ORIGINAL PAPER

A secondary phloic (bast) fibre-shy (bfs) mutant of dark jute(Corchorus olitorius L.) develops lignified fibre cells but isdefective in cambial activity

Avijit Kundu • Debabrata Sarkar •

Nur Alam Mandal • Mohit Kumar Sinha •

Bikash Sinha Mahapatra

Received: 13 November 2011 / Accepted: 19 January 2012 / Published online: 4 February 2012

� Springer Science+Business Media B.V. 2012

Abstract Bast fibre development in jute (Corchorus spp.)

is a complex process that involves the differentiation of

secondary phloic fibres (SPF) from the cambium followed

by lignification of the fibre wall. We have identified a

unique radiation-induced bast fibre-shy mutant of dark jute

(C. olitorius L.), which is concurrently defective in the

differentiation of SPF and secondary xylem (wood) but

develops lignified fibre cells. It displays the most unusual

phenotype with stunted growth and abnormal leaf shape,

matures earlier, yields significantly less bast fibres and

wood, and produces poorer quality fibres than its parental

wild-type. Cambial activities in the mutant and the normal

type were monitored by estimating the fibre content that

entails the total number of fibre cell bundles (FCBs) in an

entire transversal section. The results show that a multi-

fold reduction of bast fibre yield in the mutant is related to

development-specific loss of cambium function along the

length of the stem from to top to bottom. Since lignification

of the fibre wall in the mutant is not only normal but

also developmentally uniform, cambium function may be

unrelated to the lignification process during bast fibre

development. Lignin does not influence bast fibre strength

and fineness. The architecture of the mostly triangular FCB

wedges, which is governed by a balanced growth between

radially elongating FCBs and tangentially expanding ray

cells due to development-specific activation of the fusiform

and ray initials of the cambium, conditions fibre fineness.

Our study shows that mutation could specifically impair the

cambial activity by rendering those initials that differenti-

ate the SPF and secondary xylem nonfunctional.

Keywords Bast fibre � Cambium function � Corchorus

olitorius � Dark jute � Fibre cell bundle � Lignin � Mutant �Secondary phloic fibre

Abbreviations

bfs Bast fibre-shy

EDTA Ethylenediaminetetraacetic acid

FCB Fibre cell bundle

G Guaiacyl unit

OMU Corchorus olitorius mutant

PPF Primary phloic fibre

S Syringyl unit

SDS Sodium dodecyl sulfate

SPF Secondary phloic fibre

Introduction

The extraxylary fibres extracted from the stems of the two

cultivated Corchorus species (family Sparrmanniaceae;

Heywood et al. 2007) viz., C. capsularis L. (white jute) and

C. olitorius L. (dark jute) are known as jute (Rowell and

Stout 2007). They are phloic fibres that originate in pri-

mary and secondary phloem (Maiti 1997), but continue to

be termed as bast fibres (Summerscales et al. 2010) despite

inaccurate botanical meaning of the term bast which is

etymologically associated with the verb ‘to bind’ (Esau

1953). Yet, the term bast fibre has been used in this paper

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10725-012-9660-z) contains supplementarymaterial, which is available to authorized users.

A. Kundu � D. Sarkar (&) � N. A. Mandal �M. K. Sinha � B. S. Mahapatra

Biotechnology Unit, Division of Crop Improvement,

Central Research Institute for Jute and Allied Fibres,

(CRIJAF), Barrackpore, Kolkata 700 120, West Bengal, India

e-mail: d.sarkar@excite.com; debabrata_s@yahoo.com;

dsarkar@crijaf.org.in

123

Plant Growth Regul (2012) 67:45–55

DOI 10.1007/s10725-012-9660-z

since as compared to cotton and wool commercial phloic

fibre (flax, jute, kenaf and ramie) denotes a fibre strand

(Fahn 1990; Maiti 1979, 1997; Maiti et al. 2011).

Jute fibres develop in phloem as triangular wedges of

sclerenchymatous fibre cell bundles (FCBs) intermingled

with medullary ray cells and other soft tissues (Kundu

1944). Each wedge tapers outward and contains 8–24 FCB

layers in transversal sections of the stem (Kundu et al.

1959; Hazra and Karmakar 2008). The FCB layers form a

tubular mesh that encases the entire stem from top to

bottom, and the whole fibre sheath, after extraction through

a process known as biological retting, forms a flat ribbon in

three dimensions (Rowell and Stout 2007). A single jute

fibre is represented by an FCB that forms one of the links

of the tubular mesh; each cell of an FCB is the ultimate

fibre cell, which is polygonal in shape and characterized by

thick wall and narrow lumen (Maiti 1979; Palit 1999).

However, as compared to other bast fibre crops, such as

flax and ramie, jute is characterized by high lignin content

(ca. 13–15%) in the secondary wall of the fibre (Palit et al.

2001; Palit and Meshram 2008; Del Rio et al. 2009; Sarkar

et al. 2010).

Earlier it was suggested that all the fibres in jute including

the first-formed protophloic fibres are secondary in origin

from cambium, and therefore secondary phloic in nature

(Kundu 1956; Kundu et al. 1959). Later studies, however,

unequivocally confirmed the presence of two types of fibres-

primary phloic fibre (PPF) that develops from procambium

in the protophloem region through cell division and modi-

fication, and secondary phloic fibre (SPF) that develops from

cambium by the activity of fusiform and ray initials (Maiti

and Mitra 1972; Maiti 1980; Mitra 1984; Hazra and Kar-

makar 2008). In a mature jute plant, PPF and SPF account for

about 10 and 90% of the total FCBs, respectively (Rowell

and Stout 2007; Hazra and Karmakar 2008).

Much of our current understanding of bast fibre devel-

opment in jute comes from identification and character-

ization of a development-specific deficient lignified phloem

fibre (dlpf) mutant of white jute (Sengupta and Palit 2004).

In this mutant, lignification is specifically suppressed in the

secondary walls of the SPF at an early stage of plant

growth, but restored after the plant reaches a certain stage

of maturity (Palit et al. 2006a). As a result, secondary

FCBs, typical of jute, are replaced by comparatively thin-

walled SPF cells. The authors concluded that the lack of

sufficient lignified SPF cells might be the reason that

secondary FCBs do not develop (Sengupta and Palit 2004;

Palit et al. 2006a). Since the development of the other

vascular tissues is unaffected in the dlpf mutant irrespective

of developmental stages (Sengupta and Palit 2004; Palit

and Meshram 2008), it is assumed that lignification of the

secondary walls of the SPF bundles is one of the key

determinants for bast fibre development in jute.

We report here an induced secondary phloic mutant of

dark jute (C. olitorius), which is defective in bast fibre

development, but develops normal lignified SPF bundles

and produces lignin-rich retted fibres comparable to its

wild-type. It has been designated as bast fibre-shy (bfs)

corresponding to the gene symbols Bf and bf proposed

earlier for fibrous and non-fibrous jute stems, respectively

(Mitra 1984). Because this mutant is also defective in

secondary xylem (wood) development, it appears to rep-

resent a loss-of-function mutation of the vascular cambium.

Our study also goes in some way towards giving an insight

into bast fibre development in jute in relation to its yield

and quality characteristics.

Materials and methods

Plant material

A mutant library of Corchorus olitorius L. (2n = 2x = 14)

comprising a total of 56 mutants (OMU 001-OMU 020

and OMU 022-OMU 057) was used in the present study

(Mahapatra et al. 2006). They were developed by physical

(X-rays, gamma rays, thermal neutrons and pile neutrons)

or chemical (EMS) mutagenesis of dry seeds and isolated

either in the M2 or M3 generations. While the wild-types of

many of the mutants are unknown, C. olitorius cv. JRO 632

is the wild-type of some of the uncharacterized mutants

including the secondary phloic (bast) fibre-shy (bfs) mutant

[OMU 043 (dissected ribbon) induced by thermal neutron]

being reported here (Joshua et al. 1972; Thakare et al.

1973). Therefore, in the present study, JRO 632 was used

as a control check for bast fibre development including

lignification of the secondary walls of the fibre cells.

Phenotypic screening of the mutant library

Seeds of true-breeding lines of these 56 mutants and wild-

type JRO 632 were collected from the gene bank of the

Central Research Institute for Jute and Allied Fibres

(CRIJAF), Kolkata. They were grown in the CRIJAF

experimental field (22.45�N, 88.26�E; 3.14 m above msl)

during the summer (March-September; mean day/night

temperature: 32.7/24.2�C; RH: 68.8–93.7%) following the

recommended cultural practices. Fertilizer was applied at

the rate of 40 kg N, 20 kg P2O5 and 20 kg K2O per hectare

at sowing time, with N 50% as basal and 50% as top dress

at 30 days after sowing. Adequate measures were taken to

avoid abiotic and biotic stresses that may affect plant

growth and bast fibre yield. The experiment was conducted

in a randomized complete block design (RCBD) with three

replications over the two growing seasons. Each mutant

46 Plant Growth Regul (2012) 67:45–55

123

was represented by a single row of 10 plants each at a

spacing of 10 cm in rows 30 cm apart per replication.

Determination of bast fibre yield and quality parameters

At 120 days after sowing, the matured plants were har-

vested and steep-retted in a water tank for bast fibre

extraction. Retting was periodically assessed by inspecting

few plants each day from 10th day onwards till the fibre

releases easily from the wood. After drying, wood and bast

fibre yields (g plant-1) were determined. A bundle strength

tester (Bandopadhyay and Mukhopadhyay 1964) was used

to measure the tensile strength (g tex-1) by determining the

breaking load (kg) of clean and dry fibre strands (12.5 cm

long/200–300 mg) collected from the middle portion of the

fibre reeds and calculating the ratio of breaking load to its

linear density (tex or g km-1). An airflow fineness tester

(Sinha and Bandopadhyay 1968) was used to measure fibre

fineness (tex in a 0–5 scale with decreasing fibre fineness)

by determining the rate of airflow through a parallel fibre

plug of fixed mass and length in a cylindrical cell of fixed

dimensions at a particular pressure difference across the

fibre plug.

Bast fibre lignin estimation

The lignin content of the retted fibres was estimated as acid

detergent lignin (ADL) using FibreCapTM 2021 of the

FibertecTM 2021 system (FOSS Analytical A/S, Hilleroed,

Denmark). In brief, dried fibre sample was finely cut to a

particle size of\1mm, and 1.0 g of sample was weighed to

an accuracy of ±0.1 mg into each FibreCapTM capsule

secured with the lid. The capsules were inserted into the

capsule tray which was treated with 350 ml acetone for

5 min in an extraction beaker followed by washing with

warm water for 5 min. The capsule tray was subsequently

transferred to an extraction beaker containing 350 ml

neutral detergent solution (0.05 M EDTA disodium salt

dihydrate, 0.02 M sodium borate decahydrate, 0.1 M SDS,

0.03 M disodium hydrogen phosphate and 10 ml triethyl-

ene glycol) and placed on a hot plate to boil for 60 min.

After boiling, the capsule tray was washed twice in boiling

water followed by de-fatting of the fibre residue by

washing once with acetone, according to manufacturer’s

instructions (Anonymous 2008). The capsule tray was

transferred to a drying stand and treated with 350 ml of

72% H2SO4 (cooled to 15�C) in a de-fatting beaker fol-

lowed by several washing with boiling water to completely

remove the acid. The capsules were dried in an oven at

120�C for 3 h, cooled to room temperature for about

30 min, weighed to an accuracy of ±0.1 mg and placed in

pre-weighed ashing crucibles. The samples were ashed in a

Muffle furnace at 600�C for at least 4 h, and the ashing

crucibles were slowly cooled to room temperature. The

final weights of the ashing crucibles were recorded with a

precision of ±0.1 mg.

A blank was run through the entire estimation along

with each batch of samples to determine a correction fac-

tor, which is expressed as C = blank capsule weight after

extractions/blank capsule weight at start. The percentage of

lignin was calculated as follows:

% lignin ¼W3 � W1 � Cð Þ � W5 �W4 � Dð ÞW2

� 100

where W1 = initial capsule weight (mg), W2 = sample

weight (mg), W3 = capsule plus residue weight (mg),

W4 = empty ashing crucible, W5 = total ash (mg) and

D = capsule ash (mg).

Histochemical determination of lignification

Fresh free-hand sections were prepared from upper, middle

and lower stem segments of 70-day- and 120-day-old bfs

mutant and its wild-type JRO 632, and stained without

fixation, according to Lux et al. (2005). Native lignin of

secondary cell wall was stained with the Maule (KMnO4-

HCl) and Weisner (phloroglucinol-HCl) reagents (Dashek

1997), which give varying intensities of red coloration with

lignin syringyl monomers and cinnamaldehyde groups,

respectively. For Maule reaction, the tissue was first treated

with 1% (w/v) aqueous KMnO4 for 5 min, washed thor-

oughly with water and then treated with 3% HCl until the

color changes to beige. Finally, concentrated NH4OH was

added to develop purple-red coloration. For Weisner

reaction, a 2% (w/v) solution of phloroglucinol was pre-

pared in 95% ethanol and stored in a sealed amber color

bottle. Prior to use, two parts of phloroglucinol was mixed

with one part of concentrated HCl, and the tissue sections

were stained for 2–3 min. Observations were made under a

Zeiss Axioskop 40 (Carl Zeiss, Jena, Germany) bright field

microscope and a Canon PowerShot A80 camera system.

Histological determination of fibre content

Fresh free-hand transversal sections were prepared and

stained by the Weisner reaction as above. Histological

determination of fibre content is schematically shown in

Fig. 1. In the transversal sections of the stem, the fibre cell

bundles (FCBs) are arranged as triangular wedges, the

broader side being adjacent to the cambium. It is known

that the shape of a triangular wedge and the number of

FCBs in it depend on the number of FCB layers and the

number of FCBs in the first layer nearest the cambium

(Kundu et al. 1959). Since the number of FCB layers are

not constant over the wedges, a mean ratio (FR) of the total

number of FCBs to the number of FCBs in the first FCB

Plant Growth Regul (2012) 67:45–55 47

123

layer nearest the cambium was determined based on 10–15

wedges selected at random in the entire transversal section.

Next the total number of FCBs in the first FCB layer

nearest the cambium was counted in the entire transversal

section over all the wedges (FC). The total number of FCBs

in the entire transversal section (FT) was calculated as

FR 9 FC that measures the fibre content of the stem.

Statistical analyses

For histological analyses, there were six independent rep-

licates for each treatment. Prior to parametric statistical

analyses, data were transformed into log10x or log10(x ? 1)

as applicable. Transformed data were analyzed by one-way

(phenotypic data) or two-way (histological data) analyses

of variance (ANOVAs) or Student’s unpaired t test (phe-

notypic data). Means were separated by post-hoc least

significant difference (LSD) test or Tukey’s honestly sig-

nificant difference (HSD) test. The term significant has

been used to indicate differences for which P B 0.05.

GenStat Version 11.1.0.1504 (VSN International Ltd.,

Oxford, UK) and MSTAT-C (Michigan State University,

Ann Arbor, USA) were used for statistical analyses.

Results

Phenotypic screening of a dark jute (C. olitorius) mutant

library (56 accessions) over the two successive growing

seasons showed wide variation in bast fibre fineness, lignin

content, tensile strength, yield and wood yield (Table 1).

The maximum variation was observed for lignin content

followed by wood yield and bast fibre yield. Primarily

based on bast fibre and wood yields, one of the accessions,

namely, OMU 043 was selected from this mutant library

and designated as bast fibre-shy (bfs). The phenotypic data

for the 55 mutants, except for bfs, are shown in Table S1.

Morphology and growth

The bfs was a dwarf mutant, with a significantly shorter

plant type than its parental wild-type JRO 632 (Fig. 2;

Table 2). At 120 days after sowing it had significantly

lesser number of nodes per plant and shorter diameters

along the three spatially differentiated stem regions

(Table 2). Except for number of nodes per plant, the veg-

etative growth habit of the mutant was also similar at

70 days after sowing. However, the most important

Fig. 1 A schematic diagram

showing triangular wedges of

sclerenchymatous fibre cell

bundles (FCBs) flanked by

medullary ray cells in the

transversal stem section of

Corchorus olitorius. The

circumferential FCB layer

(ring) represents the primary

phloic fibres, while the inside

layers towards the cambium are

secondary phloic fibres.

The number of wedges in the

transversal section and the

number of FCB layers per

wedge are arbitrarily drawn,

without conforming to their

actual numbers in planta

Table 1 The range of bast fibre yield and quality, and wood yield valuesa with means, in the dark jute (Corchorus olitorius) mutant library

characterized in the study

Trait Minimum Maximum Mean SD cv (%)

Fibre fineness (tex) 1.53 3.38 2.48 0.37 14.8

Fibre yield (g plant-1) 1.60 19.60 10.27 3.06 29.8

Lignin content (%) 7.95 28.52 14.93 5.68 37.8

Tensile strength (g tex-1) 12.30 26.77 20.17 2.80 13.9

Wood yield (g plant-1) 6.00 50.60 26.44 7.93 30.0

a Pooled over 56 mutants over three replications over the two independent growing seasons

48 Plant Growth Regul (2012) 67:45–55

123

diagnostic phenotype of this mutant was dissected ribbon

leaves (Fig. 2). As compared to oblong, nearly glabrous

and serrated leaves of the wild-type, the bfs mutant could

be easily distinguished by minute trifid leaves, which, on

maturity, are discontinuous in the margins and dissected

into irregularly shaped ribbons supported by the major

veins (Fig. 3A, B). A distinct change in the root system

including architecture also occurred in the mutant (Fig. 3C,

D). It exhibited pre-mature flowering as compared to its

wild-type that usually takes 130–140 days to induce 50%

flowering (Karmakar et al. 2008). Under typical short-day

(10 h) conditions, flowering was induced in the mutant at

as early as 30–40 days after sowing (Fig. 2). Flowers were

yellow in color, two-fold smaller than that of the normal

type and pentamerous, however, with constricted petals,

which are narrower than the sepals giving an appearance

of bracts. Over the two successive growing seasons, the

mutant bred true and produced short slender capsules with

copious seeds; seed germination was comparable to the

wild-type.

Bast fibre quality and yield

After 120 days from sowing the harvested stems were

retted for the separation of fibre strands, and the optimum

retting duration was uniform in the mutant and the normal

type. The bast fibre yield of the mutant (1.64 g plant-1)

was ten-fold lesser than that of its wild-type (16.51 g

Fig. 2 Phenotypes of dark jute

bfs mutant (A) and its wild-type

JRO 632 (B) at 70 days from

sowing, with corresponding

phloroglucinol-HCl-stained

histological transversal sections

(9100) of upper, middle and

lower stem segments of

120-days-old plants. Triangular

FCB wedges are distinct in the

lower stem segment of the wild-

type. Arrows indicate flowering

induction in the mutant. cacambium, ep epidermis, FCBfibre cell bundle, PPF primary

phloic fibre bundle, mrcmedullary ray cells, pp pith

parenchyma, SPF secondary

phloic fibre bundle, sxysecondary xylem

Table 2 Morphology of dark jute bfs mutant and its wild-type JRO 632 at the two different growth stages

Genotype Plant height

(cm)

Number of nodes

(plant-1)

Lower stem diameter

(cm)

Middle stem diameter

(cm)

Upper stem diameter

(cm)

(a) 70 days after sowing

JRO 632 128.8 29.7 0.76 0.63 0.44

bfs 79.2*** 26.5ns 0.44*** 0.38*** 0.21***

(b) 120 days after sowing

JRO 632 372.7 82.7 1.80 1.36 0.46

bfs 191.0*** 66.7*** 0.96*** 0.70*** 0.30**

**, ***, ns Significantly different at P B 0.01 and 0.001, and non-significant at P B 0.05, according to Student’s unpaired t test

Plant Growth Regul (2012) 67:45–55 49

123

plant-1). There was a significant difference in wood yield

between the mutant and its wild-type; about a seven-fold

decrease in wood yield per plant occurred in the mutant at

120 days after sowing (Table 3). The bast fibre of the

mutant was significantly coarser and lower in tensile

strength than that of the normal type. However, there was

no significant difference in bast fibre lignin content

between the two genotypes (Table 3). Both of them were

characterized by about 9% bast fibre lignin, a value typical

of C. olitorius cultivars.

Fibre content

For the mutant and the normal type, the FCB parameters

were examined in the cross-sections of the three spatially

differentiated stem segments. There were significant

genotype 9 stem segment interactions for the mean FCB

layers per wedge, FC, FR, and FT, indicating that the spatial

effect of the stem segment on fibre biogenesis was not

uniform over the two genotypes. After 70 days from

sowing only one FCB layer could fully develop per wedge

at the upper stem regions of both the mutant and the normal

type (Fig. 4). Whereas a progressively higher mean FCB

layers developed per wedge at the middle and lower stem

regions of the mutant than those of the normal type. On an

average, three FCB layers developed per wedge at the

upper stem regions of the mutant and the normal type after

120 days from sowing. However, there were significantly

fewer FCB layers per wedge at the middle and lower stem

regions of the mutant than those of the normal type

(Fig. 4).

Table 4 shows the mean values of FC, FR and FT in

relation to spatially differentiated stem regions at the two

distinct plant growth stages. Irrespective of genotypes and

growth stages, a progressive increment in fibre content

Fig. 3 Diagnostic morphology of dark jute bfs mutant (A, C) in

comparison with its wild-type JRO 632 (B, D). The mutant is

characterized by trilobed dissected ribbon leaves and a spreading-type

root architecture

Table 3 Bast fibre yield and quality parameters, and wood yield of dark jute bfs mutant and its wild-type JRO 632 at 120 days after sowing

Genotype Fibre yield (g plant-1) Fibre fineness (tex) Tensile strength (g tex-1) Lignin content (%) Wood yield (g plant-1)

JRO 632 16.51 1.81 15.98 9.40 48.66

bfs 1.64*** 2.35*** 12.52*** 9.34ns 7.36***

***, ns Significantly different and non-significant at P B 0.001 and 0.05, respectively, according to Student’s unpaired t test

Fig. 4 Mean layers of fibre cell bundles per triangular wedge in the

transversal sections of the three different stem segments of dark jute

mutant bfs and its wild-type JRO 632 at the two different growth

stages (70 and 120 days after sowing). Means with common letters at

a growth stage are not significantly different at P B 0.05, according to

Tukey’s honestly significant difference (HSD) test

50 Plant Growth Regul (2012) 67:45–55

123

(FT), which is a function of FC and FR, occurred along the

length of the stem from top to bottom. After 70 days from

sowing fibre content was marginally but significantly lesser

at the middle and lower stem regions of the mutant than

that of its wild-type. However, after 120 days from sowing,

the wild-type had significantly higher fibre content than the

mutant at all the three spatially differentiated stem regions.

The magnitude of the difference in fibre content between

the two genotypes did progressively increase with

decreasing stem length (Table 4). As compared to the

normal type, there was nearly a three-fold reduction in fibre

content at the lower stem region of the mutant.

Lignification of fibre cells

The distribution of lignin in the secondary cell walls of

freshly prepared cross-sections was detected by histo-

chemical staining using the Maule (KMnO4-HCl) and

Weisner (phloroglucinol-HCl) reactions (Figs. 2 and 5).

Jute xylem was found to be highly lignified because both

stains gave an intense red coloration of the xylem in the

mutant and its wild-type. The Weisner reaction gave

characteristic pinkish coloration of jute fibre cell lignin

(Sengupta and Palit 2004), and no difference in the

phloroglucinol-HCl staining intensity for lignin could be

detected between the mutant and the normal type (Fig. 2).

Neither any difference in the phloroglucinol-HCl staining

intensity could be detected for the fibre cell lignin in cross-

sections of the three spatially differentiated stem segments.

Maule reaction gave characteristic purple-red coloration of

the fibre cell lignin in both genotypes (Fig. 5). However,

no difference in lignification of secondary fibre walls

between the mutant and the normal type could be detected

(Fig. 5C, D).

Architecture of fibre cell bundles

KMnO4-HCl- and phloroglucinol-HCl-stained stem cross-

sections revealed marked difference in the architecture of

the FCB wedges between the mutant and its wild-type

(Figs. 2 and 5). Typical narrow and triangular FCB wedges

with outward tapering apices developed at the lower stem

region of the normal type (Figs. 2 and 5B). In contrast, the

mutant had broad or irregularly shaped FCB wedges that

did not maintain a straight course and often overlapped

with each other (Fig. 5A). Atypical wide rectangular

wedges often formed in the mutant, especially at the

middle stem region (Fig. 2). Besides, the medullary ray

cells flanking the FCB wedges were more compressed and

stretched at the lower stem region in the mutant than in the

normal type (Fig. 5A, B). As compared to the normal type,

the mutant’s secondary FCBs had fewer ultimate fibre

cells, and they were often branched and fused or rearranged

into new bundles (Fig. 5C, D). In the lower stem cross-

sections of the mutant, even 2–3 ultimate fibre cells were

found to constitute a secondary FCB. In general, as com-

pared to the normal type, the ultimate fibre cells of the

mutant were slightly wider and characterized by larger

lumens with unevenly thickened walls (Fig. 5A, C).

Discussion

In parallel with a fibreless (trichomeless) mutant of cotton

(Turley 2002), the idea of an induced or spontaneous bast

fibreless mutant of jute, although much attractive, appears

to be conceptually challenging, if not botanically impos-

sible. This may be because PPF and SPF that together

constitute the bast fibres in jute (Rowell and Stout 2007)

Table 4 Fibre cell bundle (FCB) parameters and fibre contenta in the transversal sections of the three different stem segments of dark jute bfsmutant and its wild-type JRO 632 at the two different growth stages

Stem segment FR FC FT (FR 9 FC)

JRO 632 bfs JRO 632 bfs JRO 632 bfs

(a) 70 days after sowing

Upper 1.00 e 1.00 e 157.80 c 154.30 cd 157.80 e 154.30 e

Middle 1.80 d 1.90 c 220.80 a 179.70 b 396.04 c 340.61 d

Lower 4.02 a 3.42 b 147.40 e 150.50 de 592.29 a 514.74 b

(b) 120 days after sowing

Upper 2.47 f 2.70 e 302.60 c 204.40 e 746.30 e 552.20 f

Middle 5.85 b 4.90 d 516.00 b 242.20 d 3,015.50 b 1,185.90 d

Lower 6.19 a 5.55 c 774.80 a 290.40 c 4,796.86 a 1,609.94 c

Means with common letters at a growth stage are not significantly different at P B 0.05, according to Tukey’s honestly significant difference

(HSD) testa FR the mean ratio of the total number of FCBs in a wedge to the number of FCBs in the first FCB layer nearest the cambium, FC the total

number of FCBs in the first FCB layer nearest the cambium in the entire transversal section over all the wedges, FT fibre content, i.e. the total

number of FCBs in the entire transversal section (see Fig. 1 for details)

Plant Growth Regul (2012) 67:45–55 51

123

are regarded as a part of the vascular tissue phloem (Kundu

et al. 1959; Evert and Esau 2006). In the present study,

we have identified a unique radiation-induced bast fibre-

development mutant of dark jute, which is concomitantly

defective in the development of SPF and secondary xylem

(wood) without affecting lignification of the walls of either

of these tissues. Our study shows that mutation could

specifically impair the cambial activity by rendering those

initials that differentiate the SPF and secondary xylem

nonfunctional. Recent results have shown that the cambial

meristematic cells (plant stem cells) are hypersensitive to

radiation (c-irradiation) (Lee et al. 2010). This mutant is,

however, in contrast to an earlier reported dlpf mutant of

white jute (Sengupta and Palit 2004), which is normal in

cambial activity but developmentally defective in lignifi-

cation of the SPF cells at an early stage of plant growth

(Palit et al. 2006a) vis-a-vis maturation of SPF since

lignin deposition and condensation over the fibre wall is

associated with bast fibre maturation (Day et al. 2005).

Therefore, as compared to the bfs mutant under study, the

reported reduction of bast fibre yield in the dlpf mutant

accrues from immature thin-walled SPF cells that not only

fail to assemble into FCBs but also get disintegrated by

microbial degradation upon biological retting (Sengupta

and Palit 2004; Palit and Meshram 2008). It is well known

that the absence of lignin in the fibre wall renders it more

permeable to microbial degradation (Lewis and Yamamoto

1990).

Our study most convincingly demonstrated that a

marked reduction of bast fibre yield in the bfs mutant was

associated with a development-specific loss of cambium

function along the length of the stem from to top to bottom.

The difference in cambial activity, measured in terms of

histological fibre content and wood yield, between the

mutant and its wild-type was reflected in their respective

bast fibre yield. Since both normal and mutant bast fibres

Fig. 5 KMnO4-HCl-stained (Maule reaction) lower stem segments of 120-days-old dark jute mutant bfs [A (9100), C (9400)] and its wild-type

JRO 632 [B (9100), D (9400)]. Arrows indicate fibre cell ultimates with large lumens. tc tannin-containing cell; other acronyms as in Fig. 2

52 Plant Growth Regul (2012) 67:45–55

123

had similar lignin content and were characterized by the

same rate of lignification of their respective fibre wall (see

below), there are no reasons to believe that biological

retting could have compromised the results anyway. In

general, the increment percentage of FCBs in jute reaches

optimum during the period form 70–84 days (Maiti and

Mitra 1972), while bast fibres mature at 120 days after

sowing (Kundu et al. 1959; Mitra and Maiti 1974; Maiti

and Satya 2010). This was the basis for the two develop-

mental stages (70 and 120 days) of plant growth used in the

study to distinguish the intensity of cambial activity

between the mutant and the normal type.

Correlated activities of apical and intercalary meristems

regulate plant height and bast fibre production in jute

(Kundu et al. 1959; Maiti and Mitra 1972; Mitra and Maiti

1974). However, the optimum period of increment of plant

height does not coincide with that of fibre production at the

lower and middle stem regions, suggesting a possible

negative correlation between the two meristems (Maiti and

Mitra 1972). This could perhaps explain as to why the bfs

mutant despite being much shorter in height than the nor-

mal type developed significantly more number of FCB

layers per wedge at the lower and middle stem regions after

70 days from sowing. As a result, a marginal difference in

fibre content could be observed between the mutant and the

normal type at an early growth stage. However, at later

growth stages, development-specific inactivation and/or

loss of cambium initials could not allow the mutant to

support prolonged cambial activity, which is required to

foster the development of secondary FCBs (Maiti and

Mitra 1972; Hazra and Karmakar 2008), and consequently

a drastic reduction in fibre content vis-a-vis bast fibre yield

occurred. The declining cambial activity of the mutant was

coincident with its advanced reproductive maturity, which

is known to cease vertical growth and reduce the produc-

tion of FCBs in jute (Rowell and Stout 2007).

The Weisner reaction, which is exclusively used to stain

native lignin of jute fibre wall (Palit et al. 2001; Palit and

Meshram 2004; Sengupta and Palit 2004; Palit et al.

2006b), is not specific to lignin because other cell wall-

associated phenolic monomers (cinnamyl alcohols/cinn-

amaldehydes) also react positively with phloroglucinol-

HCl (Clifford 1974; Day et al. 2005). In the present study,

Maule reagent that specifically reacts with S lignin (Me-

shitsuka and Nakano 1979) positively stained cell wall

regions which were also stained positive with the Weisner

reagent, thereby confirming their lignified nature. Intense

staining of FCBs in transversal stem sections of the mutant

and the normal type using both reagents is indicative of

higher lignin content in jute fibres than in other bast fibres

(Sarkar et al. 2010). Maule-positive staining suggests that

jute xylem lignin is G-S type typical of dicotyledons (Iiy-

ama and Pant 1988), whereas its bast fibre lignin is rich in

S type. This is in agreement with the chemical profile of

jute fibre lignin, which is reported to have a relatively high

(2.1) S: G ratio (Islam and Sarkanen 1993; Jahan et al.

2007; Del Rio et al. 2009). In flax fibres, an S type-poor

lignin has been successfully detected using the Maule color

reaction (Day et al. 2005). Furthermore, no difference in

the Maule-staining intensity of FCBs between the mutant

and the normal type suggests the presence of similar lignin

types. Our study also shows that lignification of the fibre

wall in the mutant is not only normal but also uniform

along the length of the stem from top to bottom, suggesting

that the cambium function may not be related to the lig-

nification process and the signals for both of them may be

transduced through separate pathways. Sengupta and Palit

(2004) have suggested that the signal for lignification may

remain blocked in the SPF cells at an early growth stage

but starts to function at later growth stages. Taken both

results together, it is possible that there may exist a coor-

dinated regulatory pathway of cambium function and lig-

nification process in plants.

Since the mutant and the normal fibres are similar with

regard to lignin content and types, significantly lower

tensile strength of the former lends a strong support to the

view that lignin does not account for bast fibre strength

(Sengupta and Palit 2004; Palit et al. 2006b; Palit and

Meshram 2008). It is the a-cellulose (Palit and Meshram

2008) or more specifically cellulose: hemicellulose ratio

that conditions the tensile strength of the bast fibre

(Sarkar et al. 2010). The normal fibre in our study was,

however, significantly finer than the mutant one. Fibre

fineness is a complex trait that depends on the structure

and wall thickness of the ultimate fibre cells (Kundu et al.

1959; Maiti 1979; Majumdar 2002; Palit et al. 2006b;

Satya et al. 2011) and their radial and/or tangential mode

of joining to form the FCBs (Palit et al. 2006a; Hazra and

Karmakar 2008). However, the mutant fibre cells or

FCBs, as characterized in our study, are prone to be finer

upon retting. One interpretation of this ambiguity would

be a relatively higher degree of meshiness of the mutant

fibre than the normal one. Fibre meshiness results from

the deformities of mostly triangular FCB wedges (Kundu

et al. 1959) due to intrusive growth of medullary ray cells

flanking the wedges, resulting in loops of different shape

and volume upon retting (Palit et al. 2006a). Since there

exists a negative correlation between fibre meshiness and

fineness (Palit et al. 2006a), the mutant, which is char-

acterized by deformed FCB wedges at the lower and

middle stem regions, is expected to produce coarser fibre.

Tangentially expanding medullary rays upon stretching

might have deformed the FCB wedges, which are archi-

tecturally weak in the mutant due to lesser number of not

only FCBs per wedge but also fibre cell ultimates per

FCB.

Plant Growth Regul (2012) 67:45–55 53

123

An extremely unusual phenotype of the bfs mutant with

altered leaf shape and stunted growth indicates changes in

hormonal function and/or regulation, particularly of GA3,

which is known to affect the differentiation and growth of

phloic fibres (Aloni 1988; Aloni et al. 1990). In a pre-

liminary glasshouse experiment, concurrent application of

exogenous GA3 (100 ppm) to the mutant and its wild-type

removed the phenotypic difference in plant height between

them. Since exogenous GA application is a reliable test to

qualify a mutant as a GA response type (Ross 1994), we

have every reason to believe that there must be severe

disruption of endogenous GA biosynthesis and/or trans-

duction in the bfs mutant. Whether the mutant is a GA

response or synthesis type and how endogenous GA affects

its secondary phloic fibre development in relation to the

normal type will be an interesting line of research in future.

In conclusion, our results indicate that development-

specific cambium function, not lignification of the secondary

wall, is the key to the development of SPF cell bundles in

jute. Lignin may be responsible for the maturation of fibre

cells (Sengupta and Palit 2004; Day et al. 2005), but it does

not influence the quality characteristics of bast fibres in

terms of tensile strength and fibre fineness. A balanced

growth between radially elongating fibre cell bundles and

tangentially expanding medullary ray cells due to develop-

ment-specific activation of the fusiform and ray initials of

the cambium maintains the mostly triangular architecture of

fibre bundle wedges. Any imbalance in the growth of these

two tissues deforms the fibre bundle wedges, resulting in the

production of inferior quality coarser fibres upon biological

retting. The bfs mutant represents a valuable resource for

genomics-assisted dissection of SPF differentiation and

signal transduction pathways controlling its biogenesis and

maturation in planta.

Acknowledgments Comments and suggestions on the manuscript

from an anonymous reviewer and the Editor are gratefully

acknowledged.

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