Polarity and Adventitious Root Formation Polarity is the condition inherent in a cutting that exhibits different properties in opposite parts. Proximal is closest to the root-shoot junction. Distal is away from the root-shoot junction. Distal Proximal Proximal Root - Shoot Junction Distal Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 1
Polarity and Adventitious Root Formation Stem cuttings form shoots at the distal end, and roots form at the proximal end. Distal Proximal Viburnum shoot cutting showing shoots from the distal end and roots from the proximal end of the cutting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 2
Polarity and Adventitious Root Formation Root cuttings of many species form roots at the distal end and shoots at the proximal end. Proximal Distal Lilac root cutting forming shoots from the proximal end and roots from the distal end of the cutting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 3
Polarity and Adventitious Root Formation Cuttings at left were placed for rooting in the normal, upright orientation and roots formed at the usual proximal end. Upright orientation Inverted orientation Cuttings at right were placed for rooting in an inverted position, but roots still developed from the proximal end. Polarity of root formation in grape hardwood cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 4
Effect of Leaves and Buds on Adventitious Root Formation Effect of leaves and buds on adventitious root formation in auxin-treated leafy Old Home pear cuttings. With leaves No leaves No buds Half leaves Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 5
Plant Growth Regulator Effects All classes of plant growth regulators influence root initiation either directly or indirectly. However, auxin have the greatest effect on root formation in stem cuttings, while cytokinins are used to stimulate adventitious bud formation in leaf cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 6
Plant Growth Regulator Effects Plant growth regulator Adventitious root formation Adventitious shoot formation Auxins Promote Inhibit; low auxin: high cytokinin ratio promote Cytokinins Inhibit; high auxin: low cytokinin ratio promote Promote Gibberellins Inhibit Inhibit; can enhance shoot elongation after organ formation Ethylene ABA Ancillary compounds Retardants/inhibitors, polyamines, jasmonate, brassionsteroids, phenolics Can promote with auxin-induced rooting of some herbaceous plants; with woody plants generally not directly involved in rooting Inhibit; however, used in combination with auxin can promote rooting in some species Used in combination with auxin can promote rooting in some species Not promotive Inhibits; however was reported to stimulate adventitious bud formation of a herbaceous species Not promotive; may depress shoot development Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 7
Plant Growth Regulator Effects Plants can be divided into three classes with regard to growth regulator effects on rooting: Easy-to-Root Moderately-Easy-to-Root Difficult-to-Root (Recalcitrant) Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 8
Plant Growth Regulator Effects Easy-to-Root plants have all the essential endogenous substances (root morphogens) including auxin. Plants that are easy-to-root include many herbaceous plants and some woody shrubs. When cuttings are made and placed under proper environmental conditions, rapid root formation occurs. Auxin may further enhance rooting, but is generally not required. Coleus Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 9
Plant Growth Regulator Effects In easy-to-root plants like mum, auxin may further enhance rooting, but is generally not required. Untreated 100 ppm 250 ppm 500 ppm Auxin concentration Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 10
Plant Growth Regulator Effects Moderately-Easy-to-Root plants have naturally occurring root morphogens present in ample amounts, but auxin is limiting. Plants that are moderately easy-to-root include some herbaceous plants, many woody shrubs and some trees. Auxin is needed to enhance rooting. Geranium Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 11
Plant Growth Regulator Effects Difficult-to-Root (Recalcitrant) plants lack rooting morphogens and/or lack the cell sensitivity to respond to the morphogens, even though natural auxin may or may not be present in abundance. Plants that are difficult-to-root include many woody shrubs and trees, especially as they attain a mature phase. External application of auxin gives little or no rooting enhancement. Paw paw (Asimina) cuttings will only root in the juvenile phase. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 12
Plant Growth Regulator Effects - Auxin Auxin was isolated as the chemical "heteroauxin" (indole-3-acetic acid) and identified as the first plant hormone in the 1930s. Fischnich (1935) showed that IAA could induce adventitious roots to form on intact coleus (Solenostemon) stems. The first report of IAA being used to stimulate rooting in cuttings was by Cooper (1935). He applied IAA in lanolin paste to stimulate rooting in lemon (Citrus), lantana (Lantana), and chenille plant (Acalypha) stem cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 13
Plant Growth Regulator Effects - Auxin By 1935, synthetic auxins were developed that were shown to promote rooting in cuttings. These included the familiar -naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) compounds used by modern propagators. By 1937, researchers at the Boyce Thompson Institute showed that auxin stimulated rooting in over 85 genera, including woody plants that had proven too difficult to propagate in the past. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 14
Plant Growth Regulator Effects - Auxin The Boyce Thompson Institute was granted a patent for use of auxins in rooting and subsequently licensed Merck to distribute Hormodin A for commercial application. By 1947, four commercial companies were offering synthetic auxin formulations in talc for application to cuttings. Along with Merck's Hormodin formulation, they were Quick-Root from Dow Chemical, Rootone from American Chemical Paint Co., and StimRoot from Plant Products Co. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 15
Plant Growth Regulator Effects - Auxin Auxin is the most important hormone involved in rooting. Naturally occurring Indole-3-acetic acid (IAA) Indole-3-butyric acid (IBA) Synthetic -Naphthalene acetic acid (NAA) 2,4-diclorophenoxyacetic acid (2,4-D) Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 16
Plant Growth Regulator Effects - Auxin Indole-3-acetic acid (IAA) is the most abundant naturally occurring auxin. Promotes root formation. Not commercial because plant enzymes degrade it quickly. Not light stable. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 17
Plant Growth Regulator Effects - Auxin Indole-3-butyric acid (IAA) is naturally occurring, but at very low abundance. It works by being converted to IAA by the plant. It is commonly found in commercial rooting compounds. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 18
Plant Growth Regulator Effects - Auxin -Naphthalene acetic acid (NAA) is a purely synthetic auxin. It is chemically similar to IAA in structure but is a more effective auxin in promoting rooting. It is commonly found in commercial rooting compounds and is often combined with IBA. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 19
Plant Growth Regulator Effects - Auxin 2,4-diclorophenoxyacetic acid (2,4-D) is a broad leaf herbicide with auxin activity. It is not used commercially as a rooting compound. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 20
Plant Growth Regulator Effects - Auxin It is historically important because it could be chemically manipulated to move the chlorine around the phenol ring. This work demonstrated that the distance from one end of the molecule to the other was important for auxin activity. Active Inactive Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 21
Plant Growth Regulator Effects - Auxin The acid form of auxin is not water soluble. They must be dissolved in solvent (ethanol, DSMO) or a base (1N NaOH). The potassium salts of IBA and NAA are soluble in water and used commercially. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 22
Plant Growth Regulator Effects - Auxin Relative rooting response of different auxins Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 23
Plant Growth Regulator Effects - Auxin IAA is not used commercially as often as synthetic auxins. This is because it is not as stable. IAA degrades in the light and is susceptible to destruction in the plant by IAA-oxidase. Acid form of IAA CH 2 COOH N H Decarboxylated IAA IAA-oxidase removes the carboxyl group (COOH) making it ineffective as an auxin. N H CH 2 COOH CO2 + H 2 O Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 24
Plant Growth Regulator Effects - Auxin Conjugation of IAA protects it from decarboxylation. Indole-3-acetic acid (IAA) Conjugation adds a sugar or an amino acid to the carboxyl end of the molecule. The conjugated form can be metabolized back to active IAA. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 25
Plant Growth Regulator Effects - Auxin The natural conjugates of auxin are not used commercially because they are expensive and not more effective than free IBA or NAA. There are synthetic aryl esters and amides of IBA that have been used. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 26
Plant Growth Regulator Effects - Auxin These have been shown to be effective alternatives to IAA and NAA and show less toxicity in animal studies. P-ITB has label clearance from EPA, but is still not commonly available. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 27
Plant Growth Regulator Effects - Auxin Polar auxin transport Auxin is produced in the apical meristems. Distal Auxin transport is polar. It moves from distal to proximal. Proximal Proximal Root / Shoot Junction Distal Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 28
Plant Growth Regulator Effects - Auxin Polar auxin transport Auxin transport proteins are located at the base of parenchyma cells. Its transport is not sensitive to gravity. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 29
Plant Growth Regulator Effects - Auxin Polar auxin transport Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 30
Plant Growth Regulator Effects - Auxin Auxin concentration is calculated in either Parts per million (ppm) Percentage Molarity Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 31
Plant Growth Regulator Effects - Auxin Commercial auxin quick dip preparations are sold as concentrated solutions (stock solutions). These must be diluted to make your desired concentration. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 32
Plant Growth Regulator Effects - Auxin Commercial auxin quick dip preparations are sold as concentrated solutions (stock solutions). These must be diluted to make your desired concentration. They may indicate their concentration based on percentage or ppm. A one percent solution = 10,000 ppm. One part per million is equal to 1 mg per liter. Talc preparations are sold in various concentrations and not meant to be diluted. Their concentrations are based on percentages. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 33
Plant Growth Regulator Effects - Auxin Use 500 to 1,250 ppm for easy-to-root herbaceous cuttings. Use 500 to 3,000 ppm for easy-to-root woody cuttings. Use up to 10,000 ppm for difficult-to-root woody cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 34
Plant Growth Regulator Effects - Auxin A typical auxin formulation might have 1% IBA. This would be 10,000 ppm. That equals 10,000 mg per liter or 10 grams per liter. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 35
Plant Growth Regulator Effects - Auxin PPM of concentrate x Volume to be removed from concentrate = Desired PPM of new solution x Volume needed for the new solution. Make a 1 liter solution containing 250 ppm IBA from an auxin concentrate that contains 1% IBA. 1. Solve for volume 10,000 ppm X Vol (ml) = 250 ppm X 1000 ml 2. Divide both sides by 10,000 ppm Vol (ml) = 250 ppm X 1000 ml / 10,000 ppm 3. Multiply 250 ppm by 1000 ml Vol (ml) = 250,000 ppm ml / 10,000 ppm 4. Divide by 10,000 ppm Vol (ml) = 250,000 ppm ml / 10,000 ppm 5. Add 25 ml of concentrate to 1 liter Vol (ml) = 25 ml Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 36
Plant Growth Regulator Effects - Auxin In the scientific literature, auxin concentrations are reported in molarity. One mole is equal to the molecular weight of the compound in grams dissolved in one liter of solvent. It is used because it gives you the number of active molecules regardless of the weight of the compound. Different auxins have different molecular weights. Therefore, a 1000 ppm solution of IAA and IBA would have a different number of active molecules in the solution, but 100 micromolar solutions of IAA and IBA have the same number of active molecules. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 37
Plant Growth Regulator Effects - Auxin Since commercial auxin concentrations are reported in percentage of ppm, you need to be able to convert ppm to molar. ppm = micromolar x molecular weight divided by 1000. x ppm = 2000 M IBA x 203 / 1000 = 406 ppm Micromolar = (ppm x 1000) divided by molecular weight. x M IBA = 406 ppm x 1000 / 203 = 2000 M IBA Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 38
Plant Growth Regulator Effects - Cytokinin The naturally occurring cytokinins include: Isopentenyl adenosine (2iP) Zeatin (Z) The major synthetic cytokinins include: Benzyladenine (BA) or Benzylaminopurine (BAP) Kinetin (KN) Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 39
Plant Growth Regulator Effects - Cytokinin Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 40
Plant Growth Regulator Effects - Cytokinin Important in shoot initiation. Tends to be inhibitory to rooting. Ratio of auxin to cytokinin is important in determining whether tissue cultures initiate roots or shoots. Impact of increasing concentrations of cytokinin on shoot formation in begonia leaf cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 41
Cytokinin concentration Principles of Propagation by Cuttings Plant Growth Regulator Effects - Cytokinin Auxin to cytokinin ratio Root formation Shoot formation Callus formation Auxin concentration Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 42
Plant Growth Regulator Effects - Gibberellin There are over 100 naturally occurring gibberellins. GA 1, GA 3, GA 4, GA 7 are most widely occurring types of gibberellins. GA 3 is gibberellic acid and is the natural product of a rice pathogenic fungus. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 43
Plant Growth Regulator Effects - Gibberellin Developing seeds have the highest concentration of gibberellins in the plant. Gibberellins are present in the shoot, stem, leaves and roots of plants. Gibberellin is generally inhibitory to rooting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 44
Plant Growth Regulator Effects - Gibberellin Adventitious rooting in wild type and an Gibberellin mutant in tomato. Wild type Wild type plus gibberellin Gibberellin mutant Rooting % Roots per cutting Rooting % Roots per cutting Rooting % Roots per cutting 95% 14.8 65% 1.7 95% 15.8 Cuttings from stock plants producing less ethylene showed no impact on rooting. However, cuttings treated with gibberellin showed a severe reduction in rooting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 45
Plant Growth Regulator Effects - Gibberellin Inhibitors of gibberellin biosynthesis Cycocel Ancymidol Triazole inhibitors Paclobutazol - Bonzi Uniconizole - Sumagic Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 46
Plant Growth Regulator Effects - Gibberellin Inhibitors of gibberellin biosynthesis Adding these compound generally increase rooting. They are not as active as auxin. Untreated Treated Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 47
Plant Growth Regulator Effects - Gibberellin Adventitious root formation in juvenile and mature English ivy treated with paclobutrazol. Roots per cutting Treatments Juvenile Mature Untreated Paclobutrazol 25.8 56.9 5.3 4.4 Gibberellin inhibitors usually do not improve rooting in difficult-to-root cuttings. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 48
Plant Growth Regulator Effects Abscisic Acid (ABA) ABA is naturally occurring and is most important for controlling stomatal opening during drought stress and during seed development. ABA application can increase rooting possibly by countering the negative impact of gibberellin. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 49
Plant Growth Regulator Effects Abscisic Acid (ABA) Adventitious rooting in wild type and an ABA mutant in tomato. ABA mutant Wild type Untreated stock plant ABA-treated stock plant Rooting % Roots per cutting Rooting % Roots per cutting Rooting % Roots per cutting 95% 14.8 70% 9.7 90% 13.4 Cuttings from stock plants producing less ABA showed reduced rooting and this was reversed by spraying stock plants with ABA. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 50
Plant Growth Regulator Effects Ethylene Ethylene is the gaseous hormone. It is always produced when auxin is added to plant tissue. Ethylene disrupts polar auxin transport. Therefore, it is difficult to separate the auxin and ethylene effects on rooting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 51
Plant Growth Regulator Effects Ethylene Ethylene application can increase rooting in some cuttings. It is applied as ethephon. Ethylene (and auxin induced ethylene) inhibits root elongation. Rooting is reduced in ethylene mutants in tomato. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 52
Plant Growth Regulator Effects Ethylene Adventitious rooting in wild type and an ethylene mutant in tomato. Wild type Ethylene mutant Rooting % Roots per cutting Rooting % Roots per cutting 95% 14.8 65% 6.0 Cuttings from stock plants producing less ethylene showed reduced rooting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 53
Plant Growth Regulator Effects Ethylene Ethylene (applied as ethephon) is used to prevent flowering and increase branching is some stock plants. Care should be taken to observe carry-over effects that can be observed as increased cutting senescence and reduced rooting in cuttings from ethylene-treated stock plants. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 54
Plant Growth Regulator Effects Rhizocaline Auxin promotes rooting but is only one of a number of factors needed to induce rooting. The German plant physiologist, Julius Sachs (1880's) felt that there were specific root forming substances made in the leaves that moved to the base of cuttings to promote rooting. Fritz Went in 1938, again postulated the existence of this substance and termed it rhizocaline. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 55
Plant Growth Regulator Effects Rhizocaline It could be shown experimentally that cuttings with leaves rooted better than those with the leaves removed. These types of studies suggested that there was a substance (other than auxin) produced in the leaves that were essential for rooting. Orange cuttings Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 56
Plant Growth Regulator Effects Rhizocaline Van Overbeek and Gregory in 1946, compared rooting in easy and difficult-to-root cultivars of rose-of-sharon (Hibiscus rosa-sinensis). There is no rooting in white hibiscus, even with auxin treatment. No auxin treatment Auxin-treated Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 57
Plant Growth Regulator Effects Rhizocaline They observed that grafting easy-to-root red hibiscus on difficult-to-root white hibiscus induces rooting in the presence of auxin. No auxin treatment Auxin-treated Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 58
Plant Growth Regulator Effects Rhizocaline They next girdled the stem and observed that girdling prevented rooting in the grafted plants. The conclusion was there is a graft transmissible factor (not auxin) produced in the easy-to-root shoots that is important for rooting. No auxin treatment Auxin-treated Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 59
Plant Growth Regulator Effects Rhizocaline Reciprocal grafts between juvenile and mature English ivy shows that a substance from the juvenile leaf improves rooting in the mature petiole. M J J J J M M M 14 24 26 0 0 5 Roots per cutting J M Juvenile (J) Mature (M) Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 60
Plant Growth Regulator Effects Cofactors In 1959, Charles Hess detected "rooting cofactors" from extracts of the juvenile form of English ivy (Hedera helix). This was shown as increased root formation in the mung bean (Phaseolus aureus) bioassay. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 61
Plant Growth Regulator Effects Cofactors One compound was ABA. Another group were phenolics. Compounds were also found that inhibited rooting. None of these compounds were determined to be the elusive rhizocaline. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 62
Plant Growth Regulator Effects Cofactors Phenolics act to protect auxin from destruction by acting as an alternative substrate for IAA-OXIDASE. Phloroglucinol has been used to root difficult-to-root plants especially during micropropagation. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 63
Plant Growth Regulator Effects Cofactors Indole-3-acetic acid (IAA) Decarboxylation Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 64
Plant Growth Regulator Effects Cofactors The search continues for rhizocaline. For example, Max Kawase in the 1970's found that an extract from willow stems could increase rooting in stem cuttings from a variety of species. The chemical nature of willow extracts has not yet been established. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 65
Plant Growth Regulator Effects Cofactors Rhizocaline may never be found. It is more probable that there is a fundamental difference between a cell's competency to respond to auxin between easy and difficult-to-root phases of a plant's life cycle. The genes critical for initiating rooting are just starting to be discovered. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 66
Biochemical Effects on Rooting The biochemical basis for root formation implies that there are root-promoting and rootinhibiting substances produced in plants and their interaction is thought to be involved in rooting. Therefore, this theory considers that difficult-to-root cuttings either lack the appropriate root-promoting substances or are high in root-inhibiting substances. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 67
Biochemical Effects on Rooting Hypothesized scheme for the interaction between Phytohormones Phenolics IAA oxidase/peroxidase Borate in the four developmental stages of adventitious root production. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 68
Biochemical Effects on Rooting In past research, effects of experimental treatments may have been at any or all process levels, but were usually assessed only posttranslationally, in physiological and/or biochemical studies. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 69
Morphological, Physiological and Biochemical Events in Rooting Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 70
Some 220 genes are differentially expressed during the five phases (time period - days) of adventitious root development in Pinus contorta. The histogram shows the percentage of genes upregulated (increased gene expression) or downregulated (decreased) during rooting. Molecular Events during Rooting Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 71
Molecular Events during Rooting Microarray analysis was used to show changes in gene expression for different stages of adventitious root formation of Pinus contorta hypocotyl cuttings. The figure shows which classes of genes were up-regulated ( increased expression) or down-regulated ( decreased expression). Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 72
Auxin-induced Gene Expression During Rooting Given the importance of auxin in adventitious rooting, the ability for auxin to induce gene expression is necessary. It is common for auxin-inducible genes to be repressed (prevented from action) by the protein-protein interaction the Aux/IAA repressor molecule acting on the Auxin Response Factor (ARF) positioned in the promoter region of an auxin-inducible gene. Inactive gene Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 73
Auxin-induced Gene Expression During Rooting In order to activate the gene, the repressor molecule (Aux/IAA) must be removed from its interaction with ARF. In order for this to occur, auxin (IAA) must interact with its receptor complex. The auxin receptor is the F-box protein TIR1 located in the cell s nucleus. Auxin receptor complex IAA TIR1 is the auxin receptor Auxin coupled with its receptor Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 74
Auxin-induced Gene Expression During Rooting There are several proteins that associate with TIR1 to form a receptor complex. The job of the receptor complex is to locate Aux/IAA and to attach several ubiquitin molecules to the protein. This poly-ubiquitination targets Aux/IAA for proteolytic destruction. Auxin receptor complex IAA Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 75
Auxin-induced Gene Expression During Rooting Once Aux/IAA is removed from association with ARF, it acts to initiate gene transcription of the auxin-inducible gene. Active gene Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 76
Auxin-induced Gene Expression During Rooting It is evident from this discussion that Auxin Response Factors are important for initiating an auxin response including rooting. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 77
Auxin-induced Gene Expression During Rooting The ARFs important for adventitious root formation have been found in Arabidopsis. They include ARF6, ARF8, and ARF16. Additional research has shown that the level of these ARFs is under translational control by micrornas. Arabidopsis Adventitious roots A microrna is a small regulatory RNA consisting of 22 nucleotides. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 78
Auxin-induced Gene Expression During Rooting A microrna is a small regulatory RNA consisting of 22 nucleotides. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 79
Auxin-induced Gene Expression During Rooting The sequence of these nucleotides related to auxin-induced gene expression corresponds to a section of the coding region of the mrna for ARF. If a specific microrna corresponding to an ARF is produced, it will prevent or considerably reduce the production of the ARF protein. In Arabidopsis, the micrornas responsible for ARF levels are mirna 160 and mirna 167. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 80
Auxin-induced Gene Expression During Rooting In Arabidopsis, the micrornas responsible for ARF levels are mirna 160 and mirna 167. The interaction of the mirna with the coding region of the mrna causes disruption of mrna translation and no ARF protein is produced. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 81
Auxin-induced Gene Expression During Rooting Therefore, it is becoming evident that for a cutting (at least in Arabidopsis) to be competent to form adventitious roots, there must be available auxin to interact with its receptor complex as well as the production of specific Auxin Response Factors to initiate gene expression. Although, this research is yet to be accomplished, it is logical to hypothesize that one possible reason difficult-to-root, nonauxin responsive cuttings fail to root is that they fail to produce appropriate ARFs. It is also logical to assume that the deficiency in ARF production is likely due in part to gene silencing by micrornas. Hartmann and Kester s Plant Propagation, Principles and Practices 8 th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 82