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Research Article CODEN: IJPRNK ISSN: 2277-8713 Rashmi Panwar, IJPRBS, 2015; Volume 4(5): 381-404 IJPRBS

Available Online at www.ijprbs.com 381

EFFECTS OF PHYTO-HORMONES ON IN VITRO SHOOT INITIATION AND

MULTIPLCATION OF SIMMMONDSIA CHINENSIS

RASHMI PANWAR1, RAMESH JOSHI1, BHANWAR LAL JAT2

1 Department of Botany, SPC Govt. College, Ajmer, Rajasthan, India 2 Department of Botany, Dayanand College, MDS University Ajmer, Rajasthan India .

Accepted Date: 15/10/2015; Published Date: 27/10/2015

Abstract: Jojoba (Simmondsia chinensis) is economically important wind pollinated, evergreen, perennial dioecious shrub

that attains a height of 3-5 m with leathery, grayish-green leaves. Besides i ts superior lubricating properties, Jojoba has attracted interest as a landscape and soil conservation plant. Its origin is the Sonora desert region of Arizona and California in the USA and the s tates of Sonora and Baja California in Mexico. Jojoba is an industrial crop that has attracted worldwide

attention for several reasons like i ts seed contains a liquid wax which, in addition to several miscellaneous uses, can serve as a replacement of sperm whale oil, i t is an extensively drought resistant species ; it can grow in areas of marginal soil fertili ty, high atmospheric temperatures , high soil salinity, low humidity and low fertilizer requirements . The MS medium containing

81.33µM Adenine sulphate with pre-optimized concentrations of BAP (11.02µM) and kinetin (11.61µM) was found optimum for shoot regeneration potential of axillary bud explants of Simmondsia chinenesis. On which maximum number (8-9 shoots) of

shoots were obtained which attaind a hight of 42.6+ 0.05mm.The explants cul tured on the medium containing the PGRs combination of 5.55μM BA + 7.1μM IAA took the minimum days , while those cul tured on the medium containing 11.1μM

BA+12.2μM IBA took the maximum days to bud sprout. The interaction between the PGRs combinations and the genolypes was also found s tatis tically signi ficant. The explants of PKJ-3 took the minimum time to bud sprout when they were cul tured on 5.55μM BA + 7.1μM IAA, followed by those on the same PGRs combination. The explants took the maximum time when

cultured on medium containing 11.1μM BA+12.2μM IBA. The explants of PKJ-3 took the minimum time to bud sprout when they were cul tured on 5.55μM BA + 7.1μM IAA, followed by those on the same PGRs combination. The explants took the maximum time when cultured on medium containing 11.1μM BA+12.2μM IBA. The literature indicates that the number of days to bud sprout depends upon the type of cytokinings , or combination of cytokinins and auxins , thei r concentrations, composition of cul ture media , nature of explant, plant genotype and cul tural conditions .

Keywords: Simmmondsia chinensis, Jojoba, media, in vitro, axillary bud

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Rashmi Panwar, IJPRBS, 2015; Volume 4(5): 381-404



INTRODUCTION

Plants are universally recognized as a vital part of the world’s biological diversity and an

essential resource for the planet. Many thousands of wild plants have great economic and

cultural importance, providing food, medicine, fuel, clothing and shelter for humans around the

world. Plants also play a key role in maintaining the Earth’s environmental balance and

ecosystem stability. They also provide habitats for the world’s animal and insect life. Gradual

depletion of world fossil reserves and emissions of green house gasses are leading to energy

insecurity and ecological imbalance in future. Jojoba (Simmondsia chinensis) is economically

important wind pollinated, evergreen, perennial dioecious shrub that attains a height of 3-5 m

with leathery, grayish-green leaves. Besides its superior lubricating properties, Jojoba has

attracted interest as a landscape and soil conservation plant. Its origin is the Sonora desert

region of Arizona and California in the USA and the states of Sonora and Baja California in

Mexico (Dunstone and Begg, 1983). Jojoba is an industrial crop that has attracted worldwide

attention for several reasons like its seed contains a liquid wax which, in addition to several

miscellaneous uses, can serve as a replacement of sperm whale oil, it is an extensively drought

resistant species; it can grow in areas of marginal soil fertility, high atmospheric temperatures,

high soil salinity, low humidity and low fertilizer requirements (Yermanos, 1982). In all habitats,

including inland desert areas, Jojoba plants were observed to be physiologically active during

the entire year, indicating their capacity in maintaining positive carbon balance even under

severe drought at very low (-36 bar) water potential. Under green house conditions, Jojoba

was reported to maintain positive growth under stress conditions of up to- 70bars (Al-Ani et

al.,1972). A high water use efficiency (0.239 mg CO2/mg H20 per mh) for Jojoba in comparison

to wheat (0.127), sunflower (0.160) and soybean (0.182) was reported in the literature (Rawson

et al., 1977). Jojoba oil has almost the same properties as the oil obtained from the sperm

whale, which is now listed as an endangered species. Indigenous Americans and Indians used

Jojoba seeds and oil for cooking, hair care and for medicinal treatments such as poison ivy,

sores, wounds, colds, cancer and kidney malfunction (Agrawal et al., 2002). Because of all the

above properties, Jojoba oil is claimed as one of nature's gifts to human race or to be liquid gold

from the desert (Bhardwaj et al., 2010). The liquid wax, derived from the seeds, is widely used

as pharmaceutical, and plastic industries, as an additive in mineral oils. In view of its economic

value, Jojoba is gaining rapid popularity and research work on several aspects is going on. It is a

natural high temperature and high pressure lubricant. It has superior lubricating ability and

uniform viscosity over a wide range of temperatures. Jojoba oil is also useful in treatment for

skin diseases such as eczema, acute acne, skin cancers, psoriases, sores, wounds and kidney

malfunctions. It is edible and contains simmondsin, which depresses appetite. It does not grow

rancid and may become suitable for vegetable oil. Jojoba oil may also be used in disinfectants,

detergents, emulsifiers and dryers, plasticizers, protective coating and corrosion inhibitors.This



has increased interest in the agricultural production and development to improve quality and

consistency of yields. The species is dioecious and the seedlings cannot be sexed until the first

flower buds appear 9 to 24 months after sowing. Although Jojoba plants start producing fruit in

3 years, full maturity takes 10 to 12 years, with the plant's life estimated to be 100 years . The

seed is up to 2.5 Cm (1 in.) long, is green to start with and turns brown with age. Fresh seeds

give 80-90% germination, but lose viability with age. Jojoba has a deep root system and is,

therefore, drought resistant. It can also tolerate extreme temperatures ranging from -5 to 540C,

but many flower buds are killed by temperatures of -4 to-70C or lower and hence can be grown

on marginal lands that are not used for conventional agricultural crops. It can be grown in all

types of soils except heavy soils, and has a pH requirement ranging from 5 to 8 (Anonymous,

1975). Micropropagation of the species allows cloning plants of known sex and production.

Jojoba improvement programmes using advanced biotechnological approaches are of immense

value worldwide. Although vegetative propagation can be achieved by rooting semi-hard wood

cuttings (Lee 1988), this approach yields only a limited number of propagules. Plant cell and

tissue culture techniques have immense potential for the improvement of plant species (Singh

et al., 2004). These can be employed for the induction of useful and promising variability

through somaclonal variation, production of synthetic seeds, and production of disease-free

plant utilizing shoot tip and meristem culture, production of industrial compounds,

development of stress-tolerant plants, and for study of biochemical changes during

differentiation. As Jojoba is a dioecious species, usually there are an equal number of male and

female plants in the field. Hence from the viewpoint of seed production, more male plants

reduce the chance of large number of seeds being available for oil extraction. Furthermore, it

takes nearly 5 to 6 years for seeds to be available for oil extraction. Plant ti ssue culture

techniques provide an answer to this problem in two ways. Firstly, it may ensure the desired

ratio (10:90) of male/female Jojoba plants through micropropagation. Consequently, the seed

production may be enhanced. Secondly, in vitro grown callus and zygotic embryos contain oil,

which if extracted by suitable methods (Zia et al., 2007) can minimize our dependence on seeds

for oil production (Lee, 1988; Gabr, 1993). Female plants are commercially more important for

the seed production. Determination of sex at seedling stage is difficult and is possible only

when the plant starts flowering after 1½yr. In addition, a 5:1 male and female ratio, as reported

earlier (Harsh et al., 1987) is yet another major constraint that thwarted large-scale

propagation of desired plants of Jojoba. Differences in various morphological features, like

plant height and leaf size, have also been observed in Jojoba (Gentry, 1958; Kohorn, 1994). It

has also been reported that male plants have a smaller canopy and are taller than females, the

latter having larger leaves. Several attempts have been made to obtain Jojoba plants from

sexually known plants. Llorente and Apostolo (1998) have described the effect of different

growth regulators and genotypes on in vitro propagation of Jojoba. However, none of the

aforesaid findings reported any marker that could be used to differentiate male and female



plants. Chaturvedi and Sharma (1989) reported that both male and female explants behaved

alike morphologically under similar growth regulator and culture conditions. In contradiction,

the differential morphogenic response of nodal explants of male and female Jojoba clones on

media supplemented with different cytokinins has been reported earlier (Agrawal et al., 1999).

Problems and research

Jojoba is a dioecious species, i.e. having separate male and female plants. Only the females,

however, give the valuable seeds. When raised through seeds about 50% or more seedlings are

males. The sex can be recognized only when plants start flowering after 3 - 4 years of planting.

While, for commercial yield only 10% male population is required. As jojoba is a cross pollinated

crop, the progeny is highly heterozygous having tendency to generate seedlings of widely

varying size, shape and yield, which has raised doubts about the economic feasibility of

cultivating Jojoba. The success of Jojoba growers and entire Jojoba industry depends upon

selection of high yielding genotypes and their multiplication through vegetative means.

Therefore the micropropagation is urgently required for large scale propagation of Jojoba with

its known gender.

The present investigation was undertaken to develop a reproducible and reliable tissue culture

protocol for high frequency regeneration of Jojoba with following objectives –

Survey and collection of seeds of C-64, Q-104, Q-107 and C-84 varieties from Jojoba farm

Jaipur and CAZRI Jodhpur to evaluate yield.

Quantitative analysis of seeds in order to estimate wax oil present in them.

Standardization of tissue culture media for high frequency regeneration of micro-shoots

from explants.

Optimization of physio-chemical conditions for introduction of roots in micro-shoots under

in vitro as well as in vivo conditions in order to lower the cost of tissue culture plants.

Development of technology for induction, multiplication and regeneration of direct/indirect

somatic embryo in vitro and their encapsulation to produce artificial seeds for male and

female plants.

Optimization of hardening conditions for in vitro raised plants to achieve high survival rates.

Development of tissue culture protocol for high frequency regeneration of high yielding,

true-to type male female plants of all the varieties of Simmondsia chinensis(Jojoba).



MATERIALS AND METHODS

In vitro Culture Technique

Collection and preparation of explants

Plant materials of Simmondsia Chinensis were collected from surrounding areas of Dhand

village, Jaipur. The disease free, juvenile and healthy explants were selected for carrying out

study as young cells is supposed to have retained their totipotency. The nodal, inter-nodal,

axillary bud and terminal bud explants were collected for establishment of in vitro cultures. The

leaves were removed from the explants and they were cut into small pieces (2-3 nodes/piece)

and washed under running tap water in order to wash off the external dust/contaminants. The

explants were treated with tween-80 solution for 15 min. followed by several washes with tap

water and finally rinsed with distilled water.

Surface sterilization of explants

The washed explants were treated with different concentration of various sterilizing agents for

different duration of treatment. The surface sterilizing agents used were mercuric chloride(0.1-

0.5%w/v), sodium hypo-chloride(1-3%v/v), 90% ethanol and hydrogen peroxide solution(5-

10%w/v).The treatment of surface sterilization with suitable agent was followed by washing of

explants 4-5 times with autoclaved distilled water for removal of surface sterilizing agent from

the surface of explant. The explants were also treated with some specific fungicides where

fungal contaminants were observed in the explants. All surgical instruments, glass-wares and

other accessories were sterilized in autoclave at 121 ºC with 15-20 psi for 15-30 min and then

dried in hot air dryer. All operations for in vitro culture were carried out inside a laminar airflow

cabinet under aseptic conditions. The working platform of hood was wiped clean with paper

towel soaked in 90 % ethanol and sterilized by switching on germicidal ultraviolet light for at

least 10-15 minutes prior to use.

Phyto-hormones /Growth Regulators

Phyto-hormones or growth regulators are required for regulation of cell and tissue growth in

vitro as well in vivo. As an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) or Naphthalene Acetic

Acid (NAA) and Indole acetic Acid (IAA) were frequently used. The concentration of auxins in

the medium is generally between 0.1 to 50 µM. Kinetin or 6-benzyl aminopurine(BAP), as a

cytokinin was required together with auxins at concentrations of 0.1 to 10 µM. Other

derivatives of auxin and kinetin are also used in some cases. Since each plant species requires

different kinds and levels of phytohormones for induction of callus, growth and metabolites

production of cultures. it is important to select the most appropriate growth regulators and to

determine their optimal concentrations. Gibberellic acid and adenine sulfate were also added



individually or in combination with other growth regulators in the medium as and when

required.

Preparation of Stock Solutions

Stock solutions of the major components, such as macronutrients, micronutrients, vitamins,

and plant growth regulators of the media were prepared and stored in refrigerator (table.1).

Powders of different auxins (Sigma, St Louis, MO, USA) in 1N NaOH and cytokinins in 1N HCL

were dissolved and made up to the required volume with sterilized distilled water and then

stored in freezer as stock for further use. Inorganic salts, organic supplements, and vitamins

were used in basal media for initiation, establishment, multiplication and rooting of cultures

under aseptic conditions. The formulation and composition of various media is given in Table.

The concentrated stocks of the major salts, minor salts and growth regulators were prepared

and stored at - 40C.

Preparation of culture media

The composition of plant tissue culture media is inorganic salts, vitamins, amino acids, plant

growth regulators, carbohydrates and the medium matrix. All the components are soluble in

distilled water. Various types of media were used in studies (Table-6), these include MS basal

medium (Murashige and Skoog, 1962) Gamborg’s B5 medium, Liyed and McCown, 1981); WS

medium (Waltor and Skoog, 1963); Hogland nutrient solution (Hogland and Arnon, 1950) and

White medium (White, 1963). All the nutrient media sucrose was used as a source of

carbohydrate, agar- agar (6-8gm per lit) was used as gelling agent in the media. The pH of

medium was adjusted to 5.8+0.2 with 0.5N KOH and 0.1N HCL solutions and then 15 to 20 ml

and 35 to 40 ml medium was poured in tubes and conical flasks respectively followed by

autoclaving of media at 15 pound per square inch (psi) pressure for 15 minutes. Test-tubes

were placed in slanting stands to prepare the slants and transferred to the media storage room

where they were kept under aseptic conditions till their further use. These were then left to

cool and solidify.

Inoculations

The cultures inoculations were carried out under strictly aseptic conditions in laminar air flow

bench fitted with a bactericidal U. V. tube (15 W, peak emission 2637 Ao). The floor of the

chamber was thoroughly scrubbed with cotton dipped in alcohol. The surface of all the vessels

and other accessories such as instruments spatula, forceps, scalpels, blade etc. were also

cleaned with alcohol. The fresh material to be inoculated was kept in a petri dish. Alcohol was

then sprayed in the chamber with the help of an atomizer. The chamber was then sterilized

with U.V. rays continuously for 20-30 minutes. Hands and arms which were scrubbed with



alcohol before inoculation. The rims of the test tubes and the sides of the plugs were flame

sterilized. Instruments (like forceps, scalpels, spatula etc.) were all sterilized by dipping in the

alcohol and flaming a number of times. Care was taken to cool the instruments before putting

into operation. Fresh cuts were given to the stem explants after sterilization to remove

undesirable or dead portions. The explants were then planted on variously augmented media.

Sub culture and multiplication of propagules

After establishment of explants aseptically in culture media these were subcultured either on

same medium or on the modified medium with some changes in ingredients and growth

hormones like Benzyl amino purines (BAP); Kinetin (Kin); Indole 3- acetic acid (IAA), Napthalene

acetic acid (NAA). The explants were sub-cultured onto fresh media every 4-5 weeks. When the

explants started to multiply, well grown microshoots were separated with the help of a sterile

scalpel under the hood and put in the same media for further multiplication. The shoot-lets

derived from each explant were tracked individually to determine the total number of plants

produced from single seed and their subsequent genetic identity. The in vitro raised cultures

were further multiplied by sub-culturing them on to same or modified media.

Culture conditions

The cultures were maintained at the temperature of 25+2˚C with 16 hrs illumination of light of

intensity 2000 to 2500 lux by cool fluorescent tubes and incandescent bulbs and 8 hrs dark. The

temperature and light were varied according to the experiments.

Table-1 Stock solution preparation Murashinge and Skoog media (MS media)

Stocks

Components

Stock Solution (g/l)

Amount (ml/l)

Final concentration

(mg/) Major stock (I) (20X) NH4NO3

KNO3 MgSO4 7H2O KH2PO4

33.0

38.0 7.40 3.40

50

1650.0

1900.0 370.0 170.0

Major stock (II) (20X) CaCl2 2H2O

8.80

50

440.0

Major stock (III) (20X) FeSO4.7H2O

Na2EDTA

0.557

0.745

50

27.85

37.25



Experimentation for in vitro regeneration of plant

Experiments were conducted for finding out suitable season of collection, (Rainy season) type

and size of explant (Juvenile explant 1.5c.m to 2.0c.m length), and also to get optimum

conditions for regeneration of maximum number of shoots from explant, using different culture

media (Semi solid and liquid medium) namely Murashige and Skoog’s medium (MS),

supplemented with cytokinins and auxins, in various concentrations and combinations.

The following range of concentrations of auxins and cytokines were used in all the experiments.

BAP (µM) 2.21, 4.43, 8.87, 13.31, 17.75, 22.19,

Kinetin (µM) 2.32, 4.64, 9.29, 13.93, 18.58, 23.23,

Adenine sulphate (µM) 13.57, 27.14, 54.29, 69.87, 81.44, 135.74, 190.04,

RESULTS

In vitro Shoot initiation and Multiplication

Effect of BAP

Surface sterilized juvenile stem explants of Simmondsia chinensis were inoculated aseptically on

MS medium containing BAP in the concentration ranging from 1.85µM to 21.23µM. The results

showed that the number of shoots produced per explant varied in various concentration of BAP

in MS medium (Fig.-1). Shoot regeneration was achieved within 10-12 days but number and

further growth of micro-shoots were varied in different concentrations of BAP. On the MS

medium without PGR only one shoot initiated from each axillary bud of explant. The

enhancement of shoot primordial began at the BAP 4.13uM and maximum number of explants

(94.4%) showed regeneration with 4-5 of micro-shoots on 16.85uM BAP (Table-1). On the same

Minor stock (100X)

KI H3BO3

MnSO4 4H2O ZnSO4-7H2O

Na2MoO42H2O CuSO4 5H2O

CoCl2 6H2O

0.083 0.62

2.23 0.86

0.025 0.0025

0.0025

10

0.83 6.2

22.3 8.6

0.25 0.025

0.025

Vitamins (100X)

Nicotinic-acid Pyridoxine Hcl

Thiamine Hcl Glycine

0.05 0.05

0.01 0.2

10

0.5 0.5

0.1 2.0



medium the shoots attained a length of 16-17mm in 25-30 days. Further increase in the

concentration of BAP was found to be inhibitory for induction of multiple shoots and their

growth.

Table-1 Effects of BAP in MS medium on shoot induction from axillary bud explant of

Simmondsia chinensis.

Fig 1. Shoot induction from axillary bud explant of Simmondsia chinensis on BAP 16.85µM

Effect of Kinetin

The shoot formation was also achieved from juvenile explants on MS liquid medium

supplemented with various concentrations of kinetin. On medium containing kinetin (2.32µM

to 3.93µM) only one to three shoots were produced from each node with 10mm length (Table-

2). The higher concentrations of Kinetin (18.58µM to 27.87µM) in the medium inhibited the

shoot proliferation and number of shoot from explant remained 2-3 (Fig.-2)

S.No. BAP (µM) Percentage of explant sprouted (Mean+ SD)

Number of shoots per explant (Mean+ SD)

Shoot length (mm)(Mean + SD)

1 0.0 40.2 + 0.30 1.1 + 0.04 3.2 + 0.07389 2 1.85 50.2 + 0.04 1.4 + 0.05 6.4 + 0.07 3 4.13 68.7 + 0.58 2.1 + 0.07 7.3 + 0.05 4 7.97 75.1 + 0.40 2.8 + 0.05 10.9 + 0.06 5 12.50 86.4 + 0.54 3.3 + 0.07 14.8 + 0.09

6 16.85 92.4 + 0.04 4.2 + 0.07 16.4 + 0.04

7 21.23 87.0 + 0.54 3.5 + 0.05 12.7 + 0.05



Table-2 Effects of kinetin in different concentration in MS medium on shoot initiation from

axillary bud explant of Simmondsia chinensis.

Fig 2. Shoot induction from axillary bud explant of S immondsia chinensis on MS + 11.56 µM

Kinetin

Effect of BAP + IAA/NAA

In previous experiment BAP 16.85µM in medium was found suitable for regeneration of

4.2+0.07 shoots, therefore, in present experiment the concentration of BAP 16.85µM was kept

constant with varied concentrations of IAA from 0.53µM to 5.65µM (Table-3) and NAA from

0.51µM to 5.34µM (Table-4).From the results of this experiments, it was observed that the

combination of BAP with IAA/NAA was not effective to enhance percentage response of explant

for shoot induction and also number and growth of micro-shoots in vitro, even percentage

response of explant for regeneration was decreased to 96.7+0.14 on the medium containing

BAP and NAA with little callusing (Fig.- 3A&3B). The addition of auxins such as NAA and IAA with

BAP induce callusing on the surface of explant which results in the reduction of number of

multiple shoots and their further growth in subsequent sub-culturing.

S.No. Kinetin(µM) Explant response (%)for shoot initiation (Mean+ SD)

No. of shoots per explant(Mean+ SD)

Length of shoots in mm(Mean+SD)

1 2.16 60.0 + 0.58 1.2 + 0.05 5.2 + 0.01 2 4.39 69.2 + 0.70 2.4 + 0.08 6.4 + 0.07

3 9.24 76.6 + 0.54 2.6 + 0.08 7.0 + 0.03 4 11.56 92.2 + 0.39 3.8 + 0.07 10.4 + 0.05 5 18.79 87.8 + 0.70 2.2 + 0.08 8.0 + 0.05

6 23.43 84.4 + 0.34 2.4 + 0.05 8.2 + 0.03



Table-3 Effects of different concentrations of IAA with BAP (16.85µM) in MS medium on

shoots initiation from axillary bud explants of Simmondsia chinensis.

(A) (B)

Fig.3 Shoot inAduction from axillary bud explant of Simmondsia chinensis on A. BAP (16.85

µM) + IAA (2.56 µM), B. BAP (16.85 µM) + NAA (3.79 µM).

Table-4 Effects of BAP (16.85µM) with NAA in MS medium on shoot initiation from axillary

bud explant of Simmondsia chinensis.

S.No. I.A.A(µM) Explant response (%) for

shoot initiation (Mean+ SD)

No. of shoots per

explant(Mean+ SD)

Length of shoots in

mm (Mean+ SD)

1 0.53 92.3 + 0.83 3.4 + 0.05 17.2 + 0.08

2 1.69 93.1 + 0.48 3.6 + 0.08 18.8 + 0.05

3 2.56 98.5 + 0.83 4.7 + 0.08 19.3 + 0.05

4 3.89 95.7 + 0.83 3.8 + 0.08 18.5 + 0.08

5 5.65 94.1 + 0.89 3.0 + 0.07 17.3 + 0.08

S.No. NAA(µM) Explant response (%) for

shoot initiation (Mean+ SD)

No. of shoots per

explant(Mean+ SD)

Length of shoots

in mm (Mean+ SD)

1 0.51 90.6 + 0.92 3.0 + 0.07 16.3 + 0.08

2 1.63 91.0 + 0.58 3.2 + 0.05 17.5+ 0.08

3 2.65 95.3 + 0.30 3.6 + 0.08 18.1 + 0.08

4 3.79 96.7 + 0.14 4.1 + 0.08 18.7 + 0.03

5 4.86 94.9 + 0.14 3.5 + 0.03 18.3 + 0.01

6 5.34 90.2+ 0.14 2.3 + 0.04 17.6 + 0.09



Fig. 4. Shoot induction from axillary bud explant of Simmondsia chinensis on Kinetin (13.93

µM) + IAA (2.76 µM)

Effect of kinetin + IAA

Results showed that the kinetin in combination with IAA was not found as effective as BAP for

induction and growth of shoots from axillary buds. The kinetin (13.93µM) with IAA (0.53µM to

5.40µM) was incorporated in the medium. The combination of Kinetin and IAA was also found

ineffective for further increase of shoot numbers and their growth (Table-5). On this medium

again callus formation was observed on cut end of the explant as well as on basal portion of in

vitro raised shoots (Fig.- 3,4).

Table-5. Effects of different concentration of IAA + kinetin (13.93µM) in MS medium on

shoots initiation (regeneration) from axillary buds of Simmondsia chinensis.

Combined effect of BAP and kinetin

In order to further enhance the multiplication of shoot primordia from axillary buds of explants,

attempts were made by incorporating BAP and kinetin in combination in MS liquid medium in

the range of 2.27µM to 17.43µM and 2.32µM to 18.58µM respectively. A mongst all the

concentrations of BAP with kinetin used in combinations on BAP (11.02µM) + Kinetin (11.61µM)

S. No. I.A.A(µM) Percentage of shoot initiation (Mean+ S.D.)

No. of shoots per explant (Mean+ S.D.)

Length of shoots in mm Mean+ S.D.

1 0.53 92.0 + 0.58 3.1 + 0.07 10.1 + 0.04

2 1.61 93.8 + 0.30 3.6 + 0.05 10.4 + 0.08 3 2.76 97.1 + 0.70 3.9 + 0.08 11.5 + 0.01 4 3.89 95.3 + 0.58 3.6 + 0.05 11.3 + 0.08

5 5.40 92.7 + 0.30 3.1 + 0.07 10.7 + 0.08



96.6% explants (Table-6) exhibited shoot regeneration and their growth increased up to 18-19

mm but number of shoots (4-5) remained un-changed. The higher concentration of BAP +

Kinetin was found to be inhibitory for shoot regeneration and their growth in vitro (Fig.- 5). On

the basis of results of previous experiments the BAP (11.02µM) in combination with Kinetin

(11.02µM) in MS medium was found suitable for optimum growth of in vitro shoots, therefore,

this combination of BAP (11.02µM) + Kinetin (11.61µM) in MS medium was used in

combinations with auxins (IAA and NAA) for further experimentation to increase multiple

shoots and their growth from axillary bud explants of Simmondsia chinensis.

Table-6 Effects of different concentration of BAP and kinetin in MS medium on shoots

initiation from axillary bud explant of Simmondsia chinensis.

Fig.5. Shoot induction from axillary bud explant of Simmondsia chinensis on BAP(11.02 µM) +

Kinetin(11.61 µM)

Effects of IAA with BAP and kinetin

The results obtained are presented in Table-7.This experiment was conducted to enhance the

shoot numbers and their growth but 4-5 shoot were regenerated from axillary bud of each

explant and growth of shoots was further decreased with little callusing at the basal end of the

S.No. BAP + Kinetin (µM)

Percentage of shoot initiation (Mean + SD)

No. of shoots per explant (Mean + SD)

Length of shoots in mm (Mean + SD)

1 2.27 + 2.32 70.1 + 0.54 2.1 + 0.07 07.1 + 0.07 2 4.35 + 4.64 76.3 + 0.94 2.5 + 0.05 12.4 + 0.04

3 8.93 + 9.29 82.4 + 0.40 2.7 + 0.04 15.8 + 0.04 4 11.02 + 11.61 96.8 + 0.92 4.4 + 0.03 18.6 + 0.08 5 13.12 + 13.93 89.02 + 0.58 3.2 + 0.07 15.2 + 0.03

6 17.43 + 18.58 85.4 + 0.58 2.4 + 0.05 14.1 + 0.07



regenerated shoots. The maximum 14.8+0.08 mm shoot growth was achieved (Fig.-6) in 30-35

days on MS medium supplemented with BAP (11.09µM) + kinetin (11.61µM) and IAA 1.71µM.

Further increase in concentration of IAA moderate callusing was observed with minimal growth

of shoots. From the results of previous experiments it was observed that addition of IAA with

BAP and kinetin in MS medium the percentage response of explants for shoot induction from

axillary bud explant was un-changed (95% to 98%), shoot number and their growth did not

enhanced. In order to further enhance the shoot numbers, growth and to get stronger shoots

adenine sulphate was tested in different concentrations with optimized concentrations of BAP

(11.02µM) and kinetin (11.61µM) in MS media.

Table-7 Effects of different concentration of IAA in MS medium supplemented with BAP

(11.02µM) and Kinetin (11.61µM) on shoot induction from axillary bud explants of

Simmondsia chinensis.

Fig.6. Shoot induction from axillary bud explant of Simmondsia chinensis on IAA(2.76µM)

with BAP(11.02µM) + Kinetin(11.61 µM)

Effect of ADS with BAP and Kinetin

S. No. IAA (µM) Percentage of shoot initiation(Mean+ S.D.)


Length of shoots in mm (Mean+ S.D.)

1 0.53 97.0 + 0.58 4.2+ 0.01 14.2 + 0.07 * 2 1.61 97.2 + 0.83 4.6 + 0.06 14.2 + 0.08 * 3 2.76 98.3 + 0.83 4.8 + 0.05 13.6 + 0.08 **

4 3.89 97.6 + 0.14 4.0 + 0.02 12.1 + 0.05 **

5 5.40 96.2 + 0.14 3.2 + 0.08 11.1 + 0.07 **



The adenine sulphate was added in the concentration ranges between 13.37µM to 81.33µM

(Table-8). On the medium containing lower concentrations of adenine sulphate from 13.37µM

to 54.39µM, shoot regeneration was poor (4-5 shoots) with little increase in the growth of

shoots to 20mm in the period of 30-35 days. The MS medium containing 81.33µM Adenine

Sulphate with pre-optimized concentrations of BAP (11.02µM) and Kinetin (11.61µM) was

found optimum for shoot regeneration potential of axillary bud explants of Simmondsia

chinensis. On which maximum number (8-9shoots) of shoots were obtained which attained a

height of 42.6+0.05mm. (Fig.- 7).

Table-8 Effects of different concentration of ADS with BAP (11.02µM) and Kinetin (11.61µM)

in MS medium on shoots induction from axillary bud explant of Simmonsia chinensis.

Fig.7. Shoot induction from axillary bud explant of Simmondsia chinensis on ADS( 81.33µM)

with BAP(11.02µM) + Kinetin(11.61 µM).

Shoot multiplication of propagules

Once the optimum physico-chemical conditions for induction and growth of shoots from

axillary bud explant were defined, the in vitro shoot cultures were excised as such in the form

of a cluster and were cut into two or more bunches each consisted of 4-5 shoots and each

bunch was called a propagule. In this way if one mother propagule produced ten shoots (Fig.8 )

after a period of 30-35 days incubation then the same propagule was cut into two daughter

S.No. Adenine sulphate(µM)

Percentage of shoot initiation (Mean+ S.D.)


Length of shoots in mm (Mean+ S.D.)

1 13.37 91.2 + 0.14 4.2 + 0.01 18.0 + 0.07

2 27.58 91.4 + 0.83 4.4 + 0.01 18.2 + 0.06 3 40.12 92.4+ 0.48 4.8 + 0.03 18.4 + 0.06 4 54.39 94.0 + 0.96 4.6 + 0.01 20.0 + 0.02 5 67.16 98.4 + 0.83 7.6 + 0.01 26.2 + 0.01

6 81.33 97.2+ 0.34 8.2 + 0.02 42.6 + 0.05



propagules the number of daughter propagules (4-5 shoots) achieved and from each mother

propagule was presented as rate of multiplication to multiplication (M-M). At the time of sub-

culture of mother propagule the elongated shoots (3-4cm) were excised individually and were

transferred for rooting. The number of shoots harvested for rooting from each mother

propagule was presented as rate of multiplication to rooting (M-R) in the results. The

experiments for shoot multiplication were conducted in liquid as well as in MS media but

chlorosis and browning of shoots were not observed in semi solid media in multiplication

experiments, therefore, only semisolid MS media were used for subsequent experimentations

of multiplication. The M-R was 4.22+0.83 and it enhanced to 6.28+0.83 on MS medium

containing BAP 17.28µM which was further decreased on higher concentration of BAP. The

kinetin was also found to be in effective to increase rate of M-M and M-R (Fig.10) and kinetin

was found to be less effective as compare to BAP (Table-9). In previous experiment M-M and

M-R was very poor and to enhance the rate of M-M and M-R. The IAA with optimized BAP

(13.79µM) was added in the MS medium.

Table-9 Effects of different concentrations of BAP/ kinetin in MS medium on shoot

multiplication of Simmondsia chinensis.

S. No. Kinetin (µM) BAP(µM) (M-M)(Mean + SD) (M-R)(Mean + SD)

1 0.0 0.0 2.0 +0.09 4.22 + 0.83 2 - 2.11 2.0 + 0.07 4.24 + 0.54 3 - 4.26 2.4 + 0.09 5.16 + 1.14 4 - 8.38 2.6 + 0.04 5.04 + 1.14 5 - 13.79 2.6 + 0.04 6.16 + 0.54 6 - 17.28 2.2 + 0.03 6.28 + 0.83 7 - 22.35 2.0 + 0.09 5.38 + 1.48

8 2.31 - 2.6+ 0.06 2.22+ 0.09 9 4.53 - 2.2 +0.04 2.12 +0.03

10 9.42 - 2.8 +0.44 2.16 +0.04 11 13.87 - 2.2 +0.44 3.04 +0.10

12 18.62 - 2.2 +0.83 3.14 +0.14



Fig.8. Shoot multiplication in Simmondsia chinensis on BAP (8.38µM) or Kinetin (9.42 µM)

Effect of IAA with BAP 13.31µM

The addition of IAA in MS medium with BAP 13.31µM for enhancement of rate of multiplication

of propagule as well as rate of multiplication for rooting is shown in Table (10). On the medium

containing IAA 0.57µM with BAP 13.79µM the M-M was 2 fold and by increasing the

concentration of IAA the rate of M-M was further decreased to 1.62+0.12 and callusing on the

base of shoot were observed on the same medium. M-R rate was also decreased from

4.02+0.08 to 3.22+0.10. Hence, the IAA in different concentration with BAP 13.79µM was again

not found suitable for enhancement of M-M and M-R. As we have observed in previous

experiments that ADS was found suitable for induction of maximum number of shoots from

axillary bud explant of Simmondsia chinensis the ADS was again used for multiplication of in

vitro raised shoots with BAP 13.79µM. The addition of ADS in the medium with BAP 13.79µM

effectively increased the M-M rate. On lower concentration of ADS 13.57µM to 54.29µM. The

mother propagule exhibited 4 to 5 fold M-M and 6 to 7 M-R but in higher concentration of ADS

67.87µM and 81.44µM the M-M rate was increased to 5.66 fold and M-R raised to 8.9 fold

(Table-10). On the basis of results of above experiments it was observed that BAP 13.79µM

with ADS 81.44µM was found to be suitable for regeneration of propagules as well as the root

able shoots. To develop a commercially viable protocol it was found necessary to further

increase the M-M and M-R, Therefore the variable concentration of ADS with pre-optimized

concentration of BAP (13.79µM) and kinetin (13.93µM) were used.

Effect of ADS with BAP (13.31µM) and kinetin (13.93µM)

The ADS was added in concentration range of 13.57µM to 81.44µM with pre-optimized BAP

concentration 13.79µM and kinetin 13.93µM. At lower concentration of ADS 13.57µM the M-M

was 5.01+0.01 and M-R was 6.01+0.03. The rates for M-M and M-R were significantly increased

to 6.89+0.04 and 10.11+0.06 respectively in the medium containing 81.44µM ADS with BAP and

kinetin (Fig.-9).

Table-10 Influence of various concentrations of IAA and ADS with BAP (13.31µM) on shoot

multiplication of Simmondsia chinensis.

S.

No.

IAA

(µM)

Adenine sulphate(µM) Rate of M-M(Mean + SD) Rate of M-R(Mean + SD)

1 0.57 - 2.8 +0.04 4.02 +0.08 2 1.14 - 2.01 +0.07 4.22 +0.12

3 1.71 - 2.08 +0.08 3.88 +0.11 4 2.28 - 1.62 +0.12 3.08 +0.08



(A) (B)

Fig.9. Shoot multiplication in Simmondsia chinensis on BAP (13.31 µM) with. IAA(1.71µM)

A and B. ADS (81.44µM).

By further increasing the concentration of ADS, the rate of M-M and M-R was not effectively

enhanced. From the results of all the experiments conducted for multiplication of cultures from

axillary bud explants of Simmondsia chinensis, it was found that BAP 13.79µM and kinetin

13.93µM and ADS 81.44µM in MS semisolid medium was found to be most suitable for high

frequency multiplication of shoots for M-M and M-R. In present study the rate of multiplication

was designated as multiplication to multiplication (M-M) and multiplication to rooting (M-

R).Once in a series of experimentations the BAP and kinetin was standardized and after

optimization of concentration of BAP and kinetin for M-M and M-R then various concentrations

of adenine sulphate was tried with them in combinations.

Table-11 Influence of various concentrations of adenine sulphate with BAP (13.31µM) and

kinetin (13.93µM) on shoots multiplication of Simmondsia chinensis.

5 2.85 - 1.85 +0.03 3.26 +0.09 6 3.42 - 1.09 +0.01 3.22 +0.10

7 - 13.57 3.0 +0.02 6.8 +0.08 8 - 27.14 3.0 +0.03 6.28 +0.07 9 - 40.72 4.0 +0.02 6.88 +0.09 10 - 54.29 4.22 +0.05 7.22 +0.08

11 - 67.87 5.10 +0.04 8.28 +0.09 12 - 81.44 5.66 +0.06 8.92 +0.07

S. No. Adenine sulphate (µM) Rate of M-M(Mean + SD) Rate of M-R(Mean + SD)

1 13.57 5.01 +0.01 6.01 +0.03 2 27.14 5.11 +0.08 6.82 +0.01

3 40.72 5.21+0.08 8.12 +0.07 4 54.29 5.81 +0.09 8.99 +0.06



(A) (B)

Fig.10. A & B Shoot multiplication in Simmondsia chinensis on ADS (81.44µM) with A.

Kinetin(13.93µM) and B. BAP (13.31µM).

DISCUSSION

Although some protocols for in vitro culture of jojoba are known, there are still difficulties

which limit the efficiency and reliability of micro propagation. There are reports on jojoba

somatic embryogenesis (Lee & Thomas 1985; Wang & Janick 1986a,b) and both axillary and

apical buds in vitro culture (Aragao & Hogan 1976; Birnbaum 1976; Scaramuzzi & D' Ambrosio

1988). A number of workers have described culture of single node explants as reported here.

Chaturvedi & Sharma (1989) reported in vitro production of jojoba by proliferation of axillary

buds. Contamination has often a major hurdle in establishing in vitro cultures with up to 90%

losses. In our work only 20% of explants initiated were lost. We observed basal tissue

proliferation when using BA, as was also reported by Lee (1988), Scaramuzzi & D' Ambrosio

(1988.

In the present investigation, The adenine sulphate was added in the concentration ranges

between 13.37µM to 81.33µM . on the medium containing lower concentration of adenine

sulphate from 13.37µM to 54.39µM, shoot regeneration was poor (4-5 shoots) with little

increase in the growth of shoots to 20 mm in the period of 30-35 days. The MS medium

containing 81.33µM Adenine sulphate with pre-optimized concentrations of BAP (11.02µM)

and kinetin (11.61µM) was found optimum for shoot regeneration potential of axillary bud

explants of Simmondsia chinenesis. On which maximum number (8-9 shoots) of shoots were

obtained which attaind a hight of 42.6+ 0.05mm. Effect of various concentration of growth

5 67.87 6.22 +0.07 10.22 +0.05

6 81.44 6.89 +0.04 10.11 +0.06



regulators supplemented to MS medium were observed on callus induction and proliferation

from five explants,i.e. leaves, nodes, internodes, shootapices and cotyledonary segments. The

best response for callus induction from cotyledons was obtained in MS medium supplemented

with2ip and NAA (both at 10-6 M). Callus induction from various explants has also been

reported by Arce & Jordan (1988) in jojoba plants. The callus formation was 100% when nodal

segments were cultured on MS medium +0.1 mg/L GA3. Callus was also produced by from the

nodal segments of jojoba on MS +10 mg/L BA or kinetin alone or in combination with 0.2mg/L

IBA. In another study, Sardana & Batra (1998) produced callus from leaf explants of jojoba on

MS +1.0mg/L BAP. A protocol was developed for the induction, maturation and germination of

somatic embryos from leaf tissue of jojoba (Hamama et al., 2001). In their work, explants were

placed on their adaxial sides in petri dishes and maintained in darkness on half strength

Murashige and Skoog basal medium (MS/2). Combination of 2, 4-d (1.35-4.52µM ) with BAP

(1.33-4.43µM ) and 2 synthetic cytokinines, N – (2- chloro-4pyridyl)-N’- phenylurea (1.21-

4043µM ) or (E)-6-[3-(trifluoromrthyl)-but-2-enylamino] purine (1.11-3.71µM ) resulted in

formation of embryogenic cultures and somatic embryos. It is there for conclude d that auxins

and cytokinines both play an important role in regulating the induction and proliferation of

callus. It was observed in the preent investigation that different explants showed differences in

callus induction as regards time and proliferation rate. The cultured explants comprise of

various tissues, either meristematic, parenchymatous or other less differentiated cells. So, the

type of cells exposed to growth regulators perhaps determines the nature of callus. Diverse

group of cells comprising callus, on the other hand, are also triggered differently by the same

hormone. From the results of all the experiments conducted for multiplication of cultures from

axillary bud explants of Simmondsia chinensis, it was found that BAP 13.79µM and kinetin

13.93µM and ADS 81.44µM in MS semisolid medium was found to be most suitable for high

frequency multiplication of shoots for M-M and M-R. In present study the rate of multiplication

was designated as multiplication to multiplication (M-M) and multiplication to rooting (M-R).

Once in a series of experimentations the BAP and kinetin was standardized and after

optimization of concentration of BAP and kinetin for M-M and M-R then various concentrations

of adenine sulphate was tried with them in combinations. The PGRs combinations applied and

the genotypes used significantly affected the parameter. The explants cultured on the medium

containing the PGRs combination of 5.55μM BA + 7.1μM IAA took the minimum days, while

those cultured on the medium containing 11.1μMBA+12.2μM IBA took the maximum days to

bud sprout. The interaction between the PGRs combinations and the genolypes was also found

statistically significant. The explants of PKJ-3 took the minimum time to bud sprout when they

were cultured on 5.55μM BA + 7.1μM IAA, followed by those on the same PGRs combination.

The explants took the maximum time when cultured on medium containing 11.1μM

BA+12.2μM IBA. The literature indicates that the number of days to bud sprout depends upon

the type of cytokinings, or combination of cytokinins and auxins, their concentrations,



composition of culture media, nature of explant, plant genotype and cultural conditions.

Previously, Jacoboni and Standardi (1987) obtgained the best results for meristematic activity in

explants on MS medium containing 4.6μM zeatin + 0.3μMGA3. In the present study, zeatin was

replaced by BA and it was combined with auxins in various concentrations, resultantly an

improvement was recorded. The results partially supported the findings of Elhag et al. (1998),

who recorded a significant genotypic effect and a medium composition (PGRs) effect after 56

days of culture of shoot-tip explaints of jojoba on MS or B5 basal medium containing a

combination of BA at 0.0, 1.3 or 13.3μM with IAA at 0.0 1.7 or 17.1μM. These results are

partially in lines with the findings of Elhag et al. (1998), who recorded the highest number of

newly formed shoots per explants in female plants compared to make ones. They also reported

that higher BA/IAA ratio favoured shoot multiplication. BA alone has a significant role in

increasing number of shoots from jojobas explants as compared to any other cytokings

although clonal differences exist in response to various concentrations of BA, yet the

combination of BA+IAA performed well in the present study. Meyghani et al, (2005) also found

that the number of shoots produced on the medium supplemented with 8.9μM BA and 0.54μM

NAA (5.0 shoots per explant) was higher than the other PGRs combinations. According to Singh

et al, (2004), when the ratio of auxin to some plant constituents (particularly purines like

adenine i.e. BA and kinetin) is low, the meristem tends to form bud and leaf primordia.

However, the present findings contradicted to Scaramuzzi and who found that the shoot

formation was best when the MS medium was suplemented with 44.4μM BA+27.9μM IAA i.e.

the combination consisted of very higher concentrations of growth regulators. So, number of

shoots produced in vitro may depend upon source of explant (Gao and Cao, 2001; Agrawal et

al., 2002), type of explants (Hassan, 2003), composition of media, nature of growth regulators,

their concentrations, plant genotypes (Tyagi and Prakash, 2004), type of vessels and cultural

conditions (Benzioni et al., 2003; Mills et al., 2004). The above cited authors reported the

response to different growth regulators in micropropagation of jojoba but they did not study

the genotype effect. Significant differences among clones were observed in wax quality and

quantity and in seed yield (Ayerza 1996), in salt tolerance, in range of chill requirement etc. We

found considerable differences among clones in response to growth regulators at all stages of

in vitro propagation but, the average of these results were similar with those of randomly

selected population (pool). The described medium is suitable as a starting point for all the

genotypes tested but each clone needs research to optimize its in vitro propagation.



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