Biotechnological advances in jojoba [Simmondsia chinensis (Link) Schneider]: recent developments and prospects for further research
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- Kumar, S., Mangal, M., Dhawan, A.K. et al. Plant Biotechnol Rep (2012) 6: 97. doi:10.1007/s11816-011-0211-2
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Jojoba (Simmondsia chinensis), is a medicinal and oil-yielding, multi-purpose species of the family Simmondsiaceae. The most valuable product of jojoba seed is the liquid wax or jojoba oil which is used extensively in the cosmetic and bio-fuel industry. Propagation of jojoba is possible using conventional methods, but it is time consuming and cumbersome owing to long rotation periods, male-biased population, and long flowering and seed set time. The development of an efficient regeneration system is a prerequisite for a number of biotechnological interventions for the improvement of jojoba, such as genetic transformation, production of useful metabolites in vitro, etc. During the past decade, therefore, several attempts have been made for in vitro propagation of jojoba. Organogenesis has been achieved in this species from mature as well as juvenile explants. Present communication reports an overview of the in vitro regeneration of jojoba via organogenesis and somatic embryogenesis. Factors affecting organogenesis as well as production of synthetic seeds using shoot tips and axillary buds have also been discussed; however, efforts need to be made to develop an efficient genetic transformation system in jojoba. The purpose of this review is to focus upon the current information on in vitro propagation and biotechnological advances made in jojoba.
KeywordsBiochemical changesIn vitro propagationJojobaOrganogenesisSomatic embryogenesisSynthetic seeds
Simmondsia chinensis (Link) Schneider, commonly known as jojoba (pronounced as “ho-ho-ba”), is the sole species in the family Simmondsiaceae and is native to the Sonoran desert of southwestern USA and northern Mexico (Mills et al. 1997). It is an economically important wind pollinated, evergreen, perennial dioecious shrub that attains a height of 3–5 m with leathery, grayish-green leaves. The plants have a long life span (100–200 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 (Harsh et al. 2001). Jojoba has a deep root system and is, therefore, drought resistant. It can also tolerate extreme temperatures ranging from −5 to 54°C, and hence can be grown on marginal lands that are not used for conventional agricultural crops (Bhardwaj et al. 2010). It can be grown in all types of soils except heavy soils, and has a pH requirement ranging from 5 to 8 (Anonymous 1975).
The seeds of jojoba contain a light-gold-colored wax ester (jojoba oil) that makes up 50–55% of the seed weight. The oil contains only traces of saturated wax, steroids, and tocopherol, and has no resins and tars. Jojoba oil has almost the same properties as the oil obtained from the sperm whale (Low and Hackett 1981), which is now listed as an endangered species. The liquid wax and its derivatives have potential in a wide range of applications in cosmetics (lipsticks, face creams, skin fresheners, winter care lotions, shampoos, moisturizers, soaps), lubricants, anti-foaming agents, electrical insulators, and plastic industries (Reddy and Chikara 2010), and also has pharmaceutical (antibiotic production, coating of tablets) and medicinal uses (treatment of skin disorders, sores, wound, burnt skin and to remove stretch marks). Also, the wax resembles human sebum and can help dry and oily skin. It has no cholesterol or triglycerides (Undersander et al. 1990) and therefore can be used as a low calorie edible oil. 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). In the past few years, jojoba oil has emerged as an alternate fuel oil with fewer pollutants (lower emission of CO2, CO and soot because of low carbon content) and exhaust free from harmful SO2. Shelf-life studies have indicated that there is no significant change in the physico-chemical properties of oil up to 9 months (Gupta 2001). 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).
Jojoba is dioecious, and the gender influences the economic value, breeding schemes and opportunities for commercial harvest. Further, the seed maturity is non-synchronous and plants have to be harvested more than once during the ripening season. As harvesting of seeds is mechanical, harvesting costs are high, so efficiencies are relatively low. The advantage of using asexual propagules in commercial jojoba plantations is that these provide uniform and predictable plant growth and yield (Birnbaum et al. 1984; Lee 1988). Clonal propagation of elite individuals of known sexuality is thus necessary to ensure that commercial plantations will be productive (Chaturvedi and Sharma 1989).
Thus, male bias (5 male:1 female) in the population, long flowering and seed set after 3–4 years of transplantation (Sharma et al. 2008; Kumar et al. 2011), and long rotation period are serious problems of jojoba cultivation. Realising these inherent problems, jojoba improvement programmes using advanced biotechnological approaches are of immense value worldwide.
Although vegetative propagation can be achieved by rooting semi-hard wood cuttings (Low and Hackett 1981; 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 (Giri et al. 2004; 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 plants utilizing shoot tip and meristem culture, production of industrial compounds, development of stress-tolerant plants, and for study of biochemical changes during differentiation. This review highlights the major biotechnological advances made in jojoba during the past, and suggests future prospects of tissue culture and genetic engineering techniques in this important plant species.
Achievements made in jojoba through tissue culture
Summary of work done on organogenesis in jojoba (Simmondsia chinensis)
Medium + PGRs
MS + Ze + GA3 + NAA
Jacoboni and Standardi (1987)
MS + IAA + BAP
MS + IBA + IAA
Scaramuzzi and D’Ambrosio (1988)
MSH + BAP + IAA
MSH + IBA + NAA + CA
Chaturvedi and Sharma (1989)
Apical and nodal segments
MS + IAA + 2iP
MS + IBA
Mills et al. (1997)
MS + BA + NAA
MS + IBA
Sardana and Batra (1998)
MMS + BA
MMS + IBA
Llorente and Apostolo (1998)
MDK + BA + AgNO3
MDK + IBA + NAA
Roussos et al. (1999)
MS + ZT + NAA
MS + IBA + IAA
Gao and Cao (2001)
MS + BA
MS + IBA + BA + AC
Agrawal et al. (2002)
MS + BA
½ MS + IBA
Agrawal et al. (2002)
MS + BA + TIBA
½ MS + IBA
Prakash et al. (2003)
MS + BAP + IAA + Ads
MS + IBA + NAA
MS + BA
MS + IBA
Tyagi and Prakash (2004)
MS + BA
MS + IBA
Bashir et al. (2007a)
MS + TDZ + NAA
½ MS + IBA + NAA
Singh et al. (2008)
MS + BAP + NAA
½ MS + IBA
Kumar et al. (2009b)
Encapsulated shoot tips
MS + IBA
Kumar et al. (2010b)
Summary of work done on embryogenesis in jojoba (Simmondsia chinensis)
Medium + PGRs
Induction of somatic embryos
Germination of somatic embryos
Immature zygotic embryo
MS + BA + NAA
Lee and Thomas (1985)
Immature zygotic embryo
MS + 2,4-d
MS + 2,4-d + BA
Wang and Janick (1986a)
Leaf/immature zygotic embryo
MS + 2,4-d
MS + 2,4-d + BA
Wang and Janick (1986b)
½ MS + 2,4-d + BAP + N,N-phenylurea
MS + NAA + BA + IBA
Hamama et al. (2001)
Leaf/immature zygotic embryo
MS + 2,4-d + sucrose (4%)
MS + 2,4-d + BA + sucrose (6%)
Gaber et al. (2007)
Leaf/immature zygotic embryo
MS + 2,4-d + BA + sucrose (4%)
MS + Kn + sucrose
Mohammed et al. (2008)
Organogenesis involves adventitious and axillary shoot production through the formation of unipolar structures, either root or shoot derived from callus or directly from organized tissue (Singh et al. 2004). In jojoba, organogenesis has been induced in vitro both from mature explants (Chaturvedi and Sharma 1989; Mills et al. 1997; Llorente and Apostolo 1998; Tyagi and Prakash 2004; Kumar et al. 2009b) and juvenile explants (Roussos et al. 1999; Gao and Cao 2001; Kumar et al. 2010b).
Factor controlling organogenesis in jojoba
Success of in vitro regeneration depends on the control of morphogenesis, which is influenced by several factors, namely the kinds of tissues or explants, composition of media, plant growth regulators (PGRs), media additives, culture environment, etc. (Rai et al. 2010).
Nutritional requirement for optimal growth of tissue in vitro varies with species (Bhojwani and Razdan 1996), and, therefore, media composition plays a key role in morphogenesis and response of explants. Gautheret (1955) emphasized the importance of nutrition in plant tissue culture. The culture conditions, which favor callus induction, are different from those which induce organogenesis. Since the requirements differ with the type of tissue and the objective, each tissue type required a different formulation depending upon whether the objective is to maintain maximum callus growth or to induce organogenesis (Rai et al. 2010).
Attempts have been made to propagate jojoba on different culture media such as DKW (Driver and Kuniyuki 1984), SH (Schenk and Hildebrandt 1972), or MS (Murashige and Skoog 1962) medium with varying levels of success. MS medium is the most frequently used (Table 1). Several efforts have been made to propagate jojoba in vitro on MS media containing different concentrations of PGRs (Mandani et al. 1978; Lee and Palzkill 1984; Jacoboni and Standardi 1987; Mills and Benzioni 1992; Kacker et al. 1993; Benzioni 1995; Mills et al. 1997; Sardana and Batra 1998; Agrawal et al. 1999, 2002; Gao and Cao 2001; Prakash et al. 2003; Tyagi and Prakash 2004; Bashir et al. 2007a, b, 2008; Singh et al. 2008; Kumar et al. 2009b, 2010b). Chaturvedi and Sharma (1989) developed a protocol for clonal production of jojoba by using modified SH medium fortified with different PGRs, whereas in another studies, jojoba seedling explants were cultured on a modified DKW medium supplemented with various concentrations of BAP, alone and in combination with AgNO3 (Roussos et al. 1999). An increase in KNO3 concentration in the medium improved shoot multiplication rates and in vitro flowering in 20% of male cultures.
The influence of different adjuvants such as activated charcoal (AC), casein hydrolysate (CH), coconut water (CW), polyvinylpyrrolidone (PVP), and triiodobenzoic acid (TIBA) on in vitro clonal propagation of male and female jojoba plants was studied by Prakash et al. (2003), and it was observed that explants of both male and female shoots exhibited differential morphogenic behaviors under the influence of various adjuvants. Agar has most frequently been used as a gelling agent because of its desirable characteristics such as clarity, stability, and its inertness (Pati et al. 2006). Agar minimizes the water loss and allows good nutrient diffusion (Amin and Jaiswal 1984).
Plant growth regulators play an important role in plant tissue culture. The type and concentration of PGRs included in the culture medium largely determine the success of tissue culture work. They directly or indirectly affect the growth and differentiation of plant tissue. A critical role of the auxin/cytokinin ratio is inevitable for inducing root and shoot proliferation (Murashige 1974; Rout et al. 2000; Pati et al. 2006). It has been reported that low auxin and a high concentration of cytokinin in medium favored shoot induction, while a reversed proportion of these two growth regulators promoted root formation, whereas an intermediate concentration caused callus development (McCown and Amos 1979; Harris et al. 1989). The auxins differ significantly in stability, effectiveness, and their influence on the organogenesis (Rumary and Thorpe 1984). Cytokinin levels have been found to be the most critical for multiplication of many important plant species. BAP has been the most common cytokinin used for propagation of jojoba (Llorente and Apostolo 1998; Agrawal et al. 2002; Tyagi and Prakash 2004; Bashir et al. 2007a). A combined effect of BAP with NAA or IAA was also found to be very effective for the establishment and multiplication of cultures in jojoba (Scaramuzzi and D’Ambrosio 1988; Sardana and Batra 1998; Hassan 2003; Kumar et al. 2009b). The superiority of BAP for shoot induction may be attributed to the ability of plant tissues to metabolize BAP more readily than other synthetic growth regulators, or to the ability of BAP to induce production of natural hormones such as zeatin within the tissue (Malik et al. 2005; Rai et al. 2010). While working with different genotypes of jojoba, Bashir et al. (2007a, b, 2008) showed that BA alone was better than kinetin alone or BA + kinetin for in vitro shoot initiation, whereas Singh et al. (2008), working with different mature (20-year-old) genotypes of jojoba, reported that BAP in combination with adenine proved best for shoot induction and multiplication. Other growth-enhancing medium additives including sucrose (Kumar et al. 2009b), adenine sulphate (Hassan 2003) and triiodobenzoic acid (Prakash et al. 2003) also had significant effects on shoot multiplication and elongation in jojoba.
For any micropropagation protocol, successful rooting of micro-shoots is a pre-requisite to facilitate their establishment in soil (Pati et al. 2006). Among auxins, IBA has been commonly used to induce rooting (Raghava Swamy et al. 1992; Nandwani and Ramawat 1993; Shahzad and Siddiqui 2001) on MS medium. In the case of jojoba, experiments involving rooting on either auxin alone or in combination with other hormones showed significant differences (Chaturvedi and Sharma 1989; Tyagi and Prakash 2004; Bashir et al. 2008; Singh et al. 2008). Relatively low salt concentrations in medium are known to enhance rooting of micro-shoots. Several studies on jojoba have indicated that half-strength MS medium was adequate for root induction (Rost and Hinchee 1980; Llorente and Apostolo 1998; Kumar et al. 2009b). Tyagi and Prakash (2004) observed differences in the rooting behavior of male and female plants of jojoba, whereas Singh et al. (2008) could not observe any variation in the rooting response of male and female genotypes. Clonal differences in rooting and subsequent acclimatization have also been recorded in jojoba (Apostolo and Llorente 2000; Apostolo et al. 2001; Bashir et al. 2007a, b, 2008). Activated charcoal, often used in plant tissue culture to improve cell growth and differentiation, alone or sequentially after an auxin, induced rooting of micropropagated shoots (Thomas 2008; Agrawal et al. 2002). Rooting of shoots of jojoba has also been promoted by the addition of caffeic acid (Chaturvedi and Sharma 1989) and cyclodextrins (Apostolo et al. 2001).
After rooting, hardening of regenerants prior to transfer in the soil increases the survival rate of the transferred plants. Various types of substrates have been used during acclimatization such as soil vermiculite mixture (Gulati and Jaiwal 1996; Philomina and Rao 1999), sterilized sand (Thakur et al. 2001), and soil (Dewan et al. 1992). Jojoba plants have also been gradually hardened in ‘Soilrite’ and acclimatized to soil (Agrawal et al. 2002), whereas Meyghani et al. (2005) reported that peat and perlite at a ratio of 1:1 were the most suitable media for transplanting or adaptation of jojoba plantlets. Singh et al. (2008) reported the best survival rate when sand alone was used as a substrate. The survival rate was higher for plantlets which developed roots in vitro in response to IBA. However, no differences were observed during field establishment (Bashir et al. 2008). Kumar et al. (2009b, 2010b) observed 75% field establishment in jojoba plants acclimatized in pre-sterilized soil:sand (1:3) (Fig. 1f).
Somatic embryogenesis is the process by which somatic cells undergo a series of morphological and biochemical changes resulting in the formation of bipolar structures called somatic embryos (Zimmerman 1993; Komamine et al. 2005). Inductive conditions are required for this process in which somatic embryos arise from individual cells and have no vascular connection with the maternal tissue of the explants (Rai et al. 2010). Embryos may develop directly from somatic cells (direct embryogenesis), or the development of recognisable structures can be preceded by numerous, organized, non-embryogenic mitotic cycles (indirect embryogenesis). Somatic embryogenesis has a great potential for clonal multiplication. As compared to organogenesis, somatic embryogenesis provides an ideal experimental process for investigation of plant differentiation, as well as a mechanism for expression of totipotency in plant cells (Litz and Gray 1992). In addition, under controlled environmental conditions, somatic embryos germinate readily, similar to their seedling counterparts.
Besides being useful for clonal multiplication, jojoba regeneration through somatic embryogenesis may also be useful for genetic transformation (Kim and Liu 1999) and to develop new products from jojoba oil (Benzioni 1995). Only a few reports on somatic embryogenesis in jojoba are available, and most of them involve in vitro wax production from immature zygotic embryos (Lee and Thomas 1985; Wang and Janick 1986a, b). Wang and Janick (1986a) showed that immature zygotic embryos cultured in vitro could accumulate up to 39% wax on a dry weight basis. Hamama et al. (2001) developed a protocol for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba. Direct somatic embryogenesis was observed with some zygotic embryo explants, whereas leaf-derived embryogenic calli did not mature on any of the maturation/germination media examined for up to 4 weeks of culture (Mohammed et al. 2008). The comprehensive efforts made towards developing suitable protocols for somatic embryogenesis/indirect organogenesis in jojoba are summarized in Table 2.
Production of synthetic seeds
In plant species where seed propagation is not successful because of heterozygosity of seed, minute seed size, presence of reduced endosperm, and low germination percentage (Saiprasad 2001), synthetic seed technology may be an effective method of propagation. The technique has applications in the field of germplasm storage and transportation of elite germplasm (Krishna and Singh 2007; Rai et al. 2009). In this context, the most important application of synthetic seeds for these plants could be in the exchange of elite and axenic plant material between laboratories, due to small bead size and relative ease of handling these structures (Naik and Chand 2006; Rai et al. 2009, 2010; Kumar et al. 2010b). A number of problems are faced by growers involved in jojoba cultivation, such as its long rotation period, male-biased (5 male:1 female) population, and flowering and seed set after 3–4 years of transplantation (Sharma et al. 2008; Kumar et al. 2011). Therefore, it is a particularly suitable candidate for synthetic seed technology.
Only two reports are available on the production of synthetic seeds in jojoba (Hassan 2003; Kumar et al. 2010b), where shoot tips and axillary buds have been encapsulated and complete plant regeneration from encapsulated shoot tips has been recorded. Hassan (2003) encapsulated apical and axillary buds in jojoba with 6% sodium alginate and 100 mM CaCl2. Encapsulated buds exhibited the best shoot development on MS medium supplemented with BAP (1.0 mg/l) + Ads (40 mg/l) + IAA (3.0 mg/l). Recently, Kumar et al. (2010b) have also employed the encapsulation of shoot tips (excised from multiple shoots raised through nodal segments) for the development of synthetic seeds in jojoba. A gelling matrix of 3% sodium alginate and 100 mM calcium chloride was found most suitable for the formation of ideal calcium alginate beads (Fig. 1c). The best response for shoot sprouting from encapsulated shoot tips was recorded on 0.8% agar-solidified full-strength MS medium (Fig. 1d). Rooting was induced upon transfer of sprouted shoots to 0.8% agar-solidified MS medium containing 1.0 mg/l IBA (Fig. 1e). About 70% of encapsulated shoot tips were rooted and converted into plantlets.
Conservation is an important aspect of synthetic seed technology. In vitro conservation involves the maintenance of explants in a pathogen-free environment for the short, medium or long term (Engleman et al. 2003). The present encapsulation approach could also be applied as an alternative method of propagation of desirable elite genotypes of jojoba and germplasm exchange and distribution.
Biochemical changes during differentiation
Plant regeneration from cultured plant tissues is a prerequisite for the application of in vitro techniques for crop improvement. Often, it is achieved by altering the hormone composition and other components in the media (Smith and Krikorian 1991). Visible manifestation of cell differentiation includes greening of calli, variation in cell wall thickness and biogenesis of certain cytoplasmic organelles such as plastids (Kumar et al. 2009a; Singh et al. 2009). But some tissues are specifically adapted for specialized functions, such as secretion, storage, mechanical support, and protection. Differentiation in such tissues involves differences in basic metabolic pathways (Kumar et al. 2009a). Hence, there is a need to study biochemical aspects underlying the initiation of organized development in vitro.
A perusal of the literature reveals that not much work has been done on the estimation of various metabolites or on the activities of various enzymes in callus cultures during shoot differentiation in jojoba. A sole report by Kumar et al. (2009a) showed that starch content and reducing sugars were high in the calli of jojoba, which further decreased significantly in shoot differentiating cultures. In addition, total phenols were also lower in shoot differentiating cultures. The activity of a-amylase and acid-peroxidase showed an increase up to the appearance of green patches in calli (10–12 days of inoculation) and reached a peak on the 15th day of inoculation that coincided with the appearance of shoots. In conclusion, analysis of the concentrations of various metabolites and the activities of different enzymes during in vitro growth and differentiation of calli provide a reasonable and promising approach towards an understanding of the biochemical basis of various developmental pathways (Singh et al. 2009; Kumar et al. 2009a).
Achievements made in jojoba through molecular approaches
Achievement made in jojoba (Simmondsia chinensis) through molecular approaches
Detection of genetic variability in jojoba
Amarger and Mercier (1995)
Identification of sex in jojoba
Agrawal et al. (2007)
ISSR marker-assisted selection of male and female plants in jojoba
Sharma et al. (2008)
A comparative study of genetic relationships among and within male and female genotypes of jojoba
Sharma et al. (2009)
Comparative assessment of ISSR and RAPD marker assays for genetic diversity analysis in jojoba
Bhardwaj et al. (2010)
Touch-down PCR assay
Gender diagnostic PCR assay for jojoba
Ince et al. (2010)
Assessment of genetic fidelity of micropropagated plants of jojoba
Kumar et al. (2011)
Sex determination of jojoba
Hosseini et al. (2011)
Development of sex-linked AFLP markers in jojoba
Agarwal et al. (2011)
A sole report has been recently published on the assessment of the genetic fidelity of micropropagated plants using RAPD and ISSR markers in jojoba (Kumar et al. 2011). The in vitro-raised plantlets were maintained for up to 12 in vitro subcultures. During the study, a total of 48 (32 RAPD and 16 ISSR) primers were screened, out of which 24 RAPD and 13 ISSR primers produced a total of 191 (126 RAPD and 65 ISSR) clear, distinct and reproducible amplicons. The amplification products were monomorphic across all the selected micropropagated plants and were similar to the mother plant. The RAPD profile of mother plant, the in vitro-raised shoots at the 6th and 12th subculture stages, and 10 randomly selected micropropagated plants of jojoba are shown in Fig. 1g. Detection and analysis of genetic variation can help in understanding the molecular basis of various biological phenomenons in plants (Kumar et al. 2010a). Variations induced in tissue-cultured plants are most likely to be reflected in the banding profiles developed by employing different marker systems. Reports are also available on other aspects of jojoba like sex determination (Agrawal et al. 2007, 2011; Sharma et al. 2008; Hosseini et al. 2011) and genetic diversity analysis (Amarger and Mercier 1995; Sharma et al. 2009; Bhardwaj et al. 2010).
Conclusion and future prospects
Encouraging progress has been made during the recent past on in vitro propagation of jojoba via organogenesis and somatic embryogenesis by manipulation of growth media and culture conditions, and by testing a variety of explant sources. However, there is a need to exploit modern tools of biotechnology and molecular biology for further improvements in jojoba. Efforts need to be made to develop an efficient transformation system in jojoba. For instance, insertion of genes against caterpillars of the pest Heliothis could be helpful in increasing production of jojoba seeds as well as the oil. In addition, in vitro production of useful metabolites has not been attempted in this species; this aspect needs to be addressed in future research efforts. Such efforts will ultimately provide the most rapid advances in jojoba.