micropropagation of the pistachio and its rootstocks by temporary immersion system

1 23

Plant Cell, Tissue and Organ Culture(PCTOC)Journal of Plant Biotechnology ISSN 0167-6857Volume 117Number 1 Plant Cell Tiss Organ Cult (2014)117:65-76DOI 10.1007/s11240-013-0421-0

Micropropagation of the pistachio and itsrootstocks by temporary immersion system

Hülya Akdemir, Veysel Süzerer, AhmetOnay, Engin Tilkat, Yusuf Ersali & YeldaOzden Çiftçi

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media Dordrecht. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

ORIGINAL PAPER

Micropropagation of the pistachio and its rootstocksby temporary immersion system

Hulya Akdemir • Veysel Suzerer • Ahmet Onay •

Engin Tilkat • Yusuf Ersali • Yelda Ozden Ciftci

Received: 26 July 2013 / Accepted: 23 December 2013 / Published online: 1 January 2014

� Springer Science+Business Media Dordrecht 2013

Abstract Although several studies have been reported on

the micropropagation of the pistachio and its rootstocks, to

date none of them had been efficient on the mass production

of these plants in bioreactor systems. Thus, the microprop-

agation of juvenile pistachio shoot tips and nodal buds was

investigated in a temporary immersion bioreactor system

(RITA�) and on a conventional semi-solid medium. Among

the tested immersion conditions, immersion for 24 min

every 16 h reduced vitrification and improved proliferation

in the pistachio. Interactions were evident in immersion time

and frequency in nodal segments. Nodal buds were better

than shoot tips as the highest multiple shoot formation was

recorded in MS medium containing 4 mg L-1 BA and

0.1 mg L-1 GA3 in RITA�. Although shoot tip necrosis

(STN) was observed in shoots proliferated on semi-solid MS

medium, such a symptom did not occur in shoots sprouted in

the RITA�. Additionally, these optimized conditions were

applied to nodal buds of mature male pistachio ‘Atlı’ and

Pistacia rootstocks (P. khinjuk Stocks and P. atlantica

Desf.), and the micropropagation in the bioreactor system, in

comparison to the semi-solid medium, was also improved.

Furthermore, in vitro rooting of pistachio plantlets, despite

the lower range (27.5 %), was also achieved in RITA�.

However, rooting was better on semi-solid medium for all

tested species (ranged between 50 and 70 %). The results of

this study showed that RITA� could be used for the mass

propagation of pistachio and its rootstocks, as well as for

other woody plant species.

Keywords Bioreactor � In vitro proliferation � Pistacia

atlantica Desf. � Pistacia khinjuk stocks � Pistacia vera �RITA�

Abbreviations

BA 6-benzyladenine

GA3 Gibberellic acid

IBA Indole butyric acid

MS Murashige and Skoog medium

RFC Root forming capacity index

SFC Shoot forming capacity index

STN Shoot tip necrosis

Introduction

Pistachio (Pistacia vera L.) and other Pistacia species

cultivation play a vital role in the nutrition and agricultural

economy of many poor communities living in the arid and

semi-arid regions of especially Iran, Turkey, and Syria due

to the adaptation of the trees to harsh desert conditions,

where only a limited number of other Mediterranean spe-

cies can be cultivated (Padulosi et al. 1996). Different

methods of propagation can be used to assist the devel-

opment of pistachio plantations; however, tissue culture is

the most commercially feasible method for producing

uniform plantlets within a short space of time (Krishna-

pillay 2000). Therefore, various studies have been

H. Akdemir (&) � V. Suzerer � Y. O. Ciftci

Department of Molecular Biology and Genetics, Plant

Biotechnology Laboratory, Gebze Institute of Technology,

41400 Gebze, Kocaeli, Turkey

e-mail: [email protected]; [email protected]

A. Onay

Department of Biology, Dicle University, 21280 Diyarbakir,

Turkey

E. Tilkat � Y. Ersali

Department of Biology, Batman University, 72060 Batman,

Turkey

123

Plant Cell Tiss Organ Cult (2014) 117:65–76

DOI 10.1007/s11240-013-0421-0

Author's personal copy

conducted on pistachio explants excised from both in vitro-

grown juvenile seedlings and mature plants by using agar-

based systems (i.e., Barghchi 1982; Barghchi and Alderson

1985; Parfitt and Almehdi 1994; Onay 2000; Ozden-

Tokatli et al. 2005; Tilkat 2006). However, it is well known

that the commercial mass propagation of plants by tissue

culture in conventional agar-based semi-solid media is

labor intensive and costly as gelling agents not only

increase the production costs, but also limit the possibility

of automation (Quiala et al. 2012). Consequently, in vitro

studies focused on liquid cultures excluding the usage of

gelling agents in order to further optimize and reduce

production costs (Ziv 2005).

The superiority of liquid cultures includes not only the

reduction of cost and labor requirements, but also the

improved multiplication rates with scaling up, with or

without automation (Ascough and Fennel 2004). However,

the main drawback of these cultures is the incidence of

hyperhydricity which is a severe physiological disorder

involving apoplastic water accumulation that leads to the

swelling and/or glassy appearance of tissues (Ascough and

Fennel 2004; Berthouly and Etienne 2005) due to the

extended contact between the explants and the liquid

media. In order to overcome this obstacle, various proce-

dures, including the usage of shaking and non-shaking

batch cultures; bubble bioreactors; continuous and dis-

continuous gassing bioreactors; membrane rafts, and tem-

porary immersion systems (TIS) have been developed and

applied to different plant species. Among them, TIS have

been proved to be more suitable as these systems supply

temporary contact of the plant tissues with the medium by

using either the twin-flask system or the RITA� bioreactor

system. The latter was specifically designed for plant tissue

culture (Teisson et al. 1996) and recently successfully

applied to different plant species, including Musa AAB

(Roels et al. 2005); Curcuma zedoaria, Zingiber zerumbet

(Stanly et al. 2010); Siraitia grosvenorii (Yan et al. 2010);

and Dioscorea fordii and D. alata (Yan et al. 2011) for the

micropropagation of shoot microcuttings. This system was

also favorably used for the culture of somatic embryos of

different species such as Citrus (Cabasson et al. 1997);

Camellia sinensis (Akula et al. 2000); Coffea arabica

(Albarran et al. 2005); Bactris gasipaes (Steinmacher et al.

2011), and recently for the production of secondary

metabolites, especially alkaloids such as cardenolides in

Digitalis lanata (Perez Alonso et al. 2012) and galantha-

mine in Leucojum aestivum (Schumann et al. 2012).

However, the application of this system is still limited to

the micropropagation of woody plant species by using

apical and axillary bud proliferation as there are only a few

studies in the literature such as calabash tree (Murch et al.

2004), eucalyptus (McAllister et al. 2005), and teak (Quiala

et al. 2012). Moreover, to our knowledge, there are no

published studies on pistachio micropropagation using

either a liquid culture or TIS.

Various culture parameters such as the frequency and

the duration of immersions, the volume of liquid medium,

the culture container volume, the number of explants, the

aeration and forced ventilation are critically important to

optimize an efficient propagation technique using TIS

(Etienne and Berthouly 2002). Therefore, in this study in

order to develop an efficient micropropagation method for

the pistachio, using RITA�, the influences of different

immersion frequencies (8, 16 or 24 h) and their durations

(8, 16 or 24 min) together with the supplementation of

various plant growth regulators (PGRs) to the medium

were assessed. Moreover, the optimized conditions were

also tested not only on the mature pistachio ‘Atlı’ cultivar,

but also on its rootstocks, including P. atlantica Desf. and

P. khinjuk Stocks to reveal the applicability of this system

for the mass production of the Pistacia species.

Materials and methods

Plant material

Kernels of P. vera ‘Siirt’, P. atlantica Desf. and P. khinjuk

Stocks were disinfected by Ozden Tokatli et al. (2005) and

transferred to a growth regulator free MS medium (Mu-

rashige and Skoog 1962) for germination. After a 3 week

germination, shoot tips and nodal buds were transferred to

MS medium supplemented with 2 mg L-1 BA (6-benzy-

ladenine) and 0.5 mg L-1 GA3 (gibberellic acid) as a

proliferation medium. Nine-year-old in vitro cultures of

mature P. vera ‘Atlı’ were initiated by using the method

developed by Tilkat et al. (2008); and these cultures were

maintained on a modified MS medium with Gamborg

vitamins (Gamborg et al. 1968) supplemented with

1 mg L-1 BA and 0.5 mg L-1 GA3.

Apical and/or nodal buds excised from both in vitro-

germinated seeds of juvenile P. vera ‘Siirt’, P. atlantica

Desf. and P. khinjuk Stocks, and in vitro shoot cultures of

mature P. vera ‘Atlı’ were used for the experiments below.

Plantlets cultured on a semi-solid MS medium containing

different PGRs or combinations (2.0 or 4.0 g L-1 BA with/

without 0.1 or 0.5 g L-1 GA3) were used as controls. All

in vitro cultures regardless of whether on a semi-solid

medium or in RITA� were maintained in a 16 h photo-

period under cool white light (36 lmol m-2 s-1) at

25 ± 2 �C.

Culture conditions in the RITA� bioreactor system

RITA� containers composed of two separated parts, in

which the upper one is for the plant material and the other

66 Plant Cell Tiss Organ Cult (2014) 117:65–76

123


is for the liquid medium, were used as a bioreactor system.

The liquid proliferation medium (200 mL) containing

30 g L-1 sucrose at pH 5.8 and 20 explants (shoot tips or

nodal buds) were placed in each container for all treat-

ments. All in vitro cultures regardless of whether on a

semi-solid medium or in RITA� were maintained in a 16 h

photoperiod under cool white light (36 lmol m-2 s-1) at

25 ± 2 �C.

Determination of the optimum immersion frequency

in the bioreactor

To determine the optimum immersion frequency, P. vera

‘Siirt’ shoot tips and nodal buds were transferred to RITA�

containers containing proliferation medium and the system

was controlled by a timer set at a 16 min immersion every

8, 16 or 24 h.

Determination of the optimum immersion time

in the bioreactor

Following the determination of an optimum immersion

frequency, the effect of different immersion times on pis-

tachio propagation was tested by using ‘Siirt’ shoot tips

and nodal buds and the bioreactor system was set at 8, 16

or 24 min immersion every 16 h.

Influence of different PGRs and combinations

on the micropropagation in the bioreactor

As our preliminary experiments on pistachio micropropa-

gation showed that cytokinins, especially BA alone (Oz-

den-Tokatli et al. 2003) or in combination with GA3

(Ozden-Tokatli et al. 2005) were essential to obtain mul-

tiple shoot formation from nodal segments, MS medium

containing BA (2 or 4 mg L-1) with/without GA3 (0.1 or

0.5 mg L-1) was used to test the PGR influence on pista-

chio ‘Siirt’ propagation in the bioreactor system. As an

optimized condition, the system was set at a 24 min

immersion time every 16 h.

Micropropagation of P. vera ‘Atlı’, P. khinjuk Stocks and

P. atlantica Desf. nodal buds under the optimized

conditions

The optimized conditions were used for micropropagation

of mature pistachio cultivar and Pistacia species. The

bioreactor system with a liquid MS medium containing

4 mg L-1 BA and 0.1 mg L-1 GA3 was set at 24 min

immersion time every 16 h and 20 nodal buds of P. vera

‘Atlı’, P. khinjuk Stocks and P. atlantica Desf. were plated

to the system.

Rooting and acclimatization

In vitro shoots ([2.0 cm) of P. vera ‘Siirt’ from the

bioreactors were plated onto a liquid MS medium sup-

plemented with 4.0 mg L-1 BA and 0.1 mg L-1 GA3

and subcultured in RITA� set at 24 min immersion time

every 16 h for 3 times prior to root induction. Approx.

1 cm elongated shoots were transferred onto the biore-

actor system with a MS liquid medium containing dif-

ferent indole butyric acetic acid (IBA) concentrations (1,

2 or 4 mg L-1) for 4 weeks. As rooting percentage was

not very high in the bioreactor system, in vitro prolif-

erated shoots of all tested species were transferred to

Magenta GA-7 vessels, each containing 50 mL of semi-

solid MS medium with 2 mg L-1 IBA, since the best

rooting capacity in a liquid medium were obtained in

that concentration. The rooted plantlets in semi-solid

medium were washed in running tap water in order to

remove agar before being potted in a sterile 1:1:1 mix-

ture of peat, soil and perlite in plastic cups. Cups were

covered with transparent polythene bags to maintain a

high humidity during hardening for 4 weeks at

25 ± 2 �C under photoperiod of 16 h of white fluores-

cent light (40 lmol m-2 s-1). A 5 week period of

acclimatization by progressive reduction of the humidity

using sterile compost was used for higher plantlet sur-

vival rates. The acclimatized plantlets were transferred to

earthen pots in a greenhouse. After 4 months, the num-

ber of plantlets surviving in the compost was also

scored.

Statistical analysis

Each experiment was repeated twice with at least 40

explants. Following 4 weeks of the proliferation phase,

growth parameters (i.e., proliferation frequency, shoot

number per explant and shoot length) were calculated. The

shoot forming capacity (SFC) index (Lambardi et al. 1993)

of the shoot apices or nodal buds was also calculated.

Moreover, the root forming capacity index (RFC) was

calculated according to the following formula: Rooting

percentage 9 Root number per explant/100. The signifi-

cance of differences was determined by analysis of the

variance (ANOVA) and the means were separated by LSD

(P \ 0.05) multiple range test. Data presented in the pro-

liferation percentages were subjected to Chi square (v2)

analysis. Multiple regression analysis was carried out by

using SPPS 19.0 program to determine possible interac-

tions between not only tested PGRs, but also immersion

time and frequency on multiple shoot formation and shoot

length of both explant types.

Plant Cell Tiss Organ Cult (2014) 117:65–76 67

123


Results

Determination of the optimum immersion frequency

in the bioreactor

P. vera ‘Siirt’ shoot tips on semi-solid MS medium

containing 2 mg L-1 BA and 0.5 mg L-1 GA3 showed

75.6 % proliferation and an average of 2.5 shoot produc-

tion per explant (Table 1). Although relatively longer

shoots (12.4 mm) were obtained in RITA� with a 16 min

immersion frequency per 8 h, the proliferation percentage

and the SFC index were lower (30 % and 0.33, respec-

tively) in comparison to the semi-solid medium. Frequent

immersion (each 8 h) of shoots into the medium in

RITA�, irrespective of explant type, caused formation of

vitrified shoots with vitrified leaves (Fig. 1a). Through

prolonging the immersion frequency up to 16 and 24 h,

proliferation percentages significantly increased (85 and

88 %, respectively) and the vitrification of shoots was

totally prevented (0 %). Moreover, 3.20 and 2.56 shoots

were produced per explant with 16 and 24 h immersion

frequencies, respectively. The highest SFC index (2.72)

was determined when shoot tips were immersed in the

liquid MS medium every 16 h in RITA�. In the case of

the nodal buds, cultures in RITA� with longer immersion

frequencies showed relatively higher proliferation rates in

comparison to those in the conventional semi-solid med-

ium (Table 1). In the semi-solid medium, only 60 % of the

explants proliferated and the SFC index of the nodal buds

was 1.50. Among the various immersion treatments tested,

the lowest proliferation rate (50 %) and a SFC index of

1.18 was obtained with an 8 h frequent immersion time.

On the other hand, with a 16 h immersion frequency in

RITA�, 100 % of the explants produced shoots and SFC

index (3.05) was determined in its highest for this design.

By the extension of the immersion frequency to 24 h, both

the proliferation percentage (88 %) and SFC index were

decreased.

Determination of the optimum immersion time

in the bioreactor

Since 16 h was determined to be the most efficient

immersion frequency, shoot tips and nodal buds were

transferred to RITA� to study the effect of different

immersion times (8, 16 or 24 min). As shown in Table 2,

proliferation percentages of P. vera ‘Siirt’ shoot tips in

RITA� and the conventional semi-solid medium were

above 80 %. The maximum number of shoots produced per

explant and the highest SFC index (2.81) was obtained

when shoot tips were immersed in a liquid medium for

16 min every 16 h. The longest shoots (12.3 mm) were

produced in RITA� with an 8 min immersion time.

Regarding to the nodal buds, with longer immersion times,

100 % proliferation rate was observed in RITA� while

only 65 and 45 % of the nodal buds proliferated in the

semi-solid medium and RITA� with the shortest immer-

sion time, respectively (Table 2). The highest number of

shoots per explant and SFC index together with the longest

shoots was also obtained with the longest immersion time.

The propagation of both of the explant types in RITA�

caused thin shoots in the majority of the tested immersion

times; however, healthier shoots were obtained in RITA�

with a 24 min immersion time (Fig. 1b). In all tested

immersion times, vitrification was not observed in the

shoots proliferated from neither shoot tips nor nodal buds.

Table 1 Effect of different immersion frequencies in RITA� bioreactor system containing MS medium supplemented with 2 mg L-1 BA and

0.5 mg L-1 GA3 on proliferation status of P. vera ‘Siirt’ shoot tips and nodal buds

Frequency of immersion Proliferation* (%) Shoot/explant** Shoot length** (mm) SFC index Vitrification* (%)

Shoot tips

Semi-solid 75.6b 2.50 ± 0.09b*** 9.00 ± 0.50b*** 1.89 0.00b

16 min/8 h 30.0c 1.10 ± 0.40c 12.4 ± 1.8a 0.33 70.0a

16 min/16 h 85.0a 3.20 ± 0.30a 11.0 ± 0.10ab 2.72 0.00b

16 min/24 h 88.0a 2.56 ± 0.31b 11.2 ± 0.30ab 2.25 0.00b

Nodal buds

Semi-solid 60.0c 2.50 ± 0.17a*** 6.00 ± 0.40c*** 1.50 0.00b

16 min/8 h 50.0d 2.35 ± 0.67a 8.80 ± 0.90ab 1.18 50.0a

16 min/16 h 100.0a 3.05 ± 0.29a 11.5 ± 1.40a 3.05 0.00b

16 min/24 h 88.0b 3.16 ± 0.45a 7.30 ± 0.60b 2.78 0.00b

*** Mean ± standard error

* Data were subjected to Chi square (v2) analysis

** Means followed by the different lowercase letter in the column of each explant source are significantly different at P B 0.05 according to the

Post Hoc Multiple Comparisons Test


123


Fig. 1 In vitro proliferation of ‘Siirt’ explants in RITA�. a Vitrified

juvenile P. vera ‘Siirt’ shoots obtained in RITA� with frequent

immersion (each 8 h). b Proliferated P. vera ‘Siirt’ shoots in the

optimized RITA� conditions (24 min immersion every 16 h).

c P. vera ‘Siirt’ plantlets formed in RITA� containing M5 medium.

d P. vera ‘Siirt’ plantlets exhibiting STN in a semi-solid medium

(arrows indicate necrotic region of shoot tips). e–f Extended roots

through holes in the net in RITA� (arrows indicate the roots) (each

bar 0.5 cm)


123


Effect of the different PGRs and its combinations

on micropropagation in the bioreactor

After the determination of the optimum immersion fre-

quency and time (24 min immersion in every 16 h), the

shoot tips and the nodal buds were transferred to RITA�

with different PGR (or combinations) containing liquid

MS medium. The growth parameters (proliferation and,

thus, SFC indices) of the P. vera ‘Siirt’ shoot tips cul-

tured in RITA� were relatively higher than the ones

obtained in semi-solid medium (Table 3). In RITA�, all

shoot tips proliferated (100 %) and produced between

2.75 and 3.70 shoots per explant on M1, M2 or M4

medium. Although the highest multiple shoot formation

(4.50) was obtained in the liquid M6 medium that con-

tained 4 mg L-1 BA and 0.5 mg L-1 GA3, vitrification

was observed on some of the proliferated shoots (10 %).

Vitrification also occurred in 5–20 % of the shoots cul-

tured on a semi-solid medium containing higher concen-

trations of cytokinin (data not shown). In a semi-solid

medium, the highest proliferation percentage (95 %) and

the maximum shoot number per explant (3.35) were

observed on the M2 medium. However, these values were

still significantly lower than the results obtained when the

shoots were cultured in RITA� with the same PGRs

combination. In the case of the nodal buds, the efficiency

of RITA� was relatively better than that of the conven-

tional method on M3, M5 or M6 medium (Table 3).

Among them, M5 media was the optimum as all nodal

buds proliferated and the maximum number of shoots per

explant was obtained (5.25). The SFC indices of the nodal

buds cultured on MS medium with different PGR varied

from 0.87 to 5.25 in the RITA� system, whereas it ranged

from 1.00 to 3.99 in the semi-solid media. However,

relatively shorter shoots were scored in RITA� in com-

parison to the semi-solid media regardless to PGR.

Moreover, Table 3 indicated that the nodal buds were

better than the shoot tips as the highest proliferation

frequency and shoot number were scored on the M5

medium in RITA�. However, it should also be noted that

the nodal buds and the shoot tips grown in RITA� with

the M5 medium, formed thin shoots and small brown

callus (Fig. 1c). In addition, extremely extended leaves

were also obtained in RITA� with subsequent culture on

a GA3 containing medium (data not shown). On the

contrary, more calli formation was observed when

explants were cultured in a semi-solid medium compared

to explants cultured in RITA�. Shoot tip necrosis was

observed in shoots proliferated in a semi-solid medium

(Fig. 1d); however, no such symptom occurred in the

shoots sprouted in RITA�, regardless of the PGR used.

Overall these results proved that there was a significant

interaction (P B 0.05) of BA alone or in combination with

GA3 to multiple shoot formation and shoot length on both

of explants proliferated in TIS system (Table 4). Similar

significant interaction was also demonstrated in immersion

time and frequency in nodal segments whereas only

influence of immersion frequency was significant on mul-

tiple shoot formation from shoot tips.

Micropropagation of P. vera ‘Atlı’, P. khinjuk Stocks

and P. atlantica Desf. nodal buds

Once the protocol conditions were optimized with P. vera

‘Siirt’, the system was then tested with mature nodal buds

of the pistachio ‘Atlı’ cultivar and its rootstocks, including

Table 2 Effect of different immersion times in RITA� bioreactor system containing MS medium supplemented with 2 mg L-1 BA and

0.5 mg L-1 GA3 on proliferation status of P. vera ‘Siirt’ shoot tips and nodal buds

Immersion time Proliferation* (%) Shoot/explant** Shoot length** (mm) SFC index

Shoot tips

Semi-solid 80.0b 2.80 ± 0.50ab*** 10.9 ± 1.00ab*** 2.24

8 min/16 h 90.0a 2.30 ± 0.44b 12.3 ± 1.00a 2.07

16 min/16 h 85.0b 3.30 ± 0.43a 10.1 ± 0.10b 2.81

24 min/16 h 92.0a 2.83 ± 0.24ab 9.50 ± 1.10b 2.60

Nodal buds

Semi-solid 65.0b 2.75 ± 0.56ab*** 9.00 ± 0.80c*** 1.78

8 min/16 h 45.0c 1.30 ± 0.35c 10.2 ± 1.20bc 0.58

16 min/16 h 100.0a 2.65 ± 0.34b 11.0 ± 1.00b 2.65

24 min/16 h 100.0a 3.55 ± 0.29a 14.4 ± 1.40a 3.55






123


P. atlantica Desf. and P. khinjuk Stocks to demonstrate

efficiency of the system. As shown in Table 5, although

the proliferation percentage of the nodal buds (50 %) in the

semi-solid medium was relatively higher than in

the RITA� (20 %) with the nodal buds of the P. vera ‘Atlı’,the multiple shoot formation (1.30) was markedly lower

than in the RITA� (5.25). In the bioreactor system, rela-

tively longer ‘Atlı’ shoots (4.6 mm) were produced with

the higher SFC index (1.05). When P. atlantica Desf. nodal

buds were cultured in RITA�, the proliferation percentage

(40 %) and, thus, the SFC index (0.40) was doubled in

comparison to the nodal buds grown using the conventional

method. Moreover, relatively longer P. atlantica Desf.

shoots (3.8 mm) were also obtained in RITA�. Similarly,

the proliferation of P. khinjuk Stocks nodal buds was also

improved in RITA� as relatively higher proliferation per-

centage (75 %) and higher number of shoots per explant

(2.20) was obtained. Thus, the SFC index was also higher

in RITA� (1.65) in comparison to the semi-solid medium

(0.40).

Rooting and acclimatization

Shoots cultured in root induction medium containing

2 mg L-1 IBA in RITA� produced 3.72 roots on average

with 27.5 % rooting. However, lower (1 mg L-1) or higher

Table 3 Effect of different PGRs and combinations in RITA� bioreactor system set at 24 min immersion every 16 h on proliferation status of P.

vera ‘Siirt’ shoot tips and nodal buds

Medium no PGR combination Proliferation* (%) Shoot/explant** Shoot length** (mm) SFC index

Shoot tips

Semi-solid

M1 2 mg L-1 BA 90.0bc 2.90 ± 0.46b*** 8.10 ± 1.00b*** 2.61

M2 2 mg L-1 BA ? 0.1 mg L-1 GA3 95.0b 3.35 ± 0.46b 9.70 ± 0.60ab 3.18

M3 2 mg L-1 BA ? 0.5 mg L-1 GA3 80.0cd 2.40 ± 0.50bc 10.0 ± 1.00a 1.92

M4 4 mg L-1 BA 85.0c 2.05 ± 0.30d 8.80 ± 0.70b 1.74

M5 4 mg L-1 BA ? 0.1 mg L-1 GA3 90.0bc 2.70 ± 0.40bc 9.20 ± 0.80ab 2.43

M6 4 mg L-1 BA ? 0.5 mg L-1 GA3 75.0d 2.30 ± 0.36c 11.6 ± 1.00a 1.73

RITA�

M1 2 mg L-1 BA 100.0a 2.75 ± 0.26bc 7.40 ± 0.50b 2.75

M2 2 mg L-1 BA ? 0.1 mg L-1 GA3 100.0a 3.55 ± 0.29ab 6.20 ± 0.50c 3.55

M3 2 mg L-1 BA ? 0.5 mg L-1 GA3 92.0bc 2.56 ± 0.31bc 11.2 ± 1.20a 2.35

M4 4 mg L-1 BA 100.0a 3.70 ± 0.44ab 4.90 ± 0.20d 3.70

M5 4 mg L-1 BA ? 0.1 mg L-1 GA3 85.0c 3.70 ± 0.55ab 8.60 ± 1.30b 3.14

M6 4 mg L-1 BA ? 0.5 mg L-1 GA3 90.0bc 4.50 ± 0.40a 10.7 ± 1.20a 4.05

Nodal buds

Semi-solid

M1 2 mg L-1 BA 65.0g 1.55 ± 0.37d 5.70 ± 0.60cd 1.00

M2 2 mg L-1 BA ? 0.1 mg L-1 GA3 95.0b 4.20 ± 0.41b 7.50 ± 0.40b 3.99

M3 2 mg L-1 BA ? 0.5 mg L-1 GA3 65.0g 2.80 ± 0.56c 9.10 ± 0.80a 1.82

M4 4 mg L-1 BA 95.0b 2.40 ± 0.37c 8.50 ± 1.00ab 2.28

M5 4 mg L-1 BA ? 0.1 mg L-1 GA3 90.0c 3.65 ± 0.50b 8.80 ± 0.80ab 3.29

M6 4 mg L-1 BA ? 0.5 mg L-1 GA3 70.0f 2.15 ± 0.44cd 10.2 ± 1.10a 1.51

RITA�

M1 2 mg L-1 BA 60.0h 1.45 ± 0.41d 4.70 ± 0.50de 0.87

M2 2 mg L-1 BA ? 0.1 mg L-1 GA3 85.0d 1.90 ± 0.34d 4.90 ± 0.60d 1.62

M3 2 mg L-1 BA ? 0.5 mg L-1 GA3 95.0b 3.16 ± 0.45bc 7.30 ± 0.60bc 3.00

M4 4 mg L-1 BA 75.0e 1.90 ± 0.38d 4.30 ± 0.40e 1.42

M5 4 mg L-1 BA ? 0.1 mg L-1 GA3 100.0a 5.25 ± 0.34a 6.60 ± 0.60c 5.25

M6 4 mg L-1 BA ? 0.5 mg L-1 GA3 90.0c 4.65 ± 0.51ab 7.90 ± 0.70b 4.18






123


(4 mg L-1) concentrations of IBA resulted in 20 and 7.5 %

rooting percentages, respectively (Table 6). Also, as shown

in Fig. 1e–f, since some roots extended through the holes

of net in the TIS system, the excision of the rooted shoots

from the system was difficult. To overcome this problem,

the same experimental design was repeated by placing

sterile filter paper on the net in the bioreactor (data not

shown), unfortunately this attempt did not succeed.

Since the best rooting percentage was obtained in a MS

medium containing 2 mg L-1 IBA in RITA�, the best

concentration of IBA was tested in a semi-solid medium

for the rooting of both of the pistachio cultivar and its

Table 4 Assessment of interaction of different culture conditions (PGR and immersion cycle) on shoot proliferation of P. vera ‘Siirt’ shoot tips

and nodal buds

Treatment Shoot tip Nodal buds

Shoot/explant Shoot length Shoot/explant Shoot length

Semi-solid TIS Semi-solid TIS Semi-solid TIS Semi-solid TIS

PGR

BA ns s ns ns s s s ns

GA3 ns s s s ns s s s

BA 9 GA3 s s s s s s s s

IT&F

i-IT – ns – ns – s – s

i-IF – s – ns – s – s

s significant, ns not significant at P B 0.05 according to multiple regression analysis

IT&F immersion time and frequency, i-IT increased immersion time, i-IF increased immersion frequency

Table 5 Comparison of semi-solid and RITA� bioreactor system (24 min immersion every 16 h) containing MS medium supplemented with

4 mg L-1 BA and 0.1 mg L-1 GA3 on proliferation status of P. vera ‘Atlı’, P. atlantica and P. khinjuk nodal buds

Species Treatment type Proliferation* (%) Shoot/explant** Shoot length** (mm) SFC index

P. vera ‘Atlı’ Semi-solid 50a 1.30 ± 0.21b*** 2.70 ± 0.40b*** 0.65

RITA� 20b 5.25 ± 2.01a 4.60 ± 0.80a 1.05

P. atlantica Semi-solid 20b 1.00 ± 0.00a 2.50 ± 1.20b 0.20

RITA� 40a 1.00 ± 0.00a 3.80 ± 0.50a 0.40

P. khinjuk Semi-solid 30b 1.33 ± 0.21b 3.00 ± 0.60b 0.40

RITA� 75a 2.20 ± 0.24a 5.80 ± 0.70a 1.65

*Data were subjected to Chi square (v2) analysis

**Means followed by the different lowercase letter in the column are significantly different at P B 0.05 according to the Post Hoc Multiple

Comparisons Test

***Mean ± standard error

Table 6 In vitro rooting of P. vera ‘Siirt’ shoots containing various concentrations of IBA in RITA�

IBA concentration (mg/L) Rooting percentage* (%) Root/explant** Root length** (mm) RFC index

1 20.0b 1.50 ± 0.19b*** 15.3 ± 3.90a*** 0.30

2 27.5a 3.72 ± 1.77a 17.0 ± 2.00a 1.02

4 7.50c 3.66 ± 0.88a 7.60 ± 2.80b 0.27


** Means followed by the different lowercase letter in the column are significantly different at P B 0.05 according to the Post Hoc Multiple

Comparisons Test



123


rootstocks (Table 7). After 4 weeks of culture, rooting was

observed in P. vera ‘Atlı’ (Fig. 2a), ‘Siirt’, P. khinjuk

Stocks (Fig. 2c), P. atlantica Desf.. Shoots generally star-

ted to root within 2 weeks of culture in the medium above

mentioned, but the development of P. atlantica Desf.

shoots were slow. Furthermore, the rooting generally

started 4 weeks after culturing. With 4 weeks culture P.

khinjuk Stocks resulted in 70 % rooting, whereas ‘Atlı’ and

‘Siirt’ genotypes of P. vera L. and P. atlantica Desf. gave

65, 65 and 50 % rooting, respectively (Table 7). The

highest root length (21.73 mm) was obtained in P. khinjuk

Stocks with a lower number of roots (2.05), but relatively

higher number of roots was reported in P. vera ‘Siirt’ and

‘Atlı’ (3.50 and 3.05, respectively) and P. atlantica Desf.

(2.85).

Rooted shoots were transferred to pots containing peat,

soil and perlite (1:1:1) and 80 % of plantlets of P. vera

cultivars, 90 % of P. khinjuk Stocks and 70 % of P. at-

lantica Desf. plantlets survived following to 35 days of

transfer (Table 8). Plantlets regenerated from P. vera cul-

tivars and P. khinjuk Stocks tended to resume shoot growth

very quickly and there were at least two pairs of new leaves

on each plant 5 weeks after transfer to the compost, but the

growth of the P. atlantica Desf. plantlets was very slow.

The acclimatized plantlets became well established upon

transfer to a growth room. The regenerated plantlets

resumed their growth after 4 months. Figure 2b and d show

the acclimatized P. vera ‘Atlı’ and P. khinjuk Stocks

plantlets. Since more than 90 % plant survival was

obtained in the growth room for all of the genotypes

tested, the developed method for plant acclimatization was

satisfactory.

Discussion

Plant production using liquid medium in the bioreactor

systems is a complementary strategy to overcome limitations

(i.e. control of the chemical and/or physical culture

conditions in vessels) present in the agar-based conventional

propagation techniques (Aitken-Christie 1991; Paek et al.

2001, 2005). Since bioreactor systems are generally more

suitable to automation, they lead reductions in cost and labor,

and improvement in plant production with higher yield in

comparison to medium containing agar. Thus, bioreactor

systems are more appealing for scientists and commercial

producers (Preil 2005). Although the permanent contact of

plant tissues with a liquid medium causes physiological

disorders like hyperhydricity (Niemenak et al. 2008), TIS

systems such as RITA� provide temporary contact of plant

tissues with medium to reduce hyperhydricity. Moreover,

they cause gentle ventilation of the air in the bioreactor,

allow sufficient mixing of the culture medium, and the

sequential medium changing and automation (Teisson and

Alvard 1995; Etienne and Berthouly 2002; Albarran et al.

2005; Zhu et al. 2005). The utility of these systems need to be

optimized for each plant species and, sometimes, for each

sort of explant within the same species. To date, there was no

study reported on the shoot proliferation of the pistachio

either in liquid cultures or in TIS. Thus, in the current study,

we investigated the possibility of pistachio micropropaga-

tion in RITA� by assessing different immersion frequencies

and times together with various PGR combinations.

In semi-solid medium, shoots absorb nutrients through

their cut end (Guan and De Klerk 2000) and translocate the

components by water flows in xylem and floem. However,

the medium components are taken up by plants with better

translocation in a liquid medium through leaves via sto-

mata and aqueous pores (Schonherr 2006) and are trans-

ferred to shorter distanced growing regions (De Klerk and

Ter Brugge 2011). Uptake of medium ingredients and

PGRs over the whole plant surface can improve the growth

of plantlets in liquid medium with temporary immersions

(Preil 2005; Quiala et al. 2006). This fact could explain our

results, where the TIS system allowed a significantly higher

shoot proliferation from pistachio shoot tips or nodal buds

compared with that observed in semi-solid medium.

Moreover, the significant interaction obtained in immersion

Table 7 In vitro rooting of pistachio cultivars and its rootstocks in semisolid MS medium containing 2 mg L-1 IBA

Species/

cultivar

Rooting

percentage*

(%)

Root/explant** Root length**

(mm)

RFC index

P. vera ‘Siirt’ 65.0b 3.50 ± 0.66a*** 9.20 ± 1.86ab*** 2.27

P. vera ‘Atlı’ 65.0b 3.05 ± 0.39a 7.72 ± 1.03b 1.98

P. khinjuk Stocks 70.0a 2.05 ± 0.09b 21.73 ± 2.97a 1.44

P. atlantica Desf. 50.0c 2.85 ± 0.02bc 12.23 ± 1.55b 1.43

* Means followed by the different lowercase letter in the column are significantly different at P B 0.05 according to Chi square (v2) analysis

** Means followed by the different lowercase letters in the column are significantly different at P B 0.05 according to the Post Hoc Multiple

Comparisons Test



123


time and frequency on multiple shoot formation of nodal

buds could be due to breakage of apical dominance and

stimulation of lateral bud growth in TIS system (Ziv 2005).

The formation of vitrified shoots, which is one of the

main drawbacks of a liquid medium, was also reduced with

the alteration of the immersion frequency from 8 to 16 h in

the bioreactor system. However, subsequent subcultures of

shoots in semi-solid and bioreactor system containing

medium supplemented with a high level of cytokinin lead

to formation of vitrified shoots. Vitrification can occur

through a combined effect of various factors such as the

supplementation of the medium with growth regulators, the

amount of NH4? and Cl- ions, and the relative humidity in

the culture vessels (Ziv 1991; Ivanova and Van Staden

2011; Debergh et al. 1992). But it is also well-known that

cytokinins and their higher concentrations can increase the

occurrence of vitrification in many species because of their

numerous developmental and physiological influences on

plants (Ivanova and Van Staden 2011). The vitrified shoots

and leaves observed in the MS medium containing

4 mg L-1 BA and 0.1 mg L-1 GA3 in the subsequent

Fig. 2 Rooting of P. vera L. ‘Atlı’ (a, bar 2.5 cm) and P. khinjuk

Stocks (c, bar 2.1 cm) shoots in MS medium containing 2 mg L-1

IBA after 4 weeks of culture and acclimatized plantlets of P. vera L.

‘Atlı’ (b, bar 3.9 cm) and P. khinjuk Stocks (d, bar 7.5 cm) after

4 months of transfer to a peat, soil and perlite mixture 1:1:1

Table 8 Acclimatization of pistachio cultivars and its rootstocks in

sterile compost

Species/cultivar Viable plantlets* (%)

5th week** 4th month***

P. vera ‘Siirt’ 80.0b 90.0c

P. vera ‘Atlı’ 80.0b 95.0b

P. khinjuk Stocks 90.0a 100.0a

P. atlantica Desf. 70.0c 100.0a

* Means followed by the different lowercase letter in the column are

significantly different at P B 0.05 according to Chi square (v2)

analysis

** Data was recorded 35 days after acclimatization

*** Data was recorded 4 months after transfer to growth room


123


subcultures could be due to this high BA concentration, in

addition to other factors compatible with the proliferation

of teak in TIS (Quiala et al. 2012).

Gibberellins can be absorbed through the stem and the

leaves by foliar applications (Weaver et al. 1966). In bio-

reactor systems, as mentioned, whole plant parts, especially

leaves, can absorb medium components directly. Contrary

to leaves obtained from semi-solid medium, the extremely

extended leaves obtained in RITA� with MS medium

containing GA3 can be owing to the direct uptake of this

growth regulator through the leaves.

Besides vitrification, the occurrence of shoot tip necrosis

on micropropagated pistachio shoots continues to emerge

to be a limiting factor on the success of micropropagation

(Vatan Pur Azghandi et al. 2008; Akdemir et al. 2012). In

tissue culture conditions, genotype, nutrient deficiency or

immobility (weak translocation of medium components

through shoots), PGRs, medium type and strength, pH

fluctuations, duration of culture, sulphur content and the

NH4/NO3 ratio of the medium result in shoot tip necrosis

(see Bairu et al. 2009; De Klerk and Ter Brugge 2011).

Shoot tip necrosis was not observed in dahlia shoots in a

liquid medium although it occurred in a semi-solid medium

(De Klerk and Ter Brugge 2011). Similarly, shoot tip

necrosis was observed only in the pistachio shoots grown

on a semi-solid medium, while no such symptom was

evident in the microshoots proliferated in RITA�. The

absence of this disorder in a liquid medium seems to be due

to the more rapid transfer of some important medium

components leading to tip necrosis such as calcium ions to

the top of the stems.

In the literature, although there are varying rooting

responses of pistachio micropropagation in semi-solid

medium (i.e. Tilkat et al. 2008; Tilkat and Onay 2009), this

is the first study concerning the rooting of a woody plant

such as the pistachio in the bioreactor system. However,

the lower rooting percentages in RITA� obtained in our

study compared to a semi-solid medium could be due to the

necessity of negative geotropism in the pistachio plantlets.

The results show that the TIS bioreactor system can also be

used for root formation with the further optimization of the

rooting stage. Shoots of the all species tested were rooted

in semi-solid medium and successfully acclimatized in

sterilized peat, soil and perlite containing compost, with

high percentage of viability obtained in both 5 weeks and

4 months after transfer to in vivo conditions.

Conclusions

To our knowledge, this is the first study on the micro-

propagation of the pistachio and its rootstock in a biore-

actor system. Comparison of the growth parameters in a

liquid culture with that of a semi-solid medium showed that

a bioreactor system was relatively better than the conven-

tional method regarding the Pistacia species, especially

during the proliferation stages. Thus, TIS combined with

optimized immersion parameters and medium with addi-

tives such as PGRs can be an effective method in over-

coming the general obstacles especially of pistachio

micropropagation in a semi-solid medium.

Acknowledgments The study was funded by grant # TBAG-

209T030 from TUBITAK—The Scientific and Technical Research

Council of Turkey. This study was partially supported by a grant

(DUBAP-11-FF-81) from the Dicle University Research Project

Council. The authors would like to thank MSc Ibrahim Koc for

technical support, the researchers of Pistachio Research Center,

Gaziantep, Turkey and Dr. Limane Abdelkrim Mouloud Mammeri,

University of Tizi Ouzou, Algeria for seeds. The authors are also

grateful to the Linda Thain-Ali and Dr. Nezaket Turkel Sesli for

language revision of the manuscript.

References

Aitken-Christie J (1991) Automation. In: Debergh PC, Zimmerman

RH (eds) Micropropagation. Kluwer, Dordrecht, pp 342–354

Akdemir H, Yıldırım H, Tilkat E, Onay A, Ozden Ciftci Y (2012)

Prevention of shoot tip necrosis responses in in vitro-proliferated

mature pistachio plantlets. In: Vitro biology meeting of the

SIVB. Seattle, Washington, p 2

Akula A, Becker D, Bateson M (2000) High-yielding repetitive

somatic embryogenesis and plant recovery in a selected tea

clone, ‘TRI-2025’, by temporary immersion. Plant Cell Rep

19:1140–1145

Albarran J, Bertrand B, Lartaud M, Etienne H (2005) Cycle

characteristics in a temporary immersion bioreactor affect

regeneration, morphology, water and mineral status of coffee

(Coffea arabica) somatic embryos. Plant Cell Tiss Org Cult

81:27–36

Ascough GD, Fennel CW (2004) The regeneration of plant growth

and development in liquid culture. S Afr J Bot 70(2):181–190

Bairu MW, Jain N, Stirk WA, Dolezal K, Van Staden J (2009)

Solving the problem of shoot-tip necrosis in Harpagophytum

procumbens by changing the cytokinin types, calcium and boron

concentrations in the medium. S Afr J Bot 75:122–127

Barghchi M (1982) In vitro propagation of Pistacia species. PhD

Thesis, Nottingham University, UK, p 117

Barghchi M (1986) In vitro micropropagation of Pistacia rootstocks.

Proc Int Plant Prop Soc 35:334–337

Barghchi M, Alderson PG (1985) In vitro propagation of P. vera L.

and commercial varieties of Ohadi and Kelleghochi. J Hortic Sci

60:423–440

Berthouly M, Etienne H (2005) Temporary immersion system: a new

concept. In: Hvolsef-Eide A, Preil W (eds) Liquid culture systems

for in vitro plant propagation. Springer, Dordrecht, pp 165–196

Cabasson C, Alvard D, Dambier D, Ollitrault P, Teisson C (1997)

Improvement of Citrus somatic embryo development by tempo-

rary immersion. Plant Cell Tiss Org Cult 50:33–37

De Klerk GJ, Ter Brugge J (2011) Micropropagation of dahlia in

static liquid medium using slow-release tools of medium

ingredients. Sci Hortic 127:542–547

Debergh P, Aitken-Christie J, Cohen D, Grout B, Von Arnold S,

Zimmerman R, Ziv M (1992) Reconsideration of the term


123


‘vitrification’ as used in micropropagation. Plant Cell Tiss Org

Cult 30:135–140

Etienne H, Berthouly M (2002) Temporary immersion systems in

plant micropropagation. Plant Cell Tiss Org Cult 69:215–231

Gamborg OL, Constanbel F, Shyluk JP (1968) Nutrient requirements

of suspension cultures of soybean root cells. Exp Cell Res

50:151–158

Guan H, De Klerk GJ (2000) Stem segments of apple microcuttings

take up auxin predominantly via the cut surface and not via the

epidermal surface. Sci Hortic 86:23–32

Ivanova M, Van Staden J (2011) Influence of gelling agent and

cytokinins on the control of hyperhydricity in Aloe olyphylla.

Plant Cell Tiss Org Cult 104:13–21

Krishnapillay B (2000) Silviculture and management of teak planta-

tions Unasylva, No. 201 Teak. Int J Forestry Forest Ind (FAO)

51-2000/2

Lambardi M, Sharma KK, Thorpe TA (1993) Optimization of in vitro

bud induction and plantlet formation from mature embryos of

Aleppo pine (Pinus halepensis Mill.). In vitro Cell Dev-Plant

29:189–199

McAllister B, Finnie J, Watt MP, Blakeway F (2005) Use of the

temporary immersion bioreactor system (RITA�) for production

of commercial Eucalyptus clones in Mondi Forests (SA). Plant

Cell Tiss Org Cult 81:347–358

Murashige T, Skoog M (1962) A revised medium for rapid growth

and bioassays with tobacco tissue culture. Physiol Plant 15:

473–497

Murch SJ, Chunzhao L, Romero RM, Saxena PK (2004) In vitro

culture and temporary immersion bioreactor production of

Crescentia cujete. Plant Cell Tiss Org Cult 78:36–68

Niemenak N, Saare-Surminski K, Rohsius C, Ndoumou DO, Lieberei

R (2008) Regeneration of somatic embryos in Theobroma cacao

L. in temporary immersion bioreactor and analyses of free amino

acids in different tissues. Plant Cell Rep 27:667–676

Onay A (2000) Micropropagation of pistachio from mature trees.

Plant Cell Tiss Org Cult 60:159–162

Ozden-Tokatli Y, Ozudogru EA, Akcin A (2003) Enhancement of

regeneration in pistachio (Pistacia vera L.) with silver nitrate. In:

Fifth international symposium in the series ‘recent advances in

plant biotechnology’, High Tatras Slovak Republic, 7–13 Sept

2003, p 21

Ozden-Tokatli Y, Ozudogru EA, Akcin A (2005) In vitro response of

pistachio nodal explants to silver nitrate. Sci Hortic 106:415–426

Padulosi S, Caruso T, Barone E (1996) Taxonomy, distribution,

conservation and uses of Pistacia genetic resources. Report of a

workshop 29–30 June 1995, Palermo

Paek KY, Hahn EJ, Son SH (2001) Application of bioreactors of large

scale micropropagation systems of plants. In Vitro Cell Dev Biol

Plant 37:149–157

Paek KY, Chakrabarty D, Hahn EJ (2005) Application of bioreactor

systems for large scale production of horticultural and medicinal

plants. Plant Cell Tiss Org Cult 81:287–300

Parfitt DE, Almehdi A (1994) Use of high CO2 atmospheric and

medium modifications for the successful micropropagation of

pistachio. Sci Hortic 56:321–329

Perez Alonso N, Capote A, Gerth A, Jimenez E (2012) Increased

cardenolides production by elicitation of Digitalis lanata shoots

cultured in temporary immersion systems. Plant Cell Tiss Org

Cult 110:153–162

Preil W (2005) General introduction: a personal reflection on the use

of liquid media for in vitro culture. In: Preil W, Hvoslef-Eide AK

(eds) Liquid culture systems for in vitro plant propagation.

Springer, Berlin, pp 1–18

Quiala E, Barbon R, Jimenez E, de Feria M, Chavez M, Capote A,

Perez N (2006) Biomass production of Cymbopogon citratus

(DC) Stapf., a medicinal plant, in temporary immersion systems.

In vitro Cell Dev Biol Plant 42(3):298–300

Quiala E, Canal MJ, Meijon M, Rodriguez R, Chavez M, Valledor L,

Feria M, Barbon R (2012) Morphological and physiological

responses of proliferating shoots of teak to temporary immersion

and BA treatments. Plant Cell Tiss Org Cult 109:223–234

Roels S, Escalona M, Cejas I, Noceda C, Rodriguez R, Canal MJ,

Sandoval J, Debergh P (2005) Optimization of plantain (Musa

AAB) micropropagation by temporary immersion system. Plant

Cell Tiss Org Cult 82:57–66

Schonherr J (2006) Characterization of aqueous pores in plant cuticles

and permeation of ionic solutes. J Exp Bot 57:2471–2491

Schumann A, Berkov S, Claus D, Gerth A, Bastida J, Codina C

(2012) Production of galanthamine by Leucojum aestivum shoots

grown in different bioreactor systems. Appl Biochem Biotech

167(7):1907–1920

Stanly C, Bhatt A, Keng CL (2010) A comparative study of Curcuma

zedoaria and Zingiber zerumbet plantlet production using

different micropropagation systems. Afr J Biotech 9(28):

4326–4333

Steinmacher DA, Guerra MP, Saare-Surminski K, Lieberei R (2011)

A temporary immersion system improves in vitro regeneration of

peach palm through secondary somatic embryogenesis. Ann Bot

108(8):1463–1475

Teisson C, Alvard D (1995) A new concept of plant in vitro

cultivation liquid medium: temporary immersion. In: Terzi M

et al (eds) Current issues in plant molecular and cellular biology.

Kluwer, Dordrecht, pp 105–110

Teisson C, Alvard D, Berthouly B, Cote F, Escalant V, Etienne H,

Lartaud M (1996) Simple apparatus to perform plant tissue

culture by temporary immersion. Acta Hortic 440:521–526

Tilkat E (2006) Micropropagation of male Pistacia vera L. via apical

shoot tip culture (PhD thesis, in Turkish). Institute of Science,

University of Dicle, Turkey, p 142

Tilkat E, Onay A (2009) Direct shoot organogenesis from in vitro

derived mature leaf explants of pistachio. In vitro Cell Dev Biol

Plant 45(1):92–98

Tilkat E, Onay A, Yıldırım H, Ozen HC (2008) Micropropagation of

mature male pistachio Pistacia vera L. J Hortic Sci Biotech

83(3):328–333

Vatan Pur Azghandi A, Habashi AA, Taj Abadi Pur A, Mojtahedi N

et al (2008) Developing protocols for mass propagation of

important pistachio rootstocks and commercial cultivars using

tissue culture techniques. Agricultural Biotechnology Research

Institute of Iran, Karaj, p 90

Weaver RJ, Alleweldt G, Pool RM (1966) Absorption and translo-

cation of gibberellic acid. Vitis 5:446–454

Yan HB, Liang CX, Li YR (2010) Improved growth and quality of

Siraitia grosvenorii plantlets using a temporary immersion

system. Plant Cell Tiss Org Cult 103:131–135

Yan HB, Yang L, Li Y (2011) Improved growth and quality of

Dioscorea fordii Prain et Burk and Dioscorea alata plantlets

using a temporary immersion system. Afr J Bio 10(83):

19444–19448

Zhu LH, Li XY, Welander M (2005) Optimisation of growing

conditions for the apple rootstock M26 grown in RITA�

containers using temporary immersion principle. Plant Cell Tiss

Org Cult 81:313–318

Ziv M (1991) Vitrification: morphological and physiological disor-

ders of in vitro plants. In: Debergh PC, Zimmerman RH (eds)

Micropropagation: technology and application. Kluwer, Dordr-

echt, pp 45–69

Ziv M (2005) Simple bioreactors for mass propagation of plants.

In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for

in vitro plant propagation. Springer, Dordrecht, p 588


123


micropropagation of the pistachio and its rootstocks by temporary immersion system

Documents