleton such as myself can use them.  The “Rule of 3s” to determine sample size is explained (http://www.vanbelle.org/chapters%5Cwebchapter2.pdf). The equation divides 3 by an estimate. Through the literature search, 42% germination is my known estimate. I just divide 3 by 0.42 then round up to obtain 8 seeds as an estimate of a sort of guarantee to obtain 1 seed germinating per cup (Figure 1).

x=	0.42		n=	3	/	n
			n=	3	/	0.42
			n=			7.142857

Figure 1. Spreadsheet “machine” to calculate sample size using a percent estimate. In the blue box I input the estimate percent. The orange box returns the suggested sample size. I obtained the percent estimate from table 2 above.

I will further explain this witchcraft. As an example: A fisherman catches 5 fish, on average, every hour. We divide 1 hour by 5 fish to obtain the value 0.2. The 0.2 means for every 0.2 hours, a fish will be caught, on average. By multiplying 60 minutes by 0.2, we can determine that every 12 minutes a fish is caught on average. Some mad scientist came up with the idea of 3s: multiply the estimate by three and you have a good chance to obtain the expected result. We take 12 minutes and multiple it by 3 to obtain 36 minutes. If the fisherman fishes for 36 minutes, he has a good chance to catch a fish. If you gamble like a scientist, then you will put your money on the 36 minute mark that the fisherman will catch at least one fish. That was still complicated.

Example explained another way. A fisherman catches 5 fish per hour, on average. For one hour = 5 fish. We apply the rule of 3s equation. We divide 3 by 5, the result is 0.6. Meaning in 0.6 hours, you have a 95% chance to catch 1 fish, statistically speaking. Multiplying 0.6 hours by 60 minutes is 36 minutes. Between both explanations, I think the point may get through (to myself).

Now I apply the concept of the rule of 3s to my experiment. For every 100 seeds, 42 germinate (42%), my on average rough estimate taken from the literature. I take 42/100 to obtain 0.42. I divide 3 by 0.42 to obtain 7.142857. Only whole seeds can be used. No such thing exists as half a seed grows half a plant. It is one seed for one plant. Not 2 seeds for one plant. Not 0.5 seeds for one plant. Not 0.142857 seeds for one plant. Only 1 seed for one plant. So I round my 7.142857 estimate to 8 seeds. With an estimate of 42% germination, I need to plant at least 8 seeds per cup to obtain at least 1 seed germinating.

The rule of 3s calculation can also work in reverse with the same formulas. If 7.142857 is entered, then 0.42 will return. If seeds were limited, then the return on the function would estimate what percent of germination you may obtain based on seed availability.

The result of 8 is a good suggestion. “In some disciplines, group sizes of eight are almost universal and some referees may go so far as to reject a paper which deviates from this norm,” (http://www.ebd.csic.es/documents/240051/0/Sample_size_and_power_analysis.pdf). The quote references 8 for the number of samples to determine a statistical difference between groups. My application of 8 is used to obtain a statistical guarantee of germination within a cup. Applied differently the concept is the same. As a side note, understanding this explains that the “8” rule as having a slightly less than 0.4 (or slightly less than 40%) assumption on the estimate.

That is the theory, but can I apply it to actual application? 24 x 8 = 192. I have enough seeds for B. alba and B. perfoliata, but unfortunately for B. lanceolate I have 181 seeds. I will have to adjust.

With seeds, I often like to retain some in case. In time I have a collection of seeds. Older seeds are not as good as newer seeds due to decreased viability. For this I will get into the mindset to use all seeds.

Although I have gone through the process shown above, I must adjust my thoughts for the project. I determined at least 8 seeds, but I only have enough for 7 seeds per cup. I then return to the literature. I plan to use cold stratification and will need to determine how many weeks I will keep the seeds in the refrigerator. I filtered the table for cold stratified seeds in germination experiments.

Table 3. Refined Baptisia germination literature search.

Species	%	Citation	Type
B. arachnifera	90%	Pattavina within Estep 2011	Cold
B. australis	80%	Bratcher 1993	Cold 10 weekd
B. australis	80%	Bratcher 1990	Cold 10 weeks
B. australis	70%	Bratcher 1990	Cold 8 weeks
B. australis	60%	Bratcher 1990	Cold 2 weeks
B. australis	60%	Bratcher 1990	Cold 6 weeks
B. australis	40%	Bratcher 1990	Cold 4 weeks
B. tinctoria	22%	Wennerberg 2005	Cold 3 weeks + acid, in Field
B. tinctoria	13%	Wennerberg 2005	Cold 3 weeks + acid, in Greenhouse
B. leucantha	2%	Voigt 1977	Cold 8 weeks moist
Average	52%	From 25 different experiments

The estimate rose from 42% to 52%. I then used this estimate for the Rule of 3s.

x=	0.52		n=	3	/	n
	0.4		n=	3	/	0.52
			n=			5.76923077

Figure 2. Spreadsheet “machine” to calculate sample size using a percent estimate. In the blue box I input the estimate percent. The orange box returns the suggested sample size. I obtained the percent estimate from table 3 above.

For 52% estimate germination, and rounded up to a whole seed, I need 6 seeds per cup for the 24 cups. Although I didn’t have enough for 8 seeds per cup, I do for 6, but I will use 7 to use all my seeds, and more is better.

In conclusion: I will plant 7 seeds per cup, 24 cups per each species, on one shelf.

Future

I now must designate the cup distribution. Cups should be distributed across the shelves randomly. This will be for another discussion. Updated: added:

 

Figure 3. Randomized placement of Baptisia cups on shelf for 2017 planting.

Utilizing this concept I will perform a similar method to obtain how many seeds for the carnivorous plant portion of my experiment.

Thank you for viewing. Comment if you want. Enjoy the day!

Citations – Power Analysis

Van Belle, Gerald. 2008. Chapter 2 Sample size. Pages 27-51 in Statistical Rules of thumb. Wiley, Seattle. Accessed January 2017 from http://www.vanbelle.org/chapters%5Cwebchapter2.pdf.

Festing, Michael F.W. 2010. Part 1 Chapter 3 The design of animal experiments. Pages 28-29 in The UFAW handbook on the care and management of laboratory and other research animals, 8^th ed, Robert Hubrecht and James Kirkwood. Wiley-Blackwell, Singapore. Accessed January 2017 from http://www.ebd.csic.es/documents/240051/0/Sample_size_and_power_analysis.pdf.

Citations – Baptisia germination literature

Boyle, Thomas, Kristen Hladun. 2005. Influence of seed size, testa color, scarification, method, and immersion in cool or hot water on germination of Baptisia australis (L.) R. Br. Seeds. HortScience 40(6):1846-1849.

Bratcher, Carlma. 1990. Methods of producing field-grown specialty cut flowers. Oklahoma State University, Sillwater: 18-23.

Bratcher, Carlma, John Dole, Janet Cole. 1993. Stratification improves seed germination of five native wildflower species. HortScience 28(9):899-901.

Dirr, Michael. 1987. I gave it 30 minutes, I couldn’t relocate the exact citation, too bad. Probably came from: The reference manual of woody plant propagation: from seed to tissue culture, 2^nd edition. But I could be wrong. I will leave it in this post with this note. It did not change the cold stratification results.

Estep, Timothy. 2011. Evaluating restoration potential of an endangered legume, Baptisia arachnifera: shade & litter effects on early life stages. Georgia Southern University College of Graduate Studies Electronic Theses & Dissertations. 760. Accessed February 2017 from http://digitalcommons.georgiasouthern.edu/cgi/viewcontent.cgi?article=1760&context=etd.

Estes, Becky, John Kush. 2007. Longleaf pine: seeing the forest through the trees, proceedings of the Sixth Longleaf Alliance Regional Conference; November 13-16, 2006, Tifton, GA. Longleaf Alliance Report No. 10.

Ksiazek, Kelly, Jeremie Fant, Krissa Skogen. 2014. Native forbs produce high quality seeds on Chicago green roofs. Journal of Living Architecture. 1(2):21-33.

Voigt, John W. 1977. Seed Germination of True Prairie Forbs. Journal of Range Management, 30(6):439-441.

Wennerberg, Sarah. 2005. Propagation and field assessment of West Virginia native species for roadside revegetation. Eberly College of Arts and Sciences West Virginia university Thesis.