Insights into the multifaceted application of microscopictec

REVIEW

Insights into the multifaceted application of microscopic techniques in plant tissue culture systems

Mack Moyo 1?Adeyemi O.Aremu 1?Johannes Van Staden 1

Received:18February 2015/Accepted:24June 2015/Published online:11July 2015óSpringer-Verlag Berlin Heidelberg 2015

Abstract

Main conclusion Microscopic techniques remain an integral tool which has allowed for the better under-standing and manipulation of in vitro plant culture systems.The recent advancements will inevitably help to unlock the long-standing mysteries of fundamental biological mechanisms of plant cells.

Beyond the classical applications in micropropagation aimed at the conservation of endangered and elite com-mercial genotypes,plant cell,tissue and organ cultures have become a platform for elucidating a myriad of fun-damental physiological and developmental processes.In conjunction with microscopic techniques,in vitro culture technology has been at the centre of important break-throughs in plant growth and development.Applications of microscopy and plant tissue culture have included eluci-dation of growth and development processes,detection of in vitro-induced physiological disorders as well as sub-cellular localization using ?uorescent protein probes.Light and electron microscopy have been widely used in con-?rming the bipolarity of somatic embryos during somatic embryogenesis.The technique highlights basic anatomical,structural and histological evidence for in vitro-induced physiological disorders during plant growth and develop-ment.In this review,we discuss some signi?cant biological insights in plant growth and development,breakthroughs and limitations of various microscopic applications and the

exciting possibilities offered by emergent in vivo live imaging and ?uorescent protein engineering technologies.Keywords Fluorescent proteins áHistology áOrganogenesis áPhysiological disorders áSomatic embryogenesis áSubcellular localization

Introduction

The concept and discovery of microscopy date back approximately four centuries.Basically,microscopy involves the use of microscopes to enlarge samples or objects which originally are beyond the resolution of the human eye (Shur and Price 2012;Thomasson and Mac-naughtan 2013).As an indication of the great importance and value of microscopy,it remains a popular and vital tool with a wide range of applications in basic and applied sci-ences (Tran?eld and Walker 2013;Zumbusch et al.2013;El-Bakry and Sheehan 2014;Whited and Park 2014)as well as in the medical and material engineering ?elds (Torrealba and Carrasco 2004;Shur and Price 2012;De Boer et al.2013;Juszczyk et al.2013).These aforementioned publi-cations also provide excellent reviews highlighting the trends and current updates related to the speci?cs and sig-ni?cance of microscopy in these various ?elds.In addition,details on the theory and practice of speci?c microscopy-based technologies and specimen preparation protocols are well documented (Chal?e and Kain 2005;Kuo 2007;Chandler and Roberson 2009;Murphy and Davidson 2013).In plant sciences,microscopy is used in an attempt to resolve and understand various aspects of growth and developmental processes including structural and func-tional properties.It also provides insights on interactions of cells and subcellular components in plants (Chandler and

&Johannes Van Staden

rcpgd@ukzn.ac.za

1

Research Centre for Plant Growth and Development,School of Life Sciences,University of KwaZulu-Natal,

Pietermaritzburg,Private Bag X01,Scottsville 3209,South Africa

123

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DOI 10.1007/s00425-015-2359-4

Roberson2009;Domozych2012).Plant science as a?eld encompasses perse aspects of plant growth,development and ecology amongst others.With one of the main focus geared at ef?cient propagation and general plant improvement in terms of quality and quantity,plant biotechnology remains one of the fundamental?elds in plant sciences(Vasil2008).To a certain extent,plant biotechnology was established based on the principles of cellular totipotency and genetic transformation.Inevitably the use of basic in vitro plant culture techniques is essential and vital for the success of several plant biotechnology endeavours.Particularly from a conservation perspective, the value and bene?t of in vitro tissue culture systems (micropropagation)are well documented(George1993; Ramachandra Rao and Ravishankar2002;Pence2010). Besides the potential in ensuring food security via mass propagation of different staple crops and fruits(Mondal et al.2004;Dobra′nszki and Teixeira da Silva2010),many plant species with ornamental,horticulture and medicinal values are easily regenerated(Rout et al.2000,2006; Teixeira da Silva2003;Moyo et al.2011),see Fig.1.

Recent technological advances are expanding the capa-bilities of microscopy which are being used to understand and explain commonly observed morphological appearances of in vitro regenerants.Thus,coupled with complementary biochemical,histological and molecular approaches,the increasing perse microscopy-based technologies can expedite the better understanding of in vitro plant culture systems.Furthermore,physiological disorders in regener-ants are often better elucidated with the use of microscopic systems(Chakrabarty et al.2006;Jausoro et al.2010a;Bairu et al.2011).Notwithstanding these aforementioned appli-cations and bene?ts,a comprehensive review detailing the contribution of microscopy to the understanding of in vitro plant culture systems is still lacking.Thus,this review covers a brief overview on the basic microscopic principles and technological advances in the?eld as well as summarizing the present applications and gains from the use of micro-scopy in in vitro plant culture systems.In addition to iden-tifying the current knowledge gap,a critical appraisal of microscopy application in plant cell,tissue and organ culture systems was discussed.Even though the current review is not fully exhaustive of all the available literature,as much as possible,we provide representative and speci?c references to ascertain the overall objectives of the subject matter. Recent advances and general overview

on microscopic techniques

In recent times,novel and giant technological strides in the form of introduction of laser-based,vibrational,electron and X-ray systems coupled with the rapid evolution of digital image capture and analysis technologies have rev-olutionized the capabilities and applications of microscopy (Torrealba and Carrasco2004;Roberts et al.2007; Domozych2012;Picas et al.2012).As postulated by Domozych(2012),these developments have allowed for the visualization of cell dynamics with unprecedented resolution,contrast and experimental versatility.Based on the evidence of its increasing application(Jahn et al.2012; Picas et al.2012;Thomasson and Macnaughtan2013; Zumbusch et al.2013;El-Bakry and Sheehan2014;Whited and Park2014),there is no doubt that microscopy is more valuable than ever before and will remain relevant in all areas of plant science research.In the near future,modern microscopy will ultimately achieve the goal of resolving the three-dimensional(3-D)structural and functional fea-tures of cellular life(four-dimensional imaging or4-DI) (Domozych2012).

Despite the potential and advances associated with microscopic techniques for biological research,some inherent limitations still exist.The two major‘reality checks’are that(1)light microscopy and confocal laser scanning microscopy(CLSM)used to image dynamic events in live cells are inherently limited in resolution and (2)electron microscopy which possesses better resolution cannot be used to view live cells(Domozych2012). Although scanning probe microscopy exists as a different technique,optical(light)and electron microscopy are the most commonly used in in vitro culture systems(Tables1, 2,3).The conventional light microscope techniques are bright-?eld,polarized,and?uorescence light microscopy while electron microscopy includes the scanning electron microscopy and transmission electron microscopy(TEM) (Chandler and Roberson2009;Domozych2012).In addi-tion to other basic differences such as sample preparation, electron microscopy has a much higher resolution of &0.1–5nm than light microscopy with a resolution of 0.2mm(El-Bakry and Sheehan2014).Thus,the task to be performed and speci?c objective generally in?uence the choice of microscopy technique at any given time. Practical application of microscopic techniques

in in vitro plant culture systems

As a well-established system for rapid proliferation of clonal plantlets(Fig.1)for the?oricultural and ornamental industries,micropropagation allows for year-round and continuous culture(Caponetti et al.2005;Pence2010; Ruffoni and Savona2013).For plant species with medic-inal value,their mass propagation is often aimed at ensuring their conservation(Canter et al.2005).Further-more,researchers have in recent times unravelled the potential of plant tissue culture as a tool to elucidate

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metabolic pathways and to enhance the production of therapeutic phytochemicals (Ramachandra Rao and Rav-ishankar 2002;Verpoorte and Memelink 2002;Karup-pusamy 2009).In vitro cell culture systems are also valuable avenues for transformation and transgenic studies.The following subsections highlight the basic and practical application of microscopy in different in vitro systems.Elucidation of growth and development patterns Even though the frequency of application may differ,the four basic methods for micropropagation are axillary shoot proliferation,node culture,de novo formation of adventi-tious shoots through organogenesis and somatic embryo-genesis (Kane 2005).Details of these procedures are outside the scope of this review but have been well documented (Zimmerman 1993;Kane 2005;Rout et al.2006).There is no doubt that much of the available evi-dence and theories of in vitro developmental processes were achieved via histological approaches using different microscopic techniques (Trigiano et al.2005).Although tremendous advances have been recorded in recent times (Motte et al.2014),more stringent studies are required to fully understand the overall intricate events involved in in vitro plant growth and development.With the ability to generate microscopic structures and characteristics of cells through initiation,assemblage and arrangement phases,researchers have gained in-depth knowledge which allows for manipulation of plant growth using in vitro culture techniques.Both light and electron microscopic techniques have become integral components in studying plant species (Table 1).Plant growth and development processes

are

Fig.1Morphological appearance of typical high shoot proliferation of clonal plantlets with perse economical value obtained via organogenesis during micropropagation.a Amelanchier alnifolia (nutritional).b Merwilla plumbea (medicinal and ornamental).c Aloe arborescens (medicinal and ornamental).d Hypoxis hemero-callidea (medicinal and ornamental).Scale bar 10mm

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L y c a s t e h y b r i d

I n d i r e c t i n d u c t i o n o f P L B s a n d s h o o t p r o l i f e r a t i o n

P L B s w i t h l e a f p r i m o r d i a a n d a p i c a l m e r i s t e m

H u a n g a n d C h u n g (2011)

S E M

S u t h e r l a n d i a f r u t e s c e n s L .I n d i r e c t s h o o t o r g a n o g e n e s i s a n d p l a n t r e g e n e r a t i o n o f S u t h e r l a n d i a f r u t e s c e n s

N o d u l a r c a l l u s a f t e r 2w e e k s o f c u l t u r e

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S E M T e c t o n a g r a n d i s L .D e t e r m i n e w h e t h e r d i f f e r e n t c o n c e n t r a t i o n s o f b e n z y l a d e n i n e (B A )a f f e c t t e a k p l a n t d e v e l o p m e n t

S h o o t s o n e i t h e r c y t o k i n i n -f r e e m e d i u m ,2.22o r 4.44l M B A h a d e l l i p t i c a l s t o m a t a ;w h e r e a s s t o m a t a o f s h o o t s g r o w n o n 6.66l M B A w e r e r i n g -s h a p e d ,r a i s e d a n d o p e n

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M e r i s t e m o i d s t h a t o r i g i n a t e d e i t h e r d i r e c t l y o r i n d i r e c t l y f r o m c a l l i w e r e u l t r a s t r u c t u r a l l y s i m i l a r

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T a b l e 1c o n t i n u e d

M i c r o s c o p i c t e c h n i q u e

P l a n t s p e c i e s O b j e c t i v e (s )o f s t u d y O b s e r v a t i o n (s )R e f e r e n c e s

O t h e r d e v e l o p m e n t a l p a t t e r n s

L i g h t m i c r o s c o p y

P e t u n i a g r a n d i ?o r a J u s s .‘P r i m e T i m e ’E f f e c t o f ?a v o n o i d 30,50-h y d r o x y l a s e (F 3050H )d e l p h i n i d i n a c c u m u l a t i o n i n p e t a l s

C h a n g e s i n ?o w e r c o l o u r p i g m e n t a t i o n v i s u a l i z e d Q i e t a l .(2013)S E M

T h y m u s c a e s p i t i t i u s B r o t .C o m p a r i s o n o f e s s e n t i a l o i l p r o d u c t i o n i n s h o o t c u l t u r e s a n d ?e l d -g r o w n p l a n t s

S i m i l a r g l a n d u l a r t r i c h o m e s d e n s i t y f o r i n v i t r o s h o o t s a n d ?e l d -g r o w n p l a n t s M e n d e s e t a l .(2013)

S E M

P a n d a n u s a m a r y l l i f o l i u s R o x b .

D e v e l o p m e n t a l p a t t e r n o f l e a f l o w e r e p i d e r m i s p a p i l l a e

O b s e r v a t i o n o f d i f f e r e n t s t a g e s o f p a p i l l a r d e v e l o p m e n t

W a k t e e t a l .(2009)

T E M

B e t a v u l g a r i s L .E s t a b l i s h s p e c i ?c p a t t e r n s o f e x t r a c e l l u l a r p r o t e i n s a n d i d e n t i f y d i s t i n c t p r o t e i n m a r k e r s o f c o r r e s p o n d i n g p h e n o t y p e s N u m b e r o f n u c l e i i n h a b i t u a t e d n o n -o r g a n o g e n i c (H N O )[t u m o u r (T )c e l l l i n e s ;H N O a n d T c e l l s h a d e n l a r g e d ,l o b e d n u c l e i w i t h h i g h n u m b e r o f n u c l e o l i

P a v o k o v i c

′e t a l .(2012)T E M

C o c o s n u c i f e r a L .E s t a b l i s h w h e t h e r l a u r i c a c i d i m p r o v e s g r o w t h a n d d e v e l o p m e n t o f z y g o t i c c o c o n u t e m b r y o s

L a u r i c a c i d l e d t o l a r g e c i r c u l a r o i l b o d i e s ;l a r g e n u m b e r o f p l a s t i d s ,m i t o c h o n d r i a ,v a c u o l e s ,m i c r o b o d i e s a n d e x t e n s i v e e n d o p l a s m i c r e t i c u l u m

L o

′p e z -V i l l a l o b o s e t a l .(2011)T E M

S c o p o l i a p a r v i ?o r a (D u n n )

N a k a i M o r p h o l o g i c a l c h a r a c t e r i z a t i o n o f t r a n s g e n i c h a i r y r o o t s (f o r p r o d u c t i o n o f a l k a l o i d s )

P r e s e n c e o f l a r g e s p h e r i c a l -s h a p e d i d i o b l a s t c e l l s i n t r a n s g e n i c h a i r y r o o t s

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T E M

T r i t i c u m a e s t i v u m L .E f f e c t o f p o l y a m i n e s o n a n d r o g e n e s i s i n a n t h e r s o r e m b r y o -l i k e s t r u c t u r e s

C h l o r o p l a s t s (s p e r m i n e -t r e a t e d )e x h i b i t e d s l o w d e v e l o p m e n t a n d a p p e a r e d t o b e s m a l l e r i n s i z e c o m p a r e d t o p u t r e s c i n e a n d s p e r m i d i n e t r e a t m e n t s

R e d h a a n d S u l e m a n (2011)

T E M

C y m b i d i u m S o l a n a B e a c h 9C .r i n c o n R o l e o f p u t r e s c i n e i n r a p i d s h o o t r e g e n e r a t i o n i n t h e c a l l u s o f o r c h i d s p r e -t r e a t e d w i t h m a n n i t o l a n d c a d m i u m c h l o r i d e

P u t r e s c i n e -a l t e r e d C d C l 2-a n d m a n n i t o l -t r e a t e d c a l l u s h a d n u m e r o u s o i l d r o p l e t s ,r o u g h e n d o p l a s m i c r e t i c u l u m a n d d i c t y o s o m e s

G u h a a n d R a o (2010)

T E M

C u c u m i s s a t i v u s L .U l t r a s t r u c t u r a l c o m p a r i s o n o f C d -t o l e r a n t a n d -s e n s i t i v e c e l l s

T o l e r a n t c e l l s a d j u s t e d t o p r e s e n c e o f C d ,b u t w i t h o u t a n y s p e c i ?c u l t r a s t r u c t u r a l m o d i ?c a t i o n s r e l a t e d t o i n c r e a s e d t o l e r a n c e

G z y l e t a l .(2009)

778

Planta (2015)242:773–790

123

characteristically dynamic whereas histological techniques only present a narrow momentary glimpse of the process (Trigiano et al.2005).Notwithstanding,by piecing toge-ther a series of the static microscopic observations,plant biologists are able to elucidate the underlying anatomical features involved in plant development.Somatic embryogenesis

The induction of somatic embryos under in vitro culture conditions in ontogenetic steps is similar to those observed in zygotic embryogenesis and has long fascinated plant biologists (Zimmerman 1993).Somatic embryogenesis (SE)provides a model system for studying the genetic basis of early differentiation events and cellular totipotency

of somatic cells (Zimmerman 1993;Fehe

′r et al.2003;Kurczyn

′ska et al.2007).Furthermore,SE has become a widely used technique in genetic transformation and mass propagation of elite genotypes (Table 1).Light and elec-tron microscopic studies have reported on the cellular origin of somatic embryos during primary (Blazquez et al.2009;Capelo et al.2010;Lin et al.2011;Parra-Vega et al.2013)and repetitive/secondary embryogenesis (Dai et al.

2011;Pavlovic

′et al.2013;Raju et al.2013).Upon expo-sure to SE induction medium,the initial events signifying cytological changes in the formation of somatic embryos

were evident in 4–6days (Canhoto et al.1996;Kurczyn

′ska et al.2007).Induction of somatic embryos was character-ized by formation of embryonic-like centres from single or multi-cells of proto-and subprotodermal origin (Kur-czyn

′ska et al.2007),accumulation of starch grains and differentiation of mitochondria (Canhoto et al.1996).The unicellular origin of direct somatic embryos has been observed to be the main morphogenic pathway (Rugkhla

and Jones 1998;Kurczyn

′ska et al.2007).Thus,as early as 6days into the culture period,SE induction can be con-?rmed,especially for somatic embryo transformation studies.Furthermore,microscopic applications coupled with molecular techniques provides an invaluable OMICS/morphology interface for exploring plant growth and development.During the early stages of embryogen-esis,LEAFY COTYLEDON (LEC )genes play a key role in somatic embryo development (Stone et al.2001).In Ara-bidopsis thaliana ,strong promoter activity of LEC was detected at the globular somatic embryo stage after a

12-day culture period (Kurczyn

′ska et al.2007).In addition,microscopic techniques have been highly valuable in characterizing the non-bipolarity of protocorm-like bodies (Mayer et al.2010;Huang and Chung 2011),globular embryo-like structures (Woo and Wetzstein 2008;Shari?et al.2010)and nodular meristemoids (Moyo et al.2009;Rosa and Dornelas 2012),which would have been other-wise mistaken for somatic embryos (Fig.2).Thus,

T a b l e 1c o n t i n u e d

M i c r o s c o p i c t e c h n i q u e

P l a n t s p e c i e s O b j e c t i v e (s )o f s t u d y

O b s e r v a t i o n (s )

R e f e r e n c e s

T E M N i c o t i a n a t a b a c u m L .E f f e c t o f l o n g -t e r m c r y o p r e s e r v a t i o n o n e x p r e s s i o n o f p l a n t -m a d e v a c c i n e s

O r g a n e l l e s w i t h d o u b l e m e m b r a n e s h a d i n t a c t m e m b r a n e s i n b o t h t h e c o n t r o l a n d c r y o p r e s e r v e d c e l l s V a n E c k a n d K e e n (2009)

B A B e n z y l a d e n i n e ,P L B s p r o t o c o r m -l i k e b o d i e s ,S E M s c a n n i n g e l e c t r o n m i c r o s c o p y ,T E M t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y

Planta (2015)242:773–790779

123

T a b l e 2T h e r o l e o f m i c r o s c o p i c t e c h n i q u e s i n d e t e c t i o n o f i n v i t r o -i n d u c e d p h y s i o l o g i c a l d i s o r d e r s i n p l a n t c e l l ,t i s s u e a n d o r g a n c u l t u r e s

M i c r o s c o p i c t e c h n i q u e

P l a n t s p e c i e s

O b j e c t i v e (s )o f s t u d y

O b s e r v a t i o n (s )R e f e r e n c e s

C L S M M a l u s s p p c v .‘M 9E m l a ’

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o l e a f s t r u c t u r e

C o m p a r e d w i t h h e a l t h y l e a v e s ,h y p e r h y d r i c l e a v e s s h o w e d a b n o r m a l ,o f t e n d i s c o n t i n u o u s d e v e l o p m e n t o f t h e e p i d e r m i s a n d c u t i c l e .S t o m a t a w e r e m a l f o r m e d .T h e l e a f l a m i n a a p p e a r e d t h i c k e n e d a n d w a s c h a r a c t e r i z e d b y p o o r d i f f e r e n t i a t i o n b e t w e e n t h e p a l i s a d e a n d s p o n g y m e s o p h y l l t i s s u e

C h a k r a b a r t y e t a l .(2006)

L i g h t m i c r o s c o p y D i a n t h u s c a r y o p h y l l u s L .

M e a s u r e e p i d e r m a l a n d s t o m a t a l c e l l n u m b e r a n d t o v i e w s u r f a c e w a x t o p o l o g y a n d l e a f i m p r i n t s

H y p e r h y d r i c p l a n t s h a d f e w e r e p i d e r m i s c e l l a n d s t o m a t a p e r l e a f a r e a c o m p a r e d t o t h e n o r m a l o n e s

C o r r e l l a n d W e a t h e r s (2001)

L i g h t m i c r o s c o p y

V a n i l l a p l a n i f o l i a J a c k s .e x A n d r e w s

O b s e r v a t i o n o f m o r p h o l o g i c c h a n g e s t h a t o c c u r w h i l e p r o g r e s s i n g t o w a r d s h y p e r h y d r i c i t y

D e g r a d a t i o n o f t h e e n d o d e r m a l c e l l s w a s o b s e r v e d i n h y p e r h y d r i c s h o o t s ,w h e r e a s t h e l e a f s e c t i o n s s h o w e d h i g h e r d e g r a d a t i o n o f v a s c u l a r b u n d l e s ,l o s s o f ?r m n e s s o f t h e p a l i s a d e p a r e n c h y m a w i t h a b n o r m a l e n l a r g e m e n t ,a n d m o r e i n t e r c e l l u l a r s p a c e

S r e e d h a r e t a l .(2009)

L i g h t m i c r o s c o p y J a t r o p h a c u r c a s L .

E f f e c t o f b r o w n i n g o n c a l l u s m o r p h o l o g y N o n -b r o w n i n g c a l l u s h a d e v e n ,t i g h t l y a r r a n g e d c e l l s a n d b r o w n i n g c a l l u s h a d a d i s o r d e r e d c e l l p a t t e r n

H e e t a l .(2009)

L i g h t m i c r o s c o p y

S c l e r o c a r y a b i r r e a (A .R i c h .)H o c h s t .s u b s p .c a f f r a (S o n d .)K o k w a r o I n ?u e n c e o f m i c r o c u l t u r e o n l e a f a n d r o o t s t r u c t u r e

S t r u c t u r a l d i f f e r e n c e s i n t h i c k n e s s o f e p i d e r m i s a n d e n d o d e r m i s o b s e r v e d b e t w e e n p h o t o a u t o t r o p h i c a n d p h o t o m i x o t r o p h i c p l a n t s

M o y o e t a l .(2012)

S E M

A l o e p o l y p h y l l a S c h o ¨n l a n d e x P i l l a n s

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o l e a f s t r u c t u r e

F r e q u e n c y o f t h e s t o m a t a w a s h i g h e r a n d w e l l d e v e l o p e d i n n o r m a l s h o o t s .H y p e r h y d r i c l e a v e s h a d l a r g e r a n d a b n o r m a l s t o m a t a

I v a n o v a a n d V a n S t a d e n (2010)

S E M C o t o n e a s t e r w i l s o n i i N a k a i

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o l e a f s t r u c t u r e

H y p e r h y d r i c l e a v e s s h o w e d a b n o r m a l s t o m a t a w h i c h h a d d e f o r m e d g u a r d c e l l s

S i v a n e s a n e t a l .(2011)

S E M S o l a n u m m e l o n g e n a L .

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o p l a n t s

H y p e r h y d r i c l e a v e s s h o w e d a b n o r m a l s t o m a t a ,s l i g h t l y l a r g e r t h a n t h o s e o f i t s n o r m a l o n e s a n d w i t h g u a r d a n d s u b s i d i a r y c e l l s d r a s t i c a l l y d e f o r m e d

P i c o l i e t a l .(2001)

T E M A l l i u m s a t i v u m L .

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o c e l l s

H y p e r h y d r i c c e l l s h a d a b n o r m a l a n d p o o r l y d e v e l o p e d o r g a n e l l e s

W u e t a l .(2009)

T E M A n n o n a g l a b r a L .

R o l e o f c y t o k i n i n s i n t h e d i f f e r e n t i a t i o n o f t h e p h o t o s y n t h e t i c a p p a r a t u s i n i n v i t r o -d e r i v e d p l a n t s B e n z y l a d e n i n e (B A )a n d k i n e t i n -d e r i v e d p l a n t l e t s h a d c h l o r o p l a s t s w i t h w e l l -d e v e l o p e d g r a n a m a r g i n s y s t e m a n d g r e a t e r a c c u m u l a t i o n o f s t a r c h g r a i n s ;t h i d i a z u r o n c a u s e d f o r m a t i o n o f a b n o r m a l c h l o r o p l a s t s r i c h i n l a r g e ,g l o b u l a r ,e l e c t r o n d e n s e s t r u c t u r e s

d e O l i v e i r a e t a l .(2008)

780

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123

microscopy techniques have been invaluable in con?rming and ascertaining the bipolar identity of somatic embryos in SE or lack thereof in embryo-like structures during f066eba227d3240c8447efdeanogenesis

Organogenesis refers to de novo organ formation involving the processes of dedifferentiation and redifferentiation of plant cells.It is widely proven that the ratio of auxin to cytokinin in plant tissues has the ability to shift the cell physiological state.In particular,the distribution and unique movement of auxins in a polar direction from cell to cell is thought to have a major in?uence on the organogenic fate of plant tissues (Muday and DeLong 2001;Del Bianco et al.2013;Motte et al.2014).However,the underlying mechanisms involved in this process remain to be fully elucidated (Motte et al.2014).Innovative approaches using reporter genes fused to speci?c promoters,such as pep-tidyl-prolyl cis/trans isomerase (PIN1)and microscopy techniques have attempted to decipher the physiological and molecular mechanisms controlling the process of organogenesis (Vieten et al.2007).When used in con-junction with plant tissue culture model systems,the use of visual markers such as b -glucuronidase (GUS),luciferase (LUC),b -galactosidase (LacZ)and green ?uorescent pro-tein (GFP)could be useful in exploring the molecular mechanisms controlling plant growth and development.In plant transformation studies,GFP allows for non-destruc-tive direct observation of gene expression events and the

successful recovery of transgenic plants (Hras

ˇka et al.2006).Therefore,microscopy coupled with plant tissue culture offers a platform for molecular/morphology assessments in plant growth and development studies.Furthermore,regardless of the plant cell,tissue and organ culture technique,establishing the speci?c origin of structures such as adventitious shoots,roots and somatic embryos remains one of the unsolved mysteries in plant biology (Trigiano et al.2005).Numerous microscopy studies have provided signi?cant insights into the basic structural features of in vitro plant growth (Table 1).For example,using light microscopy,the regeneration rate of hypocotyl subsections (C1–C4)in Watsonia lepida showed that cell pision was highest in C2while in vitro regen-eration was signi?cantly lower than in subsection C1(Ascough et al.2009).The authors reported that subsection C1contained the apical meristem which possibly had meristematic cells that are developmentally plastic and responsive to external f066eba227d3240c8447efdeing optical microscopy da Cruz et al.(2014)showed that cell proliferation within the pericycle led to adventitious bud formation in Bixa Orel-lana root explants.Rocha et al.(2012)characterized the anatomical events and ultrastructural aspects involved in

T a b l e 2c o n t i n u e d

M i c r o s c o p i c t e c h n i q u e

P l a n t s p e c i e s

O b j e c t i v e (s )o f s t u d y

O b s e r v a t i o n (s )

R e f e r e n c e s

T E M

C a p s i c u m a n n u u m L .

E v a l u a t e s t r u c t u r a l d i f f e r e n c e s a n d s t a r c h a c c u m u l a t i o n b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o c e l l s

P a l i s a d e c h l o r o p l a s t s o f n o r m a l p l a n t s s h o w e d w e l l -d e v e l o p e d t h y l a k o i d s o r g a n i z e d i n t o m a n y g r a n a .S t a r c h a c c u m u l a t i o n i n c h l o r o p l a s t s o f h y p e r h y d r i c p l a n t s w a s s i g n i ?c a n t l y h i g h e r t h a n i n t h o s e o f n o n -h y p e r h y d r i c p l a n t s

F o n t e s e t a l .(1999)

T E M

H a n d r o a n t h u s i m p e t i g i n o s u s (M a r t .E x D C )M a t t o s E v a l u a t e s t r u c t u r a l d i f f e r e n c e s b e t w e e n h y p e r h y d r i c a n d n o r m a l i n v i t r o

D i s o r g a n i z e d c o r t e x ,e p i d e r m a l h o l e s ,e p i d e r m a l d i s c o n t i n u i t y ,c o l l a p s e d c e l l s o b s e r v e d i n h y p e r h y d r i c s h o o t s

J a u s o r o e t a l .(2010b )

T E M

U n i o l a p a n i c u l a t a L .

P h o t o s y n t h e t i c a n d c a r b o h y d r a t e s t a t u s o f a n e a s y -t o -a c c l i m a t i z e (E K 16-3)a n d a d i f ?c u l t -t o -a c c l i m a t i z e (E K 11-1)g e n o t y p e s

C h l o r o p l a s t s i n E K 11-1p l a n t l e t s e x h i b i t e d l a r g e r n u m b e r s o f p l a s t o g l o b u l i t h a n E K 16-3

V a l e r o -A r a c a m a e t a l .(2006)

B A B e n z y l a d e n i n e ,

C L S M c o n f o c a l l a s e r s c a n n i n g m i c r o s c o p y ,S E M s c a n n i n g e l e c t r o n m i c r o s c o p y ,T E M t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y

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781

123

T a b l e 3T h e r o l e o f m i c r o s c o p i c t e c h n i q u e s i n s u b c e l l u l a r l o c a l i z a t i o n ,?u o r e s c e n t p r o t e i n t a g g i n g a n d t r a n s g e n e e x p r e s s i o n i n p l a n t c e l l ,t i s s u e a n d o r g a n c u l t u r e s

M i c r o s c o p i c t e c h n i q u e

P l a n t s p e c i e s

O b j e c t i v e (s )o f s t u d y

P r o b e O b s e r v a t i o n (s )

R e f e r e n c e s

C L S M

A r a b i d o p s i s t h a l i a n a (L .)H e y n h

I d e n t i f y m o l e c u l a r f a c t o r s r e s p o n s i b l e f o r c y t o k i n i n -i n d u c e d h i g h e r c e l l d i v i s i o n r a t e i n A r a b i d o p s i s a m p 1m u t a n t

–

H i g h e r m i t o t i c i n d e x (2.5-f o l d )i n a m p 1m u t a n t c o m p a r e d t o w i l d t y p e

N o g u e ′e t a l .(2000)

C L S M

C o f f e a c a n e p h o r a P i e r r e e x A .F r o e h n e r E f f e c t o f c a l c i u m i o n o p h o r e A 23187o n s o m a t i c e m b r y o g e n e s i s –

S u b c e l l u l a r l o c a l i z a t i o n o f c a l c i u m i o n s i n c y t o p l a s m

R a m a k r i s h n a e t a l .(2011)C L S M

C o f f e a c a n e p h o r a P i e r r e e x A .F r o e h n e r

E f f e c t o f i n d o l e a m i n e s a n d c a l c i u m o n s o m a t i c e m b r y o g e n e s i s

–

L o c a l i z a t i o n o f s e r o t o n i n i n v a s c u l a r t i s s u e s o f s t e m s ,r o o t s ,a n d s o m a t i c e m b r y o s

R a m a k r i s h n a e t a l .(2012)C L S M

G o s s y p i u m h i r s u t u m L .

E v a l u a t e t h e e f f e c t o f o v e r -e x p r e s s i o n o f W U S C H E L (W U S )g e n e o n s o m a t i c e m b r y o g e n e s i s a n d /o r o r g a n o g e n e s i s

G F P

L o c a l i z a t i o n o f W U S –G F P i n e m b r y o g e n i c t i s s u e s

B o u c h a b k e ′-

C o u s s a e t a l .(2013)

C L S M H o r d e u m v u l g a r e L .

T r a n s f o r m a t i o n p r o c e d u r e f o r b a r l e y b y A g r o b a c t e r i u m i n f e c t i o n o f i n v i t r o -c u l t u r e d o v u l e s

G F P

T r a n s i e n t G F P e x p r e s s i o n i n c e l l s w i t h i n t h e o v u l e i n c l u d i n g G F P -e x p r e s s i n g c e l l s i n t h e m i c r o p y l a r

H o l m e e t a l .(2006)

C L S M

L i n d e r n i a b r e v i d e n s S k a n

A g r o b a c t e r i u m t u m e f a c i e n s -b a s e d t r a n s f o r m a t i o n p r o t o c o l f o r t h e d e s i c c a t i o n -t o l e r a n t s p e c i e s

e G F P L o c a l i z a t i o n o

f e G F P e x p r e s s i o n o b s e r v e d s p e c i ?c a l l y i n t h e

g u a r d c e l l s o f d e s i c c a t e d l e a v e s

S m i t h -E s p i n o z a e t a l .(2007)

C L S M

N i c o t i a n a t a b a c u m L .e c o t y p e ‘L i t t l e H a v a n a ’

I n v e s t i g a t i n g s u b c e l l u l a r l o c a l i z a t i o n o f A r a b i d o p s i s I C K p r o t e i n s a n d d o m a i n s r e s p o n s i b l e f o r t h i s l o c a l i z a t i o n

G F P A l l G F P –I C K f u s i o n p r o t e i n s e x c l u s i v e l y l o c a l i z e d i n t h e n u c l e u s

B i r d e t a l .(2007)

L i g h t m i c r o s c o p y A r a u c a r i a a n g u s t i f o l i a (B e r t .)O .K u n t z e

E x p r e s s i o n o f a S o m a t i c E m b r y o g e n e s i s R e c e p t o r -L i k e K i n a s e (S E R K )g e n e f a m i l y m e m b e r

–

I n s i t u h y b r i d i z a t i o n s h o w e d t h a t A a S E R K 1t r a n s c r i p t s a c c u m u l a t e i n g r o u p s o f c e l l s a t t h e p e r i p h e r y o f e m b r y o g e n i c c a l l i

S t e i n e r e t a l .(2012)

L i g h t m i c r o s c o p y

P a s s i ?o r a m o r i f o l i a L .

E f f e c t o f a u x i n ,c y t o k i n i n a n d s u c r o s e o n t h e r e g u l a t i o n o f c e l l c y c l e m a c h i n e r y

–

S p a t i a l e x p r e s s i o n o f P m C Y C D 1(D -t y p e c y c l i n g e n e )o b s e r v e d u s i n g i n s i t u h y b r i d i z a t i o n

R o s a e t a l .(2013)

T E M

A r a b i d o p s i s t h a l i a n a (L .)H e y n h

S p a t i a l a n d t e m p o r a l d i s t r i b u t i o n o f l i p i d t r a n s f e r p r o t e i n 1(L T P 1)e p i t o p e s d u r i n g i n d u c t i o n o f s o m a t i c e m b r y o g e n e s i s

G F P I m m u n o g o l d l o c a l i z a t i o n o f L T P 1e p i t o p e s i n t h e o u t e r p e r i c l i n a l ,a n t i c l i n a l ,c e l l w a l l s a n d c y t o p l a s m o f p r o t o d e r m

P o t o c k a e t a l .(2012)

G F P g r e e n ?u o r e s c e n t p r o t e i n ,I C K i n h i b i t o r /i n t e r a c t o r o f c y c l i n -d e p e n d e n t k i n a s e ,C L S M c o n f o c a l l a s e r s c a n n i n g m i c r o s c o p y ,T E M t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y

782

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Passi?ora edulis direct and indirect in vitro organogenesis.The study showed that irrespective of the organogenic process,P .edulis meristemoids had similar ultrastructural characteristics.These and other similar ?ndings provide increased knowledge and critical insights that allow a better understanding of in vitro organogenic processes.Other cellular developmental patterns

Furthermore,light and electron microscopy have con-tributed immensely in evaluating the effects of various physiological factors on cellular developmental processes (Table 1).Nakagawa et al.(2011)demonstrated that the distribution of starch grains during SE was induced by exogenous application of polyamines.In addition,sper-mine-treated Triticum aestivum plants exhibited smaller chloroplasts compared to putrescine and spermidine-treated

ones,suggesting that the response was dependent on the type of polyamine (Redha and Suleman 2011).In partic-ular,TEM provides ultrastructural details of cellular developments when plants are exposed to different physi-ological f066eba227d3240c8447efdeing TEM,Lo

′pez-Villalobos et al.(2011)showed that lauric acid induced the production of large oil bodies and a high number of organelles in Cocos nucifera zygotic embryos.The scope for elucidating the ultrastructural developmental patterns arising from physi-ological stimuli remains limitless.

Detection of in vitro-induced physiological and anatomical disorders

Despite the bene?ts of micropropagation,the process is often besieged by a number of in vitro-induced challenges which may be anatomical,physiological and

biochemical

Fig.2Induction of asynchronous nodular meristemoids on Sclero-carya birrea leaf explants under a 16-h photoperiod.a A stereomi-crograph showing somatic embryo-like globular structures (arrowhead ;bar 1.0mm).b Scanning electron micrograph of nodular meristemoids at different stages of development (solid arrow emerging shoot bud;bar 1.5mm).c Shoot bud with characteristic shoot apical meristem (A)and developing leaf primordia (L)(bar 430l m)d Longitudinal section of a nodular meristemoid showing its connection to the explant tissues (bar 200l m).Modi?ed from Moyo et al.(2009)

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in nature (Kaeppler et al.2000;Hazarika 2006;Bairu et al.2011;Neelakandan and Wang 2012;Ruffoni and Savona 2013).Several studies have demonstrated the effects of the controlled,largely arti?cial environment in plant tissue culture systems on the anatomy of in vitro plants (Figs.3,4).Researchers have continuously reviewed the subject matter (Bairu and Kane 2011)and suggested means of tackling the recurrent problems such as shoot-tip necrosis (Bairu et al.2009),hyperhydricity (Ziv 1991;Rojas-Mar-t?

′nez et al.2010),fasciation (Iliev and Kitin 2011),epige-netic (Kaeppler et al.2000;Smulders and de Klerk 2011)and somaclonal variations (Larkin and Scowcroft 1981;Bairu et al.2011).Most of these physiological disorders are not only limited to the period of in vitro growth but become more apparent upon acclimatization of the regenerants

(Kozai 1991;Hazarika 2006;Posp?

′s ˇilova ′et al.2007).As a result,the success of plant tissue culture especially on a large scale depends on how these challenges can be

alleviated or possibly eradicated (Kozai et al.1997;Hazarika 2006;Bairu and Kane 2011).A better under-standing of these multifaceted problems begins with the availability of appropriate identi?cation tools.In view of the substantial evidence (Table 2),there is no doubt that the application of microscopy remains critical in understanding these challenges.In addition,other approaches such as biochemical and molecular tools provide complementary evidence for overall elucidation of the problems.The importance of microscopy is possibly attributed to the fact that the majority of physiological disorders are often man-ifested in the anatomy of the tissue-cultured regenerants.Both light and electron microscopic techniques have demonstrated vital signi?cance in the attempt to elucidate the anatomical and histological basis for in vitro-induced physiological disorders in several plant species (Table 2).Amongst these in vitro-induced challenges,detection of hyperhydricity has received considerable success with the use of different microscopic techniques (Table 2).Con-sidering that hyperhydricity affects several organelles

in

Fig.3Effect of the controlled plant tissue culture environment on growth and development.The root structure of a ex vitro and b in vitro grown Sclerocarya birrea plants showing differences in the epidermis (white arrow )and endodermis (solid arrow ).Modi?ed from Moyo et al.(2012

)

Fig.4Scanning electron micrographs showing the effect of pho-toperiod on stomata and glandular trichome formation on Pelargo-nium sidoides leaf surfaces under in vitro conditions.a Plants growing under 16-h photoperiod after 5weeks in culture.b Plants growing under 12-h photoperiod after a 5-week culture period.Modi?ed from Moyo et al.(2014)

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the cells of regenerated plants,it becomes necessary to examine the structure for detection of possible aberrations. With the use of light microscope,parameters such as sur-face wax topology and leaf imprints are recorded while the epidermal and stomatal cell count can be easily achieved (Correll and Weathers2001).Variations in these afore-mentioned parameters afford for direct evidence on the possible underlying metabolic processes which are responsible for the incidence of hyperhydricity in regener-ated plants.Evidence from scanning electron microscopy revealed that thickening of the stem and retardation of elongation are the?rst changes observed in hyperhydric carnation plantlets(Werker and Leshem1987).Examining the ultrastructural differences between the hyperhydric and normal leaves of carnation plantlets,Olmos and Hellin (1998)observed large vacuolated mesophyll cells(showing hypertrophy of cells and large intercellular spaces),lack of cuticular wax and the presence of abundant plastoglobuli on chloroplasts in hyperhydric leaves.The authors also noted differences in the morphology of guard cells with X-ray microanalysis revealing high levels of K?on abnormal plants.Furthermore,stomatal density was signi?cantly greater in normal leaves while the crystalline structure of the epicuticular wax was absent in hyperhydric leaves.An irregular assortment of organelles and unorganized spongy mesophyll were also observed in hyperhydric leaves.

With studies involving the use of TEM for hyperhy-dricity,critical examination of the ultrastructure of plant cells and tissues remains the main objective.In such instances,organelles such as the chloroplast and mito-chondria are often the main focus of researchers.In Allium sativum,hyperhydric cells had swollen mitochondria and slender chloroplasts(Wu et al.2009).The authors also observed that the vacuole displaced the organelle to the cell wall edge and the intergranal thylakoids appeared com-pressed.While the structure of mitochondria and peroxi-somes did not change in hyperhydric Capsicum annuum plants,the number of peroxisomes was more than in nor-mal plants(Fontes et al.1999).Furthermore,the chloro-plasts in the hyperhydric plants exhibited thylakoid disorganization,low grana number as well as presence of large starch grains and a low accumulation or absence of plastoglobules.

Subcellular localization and characterization

The benchmark discovery of the wild-type green?uores-cent protein(GFP)from the jelly?sh Aequorea victoria (Shimomura et al.1962),cloning of the GFP gene(Prasher et al.1992),and its modi?cation into a functional?uo-rescent protein(Chal?e et al.1994)have revolutionized the study of plant cell biology.However,variable outcomes have been reported with the expression of the wild-type gfp gene in different plants.The expression of gfp in Ara-bidopsis and other plant species was shown to be curtailed by aberrant mRNA splicing in which an84nucleotide sequence,recognized as a cryptic intron,codes for a defective protein(Haseloff and Siemering2005).A mod-i?ed gfp gene without the cryptic intron sequence exhibited improved in vivo expression in a wide range of plant species(Reichel et al.1996).Further improvements in sensitivity of the marker protein have been achieved through modi?cations of the GFP mutant cDNA leading to single-amino acid exchanges in the chromophore region. Green?uorescent protein and its derivative?uorophores have emerged as important reporter proteins for monitoring gene expression(Tang et al.2005;Rosa et al.2013;Yang et al.2013),subcellular protein localization(Huai et al. 2009;Lai et al.2013;Liu et al.2013),organelle dynamics (Hashimoto et al.2011;Tewari et al.2013;Xu et al.2013) and cell transformation(Holme et al.2006),both in vivo and real time,as well as in?xed samples(Davidson and Campbell2009).Furthermore,a combination of plant cell, tissue and organ culture techniques and?uorescent protein tags has provided a powerful tool for unravelling funda-mental insights into mechanisms involved in plant mor-phogenesis.In vitro plant culture provides an ideal environment that can be precisely controlled and modi?ed to achieve speci?c experimental conditions.Thus,the application of?uorescent probes in plant tissue culture systems has elucidated developmental and molecular mechanisms involved in plant morphogenic processes (Table3).The most commonly used?uorescent probe application is probably protein tagging for monitoring dynamic cellular events and subcellular protein localization using confocal laser microscopy(Sirerol-Piquer et al. 2012).In addition,dynamic expression patterns of?uo-rescent probes have revealed interesting spatial and tem-poral changes in morphogenic events involving plant cell, tissue and organ culture processes such as SE(Ramakr-ishna et al.2012;Bouchabke′-Coussa et al.2013)and embryonic cell suspension cultures(Cole et al.2013).

Beyond the resolution limits of light microscopy,GFP immunogold TEM provides more detailed information on subcellular localization of proteins.The high-resolution property of TEM allows for the detection of immunogold labelled GFP-tagged proteins in the cytoplasm,organelles and plasma membrane(Boevink et al.1998;Nebenfu¨hr et al.1999;Follet-Gueye et al.2003).Using this immunocytochemical technique,Potocka et al.(2012) demonstrated spatial and temporal changes in the distri-bution of lipid transfer protein epitopes during SE.How-ever,the technique has only been sparsely applied in studying morphogenesis in plant cell,tissue and organ cultures(Table3).Notwithstanding bene?ts derived from the high resolving power of immunoelectron microscopy,

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the technique has inherent drawbacks such as preservation of GFP antigenicity and antibody speci?city,arising from denaturization and bleaching of GFP during polymeriza-tion;decreased immunogold staining with tissue depth (Sirerol-Piquer et al.2012)as well as safety concerns associated with the use of uranyl acetate in specimen preparation(Carpentier et al.2012).In attempts to?nd alternatives for uranyl acetate,polyphenolic compounds such as tannic acid(Kajikawa et al.1975)and oolong tea extracts(Sato et al.2008;Carpentier et al.2012)have been evaluated for staining ultrathin sections.Other recent pro-tocols using microwave-assisted processing resulted in good preservation of cell antigenicity and high-quality cell ultrastructure for immunocytochemical studies(Carpentier et al.2012).Polyphenol-containing extracts,for example oolong tea extracts exhibited good counterstaining prop-erties for both ultrathin sections and in block staining, making them possible alternatives for the hazardous heavy metal stains such as uranyl acetate and lead citrate. Notwithstanding,specimen?xation with glutaraldehyde and osmium tetroxide(OsO4),and double electron staining with uranyl acetate and lead salts provide excellent contrast enhancement,hence it has remained standard procedure in most microscopy laboratories(Sato et al.2008).Thus,until the discovery of suitable alternatives,common stains such as uranyl acetate,uranyl formate,methylamine tungstate and methylamine vanadate will continue to be used but with emphasis on observance of safety regulations.

The search for?uorophores with low phototoxicity and decreased auto?uorescence has advanced the boundaries of ?uorescent protein engineering.Together with the devel-opment of high-resolution imaging techniques,a range of ?uorescent protein probes with perse spectral qualities spanning the orange,red and far-red regions of the elec-tromagnetic spectrum have been developed(Davidson and Campbell2009).Some studies(Smith-Espinoza et al. 2007;Wu et al.2011;Sun et al.2013)used enhanced GFP (eGFP),a variant of the GFP mutant in which exchange of amino acid phenylalanine64to leucine(F64L)and serine 65to threonine(S65T)drastically increased brightness intensity and photostability(Reichel et al.1996;Zacharias and Tsien2005).Modi?cations of GFP have resulted in some variants with better?uorescence characteristics,for example the maturation of eGFP is four times faster than that of the wild type(Ckurshumova et al.2011). Conclusions and future perspectives

The discovery and advancement of microscopic technolo-gies have provided plant biologists with a wide array of invaluable techniques to explore cellular structures and dynamics,thereby expanding our knowledge of plant growth and development.When used in conjunction with plant cell, tissue and organ culture methods,microscopic applications have provided critical insights into the dynamics of plant growth and development.In particular,the live imaging capabilities afforded by confocal microscopy and?uores-cent protein probes(GFP and its derivatives)have further advanced the boundaries in plant morphogenesis research and expanded the possibilities of what can be achieved in the future with improved resolving power of light microscopy. Development of photostable?uorophores,especially in the red and far-red spectral regions will provide more biological insights through dynamic in vivo live imaging of cellular components.Thus,notwithstanding the limited resolving power of light microscopy,the‘illuminated plant cell’(Mathur2007)continues to contribute invaluable informa-tion on subcellular protein localization,gene expression and transport of molecules,thereby enhancing our understanding of the fundamental mechanisms involved in plant develop-mental process.Furthermore,immunoelectron microscopy and immunogold labelling have circumvented the draw-backs imposed by the limited resolving power of light microscopy.New advancements and novel innovations in specimen preparation techniques’using high-phenol content plant extracts such as OTE(in place of uranyl acetate)and microwave-assisted processing are likely to expand the uti-lization of this method.Despite having high resolving power,immunogold labelling using TEM is still limited in its deep-tissue imaging capabilities.In the future,advance-ments in microscopic technologies have the potential to unlock the fundamental biological mysteries of the plant cell,and thus provide profound insights into plant develop-mental biology.

Author contribution statement MM and AOA wrote the paper.JVS supervised the work,provided critical sugges-tions and edited the paper.

Acknowledgments The authors gratefully acknowledge?nancial support from Claude Leon Foundation,the University of KwaZulu-Natal and National Research Foundation,South Africa.We thank Dr W.A.Stirk for her valuable suggestions.

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