Conservation of Zingiber germplasm through in vitro rhizome formation

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Scientia Horticulturae 108 (2006) 210–219

Conservation of Zingiber germplasm through in vitro rhizome formation

Rishi K. Tyagi *, Anuradha Agrawal, Akkara Yusuf 1

Tissue Culture and Cryopreservation Unit, National Bureau of Plant Genetic Resources, New Delhi – 110 012, India

Received 20 February 2005; received in revised form 26 October 2005; accepted 26 January 2006

Abstract

The effects of various concentrations of maleic hydrazide (MH; 2, 4, 6, 8 mg/l) and three light treatments (16-h, 24-h, 0-h) on in vitro rhizome

formation and conservation of ginger (Zingiber officinale Rosc. cv. Rio de Janeiro) were studied. In vitro rhizome formation occurred in all the

above treatments. Addition of MH (2–8 mg/l) to the control medium (CM) comprising Murashige and Skoog’s (1962) salts, 9% sucrose, 0.8% agar-

agar, 0.1 mg/l a-naphthaleneacetic acid (NAA), 1 mg/l N6-benzyladenine (BA), did not show any significant positive effects on rhizome formation

as well as survival of cultures. A significant effect of light treatments was observed on survival of cultures but not on rhizome formation. More than

50% cultures survived up to 14 months on CM under 16-h and 24-h light conditions as compared to 20% cultures on same medium incubated under

dark. A total of 33 genotypes of cultivated and wild species of Zingiber were subsequently tested for conservation through in vitro rhizome

formation on CM under 16-h light condition. All genotypes produced rhizomes of varying size with numbers ranging from 3 to 15 per culture and

were conserved for at least 12 months; some genotypes could be conserved even up to 16–20 months. Viability of rhizomes was determined by in

vitro regeneration of shoots upon subculture and their subsequent establishment in soil. Following the protocol described in the present paper, some

160 genotypes of cultivated and wild species of Zingiber, collected from different geographical regions of India, are being conserved at In Vitro

Genebank of National Bureau of Plant Genetic Resources, New Delhi.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Ginger; Maleic hydrazide; Micro-rhizome; Multiplication; Slow growth; Storage; Tissue culture

1. Introduction

Ginger (Zingiber officinale Rosc.), a herbaceous perennial

plant of the family Zingiberaceae, is an important spice of

South Asian origin (Baily, 1949). It is cultivated extensively in

South-east Asia and Far East, and propagated vegetatively

through rhizomes. Besides culinary use as a spice, Zingiber

species are also used for their medicinal properties (Shoji et al.,

1982; Sakamura and Suga, 1989; Mustafa and Srivatava, 1990;

Srivatava and Mustafa, 1992; Mustafa et al., 1993).

Plant genetic resources (PGR), comprising cultivated, wild

and weedy relatives, are backbone of any crop improvement

programme. Therefore, conservation of PGR is of crucial

importance for mankind (Ford-Lloyd and Jackson, 1991). Plant

Abbreviations: BA, N6-benzyladenine; CM, control medium; FYM, farm

yard manure; MH, maleic hydrazide or 6-hydroxy-3-(2H)-pyridazinone; MS,

Murashige and Skoog’s (1962) medium; NAA, a-naphthaleneacetic acid

* Corresponding author. Fax: +91 112 585 2496.

E-mail address: [email protected] (R.K. Tyagi).1 Present address: Department of Biotechnology, People’s Education Society

Institute of Technology (PESIT) Campus, Banashankari III Stage, Bangalore –

560085, Karnataka, India.

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2006.01.018

tissue culture techniques have been useful in conservation of

germplasm of vegetatively propagated crops and considered as

an alternative to conventional field genebanks to safeguard

against pests and environmental vagaries (Dodds, 1991).

Application of slow growth methods (chemical and physical)

in tissue cultures is a convenient option for conservation of

germplasm of vegetatively propagated crops and is now

routinely used for conservation of such crop species (Ashmore,

1997; Mandal et al., 2000, Tyagi and Prakash, 2004; Tyagi

et al., 2004). A range of chemicals has been tested to suppress

the growth of plants (Dodds et al., 1991). MH, a commercially

available growth inhibitor, is known to inhibit growth and to

promote the tuberization in potato cultures (Harmey et al.,

1966). Photoperiodic conditions are also known to affect the

growth of the plants in tissue culture (Banerjee and DeLanghe,

1985; Jean and Cappadocia, 1991; Gopal et al., 1998).

Rapid clonal multiplication of ginger (Hosoki and Sagawa,

1977; Bhagyalakshmi and Singh, 1988; Balachandran et al.,

1990) and in vitro conservation have been reported (Dekkers

et al., 1991) only through multiple shoot induction. Induction of

in vitro storage organs is proven as a potent method for

conservation of potato and yams (Jean and Cappadocia, 1991;

Gopal et al., 1998 Garner and Blake, 1989). Though rhizome

R.K. Tyagi et al. / Scientia Horticulturae 108 (2006) 210–219 211

Table 1

Details of accessions of Zingiber germplasm used for present study

National

identity

Cultivar/Collector

Number

Botanical

name

Collected

from (State)

IC 003842 – Z. officinale Mizoram

IC 029902 150 Z. officinale Mizoram

IC 084985 DKH-27 Z. officinale Meghalaya

IC 085060 V-3906 Z. officinale Kerala

IC 085083a – Z. montanum Andhra Pradesh

IC 089782 BD-19 Z. officinale Arunachal Pradesh

IC 089789 – Z. officinale Arunachal Pradesh

IC 248818 Jamaica Z. officinale Kerala

IC 248821 M&G22 Z. officinale Kerala

IC 248823 Thingara Z. officinale Kerala

IC 248824 V-782B Z. officinale Kerala

IC 248825 Rio de Janeiro Z. officinale Kerala

IC 248844 BDJ/89-1088 Z. officinale Uttaranchal

IC 248851 BDJ/89-1156 Z. officinale Himachal Pradesh






IC 248890 3032 Z. officinale Meghalaya

IC 248893 TCR-410 Z. officinale Kerala

IC 248897 G-66 Z. officinale Kerala

IC-248898 G-42 Z. officinale Kerala



IC 248901 TCR-434 Z. officinale Kerala

IC 248902 SJ-4098 Z. officinale Andhra Pradesh

IC 248903 Dooma Z. officinale Andhra Pradesh


IC 248909a AZ/95-851 Z. wightianum Kerala

IC 248911 Wynad Z. officinale Kerala


IC 260266a YRS-120 Z. montanum Andhra Pradesh

a Wild species.

formation has also been achieved in a few genotypes of ginger

(Sakamura et al., 1986; Bhat et al., 1994; Sharma and Singh,

1995), nevertheless, the application of the developed protocols

has not been demonstrated for conservation of germplasm of

ginger.

We examined the feasibility of slow growth methods

(chemical and physical) by studying the effects of various

concentrations of MH and light treatments on in vitro rhizomes

formation and conservation in Z. officinale cv. Rio de Janeiro.

Subsequently, the optimal method was applied to 33 genotypes

of ginger, which were collected from different geographical

regions of India where variability of ginger is known to occur.

Viability of in vitro-formed and conserved rhizomes was

determined through shoot regeneration.

2. Materials and methods

2.1. Plant material

The effects of MH as well as light treatments on rhizome

formation and conservation were studied only in one

genotype of Z. officinale cv. Rio de Janeiro in different

experiments. Rhizome induction and conservation were

studied in 33 genotypes comprising 30 of Z. officinale

(common ginger), two genotypes of Z. montanum (Koenig)

I. Theilade (=Z. cassumunar Roxb.) (commonly known as Thai

ginger or Bengal ginger), and one genotype of Z. wightianum

Thw. (wild ginger). The details of the same are given in

Table 1.

2.2. Initiation of cultures and explant preparation

Rhizome buds of ginger were collected from sprouted

rhizomes of plants grown in a net house of National Bureau of

Plant Genetic Resources (NBPGR), New Delhi, India. The

buds (ca. 2 cm) were excised and thoroughly washed under

running tap water to remove the adhering soil particles. Outer

scales of the buds were removed and immersed in water

containing 2 or 3 drops of Tween-20 for 20 min and washed 5

or 6 times with distilled water and used as explants to raise the

stock cultures. Surface sterilization of the buds was done

using 0.1% mercuric chloride and 2 or 3 drops of Tween-20

for 10–15 min and subsequently thoroughly washed 7 or 8

times with sterilized distilled water to remove traces of

mercuric chloride. The sterilized buds were cultured onto

medium comprising MS (Murashige and Skoog, 1962)

salts + 3% sucrose + 0.8% agar–agar + 2.5 mg/l BA (Bala-

chandran et al., 1990). In fact, such shoots were maintained in

tissue culture on above medium for 4–10 years through

periodic subculture at 6–8 months interval on MS + 2.5 mg/l

BA (Tyagi et al., 1998). To obtain the stock of in vitro shoot

tip explants used for present study, above shoot cultures were

multiplied and subcultured every 2–3 weeks as described

below.

For all the experiments, shoots were excised from the 2- to 3-

week-old stock cultures maintained on MS + 2.5 mg/l BA and

the leaves and roots were removed. Shoot tip explants (basal 1–

2 cm swollen part of shoot containing shoot tip) were cultured

on MS + 9% sucrose + 0.8% agar-agar + 0.1 mg/l NAA + 1 mg/

l BA [referred to as control medium (CM) hereafter]; cultures

were incubated at 25 � 2 8C at a light irradiance of

40 mmol m�2 s�1 provided by cool white fluorescent lamps

(Philips, Mumbai, India) under 16-h light condition except for

the cultures tested to study the effects of light treatments.

Cultures raised on above medium and culture conditions served

as control.

2.3. Effects of MH as well as light treatments on rhizome

formation and conservation

To study the effects of MH as well as light treatments on

rhizome formation and conservation, one genotype of Z.

officinale cv. Rio de Janeiro was chosen. In one experiment,

four concentrations of MH (2, 4, 6, 8 mg/l) were incorporated in

CM and tested and in another effects of light treatments on

rhizome induction were studied by raising the cultures on CM

and incubating the cultures under three durations of light [16-h

light (16/8-h light/dark), 24-h light (continuous light) and 0-h

light (continuous dark)].

R.K. Tyagi et al. / Scientia Horticulturae 108 (2006) 210–219212

All the cultures were allowed to grow on their respective

media without transfer or subculture and studied for their

conservation.

2.4. Effects of genotypes on rhizome formation

Dictated by the results of above experiments CM and 16-h

light condition was considered best in the experiments

conducted for rhizome formation and conservation of Z.

officinale cv. Rio de Janeiro. Therefore, a total of 33 genotypes

of ginger were tested for further studies on in vitro rhizome

formation and conservation (Table 1).

2.5. Viability of rhizomes through shoot regeneration

After 12 months of conservation, the in vitro-formed

rhizomes of 11 genotypes (IC 003842, IC 084985, IC 085060,

IC 248818, IC 248824, IC 248858, IC 248890, IC, 248899, IC

248900, IC 248901 and IC 248911) that were obtained on CM

and incubated under 16-h light condition, were cultured

aseptically on to MS + 2.5 mg/l BA to test the shoot

regeneration potential. For comparison, shoot tip explants

(basal 1–2 cm swollen part of shoot containing shoot tip) of the

same 11 genotypes (as above) from 3-month-old cultures raised

on MS + 2.5 mg/l BA, were also taken. Both types of explants

(rhizomes and shoot tips) were cultured individually on

MS + 2.5 mg/l BA medium. Data on number of shoots/culture

were recorded after 3 months.

Some in vitro-formed rhizomes (10–15 of each genotype) of

three genotypes (IC 248818, IC 248851, IC 24 8863) were sown

in Petri dishes (9 cm diameter; Borosil, Mumbai, India) lined

with two discs of filter paper (Whatman No. 1) and moistened

with 1/4 strength of MS major salts; germinated rhizomes

transplanted to soil:FYM (1:1) mixture (pH 7.3). Rhizomes of

remaining genotypes were saved for further long-term

conservation experiments.

2.6. Hardening and ex vitro acclimatization

A total of 60 plantlets (30 each regenerated from rhizome

and shoot tip explants), obtained on MS + 2.5 mg/l BA, were

transferred to plastic pots (10 cm diameter) containing soil:

FYM (1:1) mixture (pH 7.3). The plants were covered with

perforated transparent polyethylene bags (20 � 30 cm) and

kept under light provided by incandescent bulbs with irradiance

of 10 mmol m�2 s�1 at 25–30 8C under 10-h photoperiod for 1

week. Polyethylene bags were removed slightly after 1 week

and completely after 2 weeks to expose the plants to ex vitro

conditions. After 3 weeks, the plantlets were transferred to

potted soil.

2.7. Culture conditions

All media were solidified with 0.8% agar-agar. The pH of all

the media was adjusted to 5.8 with 0.1 M NaOH or 0.1 M HCl

prior to addition of the agar–agar and approximately 20 ml

of the medium was dispensed into each culture tube

(25 � 150 mm, Borosil, Mumbai, India). Each culture tube

received one explant. The medium was autoclaved at 121 8Ctemperature and 1.06 kg cm�2 pressure for 20 min. Unless

otherwise stated (for the experiments on light treatment effects)

the cultures were incubated at 25 � 2 8C and a light irradiance

of 40 mmol m�2 s�1 provide by cool white fluorescent lamps

(Philips, Mumbai, India) under 16-h light condition. After

raising cultures aseptically, culture tubes were enclosed with

polypropylene caps (Tarson, India) and sealed with parafilm to

prevent dehydration of media and cultures. All the chemicals

used were of analytical grade (Hi-Media, Mumbai, India and

Sigma, St Louis, USA).

2.8. Data recording and statistical analyses

Number of shoots/culture and shoot length (cm) (between

the base and tip of longest shoot) were measured periodically.

Number of rhizomes/culture and survival percentage of cultures

were recorded periodically by visually counting the rhizomes

and estimating the health of cultures on the basis of survival of

shoots and apparently live rhizomes. For a given culture, the

period of conservation was computed from initiation of cultures

to survival of shoots when the upper leaves showed chlorosis

but the lower leaves and basal shoot portion containing shoot tip

remained green. The data were recorded at every 2-month

interval.

To test the viability and shoot regeneration potential of in

vitro-formed rhizomes and shoot tip explants, data on total

number of shoots/culture were recorded after 3 months from

implanting the explants on MS + 2.5 mg/l BA. No attempt was

made to record the data on precise number of roots/shoot, as

origin of roots from individual shoot was indistinguishable in

culture form.

To study the effects of MH as well as photoperiods on

rhizome formation and conservation, each treatment comprised

18 cultures and repeated 3 times. Data were subjected to

analyses of variance (ANOVA) and Duncan’s Multiple Range

Test (DMRT) using SPSS 10.0 programme to test the

significance of difference among the treatments using general

linear models procedures (SPSS 10.0 version). Data were

recorded for 6–8 replicates (cultures) of each genotype (total 33

genotypes) to study the effects of genotypes on rhizome

formation; mean and standard deviation (SD) were also

calculated. Three replicates were maintained for testing the

viability and to compare shoot regeneration potential of

rhizome and shoot tip explants of 11 genotypes; data were

subjected to DMRT to test the significance of difference in

shoot regeneration in both types of explants.

3. Results

3.1. Initiation of cultures

Rhizome buds sprouted within 3–4 weeks on MS + 2.5 mg/l

BA. Initially, 20–35% cultures established after first surface

sterilization; remaining contaminated cultures were re-sterilized

twice and every time re-cultured onto fresh MS + 2.5 mg/l


Table 2

Analyses of variance [mean sum of square (MSS) and F values] of the effects of MH and photoperiods on rhizome formation and conservation of Z. officinale cv. Rio

de Janeiro

Conservation period (month) Dependent variable Treatment

MH Photoperiod

MSS F-value MSS F value

2a Number of shoots/culture 1.90 2.18ns 5.41 10.93*

Number of rhizomes/culture 2.00 2.50ns 1.00 0.75ns

4a Number of shoots/culture 1.89 1.02ns 12.50 90.70**

Number of rhizomes/culture 2.85 3.97* 0.23 1.02ns

6 Number of shoots/culture 1.74 1.05ns 5.44 13.39**


Survival of cultures (%) 270.50 1.69ns 215.24 2.32ns

8 Number of shoots/culture 4.82 1.36ns 5.18 6.76*


Survival of cultures (%) 143.20 0.51ns 363.19 23.38**

10 Number of shoots/culture 10.55 12.51** 1.03 0.31ns

Number of rhizomes/culture 1.96 1.65ns 0.76 2.98ns

Survival of cultures (%) 527.67 1.34ns 363.19 23.34**

12 Number of shoots/culture 10.25 22.55** 6.20 150.83**

Number of rhizomes/culture 3.24 7.30** 1.08 50.00**

Survival of cultures (%) 2051.84 5.09* 1706.25 32.76**

14 Number of shoots/culture 11.32 7.43** 4.40 8.93*


Survival of cultures (%) 2027.13 3.94* 1706.25 32.76**

nsNon-significant.a All cultures survived up to 4 month under conservation, therefore, no values are shown for survival of cultures (%).* Significant at �0.05 level.

** Significant at � 0.01 level.

medium. Thus, finally ca. 50–60% healthy cultures could be

obtained. In another 2 weeks, 3–5 shoots (1–2 cm) regenerated

on the same medium, which were multiplied and used as stock

cultures to obtain the shoot tip explants for further experiments.

3.2. Effects of MH

The ANOVA analyses (mean sum of square and F-values) of

data recorded for the effects of various concentrations of MH as

well as light treatments on number of shoots and rhizomes/

culture, cultures forming rhizomes (%) and survival of cultures

(%) are presented in Table 2.

3.2.1. Shoot regeneration

Irrespective of the concentrations of MH incorporated in the

tested media, shoot bud formation at the base of the explants

was observed within 10–12 d in 100% cultures of Z. officinale

cv. Rio de Janeiro. Shoot buds developed into shoots in 4 weeks

from culture initiation. In 2-month-old cultures, 3.6–5.6 shoots/

culture were recorded (Fig. 1a). No significant difference was

observed up to 8 months for number of shoots/culture in all the

media tested (Table 2). Maximum number of shoots

(�8 shoots/culture) was recorded in 8-month-old cultures on

CM + 2 mg/l MH and in 10-month-old cultures on CM + 4 mg/

l MH (Fig. 1a). Number of shoots/culture decreased

significantly after 12 months of storage in all the media tested

as older shoots became chlorotic and died; higher concentra-

tions of MH (6 mg/l and 8 mg/l) were found detrimental

to growth of shoots after 10 months. Shoot length was

significantly less in the cultures grown on CM supplemented

with 6 and 8 mg/l MH even after 4 months from culture; about

75% shoots of a culture died on same media in 10- and 12-

month-old cultures, thereby, decreasing the number of shoots/

culture.

There was no significant visible effects of MH was observed

on rooting; it occurred in all the cultures within 2–3 weeks and

with the age of cultures profuse and thick roots were observed.

3.2.2. Rhizome formation

Basal part of the shoots started swelling in 1- to 2-month-old

cultures. However, rhizome formation (2 rhizomes/culture)

occurred only in cultures raised on CM at this age. Generally,

significant difference was observed in number of rhizomes/

culture in older cultures i.e. 4- to 14-month-old cultures

(Table 2). For all the MH concentrations tested, rhizome

formation was recorded in all 4-month-old cultures. However, it

was delayed by 1 month in cultures raised on CM + 8 mg/l MH.

Low concentration (2 mg/l) of MH did not show any significant

difference on rhizome formation as compared to controls, but

higher concentration of MH (4–8 mg/l) was detrimental to

formation of rhizomes. Cultures grown on medium containing

higher concentration of MH also showed rhizome formation but

number of rhizomes/culture were significantly lower than that

on CM. In general, negative effects of different MH

concentrations increased with increasing age of the cultures

but it was most pronounced after 12 months on CM + 8 mg/l. In

14-month-old cultures, rhizomes obtained from CM + 6 and

8 mg/l MH media had dried and found non-viable (Fig. 1b).


Fig. 1. Effects of MH on (a) number of shoots, (b) number of rhizomes and (c) survival of cultures (%) of Z. officinale cv. Rio de Janeiro.

MH significantly affected the percentage of cultures that

formed rhizomes at all the ages of cultures. In 6-month-old

cultures, rhizome forming cultures ranged from 10% (on

CM + 8 mg/l MH) to 65% (on CM). Maximum response for

rhizome forming cultures (65%) was observed in 4-month-old

cultures on CM and CM + 2 or 4 mg/l MH which remained

almost same up to 10 months of conservation.

3.2.3. Survival of cultures

MH significantly effected the survival of 12- and 14-month-

old cultures (Table 2). About 50–60% cultures survived up to 12

months on CM as well as on CM + 2 and 4 mg/l MH media.

Higher concentrations of MH (6 and 8 mg/l) were significantly

detrimental to survival of cultures as about 70–90% cultures

died after 12 months of conservation and the effect was further

pronounced in 14-month-old cultures (Fig. 1c). No culture

survived beyond 14 months on medium supplemented with

8 mg/l MH.

3.3. Effects of light treatments

3.3.1. Shoot regeneration

Shoot regeneration occurred in all the cultures incubated

under the three light conditions tested. Maximum shoot

regeneration (8.5 shoots/culture) occurred in 6-month-old

cultures incubated in dark (Fig. 2a). Number of shoots/

culture increased with the age of the cultures up to 6 months

of storage; it decreased significantly after 10 months of

storage under all the light treatments as older shoots became

etiolated and eventually died. Significant differences were

observed for number of shoots/culture in the cultures

incubated under three light conditions (Table 2). Cultures,

incubated under 16-h and 24-h light, retained 2 and 3 shoots/

culture, respectively even after 14 months of storage. Leaves

of the shoots were pale green and shoot length was

significantly less in 4-month-old cultures incubated in dark

in comparison to that of incubated under 16-h and 24-h light

conditions.

3.3.2. Rhizome formation

Initially, induction of rhizomes was not influenced

significantly by durations of light exposure. However, shoot

cultures incubated in dark produced highest number of

rhizomes/culture (2.8) after 6 months from culture (Fig. 2b).

Cultures, incubated under 16-h and 24-h light, formed

2 rhizomes/culture and almost all remained healthy and viable

up to 14 months whereas in cultures incubated under dark, only

33% had healthy rhizomes after 12 months of conservation.

Some 61–100% cultures formed rhizomes. Irrespective of light

treatments, maximum rhizome production (100%) was

observed in 6-month-old cultures. After 14 months of storage,

healthy and firm rhizomes were recorded in about 60% cultures

under 16-h and 24-h light conditions in comparison to 20%

(significantly different) under dark.


Fig. 2. Effects of photoperiods on (a) number of shoots/culture, (b) number of rhizomes/culture and (c) survival of cultures (%) of Z. officinale cv. Rio de Janeiro.

3.3.3. Survival of cultures

Under all the three light conditions, 100% cultures survived

up to 4 months and no significant difference was observed in

survival of cultures up to 10 months. At 14 months, about 60%

and 62% (not significantly different) cultures survived under

16-h and 24-h light conditions. In contrast, a sharp decline in

survival was recorded in the cultures incubated in dark, as only

20% (significantly different) cultures survived up to 14 months

(Fig. 2c).

3.4. Effects of genotypes on rhizome formation

The results on the effects of MH and light treatments on

rhizome formation and conservation, revealed that the shoot tip

cultures incubated under 16-h and 24-h light conditions on MS

(9% sucrose) + 0.1 mg/l NAA + 1 mg/l BA produced good

rhizomes (Fig. 3a) and such cultures could be conserved for

longer duration than those conserved in other conditions tested.

Therefore, shoot tip cultures of 33 genotypes (cultivated and

wild species) of ginger, which were maintained for 4–10 years

through periodic subculture (6–8 month interval) on

MS + 2.5 mg/l BA, were cultured on MS (9% sucro-

se) + 0.1 mg/l NAA + 1 mg/l BA. To study rhizome formation,

these cultures were incubated under 16-h light condition which

is cost-effective as compared to providing light for 24-h.

Rhizome formation was recorded in all the genotypes

studied; this phenomenon was observed with in 3–4 months

from culture initiation in 100% cultures. A great variation was

observed in number of rhizome/culture in the 33 genotypes.

On an average, it ranged from 3 rhizomes/culture to

15 rhizomes/culture in 12-month-old cultures (Table 3;

Fig. 3b, c). Rhizomes bore the small buds on the surface

covered with thin scales. About 63% genotypes produced

�6 rhizomes/culture. Considerable variation was also

recorded in size (width) of rhizomes within a genotype

(Fig. 3c) and among the genotypes. Mean width of rhizome

ranged from 5.0 to 16.4 mm. Considering 33 genotypes

together, 42% genotypes produced rhizomes of �10 mm size

(width).

3.5. Viability of rhizomes through shoot regeneration

Rhizome and shoot tip explants of all the 11 accessions

exhibited shoot bud regeneration within 1 week which further

transformed into shoots (1–3 cm long) in 4 weeks on

MS + 2.5 mg/l BA (Fig. 3d). Number of shoots regenerated

from rhizome explants was significantly higher than that

regenerated from shoot explants in IC 248858 and IC 248890;

no significant difference was observed in shoot regeneration

capacity of both types of explants in the remaining 9 genotypes

tested (Fig. 4). On the basis of mean value, maximally

7.3 shoots/rhizome in IC 248890 (collected from Meghalaya)

and 4.8 shoots/shoot tip explant in IC 248901 (collected from

Kerala) were obtained on MS + 2.5 mg/l in 3-month-old


Fig. 3. (a). Ginger cultures conserved in vitro for 12 months. Arrows indicate the rhizomes (Bar = 0.64 cm). (b) In vitro-formed rhizomes in 12-month-old cultures

(close-up). Shoots and thick roots are also seen (Bar = 0.8 cm). (c) Variation in size of in vitro-formed rhizomes harvested from single culture after 12 months

(Bar = 1.8 cm). (d) In vitro regeneration of shoots and roots (1-month-old) from rhizomes on MS + 2.5 mg/l BA (Bar = 0.68 cm). (e) Multiple shoot (8 shoots/culture)

and root formation from single rhizome on MS + 2.5 mg/l BAP (Bar = 0.8 cm). (f) Sprouting of rhizomes. Bigger rhizomes sprout early (Bar = 2.0 cm). (g) Normal

growth of plants from sprouted rhizomes, transplanted in potted soil mixture (Bar = 0.5 cm). (h) Hardened plants obtained from rhizome and shoot tip explants, and

surviving in nature (Bar = 0.25 cm).

cultures. Rooting was initiated in 2-month-old cultures.

Irrespective of the explants, profuse rooting occurred within

2 weeks in all the shoot cultures on the parent medium (Fig. 3e).

Shoot buds sprouted within 8–20 days from plating the

rhizomes in Petri dishes; bigger rhizomes sprouted earlier to the

smaller ones (Fig. 3f). Some 12 rhizomes out of 15 in IC

248818, 10 of 12 in IC 248851 and 7 of 10 in IC 248863

germinated. The germinated rhizomes grew normally upon

transfer to potted soil (Fig. 3g).

3.6. Hardening and ex vitro acclimatization

Of 60 plants (30 each from rhizome and shoot tip explants)

obtained on MS + 2.5 mg/l BA and transplanted in soil, 55


Table 3

In vitro rhizome formation on MS (9% sucrose + 1 mg/l BA + 0.1 mg/l NAA in

ginger germplasm (culture age: 12 months)

National identity Number of

rhizomes

Rhizome size

(width in mm)

Mean � S.D. Range Mean � S.D. Range

IC 003842 9.3 � 1.5 8–11 7.0 � 3.2 5–14

IC 029902 4.0 � 0.0 4–8 10.5 � 4.4 5–14

IC 084985 8.5 � 3.9 3–12 10.4 � 6.5 4–20

IC 085060 6.0 � 2.8 4–6 16.4 � 3.9 10–20

IC 085083a 7.8 � 4.3 3–13 9.0 � 5.6 5–20

IC 089782 10.0 � 2.8 8–12 5.8 � 2.0 5–20

IC 089789 4.0 � 1.3 2–5 9.5 � 1.0 8–10

IC 248818 5.0 � 0.8 5–6 7.0 � 2.1 7–10

IC 248821 8.0 � 2.2 5–10 9.0 � 4.3 2–14

IC 248823 9.5 � 2.4 6–11 7.5 � 2.2 3–10

IC 248824 5.5 � 0.7 5–6 8.5 � 2.1 7–10

IC 248825 3.3 � 1.2 2–4 7.5 � 3.3 5–18

IC 248844 15.0 � 3.4 3–5 7.7 � 3.6 4–15

IC 248851 5.0 � 1.8 5–7 8.0 � 2.1 7–10

IC 248858 9.3 � 1.5 8–11 9.0 � 1.0 8–10

IC 248859 6.4 � 1.9 5–9 8.0 � 3.2 4–13

IC 248863 3.7 � 1.5 2–5 12.3 � 6.4 5–15

IC 248868 7.5 � 2.1 6–9 7.0 � 3.2 3–10

IC 248874 10.0 � 2.8 3–10 15.3 � 4.6 9–20

IC 248890 13.5 � 7.2 4–15 15.0 � 2.2 5–20

IC 248893 4.0 � 1.5 4–8 12.5 � 4.2 7–17

IC 248897 14.0 � 1.2 3–14 11.0 � 5.6 5–20

IC 248898 3.7 � 0.8 3–4 10.9 � 4.5 8–9

IC 248899 9.3 � 1.2 8–10 11.8 � 1.9 9–14

IC 248900 4.2 � 0.8 3–5 8.0 � 2.6 4–12

IC 248901 12.5 � 3.4 9–17 5.0 � 2.0 5–18

IC 248902 10.4 � 2.6 8–14 11.0 � 1.0 10–12

IC 248903 5.8 � 1.8 3–8 9.5 � 1.0 3–20

IC 248904 8.1 � 2.7 3–11 10.5 � 5.5 5–20

IC 248909a 5.3 � 2.4 2–10 10.6 � 5.6 5–20

IC 248911 5.7 � 2.3 3–10 7.8 � 3.8 3–20

IC 248917 8.0 � 2.3 4–10 9.8 � 6.3 3–18

IC 260266a 6.6 � 2.6 5–11 13.9 � 3.2 9–18

a Wild species as in Table 1.

Fig. 4. Shoot regeneration in 3-month-old cultures raised from rhizome and

shoot tip explants on MS + 2.5 mg/l BA. Area of collection of respective

genotype is given in parentheses. Vertical bars represent standard deviation.

Values expressed in the form of bars labeled with different letters are sig-

nificantly different at P � 0.05 level using DMRT.

plants survived in nature. It indicates the successful recovery of

functional plants from rhizomes after in vitro conservation for

12 months (Fig. 3h). No significant difference was observed for

survival of plants originated from either rhizome or shoot tip

explants. All plants grew normally in pots.

4. Discussion

Methods of culture initiation and multiple shoot regenera-

tion are well established in ginger (Balachandran et al., 1990;

Tyagi et al., 1998) and similar protocols have been effectively

used for Curcuma belonging to the family Zingiberaceae (Tyagi

et al., 2004). The objective of applying in vitro slow growth

methods (chemical and physical) for germplasm conservation

is to decrease the growth rate of cultures to avoid the frequent

subculture. MH is known to suppress plant growth (Dodds,

1991), which is substantiated by our results wherein shoot

length decreased due to presence of MH in the medium.

However, MH did not enhance the subculture period of Z.

officinale cv. Rio de Janeiro, and higher concentrations of MH

(6 and 8 mg/l) were found lethal. This may be attributed to the

fact that during the long period of conservation, the endogenous

level of MH in shoots had reached to the level of lethality to the

system. Use of growth inhibitors has been found useful for non-

tuberizing wild forms of potato germplasm but the behaviour of

cultures in long-term storage greatly depends on genotypes

which develop degenerated yellow or brown shoots in potato

(Lizarraga et al., 1989; Thieme, 1992).

Photoperiod is reported to play a key role in induction of in

vitro storage organs, such as tubers in potato (Hussey and

Stacey, 1984) and bulbs in yams (Jean and Cappadocia, 1991).

Under 16-h and 24-h light, Dioscorea abysinica and D. alata

produced highest number of tubers, whereas continuous dark

affected the tuberization negatively (Jean and Cappadocia,

1991). In the present study, rhizome formation occurred under

all the three treatments of light, and no significant effect of light

treatments (tested in present study) was observed on induction

of rhizomes in ginger. This may be because unlike potato and

yams, no special physiological requirements have been

reported for rhizome formation in ginger under natural

conditions (Bhat et al., 1994). Survival of cultures (beyond

14 months) was better under 16-h and 24-h light conditions than

under dark condition. Higher rate of survival of the cultures

under light conditions is attributed simply to the efficient

photosynthesis in the cultures and etiolation and early

senescence of plantlets cultured under dark (Gopal et al., 1998).

Rhizome formation in ginger, under high intensity (9000

lux) of continuous light (Sakamura et al., 1986), in only four

genotypes (Bhat et al., 1994), and following two-step method

(Sharma and Singh, 1995) have been reported. However, rate of

success in terms of cultures forming rhizomes have not been

mentioned in earlier works. In our study, we were able to

achieve rhizome formation in 100% cultures of 33 genotypes

(cultivated and wild species) on 9% sucrose medium and under

16-h light of low intensity (1900–2100 lux) and about 63%

genotypes produced �6 rhizomes/culture. This protocol is not

only simple, economic and applicable to large array of

genotypes for in vitro conservation of ginger germplasm, but

also giving higher number of rhizomes a culture.


Considerable variation was observed in number of rhizomes/

culture and size of rhizome among the 33 genotypes tested.

Most of these genotypes belong to cultivated types except the Z.

wightianum and collected from different geographical regions

of India. Influence of genotype on production of in vitro storage

organs is reported in different genotypes of potato (Thieme,

1992) and yams (Jean and Cappadocia, 1991). It is important to

note that all the 33 genotypes produced rhizomes and

successfully conserved for more than 12 months (some

genotypes up to 16–20 months also) on the same medium,

unlike in potato where medium for tuberization was genotype-

specific (Gopal et al., 1998). Further, it is equally important

finding that shoot cultures retained the rhizome formation and

shoot regeneration potential even after maintaining them for 4–

10 years in tissue culture through periodic subculture at 6–8

month interval (see Section 2.2, Para 1). The longest storage of

microtubers of potato varies from 16 to 18 months, and a large

number of potato collections are being maintained in form of

micro-tubers (Thieme, 1992). In our studies, most of the

cultures of 33 genotypes raised for rhizome formation and

conservation were terminated after 12 months for recording the

data but some of the cultures could be conserved through in

vitro rhizome formation even up to 16–20 months. Till the

availability of this protocol, Zingiber germplasm was being

conserved in the form of shoot cultures through periodic

subculture at 6–8 months interval in our laboratory.

The main objective of present study was to develop a

protocol for conservation of ginger germplasm which is

achieved by maintaining the cultures in culture tubes (25 mm

diameter). As the rhizomes were produced aseptically, it may

prove as good source of disease-free material for planting in the

field as natural rhizomes under storage are known to be infected

with many pathogens (Sakamura and Suga, 1989; Mukherjee

et al., 1995). Irrespective of size, rhizome germinated in vitro

but delayed germination or no germination was observed in

smaller rhizomes grown in Petri dishes. Similar observations

were recorded in turmeric for regeneration of shoots from

rhizomes of various sizes, but survival of plantlets regenerated

from bigger sized rhizomes were higher and also grew faster

than that from small- and medium-sized rhizomes (Shirgurkar

et al., 2001). In our view, variation in size of rhizomes within

genotype is not desired for using it as planting material,

therefore, the size of culture vessel may play an important role

to produce the uniform size of rhizomes. In yams bigger-sized

vessels (Magenta box) than the ones used in present study

(culture tubes; 25 mm diameter), resulted in a significant

increase in micro-tuber weight (Jean and Cappadocia, 1991).

The present study describes the protocol for conservation of

diverse germplasm of ginger through rhizome formation.

Following the reported protocol, about 160 genotypes of wild

and cultivated species of Zingiber are being conserved in the In

Vitro Genebank at NBPGR, New Delhi. In vitro conservation of

ginger germplasm for extended period using this protocol allow

cost-efficient conservation as the cultures could be stored at

25 � 2 8C in the form of rhizomes. Rhizomes, maintained at

25 8C, can withstand power failures for short duration, which

frequently occur in many developing tropical countries; such

rhizomes also can be transplanted directly (without hardening

treatment) to the soil in case of such emergency. It also opens

the new opportunities to exchange of germplasm and produce

disease-free rhizomes in ginger. Future efforts are focused to

develop the cryopreservation protocol for long-term conserva-

tion and evaluate the usefulness of in vitro-formed rhizomes to

be used as disease-free planting material.

Acknowledgements

Authors are grateful to Dr. B.S. Dhillon, Director, NBPGR,

New Delhi, for providing the facilities. Sincere thanks are due

to Mr. R.P. Yadav and Ms. Poonam Dua for their technical help.

Thanks are due to Drs. Ruchira Pandey and Neelam Sharma for

critical reading of the manuscript and providing the sugges-

tions. In addition, all authors are thankful to the two reviewers

of the journal for their useful suggestions. Financial support by

Department of Biotechnology, Government of India, is also

thankfully acknowledged.

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Conservation of Zingiber germplasm through in vitro rhizome formation