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of Plants
Manuscript Draft
Manuscript Number: FLORA-D-14-00066R1
Title: The duckweed Wolffia microscopica: a unique aquatic monocot
Article Type: Original Research
Keywords: Duckweed, Pseudoroot, Vegetative propagation, Generative propagation, Wolffia
microscopica
Corresponding Author: Dr. Sowjanya Sree Kandregula, Ph.D.
Corresponding Author's Institution: Amity University UP
First Author: Sowjanya Sree Kandregula, Ph.D.
Order of Authors: Sowjanya Sree Kandregula, Ph.D.; Satish C Maheshwari, Ph.D.; Karoly Boka, Ph.D.;
Jitendra P Khurana, Ph.D.; Aron Keresztes, Ph.D.; Klaus J Appenroth, Ph.D.
Abstract: The rediscovered species Wolffia microscopica (Griff.) Kurz, endemic to India, Pakistan and
Bangladesh, shows several features that make it unique in comparison to other duckweeds, even to
other species of the genus Wolffia. During vegetative propagation, the daughter fronds are produced by
budding within a single pouch of the mother frond. Several generations of fronds and their initials exist
in union with the parent frond at a given time. Most strikingly, almost all the fronds flower, both in
nature and under controlled culture conditions. Thus, in contrast to all other duckweed species,
generative propagation is as important as vegetative propagation, which opens the opportunity for
artificial breeding. Moreover, flower development on both mother and daughter fronds was observed
at the same time. In contrast to all other duckweed species, fronds of W. microscopica often possess a
ventral projection of varying length ranging from an almost flat appearance of the ventral surface to a
length of 4 mm. Absence of root cap, root hairs and vascular tissue demonstrate that this ventral
protrusion is not a root and accordingly we name this special structure "pseudoroot". The high number
of chloroplasts in the pseudoroot may result in higher capacity of photosynthesis without increasing
the frond area which covers the water surface. Thus we propose that the pseudoroot serves to be
advantageous to W. microscopica in multiplying at a faster rate in comparison to other duckweeds.
Response to Reviewers: Ms. Ref. No.: FLORA-D-14-00066
Title: The duckweed Wolffia microscopica: A unique monocotyledonous aquatic plant#
Flora - Morphology, Distribution, Functional Ecology of Plants
Dear Dr. Kandregula,
The reviewers have commented on your above paper, although one of them was very late in
responding. They indicated that it is not acceptable for publication in its present form. One of the
reviewers ranked your manuscript high and suggested some minor changes, but the another one
requested a substantial revision in your manuscript. If you feel that you can suitably address the
reviewers' comments (included below), I invite you to revise and resubmit your manuscript.
Please carefully address the issues raised in the comments.
If you are submitting a revised manuscript, please also:
a) outline each change made (point by point) as raised in the reviewer comments
AND/OR
b) provide a suitable rebuttal to each reviewer comment not addressed
To submit your revision, please do the following:
1. Go to: http://ees.elsevier.com/flora/
2. Enter your login details
3. Click [Author Login]
This takes you to the Author Main Menu.
4. Click [Submissions Needing Revision]
PLEASE NOTE: Flora - Morphology, Distribution, Functional Ecology of Plants would like to enrich its
relevant online articles by displaying interactive phylogenetic trees that allow the reader to
interactively explore the underlying research data. Hence, if applicable, we would like to invite you to
upload your phylogenetic tree data files in the Newick or NeXML format as supplementary material to
our online submission system. Elsevier will generate interactive phylogenetic trees from these files and
include them with the online article on SciVerse ScienceDirect. If you wish, you can submit .NEW/.NWK
(Newick) or .XML (NeXML) files along with your revised submission.
I look forward to receiving your revised manuscript.
Yours sincerely,
Shahin Zarre, PhD
Subject Editor
Flora - Morphology, Distribution, Functional Ecology of Plants
Reviewers' comments:
Reviewer #1:
Overall assessment:
This is an excellent manuscript. The morphological study, microscopic detail and cytological
description are to be commended. A modern detailed description of Wolffia microscopia and its
peculiarities is timely and will be of value to the expanding duckweed community. The text is clear and
well written.
Specific minor comments:
Lines 254-255: Were seeds per se seen? if so, what do they look like. please clarify.
Reply: Seeds were not investigated in the present study as the embryology of W. microscopica
including the seed structure and embryo development were described in detail by one of the coauthors of the present manuscript (Maheshwari, 1956). This paper has been cited now at an
appropriate context in the present ms.
Line 349: Correct the publication date to "2002".
Reply: It is done.
Lines 377-387: Include mention and possibly a brief discussion of the work of Rimon & Galun
"Morphogenesis of Wolffia microscopia: frond and flower development " Phytomorphology 18(3), 364372, 1969, which is germane at this point in the manuscript. Likewise, the paper by Seth et al. "Studies
on the growth and flowering of a short day plant, Wolffia microscopia. II. role of metal ions and
chelates", Planta 90: 349-359, 1970, should be alluded to with respect to the effect of Fe on growth and
flowering.
Reply: Many thanks for this suggestion. We have cited and discussed these two papers now.
Lines 408-409: Delete "with advantages over the recently sequenced duckweed species, Spirodela
polyrhiza". The basis for this statement has not been laid out in the paper and the reference to the
sequencing work is superfluous.
Reply: We agree and have deleted this part.
Highlights: Delete "Frequent flowering overcomes disadvantages of the sequenced Spirodela
polyrhiza". There is no basis laid for this statement in the paper.
Reply: The present rephrased highlight was suggested by Reviewer no. 2 and now it does not
emphasize on the comparison with sequenced species. I hope this is acceptable.
Recommendation:
I recommend that the manuscript be accepted for publication essentially as is, with only minor
modifications.
Reply: Many thanks for this recommendation
Reviewer #2: Dear authors,
Your manuscript comprises interesting results on the structural peculiarities of one duckweed species
not exactly examined yet. However, there are some serious problems leading me against
recommending it for publication in the present form. Although the species is among the rapidly
growing angiosperms, it could be more interesting when talk about the biology of this species. The
nature of the text is too descriptive and the amount of provided images too much. You should be able to
find a way for shortening the text without losing the interesting parts.
Reply: We revised the manuscript extensively and moved the previous Fig. 1 to the supplementary
material in order to reduce the number of figures in the manuscript and also took care to shorten the
text as appropriate. We hope we have made the changes in line with the criteria suggested.
Furthermore, the presence of "Pseudoroots" has been previously reported for a group of species
belonging to Wolffia, so, it is not your finding. Please try to formulate this properly in the text and
describe shortly the internal structure.
Reply: “Pseudoroots” do not exist in any other species of Wolffia except W. microscopica. It is true that
pseudoroot is a term which already exists in the scientific literature and is used to describe root-like
structures in many other plants but with respect to W. microscopica, the root-like structure was not
named before and hence we used the term “pseudoroot” for this special structure. We have now
rephrased the text in the ms to make our intention more clear.
You know that in some water ferns there is a frond showing positive geotropism as well as root-like
appearance. These could also be called pseudoroot. They seem to be functionally very
similar to the structures you describe in Wolffia microscopica, but of course, the reduced size of the
latter do not let bearing any vascular bundle. I suggest that you compare these root-like structures in
your duckweeds with Salvinia and indicate the parallel evolution of these structures and their possible
benefit to such aquatic plants. This could make your manuscript more attractive to the readers of Flora.
How is the diversity of root-like fronds in Salvinia or among the water ferns? Are there any
phylogenetic signals indicating the possible old origin of these structures in the groups bearing them?
Reply: In the present revised form of the text, we have mentioned about the presence of root-like
structures in other plants e.g, Salvinia and discussed it in comparison to the pseudoroot in W.
microscopica. The modification of the whole leaf into a root-like structure and its non-photosynthetic
nature in Salvinia rather restricted us from drawing any conclusion of its homology or analogy with the
pseudoroot of W. microscopica. Hence, an in-detail evolutionary account of the pseudoroot of Salvinia
and that of W. microscopica could not be included in the present ms.
The another suggestion is to turn down the importance of taxonomic part in the text. The detailed
history given on the nomenclature of this species is not relevant to the subject of your study and
among your goals. I indicated the part that should be omitted in the text.
Reply: We agree and have cancelled this part of the text.
I annotated the pdf of your text and will try to pass this modified version to you.
Reply: We are very grateful for all the suggestions made directly in the pdf-file. We have accepted
almost all of them with one exception. The reviewer suggested to state that Lemnaceae is synonym
under Araceae (APG III, 2009). Majority of the members of the duckweed community are keeping the
taxonomic level of duckweed as a family separated from Araceae, i. e. Lemnaceae. In the Editorial
Letter to a special issue of “Plant Biology” (to be published in January 2015), Appenroth K-J (one of the
co-authors of the present manuscript), Crawford D and Les D wrote: “It is worth mentioning that most
(but not all) of the contributions keep the term Lemnaceae considering them as a plant family in
contrast to a subfamily (Lemnoideae). Since Lemnaceae (or Lemnoideae) are nested in Araceae in
molecular phylogenetic studies (Cusimano et al. 2011, Nauheimer et al. 2012), whether or not the
duckweeds are recognized at the familial or subfamilial level depends on whether one accepts
paraphyletic groups. To circumvent this rather philosophical debate, one can separate the small group
of Protoaraceae together with the group of Lemnaceae from the “True Araceae” (as these two groups
are the most basal elements in this group) which results in three monophyletic plant families, i.e. True
Araceae, Lemnaceae and Protoaraceae. Arguments for keeping the term Lemnaceae instead of
Lemnoideae were summarized by Appenroth et al. (2013) after thorough discussion with Elias
Landolt.” Thus, we prefer also in this manuscript the term Lemnaceae.
Kind regards
K. Sowjanya Sree
Cover Letter
5th September, 2014
Dr. K. Sowjanya Sree
Principal Investigator
(SERB-Young Scientist Project)
AIMT, Amity University Uttar Pradesh
Noida, India
The Editor,
Prof. Dr. Shahin Zarre
Flora
Dear Prof. Zarre,
We are submitting a revised version of the manuscript (No. FLORA-D-14-00066)
entitled “The duckweed Wolffia microscopica: a unique aquatic monocot” by K.
Sowjanya Sree*, Satish C Maheshwari, Karoly Boka, Jitendra P Khurana, Aron
Keresztes and Klaus-J Appenroth for publication in the journal, Flora.
We have carefully considered all suggestions given by the two reviewers and
responded to it point by point which is attached as response to reviewers. We also
considered all remarks and corrections made by the second reviewer in the
annotated pdf version of the manuscript.
We hope that the manuscript in the present form would be acceptable for
publication in Flora.
Yours sincerely
(Dr. K. Sowjanya Sree)
*Highlights (for review)
Highlights:

Vegetative and generative propagation in the fast growing W. microscopica
coexist.

A unique structure, pseudoroot, might contribute to its fast multiplication.

Knowledge of morphology and anatomy will increase its potential use in
molecular and evolutionary studies as a model.

Frequent flowering overcomes disadvantages of other model duckweeds.
Detailed Response to Reviewers
Ms. Ref. No.: FLORA-D-14-00066
Title: The duckweed Wolffia microscopica: A unique monocotyledonous aquatic plant#
Flora - Morphology, Distribution, Functional Ecology of Plants
Dear Dr. Kandregula,
The reviewers have commented on your above paper, although one of them was very late in
responding. They indicated that it is not acceptable for publication in its present form. One of the
reviewers ranked your manuscript high and suggested some minor changes, but the another one
requested a substantial revision in your manuscript. If you feel that you can suitably address the
reviewers' comments (included below), I invite you to revise and resubmit your manuscript.
Please carefully address the issues raised in the comments.
If you are submitting a revised manuscript, please also:
a) outline each change made (point by point) as raised in the reviewer comments
AND/OR
b) provide a suitable rebuttal to each reviewer comment not addressed
To submit your revision, please do the following:
1. Go to: http://ees.elsevier.com/flora/
2. Enter your login details
3. Click [Author Login]
This takes you to the Author Main Menu.
4. Click [Submissions Needing Revision]
PLEASE NOTE: Flora - Morphology, Distribution, Functional Ecology of Plants would like to enrich its
relevant online articles by displaying interactive phylogenetic trees that allow the reader to
interactively explore the underlying research data. Hence, if applicable, we would like to invite you to
upload your phylogenetic tree data files in the Newick or NeXML format as supplementary material
to our online submission system. Elsevier will generate interactive phylogenetic trees from these files
and include them with the online article on SciVerse ScienceDirect. If you wish, you can submit
.NEW/.NWK (Newick) or .XML (NeXML) files along with your revised submission.
I look forward to receiving your revised manuscript.
Yours sincerely,
Shahin Zarre, PhD
Subject Editor
Flora - Morphology, Distribution, Functional Ecology of Plants
Reviewers' comments:
Reviewer #1:
Overall assessment:
This is an excellent manuscript. The morphological study, microscopic detail and cytological
description are to be commended. A modern detailed description of Wolffia microscopia and its
peculiarities is timely and will be of value to the expanding duckweed community. The text is clear
and well written.
Specific minor comments:
Lines 254-255: Were seeds per se seen? if so, what do they look like. please clarify.
Reply: Seeds were not investigated in the present study as the embryology of W. microscopica
including the seed structure and embryo development were described in detail by one of the coauthors of the present manuscript (Maheshwari, 1956). This paper has been cited now at an
appropriate context in the present ms.
Line 349: Correct the publication date to "2002".
Reply: It is done.
Lines 377-387: Include mention and possibly a brief discussion of the work of Rimon & Galun
"Morphogenesis of Wolffia microscopia: frond and flower development " Phytomorphology 18(3),
364-372, 1969, which is germane at this point in the manuscript. Likewise, the paper by Seth et al.
"Studies on the growth and flowering of a short day plant, Wolffia microscopia. II. role of metal ions
and chelates", Planta 90: 349-359, 1970, should be alluded to with respect to the effect of Fe on
growth and flowering.
Reply: Many thanks for this suggestion. We have cited and discussed these two papers now.
Lines 408-409: Delete "with advantages over the recently sequenced duckweed species, Spirodela
polyrhiza". The basis for this statement has not been laid out in the paper and the reference to the
sequencing work is superfluous.
Reply: We agree and have deleted this part.
Highlights: Delete "Frequent flowering overcomes disadvantages of the sequenced Spirodela
polyrhiza". There is no basis laid for this statement in the paper.
Reply: The present rephrased highlight was suggested by Reviewer no. 2 and now it does not
emphasize on the comparison with sequenced species. I hope this is acceptable.
Recommendation:
I recommend that the manuscript be accepted for publication essentially as is, with only minor
modifications.
Many thanks for this recommendation
Reviewer #2: Dear authors,
Your manuscript comprises interesting results on the structural peculiarities of one duckweed species
not exactly examined yet. However, there are some serious problems leading me against
recommending it for publication in the present form. Although the species is among the rapidly
growing angiosperms, it could be more interesting when talk about the biology of this species. The
nature of the text is too descriptive and the amount of provided images too much. You should be
able to find a way for shortening the text without losing the interesting parts.
Reply: We revised the manuscript extensively and moved the previous Fig. 1 to the supplementary
material in order to reduce the number of figures in the manuscript and also took care to shorten the
text as appropriate. We hope we have made the changes in line with the criteria suggested.
Furthermore, the presence of "Pseudoroots" has been previously reported for a group of species
belonging to Wolffia, so, it is not your finding. Please try to formulate this properly in the text and
describe shortly the internal structure.
Reply: “Pseudoroots” do not exist in any other species of Wolffia except W. microscopica. It is true
that pseudoroot is a term which already exists in the scientific literature and is used to describe rootlike structures in many other plants but with respect to W. microscopica, the root-like structure was
not named before and hence we used the term “pseudoroot” for this special structure. We have now
rephrased the text in the ms to make our intention more clear.
You know that in some water ferns there is a frond showing positive geotropism as well as root-like
appearance. These could also be called pseudoroot. They seem to be functionally very
similar to the structures you describe in Wolffia microscopica, but of course, the reduced size of the
latter do not let bearing any vascular bundle. I suggest that you compare these root-like structures in
your duckweeds with Salvinia and indicate the parallel evolution of these structures and their
possible benefit to such aquatic plants. This could make your manuscript more attractive to the
readers of Flora. How is the diversity of root-like fronds in Salvinia or among the water ferns? Are
there any phylogenetic signals indicating the possible old origin of these structures in the groups
bearing them?
Reply: In the present revised form of the text, we have mentioned about the presence of root-like
structures in other plants e.g, Salvinia and discussed it in comparison to the pseudoroot in W.
microscopica. The modification of the whole leaf into a root-like structure and its non-photosynthetic
nature in Salvinia rather restricted us from drawing any conclusion of its homology or analogy with
the pseudoroot of W. microscopica. Hence, an in-detail evolutionary account of the pseudoroot of
Salvinia and that of W. microscopica could not be included in the present ms.
The another suggestion is to turn down the importance of taxonomic part in the text. The detailed
history given on the nomenclature of this species is not relevant to the subject of your study and
among your goals. I indicated the part that should be omitted in the text.
Reply: We agree and have cancelled this part of the text.
I annotated the pdf of your text and will try to pass this modified version to you.
Reply: We are very grateful for all the suggestions made directly in the pdf-file. We have accepted
almost all of them with one exception. The reviewer suggested to state that Lemnaceae is synonym
under Araceae (APG III, 2009). Majority of the members of the duckweed community are keeping the
taxonomic level of duckweed as a family separated from Araceae, i. e. Lemnaceae. In the Editorial
Letter to a special issue of “Plant Biology” (to be published in January 2015), Appenroth K-J (one of
the co-authors of the present manuscript), Crawford D and Les D wrote: “It is worth mentioning
that most (but not all) of the contributions keep the term Lemnaceae considering them as a
plant family in contrast to a subfamily (Lemnoideae). Since Lemnaceae (or Lemnoideae) are
nested in Araceae in molecular phylogenetic studies (Cusimano et al. 2011, Nauheimer et al.
2012), whether or not the duckweeds are recognized at the familial or subfamilial level
depends on whether one accepts paraphyletic groups. To circumvent this rather
philosophical debate, one can separate the small group of Protoaraceae together with the
group of Lemnaceae from the “True Araceae” (as these two groups are the most basal
elements in this group) which results in three monophyletic plant families, i.e. True Araceae,
Lemnaceae and Protoaraceae. Arguments for keeping the term Lemnaceae instead of
Lemnoideae were summarized by Appenroth et al. (2013) after thorough discussion with
Elias Landolt.” Thus, we prefer also in this manuscript the term Lemnaceae.
Kind regards
K. Sowjanya Sree
*Manuscript
1
The duckweed Wolffia microscopica: a unique aquatic monocot#
2
K. Sowjanya Sreea*, Satish C Maheshwarib, Karoly Bokac, Jitendra P Khuranad, Aron
3
Keresztesc, Klaus-J Appenrothe
4
5
a
6
201303, India; E-mail: [email protected]
7
b
8
Jaipur - 302004, India; E-mail: [email protected]
9
c
Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida-
Biotechnology Institute, Centre for Converging Technologies, University of Rajasthan,
Department of Plant Anatomy, Eötvös Loránd University, H-1117 Budapest, Hungary;
10
E-mails: [email protected] (KB), [email protected] (AK)
11
d
12
New Delhi- 110021, India; E-mail: [email protected]
13
e
14
159, D-07743 Jena, Germany; E-mail: [email protected]
15
*Corresponding author. Tel: +919999672921
16
Running head: The unique duckweed, Wolffia microscopica
17
#
18
2013)
Department of Plant Molecular Biology, University of Delhi South Campus,
Institute of General Botany and Plant Physiology, University of Jena, Dornburger Str.
Rediscovery of Wolffia microscopica: A tribute to Late Prof. Dr. Elias Landolt (1926-
19
1
20
ABSTRACT
21
The rediscovered species Wolffia microscopica (Griff.) Kurz, endemic to India, Pakistan
22
and Bangladesh, shows several features that make it unique in comparison to other
23
duckweeds, even to other species of the genus Wolffia. During vegetative propagation,
24
the daughter fronds are produced by budding within a single pouch of the mother frond.
25
Several generations of fronds and their initials exist in union with the parent frond at a
26
given time. Most strikingly, almost all the fronds flower, both in nature and under
27
controlled culture conditions. Thus, in contrast to all other duckweed species, generative
28
propagation is as important as vegetative propagation, which opens the opportunity for
29
artificial breeding. Moreover, flower development on both mother and daughter fronds
30
was observed at the same time. In contrast to all other duckweed species, fronds of W.
31
microscopica often possess a ventral projection of varying length ranging from an
32
almost flat appearance of the ventral surface to a length of 4 mm. Absence of root cap,
33
root hairs and vascular tissue demonstrate that this ventral protrusion is not a root and
34
accordingly we name this special structure “pseudoroot”. The high number of
35
chloroplasts in the pseudoroot may result in higher capacity of photosynthesis without
36
increasing the frond area which covers the water surface. Thus we propose that the
37
pseudoroot serves to be advantageous to W. microscopica in multiplying at a faster rate
38
in comparison to other duckweeds.
39
Keywords: Duckweed, Pseudoroot, Vegetative propagation, Generative propagation,
40
Wolffia microscopica
41
2
42
1 Introduction
43
44
Duckweeds represent a family of free floating monocots inhabiting the lentic
45
ecosystems distributed in almost all the continents except the Antarctic and Arctic
46
regions. Duckweeds, classified into 5 genera and 37 species belonging to the family
47
Lemnaceae (Appenroth et al., 2013; Landolt, 1986; Les et al., 2002), collected from all
48
over the world are maintained at Duckweed Stock Collection Centres situated in Zurich,
49
Switzerland; Jena, Germany; Rutgers, New Brunswick, USA and Chengdu, China. In
50
the year 2009, it was realized that the only existing clone (Clone no. 9276) of Wolffia
51
microscopica (Griff.) Kurz was lost in all the stock collections.
52
The duckweed, W. microscopica, is reported to be sporadically distributed and
53
endemic to the Indian subcontinent (Landolt, 1986). Several efforts were made since
54
2009 to collect this species. All the locations in North India and Bangladesh from where
55
this species was collected in the past (Aziz, 2001; Landolt, 1986) were screened without
56
any success until 2013. As already mentioned by Hegelmaier (1885), this species could
57
not be found for decades at places where it was found earlier. However, during our field
58
trips in the 2013 monsoon season to Gujarat, India and Bangladesh, W. microscopica
59
was rediscovered from the lakes at Patan, Ambapur, Sughad and Vadasma in Gujarat,
60
India and from that at Jessore, Bangladesh (Sree and Appenroth, 2014).
61
Originally, W. microscopica was named Grantia microscopica (Griff. ex Voigt)
62
(Griffith, 1851 a, b; Voigt, 1845) but was later identified as a member of the genus
63
Wolffia Horkel (Kurz, 1866). This plant was mainly investigated by Hegelmaier in
3
64
Germany (Hegelmaier, 1868; 1885) and by Maheshwari and his group in India (Khurana
65
and Maheshwari, 1983; Khurana et al., 1986; Maheshwari, 1956; 1958; Maheshwari
66
and Chauhan, 1963; Seth et al., 1970; Venkataraman, 1968).
67
Three unique features distinct from other duckweeds, even from other species of
68
Wolffia, make W. microscopica fascinating: 1) fastest growth amongst all angiosperms
69
(Venkataraman et al., 1970; cf. Ziegler et al., 2014) making this species a top candidate
70
for the production of biomass for energy production, animal feed or even for human
71
consumption (Anderson et al., 2011; Hillman and Culley, 1978; Xu et al., 2012); 2)
72
unusual frequent flowering in this species that opens the opportunity for artificial
73
breeding; 3) striking morphology of this species, specially the presence of a ventral
74
projection (Maheshwari and Chauhan, 1963).
75
In the present report, we describe in detail its unique morphology throughout the
76
vegetative and generative propagation cycles using light and electron microscopy. The
77
following questions were addressed: 1. What is the structural basis of the very high rate
78
of vegetative propagation? 2. How are vegetative and generative propagations related
79
to each other? 3. What is the importance of the unique downward-directed protrusion of
80
the fronds, and can this structure be considered a root?
81
82
2 Materials and Methods
83
2.1 Collection of W. microscopica
4
84
The species W. microscopica was rediscovered by two of the authors of the
85
present paper (KSS and KJA) from the lakes at Vadasma (23o 23‟ 56‟‟ N, 72o 32‟ 45‟‟ E;
86
23o 24‟01‟‟ N, 72o 32‟39‟‟E), Ambapur (23o 08‟55‟‟ N, 72o 36‟25‟‟ E) (supplementary
87
material, Fig. S1a), Sughad (23o 08‟08‟‟ N, 72o 37‟44‟‟ E) and Patan (23o 53‟03‟‟ N, 72o
88
09‟28‟‟ E) in Gujarat, India and also from a lake at Jessore, Bangladesh during the
89
monsoon season of the year 2013.
90
91
92
2.2 Culture of W. microscopica under controlled conditions
The morphological identity of all the collected clones was confirmed by the
93
authors, KSS and KJA, and the clones were registered as suggested recently
94
(International Steering Committee on Duckweed Research and Application, 2013; Zhao
95
et al. 2012). The clones were sterilized using 2.5 % sodium hypochlorite solution for 2-3
96
min and cultivated under axenic conditions in Steinberg medium (Naumann et al.,
97
2007). Plants were maintained under a daily photoperiodic cycle of 14 h white light
98
(100 µmol m-2 s-1) /10 h dark, at 25 ± 1oC.
99
100
101
2.3 Light microscopy
Whole plants at different developmental stages were fixed in 2.5%
102
glutaraldehyde in 5 mM phosphate buffer for 3 h. After thorough rinsing with the same
103
buffer, the samples were post fixed in 1 % osmium tetroxide for 2 h, rinsed with the
104
buffer and dehydrated in graded ethanol series. The samples were then treated with
5
105
absolute acetone and embedded in Spurr resin (Sigma-Aldrich Inc., St. Louis, USA)
106
after careful infiltration with it. Semi-thin sections, 1 µm thick, were prepared with
107
Microm HM 360 microtome (Microm International GmbH, Walldorf, Germany) using a
108
glass knife. These sections were then stained with toluidine blue, observed and
109
photographed by Nikon Eclipse 80i microscope (Nikon Instruments Inc., Tokyo,
110
Japan).The general morphological overview of W. microscopica plants at different
111
stages of life cycle was captured using a light microscope (Nikon SMZ1500
112
stereomicroscope with a Nikon Digital Sight DS-SMc camera using Nikon NIS-Elements
113
AR 3.0x software).
114
115
116
2.4 Scanning Electron Microscopy
The whole plants fixed in 2.5% glutaraldehyde in 5 mM phosphate buffer for 3 h
117
were rinsed with the buffer before fixing in 1% osmium tetroxide for 2 h. The samples
118
were rinsed again, dehydrated in graded ethanol series and transferred into amyl
119
acetate. The critical point drying was done using carbon dioxide. These samples were
120
then gold coated and viewed under Hitachi 2460N scanning electron microscope
121
(Hitachi Ltd, Tokyo, Japan) with 20 kV accelerating voltage using backscattered
122
electrons or secondary electrons depending on the features of interest to be observed in
123
the given samples.
124
125
3 Results
6
126
127
3.1 Habitat dominance in monsoon season
Like all other duckweed species, W. microscopica is a free floating angiosperm
128
inhabiting lakes, ponds and ditches. During the recent rediscovery of this species in
129
2013 (Sree and Appenroth, 2014), it was noted that in majority of the cases, W.
130
microscopica existed as a single species duckweed community (Supplementary
131
material, Fig. S1a) except in one case, i.e. at Vadasma, Gujarat where the territory of
132
W. microscopica was shared by its relative, Lemna aequinoctialis. In the seasons before
133
monsoon, this plant species could not be detected during several excursions in the
134
years 2010, 2011 and 2012. During our visit to the same lakes at Vadasma, Sughad
135
and Patan, Gujarat in the summer season of 2014 (June) we found them to be
136
completely dried out, consequently no duckweed could be found. However, these lakes
137
were flourishing with W. microscopica in the monsoon season of 2014 (August) similar
138
to the year before.
139
140
3.2 General structure of the fronds
141
The fronds of W. microscopica are light green in colour, 0.4–1.0 mm in length
142
and 0.3–0.8 mm in width. The frond is dorso-ventrally differentiated into a flat dorsal
143
surface and a ventral projection of varying length. Flowers appear on the dorsal surface
144
(supplementary material, Fig. S1b). A closer morphological appearance of the fronds is
145
depicted in figures 1a-c. The dorsal surface is furnished with two to three rows of
146
stomata, arranged towards the periphery and concentrated more on the posterior end of
147
the frond (Figs. 1d, f). Hardly any stomata were observed just above and anterior to the
7
148
node (Fig. 1d). In these actinocytic type of stomata, the subsidiary cells, 6-8 in number,
149
are arranged in a rosette arrangement around the guard cells and do not show
150
significant difference from the surrounding epidermal cells (Fig. 1e). The frond has an
151
irregular polygonal shape with 8-10 unequal sides stretched over the antero-posterior
152
axis (Fig. 1d) and is typically marked by a papilla at each corner (Fig. 1f). The flat dorsal
153
surface also is marked by irregularly spaced papillate cells (Fig. 1g). In general, a big
154
papilla is formed on the dorsal surface just above the vegetative pouch.
155
3.3 A mysterious structure: Pseudoroot
156
The ventral surface is characterized by the presence of a positively geotropic
157
protrusion which has not been strictly designated till now and we call it pseudoroot
158
(hence placed taxonomically under the section: Pseudorrhizae by Landolt; Landolt,
159
1986) (Figs. 1a-c). In the mature fronds, the pseudoroot is of varying length depending
160
on the culture conditions with a maximum length of 4 mm (Figs. 1a-c) and is positioned
161
slightly more towards the anterior side of the frond (Fig. 1c). It is devoid of root hairs and
162
root cap (Figs. 1c, h). It lacks stomata similar to the rest of the ventral surface (Figs. 1h,
163
i). In contrast to other places of collection, the fronds collected from the lakes at Patan
164
and Ambapur were almost without any pseudoroot (Fig. 1j) on the ventral side giving
165
them a lenticular appearance. When cultured in the laboratory in Steinberg medium,
166
these fronds started to develop pseudoroot within 24 h and grew longer over time
167
depending on the culture conditions similar to the ones shown in figures 1a-c.
168
169
3.4 Structural basis of fast asexual multiplication: vegetative pouch
8
170
The anterior end of the frond possesses a single near triangular vegetative
171
pouch, wide towards the opening and converging to the interior of the frond (Figs. 2a-c).
172
The daughter fronds are produced by vegetative budding in this special sac and are
173
held inside with the help of a stipe which connects them to the mother frond (Figs. 2b,
174
c). The stipe is continuous with the ventral epidermis of the daughter frond and as the
175
daughter frond grows the cells of the stipe elongate over their antero-posterior axes
176
(Fig. 2b). The daughter fronds are released (Figs. 1d, i) through the opening of the
177
vegetative pouch after detachment from the stipe. The remnants of the stipe are left
178
inside the pouch (Fig. 2a). Just below the opening of the pouch on the ventral surface,
179
the frond retains a prominent scar at a position where the connection to the stipe from
180
its mother existed before (Fig. 1i). It is also evident as a rupture on the ventral side of
181
the frond as depicted in Fig. 2c.
182
The meristematic zone which gives rise to daughter fronds is formed at the
183
central lower portion of the vegetative pouch. A diagrammatic representation of the
184
sequential development of primordia is shown in Fig. 2d. The primordium of a daughter
185
frond develops when the parent frond itself consists of an axial row of two to three cells
186
covered by a single layer of protodermal cells (Fig. 2d(i)). On the dorsal surface of this
187
group of undifferentiated cells, a protuberance is formed as a result of enlargement and
188
subsequent divisions of one of the proximal axial cells (Fig. 2d(ii)). At the same time, a
189
periclinal division takes place in one of the distal epidermal cells (Fig. 2d(iii)). The outer
190
cell continues to be a part of the epidermal layer whereas the inner derivative is
191
incorporated into the axial tissue. Further divisions of the primordial cells consisting of
192
axial cells and the inner derivative of the periclinal division allow the frond to grow
9
193
rapidly. The daughter frond that is initiated as a protuberance at almost right angles to
194
the parent frond (Fig. 2d(ii)) tends to face exactly opposite to its parent as it continues to
195
grow further as shown in figures 2b-2c. For instance, the orientation of frond IIb is
196
exactly opposite to its daughter IIIb (Fig. 2b). In the meantime, the daughter frond
197
produces a granddaughter frond at its proximal end (Figs. 2b, c). The fast vegetative
198
multiplication of fronds leads to the presence of at least four-five generations (up to ten
199
plantlets; Figs. 2b, c) all of them still united with the parent frond, in what is visible
200
externally as a single daughter frond attached to a mother frond; this creates spatial
201
problems to the younger sisters of the daughter frond pushing them to the interior of the
202
pouch (Figs. 2b, c). As soon as the eldest daughter (IIa) detaches from the parent (I), a
203
shift in the orientation of the sisters takes place and the next older daughter (IIb) takes
204
its position to the opening of the vegetative pouch of the mother frond (Figs. 2b, c). Over
205
time, the youngest daughter (IId) also occupies the same position as the eldest
206
daughter (IIa) at present from where it will be released to the exterior. Hence during the
207
course of development of a daughter frond starting from a primordium to its release
208
from the vegetative pouch, it undergoes a 180o shift in its axis (Figs. 2b, c).
209
210
3.5 Generative propagation: a common mode of multiplication in W. microscopica
211
In a flowering frond, a distinct elliptical furrow was observed on the dorsal surface
212
exposing the floral organs (Fig. 3), usually to one side of the median antero-posterior
213
axis. As an initial step of flowering, primordia of stamen and pistil (which might
214
represent a reduced form of male and female flowers, respectively, of an inflorescence)
10
215
develop at the posterior and anterior side, respectively, in the floral cavity formed at an
216
eccentric position. Developing primordia inside the frond give rise to the appearance of
217
mounds on the dorsal surface. A furrow on the dorsal surface which exposes these
218
immature floral organs (Figs. 3a, b) soon develops into a circular rim-like protuberance
219
which grows over the developing floral organs arching them completely and covering
220
the original opening (Fig. 3c). The stigma of the female floral organ which matures first
221
pushes apart the marginal flaps of the furrow and widens it (Figs. 3d, e) emerging to the
222
exterior (Fig. 4a), the stamen soon takes over and the anther lobes become prominent
223
on the dorsal surface of a flowering frond (Figs. 4b- d). The stigma of a mature female
224
floral organ produces an exudate which is placed like a droplet on its funnel shaped
225
upper surface (Fig. 4e). Upon maturity, the bi-lobed anther dehisces (Figs. 4d, f)
226
releasing the pollen grains (Fig. 4g) which are attracted by the stigma. A group of W.
227
microscopica fronds in a flowering state is a very frequent view (Fig. 4d). The formation
228
of organs of perenation, turions, could not be observed in this species, neither in nature
229
nor during cultivation under various controlled conditions.
230
231
232
3.6 Anatomy of frond
The epidermis on the dorsal side is interrupted by stomata and is feebly cutinised
233
(Fig. 5a). These epidermal cells are polygonal in an aerial view (Figs. 1e, f) but are
234
almost rectangular in cross section (Fig. 5a). The upper epidermis almost completely
235
lacks chloroplasts which are otherwise prominent in the lower epidermis and in the
236
ventral parenchymatic cell layers (Fig. 5a). The cells of the growing pseudoroot depict
11
237
high number of chloroplasts (Fig. 5a). On the dorsal surface, below the stomata are the
238
prominent sub-stomatal air spaces (Figs. 5a, b). The frond is multilayered at the centre
239
composed of loosely arranged parenchymatous cells interspersed with air spaces
240
towards the dorsal side and of more tightly packed parenchymatous cells with small air
241
spaces towards the ventral surface (Fig. 5a). It tapers to a one cell thick outer rim (Fig.
242
5a). The cells of the growing pseudoroot are also housed with considerable amounts of
243
starch grains in their plastids (Fig. 5a). It is tempting to speculate that these may
244
function as statoliths for the positive geotropism in the growing phase of the pseudoroot.
245
There is no hint of any vascular tissue differentiation (Fig. 5a).
246
The meristematic initial of the pseudoroot starts appearing when the frond is very
247
young and still connected with its mother (Fig. 5b). These well-staining small cells seem
248
to be in a tunica-corpus like arrangement characteristic of the shoot apical meristem,
249
with the differences that: 1) no lateral primordia appear, 2) no vascular tissues develop,
250
and 3) the pseudoroot shows a positive geotropism. Subsequently, this apical meristem
251
differentiates almost fully into epidermis and parenchyma, so only a layer consisting of a
252
few cells remains (Figs. 5a, c). This may terminate the growth of the pseudoroot. Apart
253
from the tip, the pseudoroot consists of 6-10 parenchymatous cell layers without any
254
specialization (Fig. 5c). The epidermis of the pseudoroot is in continuation with that of
255
the floating flattened part of the frond and the cells constituting the inner layers are also
256
in continuation with the corresponding inner regions (Fig. 5a). The cells close to the
257
flattened part are elongated (Fig. 1i) and the ones to the tip are much smaller and
258
roundish (Fig. 1h, 5c).
259
12
260
261
3.7 Organization of the two reproductive regions
The fronds can become fertile at a very young stage, when they are still attached
262
to their mother (Fig. 6a). Often both the mother and the daughter fronds are in a
263
flowering state (Fig. 4d) and continue to multiply vegetatively (Figs. 6a, b). The
264
epidermis of the frond is continuous with the roof and the floor of the vegetative pouch
265
(Figs. 6a, b). The vegetative pouch and the floral cavity are separated by a strip of cells
266
which constitute the nodal region of the frond (Figs. 6a, b). These two reproductive
267
regions are not on the same plane when seen from the antero-posterior axis. While the
268
vegetative pouch is on the median antero-posterior axis, the floral cavity and the dorsal
269
furrow (Fig. 6c, arrow) are more on to one side of this median axis (Figs. 6a, b).
270
271
4 Discussion
272
273
The fast growing nature of this species (Venkataraman et al., 1970), fastest of all
274
angiosperms (Ziegler et al., 2014), explains its existence as a monoculture. A similar
275
observation was made decades back by Maheshwari (1958); apparently, other
276
duckweed species are not able to compete with it. Although rare in nature, mixed
277
community of W. microscopica and Lemna aequinoctialis seems to be an exception as
278
observed in a lake at Vadasma, Gujarat, India. The investigated structure gives some
279
hints to understand the fast growth of W. microscopica as well as its strong competitive
280
power. The floating, flat structure of the frond is well adapted for photosynthesis. The
13
281
high number of stomata and the sub-stomatal air spaces beneath maintain an efficient
282
system of aeration. The presence of papillae at the rim of the polygonal frond might help
283
it to cut the surface tension of water and float steadily. The high level of reduction in the
284
whole plant size and structure and absence of higher order of tissue differentiation
285
might be a part of its high growth rate strategy.
286
The presence of pseudoroot, which does not exist in any other member of Wolffia
287
(Landolt, 1986; Maheshwari, 1956) and not even in any other species of Lemnaceae,
288
deserves attention. For this reason, it also has diagnostic value. However, sometimes
289
the fronds are visually devoid of a pseudoroot and it becomes extremely difficult to
290
identify this species in its almost flat, slightly convex form, as it was during our recent
291
rediscovery of this plant species from the lakes at Ambapur and Patan in Gujarat, India.
292
Although Hegelmaier called the pseudoroot a rhizoid and initially assumed it to be a
293
remnant of the stipe as in some species of Wolffiella (Hegelmaier, 1885), he himself
294
withdrew this notion (Hegelmaier, 1885). Our study clearly shows that a scar from
295
detachment of the stipe is present just below the vegetative pouch on the ventral side
296
as observed in other Wolffia species (Bernard et al., 1990; Lemon and Posluszny, 2000)
297
further proving that pseudoroot is not a remnant of the stipe. It is also evident from our
298
morphological and anatomical investigations that this structure is not a root: absence of
299
the typical features like root cap, root hairs and also the vascular tissue. Moreover,
300
during earlier studies on W. microscopica, no root initial or primordia could be observed
301
during embryo development (Maheshwari, 1956). However, its positioning is exactly at
302
the node, a point between the floral cavity and the vegetative pouch and it develops
303
from a mixture of exo- and endo-genously derived cells. Therefore, we use the term
14
304
pseudoroot. Such structures which are not roots but appear to be root-like are also
305
observed in some aquatic ferns, e.g. Salvinia spp., where a leaf is modified into a root-
306
like structure. However, unlike the pseudoroot of W. microscopica, the root-like structure
307
in Salvinia is non-photosynthetic (Cook, 1996). One may speculate that pseudoroot is a
308
structure formed during the time course of the evolution of Wolffia from Lemna. From a
309
phylogenetic standpoint it may be interpreted that W. microscopica acts as a link
310
between the two genera. A certain clue may be obtained from molecular systematics i.e.
311
from the Neighbour Joining tree based on the rpl 16 plastidic spacer sequences (Bog et
312
al., 2013). W. microscopica forms one of the basal groups of this tree and is placed on
313
the bottom branch of the tree in comparison to nine other species of Wolffia. However,
314
this notion needs further investigations as only one clone of W. microscopica (Clone no.
315
9276) was available for that study. A realignment using an out-group and all the clones
316
collected in the year 2013 might throw new insights into this investigation.
317
The exact function of this simple undifferentiated pseudoroot is yet another
318
mystery. As whole of the ventral surface of the frond can take up water and nutrients
319
from the aquatic environment (Cedergreen and Madson, 2002), it becomes difficult to
320
ascribe a specific function to the pseudoroot in uptake of nutrients and water. We
321
assumed that at the time of flowering, a pseudoroot is developed in the downward
322
direction in order to maintain the buoyancy which could have been disturbed by opening
323
of the floral cavity to the exterior and perhaps also due to the weight of developing floral
324
organs. In contrast to our assumption, the present study clearly shows that the
325
presence of a prominent pseudoroot is not in strong correlation with the flowering status
326
of the frond as the fronds having varying lengths of pseudoroot could develop floral
15
327
primordia and flowers. Also, the non-flowering fronds developed a pseudoroot of
328
considerable length. So, its role in stabilising a flowering frond is also equivocal.
329
However, we observed that the almost flat forms of W. microscopica which were
330
collected from the lakes at Ambapur and Patan in Gujarat, India developed pseudoroot
331
when cultured under optimal growth conditions. Hence, under favourable conditions the
332
pseudoroot formation results in enhanced number of cells available for photosynthesis.
333
We suggest that formation of the pseudoroot is connected with enhanced
334
photosynthetic capacity. It should be stressed that this happens without increasing the
335
frond area which covers the water surface. This serves to be advantageous to W.
336
microscopica in multiplying at a faster rate in comparison to other duckweeds.
337
The vegetative life cycle is known for all 37 presently defined duckweed species
338
(Appenroth et al., 2013; Landolt, 1986). In W. microscopica it was observed that
339
although the origin of daughter frond is mainly endogenous, a small part of it, formed
340
from the inner periclinal derivative of the apical epidermal cell, is exogenous but this can
341
be detected only at the very initial stages of primordial development. Flowering has
342
been reported in several species of duckweeds including species of Wolffia (Bernard et
343
al., 1990; Khurana et al., 1986; Venkataraman, 1968; White and Wise, 1998) but in
344
general it is very rare (Landolt, 1986; Landolt and Kandeler, 1987; Pieterse, 2013). In
345
W. microscopica, however, flowering is very common representing an exception in the
346
whole duckweed family (Maheshwari and Chauhan, 1963; Venkataraman et al., 1970;
347
Rimon and Galun, 1969). Rimon and Galun reported that flowers are induced by lower
348
temperature (22oC) and short-day conditions (12 h light period) whereas higher
349
temperature and longer light-periods prevent flowering. Iron seems to play an important
16
350
role in flower induction, especially in connection with a suitable chelator (Maheshwari
351
and Chauhan, 1963; Seth et al., 1970; Venkataraman et al., 1970). The use of Fe-
352
EDDHA even induced flowering under otherwise non-inductive long-day conditions
353
(Seth et al., 1970). Likewise, salicylic acid also induced profuse flowering in W.
354
microscopica (Khurana and Maheshwari, 1983). All these effects await molecular
355
analyses.
356
Apart from the vegetative propagation of non-flowering fronds, we observed
357
vegetative propagation of flowering fronds both in nature and even more frequently
358
under in vitro conditions. More surprisingly, sometimes both the mother and daughter
359
fronds were found in advanced stages of flowering. The present morphological studies
360
show that the two life cycles of W. microscopica, generative and vegetative modes, co-
361
exist in time, which may not be the case with other rarely flowering species of Wolffia.
362
However, these two processes are spatially separated occupying the floral cavity and
363
the vegetative pouch, respectively. This spatial separation is like in other species of
364
Wolffia. But in the species of Spirodela, Landoltia and Lemna, which harbour two
365
pouches for vegetative multiplication, flowers invariably originate in only one of the two
366
pouches and during flowering the vegetative mode of multiplication in that pouch is
367
halted (Landolt, 1986; Lemon and Posluszny, 2000). In rare instances, however, flowers
368
may develop in both the pouches in some of these species (Khurana, 1982).
369
The genetic variations in the fronds of one clone of duckweed under controlled
370
conditions are close to absent because in the laboratory a clone is developed from one
371
single frond. However, in a lake there is not just one but are several clones (Bog and
372
Appenroth; data unpublished). So some genetic variations can be expected amongst
17
373
the fronds collected from the same pond in spite of their dominant vegetative
374
propagation. In the frequently flowering fronds of W. microscopica, protogyny and cross
375
pollination (Maheshwari, 1954) might even enhance the extent of the genetic variations
376
amongst the fronds of the same pond population. As a consequence, W. microscopica
377
could be further exploited for studies on crossing between different clones or mutants.
378
Besides ensuring higher genetic variations, sexual propagation has the
379
advantage that the resulting seeds may help the population to overcome periods of
380
drought. The production of seeds in W. microscopica has already been reported and its
381
embryology including endosperm, seed structure and embryo development was detailed
382
by Maheshwari (1956). As observed during our field trips, W. microscopica was found in
383
the monsoon season in lakes which dry out during the rest of the year. Occurrence of a
384
new population of W. microscopica in the same lakes in the following monsoon season
385
indicates that this species survives the dry period with the help of seeds.
386
The high level of reduction, high vegetative multiplication rate and the co-
387
existence of vegetative and generative propagation modes in this species make it a
388
unique duckweed with advantages over other duckweed species. Further physiological
389
and molecular studies on this species of duckweed would also lead to deeper insights
390
into its evolutionary relationship amongst angiosperms, and also understanding what
391
the minimal requirements of a higher plant are for regulating its vegetative growth and
392
reproductive development.
393
394
5 Acknowledgements
18
395
We acknowledge the support of Dr. Ajay Gor and his colleagues from Kadi Science
396
College, Dr. P.K. Patel from PG College, Godhra, in the collection of W. microscopica
397
from Gujarat and that of Dr. A. Aziz from Dhaka University, Bangladesh. We
398
acknowledge the support of Joerg Fuchs, IPK Gatersleben, in making some of the light
399
micrographs.
400
401
6 References
402
403
Anderson, K.E., Lowman, Z., Stomp, A.M., Chang, J., 2011. Duckweed as a feed
404
ingredient in laying hen diets and its effect on egg production and composition. Int. J.
405
Poultry Sci. 10, 4-7.
406
Appenroth, K.-J., Borisjuk, N., Lam, E., 2013. Telling duckweed apart: Genotyping
407
technologies for Lemnaceae. Chin. J. Appl. Environ. Biol.19, 1-10.
408
Aziz, A., 2001. Illustrated account of Wolffia microscopica (Griff.) Kurz (Lemnaceae)
409
from Sirajganj, Bangladesh. Bangladesh J. Bot. 30, 149-151.
410
Bernard, F.A., Bernard, J.M., Denny, P., 1990. Flower structure, anatomy and life
411
history of Wolffia australiana (Benth.) den Hartog & Van der Plas. Bull. Torrey Bot. Club.
412
117, 18-26.
413
Bog, M., Schneider, P., Hellwig, F., Sachse, S., Kochieva, E.Z., Martyrosian, E.,
414
Landolt, E., Appenroth, K-J., 2013. Genetic characterization and barcoding of taxa in
19
415
the genus Wolffia Horkel ex Schleid. (Lemnaceae) as revealed by two plastidic markers
416
and amplified fragment length polymorphism (AFLP). Planta 237, 1–13.
417
Cedergreen, N., Madsen, T.V., 2002. Nitrogen uptake by the floating macrophyte
418
Lemna minor. New Phytol. 155, 285–292.
419
Cook, C.D.K., 1996. Aquatic and wetland plants of India. Oxford University Press, Delhi,
420
pp. 29-30.
421
Griffith, W., 1851a. Notulae ad plantas asiaticas, Vol. 3, Bishop college press, Calcutta,
422
India, pp. 216-229.
423
Griffith, W., 1851b. Icones plantarum asiaticarum, Vol. 3, Bishop college press,
424
Calcutta, India, p. 264.
425
Hegelmaier, F., 1868. Die Lemnaceen. Eine monographische Untersuchung,
426
Engelmann, Leipzig, Germany, p. 169.
427
Hegelmaier, F., 1885. Wolffia microscopica. Botanische Zeitung 43, 241-249.
428
Hillman, W.S., Culley, D.D. Jr., 1978. The uses of duckweed. Am. Scientist 66, 442-451.
429
International Steering committee on Duckweed Research and Application, 2013.
430
NewsLetter No. 1:
431
http://www.internationallemnaassociation.org/uploads/Intl_Duckweed_Committee_Letter
432
_No._1.pdf.
433
Khurana, J. P., 1982. In vitro control of flowering in duckweeds. Ph.D. Thesis, University
434
of Delhi, Delhi.
20
435
Khurana, J.P., Maheshwari, S.C., 1983. Floral induction in Wolffia microscopica by
436
salicylic acid and related compounds under non-inductive long days. Plant Cell Physiol.
437
24, 907-912.
438
Khurana, J.P., Tamot, B.K., Maheshwari, S.C., 1986. Induction of flowering in a
439
duckweed, Wolffia microscopica, under non-inductive long days, by 8-hydroxyquinoline.
440
Plant Cell Physiol. 27, 373-376.
441
Kurz, S., 1866. Enumeration of Indian Lemnaceae. J. Linn. Soc. Bot. 9, 264-268.
442
Landolt, E., 1986. The family of Lemnaceae- A monographic study. Veroeffentlichungen
443
des Geobotanischen Institutes ETH, Vol. 1, Stiftung Ruebel, Zurich, Switzerland.
444
Landolt, E., Kandeler, R., 1987. The family of Lemnaceae- A monographic study.
445
Veroeffentlichungen des Geobotanischen Institutes ETH, Vol. 2, Stiftung Ruebel,
446
Zurich, Switzerland.
447
Lemon, G.D., Posluszny, U., 2000. Comparative shoot development and evolution in
448
the Lemnaceae. Int. J. Plant Sci. 161, 733-748.
449
Les, D.H., Crawford, D.J., Landolt, E., Gabel, J.D., Kimball, R.T., 2002. Phylogeny and
450
systematics of Lemnaceae, the duckweed family. Syst. Bot. 27, 221-240.
451
Maheshwari, S.C., 1954. The embryology of Wolffia. Phytomorphology 4, 355-365.
452
Maheshwari, S.C., 1956. Endosperm and seed of Wolffia. Nature 178, 925-926.
453
Maheshwari, S.C., 1958. The Lemnaceae. A contribution to their biology, morphology
454
and systematics. Ph.D. Thesis, University of Delhi, Delhi, India.
21
455
Maheshwari, S.C., Chauhan, O.S., 1963. In vitro control of flowering in Wolffia
456
microscopica. Nature 198, 99-100.
457
Naumann, B., Eberius, M., Appenroth, K-J., 2007. Growth rate based dose-response
458
relationships and EC-values of ten heavy metals using the duckweed growth inhibition
459
test (ISO 20079) with Lemna minor L. clone St. J. Plant Physiol. 164, 1656-1664.
460
Pieterse, A.H., 2013. Is flowering in Lemnaceae stress-induced? A review. Aquatic
461
Bot.104, 1-4.
462
Rimon, D., Galun, E., 1969. Morphogenesis of Wolffia microscopica: Frond and flower
463
development. Phytomorphology 18, 364-372.
464
Seth, P.N., Venkataraman, R., Maheshwari, S.C., 1970. Studies on the growth and
465
flowering of a short-day plant, Wolffia microscopica. II. Role of metal ions and chelates.
466
Planta 90, 349-359.
467
Sree, K.S., Appenroth, K-J., 2014. In: International Steering committee on Duckweed
468
Research and Application NewsLetter No. 3, pp. 2-3.
469
http://www.internationallemnaassociation.org/uploads/LetterNo3_updated.pdf.
470
Venkataraman, R., 1968. Growth and flowering of Wolffia microscopica in vitro. Ph.D.
471
Thesis, University of Delhi, Delhi, India.
472
Venkataraman, R., Seth, P.N., Maheshwari, S.C., 1970. Studies on the growth and
473
flowering of a short-day plant, Wolffia microscopica. I. General aspects and induction of
474
flowering by cytokinins. Zeitschrift Pflanzenphysiologie. 62, 316-327.
22
475
Voigt, J.O., 1845. Hortus Suburbanus Calcuttensis. Bishop college press, Calcutta,
476
India, p. 692.
477
White, S.L., Wise, R.R., 1998. Anatomy and ultrastructure of Wolffia columbiana and
478
Wolffia borealis, two nonvascular aquatic angiosperms. Int. J. Plant Sci. 159, 297-304.
479
Xu, J., Cheng, J.J., Stomp, A.M., 2012. Nutrient removal from swine wastewater by
480
growing duckweed: a pilot study. Transactions ASABE. 1, 181-185.
481
Zhao, H., Appenroth, K., Landesman, L., Salmean, A.A., Lam, E., 2012. Duckweed
482
rising at Chengdu: summary of the 1st International Conference on Duckweed
483
Application and Research. Plant Mol. Biol. 78, 627-632.
484
Ziegler, P., Adelman, K., Zimmer, S., Schmidt, C., Appenroth, K.-J. 2014. Relative in
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vitro growth rates of duckweeds (Lemnaceae) – the most rapidly growing higher plants.
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Plant Biol. 17, DOI: 10.1111/plb.12184.
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Figure legends
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Fig. 1. Morphology of vegetative fronds of W. microscopica. (a, b) Mature frond with
491
pseudoroot. (c) SEM micrograph of a whole frond showing a pseudoroot. Arrows
492
indicate the elongated epidermal cells on the proximal end and smaller rounded cells at
493
the tip of the pseudoroot. (d) Dorsal surface of mother frond (M) connected to a
494
daughter frond (D). (e) Actinocytic stomata with subsidiary cells (Su) arranged in a
495
rosette and consisting of a pair of guard cells (G). (f) Angular margin of the frond
23
496
depicting two papillae (P). Arrows indicate a row of peripheral stomata. (g) Papilla (P)
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on the dorsal surface. (h) Tip of the pseudoroot showing the small meristematic cells. (i)
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Ventral surface of mother and daughter fronds. Note the scar (S) from the connection of
499
stipe to its mother. Arrow indicates the elongated cells of the pseudoroot at the proximal
500
end. (j) Almost flat form of the mature frond possessing a daughter frond. Arrow
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indicates the small bulge in place of a pseudoroot.
502
Fig. 2. Development of fronds demonstrates clonal propagation in W. microscopica. (a)
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SEM micrograph of the vegetative pouch of a flowering frond (with furrow on the dorsal
504
surface (F)) showing remnants of stipes of two detached daughter fronds (arrow) and a
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daughter frond (D) to the interior of the pouch. (b, c) Diagrammatic representation and a
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longitudinal section of the frond along the antero-posterior axis as captured under light
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microscope respectively of four subsequent generations (I-IV) of fronds living in union,
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connected to the parent frond (I) with a stipe (Sp). Arrow in „c‟ represents the cross
509
section through a scar on the ventral surface of the frond. (d) Initial stages of
510
development of a frond from its primordium. Arrow indicates a periclinal division of an
511
apical protodermal cell.
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Fig. 3. Sequential changes during emergence of floral organs. (a) Initiation of a furrow
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formation. Arrow indicates the group of small, compact cells lined close to the centre of
514
the frond. (b) A widened furrow (F) giving a glimpse of the internally developing anther
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lobes (A). (c) Overlapping borders of the furrow (F) arching the developing floral organs
516
below. (d,e) Emergence of stigma (Sg) from the furrow (F). Note the dorsal papillae (P)
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in „e‟. (f) SEM micrograph showing furrow (F) on both mother and daughter fronds
518
positioned eccentrically.
24
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Fig. 4. Flowering fronds of W. microscopica. (a) Side view of a mother and daughter
520
frond. The daughter frond depicts the stigma (Sg) which emerges out first. (b) Side view
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of a flowering frond showing Stigma (Sg) and stamen with the anther lobes (A). (c)
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Aerial view of a flowering frond showing the anther lobes (A) and the stigma (Sg). (d)
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Prominent anther lobes at different stages of dehiscence (arrows). Also note the anther
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lobes on the daughter frond (arrow-head). (e) Arrow indicates the exudate from stigma.
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(f) SEM micrograph of a flowering frond showing the dehiscing anther (A) and the
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stigma (Sg) of the female floral organ. (g) A pollen grain dispersing from the anther.
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Fig. 5. Anatomy of vegetatively propagating fronds. (a) A cross section of frond showing
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a small pseudoroot. The upper epidermis is interrupted by stomata (St).The lower
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epidermis and the ventrally located parenchymatous cells contain high number of
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chloroplasts (arrow). Also note the starch grains loaded in the plastids of the
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parenchymatous cells on the ventral side (arrow-head) and the chloroplasts in the
532
growing pseudoroot. (b) Meristematic initials of the pseudoroot in the daughter frond (D)
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still attached to the mother frond (M) with a stipe (Sp). Note the sub-stomatal air spaces
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(SSA) in the daughter frond. (c) A longitudinal section of the pseudoroot showing the
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smaller cells (arrow) at the tip and larger elongated cells (arrow-head) at the proximal
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end.
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Fig. 6. Anatomy of flowering fronds: longitudinal sections through the median antero-
538
posterior axis of the mother frond. (a) Daughter frond showing the developing male and
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female floral organs in the floral cavity (FC) and its clonal daughters in the vegetative
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pouch (VP). Arrow indicates a scar of the stipe. “N” represents the nodal strip of cells
541
between the VP and the FC. (b) Developing male floral organ (MF) and female floral
25
542
organ (FL) in the floral cavity (FC). Note the seemingly not attached daughter frond in
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the vegetative pouch (VP) showing that the floral cavity and the vegetative pouch are
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not on the same axis. Arrow-head shows the roof of the vegetative pouch which is
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continuous with the upper epidermis. (c) Immature male floral organ depicting the
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stamen (Sm) with two anther lobes (A). Arrow indicates the two unconnected cell layers
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which depict the overlapping flaps of the rim of the furrow arching the developing floral
548
organs.
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Supplementary material:
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Fig. S1. Fronds of W. microscopica (a) in natural habitat, fronds floating in pond water.
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(b) in controlled culture conditions, arrows indicate some of the anther lobes which have
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emerged out of the fronds.
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Figure 1 Part 1
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Figure 1 Part 2
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Supplementary Figure S1
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