PHILIPP AGRIC SCIENTIST ISSN 0031-7454

Vol. 101 No. 4, 369-376

December 2018

Role of Different Nutrient Elements and AgNPs for In Vitro

Shoot Proliferation of GF-677 Rootstock
Hajra Javed1*, Saima Mumtaz2*, Touqeer Ahmad1**, Muhammad Ajmal Bashir1, Ishfaq
Ahmad Hafiz1, and Abdul Razzaq3

1
Departmentof Horticulture, PMAS, Arid Agricultural University, Rawalpindi, Pakistan
2
Horticulture Research Institute, National Agricultural Research Centre, Islamabad
3
Department of Agronomy, PMAS, Arid Agricultural University, Rawalpindi, Pakistan

*
Authors equally contributed
**
Author for correspondence; e-mail: ahmadarid@gmail.com

An efficient protocol is described for in vitro shoot proliferation from apical shoot of economically important
rootstock GF-677 using varied media amalgamations, i.e., ½ Murashige and Skoog (MS), ¾ MS and Woody
Plant Medium (WPM) and different concentrations of Silver Nanoparticles (AgNPs) (5, 10, 15, 20, 25 mg L -1).
The potential of various concentrations of AgNPs on control of bacterial contamination of GF-677 cultures
was also determined. Among different media screened for shoot proliferation of GF-677 rootstock, WPM
showed best interaction for shoot number (4.5 shoots/explant) and leaf area (0.52 cm 2) with 20 mg L-1 and 25
mg L-1 AgNPs, respectively. However, ¾ MS proved to be prosperous media for shoot length (4.92 cm) with
20 mg L-1 AgNPs and fresh (755.1 mg) and dry (84.5 mg) weight on 25 mg L-1 AgNPs. Multiple shoots with
controlled bacterial contamination were also achieved as a result of direct application of AgNPs to the
culture media as compared to media devoid of AgNPs. Hence, inclusion of the appropriate AgNPs
concentration in the culture medium could provide multiple functions so as to control bacterial
contamination in the culture process, resulting in improvement in in vitro growth efficiency of GF-677
rootstock.

Key Words: AgNPs, bacterial contamination, GF-677 rootstock, MS, Woody Plant Medium, shoot proliferation

INTRODUCTION medium (Goncalves et al. 2005), as mineral nutrients play

a dynamic role in shoot proliferation of numerous plant
GF-677 (P. persica × P. amygdalus) is an important species (Aranda-Peres et al. 2009; Kassim et al. 2010).
rootstock widely used for peach and almond cultivars Mineral elements such as AgNPs (Silver nanoparticles)
globally. It is tolerant to drought, iron chlorosis, are well known for multiple shoot induction in many
calcareous and alkaline soils (Ahmad et al. 2003; plant species through inhibition of ethylene action and for
Sepahvand et al. 2012) and fairly resistant to Plum Pox regulating the production of ethylene in cultured vessels,
virus (PPV) (Elektra et al. 2013). Production of this leading to improved growth of plants in vitro (Kumar et
rootstock via hardwoods, semi-hardwoods or soft al. 2009; Sarmast et al. 2011). It is well reported in Coffea
cuttings is hindered because of very low rooting robusta (Giridhar et al. 2003), Prunus armeniaca (Petri et al.
percentage (Ahmad et al. 2003). Hence, in vitro 2005), Sinningia speciosa (Park et al. 2012), Helicteres isora
propagation provides an alternative tool to produce true (Chawla and Bansal 2014) and Malaxis acuminate (Meena
to type plants of economic significance with high et al. 2010). Moreover, AgNPs are well documented for
multiplication rate, eliminating the difficulties hampered their antimicrobial activity, more water solubility, less
by traditional propagation methods (Elsheik and phytotoxicity and low cost availability (Prabhu and
Khalafalla 2010; Victorio et al. 2012). Poulose 2012; Mahna et al. 2013), making them a most
suitable choice for safer control of contamination rate
Success of in vitro grown genotypes largely depend (Sarmast et al. 2011; Nomiya et al. 2004; Safavi et al. 2011).
upon optimized nutrient composition of the culture

The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018)

 AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

In vitro plantlets of GF-677 rootstock were established

by determining the role of different carbon sources and
growth regulators for shoot multiplication (Ahmad et al.
2003; Ahmad et al. 2007; Younas et al. 2008; Hasan et al.
2010). Considering the novel applications of AgNPs for
plant growth improvement as well as contamination
control, the role of different levels of AgNPs and varied
media compositions was explored, in this study, for in
vitro shoot proliferation of GF-677 rootstock. More
explicitly, the impact of AgNPs for the control of the
a b
bacterial contaminants was also determined from in vitro
cultures.

MATERIALS AND METHODS

Shoot tips of 1–2 cm long were collected from healthy and
disease-free peach rootstock ‚GF-677‛ from the
Horticultural Research Institute (HRI), NARC, Islamabad
during the spring season and were rinsed under running c d
tap water for half an hour. Explants were surface-
sterilized in 10% (v/v) sodium hypochlorite (NaOCl) Fig. 1. Response of GF-677 rootstock cultured for 6
wk in vitro, exhibiting shoot number on (a) ½
solution containing two drops of Tween-20 and MS + 20 mg L-1 AgNPs, (b) ¾ MS + 20 mg L-1
thoroughly agitated for 10 min before being rinsed thrice AgNPs, (c) WPM + 20 mg L-1 AgNPs, (d)
with autoclaved distilled water for 5 min each. Explants Stunted growth of explant on AgNPs free
were resized to 1.0 cm and cultured on modified MS media.
medium (Murashige and Skoog 1962) consisting of MS
macro and micro elements, MS vitamins, 100 mg L-1 myo-
RESULTS
inositol, 2 mg L-1 glycine, 30 g L-1 sucrose and 7.0 g L-1
agar. After 8 wk of establishment of cultures, varied types Different compositions of nutrient media, AgNPs and
of nutrient composition were evaluated for shoot their interactions had remarkable effect on shoot number,
proliferation viz., MS medium reduced to half-strength (½ shoot length, leaf number, leaf area, shoot fresh and dry
MS) and three quarters (¾ MS) of their original weights at p<0.05 (Fig. 2). All media compositions (½ MS,
concentration and Woody Plant Medium (WPM) (Lloyd ¾ MS and WPM) interacted significantly with 20 mg L-1
and McCown 1980) (Tables 1 and 2). Each medium was AgNPs, resulting in 3.0, 4.25 and 4.50 number of shoots/
separately fortified with 2 mg L-1 BAP, 0.75 mg L-1 IAA explant, respectively (Fig. 1. a, b, c; Fig 2a). Significant
and 0.5 mg L-1 GA3 and with different concentrations of interaction between media formulations and 20 mg L-1
AgNPs (Nanocid L-2000; average size 16.6 nm) viz., 0, 5, AgNPs was also observed for shoot length with 4 cm, 4.92
10, 15, 20, 25 mg L-1. All media containing different cm and 3.8 cm on ½ MS, ¾ MS and WPM subsequently
concentrations of AgNPs were adjusted to a pH of 5.78 ± (Fig. 2b). Mean leaf area on ½ MS, ¾ MS and WPM was
0.02 before autoclaving at 121 °C and 15 psi for 17 min. relatively higher, i.e., 0.45 cm2, 0.30 cm2 and 0.52 cm2,
Cultured explants in 10 mL media were incubated under respectively, with 25 mg L-1 AgNPs as compared to media
irradiance of 2,000 lux for 16/8h using white fluorescent compositions without AgNPs (Fig. 2c). For mean leaf
tubes (Philips TL 40W/54) at 25 ± 1 °C. Individual shoots number, ¾ MS and WPM were at par with each other
were scored for shoot length (cm), shoot number, leaf with 25 mg L-1 AgNPs exhibiting 52.7 leaf number (Fig.
number, leaf area (cm2), bacterial contamination (%), 1d). The regenerates obtained at AgNP’s free media were
shoot dry and fresh weight (g) after 6 wk of incubation. stunted and had less number of leaves (Fig. 2d). A
Leaf area (cm2) was determined by using Leaf Area Meter significant interaction was also found between media
(LI-3000C) and plantlets were dried at 80 °C for 4 d to amalgamations and the AgNP concentrations regarding
determine their dry weights. Each treatment was fresh and dry weight of shoots produced in GF-677
replicated four times (4 explants/replicates) and means of rootstock (Fig. 2e & f). Mean fresh weight of explants
treatment were separated according to Least Significance increased on ½ MS and ¾ MS included with 25 mg L-1
Difference (LSD) test at p<0.05 using Statistix 8.1 software AgNPs, except for WPM showing less weight (309.7 mg)
(Steel et al. 1997). on similar concentration of AgNPs. Among different

370 The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018)
AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

a d

b e

c f
Fig. 2. Interaction of different media compositions with AgNPs concentration for GF-677 rootstock (a) Shoot number,
(b) Shoot length (cm), (c) Leaf area (cm 2), (d) Leaf number, (e) Fresh and (f) dry weight (mg). Bars denote the
SE.

media screened, WPM resulted in more shoot number

and maximum leaf area while ¾ MS performed better for
shoot length, shoot fresh and dry weight. Thus, shoot
development and elongation varied significantly with
different nutrient media as well as with the concentration
of AgNPs.

In the present study, bacterial contaminantion was

controlled as a result of direct application of AgNPs to the
culture media, without deleterious effect on shoot
regeneration. Bacterial contamination was controlled in
plantlets on medium with 25 mg L-1 AgNPs as compared
to AgNP-free media (Fig. 3). A linear decline is shown in
contamination rate with increase in AgNPs concentration Fig. 3. Influence of various concentrations of AgNPs
on different media compositions which revealed that in different culture media on control of
addition of AgNPs to the culture media considerably bacterial contamination (%) of GF-677
plantlets.

The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018) 371
 AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

Table 1. Media composition for in vitro shoot proliferation of GF-677 root- initiation of shoots which ultimately
stock based on Murashige and Skoog (MS) and Woody Plant Medium increases the leaf number (Shaul 2002).
(WPM). Our results are in parallel with Rahman
Culture Media (mg L-1) et al. (2014) and Thakur and Kanwar
Mineral Salts ½MS ¾MS WPM (2008) who attained maximum shoot
number per explant in Pyrus pashia and
KNO3 950 1425 —-
Pyrus pyrifolia on WPM.
NH4NO3 925 1237.5 400
Ca(NO3)2.4H2O —- —- 556 Plantlets cultured on ¾ MS were long
and had fresh and dry weight more than
CaCl2.2H2O 220 330 96
the other media screened. The difference
MgSO4. 7H2 O 185 277.5 370 of ¾ MS from ½ MS and full-strength
KH2PO4 85 127.5 170 WPM is in the levels of macronutrients,
K2SO4 —- —- 990 i.e., N and K. Thus, it is very feasible to
KI 0.415 0.622 —- assume that the influence of ¾ MS on
FeSO47H2 O 13.92 20.85 27.8 biomass accumulation can be
Na2EDTA 18.65 27.97 37.3 characterized by an additional
MnSO4.4H2O 11.15 16.72 22.3 supplement of these macronutrients.
H3BO3 3.1 4.65 6.0 Potassium stimulates long distance
ZnSO4.7 H2O 4.3 6.45 8.6 nutrient flow in cell extension and helps
in stomatal movements (George et al.
Na2MoO4.2H2O 0.125 0.187 0.25
2008) while, nitrogen is an important
CuSO4.5H2O 0.0125 0.0187 0.25 constituent of many biological
CoCl2.6H2O 0.0125 0.0187 —- molecules playing a major role in plant
growth and development and is readily
available to the plants in the form of NH4+ and NO3- ions
reduces bacterial contamination, with high survival
(Krouk et al. 2011). Unek et al. 2011 reported that the
percentage of GF-677 rootstock.
increase in K concentration enhanced N metabolism and
carbohydrate activity which ultimately increased the
DISCUSSION biomass level. In chrysanthemum the biomass
Inorganic and organic nutrients are major constitutes of accumulation in terms of fresh and dry weight was found
the culture medium used for raising cultures in vitro to depend on NH4+ and NO3- concentration (Sivakumar et
(Ibanez et al. 2003; Akbas et al. 2009; Reddy et al. 2012). al. 2005). Low shoot fresh and dry weight with short
Specific nutrient compositions are being designed for in length on full-strength WPM might be due to high level of
vitro multiplication of many fruit trees (Monteiro et al. phosphorus (P) in WPM as compared to ½ MS and ¾ MS.
2000; Nas and Read 2004; Sotiropoulos et al. 2006; Phosphorus is the crucial element for plant growth,
Sotiropoulos 2007). Statistical analysis of plantlets grown photosynthesis, nutrient movement, transformation of
on ½ MS, suggested the need for more adjustment of sugar into starch and energy transfer, however; more P
some nutrients in the culture medium for multiple shoot contents affect the uptake of many essential ions
induction. Plantlets regenerated on full-strength WPM inhibiting the growth in vitro (George et al. 2008). Similar
had significantly more shoot number, leaf number and observations were carried out by Ciccoti et al. (2008) and
leaf area than all other media scrutinized. This could be Yari et al. (2014) who obtained better results on MS
attributed to high calcium (Ca) contents in full strength medium in Malus spp. and walnut than other media
WPM because Ca has a main contribution in cell division, scrutinized.
enlargement and cell signaling (Reddy 2001; George et al. Novel applications of nano-particles in in vitro
2008). Calcium also maintains the integrity of the plasma propagation are emerging increasingly. Role of AgNPs
lemma leading to nutrient retention in cells and has been explored for multiple shoot induction in many
regulating the selectivity of ion uptake (Hirschi 2004). The horticultural crops (Turhan 2004; Patil et al. 2011;
increase in leaf number on full-strength WPM could also Pratheesh and Kumar 2012) with inhibition of microbial
be accompanied by the presence of high magnesium (Mg) activity in culture medium (Abdi et al. 2008; Jo et al.
level. Magnesium plays significant role in cell elongation 2009). In this study, inclusion of AgNPs in the culture
by activating several enzymes involved in transfer of media had remarkable impact on shoot proliferation of
phosphates whereas, these phosphates are involved in the

372 The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018)
AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

contaminants of woody plants in vitro

Table 2. Comparison of mineral elements in ½ MS, ¾ MS and WPM used
(Anvari et al. 2012). The incorporation of
for in vitro shoot proliferation of GF-677 rootstock.
AgNPs in the culture medium showed
Mineral Elements positive impact toward the control of
½MS ¾MS WPM
(mg L-1) bacterial contamination in GF-677
N 421.96 632.94 205.966 rootstock cultures. Our results are in
concurrence with the earlier
P 19.37 29.06 38.75
investigators who reported that AgNPs
K 391.25 586.87 492.94
help in removal of bacterial
Ca 68.75 103.125 124.23 contamination without deleterious effect
Mg 18.04 27.06 36.09 on plant growth (Panyala et al. 2008;
S 27.74 41.61 236.80 Safavi et al. 2011). Lowest bacterial
I 0.3175 0.476 - contamination percentage was found in
Cl 120.315 180.47 45.984 peach rootstock "GxN15" at 200 mg L-1
AgNPs (Arab et al. 2014). Another
Fe 2.79 4.185 5.58
report by previous examiner showed
Na 2.515 3.77 5.08
successful disinfection of Araucaria excels
Mn 2.75 4.125 5.485
stem explants by using AgNPs in
B 0.55 0.825 1.1
culture medium (Sarmast et al. 2011).
Zn 0.975 1.4625 1.94
Dibrov et al. (2002) reported that the
Mo 0.0495 0.0742 0.099 chemosmotic activity of Ag+ may be
Cu 3.18 4.77 6.36 responsible in affecting the
Co 3.11 4.665 - microorganism. Ag+ ions interact with
MS – Murashige and Skoog, WPM – Woody Plant Medium phospholipids and may substitute the
sulphur in the –SH groups of plasma
GF-677 rootstock. With increase in concentration of membrane of microorganism to destroy them.
AgNPs, shoot number, shoot length, leaf number, mean Additionally, another study indicated that the damaging
leaf area, shoot fresh and dry weight were influenced effects of Ag+ are linked with the production of active Ag+
significantly. Our results find support from the outcomes containing organic compounds and these compounds
of Fuentes et al. (2000) who obtained improved shoot may attract the microbes and destroy their structure
growth in Coffea canephora with the addition of AgNPs in (Tang et al. 2007). AgNP-free media resulted in higher
culture medium. The stimulatory effect of AgNPs on contamination rate on all media compositions, i.e., ½ MS,
shoot induction of GF-677 might be due to the ¾ MS and full strength WPM. These findings are in
interference of silver ion (Ag+) with ethylene signaling accordance with those of Mahna et al. (2013) who
(Sarmast et al. 2015). AgNPs are involved in inhibition of achieved maximum contamination rate in Arabidopsis
ethylene regulation by reducing the receptor capacity to seeds and tomato cotyledons on control treatment.
bind ethylene thus, generating ethylene insensitivity in
plants and improving plant growth (Zhao et al. 2002). It CONCLUSION
was also noticed that the AgNO3 can inhibit the leaf and
shoot tip abscission of the developed shoots of The present work brought into focus an effective in vitro
Holostemma ada-kodien Schult. by reducing the inhibitory regeneration protocol of GF-677 rootstock by making
effect of ethylene (Martin 2002). Plantlets regenerated on adjustments to the mineral composition of the culture
nutrient media supplemented with AgNPs were healthier medium using apical shoots as explant. The outcomes of
than control and had more growth and shoot number in this study indicated that nutrient elements have a specific
Tecomella undulate, Naga Chilli, Sinningia speciosa, Vitex role in in vitro shoot regeneration of GF-677 rootstock. It
negundo, C. Arabica and C. canephora (Giridhar et al. 2003; may also be suggested that the addition of the
Steephen et al. 2010; Aghdaei et al. 2012; Park et al. 2012; appropriate AgNPs concentration in the culture medium
Bora et al. 2014). could provide an operative method to control bacterial
contamination in the culture process, thus improving the
The antimicrobial effects of AgNPs are well known
in vitro efficiency of GF-677 rootstock.
for controlling the exogenous and endogenous

The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018) 373
 AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

CICCOTI AM, BISOGIN C, BATTOCLETTI I,

SALVADORI A, HERDEMERTENS M, JARAUSCH
REFERENCES CITED W. 2008. Micropropagation of apple proliferation-
ABDI GH, SALEHI H, KHOSH-KHUI M. 2008. Nano resistant apomictic Malus sieboldin genotypes. Agron
silver: a novel nanomaterial for removal of bacterial Res 6(2): 445–458.
contaminants in valerian (Valeriana officinalis L.) tissue DIBROV P, DZIOBA J, GOSINK KK, HASE CC. 2002.
culture. Acta Physiol Plant 30: 709–714. Chemiosmotic mechanism of antimicrobial activity of
AGHDAEI M, SALEHI H, SARMAST MK. 2012. Effects of Ag+ in Vibrio cholera. Antimicrob Agents Chemother 46
silver nanoparticles on Tecomella undulata (Roxb.) (8): 2668–2670.
Seem, micropropagation. Adv Hort Sci 26(1): 21–24. ELEKTRA S, HODAJ B, RAMA P. 2013. The influence of
AHMAD T, RAHMAN H, AHMED MS, LAGHARI MH. season collection of explants on micropropagation of
2003. Effect of culture media and growth regulators on peach rootstock GF 677. Albanian J Agric Sci 12(1): 15–
micropropagation of peach rootstock GF-677. Pak J 18.
Bot 35(3): 331–338. ELSHEIK A, KHALAFALLA M. 2010. In vitro shoot
AHMAD T, ABBASI NA, HAFIZ IA, ALI A. 2007. micropropagation and plant establishment of an
Comparison of sucrose and sorbitol as main carbon ornamental plant dumb cane (Dieffenbachia compacta).
energy sources in micropropagation of peach Int J Curr Res 6: 27–32.
rootstock Gf-677. Pak J Bot 39(4): 1269–1275. FUENTES SRL, CALHEIROS MBP, FILHO JM, VIEIRA
AKBAS F, ISIKALAN C, NAMLI S AND AK BE. 2009. LGE. 2000. The effects of silver nitrate and different
Effect of plant growth regulators on in vitro shoot carbohydrate sources on somatic embryogenesis in
multiplication of Amygdalus communis L. cv. Yaltsinki. Coffea canephora. Plant Cell Tissue Organ Cult 6(1): 5–
Afr J Biotech 8(22): 6168–6174. 13.

ANVARI S. G., CARAPETIAN J, DEJAMPOUR J. 2012. GEORGE EF, HALL MA, KLERK GJD. 2008. The
Effects of Nanosilver and Vancomycin in sterilization Components of Plant Tissue Culture Media I: Macro-
of Peach x Almond hybrids in the in vitro cultures. Int and Micro-nutrients. In: George EF et al., editors. Plant
J Agric Sci 2(5): 457–465. Propagation by Tissue Culture. Springer Netherlands.
3rd ed. p. 65–113.
ARAB MM, YADOLLAHI A, MAZINANI MH,
BAGHERI S. 2014. Effects of antimicrobial activity of GIRIDHAR P, INDU EP, RAMU DV, RAVISHANKAR
silver nanoparticles on in vitro establishment of GA. 2003. Effect of silver nitrate on in vitro shoot
G•N15 (hybrid of almond•peach) rootstock. J Genet growth of coffee. Trop Sci 43: 144–146.
Eng Biotechnol 12: 103–110. GONCALVES S, CORREIA PJ, MARTINS-LOUCAO MA,
ARANDA-PERES AN, PERES LEP, HIGASHI EN, ROMANO A. 2005. A new medium formulation for in
MARTINELLI APP. 2009. Adjustment of mineral vitro rooting of carob tree based on leaf
elements in the culture medium for the macronutrients concentrations. Biol Plant 49: 277–280.
micropropagation of three Vriesea bromeliads from HASAN SZ, AHMAD T, HAFIZ IA, HUSSAIN A. 2010.
the Brazilian atlantic forest. The importance of Direct plant regeneration from leaves of Prunus
calcium. Hort Sci 44(1): 106–112. rootstock GF-677 (Prunus amygdalus X P. persica). Pak J
BORA G, GOGOI HK, HANDIQUE PJ. 2014. Effect of Bot 42(6): 3817–3830.
silver nitrate and gibberellic acid on in vitro HIRSCHI KD. 2004. The calcium conundrum. Both
regeneration, flower induction and fruit development versatile nutrient and specific signal. Plant Physiol 36:
in Naga Chilli. Asia Pac J Mol Biol Biotechnol 22(1): 2438–2442.
137–144.
IBAÑEZ A, VALERO M, MORTE A. 2003. Influence of
CHAWLA S, BANSAL YK. 2014. Enhanced effect of cytokinins and subculturing on proliferation capacity
additives on direct and adventitious shoot of single axillary bud microcuttings of Vitis vinifera L.
multiplication in Helicteres isora L. Int J Pharm Sci Rev cv. ‘Napoleon. Anales de Biologia 25: 81–90.
Res 27(1): 261–265.
JO YK, KIM BH, JUNG G. 2009. Antifungal activity of

374 The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018)
AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

silver ions and nanoparticles on phytopathogenic Synthesis and structural characterization of silver (I),
fungi. Plant Dis 93: 1037–1043. aluminium (III) and cobalt (II) complexes with -
isopropyltropolone (hinokitiol) showing noteworthy
KASSIM NE, ABOU-RAYYA SM, ALI EAM. 2010. Effect
biological activities. Action of silver (I)-oxygen
of explant types and different basal nutrient media on
bonding complexes on the antimicrobial activities. J
in vitro growth of bitter almond cuttings during
Inorganic Biochem 98(1): 46–60.
establishment and proliferation stages. J Am Sci 6: 408
–411. PANYALA NR, PENA-MENDEZ EM, HAVEL J. 2008.
Silver or silver nano particles: a hazardous threat to
KROUK G, RUFFEL S, GUTIERREZ A, GOJON A,
environment and human health. J Appl Biomed 6: 117
CRAWFORD NM, CORUZZI GM, LACOMBE BA.
–129.
2011. Framework integrating plant growth with
hormones and nutrients. Tren Pl Sci 16(4): 178-182. PARK E, BAE HH, PARK WT, KIM YB, CHAE SC, PARK
SU. 2012. Improved shoot organogenesis of gloxinia
KUMAR V, PARVATAM G, RAVISHANKAR GA. 2009.
(Sinningia speciosa) using silver nitrate and putrescine
AgNO3-a potential regulator of ethylene activity and
treatment. Pl Omics J 5: 6–9.
plant growth modulator. Elec J Biotech 12(2): 1-15.
PATIL VM, DHANDE GA, THIGALE DM, RAJPUT JM.
LLOYD G, MCCOWN B. 1980. Commercially feasible
2011. Micropropagation of pomegranate (Punica
micropropagation of mountain laurel, Kalma latifolia,
granatum L.) ‘Bhagava’ cultivar from nodal explant.
by use of shoot tip culture. Proc Int Plant Prop Soc 30:
Afr J Biotechnol 10(79): 18130–18136.
421–427.
PETRI C, ALBURQUERQUE N, PEREZ-TORNERO O,
MAHNA N, VAHED SZ, KHANI S. 2013. Plant in vitro
BURGOS L. 2005. Auxin pulses and a synergistic
culture goes nano: nano silver mediated
interaction between polyamines and ethylene
decontamination of ex vitro explants. J Nanomed
inhibitors improve adventitious regeneration from
Nanotech 4: 161.
apricot leaves and Agrobacterium-mediated
MARTIN KP. 2002. Rapid propagation of Holostemma ada- transformation of leaf tissues. Plant Cell Tissue Organ
kodien Schult., a rare medicinal plant, through axillary Cult 82: 105–111.
bud multiplication and indirect organogenesis. Plant
PRABHU S, POULOSE EK. 2012. Silver nanoparticles:
Cell Rep 21: 112–117.
mechanism of antimicrobial action, synthesis, medical
MEENA K, ABRAHAM CJ, MANI B, THOMAS TD. 2010. applications, and toxicity effects. Int Nano Lett (2012)
Adventitious shoot induction from cultured 2: 32.
internodal explants of Malaxis acuminate D. Don, a
PRATHEESH PT, KUMAR MA. 2012. In vitro flowering in
valuable terrestrial medicinal orchid. Plant Cell Tissue
Rosa indica L. Int J Pharm Bio Sci 2(1): 196–200.
Organ Cult 99: 199–208.
RAHMAN UR, GILL MIS, DHILLON WS, BEDI S. 2014.
MONTEIRO ACB, HIGASHI EN, GONCALVES AN,
Micropropagation of Patharnakh (Pyrus pyrifolia
RODRIGUEZ APM. 2000. A novel approach for the
Burm. F.) Nakai) Pear using explants obtained from
definition of the organic medium components for
forced cuttings. Int J Agric Sci VetMed 2(2): 54–65.
micropropagation of yellow passion fruit (Passiflora
edulis Sims. f. flavicarpa Deg.). In Vitro Cell Dev Biol REDDY ASN. 2001. Calcium: Silver bullet in signaling. Pl
Plant 36: 527–531. Sci 161: 381–404.

MURASHIGE T, SKOOG F. 1962. A revised medium for REDDY SH, CHAKRAVARTH M, CHANDRASHEKARA
rapid growth and bioassays with tobacco tissue KN. 2012. In vitro multiple shoot induction through
cultures. Physiol Plant 15: 473–497. axillary bud of Asclepias curassavica L. A valuable
medicinal plant. International Journal of Scientific
NAS MN, READ PE. 2004. A hypothesis for the
Research and Publications (IJSRP) 2(1): 1–7.
development of a defined tissue culture medium of
higher plants and micropropagation of hazelnuts. Sci SAFAVI K, ESFAHANIZADEH M,
Hortic 101: 189–200. MORTAZAEINEZAHAD F, DASTJERD H. 2011. The
study of nano-silver (NS) antimicrobial activity and
NOMIYA K, YOSHIZAWA A, TSUKAGOSHI K,
evaluation of using NS in tissue culture media.
KASUGA NC, HIRAKAWA S, WATANABE J. 2004.
International Conference on Life Science and

The Philippine Agricultural Scientist Vol. 101 No. 4 (December 2018) 375
 AgNPs for In Vitro Shoot Proliferation of GF-677 Rootstock Hajra Javed et al.

Technology IPCBEE Vol. 3. IACSIT Press, Singapore. Phloroglucinol and silver nitrate enhances axillary
shoot proliferation in nodal explants of Vitex negundo
SARMAST MK, NIAZI A, SALEHI H,
L. – an aromatic medicinal plant. Iran J Biotechnol 8
ABOLIMOGHADAM A. 2015. Silver nanoparticles
(2): 82–89.
affect ACS expression in Tecomella undulata in vitro
culture. Plant Cell Tissue Organ Cult 121: 227–236. TANG HJ, FENG H, ZHENG J, ZHAO J. 2007. A study on
antibacterial properties of Ag+-implanted pyrolytic
SARMAST MK, SALEHI H, KHOSH-KHUI M. 2011.
carbon. Surf Coat Technol 201(9–11): 5633–5636.
Nano silver treatment is effective in reducing bacterial
contamination of Araucaria excelsa R. BR. var. Glauca THAKUR A, KANWAR JS. 2008. Micropropagation of
explants. Acta Biol Hung 62(4): 477–484. ‘Wild Pear’ Pyrus pyrifolia (Burm F). Nakai. I. Explant
establishment and shoot multiplication. Not Bot Horti
SEPAHVAND S, EBADI A, KAMALI K,
Agrobot Cluj Napoca 36(1): 103–108.
GHAEMMAGHAMI SA. 2012. Effects of myo-inositol
and thiamine on micropropagation of GF677 (Peach × TURHAN H. 2004. The effect of Silver nitrate (ethylene
Almond Hybrid). J Agric Sci 4(2): 275–280. inhibitor) on in vitro shoot development of potato
(Solanum tuberosum). Biotech 3(1): 72–74.
SHAUL O. 2002. Magnesium transport and function in
plants the tip of iceberg. J Biometals 15(3): 307–321. UNEK C, TANRIVER E, KUDEN AB. 2011. The effects of
different cytokinins on micropropagation of ‘Garnem’
SIVAKUMAR G, KIM SJ, HAHN EJ, PACK KY. 2005.
rootstock. Acta Hort 923: 195–198.
Optimizing environmental factors for the large scale
production of chrysanthemum (Chrysanthemum VICTORIO CP, LAGE CLS, SATO A. 2012. Tissue culture
grandiflorum) in a balloon type bioreactor culture. In techniques in the proliferation of shoots and roots of
Vitro Cell Dev Biol Plant 41: 822–825. Calendula officinalis. Rev Cien Agron 43: 539–545.

SOTIROPOULOS TE. 2007. Effect of NaCl and CaCl2 on YARI MB, GHOLAMI M, KHAZAEI I. 2014. Impact of
growth and content of minerals, chlorophyll, proline media and different cytokinins concentrations on in
and sugars in the apple rootstock M4 cultured in vitro. vitro shoot multiplication of Persian walnut (Juglans
Biol Plant 51(1): 177–180. regia L.). IJFAS 3(2): 203–209.

SOTIROPOULOS TE, FOTOPOULOS S, DIMASSI KN, YOUNAS M., RAHMAN H, SIDDIQUI S, CHAUDHARY
TSIRAKOGLOU V, THERIOS IN. 2006. Response of MF. 2008. Effect of different carbon sources on in vitro
the pear rootstock to boron and salinity in vitro. Biol shoot proliferation and rooting of peach rootstock GF-
Plant 50: 779–781. 677. Pak J Bot 40(3): 1129–1134.

STEEL RGD, TORRIE JH, BOSTON MA. 1997. Principles ZHAO XC, QU, X, MATHEWS DE, SCHALLER GE. 2002.
and procedures of statistics: A Biometric Approach. Effect of ethylene-pathway mutations upon expression
3rd ed. McGraw Hill Book Co. Inc. New York, p. 178– of the ethylene receptor ETR1 from Arabidopsis. Plant
182. Physiol 130(4): 1983–1991.

STEEPHEN M, NAGARAJAN S, GANESH D. 2010.

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