Effect of temperature on floral initiation and crown formation in B. oleracea
Peter Targett1, Storcken, Mirjam1; Gracie, Alistair1, John Kingsbury1, Aaron Thomason1,
1School
of Agricultural Science, The University of Tasmania, Private Bag 54, Hobart TAS 7001, www.utas.edu.au Email:
Abstract
Global demand for broccoli is increasing and opportunities for local supply of markets are arising in many
small tropical countries. However broccoli grown in these regions show poor crown shape and size or
produce no heads at all. Genetic technology revealed that both varieties that produce crowns and varieties
that do not produce crowns in these conditions, do not have a functional BoFLC2 gene and therefore do not
require or require very little vernalisation. To ascertain whether a lack of crown formation is due to
temperature stress, two varieties that form crowns in tropical conditions Southern Comet and Southern King,
were compared to two non-crown forming varieties, Marathon and Summer King, under three different day
night temperature regimes of 20/15 oC, 25/20 oC and 30/25 oC, in climate control cabinets. Results show that
vernalisation was still a requirement for floral iniation for all varieties, despite the fact that the BoFLC2 gene
was non-functional. Crown formation was found to be directly linked to the lower vernalisation requirement
of early maturing varieties Southern Comet and Southern King, which were simply quicker to produce a
crown than the other varieties.
Key Words
Bolting, inflorescence, heat-stress, head-formation
Introduction
Factors that affect the timing, duration and size of the crown formation stage of broccoli, are of particular
importance to growers, who must ensure that a crop of adequate size and consistent quality must be delivered
to the consumer. According to Dimsey, (2009) in the last 15 years varietal development and improvements in
the overall quality of broccoli, have led to an increased consumer acceptance and demand, with the aid of
modern marketing. Consumption of fresh broccoli in the United States increased from 2.0 - 2.9 kg/capita
from 1995 – 2012 (USDA, Economic Research Service, 2013), whilst the total area of harvested broccoli and
cauliflower in the world rose steadily from 789,300 ha to 1,209,100 ha between 2000 and 2011 (USDA,
Economic Research Service, 2013). With increasing demand and the rise of globalisation, there is a
significant potential for local production of broccoli in small tropical countries near large population centres
(Jones, 2012). A recent trial in Sogeri of Papua New Guinea, by the Australian centre for international
agricultural research (ACIAR), found that varieties Marathon, Shogun and Summer King did not produce
crowns, whilst Southern Comet, Southern Star and Green King were early maturing and brought forth flat,
ricey crowns of non-marketable size (Seta-Waken, et al., 2011). Under normal circumstances broccoli has an
extended meristem proliferation period to produce the inflorescence, followed by a second pre-anthesis arrest
that allows for head enlargement without bolting (Farnham & Bjorkman, 2011). Cool temperatures are
generally required to induce, as well as maintain vernalisation (Farnham & Bjorkman, 2011), which tends to
restrict the growing regions of broccoli. Every variety has a different requirement for vernalisation and if
vernalisation fails, then either no head is formed (Wurr, et al., 1995), or if the coordination of floral
development is disrupted, then the result is an uneven crown that is unmarketable (Bjorkman & Pearson,
1998). Genetic studies conducted before the commencement of the experiment, revealed that all four
varieties had a non-functional BoFLC2 gene, which is the only gene that has been linked to vernalisation
requirement in broccoli to date (Okazaki, et al., 2007). Thus, any vernalisation requirement should
theoretically be small and the variation seen between varieties in the ACIAR trial may be due to abiotic
factors such as temperature stress instead. According to Bjorkman & Pearson (1998), once floral
development of the broccoli has been initiated, its progress can be arrested by temperatures between 30 and
34 oC, which can be optimal for vegetative growth, so long as they don’t exceed this temperature range.
Once a broccoli plant has reached the head initiation stage, any stresses related to moisture, fertiliser,
transplant shock, pests and disease can all cause buttoning plants, to prematurely increase to the next growth
phase (Valley, 2002). Tests conducted by Bagget and Mack (1970) on broccoli support this concept, as they
found that the use of large transplants as opposed to small transplants, caused an increase in the percentage
of plants forming crowns prematurely. Photoperiod was ruled out as a factor affecting broccoli by Tan, et al.
(1999), who found that photoperiod has no influence on yield and quality of broccoli. Although much is
known about quantitiative vernalisation requirements in broccoli, little is known about the effect of
temperature and heat stress on crown formation and bolting in broccoli (Farnham & Bjorkman, 2011 and
Tan, et al., 1999). The hypothesis of the experiment, was that heat stress was the cause of the lack of
harvestable heads in the two cultivars Marathon and Summer King grown in PNG. In order to test this
hypothesis four varieties of broccoli grown in PNG; Southern Comet, Green King, Summer King and
Marathon were placed in three sealed temperature cabinets with three different temperature day/night
regimes. The aim of the experiment being to determine the effect of temperature on floral initiation time and
crown formation, as well as to identify any physiological reasons for differences between the varieties. This
experiment was done in conjunction with a wider research team led by Mirjam Storcken and Alistair Gracie,
and was part of a series of experiments that attempted to examine each of the abiotic factors that may have
affected the flowering of the broccoli in PNG.
Materials and Methods
Broccoli plants were grown from seed in seedling trays (5x5 cm) on the 4th of April, at a temperature of 20 ±
4 °C, with 12 hours of light per day. After two weeks, seedlings were planted singly in 15.2 cm pots and four
plants of each variety were placed randomly in each of the three climate control cabinets. Each cabinet was
set to one of the following day/night regimes; 30/25°C, 25/20°C and 20/15°C (12 hours/ 12 hours). The
development of the plants was monitored each week by measuring the height, flower development, as well as
the number of leaves each plant. The cultivar results per unit were compared with each other and the results
from each cultivar from all the different units was compared with each other using IBM SPSS statistics
package version 22. The potting mix comprised a mixture of 80% (volume), composted Pine Bark and 20%
(volume), coarse Sand. Dolomite (4 Kg/m3), FeSO4 (0.75 Kg/m3), Wettasol granules (0.75 Kg/m3), and
Osmocote Plus (3 Kg/m3) were added. The Osmocote Plus contained: Nitrogen (16.0%), Phosphorus (3.5%),
Potassium (10.0%), Sulfur (2.4%), Magnesium (1.2%), Boron (0.02%), Copper (0.05%), Iron (0.4%),
Manganese (0.06%), Molybdenum (0.02%), Zinc (0.01%) and Organic resin coating (8.0%). Initial screening
for FLC2 gene mutation was performed using PEA genomic DNA extraction as developed by Friedrich
Miesher and Richard Altman, PCR and gel electrophoresis (Storcken & Gracie, 2013).
Results
Figure 1 (Top - Left). Average growth stage (cm) at the end
of the experiment, for each variety, in each temperature
cabinet. Growth stage 1 = vegetative, 2 = crown formation,
3 = inflorescence, 4 = seed pod formation. Error bars
indicate 2 standard errors of the mean.
Figure 2 (Top - Right). Crown diameter (cm) at full crown
maturation for each variety in each cabinet. Error bars
indicate 2 standard errors of the mean.
Figure 3 (Bottom – Right). Gel electrophoresis results
showing non-functional FLC2 genes.
Cabinet 2
Cabinet 3
Cabinet 4
Figures 4 – 6 Effect of temperature regeime on leaf count for each variety. Variety 1 = Marathon, 2 = Green
King, 3 = Summer King, 4 = Southern Comet. Error bars indicate 2 standard errors of the mean.
Table 1: Days after planting (DAP), number of leaves > 2cm, and height from the soil to the inflorescence
butt at buttoning. Values shown are means ± one standard deviation,
Temperature
Regime
20/15
25/20
30/25
Cultivar
DAP
Number of Leaves
14.3 ± 2.7
Height to Inflorescence
butt (cm)
7.0 ± 0.9
Plants with
Inflorescences
100%
Green King
97.5 ± 5.7
Southern Comet
84.0 ± 0.0
11.3 ± 0.5
5.3 ± 0.3
100%
Marathon
104.8 ± 3.1
16.5 ± 0.6
7.9 ± 0.6
100%
Summer King
110.0 ± 4.6
17.8 ± 1.8
7.1 ± 0.5
100%
Green King
140.0 ± 7.6
31.5 ± 1.7
16.5 ± 1.7
100%
Southern Comet
156.3 ± 4.7
32.3 ± 0.9
15.2 ± 1.1
75%
Marathon
145.3 ± 8.3
33.3 ± 4.0
13.4 ± 1.6
100%
Summer King
168.0 ± 0.0
46.0 ± 0.0
17.5 ± 0.0
25%
Green King
168.0
35.0
20.5
50%
Southern Comet
161.0
29.0
19.8
25%
Marathon
-
-
-
0%
Summer King
-
-
-
0%
Discussion
The FLC gene is known to produce a compound that is an inhibitor of flowering in Arabidopsis and FLC
homologs in Brassica species are also thought to control vernalisation. There are four FLC homologes in B.
oleracea as well as six quantitative loci (QTL) controling flowering time. Although the BoFLC2 gene was
the only homologe linked to any of the six QTL’s in a study by Okazaki, et al., (2007), however the authors
of the study were careful to note that their results do not offer direct evidence to show the role of BoFLC2
gene conroling vernalisation. The results of our experiment show that the flowering time of every variety
was dependant on temperature, with warmer temperatures delaying floral initaion, an observation also noted
by Wurr et. al who found that without exposure to cool temperatures, floral initation was delayed
considerably, whilst plants continued to gain mass. This suggests that either one of the four other homologes
or another gene suite altogeather may be jointly responsible for vernalisation requirement in broccoli. FLC
independent vernalisation pathways have been described for several other species (Uptmoor, et al., 2011).
A QTL-based phenology model that accounts for genotypic differences and developed by Uptmoor, et al.,
(2011) was found to be quite accurate when floral induction was prompted by low temperature (R2 = 0.81).
However unfavourably high temperatures reduced predictability of the model (R2 = 0.65). Our results as well
as the results of other studies including the model suggest that at high temperatures, other mechanisms than
the BoFLC2 gene are responsible for flower initiation in broccoli.
Figure 4 shows that the average leaf number for Marathon and Summer King was much greater than for
Green King and Southern Comet. This was because these two varieties are shorter season varieties (Storcken
& Gracie, 2013) and floral initiation commenced much sooner (table 1). There was however no differences
between the maximum crown diameters of the different varieties. Heather et al. (1992) evaluated a collection
of broccoli hybrids for heat tolerance and found that heat stress was most damaging at 3 weeks before
harvest when the inflorescence was between 5 & 10 mm in diameter. Simmliarly Bjorkman and Pearson
(1998) found that the hybrid Galaxy was highly sensitive to heat stress during floral initation, when the
apical meristem is less than 2 mm and the shoot tip is less than 5 mm. Before or after this stage Bjorkman
and Pearson found that heat stress had little effect. We hypothesise that temperature of the meristem at the
floral initiation stage has a large effect on the size and shape of the crown due to increased enzymatic and
gene regulation activity during this time. It is well known that the effeciency of these processes increases
with temperature. This hypothesis that temperature affects enzymatic activity in the meristem is partially
supported work done by Díaz-Pérez (2009), who found that root zone temperatures below 21 oC had no
effect on the broccoli and increasing the root zone temperature between 21 oC and 25 oC significantly
increased yields without any stress effects being observed. Therefore if the meristem proliferation period
could be prolonged at the critical stage by reducing the temperature then a larger and more even crown may
be the result. This could possibly be achieved in the field using evaporative cooling via irrigation or applying
shade cloths over the broccoli before the critical stage.
Conclusion
The results of the experiment confirm previous studies that broccoli have a vernalisation requirement in
order to begin floral initiation. This is despite the fact that the varieties used in the experiment had a nonfunctional BoFLC2 gene. The quality of the broccoli crowns formed in the experiment were poor and their
diameter did not vary significantly between the varieties, despite the fact that some were early season
varieties that flowered much sooner and produced a lower total leaf count than the late season varieties.
Increased enzymatic and gene regulation activity in the meristem during periods of high temperature may be
responsible for accelerating the cascade of gene expression required to change the vegetative apical meristem
into the inflorescence stage. The experiment confirms that broccoli is best grown in cooler environments and
confirms that early maturing varieties such as Green King and Southern Comet as the best varieties to be
grown in warm climates.
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