QUANTIFICATION OF POLLEN VIABILITY IN Lantana camara BY DIGITAL HOLOGRAPHIC MICROSCOPY Vipin Kumar

2025-05-02 0 0 7.59MB 13 页 10玖币
侵权投诉
QUANTIFICATION OF POLLEN VIABILITY IN Lantana camara BY
DIGITAL HOLOGRAPHIC MICROSCOPY
Vipin Kumar∗
National Institute of Plant Genome
Research, New Delhi 110067 India
environment.vip14@gmail.com
Nishant Goyal∗
Department of Physics
Indian Institute of Technology Delhi
New Delhi 110016 India
phz218060@iitd.ac.in
Abhishek Prasad
National Institute of Plant Genome
Research, New Delhi 110067 India
abhishek1992@nipgr.ac.in
Suresh Babu
School of Human Ecology,
Dr. B.R Ambedkar University Delhi,
New Delhi 110006 India
suresh@aud.ac.in
Kedar Khare
Optics and Photonics Centre
Indian Institute of Technology Delhi
New Delhi 110016 India
kedark@physics.iitd.ac.in
Gitanjali Yadav
National Institute of Plant Genome
Research, New Delhi 110067 India
gy@nipgr.ac.in
October 11, 2022
ABSTRACT
Pollen grains represent the male gametes of seed plants and their viability is critical for efficient
sexual reproduction in the plant life cycle. Pollen analysis is used in diverse research thematics
to address a range of botanical, ecological and geological questions. More recently it has been
recognised that pollen may also be a vector for transgene escape from genetically modified
crops, and the importance of pollen viability in invasion biology has also been emphasized.
In this work, we analyse and report an efficient visual method for assessing the viability of
pollen using digital holographic microscopy (DHM). We test this method on pollen grains of
the invasive Lantana camara, a well known plant invader known to most of the tropical world.
We image pollen grains and show that the quantitative phase information provided by the
DHM technique can be readily related to the chromatin content of the individual cells and
thereby to pollen viability. Our results offer a new technique for pollen viability assessment
that does not require staining, and can be applied to a number of emerging areas in plant
science.
Keywords: Pollen viability, Invasive species, Quantitative phase imaging, Classification, Lantana
camara
1 Introduction
Pollen grains commonly appear as fine dust; each grain represents a minute body of varying form and size, produced
in specialized male floral organs called stamens in seed bearing plants, and transported to the female structures for
fertilization. Viability refers to the capacity of pollen grains to mature and then fertilize, followed by the ability to
develop into seed and fruit
(
Shapiro, Eisenberg, & Binder, 1965; Faegri, Kaland, Krzywinski, et al., 1989). Pollen
analysis has long been known as a rigorous scientific method encompassing a diverse number of research disciplines
*These authors contributed equally to this work.
arXiv:2210.04421v1 [eess.IV] 10 Oct 2022
APREPRINT - OCTOBER 11, 2022
including botany, paleoentology, geology, ecology, climatology, archaeology and many more
(
Faegri et al., 1989; Dafni
& Firmage, 2000). More recently, in the modern era, pollen have been recognised as vectors for transgene escape from
genetically modified crops
(
Firmage & Dafni, 2000). Furthermore, these tiny male reproductive units of seed plants
have also been recognised for their importance in biological invasions
(
Bufford & Daehler, 2014). Ecosystem threats
by invasive plant species often pose a huge challenge, not only for conservation but also for the nursery, horticultural
and landscape industries, leading to the necessity of exploring sterile non-invasive cultivars to replace fertile invasive
ones
(
Burns et al., 2019). In the following sections, we bring together literature that strings together pollen viability
estimations, biological invasions and the importance of imaging techniques in pollen analysis. Following this discussion
of prior literature, we describe a novel quantitative phase imaging methodology for pollen using a digital holographic
microscope system (DHM). DHM is an emerging modality where the natural refractive index contrast of the sample is
imaged in a quantitative manner as explained in detail later. The DHM system we use works with a dual mode operation,
so that, a single pollen can be imaged in the usual bright-field mode as well as in the quantitative phase mode by simple
switching of illumination without disturbing the sample. This allows us to establish phase map characteristics of viable
and non-viable pollen in a quantitative manner. A DHM system was used for pollen refractive index tomography using
multiple hologram recordings of a rotating sample followed by Radon transform inversion
(
Charrière et al., 2006).
A combined fluorescence and digital holographic microscopy system was demonstrated with pollen grains as test
sample
(
Shaffer, Pavillon, & Depeursinge, 2012). Pollen imaging in quantitative phase imaging mode has also received
attention in environmental sciences literature
(
Van Hout & Katz, 2004; Berg & Videen, 2011; Wu & Ozcan, 2018;
Sauvageat et al., 2020; Kemppinen, Laning, Mersmann, Videen, & Berg, 2020). In this work a novel single-shot full
resolution DHM system that uses a sparse optimization based phase recovery
(
Khare, Ali, & Joseph, 2013; Singh &
Khare, 2017, 2018; Mangal et al., 2019) has been employed to image a large number (more than 500) of individual
pollen grain from lantana camara. The full resolution (with 40x, 0.75 NA) image plane holography of the pollen as
performed here is clearly seen to bring out morphological features of viable and non-viable pollen in their hydrated
state. We further provide initial evidence that quantitative phase mode of imaging may enable pollen imaging and
classification without the need of conventional staining procedures.
The paper is organized as follows. In Section 2, we describe the importance of pollen viability studies to plant sciences
and briefly review the existing techniques for viability estimation. Section 3 details our sample preparation and imaging
methodology with some additional details on the single-shot phase reconstruction algorithm. An illustration showing
visual morphological differences in different types of pollen as evident from their quantitative phase maps is presented.
This is followed by Section 4 with quantitative measurements and statistics performed on the phase images of over
500 lantana pollen grains. An illustration of phase imaging for unstained pollen grains is also presented. Finally in
Section 5 we provide concluding remarks and our scope for future work including new directions being undertaken in
our laboratory.
2 Pollen viability and its importance
Viable pollen is important for species dispersal, fitness, and survival of the next plant generation. It is also essential for
directed plant breeding and, consequently, crop improvement. Pollen viability comprises different aspects of pollen
performance such as fertilization ability, germinability, and stainability
(
Dafni & Firmage, 2000). Pollen viability can be
affected by drought/dehydration, heat stress and UV-B radiation. These factors can play a role after pollen dehiscence,
when pollen is exposed to the environment, or even before, during pollen development inside the anther. Viability
may also be species-specific and dependent on pollen physiology, or presence of specific structural modifications. A
complicating factor in these assessments is the lack of standardized protocols and experimental conditions for viability
assessments as described below (Dafni & Firmage, 2000; Firmage & Dafni, 2000).
2.1 Techniques for Viability Estimation
AS pollination is the primary function of the pollen grain in a plant life cycle, one way to test pollen viability is
to use the pollen for pollination and subsequently analyze seed set. However, this is time consuming and often not
feasible, thus other methods are frequently used to elucidate pollen viability, such as staining techniques, in vitro or
in-vivo germination, as well as semi-in situ germination on the excised stigma (stigmatic germination), or impedance
flow (IF) cytometry, but again, most of these are difficult to scale up and further confounded by incompatibilities,
post-fertilization barriers and limited measurability, factors that restrict the accuracy of these tests
(
Dionne & Spicer,
1958; Dafni & Firmage, 2000).
Vital staining is by far the fastest and most commonly used method of pollen viability estimation and this involves
visualization of specific compounds, contents, or cellular compartments. For example, Potassium iodide, aniline blue,
and acetocarmine stains bind to starch, callose, and chromatin, respectively, and the absence of colors indicate non-
2
APREPRINT - OCTOBER 11, 2022
viable pollen. (Stanley & Linskens, 1974). The Alexander stain discriminates aborted pollen grains from non-aborted
pollen grains by staining the cytoplasm in red, and the cell walls green. In the absence of cytoplasm (non-viable grains),
the green cell walls become visible, indicating lack of viability
(
Alexander, 1969). The last major advance in this area
was almost half a century back when the Heslop-Harrisons developed a viability test based on fluorochromatic reaction
based membrane integrity and enzyme activity
(
J. Heslop-Harrison & Heslop-Harrison, 1970; Y. Heslop-Harrison,
1977; Shivanna & Heslop-Harrison, 1981). Although staining methods offer the possibility to distinguish aborted and
non-aborted fresh pollen, they often fail to discriminate different viability levels (Ge et al., 2011).
Refined viability estimations have been attempted by comparing and combining some of these techniques. For example,
high correlations were found between viability results from IF cytometry and FDA staining in case of mature cucumber,
sweet pepper, and tomato pollen
(
Heidmann, Schade-Kampmann, Lambalk, Ottiger, & Di Berardino, 2016). However,
pollen viability assessment purely through microscopy has not been investigated up till now. Therefore, the present
study aims to assess whether DHM can be used as a standalone method for checking viability of pollen. The term
“pollen viability” has been used as an umbrella term describing the capacity of pollen to live, grow, germinate, or
develop
(
Dafni & Firmage, 2000). By manually comparing 500 individual pollen grains of Lantana camara, we selected
distinguishing features amongst over 30 parameters identified by the DHM technique. The results were compared
with the pollen viability assessed by the fastest method (acetocarmine staining) and it was found that DHM can very
successfully discern one from the other.
2.2 Invasive species pollen analysis; Case Study Lantana camara
Our interest in pollen analysis is an extension of our ongoing efforts to understand Invasive alien plants species (IAPS),
particularly Lantana camara
(
P. Mishra, Prasad, Babu, & Yadav, 2021; Chauhan, Yadav, & Babu, 2022; Davis &
Thompson, 2000). IAPS are considered to be one of the major drivers of biodiversity loss, posing severe threats to
ecosystem services, environmental quality and human health globally
(
B. A. Jones & McDermott, 2018; Bartz &
Kowarik, 2019; Pejchar & Mooney, 2009). Lantana camara (Wild Sage) is a small broadleaf flowering shrub within the
Verbenaceae family, native to American tropics
(
Sharma, Makkar, & Dawra, 1988). It is an extremely adaptable weed,
found across a wide variety of ecosystems. Once Lantana has been introduced into a habitat, it spreads rapidly, and has
already done so from it’s native Central and South America to over 50 countries, making it one of the world’s top ten
invasives (Ghisalberti, 2000).
Pollen viability data becomes important in context of both, species invasivity as well as habitat invasibility, because
it can provide important information on successful establishment of a given species in a habitat, thereby serving as
a measure of invasiveness
(
Jiang, Ma, Lin, & Ma, 2022). For this reason, production of seedless or sterile invasive
plants would be beneficial both to the nursery/horticultural industry as well as the environment
(
Beck-Pay, 2012). One
way of identifying sterile plants is to estimate viability across populations but as mentioned in earlier sections, this
is often time consuming on account of technique limitation. A study on 32 L. camara cultivars and breeding lines
revealed that stainability, the main parameter used to determine viability, is influenced by ploidy levels, indicating a
strong potential to develop genetically sterile cultivars
(
Czarnecki, Hershberger, Robacker, Clark, & Deng, 2014). Other
studies have also established that assessment of morphological and cytological differences among lantana varieties
can help in measuring invasive potential and suitability for commercial production and landscape use
(
Steppe, Wilson,
Deng, Druffel, & Knox, 2019). To date, the only method faster than vital staining appears to be in pollen imaging, but
this has not been addressed nor explored fully as described in the next section.
2.3 Imaging of pollen grains
Traditionally, palynologists have used compound light microscopy (LM) for pollen identification and interpretation
and scanning electron microscopy (SEM) for morphological comparisons and taxonomy
(
G. D. Jones & Bryant Jr,
2007). Doubtlessly, SEM offers far greater resolution, and has led to creation of new terminology for describing pollen
ornamentation, numerical approaches to pollen sculpturing and exine architectures
(
Skvarla, Rowley, & Vezey, 1989).
However, sample preparation and the time needed to count, analyze, photograph and print the micrographs, and the
consequent lack of scalability are the limiting factors in these approaches.
With the advancement of digital microscopy, palynology studies are becoming even less time-consuming and can
generate more reliable data for species taxonomy, apart from saving hours spent on manual counts of pollen grains
following the process of staining to differentiate between viable and inviable pollen
(
S. Mishra, Srivastava, et al., 2015)).
At the turn of the millenium, alternative digital methods for counting pollen were devised, but often without regard to
viable and invisible grains, and these protocols also had drawbacks of software specific to branded instruments.
(
Bechar
et al., 1997; Aronne, Cavuoto, & Eduardo, 2001).
3
摘要：

QUANTIFICATIONOFPOLLENVIABILITYINLantanacamaraBYDIGITALHOLOGRAPHICMICROSCOPYVipinKumarNationalInstituteofPlantGenomeResearch,NewDelhi110067Indiaenvironment.vip14@gmail.comNishantGoyalDepartmentofPhysicsIndianInstituteofTechnologyDelhiNewDelhi110016Indiaphz218060@iitd.ac.inAbhishekPrasadNationalIns...

展开>> 收起<<
QUANTIFICATION OF POLLEN VIABILITY IN Lantana camara BY DIGITAL HOLOGRAPHIC MICROSCOPY Vipin Kumar.pdf

共13页,预览3页

还剩页未读， 继续阅读

声明：本站为文档C2C交易模式，即用户上传的文档直接被用户下载，本站只是中间服务平台，本站所有文档下载所得的收益归上传人(含作者)所有。玖贝云文库仅提供信息存储空间，仅对用户上传内容的表现方式做保护处理，对上载内容本身不做任何修改或编辑。若文档所含内容侵犯了您的版权或隐私，请立即通知玖贝云文库，我们立即给予删除！

相关推荐

更多
立即下载
分类：图书资源 价格：10玖币 属性：13 页 大小：7.59MB 格式：PDF 时间：2025-05-02

开通VIP享超值会员特权

  • 多端同步记录
  • 高速下载文档
  • 免费文档工具
  • 分享文档赚钱
  • 每日登录抽奖
  • 优质衍生服务

作者详情

相关内容

更多

热门标签

人际关系 动力学连接体 力的合成 配电装置 高考理综 全宋诗作者索引 公务员考试
/ 13
评分 收藏
分享
立即下载
客服
关注