cones: a lesson in patience Patrick von Aderkas & Lisheng Kong - - PowerPoint PPT Presentation

How to adorn trees with more cones: a lesson in patience

Patrick von Aderkas & Lisheng Kong University of Victoria

Initial screening of hormone levels in apical buds where cone buds are formed Initially, 9 genotypes of coastal Douglas-fir 8 genotypes of lodgepole pine were tested. Over the years, we have measured hormones in at least double this number of genotypes of each species

Meiosis Microspores Fertilization

Year 2

Pollination

(pollen enters the ovule) Year 1

♂ cone bud

• nly 1 of 4

megaspores survives

Megagametophyte Fertilization

Year 2

Pollination

(pollen enters the ovule)

Seed

Year 1

Meiosis ♀ cone bud

Douglas-fir

Microspores Fertilization

Year 2

Pollination

(pollen enters the ovule) Year 3 Year 1

Meiosis ♂ cone bud

Meiosis

• nly 1 of 4

megaspores survives

Fertilization

Year 2

Pollination

(pollen enters the ovule) Year 3

Seed

Year 1

Megagametophyte ♀ cone bud

• A photo of brach with three year cones

Year 1 Year 2 Year 3

Do endogenous hormone concentrations change over the course of apical bud flush and the period of male and female cone initiation?

Resulted in very large studies with lots of repeated sampling, and lots of chemical analysis

Rather than take you through dozens and dozens of graphs, we will summarize the overall

• results. For the curious we have listed the

papers in which they appear.

Kong, L., P. von Aderkas, Zaharia, L.I. 2016. Effects of exogenously applied gibberellins and thiadiazuron

• n phytohormone profiles of long-shoot buds and cone gender determination in lodgepole pine.

Journal of Plant Growth Regulation 35: 172-182 Kong, L., P. von Aderkas, I. Zaharia, S.R. Abrams, T. Lee and J. Woods. 2012. Analysis of phytohormone profiles during male and female cone initiation and early differentiation in long-shoot buds of lodgepole pine. Journal of Plant Growth Regulation 31: 478-489. DOI 10.1007/s00344-011-9257-1 Kong, L., P. von Aderkas, S.J. Owen, B. Jaquish, J. Woods and S.R. Abrams. 2012. Effects of stem girdling

• n cone yield and endogenous phytohormones and metabolites in developing long shoots of Douglas-

fir (Pseudotsuga menziesii). New Forests 43: 491-503. DOI 10.1007/s11056-011-9294-4 Kong, L., P von Aderkas, SJ Owen, T Wagner and SA Abrams. 2011. Comparison of endogenous cytokinins, ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine. Trees - Structure and Function 25: 1103-1110. DOI 10.1007/ s00468-011- 0585-3 Kong, L., S.R. Abrams, S. J. Owen, A. van Niejenhuis, P. von Aderkas. 2009. Dynamic changes in concentrations of auxin, cytokinin, ABA and selected metabolites in multiple genotypes of Douglas-fir (Pseudotsuga menziesii) during a growing season. Tree Physiology 29: 183-190. Kong, L., S.R. Abrams, S. J. Owen, H. Graham, P. von Aderkas. 2008. Phytohormones and their metabolites during long shoot development in Douglas-fir following cone induction by gibberellin

• injection. Tree Physiology 28: 1357-1364.

Publications (BCSOA folks in bold !)

ABA catabolism

ABA catabolic changes in growing leader buds: Douglas-fir

ABA catabolic changes in growing leader buds: lodgepole pine

ABA catabolic pathways lodgepole pine & Doug-fir Doug-fir only

Concentration (ng/g DW) of endogenous hormones in lodgepole pine leader buds

np - no peak. L - below limit of quantification/calibration Late Early

Cytokinin t-Z L L dhZ L L t-ZOG L 10 t-ZR 61 + 2 43 + 7 dhZR 37 + 2 27 + 5 iPA 14 + 1 11 + 1 2iP 7 11.4 + 0.5 Auxin IAA 28 + 3 42 + 1 IAAasp L L IAAglu L L

Concentration (ng/g DW) of endogenous hormones in Douglas-fir leader buds

np - no peak. L - below limit of quantification/calibration

Cytokinin t-Z L L dhZ L L t-ZOG 11.3 + 0.3 27 + 1 t-ZR 34 + 1 172 + 1 dhZR L 9.5 + 0.5 iPA 56 + 1 31.7 + 0.8 2iP L L Auxin IAA 216 + 15 110 + 18 IAAasp L L IAAglu np np

early late

Concentration (ng/g DW) of endogenous hormones in lodgepole pine leader buds

np - no peak. L - below limit of quantification/calibration Late Early

Gibberellin GA1 np np GA3 np np GA4 np np GA7 np np

Concentration (ng/g DW) of endogenous hormones in Douglas fir leader buds

np - no peak. L - below limit of quantification/calibration

Gibberellin GA1 np np GA3 np np GA4 22 + 3 np GA7 L np

Late Early

Do endogenous hormone concentrations change over the course of apical bud flush and the period of male and female cone initiation? YES, in a quite species-specific manner ! Doug-fir lodgepole pine Cytokinins up down Auxins down up Abscisic acid down up Gibberellins not detectable not detectable

Conclusion Cone induction methods need to be designed in accordance with the temporal hormone regimes of a species

Conclusion Cone induction methods need to be designed in accordance with the temporal hormone regimes of a species BUT WAIT A MINUTE ! What about girdling and GA induction? How do they affect initiation of cones?

Douglas-fir

• 1. Optimization of female cone production
• 2. Induction of cones in cone-poor genotypes

Douglas-fir

• 1. Optimization of female cone production
• 2. Induction of cones in cone-poor genotypes

Girdling (alone) of 14-year-old Douglas-fir trees and its effect on endogenous hormones from time of treatment to end of initiation period two genotypes gibberellins (GA4,GA7) initial rise, then decline abscisic acid no change auxins no change cytokinins no change

Treatment 9550 9137 ♂ cone ♀ cone ♂ cone ♀ cone Control 572 + 143 Girdling 8723 + 1690 10 + 9 8135 + 1356 6 + 5

We then looked at the classic hormone injection treatment for Douglas-fir, GA4/7 injection

prior to injection, GAs are, if even detectable, in the ng/g DW range – an

• rder of

magnitude less 4 7 3 1

4 7 3 1 prior to injection, GAs are, if even detectable, in the ng/g DW range – an

• rder of

magnitude less endogenous gibberellins – GA1 & GA3 – are produced

ABA cytokinins

control 4 mg GA4/7 /tree 40 mg GA4/7 /tree 400mg GA4/7 /tree control 4 mg GA4/7 /tree 40 mg GA4/7 /tree 400mg GA4/7 /tree

IAA - auxin

control 4 mg GA4/7 /tree 40 mg GA4/7 /tree 400mg GA4/7 /tree

Do endogenous hormone concentrations change after GA4/7 injection (compared to controls) ? Cytokinins no change Auxins up three weeks after treatment Abscisic acid no change Gibberellins GA3 & GA1 up (endogenous) GA4 & GA7 up (endogenous & exogenous are impossible to separate)

GA4/7 (mg/tree) ♂ cone ♀ cone 2994 + 999 124 + 46 4 4367 + 1269 154 + 58 40 4917 + 1143 236 + 32 400 8166 + 1616 256 + 20

If you were given a choice of which hormones to play around with to try and improve cone production, the answer is clearly GA and auxins Cytokinins no change Auxins up three weeks after treatment Abscisic acid no change Gibberellins GA3 & GA1 up (endogenous) GA4 & GA7 up (endogenous & exogenous are impossible to separate)

Douglas-fir

• 1. Optimization of female cone production
• 2. Induction of cones in cone-poor genotypes

After years of trial and error.....

After years of trial and error, we came up with a winning combination for female cone induction – GA4/7 with an anti-auxin, 2,3,5-triiodobenzoic acid (TIBA), which we tested on six historically poor-yielding i.e. crap, genotypes of Douglas-fir

Female cone yield per ramet in six Douglas-fir genotypes with various cone induction

• treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied

to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine

Female cone yield per ramet in six Douglas-fir genotypes with various cone induction

• treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied

to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine

Female cone yield per ramet in six Douglas-fir genotypes with various cone induction

• treatments. Mean ± SE, n= 3 to 5 for each genotype/treatment. Treatments were applied

to six low yield genotypes by stem-injection. Treatment application was completed in spring 2013 in PRT seed orchard. Data was collected in spring 2014. GA- Gibberellin, MGBG- methylglyoxal bis (guanylhydrazone), TIBA- 2,3,5-triiodobenzoic acid, Put - putrescine

* * * * * *

five out of six poor- yielding genotypes did better !

*

Lodgepole pine

• 1. Hormone profiles
• 2. Optimization of female cone production

Lodgepole pine

• 1. Hormone profiles
• 2. Optimization of female cone production

Hormone 233 (low) 299 (high) ABA high low cytokinins low high

A high-yielding genotype has a different hormone profile than a low-yielding genotype

• A photo of brach with three year cones

Year 1 Year 2 Year 3

We compared three high yield genotypes with three low yield genotypes, i.e. differing 4-fold in cone yield.

cytokinins

 ☐ 



good producers apex base apex base base poor good poor producers c-zeatin riboside

Conclusion The basal part of long-shoot bud has lower cytokinins (also true for t-zeatin riboside, dh- zeatin riboside) Other hormones (ABA, IAA) show little difference between genotypes. c-zeatin riboside

cytokinins

 ☐ 



good producers apex base apex base poor producers

Next step Attempt to alter cytokinin metabolism by applying exogenous cytokinins, e.g. benzyl adenine (BA), thidiazuron

cytokinins

 ☐ 



good producers apex base apex base poor producers c-zeatin riboside

Lodgepole pine

• 1. Hormone profiles
• 2. Optimization of female cone production

GA4/7 in combination with BA resulted in higher female cone initiation than control treatment.

Response to cone induction in various lodgepole pine genotypes. Effects of the treatment was judged by cone yield using percentage of cone yield of the treated compared to untreated (control). Female cones were induced by stem-injection of GA plus BA in early summer of 2013. Data was collected in late spring 2014. Mean, n ≥ 24 buds of three ramets. Different letters indicate significant difference (P < 0.05). GA4/7 and BA: stem injection

Response to cone induction in various lodgepole pine genotypes. Effects of the treatment was judged by cone yield using percentage of cone yield of the treated compared to untreated (control). Female cones were induced by stem-injection of GA plus BA in early summer of 2013. Data was collected in late spring 2014. Mean, n ≥ 24 buds of three ramets. Different letters indicate significant difference (P < 0.05). GA4/7 and BA: stem injection

We developed a method to test whether a genotype may be sensitive to GA4/7 and BA. Long-shoot buds are sprayed with a set dosage

• f GA4/7 alone or in combination with BA. If the

needles go brown within two weeks, then the dosage injected into the tree may have to be adjusted downward.

GA4/7 alone GA4/7 + BA

Stem injection is better and more practical than application of hormones in bud paste. Both of these methods are better than spray application, which is only effective if it does not rain. GA4/7 in combination with thidiazuron proved to be the most effective for raising endogenous cytokinin levels. This combination also resulted in the highest female cone initiation

The noteworthy element of female cone initiation is that it is at the expense of male cone initiation. Female cone buds initiated in high numbers in the basal portion of the long- shoot bud. These numbers are much higher than in the apical portions.

GA4/7 + thidiazuron: paste

PGR ♀ cone cluster genotypes with ♀ cone clusters long-shoot buds with ♀ cone clusters control no 0 /6 0/60 thidiazuron no 0/6 0/60 GA4/7 yes 2/6 3/20 GA4/7 + thidiazuron yes 4/6 15/40

Effects of exogenously applied GA4/7 and TDZ on initiation of ♀ cone clusters

Bract-

• vuliferous

scale complex

* BOSC = Bract-ovuliferous scale complex

lodgepole pine genotype 2082

individual clustered

Cones Seeds # cone g DW/cone MC (%) seed/cone mg DW/seed Sinkers (%) Cluster 186 3.039 6.017 6.49 1.767 55.12 Non-cluster 156 3.79 6.647 10.8 2.045 54.42 Germination (%) sinkers floaters cluster 77 1.1 non-cluster 86 0.5

Conclusions Cone induction methods can be adapted not only to phenology of individual tree species, but to temporal hormone regimes within long-shoot buds of those species Methods can be customized to

• ptimize particular genotypes for

breeding, or to improve cone production from traditionally lousy genotypes

Perspectives Precocious induction of males and/or females As done in containerized Chamaecyparis lawsoniana (Port Orford cedar) in Dorena Lake, Oregon

Perspectives Precocious induction of males and/or females Induction of male cone buds would be useful in young lodgepole pine orchards where pollen production is limited

the late great Tim Lee

Lodgepole Pine Cone Induction Workshop Skimikin, June 15, 2016

Tia, can you believe this !?

Tia, can you believe this !?

• Nick. Those

cones are from VSOC.

Acknowledgments

FGC

A big thanks to everybody who helped at all of the orchards !

Any questions?