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Discoveries in Agriculture and Food Sciences - Vol. 12, No. 1
Publication Date: February 25, 2024
DOI:10.14738/dafs.121.16374.
Stevanato, P., Ravi, S., Townson, P., Virk, D. S., & Senior, H. (2024). Molecular Fingerprinting Confirms Pollen-Proofing of Nonwoven
Pollination Control Fabrics in Sugar Beet. Discoveries in Agriculture and Food Sciences, 12(1). 25-41.
Services for Science and Education – United Kingdom
Molecular Fingerprinting Confirms Pollen-Proofing of Nonwoven
Pollination Control Fabrics in Sugar Beet
Piergiorgio Stevanato
Department of Agronomy, Food, Natural Resources,
Animals and Environment, DAFNAE, University of Padua,
Agripolis Campus, Viale dell’Università 16, Legnaro, PD, Italy
Samathmika Ravi
Department of Agronomy, Food, Natural Resources,
Animals and Environment, DAFNAE, University of Padua,
Agripolis Campus, Viale dell’Università 16, Legnaro, PD, Italy
Paul Townson
Lion Seeds Ltd. Maldon Road, Maldon, Essex CM9 6SN, UK
Daljit Singh Virk
School of Natural Sciences, Bangor University, LL57 2UW, UK
Hannah Senior
PBS International, Salter Road, Scarborough, YO11 3UP, UK
ABSTRACT
We tested the pollen-proofing ability of three new pollination control tents (PCTs)
made from nonwoven fabrics (DWB10, DWB23, DWB24) developed to have an open
architecture to improve light and air permeability while still maintaining an
effective barrier to pollen compared to standard duraweb® (DWB01) fabric. During
2020 and 2021 two methods of evaluation were used at Lion Seeds Ltd, Essex, UK
on single potted plants of a cytoplasmic-genetic male sterile family (CMS): (a)
fingerprinting of parent and progeny genotypes of seed set from CMS plants under
PCTs, using 209 molecular markers, (b) analysis of seed-related traits. Adjacent
open-pollinated plants showed high seed set indicating abundance of ambient
pollen, while those under PCTs saw 86% less seed weight and 96% less implied seed
numbers (ISG) showing that ‘non-seeds’ were collected as seeds. Molecular markers
analysis of two years of PCT progeny showed: 1. non-significant difference between
PCTs for percent homozygosity; 2. Parent vs. progeny percent homozygosity was
significant in 2020 (85% of parent vs. 77% of progeny) but not in 2021 (73% in
parent and progeny); 3. The CMS family was not pure-breeding and the mean
homozygosity of 75% over two years was a good fit for theoretical expectations of
one generation of inbreeding. Analysis (b) of various seed-related traits showed
non-significant differences between PCTs except for 1000-seed weight and
germination percent. The implied seed number weighted by germination percent
was virtually zero for all PCTs. Both (a) and (b) confirmed that novel PCT fabrics
despite greater air permeability, were as pollen-proof as the control DWB01
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Discoveries in Agriculture and Food Sciences (DAFS) Vol 12, Issue 1, February- 2024
Services for Science and Education – United Kingdom
providing new options in sugar beet breeding to maintain plant health. Highlights:
Analysis of molecular markers and seed related traits confirmed pollen proofing of
new nonwoven fabrics with more open architecture and greater strength for
pollination control in sugar beet.
Keywords: Molecular markers, male sterility, sugar beet, nonwoven fabric, pollination
control tent.
INTRODUCTION
Townson et al. [31] compared the performance of cytoplasmic-genetic male sterile (CMS)
plants grown in open and mini-isolation tents and tested the pollen proofing and effects of
micro-climate within nonwoven pollination control tents on agronomic traits. They tested the
hypotheses: (a) the mean number of seed set on CMS plants grown in tents to return a near- zero estimate within the statistical error limits, provided the tents were pollen-proof and did
not allow foreign pollen intrusion. (b) morphological plant traits of the CMS plants in tents
performed like those in the open control (H0), provided the micro-environment within tents
did not influence plant traits differently than in the open control. In both cases, they reported
the acceptance of the null hypothesis (H0).
The present study extended the 2019 experiments of Townson et al. [31] for another two years
during 2020 and 2021 to collect further evidence for pollen proofing ability of four tent fabrics
based on molecular markers and the rate of seed set. Consistently, in all years, a commercially
exploited male sterile (CMS) family was used as it produces non-functional pollen [6]. Its male
sterility is cytoplasmic-genetic type [18] in which MS (male sterile) or A-line (family) is
maintained with an O-type maintainer family or B-line, and a pollinator with restorer gene (R- line) is used for hybrid seed production. Under circumstances of perfect pollen exclusion and
complete sterility no progeny would be expected, provided there exists no environmental
sensitivity of the male sterility gene [22]. The frequency of viable seeds would then indicate the
ability of the barrier materials to exclude pollen. However, as in other crops, the stability of
cytoplasmic male sterility expression in sugar beet (Beta vulgaris L.) is reported to be
environmentally sensitive in specific genotypes [17]. Pollen sterility may break-down under
certain environmental conditions or due to the unintended presence of fertility restoring
factors in the nuclear genome (rare off-types usually due to contamination during production
of the CMS family). A comparison of the molecular marker fingerprint between each CMS and
associated progeny would confirm if they were the product of a selfing event (CMS failure) or a
cross (pollen exclusion failure).
Clifton-Brown et al. [7] reported the average size of sugar beet pollen to be 20-25 μm but
according to Hecker [13] the mean diameter of pollen of diploid (2x) strains of sugar beet was
20.8 μm (19.3 to 22.5 μm) and that of auto-tetraploid (4x) to be 25.9 μm (23.4 to 27.4 μm). Both
wind and insects assist in sugar beet cross-pollination [3] and the wind may carry the pollen as
long as 1200 m [8]. Usually, pollination bag materials with porosity smaller than the pollen size
are used to avoid contamination [12]. More open architecture increases airflow and light
penetration, helping to improve legitimate pollen dispersal, plant vigour and reduce humidity
inside the tents and improving disease resistance and seed set. Stronger fabrics are also
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Stevanato, P., Ravi, S., Townson, P., Virk, D. S., & Senior, H. (2024). Molecular Fingerprinting Confirms Pollen-Proofing of Nonwoven Pollination
Control Fabrics in Sugar Beet. Discoveries in Agriculture and Food Sciences, 12(1). 25-41.
URL: http://dx.doi.org/10.14738/dafs.121.16374
required for larger pollination control tents. In this study, nonwoven fabrics of at least three
new tent types were deliberately chosen for improved strength and more open architecture
than the standard being used at Lion Seeds. This was envisaged to allow a trade-off between
pollen-proofing and breathability for a more ambient micro-climate adjustment within tents.
The major objectives of the present study were to: (i). Compare and identify new nonwoven
pollination control tent fabrics with pollen-proofing ability and (ii). Assess if the nonwoven
fabrics with more open architecture retain pollen-proofing ability suitable for use in sugar beet
hybrid breeding. The study uniquely employed molecular markers and seed-related traits to
test the pollen-proofing ability of synthetic fabrics.
MATERIALS AND METHODS
Genetic Material
Lion Seeds provided MS plants from families bred for commercial use. Sugar beet breeding
families are not developed to be inbred lines but to be reasonably phenotypically uniform and
true-breeding, and near-homozygous for practical purposes. Such families may depict within- family variation that is a joint effect of non-genetic or environmental and residual genetic
variation.
Experimental Details
Details of five treatments (mini tents) remain as given in Townson et al. [31]. Briefly, these
included three new nonwoven synthetic fabric treatments (DWB10, DWB23, and DWB24),
standard DWB01, and open control without any cover. The standard DWB01 has been used at
the Lion Seeds for many years. It is heat-bonded fabric which retains a flexible nature that
makes it easier to handle. The three new fabrics are spun bond and have greater strength and
air permeability combined with an architecture that impedes pollen penetration.
Figure 1: Field arrangement of tent treatments and controls (left) and a close up of a mini-tent
covering a single sugar beet plant of male sterile line (right). A window on the side of mini-tent
facilitated the view of the plant in the cover.