Efficacy and Ecological Impacts of Transgenic Containment Technologies in Poplar

Amy L. Klocko1, Kori Ault1, Haiwei Lu1, Alice Morel1, Jian Huang2, Matt Betts1, Heather Root1, Dazhong Zhao2, and Steven H. Strauss1

1Department of Forest Ecosystems and Society, Oregon State University, Corvallis OR, Amy.Klocko@oregonstate.edu2Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI

Target genes and construct information

We selected a variety of poplar genes from the floral development pathway (see Table 1). We created 19 constructs targeting floral development genes, both singly and in combination. All were transformed into female clone 6K10 and 6-25 independent transformation events obtained for each construct (see Table 2). On average, we planted 4 ramets (trees) per event. Pairs of ramets were randomized into two blocks in the field.

IR = inverted repeat (RNAi constructs), DNM = dominant negative mutant, OvExp = over expression. Target length of the IR region varies from 290-392, depending the gene(s) targeted. Transgene expression is under the control of the constitutive 35S promoter and OCS terminator.

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Flowering by Construct 2014

Female poplar trees had robust flowering

Trees initiated flowering in 2014. Trees were screened for the presence of dormant floral buds in early 2014 and 2015. Floral buds can be readily distinguished from vegetative buds due to their size, shape and color. Approximately half of all 6K10 trees flowered in 2015, representing all constructs. Branches with dormant floral buds were collected for indoor flushing and analysis of catkin phenotypes prior to catkin emergence in the field.

6K10 trees with floral buds (indicated by flagging) flowered heavily in early 2015.

Floral phenotypes were stable across two growing seasons

Trees from three constructs flowered in both 2014 and 2015, with similar phenotypes both years.

control

20152014

Pt-LFY:RNAi

20152014

Pt-AG:RNAi

20152014

This project is supported by Biotechnology Risk Assessment Grant Program competitive grant no. 2011-68005-30407 from the USDA National Institute of Food and Agriculture and the Agricultural Research Service. National Institute of Food and Agriculture and Agricultural Research Service, National Science Foundation I/UCRC Center for Advanced Forestry (grant 0736283), USDA-BRAG (grant 2010-33522-21736), USDA-IFAS (grant OREZ-FS-671-R), and the TBGRC industrial cooperative at Oregon State University. We thank Maurizio Sabatti for providing early-flowering clone 6K10 for study, and Gilles Pilate for providing clones 717 and 353.

Acknowledgements

• Trees for all constructs are growing well• Several RNAi constructs give interesting, potentially sterile phenotypes• Floral phenotypes are stable across growing seasons• Gene expression levels are associated with floral morphology but are dependent on tissue type and age• Our results suggest that disruption of LFY is a powerful tool for genetic containment of trees

Summary

Targeting LFY and/or AG gave altered floral phenotypes

Seven of our RNAi constructs had trees with altered floral morphology. All of these constructs were designed to target LFYand/or AG, sometimes in combination with other floral genes. Constructs targeting just LFY led to catkins with no externally visible carpels. Constructs targeting AG led to catkins with replicated carpels, often missing ovules. Targeting both LFY and AG led to flowering resembling those of trees with single gene targeting. No other constructs have yielded changed floral phenotypes to data.

Pt-LFY:RNAi/Pt-AG:RNAi

Pt-LFY:RNAi;Pt-AG:RNAi

Targeting LFY and AG

Pt-LFY:RNAi/Pt-AG:RNAi

control

Pt-LFY:RNAi Pt-AG:RNAi (mar) Pt-AG:RNAi Pt-AP1:RNAiPt-AG:RNAi

Targeting LFY Targeting AG

Pt-AP1:RNAiPt-LFY:RNAi

control

Targeting AP1 and LFY or AG

Tree size (calculated as diameter2) was measured after three growth cycles. On average, the Pt-LFY:RNAi trees (all TG) were similar in size the control (CTR) trees. All Pt-LFY:RNAi events grew well, none were significantly smaller than controls. Bars show average tree size by event (number of ramets is shown in the base of the bar), error bars show standard error across ramets. Tree flowering was scored in 2015. Floral phenotype (normal flowers, intermediate flowers, tiny flowers) as assessed by field phenotypes, is indicated by bar color (green, blue, or orange). Events with tiny flowers grew very well and were similar in appearance to control trees.

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142-1 35-2 47 124-1 143 194 17 120-2 96-2 118 98 152 126-3 139-1 125-1 all TG CTR

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Average Size of Pt-LFY:RNAi EventsNormal flowers

Tiny flowers

Intermediate flowers

Pt-LFY:RNAi trees had robust vegetative growth

3-12-14

control Event 17

Table 2: Constructs, and number of confirmed transgenic events in poplar clone

6K10 planted at the field site

Transgene Gene(s) Targeted No. Events No. Trees

1 PtAG-IR AGAMOUS (AG) 22 86

2 PtAG-IR (MARs) AG 13 48

3 AtAG-DNM3 AG 15 57

4 PtLFY-IR LEAFY (LFY) 18 59

5 PtAG-IR/PtLFY-IR AG / LFY 22 83

6 PtLFY-PtAG-IR LFY:AG 10 40

7 PtAGL20-IR AGL20 16 60

8 PtAGL24-OvExp AGL24 13 54

9 PtAGL24-IR AGL24 14 56

10 PtAP1-IR APETALA1 (AP1) 20 70

11 AtAP1-DNM3 AP1 15 57

12 PtAP1-PtAG-IR AP1:AG 19 67

13 PtAP1-PtLFY-IR AP1:LFY 20 70

14 PtAP1-PtLYF-PtAG-IR AP1:LFY:AG 25 88

15 PtAP3-IR APETALA3 (AP3) 13 52

17 PtFPFL2-IR FPFL2 15 57

18 PtFT-IR FT 6 23

19 PtSVP-OvExp SVP 21 73

20 None (non-Tr control) none - 24

Totals 297 1,124

Table 1: Genes targeted for suppression or modified expression in

transgenic poplar trees

Gene name(s) Location(s) in floral pathway Poplar gene(s)

FPF1 (FPFL1, FPFL2) Input from GA pathway Potri.006G276100,

Potri.018G005200

AGL20 (SOC1) Signal integration Potri.014G074200

FT (FT1, FT2) Signal integration Potri.010G179700,

Potri.008077700

AGL24 Signal integration

Meristem determination

Potri.002G105600

LFY Meristem determination Potri.015G106900

SVP Meristem determination Potri.007G010800

AP1 (AP1-1, AP1-2) Meristem determination

Floral organ determination

Potri.008G098500,

Potri.010G154100

AP3 Floral organ determination Potri.005G118000

AG (AG-1, AG-2) Floral organ determination Potri.004G064300,

Potri.011G075800

Microscopy of field collected catkins revealed that Pt-LFY:RNAi catkins from two events were tiny, and enclosed in hairy bracts (br). While catkins from control trees had stigmas (st) and ovules (ov), neither of these structures were present in the tiny Pt-LFY:RNAi catkins. The entire carpel (ca) structure from these tiny catkins was of similar size to the ovules of thecontrol catkins. Thin sectioning of control catkins showed well-developed ovules. Sectioning of Pt-LFY:RNAi catkins showed that carpels resemble a meristem-like structures that lack any internal organs. Bar = 500 µm.

Carpel dissection

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Carpel interiorCatkin exteriors

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Carpel and catkin dissection

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Tiny Pt-LFY:RNAi catkins lacked stigmas or ovules

We used quantitative real-time PCR (QPCR) to measure the relative expression level of the LFY in young floral buds and catkins of each event as compared to an ACTIN housekeeping gene. Events 17 and 139-2 have tiny catkins, event 194 has catkins with a normal appearance. Both young and mature control catkins were tested as LFY is predicted to have higher expression in younger catkins. We found that young floral buds events that developed tiny flowers had less LFY expression than floral buds of normal flowered or control trees. As predicted, the younger control catkins had more LFY expression than the mature control catkins. Event 194, which had normal catkins, has similar LFY expression as the control samples. Surprisingly, the two events with tiny catkins had much higher expression of LFY than control trees. We hypothesize that this increased LFY expression may be due to the underdeveloped state of these small catkins. At the time of collection, the young control catkins were much larger and far more developed than the catkins from events 17 and 139-1. Bars show standard error between biological replicates, asterisks show significant differences (P < .05).

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Relative Expression of LFY in Young Floral Buds

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Control Event194

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Event139-1

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Relative Expression of LFY in Catkins

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normal flowers tiny flowers

Event 139-1

Pt-LFY:RNAi trees had reduced LFY expression in floral buds

AbstractThe dispersal of transgenes from genetically engineered plants presents substantial challenges to biotechnology regulatory bodies. Because forest trees are weakly domesticated, have wild relatives, and pollen or seeds can spread widely, they are especially problematic. However, plantation trees are often vegetatively propagated, making fertile flowers unnecessary for commercial use. Thus, genes that induce complete sterility could provide strong and simple mitigation of dispersal, simplifying regulatory decisions. We are studying the efficacy, stability, and ecological impacts of floral developmental genes as tools for mitigating or preventing transgene spread. We established a plantation of transgenic Populus alba containing 19 different constructs that modify the expression of poplar orthologs of conserved floral development genes, including LEAFY (LFY), AGAMOUS (AG), and APETALA1 (AP1). Some constructs are designed to target two to three genes simultaneously. The overarching hypothesis that we are testing is that suppression of selected floral development genes from poplar are useful tools for development of effective, stable containment technologies. We screened all trees for the presence of floral buds, as well as alterations in floral morphology. We found that RNAi constructs targeting LFY and/or AG led to interesting, potentially sterile floral phenotypes, and their phenotypes were stable across two growing seasons. Detailed analysis of trees with RNAi targeting LFY (Pt-LFY:RNAi) showed that the tiny flowers from some of these events were sterile, lacking stigmas or ovules. Despite this severe reduction in reproductive growth, Pt-LFY:RNAi trees had normal rates of vegetative growth, and appeared to be morphologically identical to control trees. Analysis of gene expression showed that LFY expression was reduced in the young floral buds of Pt-LFY:RNAi trees with small flowers. Overall, our results suggest that disruption of LFY is a powerful tool for genetic containment of trees.

Project objectives• Analyze floral and vegetative phenotypes of all insertions events• Identify constructs leading to potentially sterile flowers• Monitor phenotype stability over growing seasons• Test target gene expression of events of interest• Analyze literature on ecological impacts of containment technologies on biological diversity in tree

plantations (to begin in the upcoming year)