ݺߣ

ݺߣShare a Scribd company logo

Informe Final

•Download as DOCX, PDF•
•
A
Adrianne Seiden

This study aimed to optimize the micropropagation of naranjilla plants by manipulating hormone concentrations in different explant types. Results showed that apical buds performed best with 0.05 mg/L NAA, producing the most leaves and longest stems. Upper petioles performed better than lower petioles. For hypocotyls, straight ones with 6 mg/L BAP produced the most leaves, while curved ones with 4.5 mg/L BAP had the longest stems. Overall, apical buds were the most productive explant, regenerating plants within 25 days, while petioles and hypocotyls only produced leaves on some explants without roots. Continued measurements will compare plant health and growth

1 of 6
Download to read offline
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) 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
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
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
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
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.

More Related Content

Informe Final

  • 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.