Naranjilla Micro-Propagation

Adrianne Seiden

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)

Naranjilla background

• Distribution: indigenous to Sierra of Ecuador and Colombia

• So far unable to be grown outsideof 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.

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

--------------

0.010.020.030.040.05

----------------------

Medium BAP(mg/l) NAA (mg/l) GA3 (mg/l)

MS 0.01 4.5 0.10.250.50.751.00

Medium BAP(mg/l) NAA(mg/l) GA3 (mg/l)

MS 55.255.55.756

0.01 0.1

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 ----- 0.010.020.030.040.050.2

-----

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

0.10.250.50.751.0

Middle

Inferior

Medium Type of explant

BAP (mg/L)

NAA (mg/L)

GA3 (mg/L)

MS Curved 34.555.255.55.756

0.01 0.1

Straight

Procedure: growing the plants

1) Market seeds are cleaned and disinfected1) 3 min in alcohol 70%2) 18 min in hypochlorite 2.5% and 4-5 drops of tween, stirring every 3 min3) 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

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

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:

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)

Results: Apical BudsNumber 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

0.01 0.05 0.24.78

4.8

4.82

4.84

4.86

4.88

4.9

4.92

4.94

4.96

Number of leaves

Concentration of NAA (mg/L)

num

ber o

f lea

ves

(avg

.)

0.01 0.05 0.20

0.5

1

1.5

2

2.5

3

3.5

4Stems and Roots

length of stemLength of root

Concentration of NAA (mg/L)

Len

gth

(cm

)

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