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Naranjilla Micro-Propagation

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Adrianne Seiden

This study aimed to improve micropropagation techniques for naranjilla by standardizing procedures and comparing market seeds to those from a research institute. In the original study, different explants, hormones, and concentrations were tested. In the new study, apical buds, petioles, and hypocotyls were treated with various concentrations of NAA, BAP, and GA3 hormones. Results showed apical buds treated with 0.01 mg/L NAA produced the most leaves and longest stems on average. Next steps are to collect data on petioles and hypocotyls, and use the optimized micropropagation protocol for conservation and cultivating modified naranjilla plants.

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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 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.
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
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
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
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 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
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

More Related Content

Naranjilla Micro-Propagation

  • 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

Editor's Notes

  1. Apical buds: NAA in concentration 0.03 mg/L yielded the largest leaves, highest number of leaves, and longest roots; NAA in concentration 0.02 mg/L yielded the longest stems Petioles: inferior petioles yielded the largest sprouts and leaves, GA3 in concentration 0.5 mg/L was marginally better in terms of number and size of sprouts, GA3 in concentration 1.0 mg/L yielded the greatest number of leaves Hypocotyls: curved hypocotyls produced larger sprouts; neither type of hypocotyl (curved or straight) nor the concentration of hormones in the medium had a significant effect on any other parameters