1

Elucidating the molecular control of axillary

bud outgrowth in tulip during storage

Eveline Heynen

Wageningen University and Research

Laboratory of Plant Physiology

Wageningen, January 2017

Elucidating the molecular control of axillary

bud outgrowth in tulip during storage

Major MSc thesis

Eveline Heynen MSc Student Plant Science

Registration number : 940720337050

Code : PPH-80436

Supervisor : Natalia Moreno-Pachon

Examiners : prof.dr.ir. Richard Immink

: dr. Henk Hilhorst

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Abstract Tulip (Tulipa gesneriana L.) is an important bulbous monocot worldwide and its trade in cut flowers,

ornamental plants and bulbs is a billion euro industry. Currently, the common method of tulip bulb production

is through vegetative propagation (axillary bud outgrowth). A mature flowering size bulb normally contains six

axillary buds (A, B, C, D, E and H bud). The propagation rate of tulip bulbs is low as only two to three of these

axillary buds develop into vigorous daughter bulbs at the end of the annual growth cycle. Preliminary studies in

Wageningen on the development of axillary buds in tulips showed a two directional growth gradient. The

axillary buds closer to innermost floral bud or closer to the outer H bud (the only buds that sprout and produce

leaves) appeared to grow the most while the middle buds stayed dormant. This was linked to TgTB1

(TEOSINTHE BRANCHED1) expression, which is the primary gene blocking axillary bud outgrowth. It is

suspected that TgTB1 expression is never induced or transient in axillary buds of springpartij bulbs due to

deviation in dormancy regulation. Springpartij bulbs can be defined as a cluster with many small daughter bulbs

during lifting bulbs and though they are numerous few have enough reserves to produce a flower, causing

problems in the horticultural industry. The preliminary studies focussed on tulip growth in the field from the

beginning of winter till the end of summer; however they did not look into dormancy regulation during the

storage period. The influence of the storage period could be significant as initial induction and organogenesis

generally occurs during autumn in bulbous species, which would be during storage time. The aim of this study

was to understand how dormancy is regulated during the storage period using TgTB1 as a marker for

dormancy. Tulip bulbs were stored at an ambient temperature (18°C) and a cold temperature (4°C), as cold is

known to be able to break dormancy. Weight gain and TgTB1 expression of the axillary buds were measured

overtime. TgTB1 expression of axillary buds in springpartij bulbs was measured during storage at 20°C and

compared to the TgTB1 expression of normal bulbs. At the start of the storage experiment the A bud was still

being formed in the normal bulbs; however it gained weight faster than the other buds, which correlated with

transient TgTB1 expression. The B and C bud grew at the end of the storage period, parallel with a rapid

downregulation of their TgTB1 expression. The TgTB1 expression in the H bud was low from the beginning but

it did not gain weight. The D and E bud also did not gain weight and their TgTB1 expression stayed high.

Storage at cold temperatures delayed growth and, contrary to expectations, also delayed the downregulation

of TgTB1 expression. The axillary buds of springpartij bulbs however showed, according to expectations, low

TgTB1 expression in all bulbs while in normal bulbs this was only in the A and H axillary buds. Overall, the

weight gain and TgTB1 expression found during storage is consistent with earlier results from the field. Storage

is a significant part of the annual growth cycle as downregulation of TgTB1 expression occurs during the

storage period, ensuring outgrowth of axillary buds after winter. The two directional growth gradient is likely

the result of strategic sugar partitioning throughout the annual cycle, enforced by dormancy induction. In

springpartij there is (almost) no dormancy induction during storage causing a disturbance in normal sugar

partitioning resulting in lots, but mostly unusable daughter bulbs grown from axillary buds.

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Table of contents

Abstract ................................................................................................................................................................... 1

1. Introduction ........................................................................................................................................................ 3

1.1 Morphology and current use of tulip ............................................................................................................. 3

1.2 Dormancy....................................................................................................................................................... 4

1.3 Multiple generations in one bulb .................................................................................................................. 4

1.4 Summer dormancy ........................................................................................................................................ 5

1.5 Winter dormancy ........................................................................................................................................... 6

1.6 Storage in the annual cycle of tulip ............................................................................................................... 7

1.7 Springpartij bulbs ........................................................................................................................................... 7

1.8 Aim ................................................................................................................................................................. 8

2. Material and methods ......................................................................................................................................... 9

2.1 Storage conditions and isolating plant material ............................................................................................ 9

2.2 Isolating and pooling buds of springpartij bulbs ............................................................................................ 9

2.3 RNA Isolation and cDNA synthesis ................................................................................................................. 9

2.4 Real-time PCR .............................................................................................................................................. 10

2.5 Statistical analysis ........................................................................................................................................ 10

3. Results ............................................................................................................................................................... 11

3.1 Morphological observations of tulips in storage ......................................................................................... 11

3.2 Weight gain and TgTB1 expression of axillary buds in storage ................................................................... 13

3.3 TgTB1 expression in springpartij bulbs over time ....................................................................................... 15

4. Discussion and Conclusion ................................................................................................................................ 16

4.1 Dormancy regulation of axillary buds in tulip bulbs during storage ............................................................ 16

4.2 Leaf formation and development of the floral meristem during storage ................................................... 17

4.3 TgTB1 downregulation due to cold storage ................................................................................................ 19

4.4 Dormancy regulation in springpartij bulbs .................................................................................................. 20

4.5 Conclusions .................................................................................................................................................. 21

4.6 Future directions ......................................................................................................................................... 21

Acknowledgements ............................................................................................................................................... 23

References ............................................................................................................................................................ 24

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1. Introduction

1.1 Morphology and current use of tulip

Tulip (Tulipa gesneriana L.) is a perennial bulbous monocot belonging to the Liliaceae family (Veldkamp and

Zonneveld, 2012). It originates from the Irano-Turanian region where it flowers in spring (Kamenetsky et al.,

2012). Tulip has been a culturally important flower in The Netherlands since it first arrived from Turkey around

1562 (Bianchi, 1999). Nowadays, the trade of tulip in cut flowers, ornamental plants and bulbs is a billion euro

industry (Christenhusz et al., 2013). The majority of this industry is controlled by the Dutch as the largest

productions area (88%), capable of producing 4.32 billion tulip bulbs, is located in The Netherlands (Buschman,

2005). The common method of tulip bulb production is through vegetative propagation (axillary bud

outgrowth) as it ensures homogeneity of the genotype and it takes only one year instead of the five to seven

years needed in sexual propagation (Fortanier, 1973). Tulips produce underground axillary buds which can

grow out into vegetative propagated daughter bulbs. Axillary buds are normally found in the axil of the leaf and

in tulip they are located in a similar place (Fig.1.1). A flowering size bulb normally consists of five fleshy scales

(leaves) containing six axillary buds of which one bud (H bud) is located on the outside of the bulb.

Bulbous plants like tulip can be defined as geophytes, which are plants that produce underground storage

organs to survive adverse conditions (Raunkiaer, 1934; Rees, 1989; Le Nard and De Hertogh, 1993b).

Fig. 1.1 Architecture of a tulip plant (left) and a non-bulbous plant (right). Tulips contain fleshy

scales (leaves) and axillary meristems labelled from innermost (A bud) to the outermost (H bud).

These axillary meristems can grow out into vegetative propagated daughter bulbs. Modified from

Leeggangers et al. (2013).

A C

H

B D

E

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Geophytes produce various types of underground storage organs: bulbs, corms, tubers and rhizomes (e.g:

Tulipa, Gladiolus, Solanum tuberosum and Zingiber officinale, respectively) (Kamenetsky et al., 2012). These

underground storage organs are specialized in vegetative propagation while also serving as food reserve

(Botschantzeva, 1982). Generally, they are survival structures to overcome unfavourable conditions through

entering dormancy.

1.2 Dormancy

Dormancy has many different definitions, currently the definition: “Dormancy is a temporary suspension of

visible growth of any plant structure containing a meristem.”, proposed by Lang et al. (1987), has been widely

accepted. Additionally, Lang et al. (1987) classified two types of dormancy: paradormancy and endodormancy.

Paradormancy (pre-dormancy) is caused by endogenous signalling within the plant and not by external factors

such as temperature or light. Apical dominance, the process that the main shoot apical meristem is dominant

over the lateral buds, is an example of paradormancy. Endodormancy (true dormancy) is growth suppression

caused by endogenous signals within the meristem. After breaking endodormancy the plant is capable of

growth once exposed to favourable environmental conditions (Dennis, 1996). Paradormancy in spring

flowering bulbs, such as tulip, generally occurs in summer and has also been referred to as summer dormancy

(Doorenbos, 1953). During paradormancy of spring flowering bulbs the floral meristem inhibits the growth of

the axillary meristems through apical dominance. Endodormancy of the axillary meristems occurs in autumn

and the beginning of winter and has been referred to as winter dormancy (Doorenbos, 1953). These

dormancies are clearly part of a continuum, however the exact beginning and ending of each particular

dormancy is very hard to define (Seeley, 1996). The continuum of these dormancies ensures that geophytes

grown in temperate climates survive temperature extremes of both summer and winter and greatly

contributes to their plasticity.

1.3 Multiple generations in one bulb

A vigorous mature tulip bulb consist of three generations: a differentiated floral meristem of the mother bulb

designed to flower in spring, differentiated vegetative meristems of several daughter bulbs which will form

new mother bulbs after spring and buds inside the daughter bulbs that are still in the process of formation and

that will develop a differentiated vegetative meristem in the future mother bulbs after spring (Botschantzeva,

1982) (Fig.1.2). The floral meristem of the mother bulb will flower in the first year, the daughter bulbs will

flower in the second year after initiation and the buds in the daughter bulbs, which are not yet differentiated,

will flower in the third year after initiation. In order to promote development of the floral and vegetative

meristems alongside each other until spring, two opposite but parallel dormancy reactions occur in the mother

bulb. To promote these dormancy reactions and to complete their life cycle, tulips generally need a warm-cold-

warm sequence, as do similar bulbs such as Freesia, Narcisus and Hyacinthus (Le Nard and De Hertogh, 1993b).

Summer and winter dormancy is a feature of these spring flowering geophytes. Through dormancy they survive

the warm summer underground and complete a cold requirement before above-ground growth (Rees, 1992) .

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1.4 Summer dormancy

At the end of spring the flower of the mother bulb dies and all energy is put into the formation of the daughter

bulbs (Ho and Rees, 1976). After the energy reserves of the mother bulbs are finished the newly formed

daughter bulbs enter summer dormancy. Flower initiation takes place inside vigorous daughter bulbs to

commence flowering in spring. Delay between flower initiation and emergence also occurs in other species

such as conifers, most fruit tree crops, strawberry and Paeonia (Sedgley and Griffin, 1989; Battey et al., 1998;

Kamenetsky et al., 2012). At the end of summer vigorous daughter bulbs have fully differentiated their floral

meristem and almost all of their axillary vegetative meristems (grand-daughter buds) (Le Nard and De Hertogh,

1993a). Outgrowth of the grand-daughter buds is supressed by paradormancy.

Paradormancy is caused by auxin transport from the main meristem, suppressing outgrowth of the

axillary buds, also called apical dominance. Polar auxin transport is facilitated by the PIN1 proteins activated by

the PIN1 genes (Gälweiler et al., 1998). Auxin activates AXR1-AFB (AUXIN RESISTANT1- AUXIN SIGNALING F-

BOX; (Leyser et al., 1993; Dharmasiri et al., 2005) which downregulates IPT (ISOPENTYLTRANSFERASE) genes

and therefore also the production of cytokinins, one of the main plant hormones promoting bud outgrowth

(Tanaka et al., 2006; Shimizu-Sato et al., 2009). Cytokinins enter the bud and inhibit TB1 (TEOSINTE

BRANCHED1) (Doebley et al., 1995; Kebrom et al., 2006; Cao et al., 2012) also called BRC1 (BRANCHED1;

Aguilar-Martínez et al., 2007) in dicots, which is the primary gene blocking bud outgrowth. TB1/BRC1 promotes

NCED3 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3) which is involved in the biosynthesis of abscisic acid

(ABA), a major plant hormone which induces and sustains axillary bud dormancy (Bewley, 1997; Yao and

Finlayson, 2015). Contrary to cytokinin, which is inhibited by polar auxin transport in the stem, certain genes,

CCD7 (CAROTENOID CLEAVAGE DIOXYGENASE7) and CCD8 (CAROTENOID CLEAVAGE DIOXYGENASE8) in the

strigolactone biosynthesis pathway are upregulated by AXR1-AFB (Sorefan et al., 2003; Foo et al., 2005;

Fig. 1.2 Mature tulip bulb containing three generations. The mother bulb is the

first generation. The axillary buds of the mother bulb will become the second

generation daughter bulbs. Within these second generation daughter bulbs, the

third generation is being formed (grand-daughter bulbs).

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Hayward et al., 2009). TB1/BRC1 has shown to work downstream from strigolactones, causing an increasing

TB1/BRC1 expression under increasing strigolactone levels (Brewer et al., 2009; Minakuchi et al., 2010; Braun

et al., 2012; Dun et al., 2012).

1.5 Winter dormancy

In autumn the growth in the bulb is slowed down mainly due to lower temperatures after warm temperatures

in the warm-cold-warm sequence needed in tulip bulbs. During this time no external growth can be seen but

growth and differentiation of the shoot continues slowly (intrabulb development) (Rees, 1981; Kamenetsky et

al., 2012). Endodormancy is in activated in several buds which and is regulated by TB1/BRC1 through the

strigolactone pathway (Brewer et al., 2009; Dun et al., 2012).

During winter the tulip is exposed to a period of prolonged cold which activates the growth of roots

and promotes leaf development, floral development and floral stem elongation to initiate rapid outgrowth in

spring (Ho and Rees, 1975; Khodorova and Boitel-Conti, 2013). The expression of VRN2 (VERNALIZATION2) is

also upregulated; VRN2 downregulates the expression of FLC (FLOWERING LOCUS C); the main FT (FLOWERING

LOCUS T) suppressor (Huang et al., 2013). FTs are important genes that promote processes involved in flower

differentiation, development and flowering (Suárez-López et al., 2001). In potato, FTs are also formed in the

leaves and transported to stolons to promote tuberization, suggesting a role for FTs in multiple mechanisms

apart from flowering (Rodríguez-Falcón et al., 2006). It has been shown that upregulation of FT expression

downregulates the expression of TB1/BRC1 (Hiraoka et al, 2013). Alongside upregulation of FT, prolonged cold

has also shown to upregulate the activity of alpha-amylase in tulip, lily, hyacinth and potato (Moe and

Wickstrom, 1973; Miller and Langhans, 1990; Komiyama et al., 1997; Biemelt et al., 2000; Shin et al., 2002; Sato

et al., 2006). Alpha-amylase remobilizes starch into sucrose. Sucrose has shown to downregulate the TB1/BRC1

expression and promote outgrowth (Mason et al., 2014). These combined processes break endodormancy in

the tulip bulb to ensure outgrowth in spring (Fig. 1.3).

Fig. 1.3 Proposed model for TB1(TEOSINTE BRANCHED1)/BRC1(BRANCHED1) as an integrator of

different pathways controlling axillary bud outgrowth.

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1.6 Storage in the annual cycle of tulip

After flowering of the mother bulb in spring, the daughter bulbs finalize their growth period. At the end of

summer the floral bud and axillary buds are initiated inside daughter bulbs of sufficient size (Khodorova and

Boitel-Conti, 2013). In horticulture, tulip bulbs are lifted at the end of summer and favoured natural conditions

are simulated under an ambient temperature (18°C) during storage, before planting in early winter. The

prolonged warm temperatures in the warm-cold-warm sequence simulated through storage provide a longer

period for intrabulb development. This extends the growth period of the floral meristem, the leaves and some

axillary buds. Subsequently, the prolonged cold period during winter activates the growth of roots and

promotes leaf development, floral development and floral stem elongation to initiate rapid outgrowth in spring

(Ho and Rees, 1975; Khodorova and Boitel-Conti, 2013). Among all environmental factors, temperature has

been established to play a predominant role in controlling growth and flowering in bulbs (Le Nard and De

Hertogh, 1993a). The storage period provides an opportunity for bulb organs to grow prior to the cold period.

For example, initial induction and organogenesis generally occurs during autumn in bulbous species (Ryberg,

1959), which would be during storage time. Thus, organogenesis and growth during storage is an important

part of the annual growth cycle of tulip as it can determine whether the bulb is able to produce a flower and

axillary buds of sufficient size in spring. Mutimawurugo (unpublished) found low TgTB1 expression in the non-

dormant A and H bud after storage and high TgTB1 expression in the dormant D bud. The A and H bud where

able to grow out into vigorous daughter bulbs at the end of spring, while the D bud was not. The TgTB1

expression might be downregulated in certain buds during storage enabling them to grow out into vigorous

daughter bulbs during summer.

1.7 Springpartij bulbs

During lifting, some daughter bulbs differ in their phenotype from the normal phenotype. Springpartij bulbs are

such bulbs and can be defined as a cluster with many small daughter bulbs of which the main bulb occupies

less than 50% of the total cluster weight (de Jong, n.d.). These bulbs are problematic as they are smaller

compared to normal leaving them with fewer reserves to support the growth of the vegetative and the

reproductive organs (Fig. 1.4). They first started to appear at the beginning of the 1960’s and fifteen to twenty

percent of the tulips experience the phenomenon (Dwarswaard, n.d.; Snoek and de Jong, n.d.). It is suspected

that dormancy is never induced in axillary buds or quickly downregulated during storage causing all these bulbs

to grow out.

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1.8 Aim

Storage is an important part of the annual dormancy cycle in tulip to induce early growth as organogenesis is

active during this period. Downregulated TgTB1 expression at the end of storage seems to determine which

axillary buds eventually grow out into vigorous daughter bulbs (Mutimawurugo, unpublished). Therefore in this

study TgTB1 was measured in axillary buds during storage. It is expected that the buds that normally grow out

(A, B, C and H) have their TgTB1 expression downregulated during storage or that the expression is never high.

The weight gain was measured to correlate it to TgTB1 expression. Photos were taken at each time point

(Week 0, 4, 8 and 12) to visually compare the buds. Lastly, TgTB1 expression was measured in springpartij bulbs

and compared to the TgTB1 expression of normal bulbs. TgTB1 expression is probably downregulated in all

axillary buds of springpartij bulbs instead of only the innermost and outer bud, causing outgrowth of lots of

daughter bulbs.

Fig 1.4 Phenotype of a clump of normal daughter bulbs and a clump of

springpartij daughter bulb after lifting. (a) Clump of normal daughter bulbs

grown from the A, B and C axillary buds. (b) Clump of springpartij bulbs

grown from lots of different axillary buds. Photos at the courtesy of N.

Moreno-Pachon.

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2. Material and methods

2.1 Storage conditions and isolating plant material

Tulip cultivar ‘Dynasty’ was lifted at the end of summer and immediately stored in dark crates at 4°C or 18°C.

Vermiculite was added to the crate of bulbs stored at 4°C to prevent fungal infections caused by high relative

humidity. No vermiculite was added to the crate stored at 18°C as relative humidity was not high and no fungal

infections were detected. Buds were collected at week 0, 4, 8 and 12 of the start of the experiment. Seven

bulbs were pooled in each replicate and a total of three replicates were used per time point. Buds were

weighed and the weight gain overtime was calculated using the first weight point as a reference. Pictures were

taken at each time point to visually compare the size differences between buds. Afterwards the buds were

freeze dried overnight, ground with beads and stored in -80°C until further use.

2.2 Isolating and pooling buds of springpartij bulbs

Clumps containing springpartij bulbs of cultivar ‘Prinses Catharina Amalia’ were collected and stored in a dark

room at 20°C. All buds within each bulb were pooled to test gene expression of buds. The buds in the A, B, C, D

and H bulbs were collected, three replicates were made. The buds were freeze dried overnight, ground with

beads and stored in -80°C until further use. Gene expression of the buds within the springpartij bulbs was

compared to gene expression of bulbs (cv. ‘Dynasty’) stored in 18°C at week 8 of the storage experiment.

2.3 RNA Isolation and cDNA synthesis

RNA for gene expression was extracted from approximately 60-90mg freeze dried tissue using the hot-borate

protocol. The hot-borate was heated to 80°C to ensure that any precipitation within the stock would dissolve.

52.8 mg PVP (CAS 9003-39-8) and 1.76 mg DTT (CAS 3483-12-3) were supplemented to 0.88 ml hot-borate

stock per sample and dissolved at 80°C. 800µl of this mixture was added to the freeze dried tissue.

Subsequently, 4µl proteinase K (CAS 39450-01-6) solution was added and samples were incubated at 42°C for

15 minutes. During incubation the samples were mixed every 5 minutes. Thereafter, 64µl of 2M KCL was added

to the samples and they were incubated on ice for 30 minutes. This solution was centrifuged at 12000g for 20

minutes at 4°C. 259µl of ice-cold 8M LiCl was added to the supernatant which was left to incubate on ice for 2

hours or overnight at 4°C. Afterwards, the samples were centrifuged again at 12000g at 4ᵒC for 30 minutes and

the remaining pellet was washed with 750µl of ice-cold 2M LiCl. A new centrifugation step was performed at

12000g at 4ᵒC for 10 minutes and thereafter the pellet was air-dried for maximum 20 minutes and re-

suspended in 80μl DEPC water. Samples were stored in ice overnight at 4°C. The following day, 10μl DNAse

buffer and 10μl DNAse were added to each sample and subsequently incubated at 37°C for 20 minutes.

Thereafter, 100μL phenol (CAS 108-95-2)-chloroform (CAS 67-66-3) (1:1) was added per sample and the 200μl

mixture of RNA/phenol-chloroform was centrifuged for 5 minutes at 12000g in phase-lock tubes. Around 90μL

of the upper phase was transferred to an eppendorf tube in which 1/10 volume of 3M NaAc and 2.5x volume

100% ice-cold ethanol (9 μL and 225μL, respectively) was added. The mixture was incubated on ice for at least

1 hour or overnight on ice at 4°C. After incubation the samples were centrifuged at 12000 g for 30 minutes at

4ᵒC. The pellet was washed with 250μL ice-cold 70% ethanol and centrifuged again for 10 minutes at 12000g at

4°C. The pellet was air dried for maximum 20 minutes and afterwards dissolved in 20μl DEPC MQ. The quality

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of the samples was checked on 1% agarose gel and quantity was checked with trinean Xpose (TRINEAN nv,

Gentbrugge, Belgium). 500ng RNA of each sample was used for cDNA synthesis, using M-MuLV Reverse

Transcriptase (Thermo Scientific) and Oligo dT primers. The cDNA synthesis was performed in a Bio-rad

MyCycler.

2.4 Real-time PCR

Gene expression was measured using quantitative RT-PCR (qRT-PCR) which was performed with the Bio-Rad

CFX Connect Real-Time Detection System (Bio-Rad Laboratories, Inc., Veenendaal, The Netherlands). Per

reaction 5µl SYBR Green, 2.5µl cDNA (1.25ng/µl), 0.5µl 10µM primer (forward and reverse) and 2µl DEPC water

were added. Changes in axillary bud outgrowth regulated by tulip TEOSINTHE BRANCHED1 (TgTBC1) were

measured using the TgTB1 primers. Tulip elongation factor 1α (TgEF1α) was used as a reference gene to

normalize the TgTB1 expression (Table 2.1). qRT-PCR was performed with an initial 3 min denaturation at 95°C

followed by 40 cycles of 95°C for 10s and 60°C for 30s. Final steps used for elongation were 95°C for 1 min, melt

curve 55°C for 10s and 95°C for 5s. Calculations to normalize the TgTB1 expression were made according to the

ΔΔCt method based on three technical replicates and two biological replicates for each gene.

Table 2.1 Sequence of primers used in qRT-PCR to measure expression of Elongation factor 1α and BRANCED1.

Gene Primer name Sequence

Tulip elongation factor 1α

(EF1α)

qTgEF1a_fw TGA GAA GGA GGC TGA AAT CA

qTgEF1a_rv TCA CGA TGA CCA GCA GCA TC

Tulip TEOSINTHE BRANCHED1

(TgTB1)

tTgTB1_fw ATG AGG CTC TCC CTG GAT GT

tTgTB1_rv ACA TGG TGA GAA GCC ACT GG

2.5 Statistical analysis

Statistical analyses were done using SPSS (22nd

edition). In the storage experiment, weight gain in each bud was

compared to the first weight point of that bud and tested on significance using one-way ANOVA with Tukey’s

Post Hoc test (p<0.05). Differences in TgTB1 expression where compared within each bud using all time points

and tested on significance using one-way ANOVA with Tukey’s Post Hoc test (p<0.05). The differences in weight

gain can later be correlated to differences in dormancy level regulated by TgTB1. In the springpartij

experiment, significant differences in TgTB1 expression between the pooled buds from springpartij bulb and

the buds of the normal bulb in storage were tested using the Independent T-Test.

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3. Results

3.1 Morphological observations of tulips in storage

At the end of the annual growth cycle, axillary buds have become daughter bulbs that will become mother

bulbs the following year after a storage period. Storage is an important part of the growth cycle of tulip bulbs

as it contributes to the development of its reproductive apical bud as well as the vegetative axillary buds before

entering winter dormancy. Therefore the development of the axillary buds in terms of growth and expression

of TgTB1 (TEOSINTE BRANCHED1), which is the main gene inhibiting outgrowth of axillary buds, was

investigated during the storage period. Morphological observations of the bulbs during storage showed that

the axillary buds differ in speed of growth and initial size. The H bud had the biggest size at the start of the

storage period followed by the E, D and C bud. The A and B bud were not visible to the naked eye at the start of

storage, however using a microscope it was found that the A bud was being formed during this time (Fig. 3.1;

Fig 3.2a). This illustrates a size gradient in axillary buds from the outside to the inside of the bulb at the start of

storage. After initial observations the tulip batch was split into cold storage (4°C) and normal (18°C) storage

conditions. It was hypothesized that storage at 4°C instead of the more conventional 18°C could break

dormancy faster due to rapid downregulation of TgTB1 expression. In both storage temperatures a high growth

increase was observed in the A bud (Fig. 3.1). At week 12, this increase was also observed in the B and C buds

of bulbs stored 18°C, this effect was not seen in bulbs stored at 4°C (Fig. 3.1b). The D, E and H buds did not

show any apparent growth increase during the storage period, irrespective of the temperature. This is

interesting, as they are oldest buds it seems like they reached a developmental point in which they enter a slow

growing period.

Fig. 3.1 Morphological observation of the A, B, C, D, E and H axillary buds in tulip bulbs (‘Dynasty’) during storage.

(a) Shows the axillary buds of tulip bulbs stored in 18°C at four time points and (b) shows axillary buds of tulips

bulbs stored in 4°C at four time points. Scale bars are 1 cm.

(a) (b)

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The size differences between the axillary buds of 4°C and 18°C could be caused by the slowing of metabolism

under low temperatures. These differences in metabolism become especially apparent in the development of

the floral meristem. Morphological observations of the floral meristems showed a big size difference between

the two temperatures (Fig 3.2). During week 0 a small floral meristem could be seen, the formation of the

vegetative meristem of bud A is visible at the base of the floral meristem (Fig. 3.2a). At week 4 the floral

meristem of the bulbs stored at 18°C was already twice the size of the floral meristem of bulbs stored at 4°C.

The A bud on the floral meristem from 18°C also appeared bigger in size (Fig. 3.2b). The difference got larger in

week 8 as the floral meristem from bulbs stored at 18°C did not only appear twice the height, but also twice

the width, compared to the floral meristem from bulbs stored at 4°C (Fig. 3.2c). The last big difference in the

speed of growth of the floral meristems could be seen in week 12, when the tip of floral meristem of the bulbs

stored at 18°C could be seen on the outside of the bulb. The floral meristem from bulbs stored at 18°C

appeared at least four times the size of the floral meristem from bulbs stored at 4°C (Fig. 3.2d).

Fig. 3.2 Morphological observation of floral meristems in tulip bulbs

(‘Dynasty’) stored in 4°C and 18°C over a course of 12 weeks. (a)

Floral meristem in week 0; 2.6*10 magnification, scale bar is 1 mm.

(b) Floral meristems in week 4; 1*10 magnification, scale bar is 5

mm. (c) Floral meristems in week 8; 0.6*10 magnification, scale bar

is 5 mm. (d) Floral meristems in week 12; scale bar is 10 mm.

Arrows indicate the A bud attached to the floral meristem.

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3.2 Weight gain and TgTB1 expression of axillary buds in storage

The morphological observations were quantified by measuring the fresh weight of the buds. The weight on the

first week of measurements was compared to the measurements over time to monitor the growth of the

axillary buds. The most notable results from storage in 18°C were from the A, B, C and H bud as they showed a

connection between weight gain and TgTB1 expression. The weight gain of the A, B and C buds was very

significant in week 12 compared to the first weighing point (Fig. 3.3a). Parallel to this there was a significant

decrease in TgTB1 expression in these buds at week 12 (Fig. 3.3b). The TgTB1 expression in bud H remained

low throughout the whole storage period (Fig. 3.3b) however in contrast to the A, B and C bud there was no

weight gain of the H bud over time (Fig. 3.3a). The D and E buds did not gain weight during storage and from

the appeared to diminish in growth (Fig. 3.3c). This effect was caused by variation in D and E bud sizes between

bulbs and big buds measured for the initial size in week 0 which can make the buds look like they lose weight

overtime. Still, the D and E buds did not appear to grow and consistently and their TgTB1 expression does not

seem to decrease much. However a trend can be seen in the E bud in which the TgTB1 expression seems to be

lowering and at week 12 the expression is significantly lower compared to week 0 (Fig. 3.3d).

-200

0

200

400

600

800

1000

A B C H

Wei

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** **

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Rel

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Week 0

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Week 12

*

** ** *

nd nd

(a) (c)

-120

-90

-60

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60

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D E

Rel

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Axillary bud Tulipa

Week 0

Week 4

Week 8

Week 12

(b) (d)

*

Fig. 3.3 Weight gain over time of axillary buds from bulbs (‘Dynasty’) stored in 18°C with their respective relative TgTB1

expression. (a ) Weight gain of the A, B, C and H buds compared to the first week of measurements. (b) Relative TgTB1

expression of the A, B, C and H buds. (c) Weight gain of the D and E buds compared to the first week of measurements and

lastly (d) relative TgTB1 expression of the D and E buds. The differences compared to the first week of measurements were

tested using Tukey’s test (* = p<0.05, **= p<0.01, ***= p<0.001). Values are mean ± s.e., n = 1-3 (nd = no data).

14

The weight gain of the A bud in the bulbs stored in 4°C was significant (Fig. 3.4a). However, against

expectations no significant decrease in the TgTB1 expression of the A bud was found (Fig. 3.4b). The A bud was

very small during the first two time points and therefore there was not enough tissue to extract RNA. The fact

that the first two TgTB1 measurements, and therefore an early initial measurement, are missing could

contribute to the lack of a significant TgTB1 decrease as TgTB1 levels are already low in bud A at week 8. The

data of the B bud is missing; too little material of the B bud was available for RNA isolation so it was not

possible to measure relative TgTB1 expression. Because it was not possible to link TgTB1 expression to weight

gain it was also left out. The weight gain of the C bud was variable with no clear weight gain (Fig. 3.4a). Similar

to the bulbs stored in 18°C, the H bud shows no weight gain overtime. A significant decrease was found in the

BRC1 expression of the C bud however in week 12 the TgTB1 expression increased again (Fig. 3.4b). The TgTB1

expression of the H bud stayed low, as previous seen in storage at 18°C, except for a significant higher

expression in week 4. The D and E bud did not gain weight in the bulbs stored at 4°C, similar to the bulbs stored

at 18°C (Fig. 3.4c) and consistently their TgTB1 expression did not decrease either (Fig. 3.4d).

*

*

n

-100

0

100

200

300

400

A C H

Wei

ght

gain

(%

)

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Week 12

*

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Rel

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Week 0

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*

*

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ght

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) Week 4

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ress

ion

Axillary bud Tulipa

Week 0

Week 4

Week 8

Week 12

(a)

(d) (b)

(c)

Fig. 3.4 Weight gain over time of axillary buds from bulbs (‘Dynasty’) stored in 4°C with their respective relative TgTB1

expression. (a ) Weight gain of the A, C and H buds compared to the first week of measurements. (b) Relative TgTB1 expression

of the A, C and H buds. (c) Weight gain of the D and E buds compared to the first week of measurements and lastly (d) relative

TgTB1 expression of the D and E buds. The differences compared to the first week of measurements were tested using Tukey’s

test (* = p<0.05, **= p<0.01, ***= p<0.001) Values are mean ± s.e., n = 1-3 (nd = no data).

15

3.3 TgTB1 expression in springpartij bulbs over time

Almost all axillary buds in springpartij bulbs grow out into daughter bulbs however most are small and irregular

without enough vigour to produce a commercially acceptable flower. To understand the fundamental

differences in these springpartij buds they have to be compared to axillary buds of a normal bulb. Relative

TgTB1 expression was compared between buds of normal and springpartij bulbs. It was shown that relative

TgTB1 expression was significantly lower in the pooled buds of the B and C bulbs of springpartij compared to

the normal bulb (Fig. 3.5a). This pattern seems to be followed by the D bulb of springpartij however the

difference in relative TgTB1 expression was not significant (p<0.073). The buds of the A and H springpartij bulbs

did not differ in their relative expression from the buds of the normal bulbs, their expression stayed low

overall. Relative TgTB1 expression showed a low trend in the pooled buds of all different springpartij bulbs

while the B, C and D buds in the normal bulbs showed high TgTB2 expression, similar to a parabola (Fig. 3.5b).

0

1

2

3

4

5

A B C D H

Rel

ativ

e Tg

TB1

exp

ress

ion

Axillary buds Tulipa or pooled buds from Tulipa bulb

Normal

Springpartij

** ***

0

1

2

3

4

5

A B C D H

Axillary buds Tulipa or pooled buds from Tulipa bulb

Normal

Springpartij

(b) (a)

Fig 3.5 Relative TgTB1expression in buds of normal bulbs (‘Dynasty’) and in pooled buds of springpartij bulbs (‘Prinses

Catharina Amalia’). (a) Relative TgTB1 expression of the axillary buds of normal bulbs and pooled buds of springpartij bulbs.

(b) Trendlines to illustrate the relative TgTB1 expression of normal and springpartij bulbs. The differences between each bud

of the normal and springpartij bulbs were tested using the Independent T-test (* = p<0.05, **= p<0.01, ***= p<0.001).

16

4. Discussion and Conclusion

4.1 Dormancy regulation of axillary buds in tulip bulbs during storage

The primary focus of this research was on the growth of axillary buds in the daughter bulbs during storage.

Observations at the start of the storage period showed that multiple axillary buds are still being formed and

that the floral meristem is small and further development is required to commence flowering in spring (Fig. 3.1

+ Fig.3.2a). The A bud showed the fastest growth during storage at 18°C, followed by the B and C bud (Fig 3.1a

+ Fig. 3.3a). This significant increase in bud weight, especially in week 12, correlates with a significant decrease

in relative TgTB1 (TEOSINTE BRANCHED1) expression (Fig. 3.3b). TgTB1 is the primary gene blocking axillary

bud outgrowth in tulip. TB1, also called BRC1 (BRANCHED1) in dicots, promotes NCED3 (NINE-CIS-

EPOXYCAROTENOID DIOXYGENASE3) which is involved in the biosynthesis of abscisic acid (ABA), a major plant

hormone involved in dormancy induction (Bewley, 1997; Reddy et al., 2013). Other studies found that

TB1/BRC1 acts downstream of strigolactones to inhibit bud outgrowth in pea, Arabidopsis and rice (Brewer et

al., 2009; Minakuchi et al., 2010; Braun et al., 2012; Dun et al., 2012). Downregulation of TB1/BRC1 signifies the

breaking of dormancy and the ability to grow out under favourable conditions. Further support to link TgTB1

expression to the lifting of dormancy is found in the fact that the A bud was being formed at the beginning of

storage (Fig. 3.2a), but it was able to gain weight faster than the other buds (Fig. 3.3a). This weight gain

correlated with transient TgTB1 expression in the A bud (Fig 3.3b), meaning that dormancy in the A bud was

broken almost immediately after its formation during storage. This quick lifting of dormancy could be caused

the fact that the A bud is attached to the floral meristem.

The floral meristem showed a rapid growth from the beginning of storage till the end (Fig. 3.2). This is

in agreement with results from (Leeggangers et al., 2017) where they found that the floral meristem is very

active at the transcriptional level during storage. Sugars remobilized from starch in the scales are being

partitioned to the floral meristem to facilitate its growth. Due to its attachment to the floral meristem the A

bud probably received part of these sugars. Sucrose, a major sugar, has been shown to downregulate PsBRC1

expression in axillary buds of pea and promote outgrowth (Mason et al., 2014). In rose sucrose downregulates

RhMAX2 expression, a gene involved in the strigolactone transduction pathway, and RhBRC1 expression

(Barbier et al., 2015). The immediate access to sugars after formation (Fig. 3.2a) probably downregulates genes

in the strigolactone pathway and therefore also TgTB1 expression. This likely causes transient TgTB1 expression

and subsequent lift of dormancy.

Downregulation of TgTB1 expression occurs later on in the B and C bud as well (Fig.3.3ab). The inner

scales of the tulip bulb are more active during early growth than the outer scales therefore the inner buds are

likely provided with more sugars (Ho and Rees, 1975; Ho and Rees, 1976). Even though the A, B and C buds are

not big in the beginning, their downregulated TgTb1 expression, which remains low throughout the winter

period, causes them to be able to grow out as temperatures become favourable in spring. The sugars needed

for growth are made available during the cold period. Metabolic enzymes, as alpha-amylase, are induced by a

prolonged cold period and facilitate the degradation of starch in sugars, as glucose and sucrose (Moe and

Wickstrom, 1973; Lambrechts et al., 1994; Smith et al., 2005).

17

Quick growth after the cold period in buds which had their TgTB1 expression downregulated during

storage can be seen in results from the field experiments of Moreno-Pachon (unpublished). During field

measurements she showed that the A bud will grow into the biggest vegetative propagated daughter bulb at

the end of the growing season (Fig. 4.1a). In the field the A bud is quickly followed by the B bud, the H bud and

eventually the C bud. Consequently these are the buds that show a low TgTB1 expression at the end of the

storage period in 18°C (Fig. 3.3b). TgTB1 expression measurements during the field experiment shows similar

results to the storage experiment, the A and H bud show low TgTB1 expression while the expression in the D

bud is still high (Fig. 4.1b). Here as well, the weight gain correlates with the downregulation of TgTB1 in the

buds, causing the D bud to grow into a bulb of insufficient size due to a long period of high TgTB1 expression.

The buds that show downregulated TgTB1 expression during storage are therefore able to make use of the

sugars which are partitioned in spring, while buds that are still dormant do not and will therefore only be able

to make use of the sugars later on, putting them at a disadvantage.

4.2 Leaf formation and development of the floral meristem during storage

The expression of TgTB1 in the H bud had been low throughout the whole storage period, however in contrast

to the other buds with low TgTB1 expression the H bud did not gain weight (Fig. 3.3ab). Interestingly, the H bud

already had a big size at the start of the storage period (Fig. 3.1). It might be possible that the H bud developed

Fig. 4.1 Axillary bud outgrowth into daughter bulbs (‘Dynasty’) in the field. (a) Non-dormant A and B bud grow out and

produce reasonable sized daughter bulbs, followed by C and H bud. Dormant axillary buds D and E show minor growth. (b)

Growth in the field was quantified in weight. Alongside weight, TgTB1 expression was measured to give an indication of

the dormancy levels in the A, D and H bud. Modified from Moreno-Pachon (unpublished).

(a)

(b)

TgTB

1

18

while the daughter bulb was still attached to the mother bulb and that TgTB1 expression was, if it was ever

high, quickly downregulated. During this time the H bud might have been able to reach its intended size which

is why the bud would not grow anymore during storage. Alternatively, after separation of the mother bulb, size

increase in the H bud is blocked and sugars from the daughter bulb are used for different purposes. That the H

bud is the first bud formed, and therefore the first to receive sugars, is supported by results found at the

beginning of the storage period. Buds seem to be formed from the outside to the inside (H to A) making it

plausible that the H bud would be the first bud formed and the first to receive sugars to facilitate growth (Fig.

3.1). These findings are supported by Rees (1968) who found that the first initiated axillary buds are in the axils

of the outermost scales.

The H bud appears dormant (no external growth detected) during storage, as would the bulb, while

internal processes are ongoing. This corresponds to the dormancy definition suggested for bulbs: ‘a complex

and dynamic physiological state during which there are no apparent external morphological changes or growth.

Internally, however, many physiological and/or morphological events are occurring’ (Le Nard and De Hertogh,

1993a). As many internal processes are ongoing, it has been proposed for tulip that the inactivity of meristem

can be considered as true dormancy (Rees, 1981). It seems probable that the H bud is indeed receiving sugars

and that many internal processes are ongoing, as dormancy is already broken at the start of storage. Likely the

differentiation and formation of leaves occur simultaneously in the bulb and the H bud, which is the only

axillary bud with the ability to form leaves alongside the mother bulb, so they both sprout at the end of the

storage period to facilitate rapid outgrowth at the beginning of spring. The H bud likely uses the sugars

provided from the scales, thus is active, but more for structural purposes and not as much for starch

accumulation, as the other axillary buds would (Ho and Rees, 1976). It is also possible that the H bud (partly)

uses its own scales that it had already formed when the daughter bulb was still attached to the mother bulb.

The usage of its own starch would explain why there is no weight gain when it is forming leaves. It may be

possible that the H bud is more similar to the daughter bulb in its mechanisms than to other axillary buds, as it

has the capacity to sprout.

This might be a survival strategy as the leaves formed by the main meristem in the bulb partition most

of their sugars to the flower and the floral stem during spring instead of the axillary buds, thus causing

competition between the sink organs (Ho and Rees, 1976; Leeggangers et al., 2017). Since the H bud is not in

the proximity of the leaves it probably develops leaves of its own to ensure that it would receive substantial

sugars while also increasing the overall source strength in the tulip. This reduces competition between the

axillary buds and flower as the requirement of the axillary buds is partly met through an increased source

which is in closer proximity to the axillary buds than to the flower (Ho and Rees, 1976). During the cold period

in winter the roots and leaves are heavy sinks so most sugars derived from the scales are redistributed to them

(Ho and Rees, 1976). At the end of winter photosynthesis related genes are starting to be expressed in tulip

bulbs below ground, suggesting preparation for photosynthetic activity while leaves are still beneath the soil

surface (Leeggangers et al. 2016). The leaves emerge in the beginning of spring and subsequently the sinks in

the bulb can divide themselves over two sources (Ho and Rees, 1976). The H bud can use the sugars assimilated

by its own leaves as the resources from the scales are getting depleted by now (Rees, 1968). The floral bud and

19

stem decrease in sink strength when the leaves have reached their final size and the flower is well formed.

Leaving the axillary buds as the major sink to convert the sugars produced in the leaves into starch (Ho and

Rees, 1976; Ho and Rees, 1977). Even after the leaves senesce the axillary buds will receive nutrients and

carbohydrates from the senesced leaves through remobilization caused by autophagy (Pottier et al., 2014).

During this stage the H bud is already formed in the new daughter bulbs and might have never showed high

TgTB1 expression or only transient TgTB1 expression. An overview was made highlighting the source/sink

dynamics in the annual life cycle of tulip bulbs (Fig. 4.2).

4.3 TgTB1 downregulation due to cold storage

During winter the tulip is exposed to a period of prolonged cold which has proved to upregulate the production

of gibberellic acid (GA) to promote bud break (Druart et al., 2007). The expression of VRN2 (VERNALIZATION2)

is also upregulated; VRN2 downregulates the expression of FLC (FLOWERING LOCUS C); the main FT

(FLOWERING LOCUS T) suppressor (Huang et al., 2013). Thus, a prolonged cold period upregulates FT

expression which in turn downregulates TB1/BRC1 expression (Hiraoka et al., 2013). Furthermore, metabolic

enzymes as alpha-amylase are induced by a prolonged cold period and facilitate the degradation of starch in

sugars, as glucose and sucrose (Lambrechts et al., 1994; Smith et al., 2005; Moe and Wickstrom 1973). It was

Fig. 4.2 Proposed source/sink dynamics in during the annual tulip cycle. (a) In storage the leaves of the main meristem and

H bud and the development of floral bud are the major sinks. (b) During winter, the roots become a major sink alongside

the leaves and the floral bud. (c) The leaves become a second source alongside the scales after rapid outgrowth in spring.

The floral bud is still the major sink however the axillary buds are getting more access to carbohydrates due to the

increased source strength. (d) The flower has opened and the axillary buds have overtaken the floral bud as the major sink.

(e) The scales are depleted at this point and carbohydrates produced by the leaves are the major source for the axillary

buds now. (f) The final growth period of the axillary buds is facilitated by the nutrients derived from the senesced leaves at

the end of summer.

20

therefore tested whether cold storage (4°C) would be able to downregulate TgTB1 expression in axillary buds

faster compared to storage at an ambient temperature. However the results indicate that TgTB1 was not

downregulated faster and it even appears to be downregulated slower and more inconsistent (Fig. 3.3bd + Fig.

3.4bd). The axillary buds gained much more weight in storage at ambient temperature (Fig. 3.3ac + Fig. 3.4ac).

The lowering of metabolism rate is a probable cause as the floral meristems between the temperatures show

big difference in growth (Fig. 3.2bcd). Ambient temperatures are needed for proper outgrowth of a floral

meristem during storage and to ensure bigger axillary buds. The cold temperatures only have a positive effect

after bulb organs have had the chance for differentiation and formation during storage so they are ready for

rapid outgrowth after winter.

4.4 Dormancy regulation in springpartij bulbs

Springpartij bulbs can be defined as a cluster with many small daughter bulbs of which the main bulb occupies

less than 50% of the total cluster weight (de Jong, n.d.). It is suspected that dormancy is never induced in

axillary buds or quickly downregulated causing all these bulbs to grow out. The results show that the level of

TgTB1 expression in springpartij buds seems about the same level in each bud, while in normal buds the TgTB1

expression in the B, C and D bud is much higher compared to the A and H bud (Fig 3.5a). Results show that

normal bulbs experience a trend in which the middle buds are more dormant compared to the inner and outer

buds, causing a parabola effect. In the springpartij bulbs no parabola effect can be seen as the buds all show

the same level of TgTB1 expression (Fig. 3.5b).

Due to the overall low TgTB1 expression the normal conditions during storage are not applicable

anymore. In normal bulbs the high TgTB1 expression causes the sugars to mainly partition to the floral

meristem and the leaves (Fig.4.2). This is either directly (dormancy) or indirectly (small buds and therefore low

sink strength). In springpartij bulbs it seems likely that more sugars are being partitioned to the axillary buds

during storage than normally due to low dormancy. Therefore fewer sugars are available for floral and leaf

development as the source has to divide itself over more strong sinks. After the leaves are able to produce

sugars it seems likely less sugars are partitioned to support flower development as more and bigger axillary

buds are present with higher sink strength. This ultimately causes an insufficient flower to be developed

alongside lots of small, irregular daughter bulbs as the sink strength increased whereas the source strength did

not.

The springpartij phenotype is passed along in each generation of springpartij bulb (Snoek and de Jong,

n.d.). This causes the bulbs to eventually fall into a vicious cycle where they invest less carbohydrates in leaves

but still produce lots of daughter bulbs which in turn will become smaller, thus retain less starch leaving less

sugars available to produce leaves. The leaves are an important source for development, especially for

development of daughter bulbs (Ho and Rees, 1976). The decreasing source strength of the leaves in each

generation will cause springpartij batch to eventually be unable to produce any decent sized daughter bulbs

anymore.

21

4.5 Conclusions

The A bud is formed during storage and shows quick weight gain at ambient temperatures, correlating with

transient TgTB1 expression. This transient expression is probably caused by the availability of sugars from the

floral meristem that downregulate TgTB1 expression. The B and C bud show significant weight gain during a

later period in storage which also correlates with downregulated TgTB1 expression. The H bud did not grow,

however it showed low TgTB1 expression throughout the whole storage period. It probably invested sugars

into the differentiation and formation of its leaves that will grow out at the beginning of spring instead of

further starch accumulation. The D and E bud did not show weight gain and their TgTB1 expression stayed high.

These results suggest that the main function of the dormancy gradient in axillary buds is to ensure that sugars

are re-directed to bulb organs which need them most at a certain time point during the annual growth cycle.

During storage the main meristem, axillary buds in proximity to the main meristem and the leaf formation in

the H bud appear to have priority. In a successful annual cycle, sexual and vegetative propagation occur

alongside each other to increase plasticity. This fragile balance is probably maintained due to strategic sugar

partitioning enforced by dormancy induction.

Storage at cold temperatures did not result in quicker downregulation of TgTB1 expression. It slowed

down organ formation and development due to the low temperature, causing insufficient development of the

main meristem at the end of the storage period. These bulbs are therefore probably unable to commence

flowering the following spring. These results show that storage at low temperatures has no positive effect on

the internal physiological and morphological processes.

Springpartij bulbs showed downregulated TgTB1 expression in all axillary buds. The sink strength of

the axillary buds was higher during the storage period compared to normal bulbs, possibly retaining more

sugars. This could cause a chain reaction in which the axillary buds are able to access sugars which are normally

partitioned to the leaves and floral meristem. Subsequently, the decreased source partitioning to the leaves

could cause less development of the leaves and therefore decreased source strength during spring.

Consequently, insufficient sugars are partitioned towards the flower bud because of lower source strength and

increased competition of axillary buds exhibiting high sink strength. This eventually causes the formation of an

insufficient flower and a cluster with lots of small daughter bulbs in which the main daughter bulb possesses

less than 50% of the total cluster weight. The TgTB1 expression in the axillary buds of these daughter bulbs will

already be downregulated causing them to present the same phenotype as the previous generation when

planted.

4.6 Future directions

As TB1/BRC1 is known to repress axillary bud outgrowth through increasing the abscisic acid (ABA) biosynthesis

(Bewley, 1997; Yao and Finlayson, 2015), it might be interesting to correlate them with each other in tulip.

During this research ABA2, a major gene involved in the ABA biosynthesis pathway, was sequenced for tulip.

Due to lack of time, experiments measuring ABA2 expression in tulip tissue were not included in this research.

However ABA2 expression can be measured in numerous tissues instead of only in the axillary bud like TgTB1,

making it possible to create a broader picture of how dormancy is regulated in tulip. Additionally, ABA

22

hormone measurements can be performed on those tissues to link ABA2 expression to the presence of the

actual ABA hormone.

The control used in the springpartij experiment was not ideal as it was a different cultivar which was

harvested at a different time. However this control had to be used as a no good control was available at the

time. It is recommended to re-do the experiment with a good control which is from the same cultivar but from

a different batch. Ideally, the control would be grown under similar conditions and harvested at the same time

as the springpartij bulbs.

Further, an experiment to check to the vascular connection in the different axillary buds of the

springpartij bulbs could be performed to check whether this causes the sudden increased growth capacity of

the axillary buds that are normally dormant (D and E). Preliminary results of experiments performed in

Wageningen on normal bulbs showed that both the non-dormant A bud and the dormant D bud had a vascular

connection however the vascular connection in the D bud was more irregular compared to the A bud. It would

be interesting to compare the vascular connection between the A bud and D bud of the springpartij bulb, which

could be linked to the sudden decrease in dormancy of the D bud.

Lastly, I would be interesting to look into the differences in sugar partitioning between normal bulbs

and springpartij bulbs. Normally, bulbs partition most of their sugars to leaves and the floral meristem at the

start of the annual growth cycle (Ho and Rees, 1976). It might be possible that springpartij bulbs partition more

sugars to the axillary buds than normally at the start of the annual growth cycle due to decreased dormancy.

This could cause the eventual outgrowth of numerous, small but unusable daughter bulbs seen in springpartij

bulbs, due to the lack of strategic sugar partitioning.

23

Acknowledgements I wish to thank my supervisor Natalia Moreno-Pachon for her guidance and encouragement during my thesis. I

would also like to thank Richard Immink for his constructive comments and suggestions. Additionally I want to

express my thanks to Melissa Leeggangers who helped me during the start of my thesis. Lastly, I would like to

thank the students and the members of Plant Physiology department for their support and kindness.

24

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