INFLUENCE OF CULTIVAR AND ROOTING HORMONE CONCENTRAION ON

SHOOT GROWTH AND ROOT MASS OF BASIL (OSIMUM BASILICUM)

BY

STEPHANIE PETRAKOS

Submitted to the Honors Program for the graduation requirement of

SUMMA CUM LAUDE HONORS

UNIVERSITY OF FLORIDA

College of Liberal Arts and Sciences

MAY 2016

________________________________

Student Signature: Stephanie Petrakos

Contact: spetrakos@ufl.edu

________________________________

Thesis Advisor Signature: Dr. Brian J. Pearson

Contact: bpearson@ufl.edu

________________________________

Co-Thesis Advisor Signature: Dr. Jeremy Lichstein

Contact: jlichstein@ufl.edu

________________________________

Departmental Honors Coordinator Signature: Dr. Leslie Paul Thiele

Contact: thiele@ufl.edu

Stephanie Petrakos

2

Abstract

Basil (Ocimum basilicum) is a popular aromatic herb cultivated primarily for its culinary

attributes. Recent increased demand for this crop has prompted a need for an improvement in

production technique aimed at enhancing yield and improving production efficiency. The aim of

this study is to measure and record growth of three popular basil cultivars during propagation

and identify optimal root hormone concentration for these cultivars. A secondary objective of

this work is to identify cultivars with enhanced root:shoot ratios that may be produced under

reduced irrigation regimes and possess enhanced drought tolerance. Three basil cultivars (Mrs.

Burns Lemon, Red Rubin, and Aroma 2) were chosen for the study and seventy-two cuttings

from each cultivar were taken and treated with 0%, 0.1%, 0.3%, and 0.8% indole-3-butyric acid

rooting hormone. Two weeks after vegetative clones were propagated, plant height was recorded

and root mass was measured. Mrs. Burns Lemon was significantly taller than Aroma 2 cultivars.

However, the root mass of Aroma 2 was greater than Mrs. Burns Lemon and Red Rubin. Greater

root:shoot ratios associated with Aroma 2 suggests lower irrigation needs during production and

enhanced drought tolerance for this cultivar. Root mass was similar amongst plants treated with

0%, 0.1%, and 0.3% indole-3-butyric acid. Results from this work aid in the development of

sustainable or reduced-input production strategies with specific aim at reductions in reliance on

synthetic plant hormone and irrigation use.

3

Introduction

Basil (Ocimum basilicum) is a popular, aromatic herb prized for its superior flavors and

aromas. Essential oils extracted from its fresh leaves and flowers are used as additives in

pharmaceutical, household, cosmetic, and culinary products (Loughrin and Kasperbauer, 2003).

In addition to these uses, basil is also cultivated for its neutraceutical properties. Basil can be

used as a stomachic, antihelminthic, antipyretic, diaphoretic, expectorant, carminative, stimulant,

and pectoral (Sahoo et. al, 1997). Despite the diverse applications of basil and its essential oils,

basil is an important culinary herb, marketed fresh, frozen, or dried. (Jayasinghe et. al, 2003).

Basil is often dried post-harvest rather than sold fresh (wet) to aid in storage and transport

(Bekhradi et al., 2014). However, recent interest in fresh, localized food products, has increased

demand for wet, recently harvested basil. In order to satisfy increased demand for fresh basil,

production methods which maximize production need to be established.

Although traditional basil production practices are well established, producers have

recently investigated new methods to reduce plant production time and meet consumptive

demands (Purcino et al., 2012). Despite traditional soil-based cultivation from seed, soilless basil

production utilizing clonal propagules within protected greenhouse environments has grown in

popularity. Advantages of greenhouse production include precise control of environmental

conditions, nutrient availability, and irrigation. However, high precision production requires

proper selection of cultivar and propagation technique to maximize production and efficiency.

Research focusing on the development of appropriate and efficient production practices are

needed to establish effective and sustainable production of this crop (Bekhradi et al., 2014).

Sustainable utilization of water is critical to commercial plant production and long term

protection of natural resources. In 2010, mean total water usage in the United States was 355

4

billion gallons per day. Of that volume, total irrigation withdrawals were 115,000 million gallons

per day, or 61% of total freshwater withdrawals (Maupin, et al., 2014). Besides thermoelectric

power, irrigation remained the largest single use of freshwater resources (Maupin, et al., 2014).

Research examining plant water use and drought tolerance is necessary to assist in the selection

of plant species which would help allow for reduction in consumption of freshwater for irrigation

purposes. Further, related research has observed reduced irrigation can result in higher quality

basil crop. Water stress has been shown to increase the content of phenolic acids and antioxidant

properties in green and purple varieties of sweet basil (Luna, et al., 2015). Thus, reduced

irrigation may improve plant quality while simultaneously reducing reliance on natural

resources.

Disease management is an important component in plant production. Downy mildew, a

relatively new and destructive disease in basil, was first discovered in Florida in the fall of 2007

(Wyenandt, et al., 2010). The disease is believed to have originated from Uganda (McGrath,

2015) and since its introduction into the US has been reported in commercial basil production

facilities in California and the Midwest. The first symptoms of downy mildew are yellowing in

the leaves, at first making downy mildew overlooked as a nutrient deficiency. As the disease

develops, dark, gray-purple sporangia forms on the abaxial surface of the leaves, eventually

making its way to the adaxial side of the leaves (Wyenandt, et al., 2010). This sporangia prevents

photosynthesis in the leaves, stunts plant growth, and destroys the essential oils that the basil

leaves produce, making the basil plant unviable. There is no specific treatment or cure for downy

mildew, but preventing wet, moist environments for the mildew to grow and selecting a cultivar

less susceptible to downy mildew is crucial to maximizing the prevention of the disease. Ocimum

basilicum is more susceptible to the disease than basil varieties of a different species such as

5

Ocimum citriodorum and Ocimum americanum (McGrath, 2015). However, purple or red

varieties of Ocimum basilicum such as Red Rubin and lemon varieties of Ocimum basilicum such

as Mrs. Burns Lemon are less susceptible to downy mildew. The Aroma 2 variety of basil, most

similar in aroma and taste to the popular Genovese basil, has also shown a resistance to downy

mildew that is nearly equal to that of Mrs. Burns Lemon and Red Rubin (Wyenandt, et al., 2010).

Due to their high disease resistance, these three cultivars of basil were chosen for this study.

Materials and Methods

Seed propagation. Seeds of three basil cultivars (Mrs. Burns Lemon, Red Rubin, and Aroma 2)

were purchased (Johnny’s Selected Seeds in Winslow, MA) and propagated from seed on June

17, 2014. Seeds of each cultivar were germinated in a 72 cell pack tray (n=216) containing

Fafard Growing Mix 2 (SunGro, Agawam, MA) soilless substrate in a greenhouse located in

Apopka, FL. The experiment was conducted over a duration of eight weeks starting on June 17,

2014, and concluding on August 8, 2014. Data was recorded over a six week period, starting on

June 17, 2014. Each tray received irrigated mist every 5 minutes for a duration of 15 seconds. On

June 24, one week after the seeds were planted, plant height was recorded for Mrs. Burns Lemon

and Aroma 2 varieties. Slow germination rate for Red Rubin precluded measurement of plant

height until three weeks after transplant (WAT) on June 2, 2014. Trays were irrigated for a

duration of 4 weeks with the mist irrigation system prior to container production. Fertilizer (50

ppm N) was applied through capillary tray absorption 3 weeks after transplant. After reaching a

height of 30 cm, 8 basil plugs were removed from their trays and transplanted into 7.5 L

containers (8 basil plugs per container). Each container was irrigated twice daily utilizing a low-

volume drip emitter. Plant height measurements were collected weekly beginning post-transplant

6

and terminated 6 WAT. Throughout this production period, electrical conductivity was collected

(data not shown) to ensure adequate substrate nutrient availability.

Vegetative clonal propagation. On July 27, 2016, 6 WAT, seventy-two cuttings from the three

basil cultivars were taken and treated with 0%, 0.1%, 0.3%, and 0.8% indole-3-butyric acid

(IBA) (Hormodin, OHP, Inc., Mainland, PA) rooting hormone. After application of the rooting

hormone, cuttings were transplanted into a 72 cell pack tray containing Fafard Growing Mix 2

(SunGro, Agawam, MA) soilless substrate. Cuttings were placed within the greenhouse and

irrigated every 5 minutes for a duration of 15 seconds using a mist irrigation system. Plant height

measurements were collected weekly for a duration of 2 weeks at which time the clonal

propagules were ready for transplant. Plants were destructively harvested and dried in an oven at

65 °C until a constant dry weight was obtained to quantify root mass and shoot mass.

Statistical analysis. Plants cultivated for this experiment were arranged utilizing a completely

randomized design. Plant height, root, and shoot mass data were analyzed using the PROC GLM

procedure in SAS with mean separation by Duncan’s multiple range test. Statistical tests were

considered to be significant if P<0.05.

Results and Discussion

Seed propagation. Beginning 2 WAT, mean height of Mrs. Burns Lemon and Aroma 2 were

approximately 2.14 and 1.07 cm, respectively (Figure 1). Three WAT Red Rubin varieties were

large enough to measure and had a mean height of 1.27 cm. Beginning 3 WAT, rapid growth was

observed. High growth rates between 3 and 6 WAT are likely a result of nutrient application.

Beginning 4 WAT, Mrs. Burns Lemon had greater plant height than either Aroma 2 or Red

Rubin (Figure 1). Beginning 5 WAT, Aroma 2 and Red Rubin displayed rapid growth. Aroma 2

had an average height of 9.2 cm with the largest Aroma 2 plant being 17.5 cm tall. Red Rubin

7

had an average height of 4.75 cm with the largest Red Rubin plant being 13.8 cm tall. Mrs. Burns

Lemon had an average height of 24.38 cm with the tallest being 32.7 cm (Figure 1). Rubin

remained the most compact cultivar with mean height of 9.5 cm at termination of the experiment

(6 WAT).

At termination of the experiment (6 WAT), downy mildew was present on all three

cultivars, making them unviable and ineffective for further experimentation. However, the

quantity of downy mildew and severity of the outbreak varied dependent upon cultivar. Downy

mildew was most virulent on Aroma 2 cultivars where it infected approximately 60% of plant

leaves. Disease incidence was less severe for Red Rubin and Mrs. Burns Lemon with leaf

infection of approximately 25% and 40%, respectively. These observations imply that significant

variability of disease resistance exists amongst basil cultivars. Further research is warranted to

identify and potentially breed resistance amongst popular, susceptible varieties.

Amongst the cultivars tested, greatest plant growth was observed for Mrs. Burns Lemon.

As expected, Mrs. Burns Lemon possessed the highest mean leaf count with 8 leaves on each

stem and an average height of 8 cm (Table 1). Aroma 2 had an average of at least 6 leaves on

each stem and an average height of 4 cm whereas Red Rubin had an average of at least 6 leaves

on stem and an average height of 4 cm. Greater leaf count in Mrs. Burns Lemon may be

significant to the culinary market given its application in food preparation and production.

Vegetative clonal propagation. Plant height was not influenced by hormone application.

Over the duration of the experiment, the height of Mrs. Burns Lemon was approximately 2-fold

greater and significantly different than Aroma 2 despite higher mean variability. Red Rubin had

similar plant height to both Mrs. Burns Lemon and Aroma 2 in the three weeks prior to the

8

application of fertilizer in week four. Although plant height of Mrs. Burns Lemon was greatest, it

had similar root mass to Red Rubin and significantly less root mass than Aroma 2 (Figure 2).

This suggests Aroma 2 may possess greater root:shoot ratios and greater drought tolerance when

compared to Mrs. Burns Lemon and Red Rubin cultivars. This may be beneficial when

considering selection of cultivar for transplant and establishment in the landscape. Dry root mass

amongst all three cultivars was highest when propagated using 0.3% IBA, but similar to those

treated with 0% and 0.1% (Figure 3). Root mass was lowest when treated with 0.8% IBA and

was approximately 2-fold less than propagates treated with 0.3% IBA (Figure 3). Increased root

mass as a result of IBA application was similar to findings reported by Sahoo et al. (1997). In

contrast to Sahoo et al., (1997); however, we observed deleterious effects when IBA

concentrations exceeded 0.3% (Figure 3).

Conclusion

Plant growth utilizing conventional seed germination techniques was acceptable and

similar to expectations for related ornamental and edible crops produced under a protected

greenhouse system. Although not the main focus of this thesis, information collected and

presented within this manuscript is beneficial and of value to commercial production of this

unique crop. Overall, high growth rates observed for Mrs. Burns Lemon and Aroma 2 varieties

suggest these varieties possess the greatest commercial production potential.

Although there is currently no cure for downy mildew, there are measures that can be

taken in commercial production to prevent downy mildew from occurring and to control the

severity of a downy mildew outbreak. It is increasingly common for the Ocimum basilicum

species to be marketed as resistant to downy mildew. However, additional research efforts are

needed to scientifically quantify levels of resistance and to aid in identification of cultivars that

9

may be utilized for plant breeding to confer resistance to popular, susceptible cultivars.

Currently, it is recommended that commercial cultivators of Ocimum basilicum select cultivars

crossed with disease resistant varieties of basil, such as Ocimum citriodorum or Ocimum

americanum. If a pure Ocimum basilicum cultivar must be selected for production, it is

recommended that Red Rubin or Mrs. Burns Lemon be selected based upon our experimental

observations. In order to avoid an outbreak, maintenance of a warm, dry environment and the

avoidance of moisture on the basil leaves are vital.

Overall, Mrs. Burns Lemon did not possess significantly greater root mass than Aroma 2

or Red Rubin cultivars, despite greater plant height. Even though Aroma 2 had a shorter average

height than Mrs. Burns Lemon, the greater root mass of Aroma 2 indicates that Aroma 2 may

have a greater root:shoot ratio and a greater drought tolerance when compared to Mrs. Burns

Lemon and Red Rubin cultivars. A greater root:shoot ratio and its associated effect on a drought

tolerance may be beneficial when considering selection of cultivar for cultivation within a

greenhouse or transplant and establishment in the landscape given less water would be required

to cultivate healthy, high quality plants. This would likely have significant impact on

improvements in water conservation. Therefore, out of all three basil cultivars, Aroma 2 would

most likely be the most sustainable in terms of water utilization.

In regards to root hormone concentration influence on the growth of basil root mass, root

mass is generally increased following the application of 0.1% and 0.3%IBA. However, the root

mass of hormone treated propagates was not significantly higher than the root mass of untreated

propagates. In order to avoid deleterious effects to root development in these three basil

cultivars, it is not recommended to apply a root hormone concentration that is greater than 0.3%

IBA. Despite positive correlations between IBA application and root development in related

10

research, our findings suggests root hormones are not significantly advantageous to commercial

production of basil. Additionally, our findings suggests that basil cultivar, not concentration of

root hormone, is of importance for root development. The natural tendency for increased, healthy

root development, or lack thereof, is what makes a particular basil cultivar sustainable or not

sustainable in the physical environment.

11

References

Bekhradi, F., Luna, M., Delshad, M., Jordan, M., Sotomayor, J., Martínez-Conesa, C., & Gil, M.

(2015). Effect of deficit irrigation on the postharvest quality of different genotypes of

basil including purple and green Iranian cultivars and a Genovese variety. Postharvest

Biology and Technology, 100, 127-135. Retrieved June 10, 2015, from Web of Science.

Jayasinghe, Chamila, Naohiro Gotoh, Tomoko Aoki, and Shun Wada. "Phenolics Composition

and Antioxidant Activity of Sweet Basil ( Ocimum Basilicum L.)." J. Agric. Food

Chem. Journal of Agricultural and Food Chemistry 51.15 (2003): 4442-449. Web. 11

June 2015.

Loughrin, J., & Kasperbauer, M. (2003). Aroma Content of Fresh Basil (Ocimum basilicum L.)

Leaves Is Affected by Light Reflected from Colored Mulches. Journal of Agricultural

Food Chemistry, 2272-2276. Retrieved July 12, 2015, from Web of Science.

Luna, María C., Farzaneh Bekhradi, Federico Ferreres, María J. Jordán, Mojtaba Delshad, and

María I. Gil. "Effect of Water Stress and Storage Time on Anthocyanins and Other

Phenolics of Different Genotypes of Fresh Sweet Basil." J. Agric. Food Chem. Journal

of Agricultural and Food Chemistry 63.42 (2015): 9223-231. Web.

Maupin, Molly A., Joan Kenny, Susan S. Hutson, John K. Lovelace, Nancy L. Barber, and

Kristin S. Linsey. "Estimated Use of Water in the United States in 2010." USGS (2014):

25. Web.

McGrath, M. (2015). Expect and Prepare for Downy Mildew in Basil. Department of Plant

Pathology and Plant-Microbe Biology, Cornell University, 1-6. Retrieved June 10, 2015,

from http://vegetablemdonline.ppath.cornell.edu/NewsArticles/BasilDowny.html

Purcino, M., Machado, M., & Biasi, L. (2012). Effect of Leaves on the Rooting of Cuttings of

Clove basil (Ocimum gratissimum L.) and anis (Ocimum selloi Benth). Revista De

12

Ciências Agroveterinárias, 11(2), 93-98. Retrieved June 10, 2015, from CabDirect.

Sahoo, Y., Pattnaik, S., & Chand, P. (1997). In vitro Clonal Propagation of an Aromatic

Medicinal Herb Ocimum basilicum L. (Sweet Basil)by Axillary Shoot Proliferation. In

Vitro Cellular &amp; Developmental Biology. Plant, 293-296. Retrieved July 12, 2015,

from Jstor.

Wyenandt, C., Simon, J., McGrath, M., & Ward, D. (2010). Susceptibility of Basil Cultivars and

Breeding Lines to Downy Mildew. HortScience, 45(9), 1416-1419. Retrieved June 10,

2015, from Web of Science.

13

Appendix

Figure 1. Mean height of three basil (Ocimum basilicum) cultivars (n=72) from June 22, 2014

(Week 1) through July 27, 2014 (Week 6).

0

5

10

15

20

25

30

35

40

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Hei

gh

t (c

m)

Weeks After Transplant

Height of Basil Cultivars

Red Rubin Mrs. Burns Lemon Aroma 2

14

Figure 2. Dry root mass of three basil (Ocimum basilicum) cultivars (n=24) two weeks after

transplant planted into Fafard Growing Mix 2 soilless substrate. Error bars represent the standard

error of the mean.

b

b

a

0

0.5

1

1.5

2

2.5

3

Mrs. Burns Lemon Red Rubin Aroma 2

Dry

root

mas

s (m

g)

Cultivar

15

Figure 3. Dry root mass of basil (Ocimum basilicum) (n=18) two weeks after transplant into

Fafard Growing Mix 2 soilless substrate propagated with 0%, 0.1%, 0.3%, and 0.8 indole-3-

butyric acid (IBA) rooting hormone treatments. Error bars represent the standard error of the

mean.

ab ab

a

b

0

0.5

1

1.5

2

2.5

3

0% 0.1% 0.3% 0.8%

Dry

root

mas

s (m

g)

Indole-3-butyric acid concentration

16

Table 1. Height and leaf count of three basil (Ocimum basilicum) cultivars (Mrs. Burns Lemon,

Aroma 2, and Red Rubin) four weeks after transplantz.

Cultivar Height (cm) Leaf count

Mrs. Burns Lemon 8 8

Aroma 2 4 6

Red Rubin 4 6

zMeans of 72 plants per cultivar.