Anatomy and physiology of adventitious root...

Anatomy and physiology of adventitious root formation Prof. Dr. Károly Hrotkó

SZIU Faculty of Horticultural Science

Anatomy and physiology of adventitious root formation

Autovegetative propagation in nursery 2

Widespread used autovegetative propagation methods in nurseries Cuttings (green cuttings, softwood cuttings, semi-

hardwood cuttings: leafy cuttings): ornamental shrubs and trees, conifers, some rootstocks

Hardwood cuttings: ornamental shrubs and trees, berry fruits, some rootstocks, forest plants, energy plants

Micropropagation: ornamental shrubs and trees, conifers, some rootstocks

Layerage: rootstocks, berry fruits, some ornamentals Markottage: using suckers (raspberry) Root cuttings: very few application (some berry fruits,

Rhus, etc.)

Faiskola bevezető előadás

Dr. Hrotkó Károly

Mist propagation (leafy cuttings)

Hardwood cuttings in outdoor beds

Stoolbed

Micropropagation

Possible application at plants developing propagating organs:

plantlets, (strawberry) stolons, root suckers

plants developing preformed roots (Ribes, some Prunus sp., Malus, Salix, Populus)

plants developing adventitious roots (Ligustrum, Forsythia, Philadelphus)

plants developing adventive buds, shoots on root part (root cuttings)

Growth of preformed roots on Salix caprea

Plantlets for propagfation of strawberry

Preformed roots Colt cherry rootstock

Apple (J-TE-D)

Possible application at plants developing propagating organs:

plantlets, (strawberry) stolons, root suckers

plants developing preformed roots (Ribes, some Prunus, Malus, Salix, Populus)

plants developing easily adventitious roots (Ligustrum, Forsythia, Philadelphus)

plants developing adventive buds, shoots on root part (root cuttings)

Schedule of reparative regeneration in autovegetative propagation methods Cuttings (hardwood or leafy cuttings):

regeneration after separation of propagationg material

Layerage (layering, stooling): regeneration on stockplant separation after rooting of shoots

Markottage, suckers, propagating organs: regeneration on stockplant separation after rooting of shoots

Micropropagation: regeneration after separation of propagationg material, in-vitro conditions, followed by acclimatization and hardening off

Anatomy of adventive root formation

← terminal cutting

← basal cutting

Hardwood cutting

Shoot meristem in shoot tip Section of subterminal growth

Rhytidom typical to species, leaf fall, → leafless shoot

Secondary expansion, lignification

Secondary expansion, complete cambial ring, formation of secondary xylem schlerenchyma (lignification starts)

Cross section of an angiosperm,

dicotyledonous, secondary expansion stem periderm

paracambium

yearring

Tilia cordata

littleleaf linden

MALVACEAE

parenchyma

flared

primary pith

xylem cambium

soft phloem hard phloem pith

Possible position of new roots

Hormonal compounds in root initiation

Adventive root formation in the stem

Activating factors - ceased correlative balance due to cutting separation

- accumulation of hormonal compounds

- environmental conditions (etiolation, blanching, soil cover)

Root initiation process 1. dedifferenciating cells, cell multiplication 2. formation of root initials

- auxin sensitive stage

- auxin inaktiv stage

3. formation of root primordium

4. root elongation and growth

Role of auxine compounds

Auxin - controls rooting in early stage of initiation

- bazipetal auxine transport is important

- decreasing concentration in the later stage of rooting

- interaction between native (IES) and exogen applied heteroauxins

- shoot tips, buds, leaves are necessary

Rizocalin theory (Bouillene & Bouillene-Walrand 1955)

root initiating complex consisting of:

a) specifikus factor from leaves or buds (o-dihidroxi-fenol)

b) non-specific factor (auxin)

c) specific enzim (polifenol-oxidase PPO)

Stages of root initiation

Rhizocalin theory (Bouillene & Bouillene-Walrand 1955)

Application of auxine in practice

Auxine compounds : IAA, NAA, IBA

Commercial powder preparations (talcum, bentonit)

Dilute solution Soaking method (50 – 150 ppm)

Quick dip (concentrated solution in 50% alcohol)

K + salt formulation (water soluble form)

Spray, paints.

• Turgor, water content.

• Temperature.

• Light - shading, etiolation.

• Nutrition.

• CO2 enrichment.

Stockplant management for cutting propagation

Dr. Hrotkó Károly Environmental conditions and rooting of cuttings 25

Water supply of stockplant and water content of cuttings Optimal water supply is required.

Taking cuttings earlier (before 9 am) results higher turgor.

Water deficit of stockplants results low rooting capacity and susceptibility to infections.

Water loss of hardwood cuttings improves the uptake of hormonal soultion.

16.05.2008

Érdi V. 4/24 Érdi V. 4/23

Korponay 4/8 Korponay 4/10

26.05.2008

0 time

Érdi V. 4/24 Érdi V. 4/23

Korponay 4/8 Korponay 4/10

Dynamics of transpiration of

leaves on Prunus avium

in May 2008.

(2008. 05. 16. és 2008. 05. 26.)

Water content

influence the

rooting of cuttings;

above 50% more

favorable

Sainte Julien GF 655/2

40 45 50 55 60

Water (%)

Gyökeresedés

Lineáris (Gyökeresedés)

Rooting of plum hardwood cuttings related to

water content of cuttings

activity

cold unit

content leaf fall POD-activity

phenolics

sprouting rooting

temperature

Effects on rooting of plum rootstocks hardwood cuttings

Marianna GF 8-1

-250 0 250 500 750 1000 1250 1500 1750 2000

Cold unit (CU)

gyökeresedés

Lineáris (gyökeresedés)

– No direct link

between dayly

average

temperature and

rooting

– Low cold unit

quantity befora

taking cuttings

(below 500 CU) is

more favorable

Temperature and cold unit (CU) on plum rootstocks’

hardwood cuttings

Rooting of plum rootstocks’ hardwood cuttings

Phenolics and

rooting

– favorable:

Fehér besztercei:

above 10 μg/mg

St. Julien GF 655/2:

10-30 μg/mg

Marianna GF 8-1:

15-40 μg/mg

Marianna GF 8-1

0 5 10 15 20 25 30 35 40 45 50

phenol content in buds (μg/mg)

Gyökeresedés

Polinom. (Gyökeresedés)

POD activity and

rooting

– Low activity is more

favourable

– Fehér besztercei (bud)

St. Julien GF 655/2

(cutting): under 4 U/mg,

Marianna GF 8-1

(cutting): under 3 U/mg

Marianna 8-1

0 1 2 3 4 5 6 7

POD activity (U/mg protein)

Gyökeresedés

Lineáris (Gyökeresedés)

Rooting of plum hardwood cuttings

9. 1. 10. 1. 11. 1. 12. 1. 1. 1. 2. 1. 3. 1.

taking of cuttings (date)

Fehér besztercei

Sainte Julien GF

Marianna GF 8-1

Polinom. (Fehér

besztercei)

Polinom. (Marianna

GF 8-1)

Polinom. (Sainte

Julien GF 655/2)

Effect of light on stockplants Daylength

- affetc photosysnthesis: carbohidrate accumulation - fotomorfogenetic effect : increasing daylength affect positive the vegetative growth and rooting of Chrisanthemum.

Wawelength no consistent results: red light stimulate the phytochrom system and rooting, but some results indicate that blue light also positive.

Etiolation and shading of stockplants Etiolation: excluded solar radiation. During preconditioning different level of shading

is more applied in practice. Local etiolation using black strips or Alu folie. Effect

- starch accumulation, - reduced formation of schlerenchima, - reduced lignin production – phenolics may be used for rooting, - etiolated tissue react more efficient to auxine treatments.

Effect of etiolation on stem – Tilia cordata

Rejuvenalization

grafting subsequently

on seedlings →

Etiolated and shaded

stockplants

kontroll talajjal takart Raschel háló átlátszó fólia fekete fólia

gyökeresedés %

Effect of preconditioning on rooting of MM.106 cuttings

Nutrition of stockplants Shoot mass and its nutrient content is determining

to the regeneration process (related to the anatomy structure too)

Factors of nutrient balance during rooting: + absorption from substrate (no importance!) + assimilation (only leafy cuttings!) - respiration during rooting (increase due auxine application) - leaching due to irrigation, mist application

Principles of nitrogen and carbohydrate metabolism in cuttings Lack of roots – no nitrogen uptake and assimilation

Amino acid sources for root formation: - Amino acid reserve - proteolitic processes (decomposition of proteins)

Carbohydrates: energy reserves and sources for structural carbohydrates (cellulose)

Optimal concentration of non-structural carbohydrates (sugar, starch) is required for rooting

Starch content test on hardwood cuttings (jódos-jódkáli 0,2%)

Importance of Carbohydrate / Nitrogen ratio Carbohydrates: energy reserves and sources for

structural carbohydrates (cellulose)

Indirect effects!

Higher C / N ratio in stockplant shoots is favourable.

Exceed nitrogen reduction decreases the shoot (cuttings) production.

In thin shoots, secondary shoots the C/N ratio and rooting is higher.

Management of stockplant in order to improve C/N ratio in practice Decreased N- supply; increased K and P supply

Root restriction: close planting, root pruning

Pruning back the primary shoots (increased C/N ratio in secondary shoots)

Moving away from shoot basis C/N ratio decreases

Maintaining healthy leaf surface

Positive effect of ringing, etiolation

Role of other nutrients in rooting process Ca - activates peroxidase, essential for growing of root

primordium

Zn - cathalitic effect on triptophan synthesys → formation of native auxine

Mn - activates oxidative enzymes

B - influences the permeability of membrane, sugar transport, activates enzyme, carbohydrate metabolism, metabolism of phenolics, formation of auxine, and nucleic acids

treatments Rooting (%) Cuttings’ weight

Kelpak on stockplant

+ IBA 0.2% on cuttings

77,1 a 3,77 b

Wuxal Ascofol on stockplants

+ IBA 0.2% on cuttings

66,7 a 2,86 a

Pentakeep-V on stockplants

+ IVS 0.2% on cuttings

60,4 a 2,96 ab

Untreated stockplant

+ IVS 0.2% on cuttings

75,0 a 2,88 a

Effect of biostimulator treatments of stockplants

on cuttings’ quality (Szabó et al. 2011)

Shoot position and type of cuttings is related to nutrient content Considerable morphological and nutrient content

differences along the shoot from basis to shoot tip. Secondary shoots, branches root better, but

consider plagiotropic shoots. Basal cuttings generally root better:

- more ripe and lignificated shoots, higher C/N ratio, - meristematic cell groups in branch collar, - number of preformed roots considerable decreases towards the shoot tip.

Shoots with flower buds root less (blueberry, Abelia, Ligustrum, Ilex, etc.)

CO2 enrichment Increased photosynthese – more shoots appropriate

for cuttings.

Only in closed systems (greenhouse) is possible.

In winter effectivity should be increased usin supplementary lighting.

• Mentain leaf water content and turgor.

• Water loss through transpiration activity water supply

• Photosynthetic activity and solar radiation transpiration to avoid heat stress

• Optimal temparature in rooting zone

Environmental conditions during rooting of leafy cuttings

Prof. Dr. Hrotkó Károly Seed germination….. 50

Autovegetative propagation in nursery

Difficulties in water management of leafy cuttings Water loss due to transpiration.

Water uptake - no roots , - basal part of tracheas, tracheids are blocked, no water transport, - minimal water uptake through leaves, - more water uptake through basal part of cuttings.

Water deficit→ stomatal conductance reduced→ no CO2 uptake→reduced photosyntesis

How to control water loss of cuttings? Water loss depends on difference in vapour

pressure of leaf and environmental air.

Difference could be deminish - leaf temperature and vapour pressure of leaves - increased vapour content in environmental air, - combination of both factors.

Vapour pressure of leaves is function of - leaf temperature and - difference between leaf temperature and the leaf-air temperature difference (Δt).

Importance of solar radiation When solar radiation intensity is larger than 0,8 J

cm-2 min-1 (Klougart 1975) shading is required. Large fluctuation in solar radiation

in May 250 – 540 J cm-2

in Agust 250 – 2100 J cm-2 (Klougart 1975). Shading reduces insolation at 70-80%. Long wave radiation gets throug PE tent →plant

temperature is higher than under glass, but during the night the temperature decrease more.

White plastic foil reflects 40-60% of solar radiation.

Options in reducing water loss of leafy cuttings in propagation technology Using closed propagation units

(polythene tent, closed systems inside the glasshouse, polythene tent, shading, contact plastic cover, wet cover)

Intermittent mist (open and closed propagation units)

Fog system

Closed propagation units: advantages and shortcomings Simple and cheap (glass, polythene tent, etc.) Heat trap effect → increasing leaf – air vapor pressure

difference → water loss. Shading is required:

- PE-inner cover, modifyed PE foils (vinil-acetate, Al- or Mg-silicate added) long wave radiation in reflected - flies

Additional mist or fog systems could be installed. Contact foil cover: advantageous when radiation and

temperature is controlled, condeansation of vapour contributrs to wetting of leaves. Recommended for less sensitive leaves (evergreens – thick epidermis – danger of drying out is lower).

Double cover inside the polythene tent

Dr. Hrotkó Károly

Environmental

conditions and

rooting of

cuttings 63

Shading is required !

Intermittent mist: benefits and shortcomings Water film on leaves reduces leaf temperature → reduced

leaf vapour pressure – minimal difference in vapor pressure between leaves and air → reduced leaf transpiaration.

Intermittent mist reduces the air temperature due to evaporation → advective cooling of leaves.

High air vapour content (80-95% relative humidity) reduces the Δ RH.

Reduces the temperature in rooting zone – bottom heat is required.

Leaching of nutrients from leaves and cuttings. Applyed in closed propagation units.

(Environmental factors in open systems are less controllable, nowadays are not utilized in practice.)

Propagation unit with intermittent mist and shading

Application of intermittent mist and shading in PE tent and glasshouse

Fog systems High air relative humidity.

Fog systems spray at 40-60 bar pressure water drops of 15 μm size into air, which remain flotating.

Reduces leaching of nutrients.

No temperature decrease in rooting zone.

Larger costs in investment and maintenance .

Applicable for acclimatization of in-vitro plants also.

Propagation unit with fog system

Importance of temperature Optimum temperature at temperate zone plants 18-25

˚C, at tropical and subtropical plants 25-32 °C

Higher temperature at daytime (21-27 °C), lower at night (15 °C) .

Higher temperature increases the shoot growth in contrary root formation; by using bottom heat could be balanced.

At root growth higher roote zone temperature is recommended while for formation of root initials lower temperature is favourable.

Role of light in rooting of cuttings Rooting requires relative low solar radiation.

Cuttings of some herbaceous plants (Chrisanthemum, Pelargonium, Poinsettia) root better at increased solar radiation (116 Wm-2), optimum differrs by species (20-100 Wm-2).

Cuttings of some ornamental plants root better at long-day conditions.

Long day conditons are preferable for growth of rooted cuttings eg. Rododendrons (additional lighting in wintertime is favourable).

Red and fare-red wavelength is favourable for rooting (rooting is linked to phytochrom-system!).

Photosysnthetic activity of cuttings For rooting no highest photosysnthetic activity is

required, too high PAR increase the danger of drying out of cuttings.

After rooting intensive photosynthetic activity is required for growth of cuttings.

Citokinin formation of cuttings increase the stomatal conductance and the intensity of photosynthesis.

Treatment Rooting % Cuttings’ weight (g)

Untreated 37,5 b 1,74 ab

IBA 60,4 cd 1,48 ab

IBA + Pentakeep V 81,3 e 2,53 c

IBA + Kelpak 47,9 bc 1,90 b

IBA + Wuxal Ascofol 68,8 de 2,01 b

Yeald Plus 66,7 de 1,94 b

Benziladenin 0,02% 6,3 a 1,29 a

Effect of treatments

on Prunus mahaleb ‘Bogdány’ cuttings (Szabó et al. 2011)

Benefits and shortcomings of different propagation systems in technology Working with cuttings placed on ground is difficult. Replanting, potting of rooted cuttings is not economical. Mist propagation when using high Fe and Mg containing

water cause stayning by salt – cuttings are not attractive. Bottom heat is required when using mist beacause of

temperature decrease in rooting zone. Nutrient leaching from cuttings could not be balanced by

spray with fertilizers. White (milk foil) cover reflects of 40-60% of radiation,

isothermal condition, minimal vapour condensation. Using propagatin trays nutrient sprays after rooting

improves the growth of cuttings.

Vegetatív szaporítási módok

Taking and preparation of cuttings - Timing: easy to root plants during winter dormancy, difficult to root species only in rooting optimum - taking and cutting preparation - hormonal treatment, wounding - using antitranspirants

Hardwood cuttings

Autovegetatív szaporítások

9. 1. 10. 1. 11. 1. 12. 1. 1. 1. 2. 1. 3. 1.

taking of cuttings (date)

Fehér besztercei

Sainte Julien GF

Marianna GF 8-1

Polinom. (Fehér

besztercei)

Polinom. (Marianna

GF 8-1)

Polinom. (Sainte

Julien GF 655/2)

Optimal timing of plum hardwood cuttings

straight heel mallet

Cutting types and wounding

Polarity

Storage of cuttings - temporary storage at autumn planting - Cold storage (+ 2°C) - bottom heat → storage

Planting cuttings in open field in spring - optimal soil temperature (10-12 °C) - planting by machine or by hand - soil covering, black plastic soilc covers - mounding up

Taking care of cuttings during season Lifting specialities

Major steps in rooting technology of hardwood cuttings

Storage of hardwood cuttings

Bottom heat treatment should be ceased

before root initials start grow!

Bottom heat treatment of hardwood cuttings

’St. Julien A’ hardwood cuttings in open ground conditions

Anatomy and physiology of adventitious root...

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