1. Effect of HormoneConcentration and Typeof Explant on Efficiency of Naranjilla
Micropropagation
AdrianneSeiden
Introduction:
Naranjilla is a fruiting shrub indigenous to the Sierra region of Ecuador and Colombia. Due to
low genetic diversity and sensitivities to disease and parasites, namely nematodes (AKA
roundworms), it has thus far been unable to be grown outside of South America. Due to its
myriad culinary uses and potential for commercial sale, there is growing interest in improving
propagation techniques for naranjilla. One way to do this would be to perfect the procedure for
in vitro micropropagation of naranjilla. In vitro micropropagation is a technique has been used
for various plants to allow quick regeneration of targeted plants in transgenic manipulations
(O’Connor-Sánchez et al. 2010) or agricultural improvement/conservation programs. This
project aims to standardize in vitro micropropagation of naranjilla via hypocotyls, apical buds,
and petioles, by manipulating levels of the hormones naphthaleneacetic acid, 6-
benzylaminopurine, and gibberellic acid in Murashige and Skoog medium, a commonly used
medium for plant studies, which contains water and other necessary micronutrients.
Naphthaleneacetic acid is an artificially made rooting hormone whose binding is not affected by
salinity (Ray and Dohrmann 1977). NAA is often used to encourage growth of explants but is
toxic to plants in high concentrations. Gibberellic acid has been shown to have positive effects
on the formation of juvenile and adult leaves as well as stemgrowth (Phinney 1956). 6-
benzylaminopurine is a cytokinin that encourages plant growth by stimulating cell division
(Siddiqui et al. 2011). The interaction of these hormones is necessary for plant growth, and
manipulating their relative concentrations can help improve the efficiency of naranjilla
regeneration.
General Objectives:
The general goal of the project is to regenerate naranjilla plants using three types of explants:
hypocotyls, apical buds, and petioles.
Specific Objectives:
Specifically, this project aims to determine the ideal concentration of naphthaleneacetic acid
in the medium for regeneration via apical buds, determine the superior type of petiole,
between those taken from the upper third or the lower third of the plant, for regeneration via
petioles, and determine the ideal concentration of 6-benzylaminopurine as well as the ideal
type of hypocotyl (curved or straight) for regeneration via hypocotyls.
Methods:
1) Market seeds are cleaned and disinfected
1) 3 min in alcohol 70%

2. 2) 18 min in hypochlorite 2.5% and 4-5 drops of tween, stirring every 3 min
3) 5 washes with sterile deionized water
2) Seeds are planted in autoclaved Murashige and Skoog medium, under sterile camera, 10
seeds per jar
3) Jars are covered with saran wrap and placed under light
4) Once plants have grown large enough explants are isolated and planted in petri dished
of MS with controlled concentrations of NAA, BAP, and GA3
Apical buds:
• All leaves are removed, stem is cut just below most recent leaves, planted right-side-up
in petri dishes, 5 explants/dish; moved to larger jars after 1 month
• Treatments: NAA .01 mg/L, NAA .05 mg/L, NAA .20 mg/L
Petioles:
• roots are removed, stalk is cut in 3, discarding the middle, leaves are removed, and
petioles are cut from the main stem, placed in petri dish of MS medium on their side, 5
explants/dish
• Treatments: Upper third petioles, lower third petioles
Hypocotyls:
• curved: as soon as a stem begins to emerge, the seed and any roots or leaves are cut
away, hypocotyl is planted on its side in petri dish of MS medium, 5 explants/dish
straight: the plant is allowed to grow until the stem is straight, at
which point the stem is cut directly below the first leaves and approx. 6mm down,
hypocotyl is planted on its side in petri dish of MS medium, 5 explants/dish
• Treatments: BAP 3 (mg/L), BAP 4.5 (mg/L), BAP 6 (mg/L)
Results:
All data was collected 25 days after planting of the explants. Apical buds grown in 0.01 mg/L
NAA MS medium had produced on average 4.85 leaves per explant while buds grown in 0.05
mg/L NAA MS medium had produced on average 4.95 leaves per explant and buds grown in 0.2
mg/L NAA MS medium had produced on average 4.9 leaves per explant.
0.01 mg/L NAA, 4.85 leaves/explant ; 0.05mg/L NAA, 4.95 leaves/explant ; 0.2 mg/LNAA, 4.9 leaves/explant
4.85
4.95
4.9
4.8
4.82
4.84
4.86
4.88
4.9
4.92
4.94
4.96
0.01 0.05 0.2
numberofleaves(avg.)
Concentration ofNAA (mg/L)
Apical Buds: Number of leaves

3. Stems of apical buds grown in 0.01 mg/L NAA MS medium grew to an average length of 2.5 cm,
while stems of buds grown in 0.05 mg/L NAA MS medium grew to an average length of 3.58 cm,
and stems of buds grown in 0.2 mg/L NAA MS medium grew to an average length of 2.93 cm.
Roots of buds grown in 0.01 mg/L NAA MS medium grew to an average length of 2.27 cm while
roots of buds grown in 0.05 mg/L NAA MS medium grew to an average length of 1.66 cm and
roots of buds grown in 0.2 mg/L NAA MS medium grew to an average length of 1.56 cm.
0.01 mg/L NAA, stem: 2.5cm (avg) root: 2.27cm (avg) ; 0.05mg/L NAA, stem: 3.58cm(avg) root: 1.66cm(avg) ;
0.20 mg/L NAA, stem: 2.93cm(avg) root: 1.56cm(avg)
Petioles sampled from the upper third of the plant produced on average 2 leaves per explant
while petioles sampled from the lover third of the plant produced on average 0.95 leaves per
explant. Stems of upper petioles grew to an average length of 1 cm while stems of lower
petioles grew to an average length of 0.6 cm.
Upper petioles, 2leaves/explant(avg.) 1cm stem (avg.) ; lower petioles,0.95 leaves/explant (avg.) 0.6cmstem(avg.)
0
1
2
3
4
0.01 0.05 0.2
Length(cm)
Concentration of NAA (mg/L)
Apical Buds: Stems and Roots
length of stem
Length of root
0
0.5
1
1.5
2
2.5
avg. number of leaves avg. length of stem (cm)
Petioles: Upper vs Lower
Upper third Lower third

4. Straight hypocotyls grown in 3 mg/L BAP MS medium produced on average 1.4 leaves per
explant while straight hypocotyls grown in 4.5 mg/L BAP MS medium produced on average 2.05
leaves per explant and straight hypocotyls grown in 6 mg/L BAP MS medium produced on
average 2.68 leaves per explant. Stems of straight hypocotyls grown in 3 mg/L BAP MS medium
grew to an average length of 1.05 cm while stems of straight hypocotyls grown in 4.5 mg/L BAP
MS medium grew to an average length of 1.04 cm and stems of straight hypocotyls grown in 6
mg/L BAP MS medium grew to an average length of 0.84 cm.
Curved hypocotyls grown in 3 mg/L BAP MS medium produced on average 1.65 leaves per
explant while curved hypocotyls grown in 4.5 mg/L BAP MS medium produced on average 0.9
leaves per explant and curved hypocotyls grown in 6 mg/L BAP MS medium produced on
average 1.45 leaves per explant. Stems of curved hypocotyls grown in 3mg/L BAP MS medium
grew to an average length of 1.31 cm while stems of curved hypocotyls grown in 4.5 mg/L BAP
MS medium grew to an average length of 1.34 cm and curved hypocotyls grown in 6 mg/L BAP
MS medium grew to an average length of 0.91 cm.
Straight 3 mg/LBAP, 1.4 leaves/explant (avg.) ; curved 3mg/L, 1.65leaves/explant (avg.) ; straight4.5mg/L BAP 2.05leaves/explant(avg.) ;
curved 4.5 mg/L BAP 0.9 leaves/explant (avg.) ; straight6 mg/L BAP,2.68 leaves/explant (avg.) ; curved6 mg/L BAP,1.45 leaves/explant (avg.)
0
0.5
1
1.5
2
2.5
3
3 mg/L 4.5 mg/L 6 mg/L
numberofleaves
Concentration of BAP
Hypocotyls: Number of leaves (avg.)
Straight hypocotyls Curved hypocotyls

5. Straight 3 mg/LBAP, 1.05cm stem (avg.) ; curved3 mg/L BAP,1.31 cmstem(avg.) ; straight 4.5 mg/LBAP,
1.04 cm stem(avg.) ; curved4.5mg/L BAP,1.34 cm stem (avg.) ; straight6 mg/L BAP,0.84 cmstem(avg.) ; curved 6 mg/LBAP, 0.91cm stem
(avg.)
Discussion:
Apical buds were clearly the most productive explant in micropropagation; every explant
sprouted roots and several leaves within 25 days of being planted, while neither petioles nor
hypocotyls produced any roots within the same time period, and produced only a few leaves on
some explants. Apical buds grew best on MS medium with a concentration of 0.05 mg/L NAA.
This concentration produced the greatest number of leaves (avg. 4.95) and the longest stems
(avg. 3.58 cm), while 0.01 mg/L NAA produced the longest roots (avg. 2.27 cm). Petioles from
the upper third of the plant were more successful than those from the lower third of the plant,
producing a greater number of leaves (avg. 2) and longer stems (avg. 1 cm). In terms of
hypocotyls, straight hypocotyls grown in 6 mg/L BAP MS medium produced the greatest
number of leaves (avg. 2.68), while curved hypocotyls grown in 4.5 mg/L BAP MS medium
produced the longest stems (avg. 1.34 cm).
Next Steps:
Continuing on with the project, petioles and hypocotyls will be moved to larger jars with regular
MS medium once they grow roots, and growth of all three explant types will be measured on
regular intervals. Measurements will be used to compare health and robustness of the plants
over time.
Bibliography:
O’Connor-Sánchez,Aileen,Ángel V.Domínguez-May,Miguel A.Keb-Llanes,TomásA.González-Estrada,
and Yuri J. Peña–Ramírez."EfficientPlantRegenerationfromLeaf Explantsof SolanumAmericanum."
2010.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
3 mg/L 4.5 mg/L 6 mg/L
lengthofstem(cm)
Concentration of BAP
Hypocotyls: Length of stem (avg.)
Straight hypocotyls Curved hypocotyls

6. Phinney,B.O."Growth Response Of Single-Gene Dwarf MutantsInMaize To GibberellicAcid."
Proceedingsof the National Academyof Sciences,1956, 185-89.
Ray, P.M., U. Dohrmann,andR. Hertel."Characterizationof NaphthaleneaceticAcidBindingto
ReceptorSitesonCellularMembranesof Maize ColeoptileTissue."PlantPhysiology,1977,357-64.
Siddiqui,MdWasim,AmritaBhattacharjya,Ivi Chakraborty,andRS Dhua. "6-Benzylaminopurine
ImprovesShelf Life,OrganolepticQualityandHealthpromotingCompoundsof Fresh-cutBroccoli
Florets."2011.