1. Naranjilla Micro-Propagation
Adrianne Seiden

2. Project Objectives
• Improve upon previous study (standardize procedure for micro-
propagation of naranjilla)
• Original sample size not large enough for data to be statistically supported
• Check for differences between seeds bought at market and seeds
from INIAP (Instituto Nacional de Investigaciones Agropecuarias)

3. Naranjilla background
• Distribution: indigenous to
Sierra of Ecuador and Colombia
• So far unable to be grown outside
of South America
• Characteristics: fruiting shrub that grows
up to 8ft (2.5m) tall, with a thick spiny stem (no spines when cultivated) and wooly
leaves up to 2ft (60cm) long
• covered in fine purple hair when young
• Fragrant white flowers
• Juicy orange fruit, covered in brown fur until ripe
• grows at 3,000 - 8,000 ft above sea level
• Susceptible to nematodes (roundworms)
• Low genetic diversity
• Uses: eaten raw, made into juice, jam, pies, etc.

4. Original Study
Treatments:
• Types of explants:
• Apical buds
• Petioles (superior, middle, inferior)
• Hypocotyls (curved,straight)
• Hormones being manipulated:
• Naphthalene acetic acid (NAA)
• Benzyl amino purine (BAP)
• Gibberellic acid (GA3)
Hormone concentrations: Apical buds
Hormone concentrations: Petioles
Hormone concentrations: Hypocotyls
Medium BAP(mg/l) NAA (mg/l) GA3 (mg/L)
MS --------------
0.01
0.02
0.03
0.04
0.05
----------------------
Medium BAP(mg/l) NAA (mg/l) GA3 (mg/l)
MS 0.01 4.5 0.1
0.25
0.5
0.75
1.00
Medium BAP(mg/l) NAA(mg/l) GA3 (mg/l)
MS 5
5.25
5.5
5.75
6
0.01 0.1

5. New Study: Treatments
• Types of explants:
• Apical buds
• Petioles (superior, middle, inferior)
• Hypocotyls (curved and straight)
• Hormones being manipulated:
• Naphthalene acetic acid (NAA)
• Benzyl amino purine (BAP)
• Gibberellic acid (GA3)
Medium BAP (mg/L) NAA (mg/L) GA3 (mg/L)
MS ----- 0.01
0.02
0.03
0.04
0.05
0.2
-----
Hormone concentrations: apical buds
Hormone concentrations: petioles
Hormone concentrations: hypocotyls
Medium Type of
explant
BAP
(mg/L)
NAA
(mg/L)
GA3
(mg/L)
MS Superior
4.5 0.01
0.1
0.25
0.5
0.75
1.0
Middle
Inferior
Medium Type of
explant
BAP
(mg/L)
NAA
(mg/L)
GA3
(mg/L)
MS Curved 3
4.5
5
5.25
5.5
5.75
6
0.01 0.1
Straight

6. Procedure: growing the plants
1) Market seeds are cleaned and disinfected
1) 3 min in alcohol 70%
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 MS medium, under sterile camera,
10 seeds per jar
3) Jars are covered with saran wrap and placed under light

7. Study Treatments: Apical Buds
• What they are: AKA terminal bud, the very top of a growing plant,
where new leaves emerge
• Procedure: All leaves are removed, stem is cut just below most recent
leaves, planted right-side-up
• Treatments: NAA .01 mg/L, NAA .05 mg/L, NAA .20 mg/L
0.01NAA 0.05 NAA 0.20 NAA

8. Study Treatments: Petioles
• What they are: The stalk attaching the leaf to the stem
of a plant
• Procedure: roots are removed, stalk is cut in 3, discarding the middle,
leaves are removed, and petioles are cut from the main stem, placed
in medium on their side
• Treatments: BAP 4.5 (mg/L)/NAA 0.01 (mg/L)/GA3 1.0 (mg/L)
Superior: Inferior:

9. Study Treatments: Hypocotyls
• What they are: Stem below the first leaves
• Procedure: 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
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
• Treatments: BAP 3 (mg/L), BAP 4.5 (mg/L), BAP 6 (mg/L)/NAA .01 (mg/L)/GA3 0.1 (mg/L)

10. Results: Apical Buds
Number of leaves
(avg. per explant)
Length of stem
(avg. per explant)
Length of root cm
(avg. per explant)
0.01 mg/L NAA 4.85 2.5 cm 2.27 cm
0.05 mg/L NAA 4.95 3.58 cm 1.66 cm
0.20 mg/L NAA 4.90 2.93 cm 1.56 cm
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 of NAA (mg/L)
Number of leaves
0
0.5
1
1.5
2
2.5
3
3.5
4
0.01 0.05 0.2
Length(cm)
Concentration of NAA (mg/L)
Stems and Roots
length of stem
Length of root

11. Next Steps
• Collect data on petioles and
hypocotyls
• Employ micro-propagation protocol
for conservation programs
• Micro-propagation protocol can also
be used to cultivate modified
naranjilla plants for further study